Metabolic regulation of single synaptic vesicle exo- and endocytosis in hippocampal synapses.

Glucose has long been considered a primary energy source for synaptic function. However, it remains unclear to what extent alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in hippocampal synapses, we find that mitochondrial ATP production regulates basal vesicle release probability and release location within the active zone (AZ), evoked by single action potentials. Mitochondrial inhibition shifts vesicle release closer to the AZ center and alters the efficiency of vesicle retrieval by increasing the occurrence of ultrafast endocytosis. Furthermore, we uncover that terminals can use oxidative fuels to maintain the vesicle cycle during trains of activity. Mitochondria are sparsely distributed along hippocampal axons, and we find that terminals containing mitochondria display enhanced vesicle release and reuptake during high-frequency trains. Our findings suggest that mitochondria not only regulate several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Mitochondrial pyruvate transport regulates presynaptic metabolism and neurotransmission.

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.

Circuit-based intervention corrects excessive dentate gyrus output in the fragile X mouse model.

Abnormal cellular and circuit excitability is believed to drive many core phenotypes in fragile X syndrome (FXS). The dentate gyrus is a brain area performing critical computations essential for learning and memory. However, little is known about dentate circuit defects and their mechanisms in FXS. Understanding dentate circuit dysfunction in FXS has been complicated by the presence of two types of excitatory neurons, the granule cells and mossy cells. Here we report that loss of FMRP markedly decreased excitability of dentate mossy cells, a change opposite to all other known excitability defects in excitatory neurons in FXS. This mossy cell hypo-excitability is caused by increased Kv7 function in Fmr1 knockout (KO) mice. By reducing the excitatory drive onto local hilar interneurons, hypo-excitability of mossy cells results in increased excitation/inhibition ratio in granule cells and thus paradoxically leads to excessive dentate output. Circuit-wide inhibition of Kv7 channels in Fmr1 KO mice increases inhibitory drive onto granule cells and normalizes the dentate output in response to physiologically relevant theta-gamma coupling stimulation. Our study suggests that circuit-based interventions may provide a promising strategy in this disorder to bypass irreconcilable excitability defects in different cell types and restore their pathophysiological consequences at the circuit level.

Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

Neurotransmission is an energetically expensive process that underlies cognition. During intense electrical activity or dietary restrictions, the glucose level in the brain plummets, forcing neurons to utilize alternative fuels. However, the molecular mechanisms of neuronal metabolic plasticity remain poorly understood. Here, we demonstrate that glucose-deprived neurons activate the CREB and PGC1α transcriptional program, which induces expression of the mitochondrial deacetylase Sirtuin 3 (Sirt3) both in vitro and in vivo. We show that Sirt3 localizes to axonal mitochondria and stimulates mitochondrial oxidative capacity in hippocampal nerve terminals. Sirt3 plays an essential role in sustaining synaptic transmission in the absence of glucose by providing metabolic support for the retrieval of synaptic vesicles after release. These results demonstrate that the transcriptional induction of Sirt3 facilitates the metabolic plasticity of synaptic transmission.

Metabolic Regulation of Single Synaptic Vesicle Exo- and Endocytosis in Hippocampal Synapses.

Glucose has long been considered a primary source of energy for synaptic function. However, it remains unclear under what conditions alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in cultured hippocampal synapses, we found that mitochondrial ATP production from oxidation of lactate/pyruvate regulates basal vesicle release probability and release location within the active zone (AZ) evoked by single action potentials (APs). Mitochondrial inhibition shifted vesicle release closer to the AZ center, suggesting that the energetic barrier for vesicle release is lower in the AZ center that the periphery. Mitochondrial inhibition also altered the efficiency of single AP evoked vesicle retrieval by increasing occurrence of ultrafast endocytosis, while inhibition of glycolysis had no effect. Mitochondria are sparsely distributed along hippocampal axons and we found that nerve terminals containing mitochondria displayed enhanced vesicle release and reuptake during high-frequency trains, irrespective of whether neurons were supplied with glucose or lactate. Thus, synaptic terminals can entirely bypass glycolysis to robustly maintain the vesicle cycle using oxidative fuels in the absence of glucose. These observations further suggest that mitochondrial metabolic function not only regulates several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Circuit-based intervention corrects excessive dentate gyrus output in the Fragile X mouse model.

