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Neuronal mitochondria play important roles beyond ATP generation, including Ca2+uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly in the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer- specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Camkk2- dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a new activity- dependent molecular mechanism underlying the extreme subcellular compartmentalization of mitochondrial morphology in dendrites of neuronsin vivothrough spatially precise regulation of mitochondria fission/fusion balance.

Human-specific (HS) genes are potential drivers of brain evolution, but their impact on human neuron development and disease remains unclear. Here we studied HS genes SRGAP2B/C in human cortical projection neurons (CPNs) in vivo, using xenotransplantation in the mouse cortex. Downregulation of SRGAP2B/C in human CPNs greatly accelerated synaptic development, indicating their requirement for human-specific synaptic neoteny. SRGAP2B/C acted by downregulating their ancestral paralog SRGAP2A, thereby upregulating postsynaptic levels of SYNGAP1, a major intellectual deficiency/autism spectrum disorder (ID/ASD) gene. Combinatorial genetic invalidation revealed that the tempo of synaptogenesis is set by a balance between SRGAP2A and SYNGAP1, which in human CPNs is tipped towards neoteny by SRGAP2B/C. Our results demonstrate that HS genes can modify the phenotypic expression of ID/ASD mutations through regulation of synaptic neoteny.One-Sentence SummaryHuman-specific genes SRGAP2B/C control human cortical neuron neoteny by regulating the function of neurodevelopmental disorder gene SYNGAP1

The brain of modern humans has evolved remarkable computational abilities that enable higher cognitive functions. These capacities are tightly linked to an increase in the size and connectivity of the cerebral cortex, which is thought to have resulted from evolutionary changes in the mechanisms of cortical development. Convergent progress in evolutionary genomics, developmental biology and neuroscience has recently enabled the identification of genomic changes that act as human-specific modifiers of cortical development. These modifiers influence most aspects of corticogenesis, from the timing and complexity of cortical neurogenesis to synaptogenesis and the assembly of cortical circuits. Mutations of human-specific genetic modifiers of corticogenesis have started to be linked to neurodevelopmental disorders, providing evidence for their physiological relevance and suggesting potential relationships between the evolution of the human brain and its sensitivity to specific diseases.

Local circuit architecture facilitates the emergence of feature selectivity in the cerebral cortex1. In the hippocampus, it remains unknown whether local computations supported by specific connectivity motifs2 regulate the spatial receptive fields of pyramidal cells3. Here we developed an in vivo electroporation method for monosynaptic retrograde tracing4 and optogenetics manipulation at single-cell resolution to interrogate the dynamic interaction of place cells with their microcircuitry during navigation. We found a local circuit mechanism in CA1 whereby the spatial tuning of an individual place cell can propagate to a functionally recurrent subnetwork5 to which it belongs. The emergence of place fields in individual neurons led to the development of inverse selectivity in a subset of their presynaptic interneurons, and recruited functionally coupled place cells at that location. Thus, the spatial selectivity of single CA1 neurons is amplified through local circuit plasticity to enable effective multi-neuronal representations that can flexibly scale environmental features locally without degrading the feedforward input structure. Single-cell tracing and optogenetics manipulation in mice are used to show how spatial tuning of individual pyramidal cells in CA1 can propagate to and be amplified by their local subnetwork of neurons.

Synaptic connectivity within neural circuits is characterized by high degrees of cellular and subcellular specificity. This precision arises from the combined action of several classes of molecular cues, transmembrane receptors, secreted cues and extracellular matrix components, coordinating transitions between axon guidance, dendrite patterning, axon branching and synapse specificity. We focus this review on recent insights into some of the molecular and cellular mechanisms controlling these transitions and present the results of large-scale efforts and technological developments aimed at mapping neural connectivity at single cell resolution in the mouse cortex as a mammalian model organism. Finally, we outline some of the technical and conceptual challenges lying ahead as the field is starting to explore one of the most challenging problems in neuroscience: the molecular and cellular logic underlying the emergence of the connectome characterizing specific circuits within the central nervous system of mammals.

Mitochondria-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. Here, we identify and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β

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.

