40 research outputs found

    A genetic analysis of complexin function in neurotransmitter release and synaptic plasticity

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    Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.Information transfer at neuronal synapses requires rapid fusion of docked synaptic vesicles in response to calcium influx during action potentials. The molecular nature of the fusion clamp machinery that prevents exocytosis of synaptic vesicles in the absence of a calcium signal is still unclear. Here we show that complexin, a small alpha-helical protein that binds fully assembled SNARE complexes, functions as the synaptic vesicle fusion clamp in vivo. Drosophila has a single complexin homolog that is abundantly expressed in presynaptic nerve terminals. Animals lacking complexin die throughout development, with adult escapers showing severe locomotion defects and a loss of visual function. Electrophysiological analysis at neuromuscular junctions in complexin null mutants reveals a dramatic increase in spontaneous synaptic vesicle fusion that is independent of nerve stimulation or extracellular calcium. High frequency stimulation at high calcium concentrations shows that the readily releasable pool in complexin mutants is severely depleted. Thus, complexin is required for maintenance of the readily releasable pool of vesicles at the synapse, and without it vesicles exocytose directly after priming. These data indicate that complexin interacts with assembled SNARE complexes to prevent premature vesicle fusion in the absence of calcium entry. In addition, a preliminary analysis of synaptotagmin 1; complexin double mutants reveals that the elevated mini frequency in complexin single mutants is dependent on synaptotagmin 1. This finding suggests that the dominant function of complexin at the synapse is to prevent synaptotagmin 1 from triggering fusion in the absence of calcium. Further analysis of synaptotagmin 1; complexin double mutants may reveal new aspects of the mechanism of the calcium-regulated vesicle fusion reaction. Minis have long been thought to represent background noise at the synapse, but there is now growing evidence that mini frequency is important in synaptic maintenance and plasticity. Complexin mutants display a substantial synaptic overgrowth phenotype. We hypothesized that the enhanced mini frequency in complexin mutants drives synaptic overgrowth and that complexin is phosphorylated by PKA to regulate mini frequency at Drosophila synapses in an activity-dependent retrograde signaling pathway that mediates a large increase in mini frequency and a concomitant induction of synaptic growth. Like complexin mutants, a syntaxin mutant with elevated mini frequency also displays enhanced synaptic growth, providing further evidence that an increase in mini frequency drives synaptic plasticity. S126 in complexin is phosphorylated by PKA in vitro. Future results may reveal that S126 is phosphorylated by PKA in vivo to regulate mini frequency in an activity-dependent manner. These results have the potential to reveal a new role for minis in local synaptic plasticity in response to neuronal activity.by Sarah Huntwork-Rodriguez.Ph.D

    Injured neurons pump up the volume

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    LRRK2 inhibition by BIIB122 in healthy participants and patients with Parkinson's disease

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    Background: Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising therapeutic approach for the treatment of Parkinson's disease (PD).Objective: The aim of this study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the potent, selective, CNS-penetrant LRRK2 inhibitor BIIB122 (DNL151) in healthy participants and patients with PD.Methods: Two randomized, double-blind, placebo-controlled studies were completed. The phase 1 study (DNLI-C-0001) evaluated single and multiple doses of BIIB122 for up to 28 days in healthy participants. The phase 1b study (DNLI-C-0003) evaluated BIIB122 for 28 days in patients with mild to moderate PD. The primary objectives were to investigate the safety, tolerability, and plasma pharmacokinetics of BIIB122. Pharmacodynamic outcomes included peripheral and central target inhibition and lysosomal pathway engagement biomarkers.Results: A total of 186/184 healthy participants (146/145 BIIB122, 40/39 placebo) and 36/36 patients (26/26 BIIB122, 10/10 placebo) were randomized/treated in the phase 1 and phase 1b studies, respectively. In both studies, BIIB122 was generally well tolerated; no serious adverse events were reported, and the majority of treatment-emergent adverse events were mild. BIIB122 cerebrospinal fluid/unbound plasma concentration ratio was similar to 1 (range, 0.7-1.8). Dose-dependent median reductions from baseline were observed in whole-blood phosphorylated serine 935 LRRK2 (<= 98%), peripheral blood mononuclear cell phosphorylated threonine 73 pRab10 (<= 93%), cerebrospinal fluid total LRRK2 (<= 50%), and urine bis (monoacylglycerol) phosphate (<= 74%).Conclusions: At generally safe and well-tolerated doses, BIIB122 achieved substantial peripheral LRRK2 kinase inhibition and modulation of lysosomal pathways downstream of LRRK2, with evidence of CNS distribution and target inhibition. These studies support continued investigation of LRRK2 inhibition with BIIB122 for the treatment of PD. (c) 2023 Denali Therapeutics Inc and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.</p

    The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration

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    Complexin Controls Spontaneous and Evoked Neurotransmitter Release by Regulating the Timing and Properties of Synaptotagmin Activity

