Drosophila models of motor neuron disease

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5–10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions.

We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci – TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression.

The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease. Glutamate is the major neurotransmitter in the mammalian central nervous system. Invertebrates like Drosophila and C. elegans use it as the neurotransmitter at neuro-muscular junction (NMJ). Glutamate receptors respond to release of presynaptic glutamate and are critical for function and plasticity of the nervous system. The post-synaptic membrane docking of glutamate receptor is dependent on activity and on the endocytic machinery where the recycling endosomes act as a source of the receptors. Deregulation of glutamate receptor levels has been observed in case of diseases like Down syndrome and Amyotrophic Lateral Sclerosis (ALS). Here, using Drosophila NMJ we show that two players in endocytosis, namely Mon1 and Rab7, regulate glutamate receptor levels. Mon1 is a highly conserved GEF that is essential for conversion of Rab5 positive early endosomes to Rab7 positive late endosomes. In this study, we show that Mon1 mutants have NMJ defects, reduced levels of pre-synaptic active zones and increased levels of glutamate receptor subunit IIA at the synapse. Strikingly, these mutants also show GluRIIA accumulation in the muscle. We think that there may be a pre-synaptic mechanism, and possibly a post synaptic one as well through which Mon1 is involved in regulation of glutamate receptor levels. Importantly, knock down of Rab7, which functions downstream of Mon1 in endocytosis, also leads to increased GluRIIA levels at the synapse. Mutations in Rab7 are known to cause a sensory neurodegenerative disease called Charcot- 77 Marie-Tooth disease (CMT disease). Our studies indicate a role for late endosomal machinery in regulating glutamate receptor levels at the synapse and suggest exploration of these players in CMT disease.