The InteractiveFly: Drosophila as a Model for Human Diseases



Amyotrophic Lateral Sclerosis
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Drosophila genes associated with
Amyotrophic Lateral Sclerosis
Als2
cabeza
futsch
Sod
TDPH (TDP-43)
Vap-33A
Related terms


Glia
Microtubules
Neuromuscular junction
Relevant studies of ALS

Romano, G., Appocher, C., Scorzeto, M., Klima, R., Baralle, F.E., Megighian, A. and Feiguin, F. (2015). Glial TDP-43 regulates axon wrapping, GluRIIA clustering and fly motility by autonomous and non-autonomous mechanisms. Hum Mol Genet 24: 6134-6145. PubMed ID: 26276811

Abstract
Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, this study modulated the expression levels of TDP-43 in the Drosophila glia and found that the protein was required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, it was determined that the glutamate transporter EAAT1 mediates the regulatory functions of TDP-43 in the glia and that genetic or pharmacological compensations of EAAT1 activity is sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in the glia and suggests new experimentally based therapeutic strategies in ALS (Romano, 2015).

Highlights

  • Loss of endogenous glial TDP-43 protein (TBPH) provokes wrapping defects in motor axons.
  • The glial role of TBPH promotes synaptic growth and glutamate receptor clustering.
  • The formation of GluRIIA clusters is early affected by TBPH dysfunctions in glia.
  • dEAAT1 mediates GluRIIA clustering.

Discussion
In addition to neurons, histological aberrations in the distribution of TDP-43 have also been observed in glial cells suggesting that these tissues might be associated with the neurodegenerative process present in ALS. In coincidence with this idea, this study found that the suppression of TDP-43 in the Drosophila glia provokes serious locomotive defects with functional alterations in synaptic transmission followed by early neurodegeneration and reduced life span but, without affecting the number or the survival rate of glial cells in vivo. More importantly, the acute reduction of TBPH function in the glia of adult flies is sufficient to initiate the typical locomotive problems observed in the disease, demonstrating that TBPH function is permanently required in these tissues to prevent neurodegeneration. Although, the Drosophila glia presents consistent molecular and cytological differences with the mammalian astrocytes, the functional characteristics of these tissues are well conserved. In agreement with these observations, it was found that the expression of the human TDP-43 in the Drosophila glia is sufficient to rescue the motility defects observed in TBPH minus larvae, revealing that the molecular role of this protein is well-preserved and analogous consequences could be expected in TDP-43 affected patients (Romano, 2015).

At the subcellular level, it was observed that the suppression of TBPH provokes evident defects in the anatomical organization of the peripheral glia with morphological alterations in the formation of the cytosolic projections that cover the synaptic surface of the presynaptic terminal axons that constitute the NMJs. Although, it is still unknown how these modifications may lead to neurodegeneration, the non-autonomous defects in the transmission of the evoked potentials described in presynaptic motoneurons coincides with analogous alterations found in different experimental models as well as in patients suffering of ALS, proposing that comparable modifications may autonomously initiate the neurological symptoms of the disease or induce the pathological mechanisms of neurodegeneration (Romano, 2015).

It was also observed that the glial function of TBPH is sufficient to rescue the molecular levels and the wild-type distribution of the GluRIIA clusters at the postsynaptic terminals of TBPH null L3 larvae. These molecular parameters were recovered together with the locomotive abilities both in larvae and adult flies, revealing that the glial function of TBPH may directly influence these genetic traits. In agreement with this view, it was found that the suppression of TBPH in the glia alters the formation of evoked synaptic potentials at the NMJ, without affecting the cycle of vesicle release in the presynaptic membranes. On the contrary, the anatomical distribution of the GluRIIA clusters present at the postsynaptic membranes is highly disturbed in the TBPH depleted glia, suggesting that electrophysiological problems originate from defects in the organization of the postsynaptic membranes. The genetic rescue experiments instead, reveal that the glial function of TBPH is necessary to promote the synaptic growth of the motoneurons terminal axons during larval development. This neurotrophic function of TBPH, nevertheless, is not sufficient to rescue the neuronal levels of the vesicular protein Syx. In addition, the distribution of the postsynaptic protein Dlg, whose localization largely depends on the presynaptic activity of the motoneurons, is not recovered after the activation of TBPH in the glia compared with identical genetic rescue experiments performed expressing TBPH in neurons (17), revealing tissue-specific differences in TBPH function and regulatory mechanisms. Finally, the genetic rescues generated through the late expression of TBPH in mature glia indicate that the pathological defects described in locomotive behaviors and postsynaptic distribution of the GluRIIA clusters are not permanent and can be regenerated (Romano, 2015).

Defects in the regulation of the intracellular levels of the glutamate transporter EAAT1 in the glia have been associated with several neurodegenerative diseases comprising ALS. Indeed, molecular data previously generated shows that EAAT1 and EAAT2 mRNA levels are largely modified in ALS patients and, moreover, these modifications are associated with the increased oxidative stress present in the affected cases. Recent studies performed in Drosophila have also identified that EAAT1 and EAAT2 messengers are modified in TBPH minus flies suggesting that these modifications may play a role in the mechanisms behind these phenotypes despite experimental evidences are not available. This study directly tested this hypothesis and uncovered that EAAT1 has an important role in the phenotypic mechanisms derived from the loss of TBPH function in the glia. The data also strongly suggest that the levels of the extracellular glutamate might be affected in TBPH null flies and responsible for the alterations in the localization of the GluRIIA at the postsynaptic membranes. These evidences, allow the hypothesis that the implementation of pharmacological treatments aimed to enhance the glutamate uptake or counteract the oxidative stress, produced by defects in its intracellular transport, may contribute to improve the symptoms observed in TBPH minus flies. In this direction, it was observed that nordihydroguaiaretic acid (NDGA) significantly improves the locomotive capacities of TBPH-RNAi treated larvae, demonstrating the role of this protein in the organization of the NMJs and proposing that analogous situations could be expected in human pathologies associated with TDP-43 dysfunctions like ALS or frontotemporal lobar degeneration (FTLD) (Romano, 2015).

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Coyne, A.N., Yamada, S.B., Siddegowda, B.B., Estes, P.S., Zaepfel, B.L., Johannesmeyer, J.S., Lockwood, D.B., Pham, L.T., Hart, M.P., Cassel, J.A., Freibaum, B., Boehringer, A.V., Taylor, J.P., Reitz, A.B., Gitler, A.D. and Zarnescu, D.C. (2015). Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Hum Mol Genet 24: 6886-6898. PubMed ID: 26385636

Abstract
RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). This study identifies Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. It was found that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduces lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. These data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons (Coyne, 2015).

Highlights

  • dFMRP is a potent modifier of TDP-43 neurotoxicity in vivo.
  • TDP-43 and dFMRP colocalize with PABP in neuronal RNA granules.
  • FMRP forms a complex with TDP-43 in vivo.
  • dFMRP modulates TDP-43 solubility and molecular mobility.
  • dFMRP OE restores the translation of futsch mRNA.
  • dFMRP OE restores Futsch dependent neuromuscular junction phenotypes.

Discussion
This study used a combination of genetic and molecular approaches to uncover a novel functional interaction between dFMRP and TDP-43. Taken together, the results support a model whereby dFMRP, a well-established translational regulator, can modulate the neurotoxicity caused by TDP-43 overexpression. When overexpressed, dFMRP decreases the association of TDP-43 with the aggregate-like fraction. Together with immunoprecipitation and binding experiments, these findings support a model whereby dFMRP promotes the remodeling of the RNP by ‘extracting’ TDP-43 and freeing the sequestered mRNA from the protein-RNA complex. This in turn may alleviate the negative impact that TDP-43 exerts on its mRNA targets as is the case for futsch mRNA. Indeed, dFMRP OE in the context of TDP-43 restores the expression of futsch, which is a translation target of TDP-43. While the change in Futsch expression is slight in magnitude, it is statistically significant. These findings suggest a scenario whereby the robust synaptic phenotypes observed in ALS may result from the combinatorial effect of decreased expression for multiple TDP-43 targets at the NMJ. In future studies it will be interesting to determine additional synaptic targets of TDP-43 whose expression is restored upon dFMRP OE. While futsch mRNA can be translationally controlled by both dFMRP and TDP-43, in the context of TDP-43 RNA granules, dFMRP appears to favor an association with TDP-43 protein over its translation target, leaving futsch mRNA available for protein synthesis, which explains the translation restoration that was observed in the context of dFMRP OE. Given the wide repertoire of RNA binding protein partners of TDP-43, it will be interesting in the future to determine whether others can also confer neuroprotection to TDP-43-dependent toxicity and whether they do so by a similar molecular mechanism. This would be expected given that Futsch expression is significantly increased but not fully restored by dFMRP OE at the NMJ (Coyne, 2015).

It has been previously shown that TDPWT and disease linked mutations, although expressed at comparable levels, confer differential toxicity in various phenotypic assays. This study provides evidence that TDPWT and TDPG298S also interact differentially with protein partners. TDPG298S colocalizes with PABP to a lesser extent than TDPWT. Further, TDPWT and TDPG298S exhibit distinct molecular mobilities within neurites, which is consistent with previous reports that although wild-type and disease linked variants both associate with stress granules, their dynamics, persistence and size differ dramatically. Taken together, these findings and published data suggest that ALS may be a consequence of chronic translation inhibition. This could result from dysregulation of RNA granule physiology in the context of excess cellular stress as previously suggested. This scenario is consistent with previous findings that inhibition of SG is neuroprotective and provides a plausible mechanism for how TDP-43 mutations lead to disease. Additionally, it can explain the association of wild-type TDP-43 with cytoplasmic aggregates in the majority of ALS cases, regardless of etiology. One possibility is that, in the context of aging related or other cellular stress, wild-type TDP-43 enters the RNA stress granule cycle, contributing to translation inhibition and disease pathophysiology (Coyne, 2015).

Results from this study indicate that FMRP remodels TDP-43 RNP granules and this restores futsch translation and expression at the NMJ. This in turn, can alleviate phenotypes associated with microtubule instability such as the presence of satellite boutons. Altered microtubule stability is emerging as a prominent pathological mechanism underlying the progression of ALS and may provide a useful avenue for the development of therapeutics. In addition, altered ribostasis has emerged as a major hypothesis for explaining the progression from RNA stress granules to aggregates seen in disease. This model suggests altered translational regulation as a molecular mechanism underlying disease progression. Results from this study support this model and provide evidence that mitigating translational repression can suppress disease phenotypes. In future studies it will be important to establish whether blanket approaches such as RNA SG inhibition or translation restoration offer more promise than targeted strategies based on specific targets (Coyne, 2015).

Two recent studies have shown that TDP-43 suppresses toxicity in CGG repeat expansion models of Fragile X associated tremor/ataxia syndrome (FXTAS). Removing a portion of the C-terminus of TDP-43 in which interactions with hnRNP A2/B1 typically occur, abolishes the ability of TDP-43 to suppress toxicity. These results suggest that TDP-43 may work to mitigate CGG RNA toxicity via interactions with its protein partners by preventing them from sequestration into toxic RNA foci. Thus, in the case of CGG repeat disorders, TDP-43 may alter RNP complexes similar to how dFMRP OE alters RNP complexes in the TDP-43 model of ALS used in this study. Taken together, these data provide evidence for common mechanisms underlying neurodegenerative diseases and repeat expansion disorders. In both cases, remodeling of RNP granules and the ‘freeing’ of RNA binding proteins or mRNA targets mitigates toxicity. Further, targeting RNP remodeling or translation restoration may prove useful as therapeutic strategies (Coyne, 2015).

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Zhang, K., Donnelly, C.J., Haeusler, A.R., Grima, J.C., Machamer, J.B., Steinwald, P., Daley, E.L., Miller, S.J., Cunningham, K.M., Vidensky, S., Gupta, S., Thomas, M.A., Hong, I., Chiu, S.L., Huganir, R.L., Ostrow, L.W., Matunis, M.J., Wang, J., Sattler, R., Lloyd, T.E. and Rothstein, J.D. (2015). The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525: 56-61. PubMed ID: 26308891

Abstract
The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and protein interactors for the G4C2 RNA including RanGAP1 have been previously identified. This study performed a candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats which identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention (Zhang, 2015).

Highlights

  • RanGAP suppresses HRE-mediated toxicity in Drosophila.
  • Nucleocytoplasmic transport modulates G4C2 toxicity.
  • G4C2 repeats bind RanGAP and cause NPC pathology.
  • The Ran gradient is disrupted by the C9orf72 HRE.
  • The C9orf72 HRE inhibits import of nuclear proteins.
  • Rescue of HRE-mediated neurodegeneration.

Discussion
Data from this study demonstrates that the G4C2 repeat expansion disrupts nucleocytoplasmic transport in a fly model and in human cells. While it was found that RanGAP is a key target of the G4C2 repeat expansion, other members of the NPC may also interact directly or indirectly with G4C2. Several human genetic studies have implicated nuclear transport deficits as the cause of a rare fetal motor neuron disease and infrequent cases of ALS, including studies on the role of the nucleoporin GLE1 implicated in mRNA export. In addition, irregularities of the nuclear membrane and distribution of nuclear pore proteins were recently noted in sporadic ALS tissue. A recent study has independently identified additional components of the NPC and nucleocytoplasmic trafficking pathways as dominant modifiers of G4C2 HRE toxicity in another C9-ALS fly model. Importantly, the observed NPC and nucleocytoplasmic trafficking defects in both iPS-cell-derived neurons and motor neurons in this study are relevant to both ALS and FTD. Taken together, these studies suggest that products of the C9orf72 HRE disrupt nucleocytoplasmic transport at the NPC and are a fundamental mechanism for inducing cellular injury in ALS and FTD. These defects may account for the nuclear depletion and cytoplasmic accumulation of TDP-43 widely seen in C9-ALS and FTD (Zhang, 2015).

Although the data only demonstrate a role for disruption of nuclear import in C9-ALS pathogenesis, the robust nuclear pore pathology that was detected suggests that both nuclear import and export may be affected. It is enticing to speculate that NPC dysfunction leads to age-related neurodegeneration, since many of the NPC components, including Nup205, are extremely long-lived, and NPC integrity is lost during normal ageing (Zhang, 2015).

The sense strand appears to be the cause of the described nucleocytoplasmic trafficking deficits in the human and fly model systems, as small molecules targeting the sense RNA suppress the nuclear import phenotypes, and neurodegeneration is caused by expression of G4C2 repeat RNA in C9orf72 iPSC neurons or Drosophila. While DPRs cannot be excluded as a contributor to nucleocytoplasmic trafficking defects, data in multiple model systems are most consistent with an RNA-mediated mechanism. Future studies will be required to determine the contribution of RanGAP disruption in C9-ALS pathogenesis compared with other pathogenic mechanisms implicated in C9-ALS such as nucleolar stress, which could act independently or in conjunction with nucleocytoplasmic transport disruption (Zhang, 2015).

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Machamer, J.B., Collins, S.E., Lloyd, T.E. (2014). The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction. Hum Mol Genet. 23: 3810-3822. PubMed ID: 24569165

Abstract
Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5-10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. This study investigates the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compares them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). It is found that lethality and locomotor phenotypes correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons (Machamer, 2014).

Highlights

  • Lethality and locomotor deficits correlate with FUS expression levels.
  • FUS overexpression depletes nuclear Caz.
  • FUS overexpression results in loss of presynaptic active zones and postsynaptic Discs large (Dlg).
  • FUS overexpression impairs synaptic transmission.
  • Presynaptic FUS overexpression disrupts postsynaptic glutamate receptor subunit composition.
  • Loss of Caz enhances synaptic transmission.

Discussion
A previous study concluded that ALS-linked mutations in FUS resulted in a toxic gain-of-function in Drosophila, and this conclusion was based on the finding that the wild-type and mutant UAS-HA-FUS transgenic lines expressed equivalent levels of protein in the eye. However, this study's analysis of these same UAS-HA-FUS lines demonstrated that when expressed in motor neurons, message and protein expression were 3- to 4-fold higher in the R521C mutant line than the wild-type line. Thus, these results suggest that the increased severity of phenotypes seen in the mutant line relative to wild-type is due to increased expression level rather than mutation-specific toxicity. Therefore, evidence for a gain-of-function effect of ALS-associated mutations in FUS is not found in this model (Machamer, 2014).

There are two non-mutually exclusive explanations for these dramatic differences in relative transgene expression levels observed in different tissues. First, given that HA-FUSR521C overexpression in eyes caused degeneration, the simplest explanation is that HA-FUSR521C lines express at higher levels than wild-type in the eyes during development, and this leads to cell loss or dysfunction that causes a reduction in protein expression in the adult, whereas expression in glutamatergic neurons with OK371-GAL4 does not have these effects. Indeed, morphological analysis of GMR>FUSR521C fly eyes demonstrated severe cell loss. An alternative possibility is that tissue-specific enhancers may differentially regulate gene expression from P-elements located at different genomic loci, as these position effects are well known in Drosophila (Machamer, 2014).

These observations suggest two important guidelines for analyzing overexpression disease models in Drosophila. First, given the widely available technologies for site-specific transgenesis, comparisons of gain-of-function phenotypes between wild-type and mutant proteins should utilize transgenic lines inserted at identical genomic sites. Indeed, when the lines were generated in this manner (FL-FUS) and analyzed, identical expression levels between the wild-type and mutant proteins in motor neurons were observed. Second, when comparing protein expression levels between wild-type and mutant transgenic lines, expression levels should be compared in the absence of cell loss and in the tissue most relevant to the disease (Machamer, 2014).

Recent data from mammalian cell culture suggests that disruption of FUS autoregulation leading to overexpression of wild-type FUS is sufficient to cause disease. In this study, it was shown that wild-type or mutant FUS overexpression in Drosophila motor neurons lead to severe downregulation of fly FUS. This suggests that an autoregulatory mechanism is conserved in Drosophila and occurs in motor neurons in vivo. This autoregulation likely explains why in many cases, stronger phenotypes in higher-expressing transgenic lines were not seen (Machamer, 2014).

Whether disease-associated mutations in FUS and other ALS genes cause disease through a gain- or loss-of-function is a matter of debate. Simple genetic model systems such as Drosophila are ideal for interpreting the effect of mutations on protein function; however, one must be cautious in interpreting results gained from overexpression studies, particularly when overexpressing human proteins in the fly. However, because low-level expression of human FL-FUS in neurons rescued phenotypes caused by loss of Drosophila FUS (i.e. Caz), this strongly argues for evolutionary conservation of FUS as well as a requirement for FUS in neurons. Furthermore, because FUSP525L failed to rescue Caz phenotypes, this strongly argues that the P525L mutation causes a loss-of-function (Machamer, 2014).

Consistent with this interpretation, analyses of disease-associated mutations in FUS was found to be most consistent with a partial loss-of-function, given that (a) low-level FUSWT but not FUSP525L expression caused increased larval and adult locomotor activity, and (b) expressing higher level FUSR521C in some instances caused less severe phenotypes than lower level FUSWT expression (e.g. Caz downregulation), altered EJP rise time, and GluRIIA upregulation. Nonetheless, the possibility that FUS mutations do exhibit some gain-of-toxic effects could not be excluded, particularly given that there was a greater reduction in GluRIIB levels with mutant FUS expression than with wild-type expression. Since mutations in the 3'UTR autoregulatory domain are sufficient to cause ALS in patients by upregulating wild-type FUS protein, ALS may be caused by increased levels of wild-type protein. In this context, low-level overexpression of FUS or Caz in aging flies may be a reasonable way to model the disease (Machamer, 2014).

FUS has been implicated in a wide range of processes in many cell types both within the nucleus and cytoplasm. Since this study was unable to detect significant wild-type FUS or Caz protein within motor axons or at the NMJ, it was postulated that FUS/Caz normally functions in the nucleus to regulate the expression of genes that modulate synapse function. Importantly, FUS/Caz appeared to be required within neurons to regulate synaptic transmission, as caz1 loss-of-function phenotypes were mimicked by presynaptic caz knockdown, and overexpression of FUS in motor neurons lead to altered synaptic transmission (Machamer, 2014).

The study showed that FUS expression inhibited evoked release due to both a reduction of quantal size (mEJP amplitude) and quantal content (number of quanta released per stimulus). A reduction in mEJP amplitude could be due to a decrease in synaptic vesicle size, glutamate concentration or postsynaptic currents due to alterations in glutamate receptor levels, localization or composition. It was postulated that the reduction in quantal size was due to a disruption of the spatial coupling of synaptic vesicle release sites with glutamate receptors given the disruption in the number and morphology of active zones and the postsynaptic density. Importantly, reduced GluR levels were not responsible for the decrease in mEJP amplitude observed in FUS overexpressing animals, but rather that GluR clustering at active zones might be altered. Furthermore, there was an increase in relative expression of A-type GluRs which would be expected to have the opposite effect on mEJP amplitude. This increase in ratio of A- to B-type GluRs was seen when glutamate release was blocked at larval NMJs and was a homeostatic response to reduction in glutamate-mediated synaptic transmission (Machamer, 2014).

There was also a striking reduction in the number of active zones in FUS-expressing animals. These changes likely contributed to the reduction in quantal content, as bruchpilot mutations in Drosophila have reduced synaptic transmission due to a reduction inythe readily releasable pool. The relatively subtle reduction in quantal content in FUS-expressing animals might be due to a homeostatic increase in the probability of vesicle fusion at any given active zone. Surprisingly, the reduction in active zone number was associated with a marked increase in the frequency of spontaneous release, suggesting that the remaining active zones had a marked increase in release probability. Consistent with this hypothesis, superresolution microscopic imaging of Brp demonstrated abnormal morphology of active zones in FUS-expressing animals (Machamer, 2014).

The results of this study contrast with those of a recent report analyzing larval NMJ physiology of HA-FUSR521C-expressing and caz1 animals using discontinuous single electrode voltage clamp recordings. This study did not observe a physiologic phenotype with FUSWT overexpression, and a reduction in evoked release in caz1 animals was observed; these findings were consistent with findings in zebrafish with overexpression of mutant FUS and with morpholino-mediated knockdown of wild-type FUS. Since FUS protein redistributes to cytoplasmic stress granules with various stressors, one possibility is that alterations in animal rearing or recording conditions may have large effects (Machamer, 2014).

The study also reported the occurrence of cell nonautonomous loss of Dlg from muscle postsynaptic compartments and alterations in GluRII subunit composition at the NMJ as a result of FUS overexpression in motor neurons. The mechanism of Dlg loss induced by FUS overexpression is unclear, and it is unknown whether the reduction in Dlg expression is accompanied by morphological alterations in the subsynaptic reticulum of muscle cells. Although the increase in the ratio of A- to B-type GluRs was consistent with homeostatic compensation for impaired synaptic transmission, Dlg is not known to be regulated by homeostatic feedback at the Drosophila NMJ. Thus, it is speculated that non-cell autonomous effects on Dlg are mediated through alterations in transsynaptic adhesion molecules. This notion was supported by the altered kinetics of EJP rise and decay times and disrupted apposition of GluRIIB/IIC with the active zone (Machamer, 2014).

Furthermore, several of the morphological and electrophysiological phenotypes caused by FUS overexpression overlapped with loss-of-function alleles of the neurexin (nrx1)/neuroligin (nlg1 and nlg2) transsynaptic adhesion complex in Drosophila. For example, both nrx1 and nlg1 animals showed expanded interbouton regions that lacked post-synaptic markers, a phenotype observed with FUS overexpression. nrx1 mutants showed decreased quantal content, increased mEJP frequency, and an increase in ratio of A to B-type glutamate receptors, phenotypes that were also observed with FUS overexpression (Machamer, 2014).

As the best described function of FUS is regulation of transcription and splicing, alterations in transcription and/or splicing may underlie the changes seen in synaptic function. Many cell adhesion molecules are highly alternatively spliced, and this splicing alters their function in synaptic development and differentiation. For example, mutations in beag alter splicing of fasciclin II (fasII), the Drosophila homologue of neural cell adhesion molecule (NCAM), and altered splicing leads to fewer synaptic boutons and decreased neurotransmitter release. Thus, this study hypothesizes that the changes in synaptic structure and function may be partially explained by altered expression and/or splicing of transsynaptic adhesion molecules (Machamer, 2014).

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Mann, J. R., McKenna, E. D., Mawrie, D., Papakis, V., Alessandrini, F., Anderson, E. N., Mayers, R., Ball, H. E., Kaspi, E., Lubinski, K., Baron, D. M., Tellez, L., Landers, J. E., Pandey, U. B. and Kiskinis, E. (2023). Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import. Sci Adv 9(33): eadi5548. PubMed ID: 37585529
Summary:

Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, this study found functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. α-tubulin and importin-ss1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, evidence was foundfor disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.

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Krupp, S., Tam, O., Gale Hammell, M. and Dubnau, J. (2023). TDP-43 pathology in Drosophila induces glial-cell type specific toxicity that can be ameliorated by knock-down of SF2/SRSF1. CLife Sci Alliance 6(9). bioRxiv. PubMed ID: 37205372
Summary:

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. This study investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, cell-type-specific nuclear RNA sequencing was used to characterize the transcriptional changes were identified that were induced by pathological TDP-43 expression. Numerous glial cell-type specific transcriptional changes. Notably,SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. Further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism (Krupp, 2023).

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Moron-Oset, J., Fischer, L. K., Carcole, M., Giblin, A., Zhang, P., Isaacs, A. M., Gronke, S. and Partridge, L. (2023). Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags. CLife Sci Alliance 6(9). PubMed ID: 37308278
Summary:

Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. This study used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models (Moron-Oset, 2023).

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Rossi, S., Di Salvio, M., Balì, M., De Simone, A., Apolloni, S., D'Ambrosi, N., Arisi, I., Cipressa, F., Cozzolino, M. and Cestra, G. (2023). C9orf72 Toxic Species Affect ArfGAP-1 Function. Cells 12(15). PubMed ID: 37566088
Summary:

Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. It has been reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, this study set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. This study demonstrated that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity (Rossi, 2023).

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Li, Y., Geng, J., Rimal, S., Wang, H., Liu, X., Lu, B. and Li, S. (2023). The mTORC2/AKT/VCP axis is associated with quality control of the stalled translation of poly(GR) dipeptide repeats in C9-ALS/FTD. J Biol Chem 299(3): 102995. PubMed ID: 36764521
Summary:

Expansion of G4C2 hexanucleotide repeats in the chromosome 9 ORF 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (C9-ALS/FTD). Dipeptide repeats generated by unconventional translation, especially the R-containing poly(GR), have been implicated in C9-ALS/FTD pathogenesis. Mutations in other genes, including TAR DNA-binding protein 43 KD (TDP-43), fused in sarcoma (FUS), and valosin-containing protein, have also been linked to ALS/FTD, and upregulation of amyloid precursor protein (APP) is observed at the early stage of ALS and FTD. Fundamental questions remain as to the relationships between these ALS/FTD genes and whether they converge on similar cellular pathways. In this study, using biochemical, cell biological, and genetic analyses in Drosophila disease models, patient-derived fibroblasts, and mammalian cell culture, it was shown that mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling is activated by APP, TDP-43, and FUS and that mTORC2/AKT and its downstream target valosin-containing protein mediate the effect of APP, TDP-43, and FUS on the quality control of C9-ALS/FTD-associated poly(GR) translation. This study also found that poly(GR) expression results in reduction of global translation and that the coexpression of APP, TDP-43, and FUS results in further reduction of global translation, presumably through the GCN2/eIF2α-integrated stress response pathway. Together, these results implicate mTORC2/AKT signaling and GCN2/eIF2α-integrated stress response as common signaling pathways underlying ALS/FTD pathogenesis.

