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1.
Mem Inst Oswaldo Cruz ; 115: e200007, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32935749

RESUMO

BACKGROUND: Behavioral and neurochemical alterations associated with toxoplasmosis may be influenced by the persistence of tissue cysts and activation of an immune response in the brain of Toxoplasma gondii-infected hosts. The cerebral extracellular matrix is organised as perineuronal nets (PNNs) that are both released and ensheath by some neurons and glial cells. There is evidences to suggest that PNNs impairment is a pathophysiological mechanism associated with neuropsychiatric conditions. However, there is a lack of information regarding the impact of parasitic infections on the PNNs integrity and how this could affect the host's behavior. OBJECTIVES: In this context, we aimed to analyse the impact of T. gondii infection on cyst burden, PNNs integrity, and possible effects in the locomotor activity of chronically infected mice. METHODS: We infected mice with T. gondii ME-49 strain. After thirty days, we assessed locomotor performance of animals using the open field test, followed by evaluation of cysts burden and PNNs integrity in four brain regions (primary and secondary motor cortices, prefrontal and somesthetic cortex) to assess the PNNs integrity using Wisteria floribunda agglutinin (WFA) labeling by immunohistochemical analyses. FINDINGS AND MAIN CONCLUSIONS: Our findings revealed a random distribution of cysts in the brain, the disruption of PNNs surrounding neurons in four areas of the cerebral cortex and hyperlocomotor behavior in T. gondii-infected mice. These results can contribute to elucidate the link toxoplasmosis with the establishment of neuroinflammatory response in neuropsychiatric disorders and to raise a discussion about the mechanisms related to changes in brain connectivity, with possible behavioral repercussions during chronic T. gondii infection.


Assuntos
Cerebelo/metabolismo , Matriz Extracelular/metabolismo , Neurônios Motores/citologia , Neurônios/patologia , Toxoplasmose Animal , Toxoplasmose/patologia , Animais , Cerebelo/citologia , Modelos Animais de Doenças , Camundongos , Neurônios Motores/metabolismo , Neurônios/metabolismo , Toxoplasma , Toxoplasmose/metabolismo
2.
BMC Bioinformatics ; 21(1): 269, 2020 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600248

RESUMO

BACKGROUND: High throughput RNA sequencing is a powerful approach to study gene expression. Due to the complex multiple-steps protocols in data acquisition, extreme deviation of a sample from samples of the same treatment group may occur due to technical variation or true biological differences. The high-dimensionality of the data with few biological replicates make it challenging to accurately detect those samples, and this issue is not well studied in the literature currently. Robust statistics is a family of theories and techniques aim to detect the outliers by first fitting the majority of the data and then flagging data points that deviate from it. Robust statistics have been widely used in multivariate data analysis for outlier detection in chemometrics and engineering. Here we apply robust statistics on RNA-seq data analysis. RESULTS: We report the use of two robust principal component analysis (rPCA) methods, PcaHubert and PcaGrid, to detect outlier samples in multiple simulated and real biological RNA-seq data sets with positive control outlier samples. PcaGrid achieved 100% sensitivity and 100% specificity in all the tests using positive control outliers with varying degrees of divergence. We applied rPCA methods and classical principal component analysis (cPCA) on an RNA-Seq data set profiling gene expression of the external granule layer in the cerebellum of control and conditional SnoN knockout mice. Both rPCA methods detected the same two outlier samples but cPCA failed to detect any. We performed differentially expressed gene detection before and after outlier removal as well as with and without batch effect modeling. We validated gene expression changes using quantitative reverse transcription PCR and used the result as reference to compare the performance of eight different data analysis strategies. Removing outliers without batch effect modeling performed the best in term of detecting biologically relevant differentially expressed genes. CONCLUSIONS: rPCA implemented in the PcaGrid function is an accurate and objective method to detect outlier samples. It is well suited for high-dimensional data with small sample sizes like RNA-seq data. Outlier removal can significantly improve the performance of differential gene detection and downstream functional analysis.


