Spring, A. M. | Search bioRxiv

bioRxiv2018

Comprehensive modeling of Spinal Muscular Atrophy in Drosophila melanogaster

Spinal muscular atrophy (SMA) is a neurodegenerative disorder that affects motor neurons, primarily in young children. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene. SMN functions in the assembly of spliceosomal RNPs and is well conserved in many model systems including mouse, zebrafish, fruit fly, nematode, and fission yeast. Work in Drosophila has primarily focused on loss of SMN function during larval stages, primarily using null alleles or strong hypomorphs. A systematic analysis of SMA-related phenotypes in the context of moderate alleles that more closely mimic the genetics of SMA has not been performed in the fly, leading to debate over the validity and translational value of this model. We therefore examined fourteen Drosophila lines expressing SMA patient-derived missense mutations in Smn, with a focus on neuromuscular phenotypes in the adult stage. Animals were evaluated on the basis of organismal viability and longevity, locomotor function, neuromuscular junction structure, and muscle health. In all cases, we observed phenotypes similar to those of SMA patients, including progressive loss of adult motor function. The severity of these defects is variable, and forms a broad spectrum across the fourteen lines examined, recapitulating the full range of phenotypic severity observed in human SMA. This includes late-onset models of SMA, which have been difficult to produce in other model systems. The results provide direct evidence that SMA-related locomotor decline can be reproduced in the fly and support the use of patient-derived SMN missense mutations as a comprehensive system for modeling SMA.

neuroscience↗

bioRxiv2017

Drosophila CaV2 Channels Harboring Human Migraine Mutations Cause Synapse Hyperexcitability That Can Be Suppressed By Inhibition Of A Ca2+ Store Release Pathway

Gain-of-function mutations in the human CaV2.1 gene CACNA1A cause familial hemiplegic migraine type 1 (FHM1). To characterize cellular problems potentially triggered by CaV2.1 gains of function, we engineered mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) into transgenes of Drosophila melanogaster CaV2/cacophony. We expressed the transgenes pan-neuronally. Phenotypes were mild for RQ-expressing animals. By contrast, single mutant SL- and complex allele RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. By electrophysiology, SL- and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of spontaneous events. Some spontaneous events were gigantic (10-40 mV), multi-quantal events. Gigantic spontaneous events were eliminated by application of TTX - or by lowered or chelated Ca2+ - suggesting that gigantic events were elicited by spontaneous nerve firing. A follow-up genetic approach revealed that some neuronal hyperexcitability phenotypes were reversed after knockdown or mutation of Drosophila homologs of phospholipase C{beta} (PLC{beta}), IP3 receptor, or ryanodine receptor (RyR) - all factors known to mediate Ca2+ release from intracellular stores. Pharmacological inhibitors of intracellular Ca2+ store release produced similar effects. Interestingly, however, the decreased viability phenotype was not reversed by genetic impairment of intracellular Ca2+ release factors. On a cellular level, our data suggest inhibition of signaling that triggers intracellular Ca2+ release could counteract hyperexcitability induced by gains of CaV2.1 function.\n\nAUTHOR SUMMARYPrior research has demonstrated that gain-of-function mutations in a gene important for neurotransmission (CACNA1A) are known to cause migraine in humans. We attempted to mimic some of those gain-of-function mutations in a simple genetic model organism and to examine neurotransmission by electrophysiology. Our findings yield potential clues as to how particular migraine-causing mutations may impact neurophysiology on a cellular level. We used the fruit fly Drosophila melanogaster and its model synapse, the neuromuscular junction (NMJ) to perform our studies. We document three main advances: 1) characterization of fruit fly models harboring gain-of-function calcium channel alterations known to cause human familial hemiplegic migraine type 1 (FHM1); 2) characterization of hyperactive neurotransmission caused by one of these alterations; and 3) an ability to quell hyperactive neurotransmission by impairing intracellular Ca2+ store release, through both genetic and pharmacological means. Our work contributes to a broader understanding of how pathological mutations could impact cellular physiology. More generally, the utilization of genetic model organisms promises to uncover potential ways to reverse those impacts.

neuroscience↗