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Nemtsova, Y., Steinert, B. L. and Wharton, K. A.(2023). Compartment specific mitochondrial dysfunction in Drosophila knock-in model of ALS reversed by altered gene expression of OXPHOS subunits and pro-fission factor Drp1. Mol Cell Neurosci 125: 103834. PubMed ID: 36868541
Summary:

Amyotrophic Lateral Sclerosis (ALS) is a fatal multisystem neurodegenerative disease, characterized by a loss in motor function. ALS is genetically diverse, with mutations in genes ranging from those regulating RNA metabolism, like TAR DNA-binding protein (TDP-43) and Fused in sarcoma (FUS), to those that act to maintain cellular redox homeostasis, like superoxide dismutase 1 (SOD1). Although varied in genetic origin, pathogenic and clinical commonalities are clearly evident between cases of ALS. Defects in mitochondria is one such common pathology, thought to occur prior to, rather than as a consequence of symptom onset, making these organelles a promising therapeutic target for ALS, as well as other neurodegenerative diseases. Depending on the homeostatic needs of neurons throughout life, mitochondria are normally shuttled to different subcellular compartments to regulate metabolite and energy production, lipid metabolism, and buffer calcium. While originally considered a motor neuron disease due to the dramatic loss in motor function accompanied by motor neuron cell death in ALS patients, many studies have now implicated non-motor neurons and glial cells alike. Defects in non-motor neuron cell types often preceed motor neuron death suggesting their dysfunction may initiate and/or facilitate the decline in motor neuron health. This study investigate mitochondria in a Drosophila Sod1 knock-in model of ALS. In depth, in vivo, examination reveals mitochondrial dysfunction evident prior to onset of motor neuron degeneration. Genetically encoded redox biosensors identify a general disruption in the electron transport chain (ETC). Compartment specific abnormalities in mitochondrial morphology is observed in diseased sensory neurons, accompanied by no apparent defects in the axonal transport machinery, but instead an increase in mitophagy in synaptic regions. The decrease in networked mitochondria at the synapse is reversed upon downregulation of the pro-fission factor Drp1. Furthermore, altered expression of specific OXPHOS subunits reverses ALS-associated defects in mitochondrial morphology and function (Nemtsova, 2023).

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Yusuff, T., Chang, Y. C., Sang, T. K., Jackson, G. R. and Chatterjee, S.(2023). Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD. Front Genet 14: 881638. PubMed ID: 36968586
Summary:

Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, this study reports gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on these observations, it is proposed that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets (Yusuff, 2023).

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Lee, S., Jun, Y. W., Linares, G. R., Butler, B., Yuva-Adyemir, Y., Moore, J., Krishnan, G., Ruiz-Juarez, B., Santana, M., Pons, M., Silverman, N., Weng, Z., Ichida, J. K. and Gao, F. B. (2023). Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD. Neuron. PubMed ID: 36931278
Summary:

GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, it was found that several antimicrobial peptide genes, such as Metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD (Lee, 2023).

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Borg, R., Purkiss, A., Cacciottolo, R., Herrera, P. and Cauchi, R. J. (2023). Loss of amyotrophic lateral sclerosis risk factor SCFD1 causes motor dysfunction in Drosophila. Neurobiol Aging 126: 67-76. PubMed ID: 36944290
Summary:

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. This study shows that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. These findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation (Borg, 2023).

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Fujino, Y., Ueyama, M., Ishiguro, T., Ozawa, D., Ito, H., Sugiki, T., Murata, A., Ishiguro, A., Gendron, T., Mori, K., Tokuda, E., Taminato, T., Konno, T., Koyama, A., Kawabe, Y., Takeuchi, T., Furukawa, Y., Fujiwara, T., Ikeda, M., Mizuno, T., Mochizuki, H., Mizusawa, H., Wada, K., Ishikawa, K., Onodera, O., Nakatani, K., Petrucelli, L., Taguchi, H. and Nagai, Y. (2023). FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD. Elife 12. PubMed ID: 37461319
Summary:

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, this study investigated the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, this study demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, this study found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases (Fujino, 2023).

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Borg, R., Herrera, P., Purkiss, A., Cacciottolo, R. and Cauchi, R. J. (2023). Reduced levels of ALS gene DCTN1 induce motor defects in Drosophila. Front Neurosci 17: 1164251. PubMed ID: 37360176
Summary:

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuromuscular disease that has a strong genetic component. Deleterious variants in the DCTN1 gene are known to be a cause of ALS in diverse populations. DCTN1 encodes the p150 subunit of the molecular motor dynactin which is a key player in the bidirectional transport of cargos within cells. Whether DCTN1 mutations lead to the disease through either a gain or loss of function mechanism remains unresolved. Moreover, the contribution of non-neuronal cell types, especially muscle tissue, to ALS phenotypes in DCTN1 carriers is unknown. This study shows that gene silencing of Dctn1, the Drosophila main orthologue of DCTN1, either in neurons or muscles is sufficient to cause climbing and flight defects in adult flies. Dred, a protein with high homology to Drosophila Dctn1 and human DCTN1 was identified, that on loss of function also leads to motoric impairments. A global reduction of Dctn1 induced a significant reduction in the mobility of larvae and neuromuscular junction (NMJ) deficits prior to death at the pupal stage. RNA-seq and transcriptome profiling revealed splicing alterations in genes required for synapse organisation and function, which may explain the observed motor dysfunction and synaptic defects downstream of Dctn1 ablation. These findings support the possibility that loss of DCTN1 function can lead to ALS and underscore an important requirement for DCTN1 in muscle in addition to neurons (Borg, 2023).

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Nemtsova, Y., Steinert, B. L. and Wharton, K. A. (2023). Compartment specific mitochondrial dysfunction in Drosophila knock-in model of ALS reversed by altered gene expression of OXPHOS subunits and pro-fission factor Drp1. Mol Cell Neurosci 125: 103834. PubMed ID: 36868541
Summary:
Amyotrophic Lateral Sclerosis (ALS) is a fatal multisystem neurodegenerative disease, characterized by a loss in motor function. ALS is genetically diverse, with mutations in genes ranging from those regulating RNA metabolism, like TAR DNA-binding protein (TDP-43) and Fused in sarcoma (FUS), to those that act to maintain cellular redox homeostasis, like superoxide dismutase 1 (SOD1). Although varied in genetic origin, pathogenic and clinical commonalities are clearly evident between cases of ALS. Defects in mitochondria is one such common pathology, thought to occur prior to, rather than as a consequence of symptom onset, making these organelles a promising therapeutic target for ALS, as well as other neurodegenerative diseases. Depending on the homeostatic needs of neurons throughout life, mitochondria are normally shuttled to different subcellular compartments to regulate metabolite and energy production, lipid metabolism, and buffer calcium. While originally considered a motor neuron disease due to the dramatic loss in motor function accompanied by motor neuron cell death in ALS patients, many studies have now implicated non-motor neurons and glial cells alike. Defects in non-motor neuron cell types often preceed motor neuron death suggesting their dysfunction may initiate and/or facilitate the decline in motor neuron health. This study investigate mitochondria in a Drosophila Sod1 knock-in model of ALS. In depth, in vivo, examination reveals mitochondrial dysfunction evident prior to onset of motor neuron degeneration. Genetically encoded redox biosensors identify a general disruption in the electron transport chain (ETC). Compartment specific abnormalities in mitochondrial morphology is observed in diseased sensory neurons, accompanied by no apparent defects in the axonal transport machinery, but instead an increase in mitophagy in synaptic regions. The decrease in networked mitochondria at the synapse is reversed upon downregulation of the pro-fission factor Drp1. Furthermore, altered expression of specific OXPHOS subunits reverses ALS-associated defects in mitochondrial morphology and function.

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Yusuff, T., Chang, Y. C., Sang, T. K., Jackson, G. R. and Chatterjee, S. (2023). Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD. Front Genet 14: 881638. PubMed ID: 36968586
Summary:
Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, this study reports gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on these observations, it is proposed that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.

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Lee, S., Jun, Y. W., Linares, G. R., Butler, B., Yuva-Adyemir, Y., Moore, J., Krishnan, G., Ruiz-Juarez, B., Santana, M., Pons, M., Silverman, N., Weng, Z., Ichida, J. K. and Gao, F. B. (2023). Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD. Neuron. PubMed ID: 36931278
Summary:
GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, it was found that several antimicrobial peptide genes, such as Metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

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Lee, S., Jun, Y. W., Linares, G. R., Butler, B., Yuva-Adyemir, Y., Moore, J., Krishnan, G., Ruiz-Juarez, B., Santana, M., Pons, M., Silverman, N., Weng, Z., Ichida, J. K. and Gao, F. B. (2023). Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD. Neuron. PubMed ID: 36931278
Summary:
GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, it was found that several antimicrobial peptide genes, such as Metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

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Borg, R., Purkiss, A., Cacciottolo, R., Herrera, P. and Cauchi, R. J. (2023). Loss of amyotrophic lateral sclerosis risk factor SCFD1 causes motor dysfunction in Drosophila. Neurobiol Aging 126: 67-76. PubMed ID: 36944290
Summary:
Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. This study shows that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. These findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation.

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Li, Y., Geng, J., Rimal, S., Wang, H., Liu, X., Lu, B. and Li, S. (2023). The mTORC2/AKT/VCP axis is associated with quality control of the stalled translation of poly(GR) dipeptide repeats in C9-ALS/FTD. J Biol Chem 299(3): 102995. PubMed ID: 36764521
Summary:
Expansion of G4C2 hexanucleotide repeats in the chromosome 9 ORF 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (C9-ALS/FTD). Dipeptide repeats generated by unconventional translation, especially the R-containing poly(GR), have been implicated in C9-ALS/FTD pathogenesis. Mutations in other genes, including TAR DNA-binding protein 43 KD (TDP-43), fused in sarcoma (FUS), and valosin-containing protein, have also been linked to ALS/FTD, and upregulation of amyloid precursor protein (APP) is observed at the early stage of ALS and FTD. Fundamental questions remain as to the relationships between these ALS/FTD genes and whether they converge on similar cellular pathways. In this study, using biochemical, cell biological, and genetic analyses in Drosophila disease models, patient-derived fibroblasts, and mammalian cell culture, it was shown that mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling is activated by APP, TDP-43, and FUS and that mTORC2/AKT and its downstream target valosin-containing protein mediate the effect of APP, TDP-43, and FUS on the quality control of C9-ALS/FTD-associated poly(GR) translation. This study also found that poly(GR) expression results in reduction of global translation and that the coexpression of APP, TDP-43, and FUS results in further reduction of global translation, presumably through the GCN2/eIF2α-integrated stress response pathway. Together, these results implicate mTORC2/AKT signaling and GCN2/eIF2α-integrated stress response as common signaling pathways underlying ALS/FTD pathogenesis.

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Karagas, N. E., Gupta, R., Rastegari, E., Tan, K. L., Leung, H. H., Bellen, H. J., Venkatachalam, K. and Wong, C. O. (2022). Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS/FTD Models. Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS. J Neurosci 42(42): 8019-8037. PubMed ID: 36261266
Summary:

Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S) ) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, this study demonstrate the suppression of vapbP58S) -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca(2+) release channels or the inhibition Ca(2+)/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca(2+)]. The Ca(2+) dyshomeostasis is attributed to delayed extrusion of cytosolic Ca(2+) Suggesting that this defect in Ca(2+) extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons (Karagas, 2022).

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Wang, X., Rimal, S., Tantray, I., Geng, J., Bhurtel, S., Khaket, T. P., Li, W., Han, Z. and Lu, B. (2022). Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1. Proc Natl Acad Sci U S A 119(42): e2202322119. PubMed ID: 36170200
Summary:
An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. This study shows that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

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Megat, S., Mora, N., Sanogo, J., Roman, O., Catanese, A., Alami, N. O., Freischmidt, A., Mingaj, X., De Calbiac, H., Muratet, F., Dirrig-Grosch, S., Dieterle, S., Van Bakel, N., Muller, K., Sieverding, K., Weishaupt, J., Andersen, P. M., Weber, M., Neuwirth, C., Margelisch, M., Sommacal, A., Van Eijk, K. R., Veldink, J. H., Lautrette, G., Couratier, P., Camuzat, A., Le Ber, I., Grassano, M., Chio, A., Boeckers, T., Ludolph, A. C., Roselli, F., Yilmazer-Hanke, D., Millecamps, S., Kabashi, E., Storkebaum, E., Sellier, C. and Dupuis, L. (2023). Integrative genetic analysis illuminates ALS heritability and identifies risk genes. Nat Commun 14(1): 342. PubMed ID: 36670122
Summary:
Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). This study shows that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, this study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

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Kim, S. H., Nichols, K. D., Anderson, E. N., Liu, Y., Ramesh, N., Jia, W., Kuerbis, C. J., Scalf, M., Smith, L. M., Pandey, U. B. and Tibbetts, R. S. (2023). Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2. Elife 12. PubMed ID: 37039476
Summary:
Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. This study shows that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2(ALS)) trigger heat stress-dependent neurodegeneration in Drosophila. A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2(ALS) alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2(ALS) knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2(ALS) alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC. The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

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Castelli, L. M., Lin, Y. H., Sanchez-Martinez, A., Gul, A., Mohd Imran, K., Higginbottom, A., Upadhyay, S. K., Markus, N. M., Rua Martins, R., Cooper-Knock, J., Montmasson, C., Cohen, R., Walton, A., Bauer, C. S., De Vos, K. J., Mead, R. J., Azzouz, M., Dominguez, C., Ferraiuolo, L., Shaw, P. J., Whitworth, A. J. and Hautbergue, G. M. (2023). A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins. Sci Transl Med 15(685): eabo3823. PubMed ID: 36857431
Summary:
Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. A cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

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Taminato, T., Takeuchi, T., Ueyama, M., Mori, K., Ikeda, M., Mochizuki, H. and Nagai, Y. (2023). Therapeutic reduction of GGGGCC repeat RNA levels by hnRNPA3 suppresses neurodegeneration in drosophila models of C9orf72-linked ALS/FTD. Hum Mol Genet. PubMed ID: 36611007
Summary:
The abnormal expansion of GGGGCC hexanucleotide repeats within the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of GGGGCC repeat-containing RNAs as RNA foci, and the deposition of dipeptide repeat proteins (DPR) produced from these repeat RNAs by unconventional translation are major pathological hallmarks of C9orf72-linked ALS/FTD (C9-ALS/FTD), and are both thought to play a crucial role in the pathogenesis of these diseases. Because GGGGCC repeat RNA is likely to be the most upstream therapeutic target in the pathogenic cascade of C9-ALS/FTD, lowering the cellular level of GGGGCC repeat RNA is expected to mitigate repeat RNA toxicity, and will therefore be a disease-modifying therapeutic strategy for the treatment of C9-ALS/FTD. This study demonstrated using a Drosophila model of C9-ALS/FTD that elevated expression of a subset of human RNA-binding proteins that bind to GGGGCC repeat RNA, including hnRNPA3, IGF2BP1, hnRNPA2B1, hnRNPR, and SF3B3, reduces the level of GGGGCC repeat RNA, resulting in the suppression of neurodegeneration. It was further shown that hnRNPA3-mediated reduction of GGGGCC repeat RNA suppresses disease pathology, such as RNA foci and DPR accumulation. These results demonstrate that hnRNPA3 and other RNA-binding proteins negatively regulate the level of GGGGCC repeat RNA, and mitigate repeat RNA toxicity in vivo, indicating the therapeutic potential of the repeat RNA-lowering approach mediated by endogenous RNA-binding proteins for the treatment of C9-ALS/FTD.

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Chang, Y. H. and Dubnau, J. (2023). Endogenous retroviruses and TDP-43 proteinopathy form a sustaining feedback driving intercellular spread of Drosophila neurodegeneration. Nat Commun 14(1): 966. PubMed ID: 36810738
Summary:
Inter-cellular movement of "prion-like" proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. This study demonstrates that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.

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Khalil, B., Chhangani, D., .., Rincon-Limas, D. E. and Rossoll, W. (2022). Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy. Mol Neurodegener 17(1): 80. PubMed ID: 36482422
Summary:
Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. This study examined the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Several members of the nuclear import receptor protein family were able to reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, its activity was found to depend on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. These results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. hese findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.

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Huber, R. G., Pandey, S., Chhangani, D., Rincon-Limas, D. E., Staff, N. P. and Yeo, C. J. J. (2022). Identification of potential pathways and biomarkers linked to progression in ALS. Ann Clin Transl Neurol. PubMed ID: 36533811
Summary:
To identify potential diagnostic and prognostic biomarkers for clinical management and clinical trials in amyotrophic lateral sclerosis. Proteomics data of ALS patient-induced pluripotent stem cell-derived motor neurons available through the AnswerALS consortium were analyzed. After stratifying patients using clinical ALSFRS-R and ALS-CBS scales, differentially expressed proteins indicative of ALS disease severity and progression rate were identified as candidate ALS-related and prognostic biomarkers. Pathway analysis for identified proteins was performed using STITCH. Protein sets were correlated with the effects of drugs using the Connectivity Map tool to identify compounds likely to affect similar pathways. RNAi screening was performed in a Drosophila TDP-43 ALS model to validate pathological relevance. A statistical classification machine learning model was constructed using ridge regression that uses proteomics data to differentiate ALS patients from controls. This study identified 76, 21, 71 and 1 candidate ALS-related biomarkers and 22, 41, 27 and 64 candidate prognostic biomarkers from patients stratified by ALSFRS-R baseline, ALSFRS-R progression slope, ALS-CBS baseline and ALS-CBS progression slope, respectively. Nineteen proteins enhanced or suppressed pathogenic eye phenotypes in the ALS fly model. Nutraceuticals, dopamine pathway modulators, statins, anti-inflammatories and antimicrobials were predicted starting points for drug repurposing using the connectivity map tool. Ten diagnostic biomarker proteins were predicted by machine learning to identify ALS patients with high accuracy and sensitivity. This study showcases the powerful approach of iPSC-motor neuron proteomics combined with machine learning and biological confirmation in the prediction of novel mechanisms and diagnostic and predictive biomarkers in ALS.

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Cheng, A., Liu, C., Ye, W., Huang, D., She, W., Liu, X., Fung, C. P., Xu, N., Suen, M. C., Ye, W., Sung, H. H. Y., Williams, I. D., Zhu, G. and Qian, P. Y. (2022). Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS/FTD Models. J Med Chem 65(19): 12825-12837. PubMed ID: 36226410
Summary:

The G-quadruplex (G4) forming C9orf72 GGGGCC (G4C2) expanded hexanucleotide repeat (EHR) is the predominant genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Developing selective G4-binding ligands is challenging due to the conformational polymorphism and similarity of G4 structures. Three first-in-class marine natural products were identified, chrexanthomycin A (cA), chrexanthomycin B (cB), and chrexanthomycin C (cC), with remarkable bioactivities. Thereinto, cA shows the highest permeability and lowest cytotoxicity to live cells. NMR titration experiments and in silico analysis demonstrate that cA, cB, and cC selectively bind to DNA and RNA G4C2 G4s. Notably, cA and cC dramatically reduce G4C2 EHR-caused cell death, diminish G4C2 RNA foci in (G4C2)(29)-expressing Neuro2a cells, and significantly eliminate ROS in HT22 cells. In (G4C2)(29)-expressing Drosophila, cA and cC significantly rescue eye degeneration and improve locomotor deficits. Overall, these findings reveal that cA and cC are potential therapeutic agents deserving further clinical study (Cheng, 2022).

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Loganathan, S., Wilson, B. A., Carey, S. B., Manzo, E., Joardar, A., Ugur, B. and Zarnescu, D. C. (2022). TDP-43 Proteinopathy Causes Broad Metabolic Alterations including TCA Cycle Intermediates and Dopamine Levels in Drosophila Models of ALS. Metabolites 12(2). PubMed ID: 35208176
Summary:
Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. This study used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, global metabolomic profiling was performed in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. This study reports alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. It was also shown that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43(G298S), and it was found that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.

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Kishino, Y., Matsukawa, K., Matsumoto, T., Miyazaki, R., Wakabayashi, T., Nonaka, T., Kametani, F., Hasegawa, M., Hashimoto, T. and Iwatsubo, T. (2022). Casein kinase 1delta/epsilon phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration. J Biol Chem: 102191. PubMed ID: 35753345
Summary:

Aberrant cytoplasmic accumulation of an RNA-binding protein, Fused in sarcoma (FUS), characterizes the neuropathology of subtypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, although the effects of post-translational modifications of FUS, especially phosphorylation, on its neurotoxicity have not been fully characterized. This study shows that casein kinase 1δ phosphorylates FUS at 10 serine/threonine residues in vitro using mass spectrometric analyses. Phosphorylation by casein kinase 1δ or 1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells. In transgenic Drosophila that overexpress wild-type or P525L ALS-mutant human FUS in the retina or in neurons it was found that coexpression of human casein kinase 1δ or its Drosophila isologue Dco in the photoreceptor neurons significantly ameliorated the observed retinal degeneration, and neuronal coexpression of human casein kinase 1δ extended fly lifespan. Taken together, these data suggest a novel regulatory mechanism of the assembly and toxicity of FUS through casein kinase 1δ/ε-mediated phosphorylation, which could represent a potential therapeutic target in FUS proteinopathies.

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Pant, D. C., Parameswaran, J., Rao, L., Loss, I., Chilukuri, G., Parlato, R., Shi, L., Glass, J. D., Bassell, G. J., Koch, P., Yilmaz, R., Weishaupt, J. H., Gennerich, A. and Jiang, J. (2022). ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function. EMBO Rep: e54234. PubMed ID: 35735139
Summary:

Mutations in the human kinesin family member 5A (KIF5A; see Drosophila Khc) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. This study confirms the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. A comprehensive analysis was performed of ΔExon27 at the single-molecule, cellular, and organism levels. The results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. These results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.

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François-Moutal, L., Scott, D. D., Ambrose, A. J., Zerio, C. J., Rodriguez-Sanchez, M., Dissanayake, K., May, D. G., Carlson, J. M., Barbieri, E., Moutal, A., Roux, K. J., Shorter, J., Khanna, R., Barmada, S. J., McGurk, L. and Khanna, M. (2022). Heat shock protein Grp78/BiP/HspA5 binds directly to TDP-43 and mitigates toxicity associated with disease pathology. Sci Rep 12(1): 8140. PubMed ID: 35581326
Summary:

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, this study investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. It was further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly this study observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.

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Dubey, S. K., Maulding, K., Sung, H. and Lloyd, T. E. (2022). Nucleoporins are degraded via upregulation of ESCRT-III/Vps4 complex in Drosophila models of C9-ALS/FTD . Cell Rep 40(12): 111379. PubMed ID: 36130523
Summary:

Disruption of the nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) have been implicated in the pathogenesis of neurodegenerative diseases. A GGGGCC hexanucleotide repeat expansion (HRE) in an intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, but the mechanism by which the HRE disrupts NCT is incompletely understood. This study found that expression of GGGGCC repeats in Drosophila neurons induces proteasome-mediated degradation of select nucleoporins of the NPC. This process requires the Vps4 ATPase and the endosomal-sorting complex required for transport complex-III (ESCRT-III), as knockdown of ESCRT-III/Vps4 genes rescues nucleoporin levels, normalizes NCT, and suppresses GGGGCC-mediated neurodegeneration. GGGGCC expression upregulates nuclear ESCRT-III/Vps4 expression, and expansion microscopy demonstrates that the nucleoporins are translocated into the cytoplasm before undergoing proteasome-mediated degradation. These findings demonstrate a mechanism for nucleoporin degradation and NPC dysfunction in neurodegenerative disease.

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Han, Y. J. and Kim, K. (2022). Reduced oxidative stress suppresses neurotoxicity in the Drosophila model of TAF15-associated proteinopathies. Mol Brain 15(1): 93. PubMed ID: 36411469
Summary:
TATA-binding protein associated factor 15 (TAF15) is involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TAF15 accumulates as cytoplasmic aggregates in neurons and the clearance of aggregates may be a therapeutic strategy for ALS, the underlying mechanisms of TAF15 in ALS remain poorly understood. Recently, it was found that glutathione transferase omega 2 (GstO2) expression level is significantly reduced in the brain tissue of TAF15-expressing flies. This study demonstrated that GstO2 overexpression in TAF15-induced flies rescues the locomotive activity and neuromuscular junctional defects. Furthermore, TAF15 levels in both cytoplasm and nuclear fractions significantly decreased in the heads of GstO2 co-expressing flies. GstO2-co-overexpression in neurons caused a marked decrease in intracellular reactive oxygen species generation in TAF15-induced flies. These findings demonstrated that GstO2 was a pathogenic regulator of TAF15-associated proteinopathies. They help expand understanding of TAF15-associated ALS pathogenesis.

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Kim, Y. A., Siddiqui, T., Blaze, J., Cosacak, M. I., Winters, T., Kumar, A., Tein, E., Sproul, A. A., Teich, A. F., Bartolini, F., Akbarian, S., Kizil, C., Hargus, G. and Santa-Maria, I.(2022). RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation. Acta Neuropathol. PubMed ID: 36357715
Summary:

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Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022). Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Summary:
Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. This study shows NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, decreased NSun2 protein expression and an increased ratio of pTau/NSun2 were observed in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, this study confirmed NSun2 deficiency results in tau hyperphosphorylation. It was also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avet was observed that age affects loading into RISC for a small number of miRNAs.

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Sung, H. and Lloyd, T. E. (2022). Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD.Traffic 23(9): 430-441. PubMed ID: 35908282
Summary:
A GGGGCC (G(4) C(2)) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. This study performed live imaging of Drosophila motor neurons expressing expanded G(4) C(2) repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G(4) C(2) repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G(4) C(2) repeats. Thus,these data indicate that expanded G(4) C(2) repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD.

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Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022). Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Summary:
Increasing evidence points to the involvement of cell types other than motor neurons in both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. Studies have since focused on evaluating the contribution of glia in SMA. This study made use of the Drosophila model to ask whether the survival motor neuron (Smn) protein, the causative factor for SMA, is required selectively in glia. The specific loss of Smn function in glia during development reduced survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology in flies. In contrast, gain rather than loss of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). This study shows that glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability but, similar to Smn reduction, had no negative effect on neuromuscular function. These findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.

