Regulate Synapse Formation Research Articles

A biallelic mutation in CACNA2D2 associated with developmental and epileptic encephalopathy affects calcium channel-dependent as well as synaptic functions of α2δ-2.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α2δ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α2δ-2. Our data show that the corresponding p.R596P mutation in mouse α2δ-2 drastically decreases membrane expression and synaptic targeting of α2δ-2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies.

Mecp2 knock-out astrocytes affect synaptogenesis by interleukin 6 dependent mechanisms

Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.

Sensory deprivation arrests cellular and synaptic development of the night-vision circuitry in the retina

Experience regulates synapse formation and function across sensory circuits. How inhibitory synapses in the mammalian retina are sculpted by visual cues remains unclear. By use of a sensory deprivation paradigm, we find that visual cues regulate maturation of two GABA synapse types (GABAA and GABAC receptor synapses), localized across the axon terminals of rod bipolar cells (RBCs)-second-order retinal neurons integral to the night-vision circuit. Lack of visual cues causes GABAA synapses at RBC terminals to retain an immature receptor configuration with slower response profiles and prevents receptor recruitment at GABAC synapses. Additionally, the organizing protein for both these GABA synapses, LRRTM4, is not clustered at dark-reared RBC synapses. Ultrastructurally, the total number of ribbon-output/inhibitory-input synapses across RBC terminals remains unaltered by sensory deprivation, although ribbon synapse output sites are misarranged when the circuit develops without visual cues. Intrinsic electrophysiological properties of RBCs and expression of chloride transporters across RBC terminals are additionally altered by sensory deprivation. Introduction to normal 12-h light-dark housing conditions facilitates maturation of dark-reared RBC GABA synapses and restoration of intrinsic RBC properties, unveiling a new element of light-dependent retinal cellular and synaptic plasticity.

Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function.

Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. Here, we generate null alleles of TRMT9B and find it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, we find that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

Chemogenetic manipulation of astrocyte activity at the synapse- a gateway to manage brain disease.

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.

Postnatal expression of CD38 in astrocytes regulates synapse formation and adult social memory.

Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.

Regulation of RNG105/caprin1 dynamics by pathogenic cytoplasmic FUS and TDP-43 in neuronal RNA granules modulates synaptic loss

In neurodegenerative diseases, the condensation of FUS and TDP-43 with RNA granules in neurons is linked to pathology, including synaptic disorders. However, the effects of FUS and TDP-43 on RNA granule factors remain unclear. Here, using primary cultured neurons from the mouse cerebral cortex, we show that excess cytoplasmic FUS and TDP-43 accumulated in dendritic RNA granules, where they increased the dynamics of a scaffold protein RNG105/caprin1 and dissociated it from the granules. This coincided with reduced levels of mRNA and translation around the granules and synaptic loss in dendrites. These defects were suppressed by non-dissociable RNG105, suggesting that RNG105 dissociation mediated the defects. In contrast to the model where FUS and TDP-43 co-aggregate with RNA granule factors to repress their activity, our findings provide a novel pathogenic mechanism whereby FUS and TDP-43 dissociate RNA scaffold proteins from RNA granules which are required for local translation that regulates synapse formation.

Glioma epileptiform activity and progression are driven by IGSF3-mediated potassium dysregulation

Seizures are a frequent pathophysiological feature of malignant glioma. Recent studies implicate peritumoral synaptic dysregulation as a driver of brain hyperactivity and tumor progression; however, the molecular mechanisms that govern these phenomena remain elusive. Using scRNA-seq and intraoperative patient ECoG recordings, we show that tumors from seizure patients are enriched for gene signatures regulating synapse formation. Employing a human-to-mouse invivo functionalization pipeline to screen these genes, we identify IGSF3 as a mediator of glioma progression and dysregulated neural circuitry that manifests as spreading depolarization (SD). Mechanistically, we discover that IGSF3 interacts with Kir4.1 to suppress potassium buffering and found that seizure patients exhibit reduced expression of potassium handlers in proliferating tumor cells. Invivo imaging reveals that dysregulated synaptic activity emanates from the tumor-neuron interface, which we confirm in patients. Our studies reveal that tumor progression and seizures are enabled by ion dyshomeostasis and identify SD as a driver of disease.

