Synaptic Mitochondria in Synaptic Transmission and Organization of Vesicle Pools in Health and Disease

Cell types rich in mitochondria, including neurons, display a high energy demand and a need for calcium buffering. The importance of mitochondria for proper neuronal function is stressed by the occurrence of neurological defects in patients suffering from a great variety of diseases caused by mutations in mitochondrial genes. Genetic and pharmacological evidence also reveal a role of these organelles in various aspects of neuronal physiology and in the pathogenesis of neurodegenerative disorders. Yet the mechanisms by which mitochondria can affect neurotransmission largely remain to be elucidated. In this review we focus on experimental data that suggest a critical function of synaptic mitochondria in the function and organization of synaptic vesicle pools, and in neurotransmitter release during intense neuronal activity. We discuss how calcium handling, ATP production and other mitochondrial mechanisms may influence synaptic vesicle pool organization and synaptic function. Given the link between synaptic mitochondrial function and neuronal communication, efforts toward better understanding mitochondrial biology may lead to novel therapeutic approaches of neurological disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and psychiatric disorders that are at least in part caused by mitochondrial deficits

Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer

Lysosomal storage is the most common cause of neurodegenerative brain disease in preadulthood. However, the underlying cellular mechanisms that lead to neuronal dysfunction are unknown. Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions. At the bnch larval neuromuscular junction, we observe similar inclusions and find defects in synaptic vesicle recycling at the level of endocytosis. In addition, loss of bnch slows endosome-to-lysosome trafficking in larval garland cells. In adult bnch flies, we observe age-dependent synaptic dysfunction and neuronal degeneration. Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner. We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity

HDAC6 is a bruchpilot deacetylase that facilitates neurotransmitter release

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. Here, we show that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (ALS). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. Our work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission

Studying the direct effects of forces on embryonic stem cell behaviour

Cells experience different mechanical cues from their local environment, including shear flow, forces applied by neighbouring cells, and substrate stiffness. These external signals influence cell behaviour, also in embryonic stem (ES) cells, where they could potentially affect pluripotency or differentiation. The precise effects of external forces on ES cells are confounded by forces inducing secondary changes to attachment or cell-cell signalling, which themselves can also influence cell behaviour. In this study we developed a set-up to attach cells to elastic membranes using a novel functionalisation technique, and exposed them to single or cyclic stretch. We used this method to study the mechanosensitive response of ES cells. We found that stretching caused an immediate increase in the concentration of intracellular calcium, followed by a rapid decrease in some cells. On timescales of 1 - 2 h, stretching induced an increase in the expression of the immediate and early genes, but then cells became temporarily insensitive to subsequent mechanical signals. Stretching did not have a substantial impact on pluripotency and differentiation, as we showed using gene expression studies and a Rex1 reporter. To study how ES cells' susceptibility to mechanical signals depended on media condition, stretch duration and stretch type, we performed RNA sequencing and used gene ontology techniques to investigate the involvement of specific pathways. We found that forces have a broad impact on the overall transcriptome that is highly culture media-dependent. However, a core transcriptional response, including the biosynthesis of membrane components and stress pathways, was largely preserved across the different conditions. We supplemented our experimental findings with a conceptual model of force propagation in disordered environments, such as the nucleus of a cell. Using computational simulations, we studied how the large-scale behaviour of a disordered system depends on the microscopic structure. Contrary to common wisdom, we showed that disordered systems exhibit both positive and negative Poisson's ratios with equal probability. Overall, on short timescales, stretching affected ES cells' calcium concentration and transcription. On longer timescales, ES cells' response was small in magnitude but broad in scope, with limited effects on pluripotency. As such, our results suggest that mechanosensitivity in ES cells is mediated primarily by tissue-wide changes to morphology and attachment.EPSRC Studentship, Oliver Gatty Studentshi

Structural basis for recruitment of mitochondrial fission complexes by Fis1

Mitochondrial fission controls mitochondrial shape and physiology, including mitochondrial remodeling in apoptosis. During assembly of the yeast mitochondrial fission complex, the outer membrane protein Fis1 recruits the dynamin-related GTPase Dnm1 to mitochondria. Fis1 contains a tetratricopeptide repeat (TPR) domain and interacts with Dnm1 via the molecular adaptors Mdv1 and Caf4. By using crystallographic analysis of adaptor-Fis1 complexes, we show that these adaptors use two helices to bind to both the concave and convex surfaces of the Fis1 TPR domain. Fis1 therefore contains two interaction interfaces, a binding mode that, to our knowledge, has not been observed previously for TPR domains. Genetic and biochemical studies indicate that both binding interfaces are important for binding of Mdv1 and Caf4 to Fis1 and for mitochondrial fission activity in vivo. Our results reveal how Fis1 recruits the mitochondrial fission complex and will facilitate efforts to manipulate mitochondrial fission

Chronological requirements of TDP-43 function in synaptic organization and locomotive control.

Abstract Alterations in TDP-43 are commonly found in patients suffering from amyotrophic lateral sclerosis (ALS) and the genetic suppression of the conserved homologue in Drosophila (TBPH) provokes alterations in the functional organization of motoneuron synaptic terminals, resulting in locomotive defects and reduced life span. To gain more insight into this pathological process, it is of fundamental importance to establish when during the fly life cycle the lack of TBPH affects motoneuron activity and whether this is a reversible phenomenon. To achieve this, we conditionally expressed the endogenous protein in TBPH minus Drosophila neurons and found that TBPH is a short lived protein permanently required for Drosophila motility and synaptic assembly through the direct modulation of vesicular proteins, such as Syntaxin 1A, indicating that synaptic transmission defects are early pathological consequences of TBPH dysfunction in vivo. Importantly, TBPH late induction is able to recover synaptogenesis and locomotion in adult flies revealing an unexpected late-stage functional and structural neuronal plasticity. These observations suggest that late therapeutic approaches based on TDP-43 functionality may also be successful for the human pathology

straightjacket is required for the synaptic stabilization of cacophony, a voltage-gated calcium channel α1 subunit

In a screen to identify genes involved in synaptic function, we isolated mutations in Drosophila melanogaster straightjacket (stj), an α2δ subunit of the voltage-gated calcium channel. stj mutant photoreceptors develop normal synaptic connections but display reduced “on–off” transients in electroretinogram recordings, indicating a failure to evoke postsynaptic responses and, thus, a defect in neurotransmission. stj is expressed in neurons but excluded from glia. Mutants exhibit endogenous seizure-like activity, indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions exhibit approximately fourfold reduction in synaptic release compared with controls stemming from a reduced release probability at these synapses. These defects likely stem from destabilization of Cacophony (Cac), the primary presynaptic α1 subunit in D. melanogaster. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, indicating a role for the α2δ Ca2+ channel subunit in mediating the proper localization of an α1 subunit at synapses

Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation

Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila