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
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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).
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
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
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).
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
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).
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
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).
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
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).
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).
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
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).
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).
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
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).
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
Home page: The Interactive Fly © 2015 Thomas Brody, Ph.D.
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