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Cha, S. J., Lee, S., Choi, H. J., Han, Y. J., Jeon, Y. M., Jo, M., Lee, S., Nahm, M., Lim, S. M., Kim, S. H., Kim, H. J. and Kim, K. (2022).. Therapeutic modulation of GSTO activity rescues FUS-associated neurotoxicity via deglutathionylation in ALS disease models. Dev Cell 57(6): 783-798. PubMed ID: 35320731
Summary:
Fused in sarcoma (FUS) is a DNA/RNA-binding protein that is involved in DNA repair and RNA processing. FUS is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the molecular mechanisms underlying FUS-mediated neurodegeneration are largely unknown. Using a Drosophila model, this study showed that the overexpression of glutathione transferase omega 2 (GstO2) reduces cytoplasmic FUS aggregates and prevents neurodegenerative phenotypes, including neurotoxicity and mitochondrial dysfunction. A FUS glutathionylation site was found at the 447(th) cysteine residue in the RanBP2-type ZnF domain. The glutathionylation of FUS induces FUS aggregation by promoting phase separation. GstO2 reduced cytoplasmic FUS aggregation by deglutathionylation in Drosophila brains. Moreover, this study demonstrated that the overexpression of human GSTO1, the homolog of Drosophila GstO2, attenuates FUS-induced neurotoxicity and cytoplasmic FUS accumulation in mouse neuronal cells. Thus, the modulation of FUS glutathionylation might be a promising therapeutic strategy for FUS-associated neurodegenerative diseases.

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Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022).. Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Summary:
Increasing evidence points to the involvement of cell types other than motor neurons in both ALS and SMA, the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. This study asked whether the Smn protein, the causative factor for SMA, is required selectively in glia. Loss of Smn function in glia during development was shown to reduce survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology. In contrast, gain of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal snRNPs. Glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability. These findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.

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Jeon, Y. M., Kwon, Y., Lee, S., Kim, S., Jo, M., Lee, S., Kim, S. R., Kim, K. and Kim, H. J. (2021).. Vitamin B12 Reduces TDP-43 Toxicity by Alleviating Oxidative Stress and Mitochondrial Dysfunction. Antioxidants (Basel) 11(1). PubMed ID: 35052586
Summary:
TAR DNA-binding protein 43 (TDP-43) is a member of an evolutionarily conserved family of heterogeneous nuclear ribonucleoproteins that modulate multiple steps in RNA metabolic processes. Cytoplasmic aggregation of TDP-43 in affected neurons is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Mislocalized and accumulated TDP-43 in the cytoplasm induces mitochondrial dysfunction and reactive oxidative species (ROS) production. This study shows that TDP-43- and rotenone-induced neurotoxicity in the human neuronal cell line SH-SY5Y were attenuated by hydroxocobalamin (Hb, vitamin B(12) analog) treatment. Although Hb did not affect the cytoplasmic accumulation of TDP-43, Hb attenuated TDP-43-induced toxicity by reducing oxidative stress and mitochondrial dysfunction. Moreover, a shortened lifespan and motility defects in TDP-43-expressing Drosophila were significantly mitigated by dietary treatment with hydroxocobalamin. Taken together, these findings suggest that oral intake of hydroxocobalamin may be a potential therapeutic intervention for TDP-43-associated proteinopathies.

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Lee, H., Lee, J. J., Park, N. Y., Dubey, S. K., Kim, T., Ruan, K., Lim, S. B., Park, S. H., Ha, S., Kovlyagina, I., Kim, K. T., Kim, S., Oh, Y., Kim, H., Kang, S. U., Song, M. R., Lloyd, T. E., Maragakis, N. J., Hong, Y. B., Eoh, H. and Lee, G. (2021).. Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS. Nat Neurosci. PubMed ID: 34782793

Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. This study applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1(G93A) mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.

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Licata, N. V., Cristofani, R., Salomonsson, S., Wilson, K. M., Kempthorne, L., Vaizoglu, D., D'Agostino, V. G., Pollini, D., Loffredo, R., Pancher, M., Adami, V., Bellosta, P., Ratti, A., Viero, G., Quattrone, A., Isaacs, A. M., Poletti, A. and Provenzani, A. (2021).. C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation. EMBO J: e105026. PubMed ID: 34791698
Summary:

Abstract
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). This study developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone were found to reduce DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, these results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

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Li, J., Lim, R. G., Kaye, J. A., Dardov, V., Coyne, A. N., Wu, J., Milani, P., Cheng, A., Thompson, T. G., Ornelas, L., Frank, A., Adam, M., Banuelos, M. G., Casale, M., Cox, V., Escalante-Chong, R., Daigle, J. G., Gomez, E., Hayes, L., Holewenski, R., Lei, S., Lenail, A., Lima, L., Mandefro, B., Matlock, A., Panther, L., Patel-Murray, N. L., Pham, J., Ramamoorthy, D., Sachs, K., Shelley, B., Stocksdale, J., Trost, H., Wilhelm, M., Venkatraman, V., Wassie, B. T., Wyman, S., Yang, S., Van Eyk, J. E., Lloyd, T. E., Finkbeiner, S., Fraenkel, E., Rothstein, J. D., Sareen, D., Svendsen, C. N. and Thompson, L. M. (2021).. An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients. iScience 24(11): 103221. PubMed ID: 34746695

Abstract
Neurodegenerative diseases are challenging for systems biology because of the lack of reliable animal models or patient samples at early disease stages. Induced pluripotent stem cells (iPSCs) could address these challenges. This study investigated DNA, RNA, epigenetics, and proteins in iPSC-derived motor neurons from patients with ALS carrying hexanucleotide expansions in C9ORF72. Using integrative computational methods combining all omics datasets, this study identified novel and known dysregulated pathways. A C9ORF72 Drosophila model was used to distinguish pathways contributing to disease phenotypes from compensatory ones, and alterations in some pathways were confirmed in postmortem spinal cord tissue of patients with ALS. A different differentiation protocol was used to derive a separate set of C9ORF72 and control motor neurons. Many individual -omics differed by protocol, but some core dysregulated pathways were consistent. This strategy of analyzing patient-specific neurons provides disease-related outcomes with small numbers of heterogeneous lines and reduces variation from single-omics to elucidate network-based signatures.

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Cragnaz, L., Spinelli, G., De Conti, L., Bureau, E. A., Brownlees, J., Feiguin, F., Romano, V., Skoko, N., Klima, R., Kettleborough, C. A., Baralle, F. E. and Baralle, M. (2021).. Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance. Neurobiol Dis 160: 105515. PubMed ID: 34571136

Abstract

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. The study adapted, optimised, and extensively characterised a previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. The study provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. It was then observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. These findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies (Cragnaz, 2021).

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Choi, H. J., Lee, J. Y., Cha, S. J., Han, Y. J., Yoon, J. H., Kim, H. J. and Kim, K. (2021).. FUS-induced neurotoxicity is prevented by inhibiting GSK-3 model in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 34605896

Abstract

Amyotrophic lateral sclerosis (ALS)-linked mutations in fused in sarcoma (FUS) lead to the formation of cytoplasmic aggregates in neurons. They are believed play a critical role in the pathogenesis of FUS-associated ALS. Therefore, the clearance and degradation of cytoplasmic FUS aggregates in neurons may be considered a therapeutic strategy for ALS. This study reports GSK-3β as a potential modulator of FUS-induced toxicity. RNAi-mediated knockdown of Drosophila ortholog Shaggy in FUS-expressing flies suppresses defective phenotypes, including retinal degeneration, motor defects, motor neuron degeneration, and mitochondrial dysfunction. Furthermore, it was found that cytoplasmic FUS aggregates were significantly reduced by Shaggy knockdown. In addition, studies found that the levels of FUS proteins were significantly reduced by co-overexpression of Slimb, a F-box protein, in FUS-expressing flies, indicating that Slimb is critical for the suppressive effect of Shaggy/GSK-3β inhibition on FUS-induced toxicity in Drosophila. These findings revealed a novel mechanism of neuronal protective effect through SCFSlimb-mediated FUS degradation via GSK-3β inhibition, and provided in vivo evidence of the potential for modulating FUS-induced ALS progression using GSK-3β inhibitors (Choi, 2021).

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Romano, G., Holodkov, N., Klima, R. and Feiguin, F. (2021).. TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila CNS. Sci Rep 11(1): 18761. PubMed ID: 34548578

Abstract
Alterations in the function of the RNA-binding protein TDP-43 are largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. In previous work, it was found found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible for converting glutamate to γ-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43, suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. This study observed that TBPH was required for the regulation of GAD1 pre-mRNA splicing and the levels of GABA in the Drosophila central nervous system (CNS). Interestingly, this study discovered that pharmacological treatments aimed to potentiate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies, revealing novel mechanisms and therapeutic strategies in ALS (Romano, 2021).

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Chou, C. C., Alexeeva, O. M., Yamada, S., Pribadi, A., Zhang, Y., Mo, B., Williams, K. R., Zarnescu, D. C. and Rossoll, W. (2015). PABPN1 suppresses TDP-43 toxicity in ALS disease models. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26130692

Abstract
TAR DNA-binding protein 43 (TDP-43; see Drosophila TDP-43) is a major disease protein in amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases. Both the cytoplasmic accumulation of toxic ubiquitinated and hyperphosphorylated TDP-43 fragments and the loss of normal TDP-43 from the nucleus may contribute to the disease progression by impairing normal RNA and protein homeostasis. Therefore, both the removal of pathological protein and the rescue of TDP-43 mislocalization may be critical for halting or reversing TDP-43 proteinopathies. This study reports poly(A)-binding protein nuclear 1 (PABPN1) as a novel TDP-43 interaction partner that acts as a potent suppressor of TDP-43 toxicity. Overexpression of full-length PABPN1 but not a truncated version lacking the nuclear localization signal protects from pathogenic TDP-43-mediated toxicity, promotes the degradation of pathological TDP-43 and restores normal solubility and nuclear localization of endogenous TDP-43. Reduced levels of PABPN1 enhances the phenotypes in several cell culture and Drosophila models of ALS and results in the cytoplasmic mislocalization of TDP-43. Moreover, PABPN1 rescues the dysregulated stress granule (SG) dynamics and facilitates the removal of persistent SGs in TDP-43-mediated disease conditions. These findings demonstrate a role for PABPN1 in rescuing several cytopathological features of TDP-43 proteinopathy by increasing the turnover of pathologic proteins (Chou, 2015).

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Fumagalli, L., et al. (2021). C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility. Sci Adv 7(15). PubMed ID: 33837088

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. This study shows using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, this study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

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Zhao, M. J., Yao, X., Wei, P., Zhao, C., Cheng, M., Zhang, D., Xue, W., He, W. T., Xue, W., Zuo, X., Jiang, L. L., Luo, Z., Song, J., Shu, W. J., Yuan, H. Y., Liang, Y., Sun, H., Zhou, Y., Zhou, Y., Zheng, L., Hu, H. Y., Wang, J. and Du, H. N. (2021). O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity. EMBO Rep: e51649. PubMed ID: 33855783

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. This study shows that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. This study further showed that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. This study demonstrates that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. These findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis (Zhao, 2021).

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Lehmkuhl, E. M., Loganathan, S., Alsop, E., Blythe, A. D., Kovalik, T., Mortimore, N. P., Barrameda, D., Kueth, C., Eck, R. J., Siddegowda, B. B., Joardar, A., Ball, H., Macias, M. E., Bowser, R., Van Keuren-Jensen, K. and Zarnescu, D. C. (2021). TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6. Acta Neuropathol Commun 9(1): 52. PubMed ID: 33762006

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, TDP-43 dependent translational alterations in motor neurons were identified impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.

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Kim, E. S., Chung, C. G., Park, J. H., Ko, B. S., Park, S. S., Kim, Y. H., Cha, I. J., Kim, J., Ha, C. M., Kim, H. J. and Lee, S. B. (2021). C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons. Hum Mol Genet 30(12): 1084-1100. PubMed ID: 33783499

RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). This study report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. In Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. Stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, these data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

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Atilano, M. L., Grpnke, S., Niccoli, T., Kempthorne, L., Hahn, O., Moron-Oset, J., Hendrich, O., Dyson, M., Adams, M. L., Hull, A., Salcher-Konrad, M. T., Monaghan, A., Bictash, M., Glaria, I., Isaacs, A. M. and Partridge, L. (2021). Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila. Elife 10. PubMed ID: 33739284

G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. This study shows that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. It was further demonstrated that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, these data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD (Atilano, 2021).

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Cunningham, K. M., Maulding, K., Ruan, K., Senturk, M., Grima, J. C., Sung, H., Zuo, Z., Song, H., Gao, J., Dubey, S., Rothstein, J. D., Zhang, K., Bellen, H. J. and Lloyd, T. E. (2020). TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS. Elife 9. PubMed ID: 33300868

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. A Drosophila screen identified ref(2)P/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. These data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD (Cunningham, 2020).

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Baek, M., Choe, Y. J., Bannwarth, S., Kim, J., Maitra, S., Dorn, G. W., Taylor, J. P., Paquis-Flucklinger, V. and Kim, N. C. (2021). TDP-43 and PINK1 mediate CHCHD10(S59L) mutation-induced defects in Drosophila and in vitro. Nat Commun 12(1): 1924. PubMed ID: 33772006

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. This study generate CHCH10(S59L)-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10(S59L) mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10(S59L) independently affects the TDP-43 and PINK1 pathways. CHCHD10(S59L) expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10(S59L)-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10(S59L)-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. These findings suggest that CHCHD10(S59L)-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD (Baek, 2021).

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Kamemura, K., Chen, C. A., Okumura, M., Miura, M. and Chihara, T. (2021). Amyotrophic lateral sclerosis-associated VAP33 is required for maintaining neuronal dendrite morphology and organelle distribution in Drosophila. Genes Cells. PubMed ID: 33548103

VAMP-associated protein (VAP; see Drosophila Vap33) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. This study utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of VAP33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, human VAPB-P56S was overexpressed in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. This study provides insights into the physiological and pathological functions of VAP in neurons (Kamemura, 2021).

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Kim, J., Kim, S., Nahm, M., Li, T. N., Lin, H. C., Kim, Y. D., Lee, J., Yao, C. K. and Lee, S. (2021). (2020). ALS2 regulates endosomal trafficking, postsynaptic development, and neuronal survival. J Cell Biol 220(5). PubMed ID: 33683284

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. This study demonstrates that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). It was further demonstrated that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival (Kim 2021).

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Zhang, T., Periz, G., Lu, Y. N. and Wang, J. (2020). (2020). USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway. Proc Natl Acad Sci U S A 117(45): 28114-28125. PubMed ID: 33106424

An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). This study reports the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases (Zhang, 2020).

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Mollasalehi, N., Francois-Moutal, L., Scott, D. D., Tello, J. A., Williams, H., Mahoney, B., Carlson, J. M., Dong, Y., Li, X., Miranda, V. G., Gokhale, V., Wang, W., Barmada, S. J. and Khanna, M. (2020). An Allosteric Modulator of RNA Binding Targeting the N-Terminal Domain of TDP-43 Yields Neuroprotective Properties. ACS Chem Biol 15(11): 2854-2859. PubMed ID: 33044808

This study targeted the N-terminal domain (NTD) of transactive response (TAR) DNA binding protein (TDP-43), which is implicated in several neurodegenerative diseases. In silico docking of 50K compounds to the NTD domain of TDP-43 identified a small molecule (nTRD22) that is bound to the N-terminal domain. Interestingly, nTRD22 caused allosteric modulation of the RNA binding domain (RRM) of TDP-43, resulting in decreased binding to RNA in vitro. Moreover, incubation of primary motor neurons with nTRD22 induced a reduction of TDP-43 protein levels, similar to TDP-43 RNA binding-deficient mutants and supporting a disruption of TDP-43 binding to RNA. Finally, nTRD22 mitigated motor impairment in a Drosophila model of amyotrophic lateral sclerosis. These findings provide an exciting way of allosteric modulation of the RNA-binding region of TDP-43 through the N-terminal domain (Mollasalehi, 2020).

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Lee, S., Kim, S., Kang, H. Y., Lim, H. R., Kwon, Y., Jo, M., Jeon, Y. M., Kim, S. R., Kim, K., Ha, C. M., Lee, S. and Kim, H. J. (2020). The overexpression of TDP-43 in astrocytes causes neurodegeneration via a PTP1B-mediated inflammatory response. J Neuroinflammation 17(1): 299. PubMed ID: 33054766

Cytoplasmic inclusions of transactive response DNA binding protein of 43 kDa (TDP-43; see Drosophila TBPH) in neurons and astrocytes are a feature of some neurodegenerative diseases, such as frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). However, the role of TDP-43 in astrocyte pathology remains largely unknown. To investigate whether TDP-43 overexpression in primary astrocytes could induce inflammation, primary astrocytes were transfected with plasmids encoding Gfp or TDP-43-Gfp. The inflammatory response and upregulation of PTP1B in transfected cells were examined using quantitative RT-PCR and immunoblot analysis. Neurotoxicity was analysed in a transwell coculture system of primary cortical neurons with astrocytes and cultured neurons treated with astrocyte-conditioned medium (ACM). The lifespan was analyzed, climbing assays were performed and immunohistochemical data were analyzed in pan-glial TDP-43-expressing flies in the presence or absence of a Ptp61f RNAi transgene (the Drosophila homologue of PTP1B). PTP1B inhibition suppressed TDP-43-induced secretion of inflammatory cytokines (interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) in primary astrocytes. Using a neuron-astrocyte coculture system and astrocyte-conditioned media treatment, it was demonstrated that PTP1B inhibition attenuated neuronal death and mitochondrial dysfunction caused by overexpression of TDP-43 in astrocytes. In addition, in Drosophila, neuromuscular junction (NMJ) defects, a shortened lifespan, inflammation and climbing defects caused by pan-glial overexpression of TDP-43 were significantly rescued by downregulation of ptp61f. These results indicate that PTP1B inhibition mitigates the neuronal toxicity caused by TDP-43-induced inflammation in mammalian astrocytes and Drosophila glial cells (Lee, 2020).

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Mao, D., Lin, G., Tepe, B., Zuo, Z., Tan, K. L., Senturk, M., Zhang, S., Arenkiel, B. R., Sardiello, M. and Bellen, H. J. (2019). VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway. Autophagy 15(7): 1214-1233. PubMed ID: 30741620

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. This study found that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. Loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. It is proposed that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation (Mao, 2019).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive motor neuron degeneration and muscle weakness. More than 20 ALS associated genes have been identified and these genes affect distinct cellular pathways including RNA processing, nuclear protein transport, and the unfolded protein response (UPR). One of the key pathological findings is the presence of TARDBP-positive protein aggregates in the cytoplasm of neurons in the brains and spinal cords of patients. Accumulation of protein aggregates in the ER induces a UPR, which attenuates protein translation and promotes proteasome-mediated degradation as well as expression of numerous ER chaperones. Several ALS-causing genes, including VAPB, VCP and UBQLN2, have been documented to play an important role in the ER, and the loss of these proteins promotes the UPR. In addition, ER stress has also been documented in SOD1G93A heterozygous mice. Whether ER stress is toxic or protective is still a matter of debate as some data argue that ER stress may be beneficial whereas other data dispute this. If the observed ER stress is protective, other defects may accelerate the demise of neurons given that defects in proteostasis are tightly linked to ALS (Mao, 2019).

Two major pathways regulate protein clearance: proteasome and autophagy-lysosome mediated degradation. Basal autophagy is required to maintain neuronal function, as loss of autophagy has been shown to induce neurodegeneration. Emerging evidence indicates that 2 genes associated with ALS, including TARDBP and C9orf72, play a role in autophagy but how they achieve this is not well defined (Mao, 2019).

Various mutations (P56S, T46I, A145V, S160Δ, V234I) in the gene encoding the human VAPB protein cause ALS8 (OMIM: 608627), a form of ALS and spinal muscular atrophy. Interestingly, mRNAs of VAPB are decreased in sporadic patients and in neurons derived from ALS8 patients as well as in human SOD1G93A transgenic mice, suggesting that VAPB may play a role in many forms of ALS. The VAPs belong to the VAMP-associated protein family and are highly conserved across species. There are 2 VAP homologs in mammals: VAPA and B (VAPA/B). However, Drosophila has a single VAP, Vap33 which corresponds to VPR-1 in C. elegans. Studies in Drosophila, C. elegans as well as mammalian cells have shown that VAPs (Vap33, VPR-1, VAPA/B) affect multiple cellular processes. These include the size and shape of neuromuscular junctions (NMJ), the presence of a UPR, the transfer of lipids from the ER to the Golgi, mitochondrial calcium homeostasis and muscle mitochondrial dynamics. VAPA and B share an N-terminal major sperm protein (MSP) domain followed by a coiled-coil domain and a C-terminal transmembrane domain that targets the protein to the ER. Previous work documented that Drosophila Vap33 functions in a cell non-autonomous manner by releasing and secreting the MSP domain. The MSP domain of the human VAPB is also detected in human blood and cerebrospinal fluid (CSF) and the levels of MSP in the CSF is reduced in patients with sporadic ALS, indicating that loss of MSP secretion may be associated with different forms of ALS (Mao, 2019).

In addition to the cell non-autonomous function, VAPB also functions cell autonomously in non-vesicular lipid transfer. VAP proteins directly interact with lipid transport proteins, such as OSBP (oxysterol binding protein) and COL4A3BP/CERT through a FFAT motif (2 phenylalanines in an acidic tract) to facilitate lipid transfer. Both the OSBP and COL4A3BP/CERT proteins contain a pleckstrin homology (PH) domain that interacts with PtdIns4P on the Golgi to promote membrane tethering and lipid transfer from the ER to the Golgi. The VAP-FFAT interaction is abolished in VAPBP56S, the most prevalent variant form of VAPB in ALS8 patients. This P56S variant functions as a loss-of-function mutation in some phenotypic assays and as a dominant-negative mutation as it traps endogenous wild-type VAPA and VAPB proteins in aggregates, resulting in a partial loss of function of both VAPA and VAPB. The tethering of the ER to the Golgi facilitates the transfer of PtdIns4P from the Golgi to the ER for hydrolysis and loss of VAPs affects PtdIns4P levels, including a general increase in the cytoplasm, a decrease in the Golgi, and an increase in endosomes. However, little is known about the role of PtdIns4P in the autophagic-lysosomal degradation pathway (Mao, 2019).

This study provides both in vivo and in vitro evidence that loss of VAPs impairs endo-lysosomal degradation. It was found that loss of VAPs leads to an obvious upregulation of the PtdIns4P levels in the Golgi, and a dramatic increase in the number of endosomes, lysosomes and autophagic vesicles. These compartments are defective because they do not acidify properly. Reducing the PtdIns4P levels significantly suppresses the autophagic and lysosomal defects, suggesting that the VAPs regulate autophagy-lysosomal degradation through a PtdIns4P-mediated endosomal trafficking pathway. Impairing this pathway causes a severe defect in lysosomal degradation that may play a critical role in ALS8 and other forms of ALS (Mao, 2019).

Based on the current studies, a model is proposed. VAP proteins localize to the ER and interact with lipid transfer proteins such as OSBP and COL4A3BP/CERT through their FFAT motif. The PH domains of OSBP and COL4A3BP/CERT interact with PtdIns4P anchored on the Golgi and tether the ER to the Golgi, facilitating PtdIns4P transfer from the Golgi to the ER for its hydrolysis by SACM1L. It is argued that this leads to an accumulation of PtdIns4P in the Golgi and increased production of RAB5- and RAB7-positive endosomes. These endosomes mature into lysosomes leading to an increase in the number of lysosomes with aberrant pH. These defective lysosomes affect protein degradation, and upon fusion with autophagosomes also impair autophagic degradation, resulting in an accumulation of autophagic vesicles (Mao, 2019).

The data argue that the defects in autophagic and lysosomal degradation in VAP mutant cells are due to PtdIns4P imbalance. Indeed, by reducing the PtdIns4P to more normal levels or removing one copy of the endosome proteins Rab5 or Rab7, a significant suppression of endosome and autophagy-lysosomal defects was observed in the Vap33 mutant. Modulating the PtdIns4P and endosome pathway also rescues the locomotion deficit in Vap33 mutant animals, suggesting a strategy to modify the phenotype in patients. At the root of the elevated level of PtdIns4P is the loss of ER-Golgi tethering, as promoting ER-Golgi tethering by overexpression of an OSBP that does not require VAPs significantly suppresses the motor deficit and early lethality of mutant flies (Mao, 2019).

A recent study argues that the function of VAPB is to inhibit autophagy by promoting ER-mitochondria tethering. The authors argue that siRNA-mediated knockdown of VAPB in HeLa cells disrupts ER-mitochondria tethering through VAPB and its interaction with RMDN3/PTPIP51. Loss of this interaction promotes autophagy and does not impair degradation. Given that this study observed dysfunctional autophagy in flies and mammalian cells upon loss of the VAPs it is argued that VAPA and VAPB are redundant and that removing VAPB alone appears insufficient to impair lysosomal degradation, but seems sufficient to promote autophagy induction (Mao, 2019).

The combined loss of function of VAPA and B may be relevant to ALS8. Indeed, the most prevalent form of the VAPB mutation found in these patients is the P56S mutation, which functions as a dominant negative allele in some contexts and traps both VAPA and VAPB in aggregates. Hence, reducing VAPA and B may better mimic the conditions of patients with the VAPBP56S mutation. This interpretation is also consistent with the observed accumulation of SQSTM1 in aged heterozygous VAPBP56S knockin mice (Mao, 2019).

The accumulation of lumenal tagged LAMP1-GFP argues that there is a defect in lysosomal acidification upon loss of Vap33. This phenotype needs to be reconciled with the increased LysoTracker Red staining and increased Magic Red CtsB1 staining observed in Vap33 mutant cells. LysoTracker Red is activated at pH = 6.5, a higher pH than what is required to quench GFP, which is 4.5. The lysosomal pH typically ranges from 4.5 to 5.0. Hence, LysoTracker Red should label lysosomes with a pH between 4.5 ~ 6.5, including many that may not be fully functional when VAPs are lost. Similarly, CTSB has been shown to have high proteolytic activity at pH>5. Hence, LAMP1-GFP reveals non-acidified lysosomes, whereas the increased LysoTracker Red and Magic Red CtsB1 staining indicate an expansion of lysosomes that may include acidified as well as poorly acidified lysosomes. However, the current data cannot exclude the possibility that loss of VAPs impairs the trafficking of some lysosomal proteins that are trapped in non-acidic endosomes. This trafficking defect would also result in dysfunctional lysosomes, consistent with the model (Mao, 2019).

The data show that there is an increase in the acidified lysosomal pool when VAPs are lost. Interestingly, lysosomal expansion is also observed in lysosomal storage diseases due to defects in lysosomal degradation. Indeed, lysosomal degradation defects impair the processing of cargo as well as the renewal of the lysosomal compartment, leading to the accumulation of aberrant lysosomes. Furthermore, loss of VAPs results in a significant disruption of the balance of the various hydrolases per lysosome, and are consistent with the lysosomal phenotypes observed in lysosomal storage diseases (Mao, 2019).

