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1.
Methods Mol Biol ; 2576: 437-451, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152208

RESUMO

Single-molecule localization microscopy (SMLM) opened new possibilities to study the spatial arrangement of molecular distribution and disease-associated redistribution at a previously unprecedented resolution that was not achievable with optical microscopy approaches. Recent discoveries based on SMLM techniques uncovered specific nanoscale organizational principles of signaling proteins in several biological systems including the chemical synapses in the brain. Emerging data suggest that the spatial arrangement of the molecular players of the endocannabinoid system is also precisely regulated at the nanoscale level in synapses and in other neuronal and glial subcellular compartments. The precise nanoscale distribution pattern is likely to be important to subserve several specific signaling functions of this important messenger system in a cell-type- and subcellular domain-specific manner.STochastic Optical Reconstruction Microscopy (STORM) is an especially suitable SMLM modality for cell-type-specific nanoscale molecular imaging due to its compatibility with traditional diffraction-limited microscopy approaches and classical staining methods. Here, we describe a detailed protocol for STORM imaging in mouse brain tissue samples with a focus on the CB1 cannabinoid receptor, one of the most abundant synaptic receptors in the brain. We also summarize important conceptual and methodical details that are essential for the valid interpretation of single-molecule localization microscopy data.


Assuntos
Endocanabinoides , Microscopia , Animais , Endocanabinoides/metabolismo , Camundongos , Microscopia/métodos , Receptores de Canabinoides , Receptores de Neurotransmissores , Sinapses/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-36029930

RESUMO

Deterioration of inhibitory synapse may be an essential neurological basis underlying abnormal social behaviours. Manipulations that regulate GABAergic transmission are associated with improved behavioural phenotypes in sociability. The synaptic protein, Ephrin-B2 (EB2), plays an important role in the maintenance and reconfiguration of inhibitory synapses in the medial prefrontal cortex (mPFC). However, the inhibitory cell-type specific role of EB2 in the pathophysiology and treatment of social deficits remains unknown. As expected, we revealed that tdTomato-expressing cells were only found in GABAergic neurons instead of excitatory neurons in transgenic EB2-vGATCre mice. This result indicated that depletion of EB2 would occur in those neurons, which further contribute to social deficits. In addition, specific over-expression of mPFC EB2 restored the defective social behaviour abnormalities. These results suggest that the effect of EB2 on social deficits is anatomically and cell-type specific. More importantly, the global upregulation of HDAC4 expression was found in EB2-vGATCre mice. Significant subcellular nuclear shuttling of HDAC4 in vGAT+ neurons was examined and quantified, suggesting a role of nuclear HDAC4 in mediating the mechanism underlying EB2 impairment in vGAT+ neurons. Treatment with LMK235 led to a remarkable rescue of social deficits, thus our data revealed a new domain for the potential utility of HDAC targeting agents to treat social deficits. In conclusion, these results not only revealed a novel molecular mechanism underlying the pathophysiology of social deficits, but also suggested a potential intervention avenue for the treatment of social deficits.


Assuntos
Efrina-B2 , Histona Desacetilases , Animais , Camundongos , Proteínas de Transporte , Efrina-B2/metabolismo , Neurônios GABAérgicos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Camundongos Transgênicos , Mutação , Sinapses/metabolismo
3.
J Exp Med ; 220(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36378226

RESUMO

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Enzima de Conversão de Angiotensina 2 , SARS-CoV-2 , Peptidil Dipeptidase A/metabolismo , Sinapses/metabolismo , Ligação Proteica
4.
Synapse ; 77(1): e22253, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36121749