Abnormal cellular and circuit excitability is believed to drive many core phenotypes in fragile X syndrome (FXS). The dentate gyrus is a brain area performing critical computations essential for learning and memory. However, little is known about dentate circuit defects and their mechanisms in FXS. Understanding dentate circuit dysfunction in FXS has been complicated by the presence of two types of excitatory neurons, the granule cells and mossy cells. Here we report that loss of FMRP markedly decreased excitability of dentate mossy cells, a change opposite to all other known excitability defects in excitatory neurons in FXS. This mossy cell hypo-excitability is caused by increased Kv7 function in Fmr1 KO mice. By reducing the excitatory drive onto local hilar interneurons, hypo-excitability of mossy cells results in increased excitation/inhibition ratio in granule cells and thus paradoxically leads to excessive dentate output. Circuit-wide inhibition of Kv7 channels in Fmr1 KO mice increases inhibitory drive onto granule cells and normalizes the dentate output in response to physiologically relevant theta-gamma coupling stimulation. Our study suggests that circuit-based interventions may provide a promising strategy in this disorder to bypass irreconcilable excitability defects in different cell types and restore their pathophysiological consequences at the circuit level.

Two forms of asynchronous release with distinctive spatiotemporal dynamics in central synapses.

Asynchronous release is a ubiquitous form of neurotransmitter release that persists for tens to hundreds of milliseconds after an action potential. How asynchronous release is organized and regulated at the synaptic active zone (AZ) remains debatable. Using nanoscale-precision imaging of individual release events in rat hippocampal synapses, we observed two spatially distinct subpopulations of asynchronous events, ~75% of which occurred inside the AZ and with a bias towards the AZ center, while ~25% occurred outside of the functionally defined AZ, that is, ectopically. The two asynchronous event subpopulations also differed from each other in temporal properties, with ectopic events occurring at significantly longer time intervals from synchronous events than the asynchronous events inside the AZ. Both forms of asynchronous release did not, to a large extent, utilize the same release sites as synchronous events. The two asynchronous event subpopulations also differ from synchronous events in some aspects of exo-endocytosis coupling, particularly in the contribution from the fast calcium-dependent endocytosis. These results identify two subpopulations of asynchronous release events with distinctive organization and spatiotemporal dynamics.

Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

Neurotransmission is an energetically expensive process that underlies cognition. During intense electrical activity or dietary restrictions, glucose levels in the brain plummet, forcing neurons to utilize alternative fuels. However, the molecular mechanisms of neuronal metabolic plasticity remain poorly understood. Here, we demonstrate that glucose-deprived neurons activate the CREB and PGC1α transcriptional program that induces the expression of the mitochondrial deacetylase Sirtuin 3 (Sirt3) both in vitro and in vivo . We show that Sirt3 localizes to axonal mitochondria and stimulates mitochondrial oxidative capacity in hippocampal nerve terminals. Sirt3 plays an essential role in sustaining synaptic transmission in the absence of glucose by powering the retrieval of synaptic vesicles after release. These results demonstrate that the transcriptional induction of Sirt3 ensures the metabolic plasticity of synaptic transmission. Highlights: Glucose deprivation drives transcriptional reprogramming of neuronal metabolism via CREB and PGC1α. Glucose or food deprivation trigger the neuronal expression of mitochondrial deacetylase sirtuin 3 (Sirt3) both in vitro and in vivo . Sirt3 stimulates oxidative ATP synthesis in nerve terminals.Sirt3 sustains the synaptic vesicle cycle in the absence of glucose.

Rapid astrocyte-dependent facilitation amplifies multi-vesicular release in hippocampal synapses.

Synaptic facilitation is a major form of short-term plasticity typically driven by an increase in residual presynaptic calcium. Using near-total internal reflection fluorescence (near-TIRF) imaging of single vesicle release in cultured hippocampal synapses, we demonstrate a distinctive, release-dependent form of facilitation in which probability of vesicle release is higher following a successful glutamate release event than following a failure. This phenomenon has an onset of ≤500 ms and lasts several seconds, resulting in clusters of successful release events. The release-dependent facilitation requires neuronal contact with astrocytes and astrocytic glutamate uptake by EAAT1. It is not observed in neurons grown alone or in the presence of astrocyte-conditioned media. This form of facilitation dynamically amplifies multi-vesicular release. Facilitation-evoked release events exhibit spatial clustering and have a preferential localization toward the active zone center. These results uncover a rapid astrocyte-dependent form of facilitation acting via modulation of multi-vesicular release and displaying distinctive spatiotemporal properties.

FMRP regulates GABAA receptor channel activity to control signal integration in hippocampal granule cells.

Fragile X syndrome, the most common inherited form of intellectual disability, is caused by loss of fragile X mental retardation protein (FMRP). GABAergic system dysfunction is one of the hallmarks of FXS, yet the underlying mechanisms remain poorly understood. Here, we report that FMRP interacts with GABAA receptor (GABAAR) and modulates its single-channel activity. Specifically, FMRP regulates spontaneous GABAAR opening through modulating its single-channel conductance and open probability in dentate granule cells. FMRP loss reduces spontaneous GABAAR activity underlying tonic inhibition, while N-terminal FMRP fragment (aa 1-297) is sufficient to rapidly normalize tonic inhibition in Fmr1 knockout (KO) granule cells. FMRP-GABAAR interaction is supported by co-immunoprecipitation of FMRP with at least one GABAAR subunit, the α5. Functionally, FMRP-GABAAR interaction ensures accuracy of coincidence detection of granule cells, which is markedly reduced in Fmr1 KOs. Our study reveals a mechanism underlying FMRP regulation of the GABAergic system and information processing in the hippocampus.

