RESUMO
Postsynaptic trafficking plays a key role in regulating synapse structure and function. While spiny excitatory synapses can be stable throughout adult life, their morphology and function is impaired in Alzheimer's disease (AD). However, little is known about how AD risk genes impact synaptic function. Here we used structured superresolution illumination microscopy (SIM) to study the late-onset Alzheimer's disease (LOAD) risk factor BIN1, and show that this protein is abundant in postsynaptic compartments, including spines. While postsynaptic Bin1 shows colocalization with clathrin, a major endocytic protein, it also colocalizes with the small GTPases Rab11 and Arf6, components of the exocytic pathway. Bin1 participates in protein complexes with Arf6 and GluA1, and manipulations of Bin1 lead to changes in spine morphology, AMPA receptor surface expression and trafficking, and AMPA receptor-mediated synaptic transmission. Our data provide new insights into the mesoscale architecture of postsynaptic trafficking compartments and their regulation by a major LOAD risk factor.
Assuntos
Doença de Alzheimer , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Humanos , Proteínas Nucleares , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Proteínas Supressoras de TumorRESUMO
Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. It is currently unclear how the consequences of this genetic insult unfold to impair neurodevelopment. We reasoned that by elucidating the basis of microcephaly in AS, a highly penetrant syndromic feature with early postnatal onset, we would gain new insights into the mechanisms by which maternal UBE3A loss derails neurotypical brain growth and function. Detailed anatomical analysis of both male and female maternal Ube3a-null mice reveals that microcephaly in the AS mouse model is primarily driven by deficits in the growth of white matter tracts, which by adulthood are characterized by densely packed axons of disproportionately small caliber. Our results implicate impaired axon growth in the pathogenesis of AS and identify noninvasive structural neuroimaging as a potentially valuable tool for gauging therapeutic efficacy in the disorder.SIGNIFICANCE STATEMENT People who maternally inherit a deletion or nonfunctional copy of the UBE3A gene develop Angelman syndrome (AS), a severe neurodevelopmental disorder. To better understand how loss of maternal UBE3A function derails brain development, we analyzed brain structure in a maternal Ube3a knock-out mouse model of AS. We report that the volume of white matter (WM) is disproportionately reduced in AS mice, indicating that deficits in WM development are a major factor underlying impaired brain growth and microcephaly in the disorder. Notably, we find that axons within the WM pathways of AS model mice are abnormally small in caliber. This defect is associated with slowed nerve conduction, which could contribute to behavioral deficits in AS, including motor dysfunction.
Assuntos
Síndrome de Angelman/patologia , Axônios/patologia , Microcefalia/patologia , Fibras Nervosas/patologia , Ubiquitina-Proteína Ligases/genética , Substância Branca/patologia , Síndrome de Angelman/fisiopatologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcefalia/fisiopatologia , Substância Branca/fisiopatologiaRESUMO
Appropriate excitatory/inhibitory (E/I) balance is essential for normal cortical function and is altered in some psychiatric disorders, including autism spectrum disorders (ASDs). Cell-autonomous molecular mechanisms that control the balance of excitatory and inhibitory synapse function remain poorly understood; no proteins that regulate excitatory and inhibitory synapse strength in a coordinated reciprocal manner have been identified. Using super-resolution imaging, electrophysiology, and molecular manipulations, we show that cadherin-10, encoded by CDH10 within the ASD risk locus 5p14.1, maintains both excitatory and inhibitory synaptic scaffold structure in cultured cortical neurons from rats of both sexes. Cadherin-10 localizes to both excitatory and inhibitory synapses in neocortex, where it is organized into nanoscale puncta that influence the size of their associated PSDs. Knockdown of cadherin-10 reduces excitatory but increases inhibitory synapse size and strength, altering the E/I ratio in cortical neurons. Furthermore, cadherin-10 exhibits differential participation in complexes with PSD-95 and gephyrin, which may underlie its role in maintaining the E/I ratio. Our data provide a new mechanism whereby a protein encoded by a common ASD risk factor controls E/I ratios by regulating excitatory and inhibitory synapses in opposing directions.SIGNIFICANCE STATEMENT The correct balance between excitatory/inhibitory (E/I) is crucial for normal brain function and is altered in psychiatric disorders such as autism. However, the molecular mechanisms that underlie this balance remain elusive. To address this, we studied cadherin-10, an adhesion protein that is genetically linked to autism and understudied at the cellular level. Using a combination of advanced microscopy techniques and electrophysiology, we show that cadherin-10 forms nanoscale puncta at excitatory and inhibitory synapses, maintains excitatory and inhibitory synaptic structure, and is essential for maintaining the correct balance between excitation and inhibition in neuronal dendrites. These findings reveal a new mechanism by which E/I balance is controlled in neurons and may bear relevance to synaptic dysfunction in autism.
