RESUMO
Infantile and epileptic spasms syndrome (IESS) is a childhood epilepsy syndrome characterized by infantile or late-onset spasms, abnormal neonatal EEG, and epilepsy. Few treatments exist for IESS, clinical outcomes are poor, and the molecular and circuit-level etiologies of IESS are not well understood. Multiple human IESS risk genes are linked to Wnt/ß-catenin signaling, a pathway that controls developmental transcriptional programs and promotes glutamatergic excitation via ß-catenin's role as a synaptic scaffold. We previously showed that deleting adenomatous polyposis coli (APC), a component of the ß-catenin destruction complex, in excitatory neurons (APC cKO mice, APCfl/fl x CaMKIIαCre) increased ß-catenin levels in developing glutamatergic neurons and led to infantile behavioral spasms, abnormal neonatal EEG, and adult epilepsy. Here, we tested the hypothesis that the development of GABAergic interneurons (INs) is disrupted in APC cKO male and female mice. IN dysfunction is implicated in human IESS, is a feature of other rodent models of IESS, and may contribute to the manifestation of spasms and seizures. We found that parvalbumin-positive INs (PV+ INs), an important source of cortical inhibition, were decreased in number, underwent disproportionate developmental apoptosis, and had altered dendrite morphology at P9, the peak of behavioral spasms. PV+ INs received excessive excitatory input, and their intrinsic ability to fire action potentials was reduced at all time points examined (P9, P14, P60). Subsequently, GABAergic transmission onto pyramidal neurons was uniquely altered in the somatosensory cortex of APC cKO mice at all ages, with both decreased IPSC input at P14 and enhanced IPSC input at P9 and P60. These results indicate that inhibitory circuit dysfunction occurs in APC cKOs and, along with known changes in excitation, may contribute to IESS-related phenotypes.SIGNIFICANCE STATEMENT Infantile and epileptic spasms syndrome (IESS) is a devastating epilepsy with limited treatment options and poor clinical outcomes. The molecular, cellular, and circuit disruptions that cause infantile spasms and seizures are largely unknown, but inhibitory GABAergic interneuron dysfunction has been implicated in rodent models of IESS and may contribute to human IESS. Here, we use a rodent model of IESS, the APC cKO mouse, in which ß-catenin signaling is increased in excitatory neurons. This results in altered parvalbumin-positive GABAergic interneuron development and GABAergic synaptic dysfunction throughout life, showing that pathology arising in excitatory neurons can initiate long-term interneuron dysfunction. Our findings further implicate GABAergic dysfunction in IESS, even when pathology is initiated in other neuronal types.
Assuntos
Polipose Adenomatosa do Colo , Epilepsia , Espasmos Infantis , Masculino , Animais , Feminino , Camundongos , Humanos , Criança , Espasmos Infantis/metabolismo , Parvalbuminas/metabolismo , Camundongos Knockout , beta Catenina/metabolismo , Interneurônios/fisiologia , Convulsões , Epilepsia/metabolismo , Espasmo/metabolismo , Espasmo/patologia , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologiaRESUMO
Multiple human autism risk genes are predicted to converge on the ß-catenin (ß-cat)/Wnt pathway. However, direct tests to link ß-cat up- or down-regulation with autism are largely lacking, and the associated pathophysiological changes are poorly defined. Here we identify excessive ß-cat as a risk factor that causes expression changes in several genes relevant to human autism. Our studies utilize mouse lines with ß-cat dysregulation in forebrain excitatory neurons, identified as cell types with a convergent expression of autism-linked genes in both human and mouse brains. We show that mice expressing excessive ß-cat display behavioral and molecular changes, including decreased social interest, increased repetitive behaviors, reduced parvalbumin and altered expression levels of additional genes identified as potential risk factors for human autism. These behavioral and molecular phenotypes are averted by reducing ß-cat in neurons predisposed by gene mutations to express elevated ß-cat. Using next-generation sequencing of the prefrontal cortex (PFC), we identify 87 dysregulated genes that are shared between mouse lines with excessive ß-cat and autism-like behaviors, but not mouse lines with reduced ß-cat and normal social behavior. Our findings provide critical new insights into ß-cat, Wnt pathway dysregulation in the brain causing behavioral phenotypes relevant to the disease and the molecular etiology which includes several human autism risk genes.
