Synaptic actions of amyotrophic-lateral-sclerosis-associated G85R-SOD1 in the squid giant synapse

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Song, Y. Synaptic actions of amyotrophic-lateral-sclerosis-associated G85R-SOD1 in the squid giant synapse. Eneuro, (2020): ENEURO.0369-19.2020, doi: 10.1523/ENEURO.0369-19.2020.Altered synaptic function is thought to play a role in many neurodegenerative diseases, but little is known about the underlying mechanisms for synaptic dysfunction. The squid giant synapse (SGS) is a classical model for studying synaptic electrophysiology and ultrastructure, as well as molecular mechanisms of neurotransmission. Here, we conduct a multidisciplinary study of synaptic actions of misfolded human G85R-SOD1 causing familial Amyotrophic Lateral Sclerosis (fALS). G85R-SOD1, but not WT-SOD1, inhibited synaptic transmission, altered presynaptic ultrastructure, and reduced both the size of the Readily Releasable Pool (RRP) of synaptic vesicles and mobility from the Reserved Pool (RP) to the RRP. Unexpectedly, intermittent high frequency stimulation (iHFS) blocked inhibitory effects of G85R-SOD1 on synaptic transmission, suggesting aberrant Ca2+ signaling may underlie G85R-SOD1 toxicity. Ratiometric Ca2+ imaging showed significantly increased presynaptic Ca2+ induced by G85R-SOD1 that preceded synaptic dysfunction. Chelating Ca2+ using EGTA prevented synaptic inhibition by G85R-SOD1, confirming the role of aberrant Ca2+ in mediating G85R-SOD1 toxicity. These results extended earlier findings in mammalian motor neurons and advanced our understanding by providing possible molecular mechanisms and therapeutic targets for synaptic dysfunctions in ALS as well as a unique model for further studies.Grass Foundation, HHMI, MGH Jack Satter Foundation, Harvard University ALS and Alzheimer's Endowed Research Fund, Harvard Brain Science Initiative

Simulation of neurotransmitter transport in a presynaptic bouton of a neuron

In this paper the results of numerical simulation of neuro- transmitter fast transport in a presynaptic bouton of a biological neuron are presented. A mathematical model governing the transport that is fully described in [2] is recalled. A numerical simulation scheme, used parameters and their origins are described. Finally, the results of the simulation are presented

Synaptic Vesicles, Mitochondria, and Actin Alterations in SMN-deficient Mice

Proximale Spinale Muskel Atrophie (SMA) ist eine autosomal rezessive Krankheit, charakterisiert durch eine Degeneration des zweiten Motorneurons und einer progressiven Paralyse und Atrophie proximaler Muskeln. Nach Zystischer Fibrose, ist SMA die häufigste autosomal rezessive Erkrankung bei Menschen und der häufigste genetische Grund für Säuglingssterblichkeit. SMA, monogenetisch im Ursprung, ist verursacht durch eine Mutation in einem einzelnen Gen, dem Survival Motor Neuron 1 (SMN1) Gen, was zu einer reduzierten Menge an Survival Motor Neuron (SMN) protein führt. SMN ist ein ubiquitär expremiertes Protein mit house-keeping Funktion in snRNP Biogenese und pre-mRNA splicen. Dennoch, eine reduzierte Menge an SMN beeinträchtigt vor allem Motor Neurone und Muskeln aus bisher unverständlichen Gründen. Es wurde demonstriert, dass SMN mit ß-actin mRNA interagiert und an dessen Transport entlang des Axons beteiligt ist. Funktionelle Studien an der Neuromuskulären Synapse (NMJ) haben gezeigt, dass Evozierte Neurotransmitterfreisetzung um 55 % reduziert war in den meist betroffenen Muskelgruppen, dies indiziert, dass eine verringerte Menge an Vesikeln fusioniert, währenddessen asynchrone Transmitterfreisetzung um 300 % erhöht ist aufgrund von einer abnormalen Akkumulation von Calcium in der Nervenendigung in SMA Mäusen. Eine Mögliche Erklärung für diese Calcium Erhöhung ist eine herabgesetzte Calcium Aufnahme durch Mitochondrien während Serien von Aktions Potenzialen. Diese Studie präsentiert eine umfassende Analyse mit einem Fluoreszens Konfokal Mikroskop über die Organisation und Fülle Synaptischer Vesikel (SVs), Mitochonrien und Aktin in Nervenendigungen von SMA Mäusen ( Smn -/-; SMN2; SMNdelta7). Wir visualisierten Synaptische Vesikel mit einem Antikörper gegen den Acetylcholin ( VACht) und konnten zeigen, dass im Transversus Abdominis (TVA) Muskel SV Klusters während des Reifungsprozesse klein verbleiben mit einer Reduzierung von 50% der totalen Fläche die von SVs bedeckt ist. Diese schwere Reduktion von SVs wurde auch im der kaudalen Muskelstrang des Levator auris longus (LAL) Muskel gefunden, obwohl nur leichte Veränderungen in der Postsynapse dieses Muskels festzustellen sind. Diese Ergebniss von Präsynaptischer Pathologie, neben fast normalen postsynaptischen Status, verstärkt die Hypothese dass SMN-induzierte Veränderungen im Muskel nicht auschließlich eine reine Konsequenz von Motor Neuron Degeneration sein können. Als Nächstes, haben wir Mitochondria mit Mitotracker angefärbt und haben gefunden, dass die Fläche,die von Mitochondrien in Mutanten Mäusen bedeckt ist, etwa nur die Hälfte der Fläche im Wild typ beträgt. Überraschenderweise waren SVs und Mitochondrien stak kolokalisiert. In vielen Fällen war ein Kern von Mitochondrien deutlich umgeben von einem Ring aus SVs. Diese Verteilung war unbeeinträchtig in der Mutanten Maus und könnte eine mehr generelle Bedeutung in Nervenendigungen haben. Phalloidin gefärbtes Aktin zeigte das F-aktin ringförmige Strukturen um SV Klusters formt. Diese Strukturen und der Prozentuale Anteil der Nervenendigung der von Aktin bedeckt ist, war geringer in SMA Mutanten Mäusen. Aktin ist an multiblen Schritten des Vesikel Zyklus beteiligt. Kurz Strecken-Transport von Vesikeln und Organellen, wie Mitochondrien in Wachstunskegeln und Nervendigungen ist vor allem vom aktin-myosin-basierten Transport abhängig. Weitere Arbeit ist notwendig um zu klären ob die Charakteristiken des SMA Phänotyps wie abnormales SV Klustering, Reduktion von Mitochondrien, unabhängig auftreten oder eine gemeinsame Konsequenz von einer Dysfunktion des Aktin Zytoskeleton sind, was Aktin eine Schlüsselrolle in der SMA Pathogenese verleihen würde