Assuntos
Caderinas/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Sinapses/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de AMPA/análise , Receptores de AMPA/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclases/análise , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/análise , Proteína 4 Homóloga a Disks-Large , Eletrofisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/análise , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores Adrenérgicos beta 2/análiseRESUMO
Angelman syndrome (AS) is a neurogenetic disorder caused by mutations or deletions in the maternally-inherited UBE3A allele, leading to a loss of UBE3A protein expression in neurons. The paternally-inherited UBE3A allele is epigenetically silenced in neurons during development by a noncoding transcript (UBE3A-ATS). The absence of neuronal UBE3A results in severe neurological symptoms, including speech and language impairments, intellectual disability, and seizures. While no cure exists, therapies aiming to restore UBE3A function-either by gene addition or by targeting UBE3A-ATS-are under development. Progress in developing these treatments relies heavily on inferences drawn from mouse studies about the function of UBE3A in the human brain. To aid translational efforts and to gain an understanding of UBE3A and UBE3A-ATS biology with greater relevance to human neurodevelopmental contexts, we investigated UBE3A and UBE3A-ATS expression in the developing brain of the rhesus macaque, a species that exhibits complex social behaviors, resembling aspects of human behavior to a greater degree than mice. Combining immunohistochemistry and in situ hybridization, we mapped UBE3A and UBE3A-ATS regional and cellular expression in normal prenatal, neonatal, and adolescent rhesus macaque brains. We show that key hallmarks of UBE3A biology, well-known in rodents, are also present in macaques, and suggest paternal UBE3A silencing in neurons-but not glial cells-in the macaque brain, with onset between gestational day 48 and 100. These findings support proposals that early-life, perhaps even prenatal, intervention is optimal for overcoming the maternal allele loss of UBE3A linked to AS.
RESUMO
Background: Angelman syndrome (AS), a severe neurodevelopmental disorder resulting from the loss of the maternal UBE3A gene, is marked by changes in the brain's white matter (WM). The extent of WM abnormalities seems to correlate with the severity of clinical symptoms, but these deficits are still not well characterized or understood. This study provides the first large-scale measurement of WM volume reduction in children with AS. Furthermore, we probed the underlying neuropathology by examining the progression of myelination in an AS mouse model. Methods: We conducted magnetic resonance imaging (MRI) on children with AS (n=32) and neurotypical controls (n=99) aged 0.5-12 years. In parallel, we examined myelination in postnatal Ube3a maternal-null mice (Ube3a m-/p+; AS model), Ube3a paternal-null mice (Ube3a m+/p-), and wildtype controls (Ube3a m+/p+) using immunohistochemistry, Western blotting, and electron microscopy. Results: Our data revealed that AS individuals exhibit significant reductions in brain volume by ~1 year of age, with WM reduced by 26% and gray matter by 21% by 6-12 years of age-approximately twice the reductions observed in the adult AS mouse model. In our AS mouse model, we saw a global delay in the onset of myelination, which normalized within days (likely corresponding to months or years in human development). This myelination delay is caused by the loss of UBE3A in neurons rather than UBE3A haploinsufficiency in oligodendrocytes. Interestingly, ultrastructural analyses did not reveal any abnormalities in myelinated or unmyelinated axons. Limitations: It is difficult to extrapolate the timing and duration of the myelination delay observed in AS model mice to individuals with AS. Conclusions: This study reveals WM deficits as a hallmark in children with AS, demonstrating for the first time that these deficits are already apparent at 1 year of age. Parallel studies in a mouse model of AS show that these deficits may be associated with delayed onset of myelination due to the loss of neuronal (but not glial) UBE3A. These findings emphasize the potential of WM as both a therapeutic target for interventions and a valuable biomarker for tracking the progression of AS and the effectiveness of potential treatments.
RESUMO
Glycine receptors (GlyRs) are found in most areas of the brain, and their dysfunction can cause severe neurological disorders. While traditionally thought of as inhibitory receptors, presynaptic-acting GlyRs (preGlyRs) can also facilitate glutamate release under certain circumstances, although the underlying molecular mechanisms are unknown. In the current study, we sought to better understand the role of GlyRs in the facilitation of excitatory neurotransmitter release in mouse visual cortex. Using whole-cell recordings, we found that preGlyRs facilitate glutamate release in developing, but not adult, visual cortex. The glycinergic enhancement of neurotransmitter release in early development depends on the high intracellular to extracellular Cl(-) gradient maintained by the Na(+)-K(+)-2Cl(-) cotransporter and requires Ca(2+) entry through voltage-gated Ca(2+) channels. The glycine transporter 1, localized to glial cells, regulates extracellular glycine concentration and the activation of these preGlyRs. Our findings demonstrate a developmentally regulated mechanism for controlling excitatory neurotransmitter release in the neocortex.
