Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1α and NL1 via Hevin.

Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.

Chemico-genetic discovery of astrocytic control of inhibition in vivo.

Perisynaptic astrocytic processes are an integral part of central nervous system synapses1,2; however, the molecular mechanisms that govern astrocyte-synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte-neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function.

Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome.

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity.SIGNIFICANCE STATEMENT Epilepsy Aphasia Syndromes (EAS) are at the severe end of a spectrum of cognitive-behavioral symptoms seen in childhood epilepsies, and they remain an inadequately understood disorder. The prognosis of EAS is frequently poor, and patients have life-long language and cognitive disturbances. Here we describe a genetic mouse model of EAS, based on the KO of the EAS risk gene Cnksr2 We show that these mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an important new model for future studies of EAS. We also provide insights into the molecular disturbances downstream of Cnksr2 loss by using in vivo quantitative proteomics tools.

The Arp2/3 Complex Is Essential for Distinct Stages of Spine Synapse Maturation, Including Synapse Unsilencing.

UNLABELLED: Dendritic filopodia are actin-rich structures that are thought to contribute to early spine synapse formation; however, the actin regulatory proteins important for early synaptogenesis are poorly defined. Using organotypic hippocampal slice cultures and primary neuron hippocampal cultures from Arp2/3 conditional knock-out mice, we analyze the roles of the Arp2/3 complex, an actin regulator that creates branched actin networks, and demonstrate it is essential for distinct stages of both structural and functional maturation of excitatory spine synapses. Our data show that initially the Arp2/3 complex inhibits the formation of dendritic filopodia but that later during development, the Arp2/3 complex drives the morphological maturation from filopodia to typical spine morphology. Furthermore, we demonstrate that although the Arp2/3 complex is not required for key spine maturation steps, such as presynaptic contact and recruitment of MAGUK (membrane-associated guanylate kinase) scaffolding proteins or NMDA receptors, it is necessary for the recruitment of AMPA receptors. This latter process, also known as synapse unsilencing, is a final and essential step in the neurodevelopment of excitatory postsynaptic synaptogenesis, setting the stage for neuronal interconnectivity. These findings provide the first evidence that the Arp2/3 complex is directly involved in functional maturation of dendritic spines during the developmental period of spinogenesis. SIGNIFICANCE STATEMENT: Excitatory spine synapse formation (spinogenesis) is a poorly understood yet pivotal period of neurodevelopment that occurs within 2-3 weeks after birth. Neurodevelopmental disorders such as intellectual disability and autism are characterized by abnormal spine structure, which may arise from abnormal excitatory synaptogenesis. The initial stage of spinogenesis is thought to begin with the emergence of actin-rich dendritic filopodia that initiate contact with presynaptic axonal boutons. However, it remains enigmatic how actin cytoskeletal regulation directs dendritic filopodial emergence or their subsequent maturation into dendritic spines during development and on into adulthood. In this study, we provide the first evidence that the Arp2/3 complex, a key actin nucleator, is involved in distinct stages of spine formation and is required for synapse unsilencing.

Synaptic Actin Dysregulation, a Convergent Mechanism of Mental Disorders?

Actin polymerization governs activity-dependent modulation of excitatory synapses, including their morphology and functionality. It is clear from human genetics that neuropsychiatric and neurodevelopmental disturbances are multigenetic in nature, highlighting the need to better understand the critical neural pathways associated with these disorders and how they are altered by genetic risk alleles. One such signaling pathway that is heavily implicated by candidate genes for psychiatric and neurodevelopmental disorders are regulators of signaling to the actin cytoskeleton, suggesting that its disruption and the ensuring abnormalities of spine structures and postsynaptic complexes is a commonly affected pathway in brain disorders. This review will discuss recent experimental findings that strongly support genetic evidence linking the synaptic cytoskeleton to mental disorders, such as schizophrenia and autism spectrum disorders.

Actin Out: Regulation of the Synaptic Cytoskeleton.