The importance of autophagic and lysosomal function in ALS has only recently come into focus. Loss of TARDBP was recently reported to elevate the levels of TFEB and impair the fusion of autophagosomes with lysosomes. Conversely, C9orf72, the most prevalent ALS-causing gene, has been shown to decrease autophagic flux upon its loss, whereas others have argued that loss of C9orf72 promotes autophagy. However, these studies were not performed in cells that carry the G4C2 hexanucleotide expanded repeat, and the role of autophagy in C9orf72 ALS patients therefore remains to be established. The current data in flies and human cells as well as the phenotypes associated with the mice carrying a single P56S mutation argue that autophagic and lysosomal degradation may be impaired in ALS8 patients and that the primary defect is due to the upregulation of PtdIns4P upon loss of the VAP-mediated anchoring of ER to Golgi (Mao, 2019).

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Park, J. H., Chung, C. G., Park, S. S., Lee, D., Kim, K. M., Jeong, Y., Kim, E. S., Cho, J. H., Jeon, Y. M., Shen, C. J., Kim, H. J., Hwang, D. and Lee, S. B. (2015). Cytosolic calcium regulates cytoplasmic accumulation of TDP-43 through Calpain-A and Importin alpha3. Elife 9. PubMed ID: 33305734

Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. This study identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS (Park, 2020).

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Castelli, L. M., Cutillo, L., Souza, C. D. S., Sanchez-Martinez, A., Granata, I., Lin, Y. H., Myszczynska, M. A., Heath, P. R., Livesey, M. R., Ning, K., Azzouz, M., Shaw, P. J., Guarracino, M. R., Whitworth, A. J., Ferraiuolo, L., Milo, M. and Hautbergue, G. M. (2021). SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis. Mol Neurodegener 16(1): 53. PubMed ID: 34376242
Summary:

Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. This study investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of a gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. This study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. This study also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers (Castelli, 2021).

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Loganathan, S., Ball, H. E., Manzo, E. and Zarnescu, D. C. (2021). Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy. J Vis Exp(174). PubMed ID: 34424253
Summary:
Als is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy broad ranging metabolic deficits have been identified. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700μδ6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700μδ6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43(G298S), an ALS causing variant. This study shows how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700μδ6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

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Jang, H. J., Le, M. U. T., Park, J. H., Chung, C. G., Shon, J. G., Lee, G. S., Moon, J. H., Lee, S. B., Choi, J. S., Lee, T. G. and Yoon, S. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Phospholipid Changes in a Drosophila Model of Early Amyotrophic Lateral Sclerosis. J Am Soc Mass Spectrom. PubMed ID: 34448582
Summary:
ALS is a degenerative disease caused by motor neuron damage in the central nervous system. Drosophila is widely used to investigate disease mechanisms. MALDI-MSI was performed to investigate changes in phospholipid distribution in the brain tissue of an ALS-induced Drosophila model. Fly brain tissues of several hundred micrometers or less were sampled using a fly collar to obtain reproducible tissue sections of similar sizes. MSI of brain tissues of Drosophila cultured for 1 or 10 days showed that the distribution of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), was significantly different between the control group and the ALS group. In addition, the lipid profile according to phospholipids differed as the culture time increased from 1 to 10 days. These results suggest that disease indicators based on lipid metabolites can be discovered by performing MALDI-MSI on very small brain tissue samples from the Drosophila disease model to ultimately assess the phospholipid changes that occur in early-stage ALS.

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Burguete, A. S., Almeida, S., Gao, F. B., Kalb, R., Akins, M. R. and Bonini, N. M. (2015). GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function. Elife 4. PubMed ID: 26650351

Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. This study demonstrates that GGGGCC and CAG microsatellite repeat RNAs associated with C9orf72 in ALS/FTD and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects and the repeat RNA localizes to granules that label with FMRP, a transport granule component. Using a Drosophila GGGGCC expansion disease model, this study characterized dendritic branching defects that are modulated by FMRP and Orb2. The human orthologues of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the mRNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases (Burguete, 2015).

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Hurley, E. P. and Staveley, B. E. (2021). Inhibition of Ref(2)P, the Drosophila homologue of the p62/SQSTM1 gene, increases lifespan and leads to a decline in motor function. BMC Res Notes 14(1): 53. PubMed ID: 33557921

Sequestosome 1 (p62/SQSTM1) is a multifunctional scaffold/adaptor protein encoded by the p62/SQSTM1 gene with function in cellular homeostasis. Mutations in the p62/SQSTM1 gene have been known to be associated with patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson disease (PD). The aim of the present study was to create a novel model of human neurogenerative disease in Drosophila melanogaster by altering the expression of Ref(2)P, the Drosophila orthologue of the human p62/SQSTM1 gene. Ref(2)P expression was altered in all neurons, the dopaminergic neurons and in the motor neurons, with longevity and locomotor function assessed over time. Inhibition of Ref(2)P resulted in a significantly increased median lifespan in the motor neurons, followed by a severe decline in motor skills. Inhibition of Ref(2)P in the dopaminergic neurons resulted in a significant, but minimal increase in median lifespan, accompanied by a drastic decline in locomotor function. Inhibition of Ref(2)P in the ddc-Gal4-expressing neurons resulted in a significant increase in median lifespan, while dramatically reducing motor function (Hurley, 2021).

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Sanna, S., Esposito, S., Masala, A., Sini, P., Nieddu, G., Galioto, M., Fais, M., Iaccarino, C., Cestra, G. and Crosio, C. (2020). HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo. Cell Death Dis 11(5): 369. PubMed ID: 32409664

TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, it was demonstrated that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, this study showed that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, it was observed both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene (Sanna, 2020).

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Lee, P. T., Lievens, J. C., Wang, S. M., Chuang, J. Y., Khalil, B., Wu, H. E., Chang, W. C., Maurice, T. and Su, T. P. (2020). Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models. Nat Commun 11(1): 5580. PubMed ID: 33149115

In a subgroup of patients with amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD), the (G4C2)-RNA repeat expansion from C9orf72 chromosome binds to the Ran-activating protein (RanGAP) at the nuclear pore, resulting in nucleocytoplasmic transport deficit and accumulation of Ran in the cytosol. This study found that the sigma-1 receptor (Sig-1R), a molecular chaperone, reverses the pathological effects of (G4C2)-RNA repeats in cell lines and in Drosophila. The Sig-1R colocalizes with RanGAP and nuclear pore proteins (Nups) and stabilizes the latter. Interestingly, Sig-1Rs directly bind (G4C2)-RNA repeats. Overexpression of Sig-1Rs rescues, whereas the Sig-1R knockout exacerbates, the (G4C2)-RNA repeats-induced aberrant cytoplasmic accumulation of Ran. In Drosophila, Sig-1R (but not the Sig-1R-E102Q mutant) overexpression reverses eye necrosis, climbing deficit, and firing discharge caused by (G4C2)-RNA repeats. These results on a molecular chaperone at the nuclear pore suggest that Sig-1Rs may benefit patients with C9orf72 ALS/FTD by chaperoning the nuclear pore assembly and sponging away deleterious (G4C2)-RNA repeats (Lee, 2020).

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Tazelaar, G. H. P., Boeynaems, S., De Decker, M., ...., Veldink, J. H. and van Es, M. A. (2020). CATXN1 repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization. Brain Commun 2(2): fcaa064. PubMed ID: 32954321

Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. A large-scale international study was therefore performed in 11,700 individuals, in which a significant association was shown between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis. Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis (Tazelaar, 2020).

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Park, J. H., Chung, C. G., Seo, J., Lee, B. H., Lee, Y. S., Kweon, J. H. and Lee, S. B. (2020). C9orf72-Associated Arginine-Rich Dipeptide Repeat Proteins Reduce the Number of Golgi Outposts and Dendritic Branches in Drosophila Neurons. Mol Cells 43(9): 821-830. PubMed ID: 32975212

Altered dendritic morphology is frequently observed in various neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)This study investigated dendritic morphological defects caused by dipeptide repeat protein (DPR) toxicity associated with G4C2 expansion mutation of C9orf72 (the leading genetic cause of ALS and FTD) in Drosophila neurons. Among the five DPRs produced by repeat-associated non-ATG translation of G4C2 repeats, this study found that arginine-rich DPRs (PR and GR) led to the most significant reduction in dendritic branches and plasma membrane (PM) supply in Class IV dendritic arborization (C4 da) neurons. Furthermore, expression of PR and GR reduced the number of Golgi outposts (GOPs) in dendrites. In Drosophila brains, expression of PR, but not GR, led to a significant reduction in the mRNA level of CrebA, a transcription factor regulating the formation of GOPs. Overexpressing CrebA in PR-expressing C4 da neurons mitigated PM supply defects and restored the number of GOPs, but the number of dendritic branches remained unchanged, suggesting that other molecules besides CrebA may be involved in dendritic branching. Taken together, these results provide valuable insight into the understanding of dendritic pathology associated with C9-ALS/FTD (Park, 2020).

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Otte, C. G., Fortuna, T. R., Mann, J. R., Gleixner, A. M., Ramesh, N., Pyles, N. J., Pandey, U. B. and Donnelly, C. J. (2020). Optogenetic TDP-43 nucleation induces persistent insoluble species and progressive motor dysfunction in vivo. Neurobiol Dis: 105078. PubMed ID: 32927062

TDP-43 (see Drosophila Tdp-43) is a predominantly nuclear DNA/RNA binding protein that is often mislocalized into insoluble cytoplasmic inclusions in post-mortem patient tissue in a variety of neurodegenerative disorders, most notably, Amyotrophic Lateral Sclerosis (ALS), a fatal and progressive neuromuscular disorder. The underlying causes of TDP-43 proteinopathies remain unclear, but recent studies indicate the formation of these protein assemblies is driven by aberrant phase transitions of RNA deficient TDP-43. Technical limitations have prevented an understanding of how TDP-43 proteinopathy relates to disease pathogenesis. Current animal models of TDP-43 proteinopathy often rely on overexpression of wild-type TDP-43 to non-physiological levels that may initiate neurotoxicity through nuclear gain of function mechanisms, or by the expression of disease-causing mutations found in only a fraction of ALS patients. New technologies allowing for light-responsive control of subcellular protein crowding provide a promising approach to drive intracellular protein aggregation, as has been previously demonstrated in vitro. This study presents a model for the optogenetic induction of TDP-43 aggregation in Drosophila that recapitulates key biochemical features seen in patient pathology, most notably light-inducible persistent insoluble species and progressive motor dysfunction. These data describe a photokinetic in vivo model that could be as a future platform to identify novel genetic and pharmacological modifiers of diseases associated with TDP-43 neuropathology (Otte, 2020).

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Wen, X., An, P., Li, H., Zhou, Z., Sun, Y., Wang, J., Ma, L. and Lu, B. (2020). Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS. Neurosci Bull. PubMed ID: 32500377

Expansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders, including Huntington disease [caused by expanded CAG repeats (CAGr) in the HTT gene], and amyotrophic lateral sclerosis [ALS, possibly caused by expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene], of which the molecular mechanisms remain unclear. This study demonstrated that lowering the Drosophila homologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and the shortened life-span in Drosophila expressing expanded CAGr or expanded G4C2r. Expression of human tau (htau4R) restored the disease-related phenotypes that had been mitigated by the loss of dtau, suggesting an evolutionarily-conserved role of tau in neurodegeneration. This study further revealed that G4C2r expression increased tau accumulation by inhibiting autophagosome-lysosome fusion, possibly due to lowering the level of BAG3, a regulator of autophagy and tau. Taken together, these results reveal a novel mechanism by which expanded G4C2r causes neurodegeneration via an evolutionarily-conserved mechanism. These findings provide novel autophagy-related mechanistic insights into C9ORF72-ALS and possible entry points to disease treatment (Wen, 2020).

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Zhao, M., Kao, C. S., Arndt, C., Tran, D. D., Cho, W. I., Maksimovic, K., Chen, X. X. L., Khan, M., Zhu, H., Qiao, J., Peng, K., Hong, J., Xu, J., Kim, D., Kim, J. R., Lee, J., van Bruggen, R., Yoon, W. H. and Park, J. (2020). Knockdown of genes involved in axonal transport enhances the toxicity of human neuromuscular disease-linked MATR3 mutations in Drosophila. FEBS Lett. PubMed ID: 32515490 Mutations in the nuclear matrix protein Matrin 3 (MATR3) have been identified in amyotrophic lateral sclerosis (ALS) and myopathy. To investigate the mechanisms underlying MATR3 mutations in neuromuscular diseases and efficiently screen for modifiers of MATR3 toxicity, transgenic MATR3 flies were generated. The findings indicate that expression of wildtype or mutant MATR3 in motor neurons reduces climbing ability and lifespan of flies, while their expression in indirect flight muscles results in abnormal wing positioning and muscle degeneration. In both motor neurons and indirect flight muscles, mutant MATR3 expression results in more severe phenotypes than wildtype MATR3, demonstrating that the disease-linked mutations confer pathogenicity. A targeted candidate screen was conducted for modifiers of the MATR3 abnormal wing phenotype, and multiple enhancers involved in axonal transport were identfied. Knockdown of these genes enhanced protein levels and insolubility of mutant MATR3. These results suggest that accumulation of mutant MATR3 contributes to toxicity and implicate axonal transport dysfunction in disease pathogenesis (Zhao, 2020).

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Gonzalez, A. E. and Wang, X. (2020). Drosophila VCP/p97 Mediates Dynein-Dependent Retrograde Mitochondrial Motility in Axons. Front Cell Dev Biol 8: 256. PubMed ID: 32373611

Valosin-containing protein (VCP), also called p97, is an evolutionarily conserved and ubiquitously expressed ATPase with diverse cellular functions. Dominant mutations in VCP are found in a late-onset multisystem degenerative proteinopathy. The neurological manifestations of the disorder include frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In these patients, long motor neuron axons could be particularly susceptible to defects in axonal transport. However, whether VCP has a physiological function in maintaining axonal transport and whether this role is impaired by disease-causing mutations remains elusive. By employing live-imaging methods in Drosophila larval axons and performing genetic interaction experiments, this study discovered that VCP regulates the axonal transport of mitochondria. Downregulation of VCP enhances the retrograde transport of mitochondria and reduces the density of mitochondria in larval axons. This unidirectional motility phenotype is rescued by removing one copy of the retrograde motor dynein heavy chain (DHC), or elevating Miro which facilitates anterograde mitochondrial movement by interacting with the anterograde motor kinesin heavy chain (KHC). Importantly, Miro upregulation also significantly improves ATP production of VCP mutant larvae. Human VCP pathogenic mutations were investigated in the fly system. Expressing these mutations affects mitochondrial transport in the same way as knocking down VCP. These results reveal a new role of VCP in mediating axonal mitochondrial transport, and provide evidence implicating impaired mitochondrial motility in the pathophysiology of VCP-relevant neurodegenerative diseases (Gonzalez, 2020).

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Kankel, M. W., Sen, A., Lu, L., Theodorou, M., Dimlich, D. N., McCampbell, A., Henderson, C. E., Shneider, N. A. and Artavanis-Tsakonas, S. (2020). Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target. Genetics. PubMed ID: 32345615

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a devastating neurodegenerative disorder lacking effective treatments. ALS pathology is linked to mutations in more than twenty different genes indicating a complex underlying genetic architecture that is effectively unknown. In an attempt to identify genes and pathways for potential therapeutic intervention and explore the genetic circuitry underlying Drosophila models of ALS, this study carried out two independent genome-wide screens for modifiers of degenerative phenotypes associated with the expression of transgenic constructs carrying familial ALS (fALS)-causing alleles of FUS (hFUS(R521C)) and TDP-43 (hTDP-43(M337V)). A complex array of genes was uncovered affecting either - or both - of the two strains, and their activities were investigated in additional ALS models. These studies indicate the pathway that governs Phospholipase D (PLD) activity as a major modifier of ALS-related phenotypes, a notion supported by data generated in mice and humans (Kankel, 2020).

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Duan, Y., et al. (2019). PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins. Cell Res. PubMed ID: 30728452

Abstract

Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as myotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. This study investigated the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). It was further uncovered that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, it was revealed that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, these findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation (Duan, 2019).

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Agudelo, A., St Amand, V., Grissom, L., Lafond, D., Achilli, T., Sahin, A., Reenan, R. and Stilwell, G. (2020). Age-dependent degeneration of an identified adult leg motor neuron in a Drosophila SOD1 model of ALS. Biol Open. PubMed ID: 32994185

Abstract

Mutations in superoxide dismutase 1 (SOD1) cause familial Amyotrophic lateral sclerosis (ALS) in humans. ALS is a neurodegenerative disease characterized by progressive motor neuron loss leading to paralysis and inevitable death in affected individuals. Using a gene replacement strategy to introduce disease mutations into the orthologous Drosophila sod1 (dsod1) gene, This study characterize changes at the neuromuscular junction using longer lived dsod1 mutant adults. Homozygous dsod1 (H71Y/H71Y) or dsod1 (null/null) flies display progressive walking defects with paralysis of the 3rd metathoracic leg. In dissected legs, age-dependent changes were assessed in a single identified motor neuron (MN-I2) innervating the tibia levitator muscle. At adult eclosion, MN-I2 of dsod1 (H71Y/H71Y) or dsod1 (null/null) flies is patterned similar to wild type flies indicating no readily apparent developmental defects. Over the course of 10 days post-eclosion, MN-I2 shows an overall reduction in arborization with bouton swelling and loss of the post-synaptic marker Discs-large (Dlg) in mutant dsod1 adults. In addition, increases in polyubiquitinated proteins correlate with the timing and extent of MN-I2 changes. Because similar phenotypes are observed between flies homozygous for either dsod1 (H71Y) or dsod1 (null) alleles, it is concluded these NMJ changes are mainly associated with sod loss of function. Together these studies characterize age-related morphological and molecular changes associated with axonal retraction in a Drosophila model of ALS that recapitulate an important aspect of the human disease (Agudelo, 2020).

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Li, S., Wu, Z., Li, Y., Tantray, I., De Stefani, D., Mattarei, A., Krishnan, G., Gao, F. B., Vogel, H. and Lu, B. (2020). Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD. Cell Rep 32(5): 107989. PubMed ID: 32755582

Abstract

Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. This study shows in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies (Li, 2020).

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Azuma, Y., Tokuda, T., Kushimura, Y., Yamamoto, I., Mizuta, I., Mizuno, T., Nakagawa, M., Ueyama, M., Nagai, Y., Iwasaki, Y., Yoshida, M., Pan, D., Yoshida, H. and Yamaguchi, M. (2018). Hippo, Drosophila MST, is a novel modifier of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS. Exp Cell Res. PubMed ID: 30092221

Abstract

Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). Drosophila models of ALS have been established targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. Whether Hippo pathway genes modify the ALS phenotype was determined using Caz knockdown flies. A genetic link was found between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. The results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS (Azuma, 2018).

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Gogia, N., Sarkar, A., Mehta, A. S., Ramesh, N., Deshpande, P., Kango-Singh, M., Pandey, U. B. and Singh, A. (2020). Inactivation of Hippo and cJun-N-terminal Kinase (JNK) signaling mitigate FUS mediated neurodegeneration in vivo. Neurobiol Dis 140: 104837. PubMed ID: 32199908

Abstract

Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. A screen was performed for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, was identified as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpo triggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH2) Kinase (JNK) mediated cell death. Downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. This study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders (Gogia, 2020).

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Cha, S. J., Han, Y. J., Choi, H. J., Kim, H. J. and Kim, K. (2020). Glutathione S-Transferase Rescues Motor Neuronal Toxicity in Fly Model of Amyotrophic Lateral Sclerosis, Antioxidants (Basel) 9(7). PubMed ID: 32674363

Abstract
Transactive response DNA-binding protein-43 (TDP-43; see Drosophila TDP-43) is involved in the pathology of familial and sporadic amyotrophic lateral sclerosis (ALS). TDP-43-mediated ALS models in mice, Drosophila melanogaster, and zebrafish exhibit dysfunction of locomotor function, defective neuromuscular junctions, and motor neuron defects. There is currently no effective cure for ALS, and the underlying mechanisms of TDP-43 in ALS remain poorly understood. In this study, a genetic screen was performed to identify modifiers of human TDP-43 (hTDP-43) in a Drosophila model, and glutathione S-transferase omega 2 (GstO2) was found to be involved in hTDP-43 neurotoxicity. GstO2 overexpressed on recovered defective phenotypes resulting from hTDP-43, including defective neuromuscular junction (NMJ) boutons, degenerated motor neuronal axons, and reduced larvae and adult fly locomotive activity, without modulating the levels of hTDP-43 protein expression. GstO2 modulated neurotoxicity by regulating reactive oxygen species (ROS) produced by hTDP-43 in the Drosophila model of ALS. These results demonstrated that GstO2 was a key regulator in hTDP-43-related ALS pathogenesis and indicated its potential as a therapeutic target for ALS (Cha, 2020).

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Couly, S., Khalil, B., Viguier, V., Roussel, J., Maurice, T. and Lievens, J. C. (2019). Sigma-1 receptor is a key genetic modulator in amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 31696229

Abstract

Sigma-1 receptor (S1R) is an endoplasmic reticulum (ER) chaperone that regulates mitochondrial respiration but also controls cellular defense against ER and oxidative stress. This makes S1R a potential therapeutic target in amyotrophic lateral sclerosis (ALS). Especially, as a missense mutation E102Q in S1R has been reported in few familial ALS cases. However, the pathogenicity of S1RE102Q and the beneficial impact of S1R in the ALS context remain to be demonstrated in vivo. To address this, transgenic Drosophila were generated that express human wild-type S1R or S1RE102Q. Expression of mutant S1R in fly neurons induces abnormal eye morphology and locomotor defects in a dose-dependent manner. This was accompanied by abnormal mitochondrial fragmentation, reduced ATP levels and a higher fatigability at the neuromuscular junction during high energy demand. Overexpressing IP3 receptor or glucose transporter mitigates the S1RE102Q-induced eye phenotype, further highlighting the role of calcium and energy metabolism in its toxicity. More importantly, wild-type S1R rescues locomotor activity and ATP levels of flies expressing the key ALS protein, TDP43. Moreover, overexpressing wild-type S1R enhances resistance of flies to oxidative stress. Therefore, these data provide the first genetic evidence that mutant S1R recapitulates ALS pathology in vivo while increasing S1R confers neuroprotection against TDP43 toxicity (Couly, 2019).

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Casci, I., Krishnamurthy, K., Kour, S., Tripathy, V., Ramesh, N., Anderson, E. N., Marrone, L., Grant, R. A., Oliver, S., Gochenaur, L., Patel, K., Sterneckert, J., Gleixner, A. M., Donnelly, C. J., Ruepp, M. D., Sini, A. M., Zuccaro, E., Pennuto, M., Pasinelli, P. and Bhan Pandey, U. (2019). Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization. Nat Commun 10(1): 5583. PubMed ID: 31811140

Abstract

Mutations in fused in sarcoma (FUS; see Drosophila Cabeza) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. This study shows the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. These data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.

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Moron-Oset, J., Super, T., Esser, J., Isaacs, A. M., Gronke, S. and Partridge, L. (2019). Glycine-alanine dipeptide repeats spread rapidly in a repeat length- and age-dependent manner in the fly brain. Acta Neuropathol Commun 7(1): 209. PubMed ID: 31843021

Abstract

Hexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the expansions are translated by repeat-associated non-AUG translation into five dipeptide repeat proteins (DPRs). Of these, the polyGR, polyPR and, to a lesser extent, polyGA DPRs are neurotoxic, with polyGA the most abundantly detected DPR in patient tissue. Trans-cellular transmission of protein aggregates has recently emerged as a major driver of toxicity in various neurodegenerative diseases. In vitro evidence suggests that the C9 DPRs can spread. However, whether this phenomenon occurs under more complex in vivo conditions remains unexplored. This study used the adult fly brain to investigate whether the C9 DPRs can spread in vivo upon expression in a subset of neurons. Only polyGA was found to progressively spread throughout the brain, accumulating in the shape of aggregate-like puncta inside recipient cells. Interestingly, GA transmission occurred as early as 3 days after expression induction. By comparing the spread of 36, 100 and 200 polyGA repeats, it was found that polyGA spread is enhanced upon expression of longer GA DPRs. Transmission of polyGA is greater in older flies, indicating that age-associated factors exacerbate the spread. These data highlight a unique propensity of polyGA to spread throughout the brain, which could contribute to the greater abundance of polyGA in patient tissue. In addition, a model of early GA transmission is presented that is suitable for genetic screens to identify mechanisms of spread and its consequences in vivo.

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Park, S., Park, S. K., Watanabe, N., Hashimoto, T., Iwatsubo, T., Shelkovnikova, T. A. and Liebman, S. W. (2019). Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies. PLoS Genet 15(8): e1008308. PubMed ID: 31390360

Abstract

Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons. Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets. Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. This study shows that CREST is toxic in yeast and forms nuclear and occasionally cytoplasmic foci that stain with Thioflavin-T, a dye indicative of amyloid-like protein. Like the yeast chromatin-remodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN+] prion and reduced by deletion of the HSP104 chaperone required for the propagation of many yeast prions. Likewise, deletion of PBP1 reduced CREST toxicity and aggregation. In accord with the yeast data, this study shows that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Downregulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to largely rescue the severe degenerative phenotype induced by human CREST. Overexpression caused considerable co-localization of CREST and PBP1/ATXN2 in cytoplasmic foci in both yeast and mammalian cells. Thus, co-aggregation of CREST and PBP1/ATXN2 may serve as one of the mechanisms of PBP1/ATXN2-mediated toxicity. These results extend the spectrum of ALS associated proteins whose toxicity is regulated by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases (Park, 2019).

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Chang, Y. H. and Dubnau, J. (2019). The gypsy endogenous retrovirus drives non-cell-autonomous propagation in a Drosophila TDP-43 model of neurodegeneration. Curr Biol. PubMed ID: 31495585

Abstract

A hallmark of neurodegenerative disease is focal onset of pathological protein aggregation, followed by progressive spread of pathology to connected brain regions. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), pathology is often associated with aggregation of TAR DNA-binding protein 43 (TDP-43). Although aggregated TDP-43 protein moves between cells, it is not clear whether and how this movement propagates the degeneration. A Drosophila model of human TDP-43 was established in which toxic expression of human TDP-43 was initiated focally within small groups of glial cells. This focal onset kills adjacent neurons. Surprisingly, this spreading death is caused by an endogenous retrovirus within the glia, which leads to DNA damage and death in adjacent neurons. These findings suggest a possible mechanism by which human retroviruses such as HERV-K might contribute to TDP-43-mediated propagation of neurodegeneration (Chang, 2019).

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Cha, S. J., Choi, H. J., Kim, H. J., Choi, E. J., Song, K. H., Im, D. S. and Kim, K. (2019). Parkin expression reverses mitochondrial dysfunction in fused in sarcoma-induced amyotrophic lateral sclerosis. Insect Mol Biol. PubMed ID: 31290213

Abstract
sLN2 and UBQLN4, cause familial ALS. The role of ubiquilins in proteasomal degradation is well established, but their role in autophagy-lysosomal clearance is poorly defined. This study describes a crosstalk between endoplasmic reticulum stress, mTOR signalling and autophagic flux in Drosophila and mammalian cells lacking ubiquilins. Loss of ubiquilins leads to endoplasmic reticulum stress, impairs mTORC1 activity, promotes autophagy and causes the demise of neurons. Ubiquilin mutants were shown to display defective autophagic flux due to reduced lysosome acidification. Ubiquilins are required to maintain proper levels of the V0a/V100 subunit of the vacuolar H(+)-ATPase and lysosomal pH. Feeding flies acidic nanoparticles alleviates defective autophagic flux in ubiquilin mutants. Hence, these studies reveal a conserved role for ubiquilins as regulators of autophagy by controlling vacuolar H(+)-ATPase activity and mTOR signalling (Senturk, 2019).

