An interactive deep learning-based approach reveals mitochondrial cristae topologies.

The convolution of membranes called cristae is a critical structural and functional feature of mitochondria. Crista structure is highly diverse between different cell types, reflecting their role in metabolic adaptation. However, their precise three-dimensional (3D) arrangement requires volumetric analysis of serial electron microscopy and has therefore been limiting for unbiased quantitative assessment. Here, we developed a novel, publicly available, deep learning (DL)-based image analysis platform called Python-based human-in-the-loop workflow (PHILOW) implemented with a human-in-the-loop (HITL) algorithm. Analysis of dense, large, and isotropic volumes of focused ion beam-scanning electron microscopy (FIB-SEM) using PHILOW reveals the complex 3D nanostructure of both inner and outer mitochondrial membranes and provides deep, quantitative, structural features of cristae in a large number of individual mitochondria. This nanometer-scale analysis in micrometer-scale cellular contexts uncovers fundamental parameters of cristae, such as total surface area, orientation, tubular/lamellar cristae ratio, and crista junction density in individual mitochondria. Unbiased clustering analysis of our structural data unraveled a new function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in regulating the balance between lamellar versus tubular cristae subdomains.

Correction: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

[This corrects the article DOI: 10.1371/journal.pbio.1002516.].

New insights into the regulation of synaptic transmission and plasticity by the endoplasmic reticulum and its membrane contacts.

Mammalian neurons are highly compartmentalized yet very large cells. To provide each compartment with its distinct properties, metabolic homeostasis and molecular composition need to be precisely coordinated in a compartment-specific manner. Despite the importance of the endoplasmic reticulum (ER) as a platform for various biochemical reactions, such as protein synthesis, protein trafficking, and intracellular calcium control, the contribution of the ER to neuronal compartment-specific functions and plasticity remains elusive. Recent advances in the development of live imaging and serial scanning electron microscopy (sSEM) analysis have revealed that the neuronal ER is a highly dynamic organelle with compartment-specific structures. sSEM studies also revealed that the ER forms contacts with other membranes, such as the mitochondria and plasma membrane, although little is known about the functions of these ER-membrane contacts. In this review, we discuss the mechanisms and physiological roles of the ER structure and ER-mitochondria contacts in synaptic transmission and plasticity, thereby highlighting a potential link between organelle ultrastructure and neuronal functions.

Pleiotropic Mitochondria: The Influence of Mitochondria on Neuronal Development and Disease.

Mitochondria play many important biological roles, including ATP production, lipid biogenesis, ROS regulation, and calcium clearance. In neurons, the mitochondrion is an essential organelle for metabolism and calcium homeostasis. Moreover, mitochondria are extremely dynamic and able to divide, fuse, and move along microtubule tracks to ensure their distribution to the neuronal periphery. Mitochondrial dysfunction and altered mitochondrial dynamics are observed in a wide range of conditions, from impaired neuronal development to various neurodegenerative diseases. Novel imaging techniques and genetic tools provide unprecedented access to the physiological roles of mitochondria by visualizing mitochondrial trafficking, morphological dynamics, ATP generation, and ultrastructure. Recent studies using these new techniques have unveiled the influence of mitochondria on axon branching, synaptic function, calcium regulation with the ER, glial cell function, neurogenesis, and neuronal repair. This review provides an overview of the crucial roles played by mitochondria in the CNS in physiological and pathophysiological conditions.

LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

The polycomb component Ring1B regulates the timed termination of subcerebral projection neuron production during mouse neocortical development.

In the developing neocortex, neural precursor cells (NPCs) sequentially generate various neuronal subtypes in a defined order. Although the precise timing of the NPC fate switches is essential for determining the number of neurons of each subtype and for precisely generating the cortical layer structure, the molecular mechanisms underlying these switches are largely unknown. Here, we show that epigenetic regulation through Ring1B, an essential component of polycomb group (PcG) complex proteins, plays a key role in terminating NPC-mediated production of subcerebral projection neurons (SCPNs). The level of histone H3 residue K27 trimethylation at and Ring1B binding to the promoter of Fezf2, a fate determinant of SCPNs, increased in NPCs as Fezf2 expression decreased. Moreover, deletion of Ring1B in NPCs, but not in postmitotic neurons, prolonged the expression of Fezf2 and the generation of SCPNs that were positive for CTIP2. These results indicate that Ring1B mediates the timed termination of Fezf2 expression and thereby regulates the number of SCPNs.

Up-regulation of HP1γ expression during neuronal maturation promotes axonal and dendritic development in mouse embryonic neocortex.

