Endoplasmic Reticulum Lumenal Indicators in Drosophila Reveal Effects of HSP-Related Mutations on Endoplasmic Reticulum Calcium Dynamics

Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly

How neurons maintain their axons long-term: an integrated view of axon biology and pathology

Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell bodies and up to a century in humans. This requires self-sufficient cell biology including structural proteins, organelles, and membrane trafficking, metabolic, signalling, translational, chaperone, and degradation machinery—all maintaining the homeostasis of energy, lipids, proteins, and signalling networks including reactive oxygen species and calcium. Axon maintenance also involves specialised cytoskeleton including the cortical actin-spectrin corset, and bundles of microtubules that provide the highways for motor-driven transport of components and organelles for virtually all the above-mentioned processes. Here, we aim to provide a conceptual overview of key aspects of axon biology and physiology, and the homeostatic networks they form. This homeostasis can be derailed, causing axonopathies through processes of ageing, trauma, poisoning, inflammation or genetic mutations. To illustrate which malfunctions of organelles or cell biological processes can lead to axonopathies, we focus on axonopathy-linked subcellular defects caused by genetic mutations. Based on these descriptions and backed up by our comprehensive data mining of genes linked to neural disorders, we describe the ‘dependency cycle of local axon homeostasis’ as an integrative model to explain why very different causes can trigger very similar axonopathies, providing new ideas that can drive the quest for strategies able to battle these devastating diseases

Virtual Fly Brain—An interactive atlas of the Drosophila nervous system

As a model organism, Drosophila is uniquely placed to contribute to our understanding of how brains control complex behavior. Not only does it have complex adaptive behaviors, but also a uniquely powerful genetic toolkit, increasingly complete dense connectomic maps of the central nervous system and a rapidly growing set of transcriptomic profiles of cell types. But this also poses a challenge: Given the massive amounts of available data, how are researchers to Find, Access, Integrate and Reuse (FAIR) relevant data in order to develop an integrated anatomical and molecular picture of circuits, inform hypothesis generation, and find reagents for experiments to test these hypotheses? The Virtual Fly Brain (virtualflybrain.org) web application & API provide a solution to this problem, using FAIR principles to integrate 3D images of neurons and brain regions, connectomics, transcriptomics and reagent expression data covering the whole CNS in both larva and adult. Users can search for neurons, neuroanatomy and reagents by name, location, or connectivity, via text search, clicking on 3D images, search-by-image, and queries by type (e.g., dopaminergic neuron) or properties (e.g., synaptic input in the antennal lobe). Returned results include cross-registered 3D images that can be explored in linked 2D and 3D browsers or downloaded under open licenses, and extensive descriptions of cell types and regions curated from the literature. These solutions are potentially extensible to cover similar atlasing and data integration challenges in vertebrates

Prescriptive variability of drugs by general practitioners

<div><p>Prescription drug spending is growing faster than any other sector of healthcare. However, very little is known about patterns of prescribing and cost of prescribing between general practices. In this study, we examined variation in prescription rates and prescription costs through time for 55 GP surgeries in Northern Ireland Western Health and Social Care Trust. Temporal changes in variability of prescribing rates and costs were assessed using the Mann–Kendall test. Outlier practices contributing to between practice variation in prescribing rates were identified with the interquartile range outlier detection method. The relationship between rates and cost of prescribing was explored with Spearman's statistics. The differences in variability and mean number of prescribing rates associated with the practice setting and socioeconomic deprivation were tested using t-test and <i>F</i>-test respectively. The largest between-practice difference in prescribing rates was observed for Apr-Jun 2015, with the number of prescriptions ranging from 3.34 to 8.36 per patient. We showed that practices with outlier prescribing rates greatly contributed to between-practice variability. The largest difference in prescribing costs was reported for Apr-Jun 2014, with the prescription cost per patient ranging from £26.4 to £64.5. In addition, the temporal changes in variability of prescribing rates and costs were shown to undergo an upward trend. We demonstrated that practice setting and socio-economic deprivation accounted for some of the between-practice variation in prescribing. Rural practices had higher between practice variability than urban practices at all time points. Practices situated in more deprived areas had higher prescribing rates but lower variability than those located in less deprived areas. Further analysis is recommended to assess if variation in prescribing can be explained by demographic characteristics of patient population and practice features. Identification of other factors contributing to prescribing variability can help us better address potential inappropriateness of prescribing.</p></div