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Held, A., Major, P., Sahin, A., Reenan, R., Lipscombe, D. and Wharton, K. A. (2019). Circuit dysfunction in SOD1-ALS model first detected in sensory feedback prior to motor neuron degeneration is alleviated by BMP signaling. J Neurosci. PubMed ID: 30659087

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose origin and underlying cellular defects are not fully understood. While motor neuron degeneration is the signature feature of ALS, it is not clear if motor neurons, or other cells of the motor circuit, are the site of disease initiation. To better understand the contribution of multiple cell types in ALS, use was made of a Drosophila Sod1(G85R) knock-in model, in which all cells harbor the disease allele. End-stage dSod1(G85R) animals of both sexes exhibit severe motor deficits with clear degeneration of motor neurons. Interestingly, earlier in dSod1(G85R) larvae, motor function is also compromised, but their motor neurons exhibit only subtle morphological and electrophysiological changes, that are unlikely to cause the observed decrease in locomotion. The intact motor circuit was analyzed, and a defect was identified in sensory feedback that likely accounts for the altered motor activity of dSod1(G85R). Cell-autonomous activation of BMP signaling in proprioceptor sensory neurons, critical for the relay of the contractile status of muscles back to the central nerve cord, is able to completely rescue early stage motor defects and partially rescue late stage motor function to extend lifespan. Identification of a defect in sensory feedback, as a potential initiating event in ALS motor dysfunction, coupled with the ability of modified proprioceptors to alleviate such motor deficits, underscores the critical role that non-motor neurons play in disease progression and highlights their potential as a site to identify early-stage ALS biomarkers and for therapeutic intervention (Held, 2019).

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Chaplot, K., Pimpale, L., Ramalingam, B., Deivasigamani, S., Kamat, S. S. and Ratnaparkhi, G. S. (2019). SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via ROS-induced proteasomal degradation in a Drosophila model of Amyotrophic Lateral Sclerosis. Dis Model Mech. PubMed ID: 30635270

Abstract

Familial Amyotrophic Lateral Sclerosis (F-ALS) is an incurable, late onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated Protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase understanding of the disease and lead to potential therapeutics. A quantitative high-throughput, Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy was developed to score for genes involved in the modulation of aggregates of fly ortholog, VAP(P58S), fused with GFP. A targeted RNAi screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci SOD1, TDP43 and also genes belonging to the TOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell based screen. In the larval brain, it was found that reduction of SOD1 level or decreased TOR signalling reduces aggregation, presumably by increasing levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation which appears to be triggered by an increase in ROS. This study has thus uncovered an interesting interplay between SOD1, ROS and TOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to a better understanding of initiation and progression of ALS (Chaplot, 2019).

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Kushimura, Y., Tokuda, T., Azuma, Y., Yamamoto, I., Mizuta, I., Mizuno, T., Nakagawa, M., Ueyama, M., Nagai, Y., Yoshida, H. and Yamaguchi, M. (2018). Overexpression of ter94, Drosophila VCP, improves motor neuron degeneration induced by knockdown of TBPH, Drosophila TDP-43. Am J Neurodegener Dis 7(1): 11-31. PubMed ID: 29531866

Abstract

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterized by the motor neuron degeneration that eventually leads to complete paralysis and death within 2-5 years after disease onset. One of the major pathological hallmark of ALS is abnormal accumulation of inclusions containing TAR DNA-binding protein-43 (TDP-43). TDP-43 is normally found in the nucleus, but in ALS, it localizes in the cytoplasm as inclusions as well as in the nucleus. Loss of nuclear TDP-43 functions likely contributes to neurodegeneration. TBPH is the Drosophila ortholog of human TDP-43. This study confirmed that Drosophila models harboring TBPH knockdown develop locomotive deficits and degeneration of motoneurons (MNs) due to loss of its nuclear functions, recapitulating the human ALS phenotypes. Previous work has suggested that ter94, the Drosophila ortholog of human Valosin-containing protein (VCP), is a modulator of degeneration in MNs induced by knockdown of Caz, the Drosophila ortholog of human FUS. In this study, to determine the effects of VCP on TDP-43-associated ALS pathogenic processes, genetic interactions were examined between TBPH and ter94. Overexpression of ter94 suppressed the compound eye degeneration caused by TBPH knockdown and suppressed the morbid phenotypes caused by neuron-specific TBPH knockdown, such as locomotive dysfunction and degeneration of MN terminals. Further immunocytochemical analyses revealed that the suppression is caused by restoring the cytoplasmically mislocalized TBPH back to the nucleus. Consistent with these observations, a loss-of-function mutation of ter94 enhanced the compound eye degeneration caused by TBPH knockdown and partially enhanced the locomotive dysfunction caused by TBPH knockdown. The data demonstrated that expression levels of ter94 influenced the phenotypes caused by TBPH knockdown, and indicate that reagents that up-regulate the function of human VCP could modify MN degeneration in ALS caused by TDP-43 mislocalization (Kushimura, 2018).

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Miguel, L., Avequin, T., Pons, M., Frebourg, T., Campion, D. and Lecourtois, M. (2018). FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression in Drosophila. Brain Res. PubMed ID: 29778779

Abstract

TDP-43 is a major disease-causing protein in amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Today, more than 50 missense mutations in the TARDBP/TDP-43 gene have been described in patients with FTLD/ALS. However, the functional consequences of FTLD/ALS-linked TDP-43 mutations are not fully elucidated. In the physiological state, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. This study investigated whether the FTLD/ALS-associated mutations could interfere with TDP-43 protein's capacity to modulate its own protein levels using Drosophila as an experimental model. The data show that FTLD/ALS-associated mutant proteins regulate TDP-43 production with the same efficiency as the wild-type form of the protein. Thus, FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels (Miguel, 2018).

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Wang, T., Cheng, J., Wang, S., Wang, X., Jiang, H., Yang, Y., Wang, Y., Zhang, C., Liang, W. and Feng, H. (2018). alpha-Lipoic acid attenuates oxidative stress and neurotoxicity via the ERK/Akt-dependent pathway in the mutant hSOD1 related Drosophila model and the NSC34 cell line of amyotrophic lateral sclerosis. Brain Res Bull 140:299-310. Pubmed ID: 29842900

Abstract

Amyotrophic lateral sclerosis (ALS) is a degenerative disease with a progressive loss of motor neurons in the central nervous system (CNS). However, there are unsolved problems with the therapies for this disease. alpha-Lipoic acid (LA) is a natural, universal antioxidant capable of scavenging hydroxyl radicals as well as regenerating a series of antioxidant enzymes that has been widely used in clinical settings. This study aimed to evaluate the antioxidant and neuroprotective effects of LA in ALS cell and Drosophila models with mutant G85R and G93A hSOD1 genes. The biological effects of LA and the protein levels of several antioxidant factors were examined, as were those of phospho-Akt and phospho-ERK. Furthermore, specific inhibitors of the PI3K/Akt and MEK/ERK signaling pathways were used to analyze their effects on LA-induced antioxidant expression in vivo and in vitro. Evidences showed that the mutant hSOD1 resulted in the increased oxidative stress, abnormal antioxidant signaling and pathological behaviors in motor performance and survival compared with non-mutant hSOD1 models, treatment with LA improved motor activity and survival in transgenic flies, prevented NSC34 cells from mutant hSOD1 or H2O2 induced decreased antioxidant enzymes as well as increased ROS levels. In addition, LA regulated the expression levels of antioxidant proteins in a dose- and periodical time-dependent manner, which might be mediated by ERK/Akt pathway activation and independent from the mutant hSOD1 gene. The observations suggest that LA exerts strong and positive antioxidant and neuroprotective effects through the activation of the ERK-Akt pathway in hSOD1 ALS models (Wang, 2018).

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Matsumoto, T., Matsukawa, K., Watanabe, N., Kishino, Y., Kunugi, H., Ihara, R., Wakabayashi, T., Hashimoto, T. and Iwatsubo, T. (2018). Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration. Hum Mol Genet. PubMed ID: 29425337

Abstract

Aggregation of fused in sarcoma (FUS; see Drosophila Cabeza) protein, and mutations in FUS gene, are causative to a range of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. To gain insights into the molecular mechanism whereby FUS causes neurodegeneration, transgenic Drosophila melanogaster were generated overexpressing human FUS in the photoreceptor neurons, which exhibited mild retinal degeneration. Expression of familial ALS-mutant FUS aggravated the degeneration, which was associated with an increase in cytoplasmic localization of FUS. A carboxy-terminally truncated R495X mutant FUS also was localized in cytoplasm, whereas the degenerative phenotype was diminished. Double expression of R495X and wild-type FUS dramatically exacerbated degeneration, sequestrating wild-type FUS into cytoplasmic aggregates. Notably, replacement of all tyrosine residues within the low-complexity domain, which abolished self-assembly of FUS, completely eliminated the degenerative phenotypes. Taken together, it is proposed that self-assembly of FUS through its low-complexity domain contributes to FUS-induced neurodegeneration (Matsumoto, 2018).

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Solomon, D. A., Stepto, A., Au, W. H., Adachi, Y., Diaper, D. C., Hall, R., Rekhi, A., Boudi, A., Tziortzouda, P., Lee, Y. B., Smith, B., Bridi, J. C., Spinelli, G., Dearlove, J., Humphrey, D. M., Gallo, J. M., Troakes, C., Fanto, M., Soller, M., Rogelj, B., Parsons, R. B., Shaw, C. E., Hortobagyi, T. and Hirth, F. (2018). A feedback loop between dipeptide-repeat protein, TDP-43 and karyopherin-alpha mediates C9orf72-related neurodegeneration. Brain 141(10): 2908-2924. PubMed ID: 30239641

Abstract

Accumulation and aggregation of TDP-43 is a major pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 inclusions also characterize patients with GGGGCC (G4C2) hexanucleotide repeat expansion in C9orf72 that causes the most common genetic form of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Functional studies in cell and animal models have identified pathogenic mechanisms including repeat-induced RNA toxicity and accumulation of G4C2-derived dipeptide-repeat proteins. The role of TDP-43 dysfunction in C9ALS/FTD, however, remains elusive. This study found that G4C2-derived dipeptide-repeat protein but not G4C2-RNA accumulation caused TDP-43 proteinopathy that triggered onset and progression of disease in Drosophila models of C9ALS/FTD. Timing and extent of TDP-43 dysfunction was dependent on levels and identity of dipeptide-repeat proteins produced, with poly-GR causing early and poly-GA/poly-GP causing late onset of disease. Accumulating cytosolic, but not insoluble aggregated TDP-43 caused karyopherin-alpha2/4 (KPNA2/4) pathology, increased levels of dipeptide-repeat proteins and enhanced G4C2-related toxicity. Comparable KPNA4 pathology was observed in both sporadic frontotemporal dementia and C9ALS/FTD patient brains characterized by its nuclear depletion and cytosolic accumulation, irrespective of TDP-43 or dipeptide-repeat protein aggregates. These findings identify a vicious feedback cycle for dipeptide-repeat protein-mediated TDP-43 and subsequent KPNA pathology, which becomes self-sufficient of the initiating trigger and causes C9-related neurodegeneration (Solomon, 2018).

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Goodman, L. D., Prudencio, M., Srinivasan, A. R., Rifai, O. M., Lee, V. M., Petrucelli, L. and Bonini, N. M. (2019). eIF4B and eIF4H mediate GR production from expanded G4C2 in a Drosophila model for C9orf72-associated ALS. Acta Neuropathol Commun 7(1): 62. PubMed ID: 31023341

Abstract

The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD (Amyotrophic Lateral Sclerosis/Frontotemporal Degeneration) has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the Glycine Arginine (GR) reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts (Goodman, 2019).

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Lopez-Gonzalez, R., Yang, D., Pribadi, M., Kim, T. S., Krishnan, G., Choi, S. Y., Lee, S., Coppola, G. and Gao, F. B. (2019). Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD. Proc Natl Acad Sci U S A. PubMed ID: 31019093

Abstract

GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, a number of genetic modifiers of poly(GR) toxicity were identified. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD (Lopez-Gonzalez, 2019).

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Goodman, L. D., Prudencio, M., Srinivasan, A. R., Rifai, O. M., Lee, V. M., Petrucelli, L. and Bonini, N. M. (2019). eIF4B and eIF4H mediate GR production from expanded G4C2 in a Drosophila model for C9orf72-associated ALS. Acta Neuropathol Commun 7(1): 62. PubMed ID: 31023341

Abstract

The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the GR reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts (Goodman, 2019).

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He, H., Huang, W., Wang, R., Lin, Y., Guo, Y., Deng, J., Deng, H., Zhu, Y., Allen, E. G., Jin, P. and Duan, R. (2019). Amyotrophic Lateral Sclerosis-associated GGGGCC repeat expansion promotes Tau phosphorylation and toxicity. Neurobiol Dis 130: 104493. PubMed ID: 31176718

Abstract

Microtubule-associated protein Tau (MAPT) and GGGGCC (G4C2) repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) are the major known genetic causes of frontotemporal dementia (FTD) and other neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS). Although expanded G4C2 repeats and Tau traditionally are associated with different clinical presentations, pathological and genetic studies have suggested a strong association between them. This study demonstrates a strong genetic interaction between expanded G4C2 repeats and Tau. Co-expression of expanded G4C2 repeats and Tau could produce a synergistic deterioration of rough eyes, motor function, life span and neuromuscular junction morphological abnormalities in Drosophila. Mechanistically, compared with the normal allele containing (G4C2)3 repeats, the (G4C2)30 allele increased Tau phosphorylation levels and promoted Tau R406W aggregation. These results together suggest a potential crosstalk between expanded G4C2 repeats and Tau in modulating neurodegeneration (He, 2019).

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Francois-Moutal, L., Scott, D. D., Felemban, R., Miranda, V. G., Sayegh, M. R., Perez-Miller, S., Khanna, R., Gokhale, V., Zarnescu, D. C. and Khanna, M. (2019). A small molecule targeting TDP-43's RNA recognition motifs reduces locomotor defects in a Drosophila model of ALS. ACS Chem Biol. PubMed ID: 31241884

Abstract

RNA dysregulation likely contributes to disease pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. A pathological form of the transactive response (TAR) DNA Binding Protein (TDP-43) binds to RNA in stress granules and forms membraneless, amyloid-like, TDP-43 aggregates in the cytoplasm of ALS motor neurons. In was hypothesized in this study that by targeting the RNA recognition motifs (RRM) domains of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. In silico docking of 50K compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43's RRM1 and RRM2 domains; (ii) partially disrupted TDP-43's interaction with the hexanucleotide RNA repeat of the disease-linked c9orf72 gene, but not with (UG)6 canonical binding sequence of TDP-43; and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overexpression of mutant TDP-43. These findings provide an instructive example of a chemical biology approach pivoted to discover small molecules targeting RNA-protein interactions in neurodegenerative diseases (Francois-Moutal, 2019).

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Sun, X., Duan, Y., Qin, C., Li, J. C., Duan, G., Deng, X., Ni, J., Cao, X., Xiang, K., Tian, K., Chen, C. H., Li, A. and Fang, Y. (2018). Distinct multilevel misregulations of Parkin and PINK1 revealed in cell and animal models of TDP-43 proteinopathy. Cell Death Dis 9(10): 953. PubMed ID: 30237395

Abstract

Parkin and PINK1 play an important role in mitochondrial quality control, whose malfunction may also be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Excessive TDP-43 accumulation is a pathological hallmark of ALS and is associated with Parkin protein reduction in spinal cord neurons from sporadic ALS patients. In this study, it was revealed that Parkin and PINK1 are differentially misregulated in TDP-43 proteinopathy at RNA and protein levels. Using knock-in flies, mouse primary neurons, and TDP-43(Q331K) transgenic mice, it was further unveiled that TDP-43 downregulates Parkin mRNA, which involves an unidentified, intron-independent mechanism and requires the RNA-binding and the protein-protein interaction functions of TDP-43. Unlike Parkin, TDP-43 does not regulate PINK1 at an RNA level. Instead, excess of TDP-43 causes cytosolic accumulation of cleaved PINK1 due to impaired proteasomal activity, leading to compromised mitochondrial functions. Consistent with the alterations at the molecular and cellular levels, it was shown that transgenic upregulation of Parkin but downregulation of PINK1 suppresses TDP-43-induced degenerative phenotypes in a Drosophila model of ALS. Together, these findings highlight the challenge associated with the heterogeneity and complexity of ALS pathogenesis, while pointing to Parkin-PINK1 as a common pathway that may be differentially misregulated in TDP-43 proteinopathy (Sun, 2018).

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Lo Piccolo, L., Bonaccorso, R., Attardi, A., Li Greci, L., Romano, G., Sollazzo, M., Giurato, G., Ingrassia, A. M. R., Feiguin, F., Corona, D. F. V. and Onorati, M. C. (2018). Loss of ISWI function in Drosophila nuclear bodies drives cytoplasmic redistribution of Drosophila TDP-43. Int J Mol Sci 19(4). PubMed ID: 29617352

Abstract

Over the past decade, evidence has identified a link between protein aggregation, RNA biology, and a subset of degenerative diseases. An important feature of these disorders is the cytoplasmic or nuclear aggregation of RNA-binding proteins (RBPs). Redistribution of RBPs, such as the human TAR DNA-binding 43 protein (TDP-43) from the nucleus to cytoplasmic inclusions is a pathological feature of several diseases. Indeed, sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration share as hallmarks ubiquitin-positive inclusions. Recently, the wide spectrum of neurodegenerative diseases characterized by RBPs functions' alteration and loss was collectively named proteinopathies. This study shows that TBPH (TAR DNA-binding protein-43 homolog), the Drosophila ortholog of human TDP-43 TAR DNA-binding protein-43, interacts with the 'architectural RNA' (arcRNA) hsromega and with hsromega-associated hnRNPs. Additionally, it was found that the loss of the omega speckles remodeler ISWI (Imitation SWI) changes the TBPH sub-cellular localization to drive a TBPH cytoplasmic accumulation. These results, hence, identify TBPH as a new component of omega speckles and highlight a role of chromatin remodelers in hnRNPs nuclear compartmentalization (Lo Piccolo, 2018).

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Kim, S. H., Stiles, S. G., Feichtmeier, J. M., Ramesh, N., Zhan, L., Scalf, M. A., Smith, L. M., Pandey, U. B. and Tibbetts, R. S. (2018). Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS. Hum Mol Genet 27(2):322-337. PubMed ID: 29161404

Abstract

Members of the conserved ubiquilin (UBQLN) family of ubiquitin (Ub) chaperones harbor an antipodal UBL (Ub-like)-UBA (Ub-associated) domain arrangement and participate in proteasome and autophagosome-mediated protein degradation. Mutations in a proline-rich-repeat region (PRR) of UBQLN2 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD); however, neither the normal functions of the PRR nor impacts of ALS-associated mutations within it are well understood. This study shows that ALS mutations perturb UBQLN2 solubility and folding in a mutation-specific manner. Biochemical impacts of ALS mutations were additive, transferrable to UBQLN1, and resulted in enhanced Ub association. A Drosophila melanogaster model for UBQLN2-associated ALS revealed that both wild-type and ALS-mutant UBQLN2 alleles disrupted Ub homeostasis; however, UBQLN2ALS mutants exhibited age-dependent aggregation and caused toxicity phenotypes beyond that seen for wild-type UBQLN2. Although UBQLN2 toxicity was not correlated with aggregation in the compound eye, aggregation-prone UBQLN2 mutants elicited climbing defects and NMJ abnormalities when expressed in neurons. An UBA domain mutation that abolished Ub binding also diminished UBQLN2 toxicity, implicating Ub binding in the underlying pathomechanism. It is proposed that ALS-associated mutations in UBQLN2 disrupt folding and that both aggregated species and soluble oligomers instigate neuron autonomous toxicity through interference with Ub homeostasis (Kim, 2018).

This study has characterized biochemical properties of wild-type and ALS-mutant UBQLN2 proteins and constructed Drosophila models to understand phenotypic impacts of disease-associated mutations in the UBQLN2 PRR. ALS mutations were shown to exert non-identical effects on UBQLN2 solubility and folding and cause mutation- and tissue-specific toxicities in Drosophila. These findings further implicate Ub binding as a central feature of UBQLN2 pathomechanisms (Kim, 2018).

The clustering of ALS mutations within or proximal to the functional-orphan PRR domain imply that such mutations interfere with cellular processes that are unique to UBQLN2 and/or initiate toxic folds that disrupt cellular regulation through GOF mechanisms. This study tested five clinical mutations (P497H, P497S, P506T, P509S, P525S) and found that only P497H consistently promoted insolubility beyond wild-type levels. His substitutions at disease codons Pro-506 and Pro-509 did not elicit the same changes in solubility as the P497H mutation, indicating that both the nature of the substitution (i.e. Pro to His) and its position within the PRR critically determine the extent of insolubility. Although individual P506T, P509S, and P525S mutations had minor effects on solubility, their combination in UBQLN22P3X (P506T, P509S, and P525S) and UBQLN22P4X (P497H, P506T, P509S, and P525S) proteins led to severe reductions in solubility and patent neuronal aggregation. Although results using compound mutants must be interpreted cautiously, these findings suggest that ALS mutations elicit additive or synergistic effects on UBQLN2 folding (Kim, 2018).

The increased immunoreactivity of UBQLN2P497H, UBQLN22P3X, and UBQLN22P4X with antibodies directed against amino-terminal epitope tags and endogenous UBQLN2 peptides supports the notion that localized mutations in the PRR lead to global changes in UBQLN2 folding, which is further bolstered by the enhanced chymotryptic sensitivity of UBQLN22P3X and UBQLN22P4X in cell culture and fly heads. Interestingly, age-dependent increases in chymotryptic cleavage of UBQLN2 proteins expressed in fly heads was observed, and aging dependent increases in the aggregation of UBQLN2WT and UBQLN2P497H in fly neurons suggesting that UBQLN2 folding was sensitive to the aging cellular environment. It is speculated that ALS mutations disrupt intramolecular folding between the PRR and amino-terminal domains and/or inhibit functional intermolecular oligomerization, which is thought to be mediated by centrally located STI1 repeats. Structural studies of wild-type and ALS-mutant UBQLN2 proteins should further inform these possibilities (Kim, 2018).

The Drosophila findings revealed significantly stronger degenerative phenotypes in flies expressing UBQLN2ALS alleles versus UBQLN2WT and support the idea that Ub-binding figures prominently in UBQLN2-mediated toxicity. Both wild-type and ALS-mutant UBQLN2 proteins altered Ub homeostasis in an UBA domain-dependent manner, likely contributing to non-specific impacts on eye structure, survival, and climbing behavior observed in this and other studies. Nevertheless, enhanced toxicity of UBQLN2ALS mutants was seen across a host of assays. Neuronally expressed UBQLN2P497H reduced viability, conferred NMJ abnormalities, and diminished climbing to a greater extent than UBQLN2WT; whereas eye-directed expression of UBQLN2P497H and UBQLN2P525S elicited severe bristle loss and hyperpigmented eye patches. Enhanced phenotypes in UBQLN2ALS flies may enable identification of disease-specific modifier genes (Kim, 2018).

Somewhat unexpectedly, UBQLN2P4X was less toxic than UBQLN2WT, UBQLN2P497H and UBQLN2P525S in the eye, and was homozygous viable when expressed in neurons at room temperature, indicating that cellular toxicity of UBQLN2 is not directly correlated to its aggregation potential. Nevertheless, Elav > UBQLN22P4X/UAS-UBQLN22P4X flies exhibited severe climbing defects and UBQLN22P4X conferred NMJ abnormalities that were comparable to those seen for UBQLN2P497H. From the combined findings, it is proposed that soluble, partially misfolded UBQLN2 oligomers and/or microaggregates are the primary toxic species as has been proposed for neurodegeneration-associated mutants of SOD1 and Huntingtin. UBQLN2 macroaggregates, amplified by the artificial P4X mutation, also contribute to neurodegeneration, perhaps through pathways that are distinct from those initiated by soluble proteins. Critical pathways mediating each mode of UBQLN2 toxicity should be illuminated through genetic modifier screens using the suite of Drosophila UBQLN2ALS models described in this study (Kim, 2018).

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Bogaert, E., Boeynaems, S., Kato, M., Guo, L., Caulfield, T. R., Steyaert, J., Scheveneels, W., Wilmans, N., Haeck, W., Hersmus, N., Schymkowitz, J., Rousseau, F., Shorter, J., Callaerts, P., Robberecht, W., Van Damme, P. and Van Den Bosch, L. (2018). Molecular dissection of FUS points at synergistic effect of low-complexity domains in toxicity. Cell Rep 24(3): 529-537.e524. PubMed ID: 30021151

Abstract

RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, this study investigated FUS, which is mutated and aggregated in both ALS and FTLD. This study generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, this study found that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins (Bogaert, 2018).

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McGurk, L., Gomes, E., Guo, L., Mojsilovic-Petrovic, J., Tran, V., Kalb, R. G., Shorter, J. and Bonini, N. M. (2018). Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization. Mol Cell 71(5): 703-717.e709. PubMed ID: 30100264

Abstract

In amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), cytoplasmic aggregates of hyperphosphorylated TDP-43 accumulate and colocalize with some stress granule components, but how pathological TDP-43 aggregation is nucleated remains unknown. In Drosophila, it was established that downregulation of tankyrase, a poly(ADP-ribose) (PAR) polymerase, reduces TDP-43 accumulation in the cytoplasm and potently mitigates neurodegeneration. TDP-43 non-covalently binds to PAR via PAR-binding motifs embedded within its nuclear localization sequence. PAR binding promotes liquid-liquid phase separation of TDP-43 in vitro and is required for TDP-43 accumulation in stress granules in mammalian cells and neurons. Stress granule localization initially protects TDP-43 from disease-associated phosphorylation, but upon long-term stress, stress granules resolve, leaving behind aggregates of phosphorylated TDP-43. Finally, small-molecule inhibition of Tankyrase-1/2 in mammalian cells inhibits formation of cytoplasmic TDP-43 foci without affecting stress granule assembly. Thus, Tankyrase inhibition antagonizes TDP-43-associated pathology and neurodegeneration and could have therapeutic utility for ALS and FTD (McGurk, 2018).