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.

Actin-related protein2/3 complex regulates tight junctions and terminal differentiation to promote epidermal barrier formation.

The epidermis provides an essential seal from the external environment and retains fluids within the body. To form an effective barrier, cells in the epidermis must form tight junctions and terminally differentiate into cornified envelopes. Here, we demonstrate that the branched actin nucleator, the actin-related protein (Arp)2/3 complex, is unexpectedly required for both these activities. Loss of the ArpC3 subunit of the Arp2/3 complex resulted in minimal changes in the morphogenesis and architecture of this stratified squamous epithelium, but resulted in profound defects in its physiology. Mutant embryos did not develop an effective barrier to the external environment and died within hours of birth. We discovered two underlying causes for these effects. First, ArpC3 was essential for robust assembly and function of tight junctions, specialized cell-cell adhesions that restrict water loss in the epidermis. Second, there were defects in differentiation of the epidermis and the production of cornified envelopes, structures essential for barrier activity. Underlying this defect, we found that YAP was inappropriately active not only in the ArpC3 mutant tissue, but also in cultured cells. Inhibition of YAP activity rescued the differentiation and barrier defects caused by loss of ArpC3. These results demonstrate previously unappreciated roles for the Arp2/3 complex and highlight the functions of branched actin networks in a complex tissue.

Under lock and key: spatiotemporal regulation of WASP family proteins coordinates separate dynamic cellular processes.

WASP family proteins are nucleation promoting factors that bind to and activate the Arp2/3 complex in order to stimulate nucleation of branched actin filaments. The WASP family consists of WASP, N-WASP, WAVE1-3, WASH, and the novel family members WHAMM and JMY. Each of the family members contains a C-terminus responsible for their nucleation promoting activity and unique N-termini that allow for them to be regulated in a spatiotemporal manner. Upon activation they reorganize the cytoskeleton for different cellular functions depending on their subcellular localization and regulatory protein interactions. Emerging evidence indicates that WASH, WHAMM, and JMY have functions that require the coordination of both actin polymerization and microtubule dynamics. Here, we review the mechanisms of regulation for each family member and their associated in vivo functions including cell migration, vesicle trafficking, and neuronal development.

Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells.

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.

Disruption of Arp2/3 results in asymmetric structural plasticity of dendritic spines and progressive synaptic and behavioral abnormalities.

Despite evidence for a strong genetic contribution to several major psychiatric disorders, individual candidate genes account for only a small fraction of these disorders, leading to the suggestion that multigenetic pathways may be involved. Several known genetic risk factors for psychiatric disease are related to the regulation of actin polymerization, which plays a key role in synaptic plasticity. To gain insight into and test the possible pathogenetic role of this pathway, we designed a conditional knock-out of the Arp2/3 complex, a conserved final output for actin signaling pathways that orchestrates de novo actin polymerization. Here we report that postnatal loss of the Arp2/3 subunit ArpC3 in forebrain excitatory neurons leads to an asymmetric structural plasticity of dendritic spines, followed by a progressive loss of spine synapses. This progression of synaptic deficits corresponds with an evolution of distinct cognitive, psychomotor, and social disturbances as the mice age. Together, these results point to the dysfunction of actin signaling, specifically that which converges to regulate Arp2/3, as an important cellular pathway that may contribute to the etiology of complex psychiatric disorders.

Presynaptic Rac1 in the hippocampus selectively regulates working memory.

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.

Disruption of wave-associated Rac GTPase-activating protein (Wrp) leads to abnormal adult neural progenitor migration associated with hydrocephalus.