Extracellular matrix and synapse formation.

The extracellular matrix (ECM) is a complex molecular network distributed throughout the extracellular space of different tissues as well as the neuronal system. Previous studies have identified various ECM components that play important roles in neuronal maturation and signal transduction. ECM components are reported to be involved in neurogenesis, neuronal migration, and axonal growth by interacting or binding to specific receptors. In addition, the ECM is found to regulate synapse formation, the stability of the synaptic structure, and synaptic plasticity. Here, we mainly reviewed the effects of various ECM components on synapse formation and briefly described the related diseases caused by the abnormality of several ECM components.

Amyloid precursor protein in Alzheimer's disease.

Amyloid precursor protein (APP) is a membrane protein expressed in several tissues. The occurrence of APP is predominant in synapses of nerve cells. It acts as a cell surface receptor and plays a vital role as a regulator of synapse formation, iron export and neural plasticity. It is encoded by the APP gene that is regulated by substrate presentation. APP is a precursor protein activated by proteolytic cleavage and thereby generating amyloid beta (Aβ) peptides which eventually form amyloid plaques that accumulate in Alzheimer's disease patients' brains. In this chapter, we highlight basic mechanism, structure, expression patterns and cleavage of amyloid plaques, and its diagnosis and potential treatment for Alzheimer's disease.

Hevin/Sparcl1 drives pathological pain through spinal cord astrocyte and NMDA receptor signaling.

High endothelial venule protein/SPARC-like 1 (hevin/Sparcl1) is an astrocyte-secreted protein that regulates synapse formation in the brain. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared with WT mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than WT mice from neuropathic pain after nerve injury. Intrathecal injection of WT hevin was sufficient to induce persistent mechanical allodynia in naive mice. In hevin-null mice with nerve injury, adeno-associated-virus-mediated (AAV-mediated) re-expression of hevin in glial fibrillary acidic protein-expressing (GFAP-expressing) spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin-potentiated N-Methyl-D-aspartic acid (NMDA) currents are mediated by GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing Ab against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin that was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal Ab may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.

Understanding global changes of the mouse brain proteome after vaginal infection with HSV-2 using a label-free shotgun approach.

Herpes simplex virus type 2 (HSV-2) is a common human pathogen that establishes lifelong latency in neurons of the nervous system. The number of severe central nervous system infections caused by the virus has increased recently. However, the pathogenesis of HSV-2 infection in the nervous system is not fully understood. Here, we demonstrated global proteomic changes in the brain tissue in BALB/c mice vaginally infected with HSV-2. Data are available via ProteomeXchange with identifier PXD034186. A total of 249 differentially expressed proteins were identified in infected brain tissue. The GO and KEGG enrichment analysis of these proteins indicated that they were mainly involved in the regulation of synapse formation and synaptic excitability. In addition, genes affecting autophagy, the development of other neurodegenerative diseases, and signaling pathways relevant to other neurologic diseases were identified. Additional experiments, comparing the brain tissue of asymptomatic and symptomatic mice showed a differential expression of proteins involved in synapse formation and synaptic transmission. Others were involved in autophagy, addiction, and signaling pathways of other neurologic diseases. These results suggest that changes in synaptic structure and function, as well as autophagy, may be related to the development of neurologic abnormalities that follow HSV-2 infection. We also identified a protein GluN2A encoded by Grin2a was continuously expressed at high levels after infection. We propose that GluN2A may be a key molecule in the pathogenesis of HSV-2-induced neurologic diseases.

Synaptic pathology in multiple sclerosis: a role for Nogo-A signaling in astrocytes?

