genes associated with
Amyotrophic Lateral Sclerosis
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
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).
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
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).
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
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).
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
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 in the 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).26130692
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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
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).
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
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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
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).
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
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
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).
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
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
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
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 organizationGo to top
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
Johnson, A.E., Shu, H., Hauswirth, A.G., Tong, A. and Davis, G.W. (2015). VCP-dependent muscle degeneration is linked to defects in a dynamic tubular lysosomal network in vivo. Elife 4. PubMed ID: 26167652
Sreedharan, J., Neukomm, L.J., Brown, R.H. Jr. and Freeman, M.R. (2015). Age-Dependent TDP-43-Mediated Motor Neuron Degeneration Requires GSK3, hat-trick, and xmas-2. Curr Biol 25: 2130-2136. PubMed ID: 26234214
Cheng, C.W., Lin, M.J. and Shen, C.J. (2015). Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP. J Neurogenet [Epub ahead of print]. PubMed ID: 26219309
Tran, H., Almeida, S., Moore, J., Gendron, T.F., Chalasani, U., Lu, Y., Du, X., Nickerson, J.A., Petrucelli, L., Weng, Z. and Gao, F.B. (2015). Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS. Neuron 87: 1207-1214. PubMed ID: 26402604
Di Salvio, M., Piccinni, V., Gerbino, V., Mantoni, F., Camerini, S., Lenzi, J., Rosa, A., Chellini, L., Loreni, F., Carrì, M.T., Bozzoni, I., Cozzolino, M. and Cestra, G. (2015). Pur-alpha functionally interacts with FUS carrying ALS-associated mutations. Cell Death Dis 6: e1943. PubMed ID: 26492376
Cragnaz, L., Klima, R., De Conti, L., Romano, G., Feiguin, F., Buratti, E., Baralle, M. and Baralle, F.E. (2015). An age-related reduction of brain TBPH/TDP-43 levels precedes the onset of locomotion defects in a Drosophila ALS model. Neuroscience 311: 415-421. PubMed ID: 26518462
Date revised: 4 Sep 2015
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