RESUMO
Synaptic dysfunction is a pathological feature in many neurodegenerative disorders, including Alzheimer's disease, and synaptic loss correlates closely with cognitive decline. Histone deacetylases (HDACs) are involved in chromatin remodeling and gene expression and have been shown to regulate synaptogenesis and synaptic plasticity, thus providing an attractive drug discovery target for promoting synaptic growth and function. To date, HDAC inhibitor compounds with prosynaptic effects are plagued by known HDAC dose-limiting hematological toxicities, precluding their application to treating chronic neurologic conditions. We have identified a series of novel HDAC inhibitor compounds that selectively inhibit the HDAC-co-repressor of repressor element-1 silencing transcription factor (CoREST) complex while minimizing hematological side effects. HDAC1 and HDAC2 associate with multiple co-repressor complexes including CoREST, which regulates neuronal gene expression. We show that selectively targeting the CoREST co-repressor complex with the representative compound Rodin-A results in increased spine density and synaptic proteins, and improved long-term potentiation in a mouse model at doses that provide a substantial safety margin that would enable chronic treatment. The CoREST-selective HDAC inhibitor Rodin-A thus represents a promising therapeutic strategy in targeting synaptic pathology involved in neurologic disorders.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Histona Desacetilases/metabolismo , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ratos Sprague-Dawley , Proteínas Repressoras/genéticaRESUMO
Autism spectrum disorder (ASD) is a highly prevalent and genetically heterogeneous brain disorder. Developing effective therapeutic interventions requires knowledge of the brain regions that malfunction and how they malfunction during ASD-relevant behaviors. Our study provides insights into brain regions activated by a novel social stimulus and how the activation pattern differs between mice that display autism-like disabilities and control littermates. Adenomatous polyposis coli (APC) conditional knockout (cKO) mice display reduced social interest, increased repetitive behaviors and dysfunction of the ß-catenin pathway, a convergent target of numerous ASD-linked human genes. Here, we exposed the mice to a novel social vs. non-social stimulus and measured neuronal activation by immunostaining for the protein c-Fos. We analyzed three brain regions known to play a role in social behavior. Compared with control littermates, APC cKOs display excessive activation, as evidenced by an increased number of excitatory pyramidal neurons stained for c-Fos in the medial prefrontal cortex (mPFC), selectively in the infralimbic sub-region. In contrast, two other social brain regions, the medial amygdala and piriform cortex show normal levels of neuron activation. Additionally, APC cKOs exhibit increased frequency of miniature excitatory postsynaptic currents (mEPSCs) in layer 5 pyramidal neurons of the infralimbic sub-region. Further, immunostaining is reduced for the inhibitory interneuron markers parvalbumin (PV) and somatostatin (SST) in the APC cKO mPFC. Our findings suggest aberrant excitatory-inhibitory balance and activation patterns. As ß-catenin is a core pathway in ASD, we identify the infralimbic sub-region of the mPFC as a critical brain region for autism-relevant social behavior.
RESUMO
Infantile spasms (IS) are a catastrophic childhood epilepsy syndrome characterized by flexion-extension spasms during infancy that progress to chronic seizures and cognitive deficits in later life. The molecular causes of IS are poorly defined. Genetic screens of individuals with IS have identified multiple risk genes, several of which are predicted to alter ß-catenin pathways. However, evidence linking malfunction of ß-catenin pathways and IS is lacking. Here, we show that conditional deletion in mice of the adenomatous polyposis coli gene (APC cKO), the major negative regulator of ß-catenin, leads to excessive ß-catenin levels and multiple salient features of human IS. Compared with wild-type littermates, neonatal APC cKO mice exhibit flexion-extension motor spasms and abnormal high-amplitude electroencephalographic discharges. Additionally, the frequency of excitatory postsynaptic currents is increased in layer V pyramidal cells, the major output neurons of the cerebral cortex. At adult ages, APC cKOs display spontaneous electroclinical seizures. These data provide the first evidence that malfunctions of APC/ß-catenin pathways cause pathophysiological changes consistent with IS. Our findings demonstrate that the APC cKO is a new genetic model of IS, provide novel insights into molecular and functional alterations that can lead to IS, and suggest novel targets for therapeutic intervention.
Assuntos
Proteína da Polipose Adenomatosa do Colo/deficiência , Modelos Animais de Doenças , Neurônios/metabolismo , Convulsões/metabolismo , Espasmos Infantis/metabolismo , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Animais Recém-Nascidos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Eletroencefalografia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Humanos , Lactente , Masculino , Camundongos Knockout , Movimento/fisiologia , Neurônios/patologia , Fenótipo , Convulsões/patologia , Transdução de Sinais , Espasmos Infantis/patologia , Técnicas de Cultura de TecidosRESUMO
Small conductance Ca(2+)-sensitive potassium (SK2) channels are voltage-independent, Ca(2+)-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca(2+) permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca(2+) and Ca(2+)-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.