Hippocampal place cells underlie spatial navigation and memory. Remarkably, CA1 pyramidal neurons can form new place fields within a single trial by undergoing rapid plasticity. However, local feedback circuits likely restrict the rapid recruitment of individual neurons into ensemble representations. This interaction between circuit dynamics and rapid feature coding remains unexplored. Here, we developed "all-optical" approaches combining novel optogenetic induction of rapidly forming place fields with 2-photon activity imaging during spatial navigation in mice. We find that induction efficacy depends strongly on the density of co-activated neurons. Place fields can be reliably induced in single cells, but induction fails during co-activation of larger subpopulations due to local circuit constraints imposed by recurrent inhibition. Temporary relief of local inhibition permits the simultaneous induction of place fields in larger ensembles. We demonstrate the behavioral implications of these dynamics, showing that our ensemble place field induction protocol can enhance subsequent spatial association learning.

The cognitive abilities that characterize humans are thought to emerge from unique features of the cortical circuit architecture of the human brain, which include increased cortico–cortical connectivity. However, the evolutionary origin of these changes in connectivity and how they affected cortical circuit function and behaviour are currently unknown. The human-specific gene duplication SRGAP2C emerged in the ancestral genome of the Homo lineage before the major phase of increase in brain size1,2. SRGAP2C expression in mice increases the density of excitatory and inhibitory synapses received by layer 2/3 pyramidal neurons (PNs)3–5. Here we show that the increased number of excitatory synapses received by layer 2/3 PNs induced by SRGAP2C expression originates from a specific increase in local and long-range cortico–cortical connections. Mice humanized for SRGAP2C expression in all cortical PNs displayed a shift in the fraction of layer 2/3 PNs activated by sensory stimulation and an enhanced ability to learn a cortex-dependent sensory-discrimination task. Computational modelling revealed that the increased layer 4 to layer 2/3 connectivity induced by SRGAP2C expression explains some of the key changes in sensory coding properties. These results suggest that the emergence of SRGAP2C at the birth of the Homo lineage contributed to the evolution of specific structural and functional features of cortical circuits in the human cortex. The human-specific gene duplication SRGAP2C is identified as a modifier of structural and functional features of cortical circuits leading to improved behavioural performance that may have allowed the emergence of cognitive properties characterizing the human brain.

SUMMARY Synaptic connectivity within adult circuits exhibits a remarkable degree of cellular and subcellular specificity. We report that the axon guidance receptor Robo2 plays a role in establishing synaptic specificity in hippocampal CA1. In vivo, Robo2 is present and required postsynaptically in CA1 pyramidal neurons (PNs) for the formation of excitatory (E) but not inhibitory (I) synapses, specifically in proximal but not distal dendritic compartments. In vitro approaches show that the synaptogenic activity of Robo2 involves a trans-synaptic interaction with presynaptic Neurexins, as well as binding to its canonical extracellular ligand Slit. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in awake behaving mice shows that preventing Robo2-dependent excitatory synapse formation cell autonomously during development alters place cell properties of adult CA1 PNs. Our results identify a trans-synaptic complex linking the establishment of synaptic specificity to circuit function., In brief Blockus et al. demonstrate that the axon guidance receptors Robo1/2 are synaptogenic cues required postsynaptically for the establishment of synaptic specificity between CA3 axons and dendrites of CA1 pyramidal neurons (PNs). Developmental deletion of Robo2 in CA1 PNs disrupts the emergence of place cell properties in adult hippocampus., Graphical Abstract

Dendritic Ca2+ signaling is central to neural plasticity mechanisms allowing animals to adapt to the environment. Intracellular Ca2+ release (ICR) from 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.

The Rac1-specific guanosine triphosphatase (GTPase)-activating protein Slit-Robo GAP2 (Srgap2) is dramatically upregulated during RANKL-induced osteoclastogenesis. Srgap2 interacts with the cell membrane to locally inhibit activity of Rac1. In this study, we determined the role of Srgap2 in the myeloid lineage on bone homeostasis and the osteoclastic response to TNFα treatment. The bone phenotype of mice specifically lacking Srgap2 in the myeloid lineage (Srgap2 f/f :LysM-Cre; Srgap2 conditional knockout [cKO]) was investigated using histomorphometric analysis, in vitro cultures and Western blot analysis. Similar methods were used to determine the impact of TNFα challenge on osteoclast formation in Srgap2 cKO mice. Bone parameters in male Srgap2 cKO mice were unaffected. However, female cKO mice displayed higher trabecular bone volume due to increased osteoblast surface and bone formation rate, whereas osteoclastic parameters were unaltered. In vitro, cells from Srgap2 cKO had strongly enhanced Rac1 activation, but RANKL-induced osteoclast formation was unaffected. In contrast, conditioned medium from Srgap2 cKO osteoclasts promoted osteoblast differentiation and had increased levels of the bone anabolic clastokine SLIT3, providing a possible mechanism for increased bone formation in vivo. Rac1 is rapidly activated by the inflammatory cytokine TNFα. Supracalvarial injection of TNFα caused an augmented osteoclastic response in Srgap2 cKO mice. In vitro, cells from Srgap2 cKO mice displayed increased osteoclast formation in response to TNFα. We conclude that Srgap2 plays a prominent role in limiting osteoclastogenesis during inflammation through Rac1, and restricts expression of the paracrine clastokine SLIT3, a positive regulator of bone formation. © 2019 American Society for Bone and Mineral Research.