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    Neurotransmitter release following synaptic vesicle (SV) fusion is the fundamental mechanism for neuronal communication. Synaptic exocytosis is a specialized form of intercellular communication that shares a common SNARE-mediated fusion mechanism with other membrane trafficking pathways. The regulation of synaptic vesicle fusion kinetics and short-term plasticity is critical for rapid encoding and transmission of signals across synapses. Several families of SNARE-binding proteins have evolved to regulate synaptic exocytosis, including Synaptotagmin (SYT) and Complexin (CPX). Here, we demonstrate that Drosophila CPX controls evoked fusion occurring via the synchronous and asynchronous pathways. cpx[superscript −/−] mutants show increased asynchronous release, while CPX overexpression largely eliminates the asynchronous component of fusion. We also find that SYT and CPX coregulate the kinetics and Ca[superscript 2+] co-operativity of neurotransmitter release. CPX functions as a positive regulator of release in part by coupling the Ca[superscript 2+] sensor SYT to the fusion machinery and synchronizing its activity to speed fusion. In contrast, syt[superscript −/−]; cpx[superscript −/−] double mutants completely abolish the enhanced spontaneous release observe in cpx[superscript −/−] mutants alone, indicating CPX acts as a fusion clamp to block premature exocytosis in part by preventing inappropriate activation of the SNARE machinery by SYT. CPX levels also control the size of synaptic vesicle pools, including the immediate releasable pool and the ready releasable pool—key elements of short-term plasticity that define the ability of synapses to sustain responses during burst firing. These observations indicate CPX regulates both spontaneous and evoked fusion by modulating the timing and properties of SYT activation during the synaptic vesicle cycle.National Institutes of Health (U.S.) (Grant NS40296)Pew Charitable Trusts. Pew Latin American Fellows Program in the Biomedical Science

    Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of complexin

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    Complexins are small α-helical proteins that modulate neurotransmitter release by binding to SNARE complexes during synaptic vesicle exocytosis. They have been found to function as fusion clamps to inhibit spontaneous synaptic vesicle fusion in the absence of Ca[superscript 2+], while also promoting evoked neurotransmitter release following an action potential. Complexins consist of an N-terminal domain and an accessory α-helix that regulates the activating and inhibitory properties of the protein, respectively, and a central α-helix that binds the SNARE complex and is essential for both functions. In addition, complexins contain a largely unstructured C-terminal domain whose role in synaptic vesicle cycling is poorly defined. Here, we demonstrate that the C-terminus of Drosophila complexin (DmCpx) regulates localization to synapses and that alternative splicing of the C-terminus can differentially regulate spontaneous and evoked neurotransmitter release. Characterization of the single DmCpx gene by mRNA analysis revealed expression of two alternatively expressed isoforms, DmCpx7A and DmCpx7B, which encode proteins with different C-termini that contain or lack a membrane tethering prenylation domain. The predominant isoform, DmCpx7A, is further modified by RNA editing within this C-terminal region. Functional analysis of the splice isoforms showed that both are similarly localized to synaptic boutons at larval neuromuscular junctions, but have differential effects on the regulation of evoked and spontaneous fusion. These data indicate that the C-terminus of Drosophila complexin regulates both spontaneous and evoked release through separate mechanisms and that alternative splicing generates isoforms with distinct effects on the two major modes of synaptic vesicle fusion at synapses.Pew Charitable Trusts. Latin American Fellow Program in Biomedical ScienceNational Institutes of Health (U.S.) (Grant NS40296

    A leucine‐rich repeat kinase 2 (LRRK2) pathway biomarker characterization study in patients with Parkinson's disease with and without LRRK2 mutations and healthy controls

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    Abstract Increased leucine‐rich repeat kinase 2 (LRRK2) kinase activity is an established risk factor for Parkinson's disease (PD), and several LRRK2 kinase inhibitors are in clinical development as potential novel disease‐modifying therapeutics. This biomarker characterization study explored within‐ and between‐subject variability of multiple LRRK2 pathway biomarkers (total LRRK2 [tLRRK2], phosphorylation of the serine 935 (Ser935) residue on LRRK2 [pS935], phosphorylation of Rab10 [pRab10], and total Rab10 [tRab10]) in different biological sources (whole blood, peripheral blood mononuclear cells [PBMCs], neutrophils) as candidate human target engagement and pharmacodynamic biomarkers for implementation in phase I/II pharmacological studies of LRRK2 inhibitors. PD patients with a LRRK2 mutation (n = 6), idiopathic PD patients (n = 6), and healthy matched control subjects (n = 10) were recruited for repeated blood and cerebrospinal fluid (CSF) sampling split over 2 days. Within‐subject variability (geometric coefficient of variation [CV], %) of these biomarkers was lowest in whole blood and neutrophils (range: 12.64%–51.32%) and considerably higher in PBMCs (range: 34.81%–273.88%). Between‐subject variability displayed a similar pattern, with relatively lower variability in neutrophils (range: 61.30%–66.26%) and whole blood (range: 44.94%–123.11%), and considerably higher variability in PBMCs (range: 189.60%–415.19%). Group‐level differences were observed with elevated mean pRab10 levels in neutrophils and a reduced mean pS935/tLRRK2 ratio in PBMCs in PD LRRK2‐mutation carriers compared to healthy controls. These findings suggest that the evaluated biomarkers and assays could be used to verify pharmacological mechanisms of action and help explore the dose–response of LRRK2 inhibitors in early‐phase clinical studies. In addition, comparable α‐synuclein aggregation in CSF was observed in LRRK2‐mutation carriers compared to idiopathic PD patients
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