Investigation of biogenesis of the presynaptic compartments using human iPSC-derived neurons

Synapse formation starts during development as axons establish contacts with dendrites and neuronal cell bodies. Having a single elongated axon and multiple dendrites derived from the cell body attribute neurons an exceptional polarity, resulting in synapses being located distant to the cell body where most protein synthesis takes place. Thus, to ensure stoichiometric assembly of functional presynaptic units, components of the presynaptic compartment, synaptic vesicle (SV) and active zone (AZ) proteins as well as synaptic cell adhesion molecules (sCAMs), need to be delivered to nascent synapses in a coordinated manner. Mechanisms regulating the efficient delivery of presynaptic proteins to axon terminals as well as the cellular identity of presynaptic transport organelles are incompletely understood. This thesis therefore focused on elucidating the axonal transport machinery of presynaptic protein carrying vesicles, referred to as precursor vesicles (PVs) in developing neurons. During the course of this thesis I established, that PVs transport not only SV proteins but also AZ proteins and the sCAM protein Neurexin 1β collectively to the nascent presynapse in developing human iPSC-derived neurons. Anterograde trafficking of PVs is driven by the kinesin motor protein KIF1A and is regulated by the lysosomal small GTPase Arl8. Double loss of Arl8a and Arl8b in human neurons results in a significantly decreased number of anterogradely moving PVs in developing neurons and, consequently, a drastic loss in presynaptic protein levels at mature synapses. Interestingly, despite being regulated by Arl8, PVs are distinct from mature degradative lysosomes: They are non-acidic, do not harbor cathepsin activity, and use a transport machinery that is distinct from the classical KIF5B-dependent lysosomal transport machinery. Live imaging and biochemical experiments indicate that PVs presumably originate from the endolysosomal system, as they have a specific PI(3,5)P2 lipid identity which is recognized by the PH domain of KIF1A. Arl8 and PI(3,5)P2 provide a coincidence detection mechanism for KIF1A to drive PV transport by interacting directly with Arl8 via its CC3 domain and with PI(3,5)P2 through its lipid binding PH domain. PV transport is further regulated by a multi-subunit complex named BORC, which regulates lysosomal motility upstream of Arl8. BORC activity can mediate PV transport independent of Arl8 by negatively regulating PI(3,5)P2 levels. Taken together, my data unravels the lipid identity of presynaptic transport organelles and sheds light on their biogenesis and the mechanisms that underlying the regulation of the machinery for PV transport

Molecular mechanisms of protein sorting in the synaptic vesicle life cycle.