Synaptic pathology in multiple sclerosis: a role for Nogo-A signaling in astrocytes?

Group I mGluRs in Therapy and Diagnosis of Parkinson's Disease: Focus on mGluR5 Subtype.

Metabotropic glutamate receptors (mGluRs; members of class C G-protein-coupled receptors) have been shown to modulate excitatory neurotransmission, regulate presynaptic extracellular glutamate levels, and modulate postsynaptic ion channels on dendritic spines. mGluRs were found to activate myriad signalling pathways to regulate synapse formation, long-term potentiation, autophagy, apoptosis, necroptosis, and pro-inflammatory cytokines release. A notorious expression pattern of mGluRs has been evident in several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and schizophrenia. Among the several mGluRs, mGluR5 is one of the most investigated types of considered prospective therapeutic targets and potential diagnostic tools in neurodegenerative diseases and neuropsychiatric disorders. Recent research showed mGluR5 radioligands could be a potential tool to assess neurodegenerative disease progression and trace respective drugs’ kinetic properties. This article provides insight into the group I mGluRs, specifically mGluR5, in the progression and possible therapy for PD.

Neurexins play a crucial role in cerebellar granule cell survival by organizing autocrine machinery for neurotrophins

Neurexins (NRXNs) are key presynaptic cell adhesion molecules that regulate synapse formation and function via trans-synaptic interaction with postsynaptic ligands. Here, we generate cerebellar granule cell (CGC)-specific Nrxn triple-knockout (TKO) mice for complete deletion of all NRXNs. Unexpectedly, most CGCs die in these mice, and this requirement for NRXNs for cell survival is reproduced in cultured CGCs. The axons of cultured Nrxn TKO CGCs that are not in contact with a postsynaptic structure show defects in the formation of presynaptic protein clusters and in action-potential-induced Ca2+ influxes. These cells also show impaired secretion of depolarization-induced, fluorescence-tagged brain-derived neurotrophic factor (BDNF) from their axons, and the cell-survival defect is rescued by the application of BDNF. These results suggest that CGC survival is maintained by autocrine neurotrophic factors and that NRXNs organize the presynaptic protein clusters and the autocrine neurotrophic-factor secretory machinery independent of contact with postsynaptic ligands.

PTPRM Is Critical for Synapse Formation Regulated by Zinc Ion.

In the nervous system, the trace metal ion zinc is required for normal mammalian brain development and physiology. Zinc homeostasis is essential for the control of physiological and pathophysiological brain functions. Synapses, the junctions between neurons, are the center of the brain’s information transmission. Zinc deficiency or excess leads to neurological disorders. However, it is still unclear whether and how zinc ion regulates synapse formation. Here, we investigated the effect of zinc on synapse formation in a cultured neuron system, and found that synapse formation and synaptic transmission were regulated by zinc ions. Finally, we identified that PTPRM is the key gene involved in synapse formation regulated by zinc ions. This study provides a new perspective to understanding the regulation of brain function by zinc ion.

Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis.

During mammalian brain development, how different astrocytes are specified from progenitor cells is not well understood. In particular, whether astrocyte progenitor cells (APCs) start as a relatively homogenous population or whether there is early heterogeneity remains unclear. Here, we have dissected subpopulations of embryonic mouse forebrain progenitors using single-cell transcriptome analyses. Our sequencing data revealed two molecularly distinct APC subgroups at the start of gliogenesis from both dorsal and ventral forebrains. The two APC subgroups were marked, respectively, by specific expression of Sparc and Sparcl1, which are known to function in mature astrocytes with opposing activities for regulating synapse formation. Expression analyses showed that SPARC and SPARCL1 mark APC subgroups that display distinct temporal and spatial patterns, correlating with major waves of astrogliogenesis during development. Our results uncover an early molecular divergence of APCs in the mammalian brain and provide a useful transcriptome resource for the study of glial cell specification.