Myosin V Regulates Spatial Localization of Different Forms of Neurotransmitter Release in Central Synapses.

Synaptic active zone (AZ) contains multiple specialized release sites for vesicle fusion. The utilization of release sites is regulated to determine spatiotemporal organization of the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR). We previously found that the vesicle-associated molecular motor myosin V regulates temporal utilization of release sites by controlling vesicle anchoring at release sites in an activity-dependent manner. Here we show that acute inhibition of myosin V shifts preferential location of vesicle docking away from AZ center toward periphery, and results in a corresponding spatial shift in utilization of release sites during UVR. Similarly, inhibition of myosin V also reduces preferential utilization of central release sites during MVR, leading to more spatially distributed and temporally uniform MVR that occurs farther away from the AZ center. Using a modeling approach, we provide a conceptual framework that unites spatial and temporal functions of myosin V in vesicle release by controlling the gradient of release site release probability across the AZ, which in turn determines the spatiotemporal organization of both UVR and MVR. Thus myosin V regulates both temporal and spatial utilization of release sites during two main forms of synchronous release.

Channelopathies in fragile X syndrome.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and the leading monogenic cause of autism. The condition stems from loss of fragile X mental retardation protein (FMRP), which regulates a wide range of ion channels via translational control, protein-protein interactions and second messenger pathways. Rapidly increasing evidence demonstrates that loss of FMRP leads to numerous ion channel dysfunctions (that is, channelopathies), which in turn contribute significantly to FXS pathophysiology. Consistent with this, pharmacological or genetic interventions that target dysregulated ion channels effectively restore neuronal excitability, synaptic function and behavioural phenotypes in FXS animal models. Recent studies further support a role for direct and rapid FMRP-channel interactions in regulating ion channel function. This Review lays out the current state of knowledge in the field regarding channelopathies and the pathogenesis of FXS, including promising therapeutic implications.

Disrupted Association of Sensory Neurons With Enveloping Satellite Glial Cells in Fragile X Mouse Model.

Among most prevalent deficits in individuals with Fragile X syndrome (FXS) is hypersensitivity to sensory stimuli and somatosensory alterations. Whether dysfunction in peripheral sensory system contributes to these deficits remains poorly understood. Satellite glial cells (SGCs), which envelop sensory neuron soma, play critical roles in regulating neuronal function and excitability. The potential contributions of SGCs to sensory deficits in FXS remain unexplored. Here we found major structural defects in sensory neuron-SGC association in the dorsal root ganglia (DRG), manifested by aberrant covering of the neuron and gaps between SGCs and the neuron along their contact surface. Single-cell RNAseq analyses demonstrated transcriptional changes in both neurons and SGCs, indicative of defects in neuronal maturation and altered SGC vesicular secretion. We validated these changes using fluorescence microscopy, qPCR, and high-resolution transmission electron microscopy (TEM) in combination with computational analyses using deep learning networks. These results revealed a disrupted neuron-glia association at the structural and functional levels. Given the well-established role for SGCs in regulating sensory neuron function, altered neuron-glia association may contribute to sensory deficits in FXS.

Hyperexcitability of Sensory Neurons in Fragile X Mouse Model.

Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model.

Satellite glial cells promote regenerative growth in sensory neurons.

Peripheral sensory neurons regenerate their axon after nerve injury to enable functional recovery. Intrinsic mechanisms operating in sensory neurons are known to regulate nerve repair, but whether satellite glial cells (SGC), which completely envelop the neuronal soma, contribute to nerve regeneration remains unexplored. Using a single cell RNAseq approach, we reveal that SGC are distinct from Schwann cells and share similarities with astrocytes. Nerve injury elicits changes in the expression of genes related to fatty acid synthesis and peroxisome proliferator-activated receptor (PPARα) signaling. Conditional deletion of fatty acid synthase (Fasn) in SGC impairs axon regeneration. The PPARα agonist fenofibrate rescues the impaired axon regeneration in mice lacking Fasn in SGC. These results indicate that PPARα activity downstream of FASN in SGC contributes to promote axon regeneration in adult peripheral nerves and highlight that the sensory neuron and its surrounding glial coat form a functional unit that orchestrates nerve repair.

Spatiotemporal dynamics of multi-vesicular release is determined by heterogeneity of release sites within central synapses.