Immature neurons undergo morphological and physiological changes including axonal and dendritic development to establish neuronal networks. As the transcriptional status changes at a large number of genes during neuronal maturation, global changes in chromatin modifiers may take place in this process. We now show that the amount of heterochromatin protein 1γ (HP1γ) increases during neuronal maturation in the mouse neocortex. Knockdown of HP1γ suppressed axonal and dendritic development in mouse embryonic neocortical neurons in culture, and either knockdown or knockout of HP1γ impaired the projection of callosal axons of superficial layer neurons to the contralateral hemisphere in the developing neocortex. Conversely, forced expression of HP1γ facilitated axonal and dendritic development, suggesting that the increase of HP1γ is a rate limiting step in neuronal maturation. These results together show an important role for HP1γ in promoting axonal and dendritic development in maturing neurons.

Epigenetic control of neural precursor cell fate during development.

The temporally and spatially restricted nature of the differentiation capacity of cells in the neural lineage has been studied extensively in recent years. Epigenetic control of developmental genes, which is heritable through cell divisions, has emerged as a key mechanism defining the differentiation potential of cells. Short-term or reversible repression of developmental genes puts them in a 'poised state', ready to be activated in response to differentiation-inducing cues, whereas long-term or permanent repression of developmental genes restricts the cell fates they regulate. Here, we review the molecular mechanisms that underlie the establishment and regulation of differentiation potential along the neural lineage during development.

A noncoding RNA regulates the neurogenin1 gene locus during mouse neocortical development.

The proneural basic helix-loop-helix (bHLH) transcription factor neurogenin1 (Neurog1) plays a pivotal role in neuronal differentiation during mammalian development. The spatiotemporal control of the Neurog1 gene expression is mediated by several specific enhancer elements, although how these elements regulate the Neurog1 locus has remained largely unclear. Recently it has been shown that a large number of enhancer elements are transcribed, but the regulation and function of the resulting transcripts have been investigated for only several such elements. We now show that an enhancer element located 5.8-7.0 kb upstream of the mouse Neurog1 locus is transcribed. The production of this transcript, designated utNgn1, is highly correlated with that of Neurog1 mRNA during neuronal differentiation. Moreover, knockdown of utNgn1 by a corresponding short interfering RNA inhibits the production of Neurog1 mRNA in response to induction of neuronal differentiation. We also found that production of utNgn1 is suppressed by polycomb group (PcG) proteins, which inhibit the expression of Neurog1. Our results thus suggest that a noncoding RNA transcribed from an enhancer element positively regulates transcription at the Neurog1 locus.

Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

PDZD8-FKBP8 tethering complex at ER-mitochondria contact sites regulates mitochondrial complexity.

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

Wnt signaling and its downstream target N-myc regulate basal progenitors in the developing neocortex.

Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. Here, we show that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of beta-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex.

PDK1 regulates the generation of oligodendrocyte precursor cells at an early stage of mouse telencephalic development.

During the development of the mouse telencephalon, multipotent neural precursor cells (NPCs) generate oligodendrocyte precursor cells (OPCs), progenitors restricted to the oligodendrocyte lineage, at various sites in a developmental stage-dependent manner. Although substantial progress has been made in identifying the transcription factors that control the production of OPCs, the signaling pathways that regulate these transcription factors and the spatiotemporal pattern of OPC production have been only partially clarified. Here, we show that the serine-threonine kinase 3-phosphoinositide-dependent kinase 1 (PDK1) contributes to an early wave of OPC production in the developing mouse telencephalon. Ablation of PDK1 in NPCs resulted in a reduction in the number of OPCs positive for Sox10 and platelet-derived growth factor receptor α (PDGFRα) within the neocortex and striatum at embryonic day (E) 15.5, but not at E18.5. Furthermore, pharmacological inhibition of phosphoinositide 3-kinase (PI3K) or deletion of the PDK1 gene suppressed the generation of OPCs from NPCs induced by fibroblast growth factor (FGF) 2 in culture. These results implicate the PI3K-PDK1 pathway in the physiological regulation of OPC production in a developmental context-dependent manner.

Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation.