Insights into Human-Induced Pluripotent Stem Cell-Derived Astrocytes in Neurodegenerative Disorders.

Most neurodegenerative disorders have complex and still unresolved pathology characterized by progressive neuronal damage and death. Astrocytes, the most-abundant non-neuronal cell population in the central nervous system, play a vital role in these processes. They are involved in various functions in the brain, such as the regulation of synapse formation, neuroinflammation, and lactate and glutamate levels. The development of human-induced pluripotent stem cells (iPSCs) reformed the research in neurodegenerative disorders allowing for the generation of disease-relevant neuronal and non-neuronal cell types that can help in disease modeling, drug screening, and, possibly, cell transplantation strategies. In the last 14 years, the differentiation of human iPSCs into astrocytes allowed for the opportunity to explore the contribution of astrocytes to neurodegenerative diseases. This review discusses the development protocols and applications of human iPSC-derived astrocytes in the most common neurodegenerative conditions.

A Progressive Build-up of Perineuronal Nets in the Somatosensory Cortex Is Associated with the Development of Chronic Pain in Mice.

Chronic pain is sustained by a maladaptive form of neuronal plasticity occurring in all stations of the pain neuraxis, including cortical regions of the pain matrix. We report that chronic inflammatory pain induced by unilateral injection of complete Freund's adjuvant (CFA) in the hindpaw of male mice was associated with a progressive build-up of perineuronal nets (PNNs) in the contralateral somatosensory cortex (SSC), medial prefrontal cortex (mPFC), and reticular thalamic nucleus. In the SSC, the density of PNNs labeled by Wisteria floribunda agglutinin (WFA) was increased at both 3 and 7 d following CFA injection, but only after 7 d in the mPFC. The number of parvalbumin (PV)-positive interneurons enwrapped by WFA+/PNNs was also increased in all three brain regions of mice injected with CFA. Remarkably, PNN degradation induced by intracortical infusion of chondroitinase-ABC significantly reduced mechanical and thermal pain, and also reversed the increased frequency of IPSCs recorded in layer 5 pyramidal neurons of the contralateral SSC in CFA-injected mice. These findings suggest a possible relationship between cortical PNNs and nociceptive sensitization, and support the hypothesis that PNNs maintain their plasticity in the adult life and regulate cortical responses to sensory inputs.SIGNIFICANCE STATEMENT The brain extracellular matrix not only provides structural support, but also regulates synapse formation and function, and modulates neuronal excitability. We found that chronic inflammatory pain in mice enhances the density of perineuronal nets (PNNs) in the somatosensory cortex and medial prefrontal cortex. Remarkably, enzymatic degradation of PNNs in the somatosensory cortex caused analgesia and reversed alterations of inhibitory synaptic transmission associated with chronic pain. These findings disclose a novel mechanism of nociceptive sensitization and support a role for PNNs in mechanisms of neuronal plasticity in the adult brain.

Astrocyte-neuron crosstalk through Hedgehog signaling mediates cortical synapse development.

SUMMARYNeuron-glia interactions play a critical role in the regulation of synapse formation and circuit assembly. Here we demonstrate that canonical Sonic hedgehog (Shh) pathway signaling in cortical astrocytes acts to coordinate layer-specific synaptic connectivity. We show that the Shh receptor Ptch1 is expressed by cortical astrocytes during development and that Shh signaling is necessary and sufficient to promote the expression of genes involved in regulating synaptic development and layer-enriched astrocyte molecular identity. Loss of Shh in layer V neurons reduces astrocyte complexity and coverage by astrocytic processes in tripartite synapses; conversely, cell-autonomous activation of Shh signaling in astrocytes promotes cortical excitatory synapse formation. Furthermore, Shh-dependent genes Lrig1 and Sparc distinctively contribute to astrocyte morphology and synapse formation. Together, these results suggest that Shh secreted from deep-layer cortical neurons acts to specialize the molecular and functional features of astrocytes during development to shape circuit assembly and function.