Autism-associated variants of neuroligin 4X impair synaptogenic activity by various molecular mechanisms

Abstract Background: Several genetic alterations, including point mutations and copy number variations in NLGN genes have been associated with psychiatric disorders, such as autism spectrum disorder (ASD) and X-linked mental retardation (XLMR). NLGN genes encode neuroligin (NL) proteins, which are adhesion molecules that are important for proper synaptic formation and maturation. Previously, we and others found that the expression level of murine NL1 is regulated by proteolytic processing in a synaptic activity-dependent manner. Methods: In this study, we analyzed the effects of missense variants associated with ASD and XLMR on the metabolism and function of NL4X, a protein which is encoded by the NLGN4X gene and is expressed only in humans, using cultured cells, primary neurons from rodents and human induced pluripotent stem cell-derived neurons. Results: NL4X was found to undergo proteolytic processing in human neuronal cells. Almost all NL4X variants caused a substantial decrease in the levels of mature NL4X and its synaptogenic activity in a heterologous culture system. Intriguingly, the L593F variant of NL4X accelerated the proteolysis of mature NL4X proteins located on the cell surface. In contrast, other variants decreased the cell-surface trafficking of NL4X. Notably, protease inhibitors as well as chemical chaperones rescued the expression of mature NL4X. Limitations: Our study did not reveal whether these dysfunctional phenotypes occurred in individuals carrying NLGN4X variant. Moreover, though these pathological mechanisms could be exploited as potential drug targets for ASD, it remains unclear whether these compounds would have beneficial effects on in ASD model animals and patients. Conclusions: These data suggest that reduced amounts of the functional NL4X protein on the cell surface is a common mechanism by which point mutants of the NL4X protein cause psychiatric disorders, although different molecular mechanisms are thought to be involved. Furthermore, these results highlight that the precision medicine approach based on genetic and cell biological analyses is important for the development of therapeutics for psychiatric disorders.

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Autism-associated variants of neuroligin 4X impair synaptogenic activity by various molecular mechanisms

10.21203/rs.2.19938/v1 ◽

2020 ◽

Author(s):

Takafumi Yumoto ◽

Misaki Kimura ◽

Ryota Nagatomo ◽

Hirotaka Watanabe ◽

Hideyuki Okano ◽

...

Keyword(s):

Cell Surface ◽

Psychiatric Disorders ◽

Molecular Mechanisms ◽

Genetic Alterations ◽

Autism Spectrum ◽

Copy Number Variations ◽

Proteolytic Processing ◽

Synaptic Activity ◽

Common Mechanism ◽

Dependent Manner

Abstract Several genetic alterations, including point mutations and copy number variations in NLGN genes have been associated with psychiatric disorders, such as autism spectrum disorder (ASD) and X-linked mental retardation (XLMR). NLGN genes encode neuroligin (NL) proteins, which are adhesion molecules that are important for proper synaptic formation and maturation. Previously, we and others found that the expression level of murine NL1 is regulated by proteolytic processing in a synaptic activity-dependent manner. In this study, we analyzed the effects of missense variants associated with ASD and XLMR on the metabolism and function of NL4X, a protein which is encoded by the NLGN4X gene and is expressed only in humans. NL4X was found to undergo proteolytic processing in human neuronal cells. Almost all NL4X variants caused a substantial decrease in the levels of mature NL4X and its synaptogenic activity in a heterologous culture system. Intriguingly, the L593F variant of NL4X accelerated the proteolysis of mature NL4X proteins located on the cell surface. In contrast, other variants decreased the cell-surface trafficking of NL4X. Notably, protease inhibitors as well as chemical chaperones rescued the expression of mature NL4X. These data suggest that reduced amounts of the functional NL4X protein on the cell surface is a common mechanism by which point mutants of the NL4X protein cause psychiatric disorders, although different molecular mechanisms are thought to be involved. Furthermore, these results highlight that the precision medicine approach based on genetic and cell biological analyses is important for the development of therapeutics for psychiatric disorders.

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Neuroligin3 Splice Isoforms Shape Mouse Hippocampal Inhibitory Synaptic Function

10.1101/2020.01.22.915801 ◽

2020 ◽

Author(s):

Motokazu Uchigashima ◽

Ming Leung ◽

Takuya Watanabe ◽

Amy Cheung ◽

Masahiko Watanabe ◽

...