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Yamamoto, I., Azuma, Y., Kushimura, Y., Yoshida, H., Mizuta, I., Mizuno, T., Ueyama, M., Nagai, Y., Tokuda, T. and Yamaguchi, M. (2018). NPM-hMLF1 fusion protein suppresses defects of a Drosophila FTLD model expressing the human FUS gene. Sci Rep 8(1): 11291. PubMed ID: 30050143

Abstract
Fused in sarcoma (FUS) was identified as a component of typical inclusions in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In FTLD, both nuclear and cytoplasmic inclusions with wild-type FUS exist, while cytoplasmic inclusions with a mutant-form of FUS occur in many ALS cases. These observations imply that FUS plays a role across these two diseases. This study examined the effect of several proteins including molecular chaperons on the aberrant eye morphology phenotype induced by overexpression of wild-type human FUS (hFUS) in Drosophila eye imaginal discs. By screening, it was found that the co-expression of nucleophosmin-human myeloid leukemia factor 1 (NPM-hMLF1) fusion protein could suppress the aberrant eye morphology phenotype induced by hFUS. The driving of hFUS expression at 28 degrees C down-regulated levels of hFUS and endogenous cabeza, a Drosophila homolog of hFUS. The down-regulation was mediated by proteasome dependent degradation. Co-expression of NPM-hMLF1 suppressed this down-regulation. In addition, co-expression of NPM-hMLF1 partially rescued pharate adult lethal phenotype induced by hFUS in motor neurons. These findings with a Drosophila model that mimics FTLD provide clues for the development of novel FTLD therapies (Yamamoto, 2018).

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Xu, W. and Xu, J. (2018). 9orf72 dipeptide repeats cause selective neurodegeneration and cell-autonomous excitotoxicity in Drosophila glutamatergic neurons. J Neurosci. PubMed ID: 30037833

Abstract

The arginine-rich dipeptide repeats (DPRs) are highly toxic products from the C9orf72 repeat expansion mutations, which are the most common causes of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the effects of DPRs in the synaptic regulation and excitotoxicity remain elusive, and how they contribute to the development of FTD is largely unknown. By expressing DPRs with different toxicity strength in various neuronal populations in a Drosophila model, it was unexpectedly found that GR/PR with 36 repeats could lead to neurodegenerative phenotypes only when they were expressed in glutamatergic neurons, including motor neurons. Increased extracellular glutamate and intracellular calcium levels were detected in GR/PR-expressing larval ventral nerve cord and/or adult brain, accompanied by significant increase of synaptic boutons and active zones in larval neuromuscular junctions. Inhibiting the vesicular glutamate transporter (vGlut) expression or blocking the NMDA receptor in presynaptic glutamatergic motor neurons could effectively rescue the motor deficits and shortened life span caused by poly GR/PR, thus indicating a cell-autonomous excitotoxicity mechanism. Therefore, these results have revealed a novel mode of synaptic regulation by arginine-rich C9 DPRs expressed at more physiologically relevant toxicity levels and provided a mechanism that could contribute to the development of C9-related ALS and FTD (Xu, 2018).

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Jantrapirom, S., Lo Piccolo, L., Yoshida, H. and Yamaguchi, M. (2018). A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities. Exp Cell Res 362(2): 461-471. PubMed ID: 29247619

Abstract

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. This study performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. The depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. The results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD (Jantrapirom, 2018).

Protein homeostasis is finely regulated by an extensive network of components and plays a critical role in maintaining normal cellular processes, and its deficiency, such as with aging, results in abnormal protein functionality. Apart from loss-of-function effects, a critical consequence of impaired proteostasis is the accumulation of cytotoxic protein aggregates, which have also been implicated in neurodegenerative diseases (Jantrapirom, 2018).

UBQLN is a key component in maintaining appropriate proteostasis because it is involved in the transportation of polyubiquitinated proteins to proteasomes and autophagosomes. UBQLN was recently linked to ALS/FTLD because of its engagement in TDP43 and/or FUS-containing aggregates. Moreover, UBQLN2 mutations have been detected in ALS patients. This evidence confirmed the critical involvement of proteostasis in a wide spectrum of neurodegenerative diseases and suggests an important role for UBQLN in these diseases. However, the mechanistic involvement of UBQLN in ALS and FTLD has not yet been elucidated in detail (Jantrapirom, 2018).

Drosophila carries a single dUbqn gene that is the only human UBQLN orthologue. dUbqn shows high similarity to human UBQLN1 and UBQLN2, both of which have been implicated in ALS/FTLD. No Drosophila model of ALS/FTLD targeting of dUbqn has yet been established (Jantrapirom, 2018).

UBQLN is an UBL protein that binds ubiquitinated proteins and proteasome, thereby acting as a chaperone for protein degradation. Insufficient proteasome degradation of abnormal protein aggregates has been proposed to be a contributory factor to neuropathogenesis. Several studies have reported on the functional roles of UBQLN/dUbqn on neuronal functions such as RNAi silencing of dUbqn in CNS led to age-dependent neurodegeneration and shortened life span in flies, the overexpression of dUbqn in Drosophila eye-imaginal disc resulted in age-dependent retinal degeneration, mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have shown cognitive deficits, shortened life span and motor neuron degenerations. The present study confirmed a significant increase in ubiquitin chains and polyubiquitinated proteins as a consequence of the dUbqn knockdown, indicating that dUbqn is needed to maintain normal in the Drosophila brain. However, from these data it cannot be evaluated whether the effect of dUbqn knockdown on proteostasis is cell autonomous or not. Further analysis is necessary to clarify this point. The effects were examined of dUbqn depletion on locomotive abilities and learning/memory functions in the fly, which represent two distinctive features of ALS/FTLD. This study reports that neuron-specific dUbqn knockdown reduced locomotive abilities and induced a marked decline in cognitive activities and dUbqn loss-of-function could be responsible for these defects (Jantrapirom, 2018).

Moreover, this study also demonstrated that the depletion of dUbqn caused morphological alterations in the neuronal structures designed for locomotion and learning/memory. For example, neuron-specific dUbqn knockdown resulted in an abnormal arrangement of abs, which are the primary neurons that form junctions with muscle-interacting secondary neurons. NMJs, which are required for appropriate synaptic transmission to muscles, also showed an aberrant morphology. MBs, which are the brain region required for learning and memory in the fly, also exhibited an altered morphology. Collectively, these results suggest that dUbqn is involved in neuronal dysfunction because of its role in the formation or maintenance of critical neuronal circuits; therefore, impaired proteostasis may alter the structures of fundamental synapses, thereby driving the distinctive deficits observed in ALS/FTLD (Jantrapirom, 2018).

This study provides a new model to examine the role of impaired proteostasis in neurodegenerative diseases, which may expand knowledge on the critical functions of UBQLN in the nervous system. Due to the key role of UBQLN in the fate of a number of proteins, future studies are needed in order to examine whether the targets of UBQLN are associated with aberrant neurological functions. Therefore, the use of the novel Drosophila model described herein may provide interesting data and be applicable to the screening of promising ALS/FTLD therapies (Jantrapirom, 2018).

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Jantrapirom, S., Piccolo, L. L., Yoshida, H. and Yamaguchi, M. (2018). Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly. Biochim Biophys Acta. PubMed ID: 29936333

Abstract

The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. Previous work has characterized new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. The present study demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub(+)) TBPH pathology. Although it was found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, this study showed that an augmented soluble cytoplasmic Ub(+)-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. It was demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases (Jantrapirom, 2018).

The protein turnover and disposal of misfolded proteins are fundamental processes that support normal cell functions. Protein synthesis, folding, conformational maintenance, and degradation require precise control in order to ensure normal cellular protein homeostasis. Post-mitotic cells, such as neurons, are markedly affected by aberrant protein turnover or the altered degradation of misfolded proteins, which are hallmarks of aging-associated proteinopathies, including neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's (PD) diseases (Jantrapirom, 2018b).

Previous studies reported that the cellular capacity to maintain proteostasis decreases with aging, rendering the organism susceptible to proteinopathies. However, mutations in genes that regulate protein homeostasis, such as valosin-containing protein (VCP) and p62/SQSTM1, which are involved in proteasomal and autophagosomal protein degradation; Tank-binding kinase 1 (TBK1) and optineurin (OPTN), which regulate autophagy; and VAPB, CHMP2B, and Alsin, which are critical for endosome trafficking and membrane remodeling , have also been associated with the early onset of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD). Furthermore, mutations in the ubiquitin (Ub) chaperone ubiquilin 2 (UBQLN2) have been identified as a cause of the X-linked forms of ALS/FTD. Collectively, these findings strongly suggest that the Ub pathology is a common pathway in several neurodegenerative diseases and aging (Jantrapirom, 2018b).

UBQLN2 belongs to a family of UBQLN proteins and plays a critical role in both proteasomal and autophagosomal protein degradation as a Ub receptor with the ability to recognize and bind polyubiquitinated substrates in order to target them for degradation. Different ALS-related UBQLN2 mutants show an aberrant trend towards accumulating in the cytosolic aggregates of degenerating neurons, which suggests the ability of these mutants to induce proteinopathy. Furthermore, recent studies demonstrated that some ALS-related UBQLN2 mutants exhibit defective Ub-binding abilities because they are unable to bind ubiquitinated substrates and fail to deliver them to proteasomes for degradation. A loss-of-function (LOF) and/or haploinsufficiency have been proposed as the predominant disease mechanisms for UBQLN2 mutations in ALS patients (Jantrapirom, 2018b).

The 43-kDa TAR DNA-binding protein (TDP-43) is a component of cytosolic inclusions in patients with ALS, Ub-positive frontotemporal lobar degeneration (FTLD-U) and another related disorder. The reduction of TDP-43 in the nucleus and its accumulation as the Ub-positive (Ub+) hyperphosphorylated cytoplasmic inclusions in spinal motor neurons have since been recognized as a common pathological feature of approximately 97% of ALS cases. Therefore, the pathomechanisms of ALS and aging-related dementia are now considered to be directly or indirectly linked to the TDP-43 pathology. The emerging role of the Ub-related pathology in neurodegenerative diseases, such as evidence for TDP-43 UBQLN2-positive inclusions and TDP-43 aggregates in ALS and FTLD patients with or without UBQLN2 and TDP-43 mutations, has revealed a relationship between UBQLN and TDP-43. Moreover, studies on yeast and HeLa cells have shown that UBQLN2 is a polyubiquitin-TDP-43 co-chaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates. However, the mechanisms by which UBQLN2 functions in TDP-43 proteinopathies currently remain unclear (Jantrapirom, 2018b).

As described above, VCP is a highly conserved mechanoenzyme that contributes to the maintenance of protein homeostasis and has specialized functions in distinct cell types. VCP plays a role in essential cellular processes, and dysfunctional VCP was shown to be involved in pathophysiological states such as cancer, neurodegenerative disorders, and premature aging. Drosophila models have recently been developed to show that VCP physically and genetically interacts with TDP-43, and disease-causing mutations in VCP also affect the sub-cellular localization of TDP-43, similar to the above described effect induced by UBQLN2 mutations (Jantrapirom, 2018b).

Drosophila melanogaster is proving to be a very useful model for deepening understanding of UBQLN-associated neurological diseases because its genome carries only one human UBQLN orthologue (dUbqn), which shows highly conserved functional domains that are similar to human UBQLN1 and UBQLN2. A Drosophila UBQLN knockdown pathology has been modeled by the depletion of dUbqn, which induced locomotive dysfunctions and cognitive impairments in combination with severe structural defects in neuromuscular junctions (NMJs) and mushroom bodies (MBs). Therefore, this model can be used to gain insights into neurotoxicity associated with the knockdown of UBQLN and ultimately the TDP-43-related pathology. Furthermore, this study investigated the role of Drosophila VCP (dVCP) in dUbqn knockdown neurotoxicity, with a focus on its involvement in aberrantly ubiquitinated and mislocated TBPH (Jantrapirom, 2018b).

Depletion of dUbqn in the fly recapitulates a TDP-43 pathology, since TBPH was found to be aberrantly located in the cytoplasm as Ub+ partitions. Of relevance to the field, this study showed that a decline in dUbqn functions allowed the formation of at least two different Ub+ TBPH partitions, with the soluble partition being neurotoxic and the insoluble not being neurotoxic or potentially being tolerated by neurons. Moreover, by down-regulating the expression of TBPH in a proteostasis-impaired background with the TBPH pathology, this study rescued the locomotive abilities of flies. Under these conditions, the depletion of TBPH exerted positive effects by reducing the amount of soluble Ub+ TBPH (Jantrapirom, 2018b).

This study has highlighted the ability of dVCP to restore ubiquitin-dependent proteostasis that is impaired by the depletion of dUbqn and, more importantly, its ability to protectively restore proper TBPH turnover in order to reduce neurotoxic soluble Ub+ TBPH partitions. The expression of dVCP rescued dUbqn knockdown toxicity without recovering the nuclear localization of TBPH. This is of interest and warrants future extensive characterization. The present results suggest that in a proteostasis-impaired background, cytoplasmic TBPH gain-of-function may be more critical than nuclear TBPH LOF (Jantrapirom, 2018b).

Although the knockdown of dUbqn widely alters the turnover of several proteins, the present study revealed that the TBPH pathology is a crucial source of toxicity associated with the knockdown of dUbqn. Future investigations may benefit from these results, potentially leading to the development of therapeutic treatments for dysfunctional proteostasis in aging-associated neurodegenerative diseases (Jantrapirom, 2018b).

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Manzo, E., O'Conner, A. G., Barrows, J. M., Shreiner, D. D., Birchak, G. J. and Zarnescu, D. C. (2018). Medium-chain fatty acids, beta-hydroxybutyric acid and genetic modulation of the carnitine shuttle are protective in a Drosophila model of ALS Based on TDP-43. Front Mol Neurosci 11: 182. PubMed ID: 29904341

Abstract

ALS patients exhibit dyslipidemia, hypermetabolism and weight loss; in addition, cellular energetics deficits have been detected prior to denervation. Although evidence that metabolism is altered in ALS is compelling, the mechanisms underlying metabolic dysregulation and the contribution of altered metabolic pathways to disease remain poorly understood. This study used a Drosophila model of ALS based on TDP-43 that recapitulates hallmark features of the disease including locomotor dysfunction and reduced lifespan. A global, unbiased metabolomic profiling of larvae expressing TDP-43 (wild-type, TDPWT or disease-associated mutant, TDPG298S) and identified several lipid metabolism associated alterations. Among these, a significant increase was found in carnitine conjugated long-chain fatty acids and a significant decrease in carnitine, acetyl-carnitine and beta-hydroxybutyrate, a ketone precursor. Taken together these data suggest a deficit in the function of the carnitine shuttle and reduced lipid beta oxidation. To test this possibility a combined genetic and dietary approach was used in Drosophila. The findings indicate that components of the carnitine shuttle are misexpressed in the context of TDP-43 proteinopathy and that genetic modulation of CPT1 or CPT2 expression, two core components of the carnitine shuttle, mitigates TDP-43 dependent locomotor dysfunction, in a variant dependent manner. In addition, feeding medium-chain fatty acids or beta-hydroxybutyrate improves locomotor function, consistent with the notion that bypassing the carnitine shuttle deficit is neuroprotective. Taken together, these findings highlight the potential contribution of the carnitine shuttle and lipid beta oxidation in ALS and suggest strategies for therapeutic intervention based on restoring lipid metabolism in motor neurons (Manzo, 2018).

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Hautbergue, G. M., et al. (2017). SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits. Nat Commun 8: 16063. PubMed ID: 28677678

Abstract

Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. This study shows that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. it was further demonstrated that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, this study shows that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection (Hautbergue, 2017).

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M'Angale, P. G. and Staveley, B. E. (2017). A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy. BMC Res Notes 10(1): 205. PubMed ID: 28606139

Abstract

The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene has led to a study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of Drosophila. The multitude of biological functions attributable to the vitamers of vitamin B6 catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk, and this study used RNAi to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. Any association was further investigated between this enhanced disease risk gene with the established PD model induced by expression of alpha-synuclein in the same neurons. The pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, were relied on to rescue the Pdxk-induced phenotypes. Ddc-Gal4, which drives expression in both dopaminergic and serotonergic neurons, was used to drive the expression of Pdxk RNA interference in DA neurons of Drosophila. The inhibition of Pdxk in the alpha-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when Pdxk was inhibited in the developing eye using GMR-Gal4, it was found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the alpha-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. It is concluded that though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD (M'Angale, 2017).

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Coyne, A. N., Lorenzini, I., Chou, C. C., Torvund, M., Rogers, R. S., Starr, A., Zaepfel, B. L., Levy, J., Johannesmeyer, J., Schwartz, J. C., Nishimune, H., Zinsmaier, K., Rossoll, W., Sattler, R. and Zarnescu, D. C. (2017). Post-transcriptional inhibition of Hsc70-4/HSPA8 expression leads to synaptic vesicle cycling defects in multiple models of ALS. Cell Rep 21(1): 110-125. PubMed ID: 28978466

Abstract

Amyotrophic lateral sclerosis (ALS) is a synaptopathy accompanied by the presence of cytoplasmic aggregates containing TDP-43, an RNA-binding protein linked to approximately 97% of ALS cases. Using a Drosophila model of ALS, this study shows that TDP-43 overexpression (OE) in motor neurons results in decreased expression of the Hsc70-4 chaperone at the neuromuscular junction (NMJ). Mechanistically, mutant TDP-43 sequesters hsc70-4 mRNA and impairs its translation. Expression of the Hsc70-4 ortholog, HSPA8, is also reduced in primary motor neurons and NMJs of mice expressing mutant TDP-43. Electrophysiology, imaging, and These deficits can be partially restored by OE of Hsc70-4, cysteine-string protein (Csp), or dynamin. This suggests that TDP-43 toxicity results in part from impaired activity of the synaptic CSP/Hsc70 chaperone complex impacting dynamin function. Finally, Hsc70-4/HSPA8 expression is also post-transcriptionally reduced in fly and human induced pluripotent stem cell (iPSC) C9orf72 models, suggesting a common disease pathomechanism (Coyne, 2017).

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Perry, S., Han, Y., Das, A. and Dickman, D. (2017). Homeostatic plasticity can be induced and expressed to restore synaptic strength at neuromuscular junctions undergoing ALS-related degeneration. Hum Mol Genet 26(21): 4153-4167. PubMed ID: 28973139

Abstract

Amyotrophic lateral sclerosis (ALS) is debilitating neurodegenerative disease characterized by motor neuron dysfunction and progressive weakening of the neuromuscular junction (NMJ). Hereditary ALS is strongly associated with variants in the human C9orf72 gene. This study characterized C9orf72 pathology at the Drosophila NMJ and utilized several approaches to restore synaptic strength in this model. First, a dramatic reduction was demonstrated in synaptic arborization and active zone number at NMJs following C9orf72 transgenic expression in motor neurons. Further, neurotransmission is similarly reduced at these synapses, consistent with severe degradation. However, despite these defects, C9orf72 synapses still retain the ability to express presynaptic homeostatic plasticity, a fundamental and adaptive form of NMJ plasticity in which perturbation to postsynaptic neurotransmitter receptors leads to a retrograde enhancement in presynaptic release. Next, it was shown that these endogenous but dormant homeostatic mechanisms can be harnessed to restore synaptic strength despite C9orf72 pathogenesis. Finally, activation of regenerative signaling is not neuroprotective in motor neurons undergoing C9orf72 toxicity. Together, these experiments define synaptic dysfunction at NMJs experiencing ALS-related degradation and demonstrate the potential to activate latent plasticity as a novel therapeutic strategy to restore synaptic strength (Perry, 2017).

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Byrne, D. J., Harmon, M. J., Simpson, J. C., Blackstone, C. and O'Sullivan, N. C. (2017). Roles for the VCP co-factors Npl4 and Ufd1 in neuronal function in Drosophila melanogaster. J Genet Genomics 44(10): 493-501. PubMed ID: 29037990

Abstract

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD). Extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, it is inferred that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, this study finds that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo (Byrne, 2017).

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Berson, A., Sartoris, A., Nativio, R., Van Deerlin, V., Toledo, J. B., Porta, S., Liu, S., Chung, C. Y., Garcia, B. A., Lee, V. M., Trojanowski, J. Q., Johnson, F. B., Berger, S. L. and Bonini, N. M. (2017). TDP-43 promotes neurodegeneration by impairing chromatin remodeling. Curr Biol 27(23):3579-3590. PubMed ID: 29153328

Abstract

Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. This study found, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (Berson, 2017).

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Wu, C. H., Giampetruzzi, A., Tran, H., Fallini, C., Gao, F. B. and Landers, J. E. (2017). A Drosophila model of ALS reveals a partial loss of function of causative human PFN1 mutants. Hum Mol Genet. PubMed ID: 28379367

Abstract

Mutations in the profilin 1 (PFN1) gene are causative for familial amyotrophic lateral sclerosis (fALS). However, it is still not fully understood how these mutations lead to neurodegeneration. To address this question, a novel Drosophila model was generated expressing human wild-type and ALS-causative PFN1 mutants. At larval neuromuscular junctions (NMJ), motor neuron expression of wild-type human PFN1 increases the number of ghost boutons, active zone density, F-actin content, and the formation of filopodia. In contrast, the expression of ALS-causative human PFN1 mutants causes a less pronounced phenotype, suggesting a loss of function of these mutants in promoting NMJ remodeling. Importantly, expression of human PFN1 in motor neurons results in progressive locomotion defects and shorter lifespan in adult flies, while ALS-causative PFN1 mutants display a less toxic effect. In summary, this study provides evidence that PFN1 is important in regulating NMJ morphology and influences survival and locomotion in Drosophila. Furthermore, the results suggest ALS-causative human PFN1 mutants display a partial loss-of-function relative to wild-type hPFN1 that may contribute to human disease pathogenesis (Wu, 2017).

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Feuillette, S., Delarue, M., Riou, G., Gaffuri, A.L., Wu, J., Lenkei, Z., Boyer, O., Frébourg, T., Campion, D. and Lecourtois, M. (2017). Neuron-to-Neuron Transfer of FUS in Drosophila primary neuronal culture Is enhanced by ALS-associated mutations. J Mol Neurosci [Epub ahead of print]. PubMed ID: 28429234

Abstract

The DNA- and RNA-binding protein fused in sarcoma (FUS; see Drosophila Cabeza) has been pathologically and genetically linked to amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Cytoplasmic FUS-positive inclusions have been identified in the brain and spinal cord of a subset of patients suffering with ALS/FTLD. An increasing number of reports suggest that FUS protein can behave in a prion-like manner. However, no neuropathological studies or experimental data are available regarding cell-to-cell spread of these pathological protein assemblies. This study investigated the ability of wild-type and mutant forms of FUS to transfer between neuronal cells. The study combined the use of Drosophila models for FUS proteinopathies with that of the primary neuronal cultures to address neuron-to-neuron transfer of FUS proteins. Using conditional co-culture models and an optimized flow cytometry-based methodology, it was demonstrated that ALS-mutant forms of FUS proteins can transfer between well-differentiated mature Drosophila neurons. These new observations support that a propagating mechanism could be applicable to FUS, leading to the sequential dissemination of pathological proteins over years (Feuillette, 2017).

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Khalil, B., Cabirol-Pol, M. J., Miguel, L., Whitworth, A. J., Lecourtois, M. and Lievens, J. C. (2017). Enhancing Mitofusin/Marf ameliorates neuromuscular dysfunction in Drosophila models of TDP-43 proteinopathies. Neurobiol Aging 54: 71-83. PubMed ID: 28324764

Abstract

Transactive response DNA-binding protein 43 kDa (TDP-43) is considered a major pathological protein in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The precise mechanisms by which TDP-43 dysregulation leads to toxicity in neurons are not fully understood. Using TDP-43-expressing Drosophila, this study examined whether mitochondrial dysfunction is a central determinant in TDP-43 pathogenesis. Expression of human wild-type TDP-43 in Drosophila neurons results in abnormally small mitochondria. The mitochondrial fragmentation is correlated with a specific decrease in the mRNA and protein levels of the Drosophila profusion gene mitofusin/marf. Importantly, overexpression of Marf ameliorates defects in spontaneous walking activity and startle-induced climbing response of TDP-43-expressing flies. Partial inactivation of the mitochondrial profission factor, dynamin-related protein 1, also mitigates TDP-43-induced locomotor deficits. Expression of TDP-43 impairs neuromuscular junction transmission upon repetitive stimulation of the giant fiber circuit that controls flight muscles, which is also ameliorated by Marf overexpression. Enhancing the profusion gene mitofusin/marf is shown to be beneficial in an in vivo model of TDP-43 proteinopathies, serving as a potential therapeutic target (Khalil, 2017).

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Krug, L., Chatterjee, N., Borges-Monroy, R., Hearn, S., Liao, W. W., Morrill, K., Prazak, L., Rozhkov, N., Theodorou, D., Hammell, M. and Dubnau, J. (2017). Retrotransposon activation contributes to neurodegeneration in a Drosophila TDP-43 model of ALS. PLoS Genet 13(3): e1006635. PubMed ID: 28301478

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43; see Drosophila TBPH), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. This study expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. Such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. Gypsy causes the degenerative phenotypes in these flies because it was possilble to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, evidence is provided that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. These findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD (Krug, 2017).

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Şahin, A., Held, A., Bredvik, K., Major, P., Achilli, T.M., Kerson, A.G., Wharton, K., Stilwell, G. and Reenan, R. (2016). The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity. Hum Mol Genet [Epub ahead of print]. PubMed ID: Human SOD1 ALS mutations in a Drosophila knock-in model cause severe phenotypes and reveal dosage-sensitive gain and loss of function components. Genetics [Epub ahead of print]. PubMed ID: 27974499

Abstract
Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc Superoxide Dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, this study genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical and behavioral phenotypes. Contrary to previous transgenic models, ALS-like phenotypes recapitulate without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R and H71Y) exhibits neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) is phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes are recessive, a gain of function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which fail to show severe locomotor defects or nerve degeneration. The study concludes that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies (Şahin, A., 2016).

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De Rose, F., Marotta, R., Talani, G., Catelani, T., Solari, P., Poddighe, S., Borghero, G., Marrosu, F., Sanna, E., Kasture, S., Acquas, E. and Liscia, A.(2017). Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS. Sci Rep 7: 41059. PubMed ID: 28102336

Abstract

Anti-inflammatory extracts of Withania somnifera (Wse) and Mucuna pruriens (Mpe) were tested on a Drosophila model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. These results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms (De Rose, 2017).

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Crippa, V., et al. (2016). The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27466192

Abstract
Aggregation of TAR-DNA binding protein 43 (TDP-43; see Drosophila TBPH) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragment of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. This study reports that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the Drosophila functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, a novel Drosophila model expressing TDP-35 is reported, and it was shown that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively these data demonstrate that HSPB8 upregulation mitigates TDP fragment mediated toxicity, in mammalian neuronal cells and flies (Crippa, 2016).

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Lee, K. H., et al. (2016). C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167: 774-788 e717. PubMed ID: 27768896

Abstract

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. This study identified the interactome of all DPRs and found that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, it was shown that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles (Lee, 2016).

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Kramer, N. J., et al. (2016). Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science 353: 708-712. PubMed ID: 27516603

Abstract

An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. This study found that targeting the transcription elongation factor Spt4 (see Drosophila Spt4) selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. In Drosophila, Spt4 RNAi partially suppressed the degenerative phenotype of the external and internal eye in (GGGGCC)49-expressing flies and almost completely suppressed the retinal thinning normally observed in (GGGGCC)29-expressing flies. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately (Kramer, 2016).