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported along the axons in precursor vesicles which undergo cycles of exo-endocytosis in transit to either the distal growth cone or the nerve terminal. I sought to investigate the mechanisms underlying the sorting of SV components and to determine whether and how SV exocytosis is regulated prior to synaptogenesis. SV fusion is underlain by the interaction of vesicle-associated membrane protein (VAMP) 2 with plasma membrane partners. Fluorescence resonance energy transfer analysis reveals that VAMP2 interacts with Synaptophysin I (SypI), a major resident of SVs, on the SV membrane and that exocytotic stimuli cause dissociation of this complex at a stage preceding SV fusion. This observation is consistent with the idea that SypI limits VAMP2 availability for fusogenic complexes. The interaction between SypI and VAMP2 occurs early along the exocytotic pathway and is required in order for SypI to govern the sorting of VAMP2 to SVs. The control of VAMP2 sorting by its negative regulator SypI establishes a mechanism which prevents the fusion at inappropriate sites of SVs directed to the nerve terminal. The study of SV dynamics in developing hippocampal neurons reveals that cAMP-dependent pathways affect SV distribution and recycling in the axonal growth cone and that these effects are mediated by the SV-associated phosphoprotein synapsin I. Synapsin I and its phosphorylation by cAMP-dependent protein kinase A play a pivotal role in regulating SV organization and dynamics in neuronal growth cones and in determining the formation of synaptic contacts. These results provide new clues as to the bases of the well known activity of synapsin I in synapse maturation and indicate that molecular mechanisms similar to those operating at mature nerve terminals are active in developing neurons to regulate the SV life cycle prior to synaptogenesis

Restraint of the Wallenda/DLK MAP Kinase cascade by the Kinesin-3 motor regulates the assembly of synapses

Synaptic connections are fundamental units of neuronal communication in the brain. They are composed of precisely opposed pre- and postsynaptic specializations, and these structures are dynamically regulated to adapt to changing needs of neuronal circuits. While mechanisms that regulate the postsynaptic composition of synapses are highly studied, less is known about presynaptic regulation. Within presynaptic terminals, synapse assembly requires the formation of active zones (AZs) and synaptic vesicle (SV) release machinery at synapses. An important role in presynaptic assembly has been assigned to a kinesin-3 family member, Unc-104/Imac/KIF1A. Unc-104/Imac/KIF1A is required for the delivery of synaptic components and SVs to nascent synapses. However, its distinct synaptic phenotype from other kinesins and the complexity of the phenotype is not well understood. This thesis work describes how the synaptic defects of Drosophila unc-104 mutants can be rescued by inhibiting the Wallenda (Wnd)/DLK MAP kinase signaling pathway. This pathway has been previously identified as a regulator of axonal damage signaling and presynaptic terminal morphology. The accessible genetic tools in Drosophila (reviewed in Chapter II) allow for characterization of the mechanistic relationship between Wnd/DLK and Unc-104. Wnd/DLK signaling becomes activated in unc-104 mutants, and inhibits synapse formation independently of Unc-104’s transport functions by controlling the levels and timing of the expression of AZ and SV components (Chapter III). In order to understand the activation mechanism of Wnd signaling, multiple possibilities have been examined (Chapter IV). Cumulative findings lead to a model that accumulated presynaptic proteins in the cell body of unc-104 mutants triggers the Wnd signaling pathway, which then down-regulates presynaptic protein levels. In this fashion Wnd signaling may function as a stress response pathway that regulates the expression level of synaptic proteins according to their ability to be transported in axons. This model also raises an interesting possibility that DLK activation may contribute to synapse malfunction and loss in the aged or diseased nervous system.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/137169/1/jiaxing_1.pd

Characterizing Neurotransmitter Receptor Activation with a Perturbation Based Decomposition Method

The characterization of postsynaptic potentials, in terms of neurotransmitter receptor activation, is of clinical significance because information associated with receptor activation can be used in the diagnosis and study of neurological disorders. Single-unit recordings provide a method of measuring postsynaptic potentials in neurons using a microelectrode system, but yield no detailed information regarding the neurotransmitter receptors that contribute to the potential. To determine the types of neurotransmitter receptors that result in a compound postsynaptic potential from a microelectrode reading, decomposition of the potential is necessary. In this work, a perturbation-based decomposition method developed by R. Szlavik is evaluated for this application, and compared to a generalized Fourier series approach. The resultant estimator is valid for decomposition of multiple-receptor compound postsynaptic potentials as well as single-receptor compound postsynaptic potentials. The estimator also yields a satisfactory decomposition of experimental postsynaptic potential data found in the literature