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BioRxiv : the Preprint Server For... Mar 2024Glucose has long been considered the primary fuel source for the brain. However, glucose levels fluctuate in the brain during sleep, intense circuit activity, or dietary...
Glucose has long been considered the primary fuel source for the brain. However, glucose levels fluctuate in the brain during sleep, intense circuit activity, or dietary restrictions, posing significant metabolic stress. Here, we demonstrate that the mammalian brain utilizes pyruvate as a fuel source, and pyruvate can support neuronal viability in the absence of glucose. Nerve terminals are sites of metabolic vulnerability within a neuron and we show that mitochondrial pyruvate uptake is a critical step in oxidative ATP production in hippocampal terminals. We find that the mitochondrial pyruvate carrier is post-translationally modified by lysine acetylation which in turn modulates mitochondrial pyruvate uptake. Importantly, our data reveal that the mitochondrial pyruvate carrier regulates distinct steps in synaptic transmission, namely, the spatiotemporal pattern of synaptic vesicle release and the efficiency of vesicle retrieval, functions that have profound implications for synaptic plasticity. In summary, we identify pyruvate as a potent neuronal fuel and mitochondrial pyruvate uptake as a critical node for the metabolic control of synaptic transmission in hippocampal terminals.
PubMed: 38562794
DOI: 10.1101/2024.03.20.586011 -
BioRxiv : the Preprint Server For... Mar 2024One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation...
One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory is selectively impaired following the expression of a genetically encoded Rac1-inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.
PubMed: 38562715
DOI: 10.1101/2024.03.18.585488 -
Current Biology : CB Apr 2024Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron...
Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.
Topics: Spastin; Microtubules; Humans; Axons; Induced Pluripotent Stem Cells; Synaptic Vesicles; Presynaptic Terminals; Neurons; Synapses
PubMed: 38554708
DOI: 10.1016/j.cub.2024.03.010 -
Brain, Behavior, and Immunity Jul 2024Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple...
Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.
Topics: Animals; Humans; Mice; Brain; Cells, Cultured; Complement C3; Complement System Proteins; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Microglia; Neurons; Synapses; Thalamus
PubMed: 38552925
DOI: 10.1016/j.bbi.2024.03.041 -
Cell Apr 2024In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional...
In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca and via its C2A domain-mediated Ca sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.
Topics: Animals; Synaptic Vesicles; COP-Coated Vesicles; Endoplasmic Reticulum; Calcium; Golgi Apparatus; Rats; Biological Transport; Presynaptic Terminals; Synapsins; Biomolecular Condensates; Cytoskeletal Proteins; Phase Separation
PubMed: 38552623
DOI: 10.1016/j.cell.2024.03.003 -
Biomedicines Feb 2024Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural...
Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural compound with neuroprotective potential, on glutamate release in isolated nerve terminals (synaptosomes) prepared from the rat cerebral cortex were examined. In this study, 4-aminopyridine (4-AP), a potassium channel blocker, was utilized to induce the release of glutamate, which was subsequently quantified via a fluorometric assay. Our findings revealed that phillygenin reduced 4-AP-induced glutamate release, and this inhibitory effect was reversed by removing extracellular Ca or inhibiting vesicular transport with bafilomycin A1. However, exposure to the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate (dl-TOBA) did not influence the inhibitory effect. Moreover, phillygenin did not change the synaptosomal membrane potential but lowered the 4-AP-triggered increase in intrasynaptosomal Ca concentration ([Ca]). Antagonizing Ca2.2 (N-type) calcium channels blocked the inhibition of glutamate release by phillygenin, whereas pretreatment with the mitochondrial Na/Ca exchanger inhibitor, CGP37157 or the ryanodine receptor inhibitor, dantrolene, both of which block intracellular Ca release, had no effect. The effect of phillygenin on glutamate release triggered by 4-AP was completely abolished when MAPK/ERK inhibitors were applied. Furthermore, phillygenin attenuated the phosphorylation of ERK1/2 and its major presynaptic target, synapsin I, a protein associated with synaptic vesicles. These data collectively suggest that phillygenin mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca through Ca2.2 calcium channels, thereby subsequently suppressing the MAPK/ERK/synapsin I signaling cascade.
PubMed: 38540109
DOI: 10.3390/biomedicines12030495 -
Cell Reports Apr 2024Fear overgeneralization is a maladaptive response to traumatic stress that is associated with the inability to discriminate between threat and safety contexts, a...