α-Synuclein impairs macroautophagy: implications for Parkinson’s disease

α-Synuclein impairs autophagosome formation and mislocalizes Atg9 by inhibiting Rab1a

Axonal Endoplasmic Reticulum Dynamics and Its Roles in Neurodegeneration

The physical continuity of axons over long cellular distances poses challenges for their maintenance. One organelle that faces this challenge is endoplasmic reticulum (ER); unlike other intracellular organelles, this forms a physically continuous network throughout the cell, with a single membrane and a single lumen. In axons, ER is mainly smooth, forming a tubular network with occasional sheets or cisternae and low amounts of rough ER. It has many potential roles: lipid biosynthesis, glucose homeostasis, a Ca2+ store, protein export, and contacting and regulating other organelles. This tubular network structure is determined by ER-shaping proteins, mutations in some of which are causative for neurodegenerative disorders such as hereditary spastic paraplegia (HSP). While axonal ER shares many features with the tubular ER network in other contexts, these features must be adapted to the long and narrow dimensions of axons. ER appears to be physically continuous throughout axons, over distances that are enormous on a subcellular scale. It is therefore a potential channel for long-distance or regional communication within neurons, independent of action potentials or physical transport of cargos, but involving its physiological roles such as Ca2+ or organelle homeostasis. Despite its apparent stability, axonal ER is highly dynamic, showing features like anterograde and retrograde transport, potentially reflecting continuous fusion and breakage of the network. Here we discuss the transport processes that must contribute to this dynamic behavior of ER. We also discuss the model that these processes underpin a homeostatic process that ensures both enough ER to maintain continuity of the network and repair breaks in it, but not too much ER that might disrupt local cellular physiology. Finally, we discuss how failure of ER organization in axons could lead to axon degenerative diseases, and how a requirement for ER continuity could make distal axons most susceptible to degeneration in conditions that disrupt ER continuity

GAL4 Drivers Specific for Type Ib and Type Is Motor Neurons in Drosophila

The Drosophila melanogaster larval neuromuscular system is extensively used by researchers to study neuronal cell biology, and Drosophila glutamatergic motor neurons have become a major model system. There are two main Types of glutamatergic motor neurons, Ib and Is, with different structural and physiological properties at synaptic level at the neuromuscular junction. To generate genetic tools to identify and manipulate motor neurons of each Type, we screened for GAL4 driver lines for this purpose. Here we describe GAL4 drivers specific for examples of neurons within each Type, Ib or Is. These drivers showed high expression levels and were expressed in only few motor neurons, making them amenable tools for specific studies of both axonal and synapse biology in identified Type I motor neurons

Endoplasmic Reticulum Lumenal Indicators in Drosophila Reveal Effects of HSP-Related Mutations on Endoplasmic Reticulum Calcium Dynamics

Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly

NeuroGeM, a knowledgebase of genetic modifiers in neurodegenerative diseases

Background: Neurodegenerative diseases (NDs) are characterized by the progressive loss of neurons in the human brain. Although the majority of NDs are sporadic, evidence is accumulating that they have a strong genetic component. Therefore, significant efforts have been made in recent years to not only identify disease-causing genes but also genes that modify the severity of NDs, so-called genetic modifiers. To date there exists no compendium that lists and cross-links genetic modifiers of different NDs. Description: In order to address this need, we present NeuroGeM, the first comprehensive knowledgebase providing integrated information on genetic modifiers of nine different NDs in the model organisms D. melanogaster, C. elegans, and S. cerevisiae. NeuroGeM cross-links curated genetic modifier information from the different NDs and provides details on experimental conditions used for modifier identification, functional annotations, links to homologous proteins and color-coded protein-protein interaction networks to visualize modifier interactions. We demonstrate how this database can be used to generate new understanding through meta-analysis. For instance, we reveal that the Drosophila genes DnaJ-1, thread, Atx2, and mub are generic modifiers that affect multiple if not all NDs. Conclusion: As the first compendium of genetic modifiers, NeuroGeM will assist experimental and computational scientists in their search for the pathophysiological mechanisms underlying NDs. http://chibi.ubc.ca/neurogem .Biochemistry and Molecular Biology, Department ofComputer Science, Department ofMedicine, Faculty ofScience, Faculty ofOther UBCNon UBCReviewedFacult