RESUMO

Anorexia nervosa (AN) is a mental illness with the highest rates of mortality and relapse, and no approved pharmacological treatment. Using an animal model of AN, called activity-based anorexia (ABA), we showed earlier that a single intraperitoneal injection of ketamine at a dose of 30 mg/kg (30mgKET), but not 3 mg/kg (3mgKET), has a long-lasting effect upon adolescent females of ameliorating anorexia-like symptoms through the following changes: enhanced food consumption and body weight; reduced running and anxiety-like behavior. However, there were also individual differences in the drug's efficacy. We hypothesized that individual differences in ketamine's ameliorative effects involve drebrin A, an F-actin-binding protein known to be required for the activity-dependent trafficking of NMDA receptors (NMDARs). We tested this hypothesis by electron microscopic quantifications of drebrin A immunoreactivity at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (GABA-IN) in deep layer 1 of prefrontal cortex (PFC) of these mice. Results reveal that (1) the areal density of excitatory synapses on GABA-IN is greater for the 30mgKET group than the 3mgKET group; (2) the proportion of drebrin A+ excitatory synapses is greater for both PN and GABA-IN of 30mgKET than 3mgKET group. Correlation analyses with behavioral measurements revealed that (3) 30mgKET's protection is associated with reduced levels of drebrin A in the cytoplasm of GABA-IN and higher levels at extrasynaptic membranous sites of PN and GABA-IN; (5) altogether pointing to 30mgKET-induced homeostatic plasticity that engages drebrin A at excitatory synapses of both PN and GABA-IN.


Assuntos
Anorexia Nervosa , Ketamina , Camundongos , Feminino , Animais , Ketamina/farmacologia , Anorexia Nervosa/tratamento farmacológico , Anorexia Nervosa/metabolismo , Anorexia/tratamento farmacológico , Anorexia/metabolismo , Individualidade , Sinapses/metabolismo , Modelos Animais de Doenças , Córtex Pré-Frontal/metabolismo , Citoplasma/metabolismo , Ácido gama-Aminobutírico/metabolismo
5.
Behav Brain Res ; 437: 114104, 2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36100011

RESUMO

Post-stroke depression (PSD) is a common neuropsychiatric complication of stroke, which seriously affects the quality of life and prognosis of patients. Nevertheless, the pathogenesis of PSD remains unclear. In our study, a PSD rat model was established by chronic restraint stress (CRS) combined with middle cerebral artery occlusion (MCAO). Depressive and anxiety-like behaviors were tested, as well as Neuronal loss and Apoptosis. The expression of synapse and p38 MAPK signaling pathway -relevant proteins was detected. Our data indicated that CRS combined with MCAO could induce depression-like and anxiety-like behaviors, which led to neuronal damage, apoptosis, and cellular loss in the left parietal cortex and left hippocampus. Furthermore, CRS combined with MCAO decreased synaptic plasticity in the parietal cortex and left hippocampus. We found that CRS combined with MCAO had activated the p38 MAPK signaling pathway, and decreased the expression of pathway-related proteins MKK6 and MKK3. These results suggested that CRS combined with MCAO could lead to depression-like behavior via neuronal damage, apoptosis and reduced synaptic plasticity, which might be related to the activation of the p38 MAPK pathway. Therefore, it provides novel ideas for the research on the intervention and prevention mechanisms of PSD.


Assuntos
Arteriopatias Oclusivas , Depressão , Infarto da Artéria Cerebral Média , Estresse Psicológico , Acidente Vascular Cerebral , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Ratos , Depressão/etiologia , Depressão/metabolismo , Depressão/psicologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/psicologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Qualidade de Vida , Ratos Sprague-Dawley , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/psicologia , Arteriopatias Oclusivas/etiologia , Arteriopatias Oclusivas/metabolismo , Sinapses/metabolismo , Transdução de Sinais , Restrição Física/efeitos adversos , Restrição Física/fisiologia , Restrição Física/psicologia , Doença Crônica , Estresse Psicológico/etiologia , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologia , Apoptose , Ansiedade/etiologia , Ansiedade/metabolismo , Ansiedade/psicologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Neurônios/metabolismo , Neurônios/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
6.
Front Immunol ; 13: 983569, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36405708