Summary The mechanisms controlling the post-natal maturation of astrocytes play a crucial role in ensuring correct synaptogenesis. We show that mitochondrial biogenesis in developing astrocytes is necessary for coordinating post-natal astrocyte maturation and synaptogenesis. The astrocytic mitochondrial biogenesis depends on the transient upregulation of metabolic regulator peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α), which is controlled by metabotropic glutamate receptor 5 (mGluR5). At tissue level, the loss or downregulation of astrocytic PGC-1α sustains astrocyte proliferation, dampens astrocyte morphogenesis, and impairs the formation and function of neighboring synapses, whereas its genetic re-expression is sufficient to restore the mitochondria compartment and correct astroglial and synaptic defects. Our findings show that the developmental enhancement of mitochondrial biogenesis in astrocytes is a critical mechanism controlling astrocyte maturation and supporting synaptogenesis, thus suggesting that astrocytic mitochondria may be a therapeutic target in the case of neurodevelopmental and psychiatric disorders characterized by impaired synaptogenesis.

Hippocampal place cells form a physiological substrate for spatial navigation and memory in the brain. Remarkably, CA1 pyramidal neurons can acquire stable place fields in a few trials through a novel behavioral timescale" synaptic plasticity rule (BTSP). While this process can enable rapid learning, local feedback circuits likely tightly restrict the recruitment of individual cells into ensemble representations; this interaction between local circuit dynamics and BTSP remains unexplored. Here we developed \all-optical" approaches that combine novel optogenetic induction of place fields via BTSP in CA1 pyramidal cells with 2-photon calcium activity imaging during spatial navigation. We find that induction efficacy depends strongly on the density of co-activated neurons. Place fields can be reliably induced in single cells, but induction fails during co-activation of larger CA1 populations. Our results implicate local circuit constraints on the induction of place fields, whereby recurrent inhibition regulates the prevalence of BTSP across the ensemble. Consistent with this interpretation, we found that temporary relief of local inhibition permitted the simultaneous induction of place fields in larger CA1 ensembles. We demonstrate the behavioral consequences of these dynamics, as applying our ensemble optogenetic induction protocol at an arbitrary location enhanced animals' subsequent performance in a spatial reward learning task involving that location.

The complex cellular architecture of neurons combined with their longevity makes maintaining a healthy mitochondrial network particularly important and challenging. One of the many roles of mitochondrial-ER contact sites (MERCs) is to mediate mitochondrial quality control through regulating mitochondrial turn over. Pdzd8 is a newly discovered MERC protein, the organismal functions of which have not yet been explored. Here we identify and provide the first functional characterization of theDrosophila melanogasterortholog of Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan inDrosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion and reduces lifespan. We also show that depletion of pdzd8 rescues the locomotor defects characterizing an Alzheimer’s disease (AD) fly model over-expressing Amyloidβ1–42(Aβ42) and prolongs the survival of flies fed with mitochondrial toxins. Together, our results provide the firstin vivoevidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

SummaryWe present a semi-automated reconstruction of L2/3 mouse primary visual cortex from 3 million cubic microns of electron microscopic images, including pyramidal and inhibitory neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are being made publicly available, along with tools for programmatic and 3D interactive access. The density of synaptic inputs onto inhibitory neurons varies across cell classes and compartments. We uncover a compartment-specific correlation between mitochondrial coverage and synapse density. Frequencies of connectivity motifs in the graph of pyramidal cells are predicted quite accurately from node degrees using the configuration model of random graphs. Cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. These example findings illustrate the resource’s utility for relating structure and function of cortical circuits as well as for neuronal cell biology.