Keyword(s):

Synaptic Transmission ◽

Molecular Mechanisms ◽

Splice Variants ◽

Autism Spectrum ◽

Synaptic Function ◽

Synaptic Activity ◽

Sequencing Analysis ◽

Dependent Manner ◽

Splice Isoforms ◽

Inhibitory Synaptic Transmission

ABSTRACTSynapse formation is a dynamic process essential for neuronal circuit development and maturation. At the synaptic cleft, trans-synaptic protein-protein interactions constitute major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity. Dysregulation of the E-I balance has been implicated in neurodevelopmental disorders including autism spectrum disorders. However, the molecular mechanisms underlying E-I balance remain to be elucidated. Here, we investigate Neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that Nlgn3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. Distinct subcellular localization patterns of Nlgn3 isoforms contribute to the functional differences observed among splice variants. Finally, single-cell sequencing analysis reveals that Nlgn1 and Nlgn3 are the major Nlgn genes and that expression of Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons.

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Cross-species Transcriptomes Reveal Species-specific and Shared Molecular Adaptations for Plants Development on Iron-rich Rocky Outcrops Soils

10.21203/rs.3.rs-929174/v1 ◽

2021 ◽

Author(s):

Mariana Costa Dias ◽

Cecílio Caldeira ◽

Markus Gastauer ◽

Silvio Ramos ◽

Guilherme Oliveira

Keyword(s):

Circadian Rhythm ◽

Differential Expression ◽

Native Species ◽

Molecular Mechanisms ◽

Light Stimulus ◽

Differential Expression Analysis ◽

Common Mechanism ◽

Dependent Manner ◽

Rocky Outcrops ◽

Species Specific

Abstract BackgroundCanga is the Brazilian term for the savanna-like vegetation harboring several endemic species on iron-rich rocky outcrops, usually considered for mining activities. Parkia platycephala Benth. and Stryphnodendron pulcherrimum (Willd.) Hochr. naturally occur in the cangas of Serra dos Carajás (eastern Amazonia, Brazil) and the surrounding forest, indicating high phenotypic plasticity. The morphological and physiological mechanisms of the plants’ establishment in the canga environment are well studied, but the molecular adaptative responses are still unknown. We aimed to identify molecular mechanisms that allow the establishment of these plants in the canga environment.ResultsPlants were grown in canga and forest substrates collected in the Carajás Mineral Province. RNA was extracted from pooled leaf tissue, and RNA-seq paired-end reads were assembled into representative transcriptomes for P. platycephala and S. pulcherrimum containing 31,728 and 31,311 primary transcripts, respectively. We identified both species-specific and core molecular responses in plants grown in the canga substrate using differential expression analyses. In the species-specific analysis, we identified 1,112 and 838 differentially expressed genes for P. platycephala and S. pulcherrimum, respectively. Enrichment analyses showed unique biological processes and metabolic pathways affected for each species. Comparative differential expression analysis was based on shared single-copy orthologs. The overall pattern of ortholog expression was species-specific. Even so, almost 300 altered genes were identified between plants in canga and forest substrates, responding the same way in both species. The genes were functionally associated with the response to light stimulus and the circadian rhythm pathway.ConclusionsPlants possess species-specific adaptative responses to cope with the substrates. Our results also suggest that plants adapted to both canga and forest environments can adjust the circadian rhythm in a substrate-dependent manner. The circadian clock gene modulation might be a central mechanism regulating the plants’ development in the canga substrate in the studied legume species. The mechanism may be shared as a common mechanism to abiotic stress compensation in other native species.

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ASTN2 modulates synaptic strength by trafficking and degradation of surface proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809382115 ◽

2018 ◽

Vol 115 (41) ◽

pp. E9717-E9726 ◽

Cited By ~ 6

Author(s):

Hourinaz Behesti ◽

Taylor R. Fore ◽

Peter Wu ◽

Zachi Horn ◽

Mary Leppert ◽

...

Keyword(s):

Surface Protein ◽

Surface Expression ◽

Autism Spectrum ◽

Copy Number Variations ◽

Surface Proteins ◽

Synaptic Activity ◽

Synaptic Proteins ◽

Immunogold Electron Microscopy ◽

Dynamic Regulation ◽

Cerebellar Purkinje Cells

Surface protein dynamics dictate synaptic connectivity and function in neuronal circuits. ASTN2, a gene disrupted by copy number variations (CNVs) in neurodevelopmental disorders, including autism spectrum, was previously shown to regulate the surface expression of ASTN1 in glial-guided neuronal migration. Here, we demonstrate that ASTN2 binds to and regulates the surface expression of multiple synaptic proteins in postmigratory neurons by endocytosis, resulting in modulation of synaptic activity. In cerebellar Purkinje cells (PCs), by immunogold electron microscopy, ASTN2 localizes primarily to endocytic and autophagocytic vesicles in the cell soma and in subsets of dendritic spines. Overexpression of ASTN2 in PCs, but not of ASTN2 lacking the FNIII domain, recurrently disrupted by CNVs in patients, including in a family presented here, increases inhibitory and excitatory postsynaptic activity and reduces levels of ASTN2 binding partners. Our data suggest a fundamental role for ASTN2 in dynamic regulation of surface proteins by endocytic trafficking and protein degradation.