RESUMO

Background: Synapse-associated proteins (SAPs) play important roles in central nervous system (CNS) tumors. Recent studies have reported that γ-aminobutyric acidergic (GABAergic) synapses also play critical roles in the development of gliomas. However, biomarkers of GABAergic synapses in low-grade gliomas (LGGs) have not yet been reported. Methods: mRNA data from normal brain tissue and gliomas were obtained from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases, respectively. A validation dataset was also obtained from the Chinese Glioma Genome Atlas (CGGA) database. The expression patterns of GABAergic synapse-related genes (GSRGs) were evaluated with difference analysis in LGGs. Then, a GABAergic synapse-related risk signature (GSRS) was constructed with least absolute shrinkage and selection operator (LASSO) Cox regression analysis. According to the expression value and coefficients of identified GSRGs, the risk scores of all LGG samples were calculated. Univariate and multivariate Cox regression analyses were conducted to evaluate related risk scores for prognostic ability. Correlations between characteristics of the tumor microenvironment (TME) and risk scores were explored with single-sample gene set enrichment analysis (ssGSEA) and immunity profiles in LGGs. The GSRS-related pathways were investigated by gene set variation analysis (GSVA). Real-time PCR and the Human Protein Atlas (HPA) database were applied to explore related expression of hub genes selected in the GSRS. Results: Compared with normal brain samples, 25 genes of 31 GSRGs were differentially expressed in LGG samples. A constructed five-gene GSRS was related to clinicopathological features and prognosis of LGGs by the LASSO algorithm. It was shown that the risk score level was positively related to the infiltrating level of native CD4 T cells and activated dendritic cells. GSVA identified several cancer-related pathways associated with the GSRS, such as P53 pathways and the JAK-STAT signaling pathway. Additionally, CA2, PTEN, OXTR, and SLC6A1 (hub genes identified in the GSRS) were regarded as the potential predictors in LGGs. Conclusion: A new five-gene GSRS was identified and verified by bioinformatics methods. The GSRS provides a new perspective in LGG that may contribute to more accurate prediction of prognosis of LGGs.


Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Prognóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Ácido gama-Aminobutírico/metabolismo , Sinapses/metabolismo , Microambiente Tumoral/genética
7.
Biomolecules ; 12(11)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36358985

RESUMO

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequent causes of familial Parkinson's Disease (PD), an increasingly prevalent neurodegenerative disease that affects basal ganglia circuitry. The cellular effects of the G2019S mutation in the LRRK2 gene, the most common pathological mutation, have not been thoroughly investigated. In this study we used middle-aged mice carrying the LRRK2-G2019S mutation (G2019S mice) to identify potential alterations in the neurophysiological properties and characteristics of glutamatergic synaptic transmission in basal ganglia output neurons, i.e., substantia nigra pars reticulata (SNr) GABAergic neurons. We found that the intrinsic membrane properties and action potential properties were unaltered in G2019S mice compared to wild-type (WT) mice. The spontaneous firing frequency was similar, but we observed an increased regularity in the firing of SNr neurons recorded from G2019S mice. We examined the short-term plasticity of glutamatergic synaptic transmission, and we found an increased paired-pulse depression in G2019S mice compared to WT mice, indicating an increased probability of glutamate release in SNr neurons from G2019S mice. We measured synaptic transmission mediated by NMDA receptors and we found that the kinetics of synaptic responses mediated by these receptors were unaltered, as well as the contribution of the GluN2B subunit to these responses, in SNr neurons of G2019S mice compared to WT mice. These results demonstrate an overall maintenance of basic neurophysiological and synaptic characteristics, and subtle changes in the firing pattern and in glutamatergic synaptic transmission in basal ganglia output neurons that precede neurodegeneration of dopaminergic neurons in the LRRK2-G2019S mouse model of late-onset PD.


Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Parte Reticular da Substância Negra , Camundongos , Animais , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Parte Reticular da Substância Negra/metabolismo , Parte Reticular da Substância Negra/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Camundongos Transgênicos , Neurônios Dopaminérgicos/metabolismo , Mutação , Sinapses/metabolismo
8.
Int J Mol Sci ; 23(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36362317

RESUMO

Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.


Assuntos
Hipocampo , Receptores de AMPA , Camundongos , Animais , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Camundongos Transgênicos , Hipocampo/metabolismo , Sinapses/metabolismo , Dendritos/metabolismo
9.
Nat Commun ; 13(1): 6836, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369219

RESUMO

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Nevertheless, spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence can occur in many disorders, suggesting that brain circuits possess an intrinsic capacity to overcome the deficits arising from some germline mutations. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the molecular composition of excitatory synapses, the postnatal expansion of synapse diversity and the acquisition of normal synaptome architecture were delayed in all brain regions, interfering with networks and electrophysiological simulations of cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synapse diversity and synaptome architecture to the normal developmental trajectory. Synapse subtypes with rapid protein turnover mediated the synaptome remodeling. This brain-wide capacity for remodeling of synapse molecular composition to recover and maintain the developmental trajectory of synaptome architecture may help confer resilience to neurodevelopmental genetic disorders.