Hydrocephalus is the most common developmental disability and leading cause of brain surgery for children. Current treatments are limited to surgical intervention, as the factors that contribute to the initiation of hydrocephalus are poorly understood. Here, we describe the development of obstructive hydrocephalus in mice that are null for Wrp (Srgap3). Wrp is highly expressed in the ventricular stem cell niche, and it is a gene required for cytoskeletal organization and is associated with syndromic and psychiatric disorders in humans. During the postnatal period of progenitor cell expansion and ventricular wall remodeling, loss of Wrp results in the abnormal migration of lineage-tagged cells from the ventricular region into the corpus callosum. Within this region, mutant progenitors appear to give rise to abnormal astroglial cells and induce periventricular lesions and hemorrhage that leads to cerebral aqueductal occlusion. These results indicate that periventricular abnormalities arising from abnormal migration from the ventricular niche can be an initiating cause of noncommunicating hydrocephalus.

A Ventromedial Prefrontal-to-Lateral Entorhinal Cortex Pathway Modulates the Gain of Behavioral Responding During Threat.

BACKGROUND: The ability to correctly associate cues and contexts with threat is critical for survival, and the inability to do so can result in threat-related disorders such as posttraumatic stress disorder. The prefrontal cortex (PFC) and hippocampus are well known to play critical roles in cued and contextual threat memory processing. However, the circuits that mediate prefrontal-hippocampal modulation of context discrimination during cued threat processing are less understood. Here, we demonstrate the role of a previously unexplored projection from the ventromedial region of PFC (vmPFC) to the lateral entorhinal cortex (LEC) in modulating the gain of behavior in response to contextual information during threat retrieval and encoding. METHODS: We used optogenetics followed by in vivo calcium imaging in male C57/B6J mice to manipulate and monitor vmPFC-LEC activity in response to threat-associated cues in different contexts. We then investigated the inputs to, and outputs from, vmPFC-LEC cells using Rabies tracing and channelrhodopsin-assisted electrophysiology. RESULTS: vmPFC-LEC cells flexibly and bidirectionally shaped behavior during threat expression, shaping sensitivity to contextual information to increase or decrease the gain of behavioral output in response to a threatening or neutral context, respectively. CONCLUSIONS: Glutamatergic vmPFC-LEC cells are key players in behavioral gain control in response to contextual information during threat processing and may provide a future target for intervention in threat-based disorders.

Identification of new ciliary signaling pathways in the brain and insights into neurological disorders.

Primary cilia are conserved sensory hubs essential for signaling transduction and embryonic development. Ciliary dysfunction causes a variety of developmental syndromes with neurological features and cognitive impairment, whose basis mostly remains unknown. Despite connections to neural function, the primary cilium remains an overlooked organelle in the brain. Most neurons have a primary cilium; however, it is still unclear how this organelle modulates brain architecture and function, given the lack of any systemic dissection of neuronal ciliary signaling. Here, we present the first in vivo glance at the molecular composition of cilia in the mouse brain. We have adapted in vivo BioID (iBioID), targeting the biotin ligase BioID2 to primary cilia in neurons. We identified tissue-specific signaling networks enriched in neuronal cilia, including Eph/Ephrin and GABA receptor signaling pathways. Our iBioID ciliary network presents a wealth of neural ciliary hits that provides new insights into neurological disorders. Our findings are a promising first step in defining the fundamentals of ciliary signaling and their roles in shaping neural circuits and behavior. This work can be extended to pathological conditions of the brain, aiming to identify the molecular pathways disrupted in the brain cilium. Hence, finding novel therapeutic strategies will help uncover and leverage the therapeutic potential of the neuronal cilium.

Training-induced circuit-specific excitatory synaptogenesis in mice is required for effort control.

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC→DMS). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ-1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ-1 in the adult ACC→DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC→DMS neurons rescues or phenocopies the behaviors of the α2δ-1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion.

WRP/srGAP3 facilitates the initiation of spine development by an inverse F-BAR domain, and its loss impairs long-term memory.

