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Mutations in the EFHC1 gene have been linked to juvenile myoclonic epilepsy. To understand EFHC1 function in vivo, we generated knockout Drosophila for the fly homolog Defhc1.1. We found that the neuromuscular junction synapse of Defhc1.1 mutants displays an increased number of satellite boutons resulting in increased spontaneous neurotransmitter release. Defhc1.1 binds to microtubules in vitro and overlaps in vivo with axonal and synaptic microtubules. Elimination of Defhc1.1 from synaptic terminals reduces the number of microtubule loops, suggesting that Defhc1.1 is a negative regulator of microtubule dynamics. In fact, pharmacological treatment of Defhc1.1 mutants with vinblastine, an inhibitor of microtubule dynamics, suppresses the satellite bouton phenotype. Furthermore, Defhc1.1 mutants display overgrowth of the dendritic arbor and Defhc1.1 overexpression reduces dendrite elaboration. These results suggest that Defhc1.1 functions as an inhibitor of neurite growth by finely tuning the microtubule cytoskeleton dynamics and that EFHC1-dependent juvenile myoclonic epilepsy may result from augmented spontaneous neurotransmitter release due to overgrowth of neuronal processes.

3'-5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous intracellular second messenger that mediates the action of various hormones and neurotransmitters and influences a plethora of cellular functions. In particular, multiple neuronal processes such as synaptic plasticity underlying learning and memory are dependent on cAMP signalling cascades. It is now well recognized that the specificity and fidelity of cAMP downstream effects are achieved through a tight temporal as well as spatial control of the cAMP signals. Approaches relying on real-time imaging and Fluorescence Resonance Energy Transfer (FRET)-based biosensors for direct visualization of cAMP changes as they happen in intact living cells have recently started to uncover the fine details of cAMP spatio-temporal signalling patterns. Here we report the generation of transgenic fruit-flies expressing a FRET-based, GFP-PKA sensor and their use in real-time optical recordings of cAMP signalling both ex vivo and in vivo in adult and developing organisms. These transgenic animals represent a novel tool for understanding the physiology of the cAMP signalling pathway in the context of a functioning body.

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative diseases characterized by progressive weakness and spasticity of the lower limbs. Dominant mutations in the human SPG4 gene, encoding spastin, are responsible for the most frequent form of HSP. Spastin is an ATPase that binds microtubules and localizes to the spindle pole and distal axon in mammalian cell lines. Furthermore, its Drosophila homolog, Drosophila spastin (Dspastin), has been recently shown to regulate microtubule stability and synaptic function at the Drosophila larval neuromuscular junction. Here we report the generation of a spastin-linked HSP animal model and show that in Drosophila, neural knockdown of Dspastin and, conversely, neural overexpression of Dspastin containing a conserved pathogenic mutation both recapitulate some phenotypic aspects of the human disease, including adult onset, locomotor impairment, and neurodegeneration. At the subcellular level, neuronal expression of both Dspastin RNA interference and mutant Dspastin cause an excessive stabilization of microtubules in the neuromuscular junction synapse. In addition, we provide evidence that administration of the microtubule targeting drug vinblastine significantly attenuates these phenotypes in vivo. Our findings demonstrate that loss of spastin function elicits HSP-like phenotypes in Drosophila, provide novel insights into the molecular mechanism of spastin mutations, and raise the possibility that therapy with Vinca alkaloids may be efficacious in spastin-associated HSP and other disorders related to microtubule dysfunction.

Background: Hereditary Spastic Paraplegia (HSP) is a devastating neurological disease causing spastic weakness of the lower extremities and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans; however, mutations in one gene, spastin (SPG4), are the cause of >40% of all cases. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) protein family, and contains a microtubule interacting and organelle transport (MIT) domain. Previous work in cell culture has proposed a role for Spastin in regulating microtubules., Results: Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), we have investigated the role of Spastin in vivo. We show that Drosophila Spastin (D-Spastin) is enriched in axons and synaptic connections. At neuromuscular junctions (NMJ), Dspastin RNAi causes morphological undergrowth and reduced synaptic area. Moreover, Dspastin overexpression reduces synaptic strength, whereas Dspastin RNAi elevates synaptic currents. By using antibodies against posttranslationally modified alpha-Tubulin, we find that Dspastin regulates microtubule stability. Functional synaptic defects caused by Dspastin RNAi and overexpression were pharmacologically alleviated by agents that destabilize and stabilize microtubules, respectively., Conclusions: Loss of Dspastin in Drosophila causes an aberrantly stabilized microtubule cytoskeleton in neurons and defects in synaptic growth and neurotransmission. These in vivo data strongly support previous reports, providing a probable cause for the neuronal dysfunction in spastin-linked HSP disease. The role of Spastin in regulating neuronal microtubule stability suggests therapeutic targets for HSP treatment and may provide insight into neurological disorders linked to microtubule dysfunction.