Assuntos
Transtornos do Neurodesenvolvimento , Sinapses , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Fenótipo , Sinapses/metabolismo
10.
Int J Mol Sci ; 23(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36361981

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach.


Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hipocampo , Proteínas Proto-Oncogênicas c-ret , Células Piramidais , Animais , Humanos , Camundongos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Sinapses/metabolismo , Células Piramidais/metabolismo
11.
Cells ; 11(22)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36429079

RESUMO

The AMPA glutamate receptor (AMPAR) is the major type of synaptic excitatory ionotropic receptor in the brain. AMPARs have four different subunits, GluA1-4 (each encoded by different genes, Gria1, Gria2, Gria3 and Gria4), that can form distinct tetrameric assemblies. The most abundant AMPAR subtypes in the hippocampus are GluA1/2 and GluA2/3 heterotetramers. Each subtype contributes differentially to mechanisms of synaptic plasticity, which may be in part caused by how these receptors are regulated by specific associated proteins. A broad range of AMPAR interacting proteins have been identified, including the well-studied transmembrane AMPA receptor regulatory proteins TARP-γ2 (also known as Stargazin) and TARP-γ8, Cornichon homolog 2 (CNIH-2) and many others. Several interactors were shown to affect biogenesis, AMPAR trafficking, and channel properties, alone or in distinct assemblies, and several revealed preferred binding to specific AMPAR subunits. To date, a systematic specific interactome analysis of the major GluA1/2 and GluA2/3 AMPAR subtypes separately is lacking. To reveal interactors belonging to specific AMPAR subcomplexes, we performed both expression and interaction proteomics on hippocampi of wildtype and Gria1- or Gria3 knock-out mice. Whereas GluA1/2 receptors co-purified TARP-γ8, synapse differentiation-induced protein 4 (SynDIG4, also known as Prrt1) and CNIH-2 with highest abundances, GluA2/3 receptors revealed strongest co-purification of CNIH-2, TARP-γ2, and Noelin1 (or Olfactomedin-1). Further analysis revealed that TARP-γ8-SynDIG4 interact directly and co-assemble into an AMPAR subcomplex especially at synaptic sites. Together, these data provide a framework for further functional analysis into AMPAR subtype specific pathways in health and disease.


Assuntos
Proteômica , Receptores de AMPA , Animais , Camundongos , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Plasticidade Neuronal/fisiologia , Hipocampo/metabolismo , Camundongos Knockout
12.
Elife ; 112022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36378164

RESUMO

Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in Caenorhabditis elegans, in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sinapses/metabolismo , Neurônios Motores/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
13.
Proc Natl Acad Sci U S A ; 119(45): e2119044119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322725

RESUMO

Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, we found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. We reveal distinct phases of structural transsynaptic homeostatic plasticity, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy.


Assuntos
Proteínas de Drosophila , Junção Neuromuscular , Animais , Junção Neuromuscular/metabolismo , Drosophila/metabolismo , Transmissão Sináptica , Sinapses/metabolismo , Receptores de Glutamato/metabolismo , Proteínas de Drosophila/metabolismo , Neurotransmissores/metabolismo , Proteínas de Membrana/metabolismo
14.
Proc Natl Acad Sci U S A ; 119(45): e2210645119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322758

RESUMO

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca2+ condition. Confocal calcium imaging revealed that dendritic Ca2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.