The WAVE-associated Rac GAP, WRP, is thought to regulate key aspects of synapse development and function and may be linked to mental retardation in humans. WRP contains a newly described inverse F-BAR (IF-BAR) domain of unknown function. Our studies show that this domain senses/facilitates outward protrusions analogous to filopodia and that the molecular basis for this is likely explained by a convex lipid-binding surface on the WRP IF-BAR domain. In dendrites the IF-BAR domain of WRP forms a bud on the shaft from which precursors to spines emerge. Loss of WRP in vivo and in vitro results in reduced density of spines. In vivo this is primarily a loss of mushroom-shaped spines. Developmentally, WRP function is critical at the onset of spinogenesis, when dendritic filopodia are prevalent. Finally, because WRP is implicated in mental retardation, behaviors of WRP heterozygous and null mice have been evaluated. Results from these studies confirm that loss of WRP is linked to impaired learning and memory.

Bi-modal regulation of a formin by srGAP2.

The maintenance of rapid and efficient actin dynamics in vivo requires coordination of filament assembly and disassembly. This regulation requires temporal and spatial integration of signaling pathways by protein complexes. However, it remains unclear how these complexes form and then regulate the actin cytoskeleton. Here, we identify a srGAP2 and formin-like 1 (FMNL1, also known as FRL1 or FRLα) complex whose assembly is regulated by Rac signaling. Our data suggest srGAP2 regulates FMNL1 in two ways; 1) Rac-mediated activation of FMNL1 leads to the recruitment of srGAP2, which contains a Rac-specific GAP domain; 2) the SH3 domain of srGAP2 binds the formin homology 1 domain of FMNL1 to inhibit FMNL1-mediated actin severing. Thus, srGAP2 can efficiently terminate the upstream activating Rac signal while also opposing an important functional output of FMNL1, namely actin severing. We also show that FMNL1 and srGAP2 localize to the actin-rich phagocytic cup of macrophage-derived cells, suggesting the complex may regulate this Rac- and actin-driven process in vivo. We propose that after Rac-dependent activation of FMNL1, srGAP2 mediates a potent mechanism to limit the duration of Rac action and inhibit formin activity during rapid actin dynamics.

Regulation of the postsynaptic cytoskeleton: roles in development, plasticity, and disorders.

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during experience-dependent plasticity. This mini-symposium review will feature ongoing research into how spines are regulated by actin-signaling pathways during development and plasticity. It will also highlight evolving studies into how disruptions to these pathways might be functionally coupled to congenital disorders such as mental retardation.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling.

WAVE-1, which is also known as Scar, is a scaffolding protein that directs actin reorganization by relaying signals from the GTPase Rac to the Arp2/3 complex. Although the molecular details of WAVE activation by Rac have been described, the mechanisms by which these signals are terminated remain unknown. Here we have used tandem mass spectrometry to identify previously unknown components of the WAVE signalling network including WRP, a Rac-selective GTPase-activating protein. WRP binds directly to WAVE-1 through its Src homology domain 3 and specifically inhibits Rac function in vivo. Thus, we propose that WRP is a binding partner of WAVE-1 that functions as a signal termination factor for Rac.

Tripartite synaptomics: Cell-surface proximity labeling in vivo.

The astrocyte is a central glial cell and plays a critical role in the architecture and activity of neuronal circuits and brain functions through forming a tripartite synapse with neurons. Emerging evidence suggests that dysfunction of tripartite synaptic connections contributes to a variety of psychiatric and neurodevelopmental disorders. Furthermore, recent advancements with transcriptome profiling, cell biological and physiological approaches have provided new insights into the molecular mechanisms into how astrocytes control synaptogenesis in the brain. In addition to these findings, we have recently developed in vivo cell-surface proximity-dependent biotinylation (BioID) approaches, TurboID-surface and Split-TurboID, to comprehensively understand the molecular composition between astrocytes and neuronal synapses. These proteomic approaches have discovered a novel molecular framework for understanding the tripartite synaptic cleft that arbitrates neuronal circuit formation and function. Here, this short review highlights novel in vivo cell-surface BioID approaches and recent advances in this rapidly evolving field, emphasizing how astrocytes regulate excitatory and inhibitory synapse formation in vitro and in vivo.