Shawn, the Drosophila Homolog of SLC25A39/40, Is a Mitochondrial Carrier That Promotes Neuronal Survival.

Mitochondria play an important role in the regulation of neurotransmission, and mitochondrial impairment is a key event in neurodegeneration. Cells rely on mitochondrial carrier proteins of the SLC25 family to shuttle ions, cofactors, and metabolites necessary for enzymatic reactions. Mutations in these carriers often result in rare but severe pathologies in the brain, and some of the genes, including SLC25A39 and SLC25A40, reside in susceptibility loci of severe forms of epilepsy. However, the role of most of these carriers has not been investigated in neurons in vivo. We identified shawn, the Drosophila homolog of SLC25A39 and SLC25A40, in a genetic screen to identify genes involved in neuronal function. Shawn localizes to mitochondria, and missense mutations result in an accumulation of reactive oxygen species, mitochondrial dysfunction, and neurodegeneration. Shawn regulates metal homeostasis, and we found in shawn mutants increased levels of manganese, calcium, and mitochondrial free iron. Mitochondrial mutants often cannot maintain synaptic transmission under demanding conditions, but shawn mutants do, and they also do not display endocytic defects. In contrast, shawn mutants harbor a significant increase in neurotransmitter release. Our work provides the first functional annotation of these essential mitochondrial carriers in the nervous system, and the results suggest that metal imbalances and mitochondrial dysfunction may contribute to defects in synaptic transmission and neuronal survival. SIGNIFICANCE STATEMENT: We describe for the first time the role of the mitochondrial carrier Shawn/SLC25A39/SLC25A40 in the nervous system. In humans, these genes reside in susceptibility loci for epilepsy, and, in flies, we observe neuronal defects related to mitochondrial dysfunction and metal homeostasis defects. Interestingly, shawn mutants also harbor increased neurotransmitter release and neurodegeneration. Our data suggest a connection between maintaining a correct metal balance and mitochondrial function to regulate neuronal survival and neurotransmitter release.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.

Drosophila rugose is a functional homolog of mammalian Neurobeachin and affects synaptic architecture, brain morphology, and associative learning.

Neurobeachin (Nbea) is implicated in vesicle trafficking in the regulatory secretory pathway, but details on its molecular function are currently unknown. We have used Drosophila melanogaster mutants for rugose (rg), the Drosophila homolog of Nbea, to further elucidate the function of this multidomain protein. Rg is expressed in a granular pattern reminiscent of the Golgi network in neuronal cell bodies and colocalizes with transgenic Nbea, suggesting a function in secretory regulation. In contrast to Nbea(-/-) mice, rg null mutants are viable and fertile and exhibit aberrant associative odor learning, changes in gross brain morphology, and synaptic architecture as determined at the larval neuromuscular junction. At the same time, basal synaptic transmission is essentially unaffected, suggesting that structural and functional aspects are separable. Rg phenotypes can be rescued by a Drosophila rg+ transgene, whereas a mouse Nbea transgene rescues aversive odor learning and synaptic architecture; it fails to rescue brain morphology and appetitive odor learning. This dissociation between the functional redundancy of either the mouse or the fly transgene suggests that their complex composition of numerous functional and highly conserved domains support independent functions. We propose that the detailed compendium of phenotypes exhibited by the Drosophila rg null mutant provided here will serve as a test bed for dissecting the different functional domains of BEACH (for beige and human Chediak-Higashi syndrome) proteins, such as Rugose, mouse Nbea, or Nbea orthologs in other species, such as human.

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency.

Human UBIAD1 localizes to mitochondria and converts vitamin K(1) to vitamin K(2). Vitamin K(2) is best known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier. Whether vitamin K(2) exerts a similar carrier function in eukaryotic cells is unknown. We identified Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects mitochondrial function. We found that vitamin K(2) was necessary and sufficient to transfer electrons in Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that were rescued by vitamin K(2), and, similar to ubiquinone, vitamin K(2) transferred electrons in Drosophila mitochondria, resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was rescued by vitamin K(2) that serves as a mitochondrial electron carrier, helping to maintain normal ATP production.

Phosphoinositides at the neuromuscular junction of Drosophila melanogaster: a genetic approach.

Phosphoinositides are critically important for numerous cellular signaling pathways such as membrane trafficking, cytoskeleton rearrangement, and ion channel regulation in eukaryotic organisms. The physiological relevance of phosphoinositide metabolism at the Drosophila neuromuscular junction (NMJ) has been illustrated using several mutants that lack crucial factors of phosphoinositide signaling. Although several decades of research in both in vitro and in vivo models have led to an understanding of the mechanisms of lipid-protein interactions and downstream signaling, the details on how their temporal and spatial distribution is regulated at the sub-cellular level in vivo remains poorly understood. To obtain a better understanding of phosphoinositide signaling, detailed biochemical and cell biological approaches can best be combined with genetics. In this review, we present an overview of the methodologies available in the fruit fly Drosophila melanogaster, to genetically dissect the complex regulation of signaling pathways involving phosphoinositides.

WASP is activated by phosphatidylinositol-4,5-bisphosphate to restrict synapse growth in a pathway parallel to bone morphogenetic protein signaling.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] is a membrane lipid involved in several signaling pathways. However, the role of this lipid in the regulation of synapse growth is ill-defined. Here we identify PI(4,5)P(2) as a gatekeeper of neuromuscular junction (NMJ) size. We show that PI(4,5)P(2) levels in neurons are critical in restricting synaptic growth by localizing and activating presynaptic Wiscott-Aldrich syndrome protein/WASP (WSP). This function of WSP is independent of bone morphogenetic protein (BMP) signaling but is dependent on Tweek, a neuronally expressed protein. Loss of PI(4,5)P(2)-mediated WSP activation results in increased formation of membrane-organizing extension spike protein (Moesin)-GFP patches that concentrate at sites of bouton growth. Based on pharmacological and genetic studies, Moesin patches mark polymerized actin accumulations and correlate well with NMJ size. We propose a model in which PI(4,5)P(2)- and WSP-mediated signaling at presynaptic termini controls actin-dependent synapse growth in a pathway at least in part in parallel to synaptic BMP signaling.