Despite the discovery of several missense mutations in the RNA-binding protein TDP-43, the mechanisms of neurodegeneration involving the individual alleles remain poorly understood. To uncover the consequences of TDP-43 mutations linked to ALS, we performed a direct in vivo
comparison of the neuronal and functional phenotypes resulting from overexpression of wild-type and the A315T mutant TDP-43 in the Drosophila
nervous system. Expression of either allele (wild-type or A315T) of TDP-43 in photoreceptor and motor neurons led to effects on viability, locomotor function and survival. In the developing retina, overexpression of the TDP-43 variants used in this study resulted in the formation of cytoplasmic and axonal aggregates accompanied by neuronal loss. The eye tissue showed the highest sensitivity to overexpression of TDP-43 and revealed the most dramatic alterations in the subcellular localization of TDP-43, resembling those found in postmortem ALS tissues (9
). When TDP-43 variants were expressed in motor neurons, we also found several hallmark features of ALS, including cytoplasmic aggregates, cell death and defects in locomotor ability (Table ). To our surprise, when expressed at comparable levels, wild-type TDP-43 was more toxic than the A315T mutant in regard to viability, survival, NMJ anatomy and adult climbing behavior but not for larval turning behavior. We also found that by simply increasing expression of the mutant allele we were able to recapitulate several, although not all, phenotypes resulting from overexpression of wild-type TDP-43. These include effects on viability, and select NMJ features such as decreased synaptic area and malformed synaptic boutons that may be more sensitive to mutations in TDP-43 than others (e.g. axon branching). This is particularly interesting because, on the one hand, it shows that TDP-43 is inherently toxic, consistent with previous in vivo
and in vitro
), whereas, on the other hand, our data show that wild-type and A315T mutant TDP-43 exert differential toxicity when evaluated by a repertoire of cellular and behavioral assays. These results emphasize the need for detailed studies of individual missense mutations in TDP-43, in particular those that mimic the mutations identified in ALS patients. Such studies are needed to identify allele-specific neuronal and synaptic defects that could provide clues to their mechanism of action and lead to the development of personalized therapeutic interventions.
We found that when expressed at comparable levels, the TBPH variants were more toxic than their human counterparts. A possible explanation for this difference is provided by the fact that the Drosophila genome encodes an additional, previously unreported homolog of TDP-43, namely CG7804. This locus encodes a second, highly homologous TDP-43 protein in Drosophila that contains the RNA-binding domains RRM1/2 but lacks the C-terminal glycine-rich region. The function of this predicted, truncated, additional TDP-43 protein, as well as the significance of its loss during evolution, remains to be investigated. Although the fly and hTDP-43 pathways may not be identical, most differences seem to be mitigated by the fact that by simply expressing higher levels of hA315T (hA315T HE) we can recapitulate several of the neuronal and functional phenotypes resulting from wild-type TBPH overexpression, the most toxic of the transgenes in this study. Thus, dosage appears to directly correlate with toxicity.
TDP-43 has been previously shown to be required for the proper morphology of the NMJ synapse (23
) and our findings further support this. In addition, our morphological analyses revealed novel phenotypes, including the reduced area occupied by synaptic vesicles, which appears to be most sensitive to alterations in TDP-43 (both wild-type and A315T) and suggests the presence of functional defects at the synapse. Indeed, larval turning assays, which rely on complex motor coordination along the anterior–posterior and dorsal–ventral axes of the larvae, showed that both the wild-type and A315T alleles affect locomotor ability. It is intriguing that larval turning is the only assay in which the A315T mutant shows higher toxicity than the wild-type allele and it remains to be seen what are the molecular mechanisms underlying this effect. Additional evidence for TDP-43′s effect on locomotor function is provided by the adult climbing assays. Although these results suggest that TDP-43 overexpression affects synaptic function, more work is needed to determine the underlying defects at the NMJ synapse. For example, it would be interesting to determine what specific aspects of synaptic transmission may be affected by altering TDP-43 function in presynaptic motor neurons. Knowing which parameters of synaptic function are impaired due to expression of missense mutations in TDP-43 will provide important clues to the mechanisms underlying the pathology of ALS.