Assuntos
Potenciais Pós-Sinápticos Excitadores , Neurotransmissores/metabolismo , Receptores de Glicina/metabolismo , Córtex Visual/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Cloro/metabolismo , Exocitose , Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Sinapses/fisiologia , Sinapses/ultraestrutura , Córtex Visual/crescimento & desenvolvimentoRESUMO
Background: Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A's localization is integral to its function. Methods: We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals. Results: We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals. Conclusions: By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder.
Assuntos
Córtex Cerebral/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndrome de Angelman/metabolismo , Córtex Cerebral/ultraestrutura , Epilepsia/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Adulto Jovem , Ácido gama-Aminobutírico/metabolismoRESUMO
Ubiquitination regulates a broad array of cellular processes, and defective ubiquitination is implicated in several neurological disorders. Loss of the E3 ubiquitin-protein ligase UBE3A causes Angelman syndrome. Despite its clinical importance, the normal role of UBE3A in neurons is still unclear. As a step toward deciphering its possible functions, we performed high-resolution light and electron microscopic immunocytochemistry. We report a broad distribution of UBE3A in neurons, highlighted by concentrations in axon terminals and euchromatin-rich nuclear domains. Our findings suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. J. Comp. Neurol. 525:233-251, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Animais , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de TransmissãoRESUMO
Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remain poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. Clustered regularly interspaced short palindromic repeats (CRISPR) depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced the frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders.
Assuntos
Encefalopatias/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural , Densidade Pós-Sináptica/metabolismo , Proteoma/metabolismo , Animais , Encefalopatias/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/genéticaRESUMO
Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs)-all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.
Assuntos
Síndrome de Angelman/genética , Epilepsia/genética , Neurônios GABAérgicos/metabolismo , Neocórtex/metabolismo , Células Piramidais/metabolismo , Convulsões/genética , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/fisiopatologia , Animais , Vesículas Revestidas por Clatrina/metabolismo , Eletroencefalografia , Epilepsia/fisiopatologia , Ácido Glutâmico/metabolismo , Camundongos , Neocórtex/fisiopatologia , Inibição Neural , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Convulsões/fisiopatologiaRESUMO
Tumor necrosis factor receptor-associated factor 2 (TRAF2)- and noncatalytic region of tyrosine kinase (NCK)-interacting kinase (TNIK) has been identified as an interactor in the psychiatric risk factor, Disrupted in Schizophrenia 1 (DISC1). As a step toward deciphering its function in the brain, we performed high-resolution light and electron microscopic immunocytochemistry. We demonstrate here that TNIK is expressed in neurons throughout the adult mouse brain. In striatum and cerebral cortex, TNIK concentrates in dendritic spines, especially in the vicinity of the lateral edge of the synapse. Thus, TNIK is highly enriched at a microdomain critical for glutamatergic signaling.
Assuntos
Encéfalo/citologia , Espinhas Dendríticas/metabolismo , Regulação da Expressão Gênica/genética , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Espinhas Dendríticas/genética , Espinhas Dendríticas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/ultraestrutura , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The 53 kDa insulin receptor substrate protein (IRSp53) is highly enriched in the brain. Despite evidence that links mutations of IRSp53 with autism and other neuropsychiatric problems, the functional significance of this protein remains unclear. We used light and electron microscopic immunohistochemistry to demonstrate that IRSp53 is expressed throughout the adult rat brain. Labeling concentrated selectively in dendritic spines, where it was associated with the postsynaptic density (PSD). Surprisingly, its organization within the PSD of spiny excitatory neurons of neocortex and hippocampus differed from that within spiny inhibitory neurons of neostriatum and cerebellar cortex. The present data support previous suggestions that IRSp53 is involved in postsynaptic signaling, while hinting that its signaling role may differ in different types of neurons.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Densidade Pós-Sináptica/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/ultraestrutura , Espinhas Dendríticas/ultraestrutura , Imunofluorescência , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Neurônios/ultraestrutura , Densidade Pós-Sináptica/ultraestrutura , Ratos Sprague-Dawley , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/metabolismoRESUMO
Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3A. AS model mice, which carry a maternal Ube3a null mutation (Ube3a(m-/p+)), recapitulate major features of AS in humans, including enhanced seizure susceptibility. Excitatory neurotransmission onto neocortical pyramidal neurons is diminished in Ube3a(m-/p+) mice, seemingly at odds with enhanced seizure susceptibility. We show here that inhibitory drive onto neocortical pyramidal neurons is more severely decreased in Ube3a(m-/p+) mice. This inhibitory deficit follows the loss of excitatory inputs and appears to arise from defective presynaptic vesicle cycling in multiple interneuron populations. In contrast, excitatory and inhibitory synaptic inputs onto inhibitory interneurons are largely normal. Our results indicate that there are neuron type-specific synaptic deficits in Ube3a(m-/p+) mice despite the presence of Ube3a in all neurons. These deficits result in excitatory/inhibitory imbalance at cellular and circuit levels and may contribute to seizure susceptibility in AS.