mRNA localization and local translation enable exquisite spatial and temporal control of gene expression, particularly in highly polarized and elongated cells. These features are especially prominent in radial glial cells (RGCs), which serve as neural and glial precursors of the developing cerebral cortex, and scaffolds for migrating neurons. Yet the mechanisms by which distinct sub-cellular compartments of RGCs accomplish their diverse functions are poorly understood. Here, we demonstrate that subcellular RNA localization and translation of the RhoGAP Arhgap11a controls RGC morphology and mediates cortical cytoarchitecture. Arhgap11a mRNA and protein exhibit conserved localization to RGC basal structures in mice and humans, conferred by a 5′UTR cis-element. Proper RGC morphology relies upon active Arhgap11a mRNA transport and localization to basal structures, where ARHGAP11A is locally synthesized. Thus, RhoA activity is spatially and acutely activated via local translation in RGCs to promote neuron positioning and cortical cytoarchitecture. Altogether, our study demonstrates that mRNA localization and local translation mediate compartmentalization of neural progenitor functions to control brain development. Highlights Arhgap11a in radial glia non-cell autonomously promotes neuronal migration and lamination Arhgap11a mRNA localizes to radial glial endfeet via a 5’ UTR cis element ARHGAP11A expression in basal process and endfeet depends upon its localized mRNA Localized mRNA and RhoA-GAP activity in endfeet control radial glial morphology

The balance between excitatory and inhibitory (E and I) synapses is thought to be critical for information processing in neural circuits. However, little is known about the spatial principles of E and I synapses organization across the entire dendritic tree of mammalian neurons. We developed a new, open-source, reconstruction platform for mapping the size and spatial distribution of E and I synapses received by individual, genetically-labeled, layer 2/3 cortical pyramidal neurons (PNs) in vivo. We mapped over 90,000 E and I synapses across twelve L2/3 PNs and uncovered structured organization of E and I synapses across dendritic domains as well as within individual dendritic segments in these neurons. Despite significant, domain-specific, variations in the absolute density of E and I synapses, their ratio is strikingly balanced locally across dendritic segments. Computational modeling indicates that this spatially-precise E/I balance dampens dendritic voltage fluctuations and strongly impacts neuronal firing output.

We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ~250×140×90 μm(3) of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.

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.

Mitochondria play numerous critical physiological functions in neurons including ATP production, Ca(2+) 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 Ca(2+), 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.

Pattern formation in the brain Neurons in the developing brain cooperate to build circuits. Mountoufaris et al. found that ∼50 variable protocadherin genes support a combinatorial identity code that allows millions of olfactory neuron axons to sort into ∼2000 glomeruli. Sharing olfactory receptors drives axons to one glomerulus, and protocadherin diversity allows the multiple axons to touch each other as they converge. On the other hand, Chen et al. found that a single C-type protocadherin underlies the tiled distribution of serotonergic neurons throughout the central nervous system. These neurons, which share protocadherin identity, enervate broad swaths evenly without touching neighboring neurons. Science , this issue p. 411 , p. 406

During the early stages of Alzheimer’s disease (AD) in both mouse models and human patients, soluble forms of Amyloid-β 1–42 oligomers (Aβ42o) trigger loss of excitatory synapses (synaptotoxicity) in cortical and hippocampal pyramidal neurons (PNs) prior to the formation of insoluble amyloid plaques. In a transgenic AD mouse model, we observed a spatially restricted structural remodeling of mitochondria in the apical tufts of CA1 PNs dendrites corresponding to the dendritic domain where the earliest synaptic loss is detected in vivo. We also observed AMPK over-activation as well as increased fragmentation and loss of mitochondrial biomass in Ngn2-induced neurons derived from a new APPSwe/Swe knockin human ES cell line. We demonstrate that Aβ42o-dependent over-activation of the CAMKK2-AMPK kinase dyad mediates synaptic loss through coordinated phosphorylation of MFF-dependent mitochondrial fission and ULK2-dependent mitophagy. Our results uncover a unifying stress-response pathway causally linking Aβ42o-dependent structural remodeling of dendritic mitochondria to synaptic loss.

