Analysis of the interaction between DVAP, the orthologue of human VAPB, and Drosophila Sac1
Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease characterizeby devastating symptoms, such as muscle weakness, paralysis, and death within 5 years of disease onset. Mutations in human (VAMP)-associated protein B (hVAPB) gene cause ALS8. Interestingly, the Drosophila VAPB (DVAP) is required at the Drosophila larval neuromuscular junction (NMJ), to control bouton morphology. In Pennetta’s lab Drosophila Sac1, a phosphoinositide-4- phosphate (PI4P) phosphatase, was identified during a genome-wide yeast twohybrid screen, as a DVAP binding partner. VAP plays a role in regulating PI4P turnover in yeast and phosphoinositides are implicated in some neurodegenerative processes. In this PhD thesis, Sac1-DVAP interaction is used as the starting point to identify the mechanism that is altered when DVAP function is impaired. Thus, the possibility that the cellular pathways regulated by PI4P are affected in VAPB-mediated neurodegeneration was explored. First, the Sac1-DVAP association was confirmed in vitro by coimmunoprecipitation. Subsequently, we found that these two proteins colocalize in vivo at the ER membranes. Then, immunohistochemical analysis of Drosophila larval NMJ revealed that Sac1 and DVAP are involved in similar pathways. They both have a function in microtubule stabilization in the presynaptic boutons and axonal vesicle transport at the presynaptic compartment. They also seem to contribute to the spectrin-actin cytoskeleton stabilization at the postsynaptic compartment of the NMJ. Lastly, we reported that reduced levels of Sac1 phosphatase cause progressive neurodegeneration. Moreover, Sac1 is trapped into cytosolic aggregates induced by the expression of the ALS8-mutant allele of DVAP, and it does not localize to its original place in the cell. All together these results suggest that in ALS8 hVAPB seems to have a dominant negative effect on Sac1. Sac1 mislocalization could inhibit the dephosphorylation of PI4P. This PhD work further confirms Sac1-DVAP interaction and it suggests a mechanism underlying ALS8 pathogenesis, supporting the idea that altered metabolism of phosphoinositides can cause neurodegeneration.
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