Assuntos
Hipotireoidismo Congênito , Células de Purkinje , Camundongos , Animais , Células de Purkinje/metabolismo , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Cálcio/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Depressão , Hipotireoidismo Congênito/metabolismo , Sinapses/metabolismo , Cerebelo/fisiologia
15.
Cell Rep ; 41(5): 111574, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36323257

RESUMO

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of AKT/mTOR signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten loss results in neuronal hypertrophy, hyperexcitability, seizures, and ASD-like behaviors. The underlying molecular mechanisms of these phenotypes are not well delineated. We determined which of the Pten loss-driven aberrations in neuronal form and function are orchestrated by downstream mTOR complex 1 (mTORC1). Rapamycin-mediated inhibition of mTORC1 prevented increase in soma size, migration, spine density, and dendritic overgrowth in Pten knockout dentate gyrus granule neurons. Genetic knockout of Raptor to disrupt mTORC1 complex formation blocked Pten loss-mediated neuronal hypertrophy. Electrophysiological recordings revealed that genetic disruption of mTORC1 rescued Pten loss-mediated increase in excitatory synaptic transmission. We have identified an essential role for mTORC1 in orchestrating Pten loss-driven neuronal hypertrophy and synapse formation.


Assuntos
Neurônios , Sinapses , Animais , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Sinapses/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Hipertrofia/metabolismo
16.
Int J Mol Sci ; 23(19)2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36232725

RESUMO

Depressive disorders (DDs) are an increasingly common health problem that affects all age groups. DDs pathogenesis is multifactorial. However, it was proven that stress is one of the most important environmental factors contributing to the development of these conditions. In recent years, there has been growing interest in the role of the glutamatergic system in the context of pharmacotherapy of DDs. Thus, it has become increasingly important to explore the functioning of excitatory synapses in pathogenesis and pharmacological treatment of psychiatric disorders (including DDs). This knowledge may lead to the description of new mechanisms of depression and indicate new potential targets for the pharmacotherapy of illness. An excitatory synapse is a highly complex and very dynamic structure, containing a vast number of proteins. This review aimed to discuss in detail the role of the key postsynaptic proteins (e.g., NMDAR, AMPAR, mGluR5, PSD-95, Homer, NOS etc.) in the excitatory synapse and to systematize the knowledge about changes that occur in the clinical course of depression and after antidepressant treatment. In addition, a discussion on the potential use of ligands and/or modulators of postsynaptic proteins at the excitatory synapse has been presented.


Assuntos
Transtorno Depressivo , Sinapses , Encéfalo/metabolismo , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Humanos , Ligantes , Sinapses/metabolismo
17.
Proc Natl Acad Sci U S A ; 119(42): e2211572119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36215504

RESUMO

Activation of Ca2+/calmodulin-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a long accepted cellular model for learning and memory. However, how LTP and memories survive the turnover of synaptic proteins, particularly CaMKII, remains a mystery. Here, we take advantage of the finding that constitutive Ca2+-independent CaMKII activity, acquired prior to slice preparation, provides a lasting memory trace at synapses. In slice culture, this persistent CaMKII activity, in the absence of Ca2+ stimulation, remains stable over a 2-wk period, well beyond the turnover of CaMKII protein. We propose that the nascent CaMKII protein present at 2 wk acquired its activity from preexisting active CaMKII molecules, which transferred their activity to newly synthesized CaMKII molecules and thus maintain the memory in the face of protein turnover.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Calmodulina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Hipocampo/metabolismo , Aprendizagem/fisiologia , Potenciação de Longa Duração/fisiologia , Fosforilação , Sinapses/metabolismo
18.
Neurobiol Dis ; 174: 105887, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36209950

RESUMO

We have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Recently, we have described novel structures in hippocampal area CA1 in aging mice, apparently derived from degenerating presynaptic terminals, that are significantly more prevalent in DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appear as clusters of large puncta that colocalize NeuN, synaptophysin, and chromogranin A, and also partially label for MAP2, amyloid ß, APP, α-synuclein, and phosphorylated tau, with synapsin-1 and VGluT1 labeling on their periphery. In this current study, using immunofluorescence and electron microscopy, we confirm that both APP and amyloid ß are prevalent in these structures; and we show with immunofluorescence the presence of similar structures in humans with Alzheimer's disease. Here we also found evidence that aging DPP6-KO mutants show additional changes related to Alzheimer's disease. We used in vivo MRI to show reduced size of the DPP6-KO brain and hippocampus. Aging DPP6-KO hippocampi contained fewer total neurons and greater neuron death and had diagnostic biomarkers of Alzheimer's disease present including accumulation of amyloid ß and APP and increase in expression of hyper-phosphorylated tau. The amyloid ß and phosphorylated tau pathologies were associated with neuroinflammation characterized by increases in microglia and astrocytes. And levels of proinflammatory or anti-inflammatory cytokines increased in aging DPP6-KO mice. We finally show that aging DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimer's disease. Together these results indicate that aging DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of dementia associated with a novel structure resulting from synapse loss and neuronal death. This study continues our laboratory's work in discerning the function of DPP6 and here provides compelling evidence of a direct role of DPP6 in Alzheimer's disease.


