Spinal and bulbar muscular atrophy (SBMA) impairs motor function in men and is linked to a CAG repeat mutation in the androgen receptor (AR) gene. Defects in motoneuronal retrograde axonal transport may critically mediate motor dysfunction in SBMA, but the site(s) where AR disrupts transport is unknown. We find deficits in retrograde labeling of spinal motoneurons in both a knock-in (KI) and a myogenic transgenic (TG) mouse model of SBMA. Likewise, live imaging of endosomal trafficking in sciatic nerve axons reveals disease-induced deficits in the flux and run length of retrogradely transported endosomes in both KI and TG males, demonstrating that disease triggered in muscle can impair retrograde transport of cargo in motoneuron axons, possibly via defective retrograde signaling. Supporting the idea of impaired retrograde signaling, we find that vascular endothelial growth factor treatment of diseased muscles reverses the transport/trafficking deficit. Transport velocity is also affected in KI males, suggesting a neurogenic component. These results demonstrate that androgens could act via both cell autonomous and non-cell autonomous mechanisms to disrupt axonal transport in motoneurons affected by SBMA.

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The molecular mechanisms influencing healthspan are unclear but mitochondrial function, resistance to oxidative stress and proteostasis are recurring themes. Tumor necrosis factor Receptor Associated Protein 1 (TRAP1), the mitochondrial analogue of Hsp75, regulates levels of reactive oxygen species in vitro and is found expressed at higher levels in tumor cells where it is thought to play a pro-survival role. While TRAP1-directed compartmentalized protein folding is a promising target for cancer therapy, its role at the organismal level is unclear. Here we report that overexpression of TRAP1 in Drosophila extends healthspan by enhancing stress resistance, locomotor activity and fertility while depletion of TRAP1 has the opposite effect, with little effect on lifespan under both conditions. In addition, modulating TRAP1 expression promotes the nuclear translocation of homeobox protein Dve and increases expression of genes associated with the mitochondrial unfolded protein response (UPRmt), indicating an activation of this proteostasis pathway. Notably, independent genetic knockdown of components of the UPRmt pathway dampen the enhanced stress resistance observed in TRAP1 overexpression flies. Together these studies suggest that TRAP1 regulates healthspan, potentially through activation of the UPRmt.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors.

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

The Drosophila melanogaster (Dmel) eye is an ideal model to study development, intracellular signaling, behavior, and neurodegenerative disease. Interestingly, dynamic data are not commonly employed to investigate eye-specific disease models. Using axonal transport of the morphogen Hedgehog (Hh), which is integral to Dmel eye-brain development and implicated in stem cell maintenance and neoplastic disease, we demonstrate the ability to comprehensively quantify and characterize its trafficking in various neuron types and a neurodegeneration model in live early third-instar larval Drosophila. We find that neuronal Hh, whose kinetics have not been reported previously, favors fast anterograde transport and varies in speed and flux with respect to axonal position. This suggests distinct trafficking pathways along the axon. Lastly, we report abnormal transport of Hh in an accepted model of photoreceptor neurodegeneration. As a technical complement to existing eye-specific disease models, we demonstrate the ability to directly visualize transport in real time in intact and live animals and track secreted cargoes from the axon to their release points. Particle dynamics can now be precisely calculated and we posit that this method could be conveniently applied to characterizing disease pathogenesis and genetic screening in other established models of neurodegeneration.