Axonal transport defects are a common phenotype in Drosophila models of ALS

Human Molecular Genetics, 2016, Vol. 25, No. 12 2378–2392 doi: 10.1093/hmg/ddw105 Advance Access Publication Date: 7 April 2016 Original Article ORIGINAL ARTICLE Axonal transport defects are a common phenotype in Drosophila models of ALS 1 Department of Biomedical Sciences, University of Sheffield, Sheffield S10 2TN, UK and 2Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK *To whom correspondence should be addressed. Tel: þ44 1223252910; Fax: þ44 1223252715; Email: Abstract Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. Here, we assessed the axonal transport of different cargos in multiple Drosophila models of ALS. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models and generally worsened with age, though surprisingly there was not 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. Introduction Amyotrophic lateral sclerosis (ALS) is a typically adult onset progressive neurodegenerative disorder and the most common form of motor neuron disease. It is characterized by the loss of both the upper and lower motor neurons representing a catastrophic loss of motor function. The condition is fatal, usually due to respiratory failure, with an average life expectancy of 3–5 years from diagnosis (1). There is no cure and current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Although the majority of ALS cases is sporadic, 10% are monogenic, familial forms. 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 pathogenic mutations have provided great insight into the underlying disease mechanisms (1,2). TDP-43 is a multi-functional DNA/RNA-binding protein that shuttles between the nucleus and cytoplasm (3). In the nucleus, it plays many roles in transcription and RNA processing (4–7). In the cytoplasm, TDP-43 localizes to stress granules, P-bodies and RNA transport granules, and is involved in the regulation and spatial distribution of RNAs (8–12). FUS is also a DNA/RNA-binding protein that undergoes nuclear/cytoplasmic shuttling, with Received: February 9, 2016. Revised: March 10, 2016. Accepted: March 29, 2016 C The Author 2016. Published by Oxford University Press. V This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2378 Katie R. Baldwin1, Vinay K. Godena1, Victoria L. Hewitt2 and Alexander J. Whitworth1,2,* Human Molecular Genetics, 2016, Vol. 25, No. 12 Results Gain and loss of TDP-43 function have differential effects on axonal transport Mutations in TDP-43 cause ALS in an autosomal dominant manner and likely impact on neuronal function in a multitude of ways. We first analyzed whether ectopic expression of a pathogenic variant of TDP-43, M337V, caused any disruption of axonal transport in Drosophila motor nerves in comparison to TDP43WT. To minimize the likelihood of artifacts from different expression levels or from disruption of genomic insertion, we made use of transgenes generated in the same integration site and expressing at equivalent levels (see Materials and Methods). Somewhat surprisingly no significant effect on mitochondrial transport was observed for any of the transgenes (Fig. 1A). However, when we analyzed a different transport cargo—vesicles loaded with GFP-fused neuropeptide Y (NPY.GFP)—although TDP-43WT did not cause any significant defects, TDP-43M337V expression caused a decrease in motility, with a concomitant significant increase in the stationary fraction (Fig. 1B). Overexpression of the fly homolog of TDP-43, named TBPH, also interfered with the transport of vesicles but not mitochondria (Fig. 1). We next addressed whether TDP-43 normally plays a role in axonal transport by examining whether loss of the endogenous TBPH disrupted transport. We used a trans-heterozygous combination of the independently derived TBPH1 and the TBPHD23 deletion mutants to avoid potential genetic background effects. Loss of TBPH caused an overall increase in the stationary fraction of mitochondria (Fig. 2A). Although there was an observable decrease in both anterograde and retrograde transport, this only reached significance in the anterograde direction. In contrast, we found no impact of loss of TBPH on vesicle transport (Fig. 2B). Importantly, the disruption of mitochondrial transport could be completely rescued by re-expression of a TBPHWT transgene (Fig. 2A), verifying that this effect is a direct consequence of the genetic loss of TBPH. Interestingly, the loss of mitochondrial transport was also rescued by the ectopic expression of human TDP-43WT and by the ALS-linked variant TDP-43M337V. This crossspecies rescue is consistent with previous reports and reflects their close homology, but also reveals that the pathogenic variant can broadly function as normal. Overall, these results indicate that dysregulated TDP-43 expression can affect axonal transport but cargoes are affected differently, and suggest that the pathogenic mutation may increase the susceptibility to disease. Axonal transport disruption in TDP-43 models correlates with behavioral deficits A prime motivation for this study was to correlate disruptions in axonal transport to decline in motor neuron function; the cardinal clinical feature of ALS. Thus, flies were examined for their neuromuscular function using a number of assays. One indicator of severe motor dysfunction is th (...truncated)