Assuntos
Doença de Alzheimer , Humanos , Camundongos , Animais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Envelhecimento/patologia , Hipocampo/metabolismo , Sinapses/metabolismo , Camundongos Transgênicos , Proteínas tau/genética , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo
19.
Proc Natl Acad Sci U S A ; 119(44): e2205264119, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36282913

RESUMO

Brain oscillations have long-lasting effects on synaptic and cellular properties. For instance, synaptic stimulation at theta (θ) frequency induces persistent depression of both excitatory synaptic transmission and intrinsic excitability in CA1 principal neurons. However, the incidence of θ activity on synaptic transmission and intrinsic excitability in hippocampal GABAergic interneurons is unclear. We report here the induction of both synaptic and intrinsic potentiation in oriens-lacunosum moleculare (O-LM) interneurons following stimulation of afferent glutamatergic inputs in the θ frequency range (∼5 Hz). Long-term synaptic potentiation (LTP) is induced by synaptic activation of calcium-permeable AMPA receptors (CP-AMPAR), whereas long-term potentiation of intrinsic excitability (LTP-IE) results from the mGluR1-dependent down-regulation of Kv7 voltage-dependent potassium channel and hyperpolarization activated and cyclic nucleotide-gated (HCN) channel through the depletion of phosphatidylinositol-4,5-biphosphate (PIP2). LTP and LTP-IE are reversible, demonstrating that both synaptic and intrinsic changes are bidirectional in O-LM cells. We conclude that synaptic activity at θ frequency induces both synaptic and intrinsic potentiation in O-LM interneurons, i.e., the opposite of what is typically seen in glutamatergic neurons.


Assuntos
Cálcio , Receptores de AMPA , Receptores de AMPA/metabolismo , Cálcio/metabolismo , Sinapses/metabolismo , Fosfatidilinositol 4,5-Difosfato , Hipocampo/metabolismo , Interneurônios/metabolismo , Potenciação de Longa Duração/fisiologia , Canais de Potássio , Nucleotídeos Cíclicos/farmacologia , Estimulação Elétrica
20.
Neurosci Lett ; 790: 136896, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36202195

RESUMO

Zinc, loaded into glutamate-containing presynaptic vesicles and released into the synapse in an activity-dependent manner, modulates neurotransmission through its actions on postsynaptic targets, prominently via high-affinity inhibition of GluN2A-containing NMDA receptors. Recently, we identified a postsynaptic transport mechanism that regulates endogenous zinc inhibition of NMDARs. In this new model of zinc regulation, the postsynaptic transporter ZnT1 mediates zinc inhibition of NMDARs by binding to GluN2A. Through this interaction, ZnT1, a transporter that moves zinc from the cytoplasm to the extracellular domain, generates a zinc microdomain that modulates NMDAR-mediated neurotransmission. As ZnT1 expression is transcriptionally driven by the metal-responsive transcription factor 1 (MTF-1), we found that intracellular zinc strongly drives MTF-1 in cortical neurons in vitro and increases the number of GluN2A-ZnT1 interactions, thereby enhancing tonic zinc inhibition of NMDAR-mediated currents. Importantly, this effect is absent when the interaction between GluN2A and ZnT1 is disrupted by a cell-permeable peptide. These results suggest that zinc-regulated gene expression can dynamically regulate NMDAR-mediated synaptic processes.


Assuntos
Receptores de N-Metil-D-Aspartato , Zinco , Receptores de N-Metil-D-Aspartato/metabolismo , Zinco/farmacologia , Zinco/metabolismo , Sinapses/metabolismo , Ácido Glutâmico/metabolismo , Fatores de Transcrição/metabolismo
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