Assuntos
Potenciais Pós-Sinápticos Inibidores/genética , Inibição Neural/genética , Neurônios/classificação , Neurônios/fisiologia , Ubiquitina-Proteína Ligases/deficiência , Córtex Visual/citologia , Fatores Etários , Animais , Animais Recém-Nascidos , Biofísica , Calbindina 2 , Estimulação Elétrica , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Rede Nervosa/citologia , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/metabolismo , Plasticidade Neuronal/genética , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Proteína G de Ligação ao Cálcio S100/metabolismo , Somatostatina/metabolismo , Córtex Visual/crescimento & desenvolvimento , Córtex Visual/metabolismoRESUMO
The sodium-driven chloride/bicarbonate exchanger (NDCBE), a member of the SLC4 family of bicarbonate transporters, was recently found to modulate excitatory neurotransmission in hippocampus. By using light and electron microscopic immunohistochemistry, we demonstrate here that NDCBE is expressed throughout the adult rat brain, and selectively concentrates in presynaptic terminals, where it is closely associated with synaptic vesicles. NDCBE is in most glutamatergic axon terminals, and is also present in the terminals of parvalbumin-positive γ-aminobutyric acid (GABA)ergic cells. These findings suggest that NDCBE can regulate glutamatergic transmission throughout the brain, and point to a role for NDCBE as a possible regulator of GABAergic neurotransmission.
Assuntos
Antiportadores de Cloreto-Bicarbonato/biossíntese , Neurônios GABAérgicos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Animais , Antiportadores de Cloreto-Bicarbonato/análise , Neurônios GABAérgicos/ultraestrutura , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Synaptic function depends on interactions among sets of proteins that assemble into complex supramolecular machines. Molecular biology, electrophysiology, and live-cell imaging studies have provided tantalizing glimpses into the inner workings of the synapse, but fundamental questions remain regarding the functional organization of these "nano-machines." Electron tomography reveals the internal structure of synapses in three dimensions with exceptional spatial resolution. Here we report results from an electron tomographic study of axospinous synapses in neocortex and hippocampus of the adult rat, based on aldehyde-fixed material stabilized with tannic acid in lieu of postfixation with osmium tetroxide. Our results provide a new window into the structural basis of excitatory synaptic processing in the mammalian brain.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Sinapses/ultraestrutura , Animais , Artefatos , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Masculino , Tetróxido de Ósmio , Densidade Pós-Sináptica/fisiologia , Densidade Pós-Sináptica/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologiaRESUMO
Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca(2+) extrusion from the cell, the plasma membrane calcium-dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a "housekeeping" function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca(2+) transients.
Assuntos
Encéfalo/enzimologia , Neurônios/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Isoformas de Proteínas/metabolismo , Processamento Alternativo , Animais , Encéfalo/citologia , Cálcio/metabolismo , Imuno-Histoquímica , Masculino , Neurônios/citologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-DawleyRESUMO
The plasma membrane Ca(2+)-ATPases (PMCA) represent the major high-affinity Ca(2+) extrusion system in the brain. PMCAs comprise four isoforms and over 20 splice variants. Their different functional properties may permit different PMCA splice variants to accommodate different kinds of local [Ca(2+)] transients, but for a specific PMCA to play a unique role in local Ca(2+) handling it must be targeted to the appropriate subcellular compartment. We used immunohistochemistry to study the spatial distribution of PMCA2a-one of the two major carboxyl-terminal splice variants of PMCA2-in the adult rat brain, testing whether this isoform, with especially high basal activity, is targeted to specific subcellular compartments. In striking contrast to the widespread distribution of PMCA2 as a whole, we found that PMCA2a is largely restricted to parvalbumin-positive inhibitory presynaptic terminals throughout the brain. The only major exception to this targeting pattern was in the cerebellar cortex, where PMCA2a also concentrates postsynaptically, in the spines of Purkinje cells. We propose that the fast Ca(2+) activation kinetics and high V(max) of PMCA2a make this pump especially suited for rapid clearance of presynaptic Ca(2+) in fast-spiking inhibitory nerve terminals, which face severe transient calcium loads.