A synaptic active zone (AZ) can release multiple vesicles in response to an action potential. This multi-vesicular release (MVR) occurs at most synapses, but its spatiotemporal properties are unknown. Nanoscale-resolution detection of individual release events in hippocampal synapses revealed unprecedented heterogeneity among vesicle release sites within a single AZ, with a gradient of release probability decreasing from AZ center to periphery. Parallel to this organization, MVR events preferentially overlap with uni-vesicular release (UVR) events at sites closer to an AZ center. Pairs of fusion events comprising MVR are also not perfectly synchronized, and the earlier event tends to occur closer to AZ center. The spatial features of release sites and MVR events are similarly tightened by buffering intracellular calcium. These observations revealed a marked heterogeneity of release site properties within individual AZs, which determines the spatiotemporal features of MVR events and is controlled, in part, by non-uniform calcium elevation across the AZ.

Nanoscale Organization of Vesicle Release at Central Synapses.

Presynaptic boutons support neurotransmitter release with nanoscale precision at sub-millisecond timescales. Studies over the past two decades have revealed a rich tapestry of molecular players governing synaptic vesicle fusion at highly specialized release sites in the active zone (AZ). However, the spatiotemporal organization of release at active synapses remains elusive, in part owing to the extremely small size of the AZ and the limited resolution of conventional approaches. Recent advances in fluorescence nanoscopy have revolutionized direct investigation of presynaptic release organization and dynamics. We discuss here recent nanoscopy-based studies of the molecular architecture, the spatial organization and dynamic regulation of release sites, and the mechanisms of release site replenishment. These findings have uncovered previously unknown levels of structural and functional organization at central synapses, with important implications for synaptic transmission and plasticity.

Voltage-Independent SK-Channel Dysfunction Causes Neuronal Hyperexcitability in the Hippocampus of Fmr1 Knock-Out Mice.

Neuronal hyperexcitability is one of the major characteristics of fragile X syndrome (FXS), yet the molecular mechanisms of this critical dysfunction remain poorly understood. Here we report a major role of voltage-independent potassium (K+)-channel dysfunction in hyperexcitability of CA3 pyramidal neurons in Fmr1 knock-out (KO) mice. We observed a reduction of voltage-independent small conductance calcium (Ca2+)-activated K+ (SK) currents in both male and female mice, leading to decreased action potential (AP) threshold and reduced medium afterhyperpolarization. These SK-channel-dependent deficits led to markedly increased AP firing and abnormal input-output signal transmission of CA3 pyramidal neurons. The SK-current defect was mediated, at least in part, by loss of FMRP interaction with the SK channels (specifically the SK2 isoform), without changes in channel expression. Intracellular application of selective SK-channel openers or a genetic reintroduction of an N-terminal FMRP fragment lacking the ability to associate with polyribosomes normalized all observed excitability defects in CA3 pyramidal neurons of Fmr1 KO mice. These results suggest that dysfunction of voltage-independent SK channels is the primary cause of CA3 neuronal hyperexcitability in Fmr1 KO mice and support the critical translation-independent role for the fragile X mental retardation protein as a regulator of neural excitability. Our findings may thus provide a new avenue to ameliorate hippocampal excitability defects in FXS.SIGNIFICANCE STATEMENT Despite two decades of research, no effective treatment is currently available for fragile X syndrome (FXS). Neuronal hyperexcitability is widely considered one of the hallmarks of FXS. Excitability research in the FXS field has thus far focused primarily on voltage-gated ion channels, while contributions from voltage-independent channels have been largely overlooked. Here we report that voltage-independent small conductance calcium-activated potassium (SK)-channel dysfunction causes hippocampal neuron hyperexcitability in the FXS mouse model. Our results support the idea that translation-independent function of fragile X mental retardation protein has a major role in regulating ion-channel activity, specifically the SK channels, in hyperexcitability defects in FXS. Our findings may thus open a new direction to ameliorate hippocampal excitability defects in FXS.

Characterization of a Mouse Model of Börjeson-Forssman-Lehmann Syndrome.

Mutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

Myosin V functions as a vesicle tether at the plasma membrane to control neurotransmitter release in central synapses.

Synaptic vesicle fusion occurs at specialized release sites at the active zone. How refilling of release sites with new vesicles is regulated in central synapses remains poorly understood. Using nanoscale-resolution detection of individual release events in rat hippocampal synapses we found that inhibition of myosin V, the predominant vesicle-associated motor, strongly reduced refilling of the release sites during repetitive stimulation. Single-vesicle tracking revealed that recycling vesicles continuously shuttle between a plasma membrane pool and an inner pool. Vesicle retention at the membrane pool was regulated by neural activity in a myosin V dependent manner. Ultrastructural measurements of vesicle occupancy at the plasma membrane together with analyses of single-vesicle trajectories during vesicle shuttling between the pools suggest that myosin V acts as a vesicle tether at the plasma membrane, rather than a motor transporting vesicles to the release sites, or directly regulating vesicle exocytosis.