Fear overgeneralization is a maladaptive response to traumatic stress that is associated with the inability to discriminate between threat and safety contexts, a hallmark feature of post-traumatic stress disorder (PTSD). However, the neural mechanisms underlying this deficit remain unclear. Here, we show that traumatic stress exposure impairs contextual discrimination between threat and safety contexts in the learned helplessness (LH) model. Mossy cells (MCs) in the dorsal hippocampus are suppressed in response to traumatic stress. Bidirectional manipulation of MC activity in the LH model reveals that MC inhibition is causally linked to impaired contextual discrimination. Mechanistically, MC inhibition increases the number of active granule cells in a given context, significantly overlapping context-specific ensembles. Our study demonstrates that maladaptive inhibition of MCs after traumatic stress is a substantial mechanism underlying fear overgeneralization with contextual discrimination deficit, suggesting a potential therapeutic target for cognitive symptoms of PTSD.
Topics: Animals; Dentate Gyrus; Male; Stress Disorders, Post-Traumatic; Mice; Mice, Inbred C57BL; Fear; Mossy Fibers, Hippocampal; Helplessness, Learned
PubMed: 38527063
DOI: 10.1016/j.celrep.2024.114000 -
Cell Reports Methods Apr 2024Intracellular signaling plays essential roles in various cell types. In the central nervous system, signaling cascades are strictly regulated in a spatiotemporally...
Intracellular signaling plays essential roles in various cell types. In the central nervous system, signaling cascades are strictly regulated in a spatiotemporally specific manner to govern brain function; for example, presynaptic cyclic adenosine monophosphate (cAMP) can enhance the probability of neurotransmitter release. In the last decade, channelrhodopsin-2 has been engineered for subcellular targeting using localization tags, but optogenetic tools for intracellular signaling are not well developed. Therefore, we engineered a selective presynaptic fusion tag for photoactivated adenylyl cyclase (bPAC-Syn1a) and found its high localization at presynaptic terminals. Furthermore, an all-optical electrophysiological method revealed rapid and robust short-term potentiation by bPAC-Syn1a at brain stem-amygdala synapses in acute brain slices. Additionally, bPAC-Syn1a modulated mouse immobility behavior. These results indicate that bPAC-Syn1a can manipulate presynaptic cAMP signaling in vitro and in vivo. The all-optical manipulation technique developed in this study can help further elucidate the dynamic regulation of various cellular functions.
Topics: Animals; Male; Mice; Adenylyl Cyclases; Cyclic AMP; HEK293 Cells; Mice, Inbred C57BL; Neuronal Plasticity; Optogenetics; Presynaptic Terminals; Rats
PubMed: 38521059
DOI: 10.1016/j.crmeth.2024.100740 -
Cold Spring Harbor Protocols Mar 2024Chemical synaptic transmission is an important means of neuronal communication in the nervous system. Upon the arrival of an action potential, the nerve terminal...
Chemical synaptic transmission is an important means of neuronal communication in the nervous system. Upon the arrival of an action potential, the nerve terminal experiences an influx of calcium ions, which in turn trigger the exocytosis of synaptic vesicles (SVs) and the release of neurotransmitters into the synaptic cleft. Transmitters elicit synaptic responses in the postsynaptic cell by binding to and activating specific receptors. This is followed by the recycling of SVs at presynaptic terminals. The larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic features of synaptic transmission, as well as the molecular mechanisms regulating the SV cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ is an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Here, we describe the theory and practice of electrophysiology as applied to the larval NMJ preparation. We introduce the basics of membrane potentials, with an emphasis on the resting potential and synaptic potential. We also describe the equipment and methods required to set up an electrophysiology rig.
PubMed: 38519095
DOI: 10.1101/pdb.top107820 -
Methods in Molecular Biology (Clifton,... 2024Tau protein has important physiological functions at both presynaptic and postsynaptic terminals. Pathological tau species are also associated with synaptic dysfunctions...
Tau protein has important physiological functions at both presynaptic and postsynaptic terminals. Pathological tau species are also associated with synaptic dysfunctions in several neurodegenerative disorders, especially Alzheimer's disease. To understand tau distribution inside synaptic compartments, super-resolution imaging is required. Here, we describe a facile protocol to immobilize and image brain synaptosomes without aggregation artefacts, by substituting the standard fixative paraformaldehyde with ethylene glycol bis(succinimidyl succinate) (EGS). Super-resolution imaging of tau proteins is achieved through three-color direct stochastic optical reconstruction microscopy (dSTORM). Tau protein is found to colocalize with synaptic vesicles as well as postsynaptic densities.
Topics: Humans; Synaptosomes; tau Proteins; Alzheimer Disease; Synaptic Vesicles; Brain
PubMed: 38512681
DOI: 10.1007/978-1-0716-3629-9_24