SUMMARYThe remarkable cognitive abilities characterizing humans are thought to emerge from our unique features of cortical circuit architecture, including increased feedforward and feedback connectivity. However, our understanding of the evolutionary origin and nature of these changes in circuit connectivity, and how they impact cortical circuit function and behavior is currently lacking. Here, we demonstrate that expression of the human-specific gene duplication SRGAP2C leads to a specific increase in feedforward and feedback cortico-cortical connectivity. Moreover, humanized SRGAP2C mice display improved cortical sensory coding, and an enhanced ability to learn a cortex-dependent sensory discrimination task. Our results identify a novel substrate for human brain evolution whereby the emergence of SRGAP2C led to increased feedforward and feedback cortico-cortical connectivity, improved cortical sensory processing and enhanced behavioral performance.

The developmental transition between axon guidance and synapse formation is critical for circuit assembly but still poorly understood at the molecular level. We hypothesized that this key transition could be regulated by axon guidance cues switching their function to regulate synaptogenesis with subcellular specificity. Here, we report evidence for such a functional switch, describing a novel role for the axon guidance molecule Robo2 in excitatory synapse formation onto dendrites of CA1 pyramidal neurons (PNs) in the mouse hippocampus. Cell-autonomous deletion of Robo2 from CA1 PNs leads to a drastic reduction of the number of excitatory synapses specifically in proximal dendritic compartments. At the molecular level, we show that this novel postsynaptic function of Robo2 depends on both its canonical ligand Slit and a novel interaction with presynaptic Neurexins. Biophysical analysis reveals that Robo2 binds directly to Neurexins via its Ig4-5 domains. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in mice shows that sparse deletion of Robo2 during development drastically reduces the likelihood of place cell emergence and alters spatial coding properties of the remaining place cells. Our results identify Robo2 as a novel molecular effector linking synaptic specificity to the acquisition of spatial coding properties characterizing hippocampal circuits.

During the early stages of Alzheimer’s disease (AD) in both mouse models and human patients, soluble forms of Amyloid-β1-42 oligomers (Aβ42o) trigger loss of excitatory synapses (synaptotoxicity) in cortical and hippocampal pyramidal neurons (PNs) prior to the formation of insoluble Aβ plaques. We observed a spatially restricted structural remodeling of mitochondria in the apical tufts of CA1 PNs dendrites in the hAPPSWE,IND transgenic AD mouse model (J20), corresponding to the dendritic domain receiving presynaptic inputs from the entorhinal cortex and where the earliest synaptic loss is detected in vivo. We also observed significant loss of mitochondrial biomass in human neurons derived from a new model of human ES cells where CRISPR-Cas9-mediated genome engineering was used to introduce the ‘Swedish’ mutation bi-allelically (APPSWE/SWE). Recent work uncovered that Aβ42o mediates synaptic loss by over-activating the CAMKK2-AMPK kinase dyad, and that AMPK is a central regulator of mitochondria homeostasis in non-neuronal cells. Here, we demonstrate that Aβ42o-dependent over-activation of CAMKK2-AMPK mediates synaptic loss through coordinated MFF-dependent mitochondrial fission and ULK2-dependent mitophagy in dendrites of PNs. We also found that the ability of Aβ42o-dependent mitochondrial remodeling to trigger synaptic loss requires the ability of AMPK to phosphorylate Tau on Serine 262. Our results uncover a unifying stress-response pathway triggered by Aβo and causally linking structural remodeling of dendritic mitochondria to synaptic loss.

Human-specific gene duplications (HSGD) have recently emerged as key modifiers of brain development and evolution. However, the molecular mechanisms underlying the function of HSGDs remain often poorly understood. In humans, a truncated duplication of SRGAP2A led to the emergence of two human-specific paralogs: SRGAP2B and SRGAP2C. The ancestral copy SRGAP2A limits synaptic density and promotes maturation of both excitatory (E) and inhibitory (I) synapses received by cortical pyramidal neurons (PNs). SRGAP2C binds to and inhibits all known functions of SRGAP2A leading to an increase in E and I synapse density and protracted synapse maturation, traits characterizing human cortical neurons. Here, we demonstrate how the evolutionary changes that led to the emergence of SRGAP2 HSGD generated proteins that, in neurons, are intrinsically unstable and upon hetero-dimerization with SRGAP2A, reduces SRGAP2A levels in a proteasome-dependent manner. Moreover, we show that, compared to SRGAP2B, and despite only a few non-synonymous mutations specifically targeting arginine residues, SRGAP2C is unique, compared to SRGAP2B, in its ability to induce long-lasting changes in synaptic density throughout adulthood. The non-synonymous mutations specifically targeting arginine residues led to the unique ability of SRGAP2C to inhibit SRGAP2A function and thereby contribute to the emergence of human-specific features of synaptic development during evolution.