Our results indicate that cytoplasmic aggregates are not required for motor neuron death, consistent with previous findings using hwt (24
). Upon wild-type TBPH overexpression, motor neuron death was sparsely detected in larval ventral ganglia before lethality ensued. When hwt was overexpressed, adult motor neuron numbers were significantly reduced in thoracic ganglia and we found strong evidence for apoptotic death. In contrast, overexpression of A315T TBPH led to barely detectable apoptosis, and hA315T resulted in some cells positive for TUNEL staining. Surprisingly, apoptosis detection was also marginal in the ventral ganglia of the higher expressing hA315T HE larvae. Although our findings on the extent of apoptotic death may not be in full agreement with previous reports (22
), it is likely that differences in expression levels and type of neuron or animal model studied may account for the differences observed. We would also like to note that in the case of the Drosophila
transgenes, their effect on viability may have overshadowed the motor neuron phenotypes. For example, overexpression of TBPH, which overall has more pronounced effects than the hTDP-43 variants, may have led to lethality due to the inability of the transgenic larvae to feed properly. Indeed, the wild-type TBPH expressing larvae appeared ‘skinny’ and developmentally delayed. The TBPH transgenics that died before reaching adulthood may have succumbed to starvation and/or delayed growth, thus precluding us from detecting extensive motor neuron death, which may otherwise have been observed later in development. This scenario is further substantiated by our findings with the hTDP-43 variants, which exhibited motor neuron apoptosis in the adult but not larval nervous system.
Several questions remain regarding the molecular mechanisms that mediate the dominant effect of TDP-43 overexpression. How can overexpression of wild-type TDP-43 mimic a loss-of-function phenotype? One possibility is that excess protein is sequestered in cytoplasmic or nuclear aggregates, which may affect the stoichiometry of physiological TDP-43 complexes, thus mimicking a loss-of-function phenotype (49
). Overall, our data suggest that the precise amount of TDP-43 expression is critical to proper neuronal and synaptic function and alterations in TDP-43 levels and/or function are responsible for the observed phenotypes. The dominant effects of TDP-43 overexpression are not unlike those obtained with other RNA-binding proteins including FMRP and Argonaute 1, which result in visible phenotypes when overexpressed in the eye (50
). Thus, we suggest that the phenotypes resulting from wild-type and mutant TDP-43 overexpression stem from defects in gene expression, in particular the dysregulation of RNA targets that are just beginning to be discovered (52
The genetic interactions with the proteasome support the notion that the insoluble TDP-43 products that arise from overexpression or the presence of missense mutations (3
) may normally be processed by the proteasome degradation machinery. By impairing the function of the proteasome, insoluble TDP-43 products may not be cleared efficiently, which could account for the enhanced toxicity we observed in the eye. Although this result is not entirely unexpected and previous studies have shown that pharmacological inhibition of the proteasome enhances the accumulation of C-terminal fragments of TDP-43 (3
), our results provide an in vivo
demonstration for the involvement of the proteasome in TDP-43 neurotoxicity. Future biochemical studies will focus on whether the amount of insoluble TDP-43 products is increased upon proteasome impairment or whether additional mechanisms account for the enhanced neurotoxicity we observed in the eye. Although it remains to be seen whether cytoplasmic and axonal TDP-43 aggregates are cleared by HSP70 coexpression, our discovery that excess HSP70 chaperone alleviates the phenotype of TDP-43 in the eye supports the notion that TDP-43 misfolding underlies its toxicity in the eye. Not surprisingly, given the dramatic cell loss evident in the retina, we found that inhibition of caspases through overexpression of the p35 baculovirus protein also alleviates, although does not completely rescue, the phenotype of TDP-43 overexpression in the eye. Taken together, these findings indicate that commonalities may exist between TDP-43 and other known proteinopathies and suggest that therapies based on targeting the proteasome function, HSP70 chaperone activity or caspases may be effective for ALS as well as other neurodegenerative disorders in which these pathways have been implicated.
In summary, our results support previous findings and provide further evidence that wild-type and mutant TDP-43 overexpression in Drosophila
recapitulates hallmark aspects of ALS pathology. Perhaps the most surprising result is that, when expressed at comparable levels, wild-type TDP-43 exerted higher toxicity than the A315T allele, in regard to viability, survival, neuronal loss and adult locomotor function but not larval locomotor behavior. Although a mouse model of A315T overexpression exhibits ubiquitin-positive cytoplasmic aggregates and other features of ALS pathology (28
), a recent article reports the presence of rather weak pathological hallmarks in an ALS patient carrying this allele (54
). These findings suggest that the genetic background may influence TDP-43 phenotypes and are consistent with a recent report that ataxin 2 constitutes a risk factor for ALS (47
). Taken together, our results and these published data indicate that detailed phenotypic analyses of different mutants in animal models such as Drosophila
are critical for elucidating the biology of TDP-43 and the mechanisms of neurodegenerative disease. In addition, our work suggests that perhaps animal models based on individual missense mutations that mimic those found in human patients will serve as better models for genetic and drug screens that can lead to novel effective and potentially personalized therapies.