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Matsukawa, K., Hashimoto, T., Matsumoto, T., Ihara, R., Chihara, T., Miura, M., Wakabayashi, T. and Iwatsubo, T. (2016). Familial ALS-linked mutations in Profilin 1 exacerbate TDP-43-induced degeneration in the retina of Drosophila melanogaster through an increase in the cytoplasmic localization of TDP-43. J Biol Chem [Epub ahead of print]. PubMed ID: 27634045

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons. Causative genes for familial ALS (fALS) include mutations within profilin 1 (PFN1; see Drosophila Chickadee) have recently been identified in ALS18. Transgenic Drosophila melanogaster were generated overexpressing human PFN1 in the retinal photoreceptor neurons. Overexpression of wild-type or fALS mutant PFN1 caused no degenerative phenotypes in the retina. Double overexpression of fALS mutant PFN1 and human TDP-43 (see Drosophila TDP-43) markedly exacerbated the TDP-43-induced retinal degeneration, i.e., vacuolation and thinning of the retina, whereas co-expression of wild-type PFN1 did not aggravate the degenerative phenotype. Notably, co-expression of TDP-43 with fALS mutant PFN1 increased the cytoplasmic localization of TDP-43, the latter being remained in nuclei upon co-expression with wild-type PFN1, whereas co-expression of TDP-43 lacking the nuclear localization signal with fALS mutant PFN1 did not aggravate the retinal degeneration. Knockdown of endogenous Drosophila PFN1 did not alter the degenerative phenotypes of the retina in flies overexpressing wild-type TDP-43. These data suggest that ALS-linked PFN1 mutations exacerbate TDP-43-induced neurodegeneration in a gain-of-function manner, possibly by shifting the localization of TDP-43 from nuclei to cytoplasm (Matsukawa, 2016).

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Deshpande, M., Feiger, Z., Shilton, A. K., Luo, C. C., Silverman, E. and Rodal, A. A. (2016). Role of BMP receptor traffic in synaptic growth defects in an ALS model. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27535427

Abstract

TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to Amyotrophic Lateral Sclerosis (ALS), and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is thought to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it remains unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. This study found that both loss- and gain-of-function of TDP-43 in Drosophila cause a reduction of synaptic-growth-promoting Bone Morphogenic Protein (BMP) signaling at the neuromuscular junction (NMJ). Further, a shift of BMP receptors from early to recycling endosomes was observed along with increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution, and suppressed BMP signaling, synaptic growth, and larval crawling defects. These results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation, and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment (Deshpande, 2016).

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Baldwin, K.R., Godena, V.K., Hewitt, V.L. and Whitworth, A.J. (2016). Axonal transport defects are a common phenotype in Drosophila models of ALS. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27056981

Abstract

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. This study assessed the axonal transport of different cargos in multiple Drosophila models of ALS. It was found that axonal transport defects are common across all models tested, although they often show a differential effect between mitochondria and vesicle cargos. Motor deficits are also common across the models and generally worsen with age, though surprisingly there isn't a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process (Whitworth, 2016).

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Coyne, A.N., Siddegowda, B.B., Estes, P.S., Johannesmeyer, J., Kovalik, T., Daniel, S.G., Pearson, A., Bowser, R. and Zarnescu, D.C. (2014). Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of Amyotrophic Lateral Sclerosis. J Neurosci 34: 15962-15974. PubMed ID: 25429138

Abstract
TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. This study provides evidence that Drosophila TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there was a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where higher levels of transcript were found compared with controls. TDP-43 also lead to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicated that TDP-43 lead to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulated the translation of futsch mRNA. futsch overexpression was shown to be neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, was altered in ALS spinal cords in a manner similar to these observations in Drosophila motor neurons. Together, these results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo (Coyne, 2014).

Highlights

  • futsch mRNA associates with TDP-43 in a complex in vivo.
  • TDP-43 alters futsch mRNA localization and inhibits Futsch expression post-transcriptionally.
  • futsch mRNA translation is inhibited in the context of TDP-43 proteinopathy in motor neurons.
  • Futsch is neuroprotective in the context of TDP-43 overexpression.
  • Futsch mitigates architectural defects at the neuromuscular junction by increasing microtubule stability.
  • TDP-43 aggregates are significantly decreased by futsch overexpression. Futsch/MAP1B localization is altered in ALS spinal cords.

Discussion
TDP-43, an RNA-binding protein linked to a significant fraction of ALS cases, associates with futsch mRNA in a complex in vivo and regulates its localization and translation in Drosophila motor neurons. Using polysome fractionations, this study showed that wild-type and disease-associated mutant TDP-43 co-fractionated with both the untranslated fractions, namely RNPs and ribosomal subunits, and actively translating polyribosomes. These results add translation regulation to TDP-43’s plethora of known roles in RNA processing, such as transcription, splicing, and mRNA transport, and suggest that TDP-43 contributes to the pathophysiology of ALS via multiple RNA-based mechanisms. These data provide the first in vivo demonstration that TDP-43 associates with polysomes and regulates the translation of futsch mRNA (Coyne, 2014).

A decrease in Futsch levels at the NMJ and an increase in Futsch levels in motor neuron cell bodies was shown that suggested a model whereby futsch/MAP1B mRNA may not be properly transported into axons. This was substantiated by qPCR from ventral ganglia where futsch mRNA was found at higher levels than at the NMJ compared with controls. Although the possibility that TDP-43 regulated futsch mRNA stability could not be excluded, given the more pronounced reduction in protein versus transcript levels at the NMJ compared with cell bodies and the shift to untranslated fractions in polysomes, the data suggested TDP-43-dependent defects in futsch/MAP1B mRNA transport and protein expression at the NMJ (Coyne, 2014).

Since futsch is the Drosophila homolog of MAP1B and MAP1B mRNA has been identified in TDP-43-containing RNP complexes in mouse models, it was predicted that MAP1B and microtubule-based processes might also be affected in ALS patient tissues. Indeed, similar to their results in the fly, immunohistochemistry experiments revealed a significant accumulation of MAP1B in motor neuron cell bodies in ALS spinal cords compared with controls but not in the hippocampus. Although these alterations may be the result of ongoing neurodegeneration, the remarkable similarities with the fly model suggest that comparable defects in transport and translation processes may occur in the human disease. Interestingly, Futsch protein expression was similarly inhibited by wild-type or mutant TDP-43, supporting a scenario in which MAP1B dysregulation might be a shared feature of ALS cases with TDP-43-positive pathology, regardless of etiology (Coyne, 2014).

Using genetic interaction approaches, it was shown that futsch is a physiologically significant RNA target of TDP-43 and can alleviate locomotor dysfunction and increase life span. Given Futsch’s known requirement in axonal and dendritic development and the organization of microtubules at the synapse, it was suggested that these processes may be involved in the pathophysiology of ALS. Consistent with previous studies in which tubulin acetylation was shown to rescue transport defects in neurodegeneration, it was shown that TDP-43 lead to reduced levels of acetylated tubulin, and this was rescued by futsch overexpression. Other TDP-43 targets such as HDAC6, which is regulated by TDP-43 at the level of transcription, were also linked to microtubule stability, providing additional support to the notion that microtubule stability is an important factor mediating TDP-43 toxicity. It is possible that microtubule stability is regulated locally by an interplay between Futsch and HDAC6 at the NMJ (Coyne, 2014).

In conclusion, this study identifies futsch as a disease-relevant and functionally significant post-transcriptional target of TDP-43. Given the role of futsch/MAP1B in microtubule and synaptic stabilization, this points to microtubule-based processes as targets for the development of therapeutic strategies for TDP-43 proteinopathies (Coyne, 2014).

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Joardar, A., Menzl, J., Podolsky, T. C., Manzo, E., Estes, P. S., Ashford, S. and Zarnescu, D. C. (2014). PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43. Hum Mol Genet. 24: 1741-1754. PubMed ID: 25432537

Abstract
Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. A Drosophila model has been developed of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, a drug screening strategy was designed based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, the PPARgamma agonist pioglitazone was found to be neuroprotective in Drosophila. It was shown that pioglitazone could rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS, it was found that pioglitazone was also neuroprotective when FUS, but not SOD1, was expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models showed no increase in lifespan, which was consistent with recent clinical trials. Using a pharmacogenetic approach, it was shown that the predicted Drosophila PPARgamma homologs, E75 and E78 were in vivo targets of pioglitazone. Finally, using a global metabolomic approach, a set of metabolites was identified that pioglitazone could restore in the context of TDP-43 expression in motor neurons. Taken together, the study provides evidence that modulating PPARgamma activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, it also identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials (Joardar, 2014).

Highlights

  • Drug screening in a Drosophila model of ALS based on TDP-43 identifies pioglitazone, a PPARgamma agonist as neuroprotective.
  • Larval locomotor deficits caused by TDP-43 expression in motor neurons are rescued by pioglitazone.
  • Pioglitazone exerts no protective effect on lifespan in the context of TDP-43 expression in motor neurons.
  • Glial toxicity caused by TDP-43 is partially mitigated by pioglitazone.
  • Locomotor defects caused by TDP-43 in muscles are not rescued by pioglitazone.FUS-dependent toxicity in motor neurons is partially mitigated by pioglitazone.
  • Pioglitazone is not protective in a Drosophila model of ALS based on SOD1.
  • PPARgamma acts as the molecular target of pioglitazone in vivo, in Drosophila.
  • Pioglitazone restores a subset of metabolites dysregulated in the context of TDP-43 proteinopathy.
     

Discussion
Using a previously generated Drosophila model of ALS based on TDP-43, this study showed that the antidiabetic drug pioglitazone acted as a neuroprotectant for aspects of TDP-43 proteinopathy by activating the putative Drosophila PPARgamma homologs E75 and E78. Pioglitazone mitigated FUS but not SOD1-dependent toxicity in Drosophila, consistent with previous published work showing that distinct mechanisms were likely at work in the context of these different models of ALS. Interestingly, pioglitazone did not improve, and in some cases worsened, the lifespan of TDP-43-expressing flies, when administered either during development, or after ‘disease onset’, which was consistent with results from recent clinical trials. This apparent disconnect was consistent with the effects of pioglitazone on cellular metabolism. While pioglitazone treatment restored some metabolites altered owing to TDP-43 overexpression in motor neurons, others were unchanged or even worsened. This provided a potential explanation for why some phenotypes but not others were rescued by pioglitazone. Aside from the possibility that different drug concentrations might be needed, it remains unclear why pioglitazone is protective in mouse but not fly SOD1 models and, in retrospect, given the similarities between the effect of pioglitazone in Drosophila models of ALS and humans, the fly appears to be a more accurate predictor of clinical trial outcomes (Joardar, 2014).

It is tempting to speculate that the predictive power of the Drosophila model may lie in the tools that enable motor neuronal versus glial versus muscle-specific expression of the toxic TDP-43 protein. It was shown that pioglitazone mitigated neuronal and glial TDP-43-dependent toxicity but had no effect on the locomotor dysfunction caused by muscle-specific expression of TDP-43. The protective effects of pioglitazone were specific to the nervous system and were not observed in muscles, at least within the limits of experimental conditions (i.e. tissue-specific levels of expression and drug concentration). These findings suggest that future preclinical studies may benefit from testing candidate therapies in multiple disease models in which tissue specificity and several phenotypic outcomes are easily ascertained (Joardar, 2014).

Pioglitazone has been originally developed for the treatment of type 2 diabetes as PPARgamma activation in the liver improves glucose metabolism systemically. In the nervous system, activation of the nuclear hormone receptor PPARgamma has been shown to have anti-inflammatory and neuroprotective effects. In the model used it this study, it was found that pioglitazone could restore a rather limited set of metabolites altered in a TDP-43-dependent manner. Evidence for altered glutamine/glutamate metabolism in TDPWT flies was found, as displayed by elevated levels of N-acetylglutamine, which was restored by pioglitazone. Excessive levels of extracellular glutamate in the central nervous system cause hyperexcitability of neurons, ultimately leading to their death. The glutamate transporter GLT1/EAAT2 plays a major role in maintaining extracellular glutamate levels below the excitotoxic concentrations by efficiently transporting this metabolite. Interestingly, astrocytic GLT1/EAAT2 gene is a target of PPARgamma, leading to neuroprotection by increasing glutamate uptake (Joardar, 2014).

Furthermore, pyruvate, which was significantly high in both TDPWT and TDPG298S, showed a trend toward reduction upon pioglitazone treatment for TDPWT. Pyruvate is a central metabolite that lies at the junction of several intersecting cellular pathways including glucose and fatty acid metabolism. It is converted to oxaloacetate by the enzyme pyruvate carboxylase, which is a key step in lipogenesis. Interestingly, PPARgamma, the target of pioglitazone, is a direct transcriptional modulator of the pyruvate carboxylase gene (Joardar, 2014).

Given the fact that ALS patients suffer from massive weight loss, results from this study provide a possible explanation for the potential protective effects of pioglitazone through increased lipogenesis. Taken together, the metabolomics approach of this study provides useful insights for understanding the molecular mechanisms underlying ALS pathophysiology. Notably, the fly model used in this study also showed signs of hypermetabolism including an increase in pyruvate, a key metabolite linking glucose metabolism to the TCA cycle. Additionally, the ketone body GHB was reduced in the context of TDPWT, consistent with a clinical study showing that a ketogenic diet slowed ALS disease progression. Given the similarities between the metabolic profile of the Drosophila model and human samples, it will be interesting in the future, to design therapeutic approaches aimed at restoring these common metabolic changes using nutritional supplementation (Joardar, 2014).

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Forrest, S., Chai, A., Sanhueza, M., Marescotti, M., Parry, K., Georgiev, A., Sahota, V., Mendez-Castro, R. and Pennetta, G. (2013). Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet 22(13): 2689-2704. PubMed ID: 23492670

Abstract

The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. This study has identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, this study found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, this study reports that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP-Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS (Forrest, 2013).

Amyotrophic lateral sclerosis (ALS) is a progressive, degenerative disorder characterized by the selective loss of motor neurons in the brain and spinal cord leading to paralysis, muscle atrophy and eventually, death. Two missense mutations in the gene encoding the human Vesicle-associated membrane protein (VAMP)-Associated Protein B (hVAPB) causes a range of dominantly inherited motor neuron diseases including ALS8. VAP family proteins are characterized by an N-terminal major sperm protein (MSP) domain, a coiled-coil (CC) motif and a transmembrane (TM)-spanning region. They are implicated in several biological processes, including regulation of lipid transport, endoplasmic reticulum (ER) morphology and membrane trafficking. Drosophila Vap-33-1 (hereafter, DVAP) regulates synaptic structure, synaptic microtubule (MT) stability and the composition of postsynaptic glutamate receptors. MSP domains in DVAP are cleaved and secreted into the extracellular space where they bind Ephrin receptors. MSPs also bind postsynaptic Roundabout and Lar-like receptors to control muscle mitochondria morphology, localization and function. Transgenic expression of the disease-linked alleles (DVAP-P58S and DVAP-T48I) in the larval motor system recapitulates major hallmarks of the human disease, including aggregate formation, locomotion defects and chaperone upregulation. Several studies have also implicated the ALS mutant allele in abnormal unfolded protein response (UPR) and in the disruption of the anterograde axonal transport of mitochondria. However, it is unclear how these diverse VAP functions are achieved and which mechanisms underlie the disease pathogenesis in humans. One way to address these questions is to search for DVAP-interacting proteins. This study identified Sac1 (Suppressor of Actin 1), an evolutionarily conserved phosphoinositide phosphatase, as a DVAP-binding protein. Phosphoinositides are low-abundance lipids that localize to the membrane-cytoplasm interface and function by binding various effector proteins. The inositol group can be reversibly phosphorylated at the 3', 4' and 5' positions to generate seven possible phosphoinositide derivatives, each with a specific intracellular dynamic distribution. Sac1 predominantly dephosphorylates PtdIns4P pools, although PtdIns3P and PtdIns(3,5)P2 can also function as substrates. In yeast, Sac1 has been linked to several processes, including actin organization, vacuole morphology and sphingomyelin synthesis. Drosophila Sac1 mutants die as embryos and exhibit defects in dorsal closure and axonal pathfinding. Mouse lines deficient for Sac1 are cell lethal, whereas Sac1 downregulation in mammalian cell cultures results in disorganization of Golgi membranes and mitotic spindles. Interestingly, SAC3 (also known as FIG4), another member of the Sac phosphatase family, is mutated in familial and sporadic cases of ALS. Inactivation of SAC3 in mice also results in extensive degeneration and neuronal vacuolization in the brain, most relevantly in the motor cortex. This study identified Sac1 and DVAP as binding partners and shows that DVAP is required to maintain normal levels of PtdIns4P. Loss of either Sac1 or DVAP function disrupts axonal transport, MT stability, synaptic growth and the localization of a number of postsynaptic markers. Rhe disease-causing mutation (DVAP-P58S) induces neurodegeneration and displays synaptic phenotypes similar to those of either Sac1 or DVAP loss-of-function, including an increase in PtdIns4P levels. Importantly, reducing PtdIns4P levels rescues the neurodegeneration associated with DVAP-P58S and suppresses the synaptic phenotypes associated with DVAP-P58S and DVAP loss-of-function alleles. Consistent with these observations, Sac1 is sequestered into DVAP-P58S-mediated aggregates and downregulation of Sac1 in neurons induces increased PtdIns4P levels and degeneration. These data highlight the crucial role of DVAP and Sac1 in regulating phosphoinositides and support a causative role for PtdIns4P levels in ALS8 pathogenesis (Forrest, 2013).

This study has identified Sac1 as a DVAP-binding protein and uncovered a hitherto unknown function of Sac1 in postembryonic synaptic maturation and neurodegeneration. Presynaptic reduction of either DVAP or Sac1 levels induces structural changes, disruption of the synaptic MT cytoskeleton and accumulation of clusters of proteins and vesicles along the axons. In addition, muscle down-regulation of either Sac1 or DVAP leads to a strikingly aberrant synaptic morphology and abnormal localization and distribution of several postsynaptic markers, including adducin and β-spectrin. Depletion of DVAP as well as Sac1 expression induces an increase in PtdIns4P levels. Sac1 downregulation in the adult nervous system was shown to cause early death and neurodegeneration in a dosage-dependent manner, a phenotype similar to that of DVAP-P58S transgenic expression. This analysis indicates that the DVAP-P58S allele has a dominant negative effect, as its transgenic expression leads to an upregulation of PtdIns4P and its mutant phenotypes are similar to those associated with either DVAP or Sac1 loss-of-function. In agreement with the hypothesis that neurodegeneration in the DVAP-P58S context is due to a loss-of-function of both DVAP and Sac1, it is reported that both wild-type DVAP and Sac1 are depleted from their normal localization and are sequestered into DVAP-P58S-mediated aggregates. Altogether, these data are consistent with a model in which DVAP is required for Sac1 activity and for the regulation of intracellular PtdIns4P levels. Loss-of-function of DVAP and Sac1 by a DVAP-P58S-mediated dominant-negative mechanism induces cell degeneration by an upregulation of PtdIns4P, which is also responsible for the observed disruption of fundamental biological processes at the NMJs . It has been previously shown that transgenic expression of DVAP proteins carrying the equivalent ALS8 mutations in Drosophila mimic the human disease. Notably, expression of hVAPB in flies rescues the lethality and the phenotypes associated with DVAP mutants, indicating an evolutionarily conserved function for VAP proteins. Collectively, these data indicate that DVAP-mediated molecular pathways are likely to be important for understanding of the disease pathogenesis in humans (Forrest, 2013).

There is evidence supporting that DVAP functions to maintain normal cellular levels of PtdIns4P by interacting with Sac1. First, Sac1 and DVAP bind to each other and colocalize in many different tissues. It has been reported that phosphoinositol transfer proteins/phosphoinositide-binding proteins associate directly with phosphatases and kinases to control their activities. Specifically, VAP has been shown to bind PtdIns4P in vitro and to be required for Sac1 activity in yeast. Second, PtdIns4P levels are upregulated in DVAPRNAi mutants, suggesting that DVAP function is required for normal PtdIns4P levels. Similarly, in yeast, inactivation of Scs2/Scs22 VAP genes induces an increase in the levels of PtdIns4P. Third, the phenotypic similarity associated with either DVAP or Sac1 loss-of-function mutations supports the idea that the pool of PtdIns4P that is upregulated in DVAP mutants is the same as the one dephosphorylated by Sac1. Previous studies attributed a prominent functional role to the N-terminal MSP domain of DVAP. The MSP domain is cleaved and secreted and binds to the extracellular domain of Ephrin receptors. Secreted MSP also binds to Robo and Lar-like receptors to control mitochondria morphology, localization and function in muscles. A new DVAP-binding activity was identified that is MSP-independent and involves a C-terminal fragment encompassing the TM domain. Interestingly, a new hVAPB mutation replacing valine at position 234 with an isoleucine in the conserved TM domain of hVAPB has been shown to cause ALS8 in humans. These data may provide direct evidence of a role of hVAPB-Sac1 interaction in the disease pathogenesis in humans (Forrest, 2013).

In yeast and mammalian cells, Sac1 is an integral membrane protein localized to the ER and the Golgi. This study reports a similar localization for the Drosophila homologue of Sac1. In yeast and mammalian cells, Sac1 localization appears to be very dynamic, as this protein shuttles between ER and Golgi upon nutrient conditions. Specifically, glucose starvation in yeast or growth factor deprivation in mammalian cells causes relocalization of Sac1 from the ER to the Golgi complex, where it reduces PtdIns4P levels and slows protein trafficking. The ER-Golgi shuttling ability of Sac1 is reversed when nutrients or growth factors are added back to the growth medium. The growth factor-induced translocation of Sac1 from the Golgi to the ER requires p38 MAPK (mitogen-activated protein kinase) activity. These data suggest that Sac1 trafficking may be regulated by stressors that activate p38 MAPK. Some of these stressors such as oxidative damage and ER stress are triggers of neurodegeneration. This raises the intriguing possibility that a p38 MAPK-activated mechanism of PtdIns4P spatial regulation may be implicated in neurodegenerative processes (Forrest, 2013).

Scs2/Scs22 VAP proteins in yeast play a pivotal role in tethering the ER to the PM to form ER/PM contact sites. Studies have highlighted the role of membrane junctions between organelles as important sites for lipid metabolism and intracellular signalling controlled by PtdIns4P. Depletion of Scs2/Scs22 VAP proteins located to the ER/PM contact sites leads to a retraction of the ER into internal structures, elevated levels of PtdIns4Ps at the PM and induction of the UPR. At the ER/PM contact sites, Sac1 dephosphorylates PtdIns4P on the PM in trans from the ER. This reaction requires the Scs2/Sc22p VAP proteins and the oxysterol-binding homology proteins that act as PtdIns4P sensors and activates Sac1 phosphatase activity. ER/PM junctions have been described in many organisms and cell types, including neurons and Drosophila photoreceptors. In addition, VAP proteins have been implicated in ER-Golgi, ER-endosomes and ER-mitochondria contacts in mammalian cells, suggesting that they may function as a tether for several organelle/membrane contact sites. In conclusion, emerging evidence suggests that VAP proteins may be a crucial component of a hub controlling PtdIns4P metabolism in yeast and possibly, in higher eukaryotes as well (Forrest, 2013).

The ability of either PI4KIIIα or four wheel drive fwd, a Golgi-localized lipid kinase that synthesizes phosphatidylinositol 4-phosphate from phosphatidylinositol, to suppress the synaptic and neurodegenerative phenotypes associated with transgenic expression of DVAP-P58S is somewhat surprising, as their yeast homologues (Stt4 and Pik1, respectively) are supposed to play non-redundant functions and to control spatially separate pools of PtdIns4P. This is based on previously published data showing that, in yeast, Stt4 and Pik1 are both essential for cell viability but control different cellular processes. Pik1 is essential for anterograde vesicular trafficking, whereas Stt4 plays a role in actin cytoskeleton organization and protein kinase C signalling. Both Pik1 and Stt4 play distinct roles in regulating MAPK signalling. Localization studies further suggest that Pik1p is primarily present in the nucleus and in the Golgi, whereas Stt4p is mainly cytoplasmic and is recruited to the PM for localized synthesis of PtdIns4P (Forrest, 2013).

However, at present, the precise degree to which Stt4 and Pik1 functions have been conserved and apportioned among their homologues in flies remains unclear. In Drosophila, the Stt4 homologue PI4KIIIα is required for oocyte polarization and its intracellular localization has not been determined. On the other hand, previous studies revealed that the fly Pik1 homologue Fwd is required for male germ-line cytokinesis. In spermatocytes, Fwd localizes to the Golgi and it is required for the accumulation of PtdIns4P on this organelle, implying that its function in providing PtdIns4P in the Golgi is evolutionarily conserved with yeast. However, whereas Pik1 is required for cell viability, Fwd appears to be dispensable for normal development, suggesting that it is redundant with similar genes in carrying out its function (Forrest, 2013).

Another way to explain the rescue data would be to admit that upregulation of PtdIns(4,5P)2 and not PtdIns4P is responsible for DVAP-P58S mutant phenotypesPtdIns4P formed by the PM-associated STT4 can function as a substrate of PI4P 5-kinase to generate PtdIns(4,5)P2 at the cell cortex. It is also possible that PtdIns4P that is phosphorylated by plasmalemmal PI4P 5-kinase originates from intracellular sources. In the Golgi, PtdIns4P levels play a central role in the formation of vesicles delivered from the trans-Golgi network to the PM and their lipid cargo could be the substrate for the plasmalemmal PtdIns(4,5)P2 synthesis (676767). As PtdIns4P in the Golgi is mainly produced by Pik1 is therefore possible that PtdIns(4,5)P2 associated with the PM and its effector proteins are downstream of both Stt4 and Pik1. Moreover, upregulation of PtdIns(4,5)P2 would explain the MT phenotypes, the mislocalization of post-synaptic markers and the axonal transport defects. Indeed, PtdIns(4,5)P2-enriched microdomains in the PM have been shown to participate in the regulation of MT plus-end capture and stabilization during polarized mobility. In Caenorhabditis elegans, the microtubular motor UNC-4 gene was shown to be anchored to synaptic vesicles, using a pleckstrin homology domain, thus implicating PtdIns(4,5)P2 in MT-based intracellular motility . Finally, spectrin proteins and adducin require PtdIns(4,5)P2 for their correct localization to the cell cortex (Forrest, 2013).