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Wnt5a signaling promotes apical and basolateral polarization of single epithelial cells

Molecular Biology of the Cell ◽

10.1091/mbc.e13-07-0357 ◽

2013 ◽

Vol 24 (23) ◽

pp. 3764-3774 ◽

Cited By ~ 17

Author(s):

Hidetoshi Gon ◽

Katsumi Fumoto ◽

Yonson Ku ◽

Shinji Matsumoto ◽

Akira Kikuchi

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Molecular Mechanisms ◽

Type I Collagen ◽

Single Cells ◽

Surface Region ◽

Type I ◽

Dependent Manner ◽

Intestinal Epithelial ◽

Rac1 Activity

Single epithelial-derived tumor cells have been shown to induce apical and basolateral (AB) polarity by expression of polarization-related proteins. However, physiological cues and molecular mechanisms for AB polarization of single normal epithelial cells are unclear. When intestinal epithelial cells 6 (IEC6 cells) were seeded on basement membrane proteins (Matrigel), single cells formed an F-actin cap on the upper cell surface, where apical markers accumulated, and a basolateral marker was localized to the rest of the cell surface region, in a Wnt5a signaling–dependent manner. However, these phenotypes were not induced by type I collagen. Rac1 activity in the noncap region was higher than that in the cap region, whereas Rho activity increased toward the cap region. Wnt5a signaling activated and inhibited Rac1 and RhoA, respectively, independently through Tiam1 and p190RhoGAP-A, which formed a tertiary complex with Dishevelled. Furthermore, Wnt5a signaling through Rac1 and RhoA was required for cystogenesis of IEC6 cells. These results suggest that Wnt5a promotes the AB polarization of IEC6 cells through regulation of Rac and Rho activities in a manner dependent on adhesion to specific extracellular matrix proteins.

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Associations between Familial Rates of Psychiatric Disorders and De Novo Genetic Mutations in Autism

Autism Research and Treatment ◽

10.1155/2017/9371964 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Kyleen Luhrs ◽

Tracey Ward ◽

Caitlin M. Hudac ◽

Jennifer Gerdts ◽

Holly A. F. Stessman ◽

...

Keyword(s):

Psychiatric Disorders ◽

Copy Number ◽

Depressive Disorders ◽

De Novo ◽

Familial Risk ◽

Autism Spectrum ◽

Copy Number Variations ◽

Genetic Etiology ◽

Additive Contribution ◽

Familial Risk Factors

The purpose of this study was to examine the confluence of genetic and familial risk factors in children with Autism Spectrum Disorder (ASD) with distinct de novo genetic events. We hypothesized that gene-disrupting mutations would be associated with reduced rates of familial psychiatric disorders relative to structural mutations. Participants included families of children with ASD in four groups: de novo duplication copy number variations (DUP, n=62), de novo deletion copy number variations (DEL, n=74), de novo likely gene-disrupting mutations (LGDM, n=267), and children without a known genetic etiology (NON, n=2111). Familial rates of psychiatric disorders were calculated from semistructured interviews. Results indicated overall increased rates of psychiatric disorders in DUP families compared to DEL and LGDM families, specific to paternal psychiatric histories, and particularly evident for depressive disorders. Higher rates of depressive disorders in maternal psychiatric histories were observed overall compared to paternal histories and higher rates of anxiety disorders were observed in paternal histories for LGDM families compared to DUP families. These findings support the notion of an additive contribution of genetic etiology and familial factors are associated with ASD risk and highlight critical need for continued work targeting these relationships.

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ASTN2 modulates synaptic strength by trafficking and degradation of surface proteins

10.1101/337618 ◽

2018 ◽

Author(s):

Hourinaz Behesti ◽

Taylor Fore ◽

Peter Wu ◽

Zachi Horn ◽

Mary Leppert ◽

...