Synaptic vesicle (SV) clusters, which reportedly result from synapsin's capacity to undergo liquid-liquid phase separation (LLPS), constitute the structural basis for neurotransmission. Although these clusters contain various endocytic accessory proteins, how endocytic proteins accumulate in SV clusters remains unknown. Here, we report that endophilin A1 (EndoA1), the endocytic scaffold protein, undergoes LLPS under physiologically relevant concentrations at presynaptic terminals. On heterologous expression, EndoA1 facilitates the formation of synapsin condensates and accumulates in SV-like vesicle clusters via synapsin. Moreover, EndoA1 condensates recruit endocytic proteins such as dynamin 1, amphiphysin, and intersectin 1, none of which are recruited in vesicle clusters by synapsin. In cultured neurons, like synapsin, EndoA1 is compartmentalized in SV clusters through LLPS, exhibiting activity-dependent dispersion/reassembly cycles. Thus, beyond its essential function in SV endocytosis, EndoA1 serves an additional structural function by undergoing LLPS, thereby accumulating various endocytic proteins in dynamic SV clusters in concert with synapsin.

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release.

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.

Compartment-specific tuning of dendritic feature selectivity by intracellular Ca2+ release.

Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.

Endoplasmic Reticulum-Mitochondria Contact Sites-Emerging Intracellular Signaling Hubs.

It has become apparent that our textbook illustration of singular isolated organelles is obsolete. In reality, organelles form complex cooperative networks involving various types of organelles. Light microscopic and ultrastructural studies have revealed that mitochondria-endoplasmic reticulum (ER) contact sites (MERCSs) are abundant in various tissues and cell types. Indeed, MERCSs have been proposed to play critical roles in various biochemical and signaling functions such as Ca2+ homeostasis, lipid transfer, and regulation of organelle dynamics. While numerous proteins involved in these MERCS-dependent functions have been reported, how they coordinate and cooperate with each other has not yet been elucidated. In this review, we summarize the functions of mammalian proteins that localize at MERCSs and regulate their formation. We also discuss potential roles of the MERCS proteins in regulating multiple organelle contacts.

Diverse gene regulatory mechanisms mediated by Polycomb group proteins during neural development.

While all the developmental genes are temporarily repressed for future activation in the pluripotent stem cells, non-neural genes become persistently repressed in the course of commitment to the neuronal lineage. Although Polycomb group proteins (PcG) are key factors for both temporary and persistent repression of the developmental genes, how the same group of proteins can differentially repress target genes remains unclarified. The identification of a variety of PcG complexes and activities sheds light on these issues. In this review, based on the recent findings including those with the use of interactome and Chromosome Conformation Capture (3C)-type analyses, we summarize the molecular mechanisms of PcG-mediated gene regulation and discuss how PcG regulates cell fate specification during neural development.

Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo.

A challenging aspect of neuroscience revolves around mapping the synaptic connections within neural circuits (connectomics) over scales spanning several orders of magnitude (nanometers to meters). Despite significant improvements in serial section electron microscopy (SSEM) technologies, several major roadblocks have impaired its general applicability to mammalian neural circuits. In the present study, we introduce a new approach that circumvents some of these roadblocks by adapting a genetically-encoded ascorbate peroxidase (APEX2) as a fusion protein to a membrane-targeted fluorescent reporter (CAAX-Venus), and introduce it in single pyramidal neurons in vivo using extremely sparse in utero cortical electroporation. This approach allows us to perform Correlated Light-SSEM (CoLSSEM), a variant of Correlated Light-EM (CLEM), on individual neurons, reconstructing their dendritic and axonal arborization in a targeted way via combination of high-resolution confocal microscopy, and subsequent imaging of its ultrastructural features and synaptic connections with ATUM-SEM (automated tape-collecting ultramicrotome - scanning electron microscopy) technology. Our method significantly will improve the feasibility of large-scale reconstructions of neurons within a circuit, and permits the description of some ultrastructural features of identified neurons with their functional and/or structural connectivity, one of the main goal of connectomics.

Emerging roles of mitochondria in synaptic transmission and neurodegeneration.

Mitochondria play numerous critical physiological functions in neurons including ATP production, Ca2+ regulation, lipid synthesis, ROS signaling, and the ability to trigger apoptosis. Recently developed technologies, including in vivo 2-photon imaging in awake behaving mice revealed that unlike in the peripheral nervous system (PNS), mitochondrial transport decreases strikingly along the axons of adult neurons of the central nervous system (CNS). Furthermore, the improvements of genetically-encoded biosensors have enabled precise monitoring of the spatial and temporal impact of mitochondria on Ca2+, ATP and ROS homeostasis in a compartment-specific manner. Here, we discuss recent findings that begin to unravel novel physiological and pathophysiological properties of neuronal mitochondria at synapses. We also suggest new directions in the exploration of mitochondrial function in synaptic transmission, plasticity and neurodegeneration.