Phenotypic and molecular characterization of human SPG10 model in Drosophila melanogaster and its link to BMP signaling

Abstract

Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity of lower extremities as a consequence of axonopathy of the longest cortico spinal tract. Defects in axonal transport, ER membrane modeling, mitochondrial function, DNA repair, autophagy, lipid metabolism and myelination have been linked to HSP. SPG10, a subtype of HSP is caused due to mutations in the anterograde microtubular motor protein kinesin. Kinesin mutation impairs axonal transport leading to synaptic dysfunction. My study was aimed at the phenotypic and molecular characterization of the human SPG10 model in Drosophila melanogaster and to investigate its suspected interplay with BMP signaling. The mutation in kinesin protein in human SPG10 has been mapped to position N256S. This point mutation was inserted into Drosphila kinesin heavy chain (Khc) at the N262S position and the mutated kinesin protein was ectopically expressed using tissue specific drivers. The Drosophila KhcN262S mutants were characterized by reduced survival, behavioral impairments, axonal swellings, synaptic protein depletion at distal NMJs, cytoskeletal disability, developmental delay, and synaptic degeneration. Ectopic overexpression of wild type kinesin alongside mutated kinesin (KhcN262S+wt) partially rescued the HSP pathology thus revealing the dominant negative nature of this mutation. KhcN262S mutation affected both anterograde and retrograde transport in Drosophila larvae. Impairment of long distance transport within axons directly contributes to synaptic defects by perturbing the BMP signaling pathway which is essential for synapse maintenance and function. Till date a common pathological mechanism in HSPs remains to be identified. Atleast 4 human SPGs have been linked to altered BMP signaling. The key pathological features of downregulated BMP signaling in Drosophila resemble that of the KhcN262S Drosophila model. A previous study in the lab show reduced pMad (transcription factor of BMP target gene, Trio) levels in KhcN262S larval motor neuron cell bodies. Assuming that the phenotypical defects in the KhcN262S Drosophila model may be due to altered BMP signaling, we hypothesized that upregulation of BMP signaling in KhcN262S mutants could rescue the synaptic and behavioral defects via cytoskeletal stabilization. However, neither the overexpression of constitutively active BMP receptor Tkv-CA nor the overexpression of target gene Rac-GEF Trio was able to rescue the KhcN26S pathology significantly. In- vivo study using YFP tagged Tkv showed impairments in retrograde transport of Tkv which was consistent with reduced pMad levels observed in motor neuron cell bodies of KhcN262S+TkV-CA larvae. Though overexpression of Trio in mutants partially rescued the cargo trafficking it did not suffice synaptic or behavioral rescue owing to its tight regulation and influence on numerous other genes. Since Trio is the only BMP target gene known, it does not rule out regulation of other unidentified genes. Finally, molecular mechanism involved in KhcN262S induced SPG10 pathology is quite complex since both anterograde and retrograde transport is impaired, hence BMP signaling could only be one among the many signaling pathways affected, and sole upregulation of which alone does not suffice rescue. My study has successfully characterized the severity of the SPG10 model in Drosophila at the molecular level as well as behavioral level. After testing our hypothesis, arising from similarity of phenotypes and previously described interplay at molecular level, a connection between SPG10 pathology and BMP signaling seems highly plausible, yet our proposed mechanism of rescue was only partially successful.