Summary Cell polarization is important for various biological processes. However, its regulation, particularly initiation, is incompletely understood. Here, we investigated mechanisms by which neutrophils break their symmetry and initiate their cytoskeleton polarization from an apolar state in circulation for their extravasation during inflammation. We show here that a local increase in plasma membrane (PM) curvature resulting from cell contact to a surface triggers the initial breakage of the symmetry of an apolar neutrophil and is required for subsequent polarization events induced by chemical stimulation. This local increase in PM curvature recruits SRGAP2 via its F-BAR domain, which in turn activates PI4KA and results in PM PtdIns4P polarization. Polarized PM PtdIns4P is targeted by RPH3A, which directs PIP5K1C90 and subsequent phosphorylated myosin light chain polarization, and this polarization signaling axis regulates neutrophil firm attachment to endothelium. Thus, this study reveals a mechanism for the initiation of cell cytoskeleton polarization.

Summary The importance of mitochondria for neuronal function is evident by the large number of neurodegenerative diseases that have been associated with a disruption of mitochondrial function or transport (reviewed in [1, 2]). Mitochondria are essential for proper biological function as a result of their ability to produce ATP through oxidative phosphorylation, buffer cytoplasmic calcium, regulate lipid biosynthesis, and trigger apoptosis (reviewed in [2]). Efficient transport of mitochondria is thought to be particularly important in neurons in light of their compartmentalization, length of axonal processes, and high-energy requirements (reviewed in [3]). However, the majority of these results were obtained using short-term, in vitro neuronal culture models, and very little is currently known about mitochondrial dynamics in mature axons of the mammalian CNS in vitro or in vivo. Furthermore, recent evidence has demonstrated that mitochondrial immobilization at specific points along the axon, such as presynaptic boutons, play critical roles in axon morphogenesis [4, 5]. We report that as cortical axons mature, motility of mitochondria (but not other cargoes) is dramatically reduced and this coincides with increased localization to presynaptic sites. We also demonstrate using photo-conversion that in vitro mature axons display surprisingly limited long-range mitochondrial transport. Finally, using in vivo two-photon microscopy in anesthetized or awake-behaving mice, we document for the first time that mitochondrial motility is also remarkably low in distal cortical axons in vivo. These results argue that mitochondrial immobilization and presynaptic localization are important hallmarks of mature CNS axons both in vitro and in vivo.

Unnatural amino acid (UAA) mutagenesis of recombinant proteins in live mammalian cells requires coexpression of the mutant target, as well as an engineered tRNA/aminoacyl-tRNA synthetase pair. The ability to readily determine the optimal relative expression levels of these three genetic components for efficient expression of the UAA-modified target is highly desirable, but remains challenging to accomplish. Here we report a facile strategy to achieve this by taking advantage of the efficient gene-delivery by a baculovirus vector, which enables systematic variation of the expression level of each genetic component in a population-wide manner. Insights gained from this study led to the design of an optimal expression system, which can be delivered into mammalian cells by a single baculovirus vector to provide significantly improved UAA incorporation efficiency at a low virus load. Furthermore, this optimized baculovirus vector was shown to enable efficient UAA mutagenesis of proteins expressed in mouse brain tissue.

The proper function of neural circuits requires spatially and temporally balanced development of excitatory and inhibitory synapses. However, the molecular mechanisms coordinating excitatory and inhibitory synaptogenesis remain unknown. Here we demonstrate that SRGAP2A and its human-specific paralog SRGAP2C co-regulate the development of excitatory and inhibitory synapses in cortical pyramidal neurons in vivo. SRGAP2A promotes synaptic maturation, and ultimately the synaptic accumulation of AMPA and GABAA receptors, by interacting with key components of both excitatory and inhibitory postsynaptic scaffolds, Homer and Gephyrin. Furthermore, SRGAP2A limits the density of both types of synapses via its Rac1-GAP activity. SRGAP2C inhibits all identified functions of SRGAP2A, protracting the maturation and increasing the density of excitatory and inhibitory synapses. Our results uncover a molecular mechanism coordinating critical features of synaptic development and suggest that human-specific duplication of SRGAP2 might have contributed to the emergence of unique traits of human neurons while preserving the excitation/inhibition balance.