Keyword(s):

Surface Protein ◽

Surface Expression ◽

Autism Spectrum ◽

Copy Number Variations ◽

Surface Proteins ◽

Synaptic Activity ◽

Synaptic Proteins ◽

Dynamic Regulation ◽

Cerebellar Purkinje Cells ◽

And Function

AbstractSurface protein dynamics dictate synaptic connectivity and function in neuronal circuits. ASTN2, a gene disrupted by copy number variations (CNVs) in neurodevelopmental disorders, including autism spectrum, was previously shown to regulate the surface expression of ASTN1 in glial-guided neuronal migration. Here, we demonstrate that ASTN2 binds to and regulates the surface expression of multiple synaptic proteins in post-migratory neurons by endocytosis, resulting in modulation of synaptic activity. In cerebellar Purkinje cells (PCs), by immuno-gold electron microscopy, ASTN2 localizes primarily to endocytic and autophagocytic vesicles in the cell soma and in subsets of dendritic spines. Overexpression of ASTN2 in PCs, but not of ASTN2 lacking the FNIII-domain commonly disrupted by CNVs in patients including in a family presented here, increases inhibitory and excitatory postsynaptic activity and reduces levels of ASTN2 binding partners. Our data suggest a fundamental role for ASTN2 in dynamic regulation of surface proteins by endocytic trafficking and protein degradation.

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Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms21145101 ◽

2020 ◽

Vol 21 (14) ◽

pp. 5101

Author(s):

Jyoji Morise ◽

Saki Yamamoto ◽

Ryosuke Midorikawa ◽

Kogo Takamiya ◽

Motohiro Nonaka ◽

...

Keyword(s):

Cell Surface ◽

Glutamate Receptor ◽

Expression Patterns ◽

Surface Expression ◽

Glycosylation Site ◽

Synaptic Activity ◽

Cell Surface Expression ◽

Dependent Manner ◽

Distinct Cell

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1–4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits’ ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

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Neuroligin3 splice isoforms shape inhibitory synaptic function in the mouse hippocampus

Journal of Biological Chemistry ◽

10.1074/jbc.ac120.012571 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8589-8595 ◽

Cited By ~ 3

Author(s):

Motokazu Uchigashima ◽

Ming Leung ◽

Takuya Watanabe ◽

Amy Cheung ◽

Timmy Le ◽

...

Keyword(s):

Synaptic Transmission ◽

Single Cell ◽

Pyramidal Neurons ◽

Autism Spectrum ◽

Synaptic Function ◽

Synaptic Activity ◽

Dependent Manner ◽

Splice Isoforms ◽

Inhibitory Synaptic Transmission ◽

Ca1 Pyramidal Neurons

Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein–protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform–dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus.

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Whole Exome Sequencing Reveals a Novel AUTS2 In-Frame Deletion in a Boy with Global Developmental Delay, Absent Speech, Dysmorphic Features, and Cerebral Anomalies

Genes ◽

10.3390/genes12020229 ◽

2021 ◽

Vol 12 (2) ◽

pp. 229

Author(s):

Pietro Palumbo ◽

Ester Di Muro ◽

Maria Accadia ◽

Mario Benvenuto ◽

Marilena Carmela Di Giacomo ◽

...

Keyword(s):

Corpus Callosum ◽

Exome Sequencing ◽

Developmental Delay ◽

Whole Exome Sequencing ◽

Molecular Mechanisms ◽

Autism Spectrum ◽

Copy Number Variations ◽

Cerebellar Vermis ◽

Global Developmental Delay ◽

Whole Exome

Neurodevelopmental disorders (NDDs) are a group of highly prevalent, clinically and genetically heterogeneous pediatric disorders comprising, according to the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-V), intellectual disability, developmental delay, autism spectrum disorders, and other neurological and cognitive disorders manifesting in the developmental age. To date, more than 1000 genes have been implicated in the etiopathogenesis of NNDs. Among them, AUTS2 (OMIM # 607270) encodes a protein involved in neural migration and neuritogenesis, and causes NNDs with different molecular mechanisms including copy number variations, single or multiple exonic deletion and single nucleotide variants. We describes a 9-year-old boy with global developmental delay, absent speech, minor craniofacial anomalies, hypoplasia of the cerebellar vermis and thinning of the corpus callosum, resulted carrier of the de novo AUTS2 c.1603_1626del deletion at whole exome sequencing (WES) predicted to cause the loss of eight amino acids [p.(His535_Thr542del)]. Notably, our patient is the first reported so far in medical literature carrying an in-frame deletion and the first in which absent language, hypoplasia of the cerebellar vermis and thinning of the corpus callosum has been observed thus useful to expand the molecular spectrum of AUTS2 pathogenic variants and to broaden our knowledge on the clinical phenotype associated.

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