Assuntos
Receptores Nicotínicos/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Processamento Alternativo , Animais , Biotinilação , Encéfalo/fisiologia , Cálcio/fisiologia , Calmodulina/fisiologia , Galinhas , Cóclea/fisiologia , Endocitose , Células Ciliadas Auditivas/fisiologia , Camundongos , Neurônios/fisiologia , Oócitos , Isoformas de Proteínas/fisiologia , Xenopus laevisRESUMO
The auxiliary subunit α2δ3 modulates the expression and function of voltage-gated calcium channels. Here we show that α2δ3 mRNA is expressed in spiral ganglion neurons and auditory brainstem nuclei and that the protein is required for normal acoustic responses. Genetic deletion of α2δ3 led to impaired auditory processing, with reduced acoustic startle and distorted auditory brainstem responses. α2δ3(-/-) mice learned to discriminate pure tones, but they failed to discriminate temporally structured amplitude-modulated tones. Light and electron microscopy analyses revealed reduced levels of presynaptic Ca(2+) channels and smaller auditory nerve fiber terminals contacting cochlear nucleus bushy cells. Juxtacellular in vivo recordings of sound-evoked activity in α2δ3(-/-) mice demonstrated impaired transmission at these synapses. Together, our results identify a novel role for the α2δ3 auxiliary subunit in the structure and function of specific synapses in the mammalian auditory pathway and in auditory processing disorders.
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Transtornos da Percepção Auditiva/metabolismo , Canais de Cálcio/metabolismo , Nervo Coclear/metabolismo , Aprendizagem por Discriminação/fisiologia , Sinapses/metabolismo , Animais , Transtornos da Percepção Auditiva/genética , Transtornos da Percepção Auditiva/fisiopatologia , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Canais de Cálcio/genética , Nervo Coclear/patologia , Eletrofisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/fisiologia , Sinapses/patologia , Transmissão Sináptica/fisiologiaRESUMO
Voltage-gated Ca(2+) (Ca(v))1.3 α-subunits of high voltage-activated Ca(2+) channels (HVACCs) are essential for Ca(2+) influx and transmitter release in cochlear inner hair cells and therefore for signal transmission into the central auditory pathway. Their absence leads to deafness and to striking structural changes in the auditory brain stem, particularly in the lateral superior olive (LSO). Here, we analyzed the contribution of various types of HVACCs to the total Ca(2+) current (I(Ca)) in developing mouse LSO neurons to address several questions: do LSO neurons express functional Ca(v)1.3 channels? What other types of HVACCs are expressed? Are there developmental changes? Do LSO neurons of Ca(v)1.3(-/-) mice show any compensatory responses, namely, upregulation of other HVACCs? Our electrophysiological and pharmacological results showed the presence of functional Ca(v)1.3 and Ca(v)1.2 channels at both postnatal days 4 and 12. Aside from these L-type channels, LSO neurons also expressed functional P/Q-type, N-type, and, most likely, R-type channels. The relative contribution of the four different subtypes to I(Ca) appeared to be 45%, 29%, 22%, and 4% at postnatal day 12, respectively. The physiological results were flanked and extended by quantitative RT-PCR data. Altogether, LSO neurons displayed a broad repertoire of HVACC subtypes. Genetic ablation of Ca(v)1.3 resulted in functional reorganization of some other HVACCs but did not restore normal I(Ca) properties. Together, our results suggest that several types of HVACCs are of functional relevance for the developing LSO. Whether on-site loss of Ca(v)1.3, i.e., in LSO neurons, contributes to the recently described malformation of the LSO needs to be determined by using tissue-specific Ca(v)1.3(-/-) animals.
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Envelhecimento/fisiologia , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Neurônios/fisiologia , Núcleo Olivar/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , OleaRESUMO
Within the family of voltage-gated calcium channels (VGCCs), L-type channels (L-VGCCs) represent a well-established therapeutic target for calcium channel blockers, which are widely used to treat hypertension and myocardial ischemia. L-VGCCs outside the cardiovascular system also control key physiological processes such as neuronal plasticity, sensory cell function (e.g. in the inner ear and retina) and endocrine function (e.g. in pancreatic beta cells and adrenal chromaffin cells). Research into L-VGCCs was stimulated by the discovery that the known L-VGCC isoforms (Ca(V)1.1, Ca(V)1.2, Ca(V)1.3 and Ca(V)1.4) possess different biophysical properties. However, no L-VGCC-isoform-selective drugs have yet been identified. In this review, we examine Ca(V)1.2 and Ca(V)1.3 isoforms at the level of genetic structure, splice variants, post-translational modifications and functional protein coupling. We discuss candidate Ca(V)1.2- and Ca(V)1.3-specific characteristics as future therapeutic targets in individual organs.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L , Proteínas do Tecido Nervoso/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas , Alinhamento de Sequência , Homologia de Sequência , Transdução de SinaisRESUMO
To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca(2+) channel (Ca(V)) α(1S)-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of Ca(V)α(1S) in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged α(1S)T1354S in dysgenic (α(1S)-null) myotubes. Whole cell patch-clamp analysis revealed that α(1S)T1354S produced significantly faster activation of L-type Ca(2+) currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α(1S) (α(1S)WT). In addition, α(1S)T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca(2+) release and to larger action-potential-induced intracellular Ca(2+) transients under low (≤ 2 mM) caffeine concentrations compared with α(1S)WT. Thus our data suggest that an additional influx of Ca(2+) due to faster activation of the α(1S)T1354S L-type Ca(2+) current, in concert with higher caffeine sensitivity of Ca(2+) release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.