However, if this were true, an increase in PtdIns(4,5)P2 levels whould be observed wherever an upregulation of PtdIns4P is observed. By using an antibody specific for PtdIns(4,5)P2, PtdIns(4,5)P2 levels were quantified in tissues in which either Sac1 or DVAP were downregulated as well as in tissues expressing the DVAP-P58S transgene. Surprisingly, PtdIns(4,5)P2 levels were not affected by the dramatic upregulation of PtdIns4P in any of the genotypes described earlier. Consistent with these data, it was previously reported that, in Drosophila eye imaginal discs, depletion of Sac1 exhibits a dramatic increase in PtdIns4P levels, whereas PtdIns(4,5)P2 and PtdIns3P levels remain similar to wild-type. In addition, loss of PM PtdIns4P by downregulation of PI4KIIIα was not matched by a decrease in PtdIns(4,5)P2 levels. It was shown that the major function of PtdIns4P is not to generate the pool of PtdIns(4,5)P2 on the PM but rather to contribute to the generation of a polyanionic lipid environment in the inner leaflet of the PM. PtdIns4P would then function in recruiting soluble proteins to the PM by electrostatic interaction with their polycationic surface. It was also shown that PtdIns4P contributes to processes such as modulation of ion channel activity that have been traditionally associated with changes in PtdIns(4,5)P2. Finally, upregulation of PtdIns(4,5)P2 by inactivation of the Drosophila PI(4,5)P2 5-phosphatase synaptojanin leads to a distinct endocytotic phenotype due to defects in synaptic vesicle recycling. In synaptojanin mutants, synaptic vesicles are severely depleted and those remaining are clearly clathrin-coated. Intracellular recordings revealed enhanced synaptic depression during prolonged high-frequency stimulation. Ultrastructural and electrophysiological analysis of DVAP mutants do not exhibit a synaptojanin-like phenotype, indicating that upregulation of PtdIns(4,5)P2 does not mimic the phenotype associated with increased levels of PtdIns4P. Taken together, these considerations suggest that increased levels of PtdIns4P could be the main factor determining the observed synaptic and neurodegenerative phenotypes. Further studies using fluorescent phosphoinositide probes and genetic analyses will be needed to fully clarify the contribution of PtdIns4P versus PtdIns(4,5)P2 pools to NMJ physiology and neurodegeneration (Forrest, 2013).

Emerging evidence indicates that VAP and Sac1 may also play an important and specific role in membrane homeostasis. Biogenesis of sphingolipids, sterols and phosphoinositides that together determine the structural and functional properties of cell membranes must be closely coordinated. VAP interacts with both oxysterol-binding protein (OSBP) and ceramide transfer protein (CERT), recruiting them to contact sites between the ER and the Golgi complex. CERT has a FFAT (diphenylalanine in an acidic tract) motif that mediates its binding to ER-localized VAP and a PH domain that recognizes the PtdIns4P-enriched Golgi membrane. It has been proposed that CERT, because of its dual-binding ability, shuttles ceramide from the ER to the Golgi, where it is converted into sphingomyelin. Sphingomyelin continues to move through the secretory pathway to the PM, where it is most abundant. OSBP has an analogous function to CERT but instead mediates inter-membrane sterol transfer. This functional similarity is also reflected in OSBP's domain architecture: like CERT, it contains a PtdIns4P-binding PH domain and a VAP-binding FFAT motif. It has been shown that sterols regulate sphingolipid metabolism by inducing a significant increase in SM synthesis that is dependent on OSBP, CERT and their shared binding partner VAP. The precise mechanism is not yet known but OSBP appears to activate CERT by promoting its recruitment to membranes and its binding to VAP. It is likely that disruption of the VAP-Sac1 interaction may have profound effects on the lipid composition of the PM, affecting its curvature and thickness and by consequence, vesicle budding and membrane remodelling. Interestingly, synaptic growth requires membrane remodelling and, at the Drosophila NMJs, occurs mainly by the budding of new boutons from pre-existing ones (Forrest, 2013).

VAPB is involved in the IRE1/XBP1 signalling pathway of the UPR, an ER reaction inhibiting the accumulation of unfolded/misfolded proteins. In the disease-context, the hVAPB-P56S protein recruits its wild-type counterpart into the aggregates and it attenuates its ability to induce the UPR. This together with the observation that, in yeast, depletion of VAP proteins from the ER/PM contact sites induces a constitutive activation of the UPR suggests that motor neurons in ALS8 could be particularly vulnerable to cell death-induced ER stress. In addition, VAP proteins have been shown to be involved in lipid transfer and metabolism and accumulation of lipids and intermediates of lipid biosynthetic pathways are potent inducers of apoptosis. Finally, recent studies in yeast have shown that defects in the PtdIns4K Pik1 activity lead to a blockage of autophagy, a process controlling the degradation of long-lived proteins, damaged organelles and bulk cytoplasm in response to various types of stress. Many questions remain to be explored concerning the precise molecular mechanism underlying neurodegeneration in ALS. However, over the last few years, an increasing number of experimental models have been generated and they represent an excellent tool for identifying molecular pathways in ALS and for evaluating their contribution to the disease pathogenesis (Forrest, 2013).

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He, F., Krans, A., Freibaum, B. D., Taylor, J. P., Todd, P. K. (2014). TDP-43 suppresses CGG repeat-induced neurotoxicity through interactions with HnRNP A2/B1. Hum Mol Genet. 23: 5036-5051. PubMed ID: 24920338

Abstract
Nucleotide repeat expansions can elicit neurodegeneration as RNA by sequestering specific RNA-binding proteins, preventing them from performing their normal functions. Conversely, mutations in RNA-binding proteins can trigger neurodegeneration at least partly by altering RNA metabolism. In Fragile X-associated tremor/ataxia syndrome (FXTAS), a CGG repeat expansion in the 5'UTR of the fragile X gene (FMR1) leads to progressive neurodegeneration in patients and CGG repeats in isolation elicit toxicity in Drosophila and other animal models. This study identified the amyotrophic lateral sclerosis (ALS)-associated RNA-binding protein TAR DNA-binding protein (TDP-43) as a suppressor of CGG repeat-induced toxicity in a Drosophila model of FXTAS. The rescue appeared specific to TDP-43, as co-expression of another ALS-associated RNA-binding protein, FUS, exacerbated the toxic effects of CGG repeats. Suppression of CGG RNA toxicity was abrogated by disease-associated mutations in TDP-43. TDP-43 did not co-localize with CGG RNA foci and its ability to bind RNA was not required for rescue. TDP-43-dependent rescue did, however, require fly hnRNP A2/B1 homologues Hrb87F and Hrb98DE. Deletions in the C-terminal domain of TDP-43 that precluded interactions with hnRNP A2/B1 abolished TDP-43-dependent rescue of CGG repeat toxicity. In contrast, suppression of CGG repeat toxicity by hnRNP A2/B1 was not affected by RNAi-mediated knockdown of the fly TDP-43 orthologue, TBPH. Lastly, TDP-43 suppressed CGG repeat-triggered mis-splicing of an hnRNP A2/B1-targeted transcript. These data support a model in which TDP-43 suppresses CGG-mediated toxicity through interactions with hnRNP A2/B1 and suggest a convergence of pathogenic cascades between repeat expansion disorders and RNA-binding proteins implicated in neurodegenerative disease (He, 2014).

Highlights

  • Overexpression of TDP-43 suppresses CGG repeat toxicity.
  • CGG repeat RNA levels and RAN translation are not altered by TDP-43.
  • TDP-43 suppression of CGG repeat toxicity is dependent on hnRNP A2/B1 homologues.
  • Normal TBPH expression is not required for hnRNP A2/B1 suppression of CGG toxicity.
  • TDP-43 restores alternative splicing of EPH, which is perturbed by CGG repeat expression.

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Watson, M. R., Lagow, R. D., Xu, K., Zhang, B., Bonini, N. M. (2008). A Drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1. J Biol Chem. 283: 24972-29481. PubMed ID: 18596033

Abstract
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to loss of motor function and early death. About 5% of cases are inherited, with the majority of identified linkages in the gene encoding copper, zinc-superoxide dismutase (SOD1). Strong evidence indicates that the SOD1 mutations confer dominant toxicity on the protein. To provide new insight into mechanisms of ALS, this study generated and characterized a model for familial ALS in Drosophila with transgenic expression of human SOD1. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits. These effects were accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia. However, toxicity was not associated with oligomerization of SOD1 and did not lead to neuronal loss. These data uncover cell-autonomous injury by SOD1 to motor neurons in vivo, as well as non-autonomous effects on glia, and provide the foundation for new insight into injury and protection of motor neurons in ALS (Watson, 2008).

Highlights

  • Expression of hSOD1 but not dSOD1 in motor neurons causes progressive motor dysfunction.
  • No apparent loss of motor neurons.
  • Biochemical oligomerization of hSOD1 is not linked to neuronal loss or dysfunction.
  • Synaptic transmission along the giant fiber motor pathway is abnormal.
  • hSOD1 progressively accumulates in motor neuron somata and processes.
  • Expression of hSOD1 in motor neurons produces a stress response in glia.

Discussion
This study presents a model for SOD-linked fALS in Drosophila that displays motor dysfunction, a defining feature of the human disease. Motor dysfunction in flies was accompanied by failure in high frequency synaptic transmission, focal accumulation of hSOD1 in motor neurons, and up-regulation of heat shock protein in glia. These findings suggest that SOD can cause cell-autonomous damage to motor neurons, and highlight that expression of hSOD1 selectively in motor neurons induces a change in glia (Watson, 2008).

It was shown that a motor neuron-restricted expression pattern conferred behavioral compromise in climbing ability. This suggests that hSOD1 may have an intrinsic toxicity to motor neurons, which can be defined in the Drosophila system. Previous models in mice have demonstrated a dependence of toxicity on widespread tissue expression, specifically with the genes under control of the endogenous hSOD1 enhancer/promoter elements. Several studies with mice have reported no toxicity with neuron-restricted expression using the Thy1 or the neurofilament light chain promoters. This idea was expanded when another study demonstrated in chimeric mice that motor neurons can display ALS-like pathology when they are not expressing the mutant protein themselves but rather are surrounded by other cell types that are expressing the mutant protein. The model presented in this study, on the other hand, provides an approach to define toxic properties of hSOD1 specifically in motor neurons that can lead to a motor deficit (Watson, 2008).

Upon expressing hSOD1 in the fly, deficits were seen with expression restricted to motor neurons which supported a role for cell-autonomous damage to motor neurons by hSOD1. Within motor neurons, there was progressive accumulation of hSOD1, both in the somata surrounding the nucleus, as well as in neurites. These focal accumulations may both cause and result from hindrances in trafficking and axonal transport or insufficient protein degradation. It is known that disruption of anterograde and retrograde axonal movement of synaptic proteins and neurotrophic entities can negatively affect neuronal function. The p150glued mutation in dynactin-1, which severely disrupts axonal transport, causes a progressive, late onset motor phenotype in mice. Mice expressing mutant SOD1 also have compromised axonal transport. The flies displayed electrophysiological defects reflective of impaired motor neuron function, indicating that the fly may provide a sensitive system for the detection of subtle motor neuron defects caused by hSOD1 and disease-linked forms. Despite a progressive motor phenotype, there was no change in numbers of neuronal nuclei, excluding widespread loss of cells. The electrical features of the motor pathway also indicated that it could function fine at low activity levels, suggesting that synapses may be the primary site of dysfunction of SOD1 flies (Watson, 2008).

WT hSOD1 imparted toxicity nearly on a par with either A4V or G85R mutant forms; WT hSOD1 even showed a tendency to accumulate in foci, a feature generally expected of a mutant but not normal hSOD1. It was hypothesized that WT hSOD1 may function as a conformational mutant protein in the context of Drosophila neurons for the following reasons. Toxicity can be conferred onto hSOD1 by any one of more than a hundred distinct amino acid substitutions, which implies an exquisite dependence upon conformation. This raises the possibility that any sequence other than the wild type Drosophila SOD1 conformation in the context of the SOD1 protein may appear abnormal to the fly. Although Drosophila SOD1 and hSOD1 are very similar in sequence, and hSOD1 can even functionally replace the Drosophila gene, the enzymes do differ in many amino acids, including locations where mutations occur that are associated with fALS. Importantly, overexpression of dSOD1 did not mimic the effects of hSOD1 expression in the fly. This finding also fails to support the idea that SOD1 toxicity may be related to dismutase activity of the enzyme as both dSOD1 and hSOD1 would presumably result in the overabundance of hydrogen peroxide, yet there was selective toxicity of hSOD1 (Watson, 2008).

Affected tissues in neurodegenerative diseases often exhibit the induction of a chaperone stress response. The heat shock protein immunoreactivity that was observed in fly thoracic ganglion did not overlap with hSOD1 staining. Rather, it was present exclusively in cells that were positive for the glial-specific marker protein Repo. Thus, in the fly model, the motor neurons contained the toxic protein, but the glia appeared to initiate a stress response. It was unlikely that exogenous SOD1 induced a stress response due to SOD1 expression in glia themselves since the D42 motor neuron driver is specific, and SOD1 was not detected in glia by immunofluorescence using a variety of primary antibodies, despite robust SOD1 levels. Leaky expression due to the genomic insertion sites of the transgenes could result in glial expression of the exogenous proteins, although analysis of flies lacking the GAL4 driver revealed no detectable hSOD1 protein. Furthermore, expanded polyglutamine protein in flies with the same motor neuron driver was only observed in neurons (Watson, 2008).

The glial chaperone up-regulation may be a reaction to the toxic protein or a signal secondary to effects of SOD1 in motor neurons. Motor neuron expression of dSOD1, but not of a pathogenic polyglutamine protein by the same driver, also resulted in a glial response, indicating that the response occurs with SOD1. Flies with greater chaperone induction showed more severe indicators of motor dysfunction. Thus, the degree of stress response in glia may serve as a measure of neuronal dysfunction or a measure of the extent to which glia are attempting to combat problems in motor neurons (Watson, 2008).

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Huang, Y., Wu, Z., Zhou, B. (2015). hSOD1 promotes Tau phosphorylation and toxicity in the Drosophila model. J Alzheimers Dis. 45: 235-244. PubMed ID: 25524953

Abstract
Tau hyperphosphorylation has been found in several neurodegenerative diseases such as Alzheimer's disease (AD), Down syndrome, and amyotrophic lateral sclerosis (ALS). However, factors affecting tau hyperphosphorylation are not yet clearly understood. SOD1, a Cu/Zn superoxide dismutase whose mutations can cause adult-onset ALS, is believed to be involved in the pathology of Down syndrome. In this work, the model organism Drosophila was used to study the possible link between hSOD1 and tau. It was shown that hSOD1, and to a higher degree hSOD1(A4V), could increase tau toxicity in Drosophila and exacerbate the corresponding neurodegeneration phenotype. The increased tau toxicity appeared to be explainable by elevated tau phosphorylation. Tau(S2A), a tau mutant with impaired phosphorylation capabilities, did not respond to expression of hSOD1 and hSOD1(A4V). The study suggests that increased SOD1 expression can lead to tau hyperphosphorylation, which might serve as an important contributing factor to the etiology of Down syndrome and SOD1-related ALS disease (Huang, 2015).

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Takayama, Y., Itoh, R.E., Tsuyama, T. and Uemura, T. (2014). Age-dependent deterioration of locomotion in Drosophila melanogaster deficient in the homologue of amyotrophic lateral sclerosis 2. Genes Cells: 464-477. PubMed ID: 24702731

Abstract
Recessive mutations in the amyotrophic lateral sclerosis 2 (ALS2) gene have been linked to juvenile-onset ALS2. Although one of the molecular functions of the ALS2 protein is clearly the activation of Rab5, the mechanisms underlying the selective dysfunction and degeneration of motor neurons in vivo remain to be fully understood. This study focused on the ALS2 homologue of Drosophila melanogaster, isolated two independent deletions, and systematically compared phenotypes of the mutants with those of animals in which Rab5 function in identified neurons was abrogated. In the dALS2 mutant flies, it was found that the stereotypic axonal and dendritic morphologies of neurons shared some features with those in Rab5-deficient flies, but the dALS2 mutant phenotypes were much milder. It was also found that the abrogation of Rab5 function in motor neurons strongly depressed the locomotion activity of adults, resembling the behavior of aged dALS2 mutants. Importantly, this age-dependent locomotion deficit of dALS2 mutants was restored to normal by expressing the dALS2 transgene in a wide range of tissues. This finding provided a platform where particular cell types responsible for the phenotype by tissue-specific rescue experiments coule be potentially identified. The study also discussed the future usage of the dALS2 mutant as a new ALS model (Takayama, 2014).

Highlights

  • CG7158 encodes a homologue of ALS2.
  • CG7158/dALS2 has a GEF activity for Rab5.
  • dALS2 mutants are viable and fertile.
  • Morphological analysis of axon terminals and dendritic arborization of identified neurons in the dALS2 mutant and in wild-type flies expressing a dominant negative form of Rab5 .
  • Mutant dALS2 adults show a lowered locomotion activity.
  • Locomotion deficit of the dALS2 mutant is rescued by dALS2 transgene expression.

Discussion
Annotated genes in the genome of Drosophila melanogaster include homologues of four human GEFs and two GTPase activating proteins (GAPs). Among them, the predicted protein product of a single gene CG7158 shows similarities to ALS2/Alsin. This study designated CG7158 and its protein product as dALS2 and dALS2. Both ALS2/Alsin and dALS2 contain four domains in the same order from their amino-terminals, among which the ‘membrane occupation and recognition nexus (MORN)’ motifs and the carboxyl-terminal ‘vacuolar protein sorting 9 (VPS9)’ domain in human ALS2 are necessary for its selective GEF activity for Rab5 in vitro. One distinctive difference between dALS2 and ALS2/Alsin is that dALS2 lacks a DH domain, which is adjacent to the PH domain in human ALS2 and provides a basis of its GEF activity for Rac1 (Kunita et al. 2007). Thus, dALS2 could be a more Rab5-specific GEF compared with ALS2 (Takayama, 2014).

To examine whether dALS2 does possess GEF activity or not, dALS2 and a fluorescence resonance energy transfer (FRET) probe, Raichu-Rab5, were co-expressed in Drosophila S2 cells. The probe comprised Venus (a modified YFP), the amino-terminal Rab5-binding domain of EEA1, SECFP (a modified CFP) and Rab5. In this probe design, the increase in the emission ratio reflected an increase in the active GTP-bound form of Rab5 relative to the inactive GDP-bound form in living cells. The emission ratio was increased significantly when dALS2 was co-expressed compared with the transfection of the vector. This increase in the ratio was indeed dependent on the conversion from the GDP-bound form to the GTP-bound one, as shown by the fact that the emission ratio of Raichu-Rab5[S34N], a constitutive GDP-bound form, was unchanged even in the presence of dALS2. The effects of substitutions of two conserved amino acid residues in the VPS9 domain, which are necessary for full GEF activity of ALS2 in vitro were also studied. The two mutant forms of dALS2 (dALS2[P1425A] and dALS2[L1439A]) increased the FRET efficiency of Raichu-Rab5, but not to the same extent as the wild-type form. Collectively, these results showed that the wild-type form of dALS2 has GEF activity for Rab5 (Takayama, 2014).

To study the in vivo consequences of dALS2 dysfunction, an existing transposable element that was inserted 120-bp upstream of the 1st ATG of the dALS2 coding sequence was mobilized and two independent alleles (Ex44 and Ex54) that delete approximately 30% of the coding sequence, including the start codon and the entire RLD domain, were isolated. Further, precise jumpers, where the transposon excision restored the exact contiguous WT sequence were also obtained, and homozygotes of two of these (Ex101/Ex101 and Ex95/Ex95) were used for subsequent analysis as controls or the wild-type animals. Homozygotes of either Ex44 or Ex54 were viable and fertile; and the adults looked morphologically normal. Thus, dALS2 may be dispensable for viability in flies, as is the case in mice. RT-PCR analysis confirmed the deletion of the amino-terminal coding sequence of dALS2 in adult dALS2−/− flies. To address whether truncated polypeptides might be made by translation initiation from internal ATG codons downstream of the deletion in dALS2−/−, antibodies to the carboxyl-terminal VPS9 domain were generated. Unfortunately however, the antibodies failed to detect endogenous dALS2 with high sensitivity and thus, the possibility of the generation of truncated polypeptides could not be excluded. Nonetheless, it is known that ALS2 without the RLD domain no longer associates with endosomes, so the truncated dALS2 polypeptides, if synthesized from Ex44 and Ex54 alleles, would most likely not be functional (Takayama, 2014).

It was found that Rab5[S34N] expression using OK371-Gal4 in the motor neurons resulted in an increase in the bouton number of presynaptic terminals of motor axons in larvae and adults, and the Rab5[S34N] effect was more dramatic than the phenotype in the dALS2 mutant. In addition to the increase in the number of boutons, each bouton became smaller than that of the control axon terminals at larval NMJs. From an earlier study with Rab5[S34N], it is known that Rab5 controls synaptic transmission at larval NMJs; however, in that study Rab5[S34N] expression was kept low during embryonic and early larval stages so that it did not affect morphological development of NMJs. Further, Rab5[S34N] expression strongly depressed the climbing ability of adults. In the climbing assay with the dALS2−/− mutant adults, a more prominent phenotype, age-dependent locomotion deficit, was observed, which was causally related to loss of dALS2 function (Takayama, 2014).

To realize a broader expression, Ubiquitin (Ubi)-Gal4 was used to drive expression of the wild-type dALS2 transgene in a wide range of tissues. Two-week-old dALS2−/− adults showed lowered climbing ability, compared with wild type, and this phenotype was restored to normal by dALS2 transgene expression in both females and males. These results showed that the age-dependent locomotion deficit is indeed a loss-of-function phenotype of dALS2. This phenotype is reminiscent of the moderate, age-dependent deficit in motor coordination in ALS2-null mice (Takayama, 2014).

The observations from this study can be interpreted in several ways: First, dALS2 is indeed required in the motor neuron; however, the motor neuron GAL4 driver (OK371-Gal4) failed to correct the phenotype significantly because this Gal4-driven expression of dALS2 far exceeds the physiological range and disturbs precise spatial-temporal regulation of Rab5 activity. Second, dALS2 is supplied in the motor neuron at larval stages by Ubi-GAL4 and a portion of the proteins persist and function at the adult stage (Ubi-GAL4 was found to be expressed in the motor neuron in larvae, but not in adults). Third, dALS2 is critically required in cell types other than the motor neuron to prevent the deterioration of locomotion during aging (e.g., the presumptive ‘upper’ motor neuron in flies); and Ubi-GAL4, not OK371-Gal4, is expressed in that cell type. Use of the rich resource of GAL4 stocks and searches for the stocks that realize appropriate expression levels of dALS2 would allow to distinguish these possibilities (Takayama, 2014).

In addition to animal behaviors and neuronal cell morphologies, the absence of dALS2 function could impact synaptic transmission. Control of Rab5 activity is required for normal development of NMJ; in addition, Rab5 regulates the efficacy of the evoked neurotransmitter release once the NMJ is formed. So NMJs in the dALS2 mutant could be a target of physiological and ultrastructural investigations. Other future targets are premotor interneurons that control the neurotransmitter release at NMJ and further upstream neural circuits, which are functional counterparts of UMNs in mammals. Identification of such neurons and technical accessibility to those would allow to readdress whether the markers of neuronal aging and/or the Drosophila homologue of TDP-43 are accumulated in those particular neuronal classes, and this approach may validate Drosophila as a tractable model of not only ALS2 but also other genetic causes of ALS (Takayama, 2014).

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Sanhueza, M., Chai, A., Smith, C., McCray, B.A., Simpson, T.I., Taylor, J.P., Pennetta G., et al. (2015). Network analyses reveal novel aspects of ALS pathogenesis. PLoS Genet. 11: e1005107. PubMed ID: 25826266

Abstract
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons, muscle atrophy and paralysis. Mutations in the human VAMP-associated protein B (hVAPB) cause a heterogeneous group of motor neuron diseases including ALS8. Despite extensive research, the molecular mechanisms underlying ALS pathogenesis remain largely unknown. Genetic screens for key interactors of hVAPB activity in the intact nervous system, however, represent a fundamental approach towards understanding the in vivo function of hVAPB and its role in ALS pathogenesis. Targeted expression of the disease-causing allele leads to neurodegeneration and progressive decline in motor performance when expressed in the adult Drosophila, eye or in its entire nervous system, respectively. By using these two phenotypic readouts, this study carried out a systematic survey of the Drosophila genome to identify modifiers of hVAPB-induced neurotoxicity. Modifiers clustered in a diverse array of biological functions including processes and genes that had been previously linked to hVAPB function, such as proteolysis and vesicular trafficking. In addition to established mechanisms, the screen identified endocytic trafficking and genes controlling proliferation and apoptosis as potent modifiers of ALS8-mediated defects. Surprisingly, the list of modifiers was mostly enriched for proteins linked to lipid droplet biogenesis and dynamics. Computational analysis revealed that most modifiers could be linked into a complex network of interacting genes, and that the human genes homologous to the Drosophila modifiers could be assembled into an interacting network largely overlapping with that in flies. Identity markers of the endocytic process were also found to abnormally accumulate in ALS patients, further supporting the relevance of the fly data for human biology. Collectively, these results not only lead to a better understanding of hVAPB function but also point to potentially relevant targets for therapeutic intervention (Sanhueza, 2015).

Highlights

  • A large-scale screen in Drosophila identifies modifiers of the DVAP-P58S-induced eye phenotype.
  • Genetic validation of the identified modifiers.
  • The modifying effect of DVAP-P58S interacting genes is extended to the adult nervous system.
  • Building the human interactome of DVAP-P58S genetic modifiers.
  • Computational and experimental analysis identifies endocytosis as a process implicated in ALS8 pathogenesis.
  • Rab5 accumulates abnormally in motor neurons of patients affected by ALS.

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Deivasigamani, S., Verma, H.K., Ueda, R., Ratnaparkhi, A., Ratnaparkhi, G.S. (2014). A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis. Biol Open. 3: 1127-1138. 25361581

Abstract
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. This study conducted a reverse genetic screen to identify interactors of Drosophila VAPB. By screening 2635 genes, 103 interactors were identified, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression. The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease (Deivasigamani, 2014).

Highlights

  • Known ALS loci and physical interactors of VAP act as modifiers.
  • Modifiers identified in screen alter VAP(P58S) induced bouton size.
  • Modulation of TOR pathway components suppresses VAP(P58S) bouton phenotypes.
  • Modulation of Tor pathway components suppresses VAP(wt) bouton phenotypes.
  • Rapamycin feeding mitigates VAP(P58S) phenotype.

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Reviews

Ambegaokar, S.S., Roy, B., Jackson, G.R. (2010). Neurodegenerative models in Drosophila: polyglutamine disorders, Parkinson disease, and amyotrophic lateral sclerosis. Neurobiol Dis. 40: 29-39. PubMed ID: 20561920

Lloyd, T.E., Taylor, J.P. (2010). Flightless flies: Drosophila models of neuromuscular disease. Ann N Y Acad Sci. 1184: e1-20. PubMed ID: 20329357

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More in IF

The Amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors

hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction

TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization

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Recent Updates

Yang, D., Abdallah, A., Li, Z., Lu, Y., Almeida, S. and Gao, F.B. (2015). FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol [Epub ahead of print]. PubMed ID: 26031661

Freibaum, B.D., Lu, Y., Lopez-Gonzalez, R., Kim, N.C., Almeida, S., Lee, K.H., Badders, N., Valentine, M., Miller, B.L., Wong, P.C., Petrucelli, L., Kim, H.J., Gao, F.B. and Taylor, J.P. (2015). GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature [Epub ahead of print]. PubMed ID: 26308899

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Date revised: 10 July 2021

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