Assuntos
Análise de Componente Principal , RNA-Seq/métodos , Animais , Cerebelo/metabolismo , Feminino , Masculino , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
PLoS One ; 15(7): e0230400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639965

RESUMO

Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cerebelo/metabolismo , Esquizofrenia/patologia , Adulto , Calmodulina/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Parvalbuminas/metabolismo , Proteoma/análise , Esquizofrenia/metabolismo , Tentativa de Suicídio , Espectrometria de Massas em Tandem , Regulação para Cima
4.
PLoS Genet ; 16(7): e1008901, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645003

RESUMO

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.


Assuntos
Doenças Cerebelares/genética , Proteínas do Citoesqueleto/genética , Drosophila melanogaster/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Substituição de Aminoácidos/genética , Animais , Sistemas CRISPR-Cas/genética , Doenças Cerebelares/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Exossomos/genética , Humanos , Mutação/genética , Neurônios/patologia , RNA/genética
5.
Proc Natl Acad Sci U S A ; 117(20): 11097-11108, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358199

RESUMO

It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.


Assuntos
Potenciais de Ação/fisiologia , Axônios/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Células de Purkinje/metabolismo , Cálcio/metabolismo , Cerebelo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Purinérgicos P2Y1 , Fosfolipases Tipo C/metabolismo
6.
Toxicology ; 440: 152492, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32407874

RESUMO

Neurotoxicity induced by exposure to heavy metal lead (Pb) is a concern of utmost importance particularly for countries with industrial-based economies. The developing brain is especially sensitive to exposure to even minute quantities of Pb which can alter neurodevelopmental trajectory with irreversible effects on motor, emotive-social and cognitive attributes even into later adulthood. Chemical synapses form the major pathway of inter-neuronal communications and are prime candidates for higher order brain (motor, memory and behavior) functions and determine the resistance/susceptibility for neurological disorders, including neuropsychopathologies. The synaptic pathways and mechanisms underlying Pb-mediated alterations in neuronal signaling and plasticity are not completely understood. Employing a biochemically isolated synaptosomal fraction which is enriched in synaptic terminals and synaptic mitochondria, this study aimed to analyze the alterations in bioenergetic and redox/antioxidant status of cerebellar synapses induced by developmental exposure to Pb (0.2 %). Moreover, we test the efficacy of vitamin C (ascorbate; 500 mg/kg body weight), a neuroprotective and neuromodulatory antioxidant, in mitigation of Pb-induced neuronal deficits. Our results implicate redox and bioenergetic disruptions as an underlying feature of the synaptic dysfunction observed in developmental Pb neurotoxicity, potentially contributing to consequent deficits in motor, behavioral and psychological attributes of the organisms. In addition, we establish ascorbate as a key ingredient for therapeutic approach against Pb induced neurotoxicity, particularly for early-life exposures.


Assuntos
Antioxidantes/uso terapêutico , Ácido Ascórbico/uso terapêutico , Cerebelo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Intoxicação do Sistema Nervoso por Chumbo/patologia , Sinapses/metabolismo , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Cerebelo/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Chumbo/sangue , Intoxicação do Sistema Nervoso por Chumbo/psicologia , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
7.
Toxicology ; 440: 152500, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32428529

RESUMO

Autism spectrum disorders (ASD) include neurodevelopmental disorders in which behavioral deficits can result from neuronal imbalance of excitation to inhibition (E/I) in the brain. Here we used RT-qPCR to screen for the expression of 99 genes associated with excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmission in the cerebral cortex, hippocampus and cerebellum of rats in an established VPA model of ASD. The largest changes in the expression of glutamatergic genes were found in the cerebral cortex, where 12 genes including these encoding some of the subunits of the ionotropic glutamate receptors, were upregulated, while 2 genes were downregulated. The expression of genes encoding the presynaptic glutamatergic proteins vGluT1 and mGluR7 and PKA, involved in downstream glutamatergic signaling, was elevated more than 100-fold. Changes in GABAergic gene expression were found in the cortex, cerebellum and hippocampus; 3 genes were upregulated, and 3 were downregulated. In conclusion, these results revealed that, in the ASD model, several glutamatergic genes in the rat cerebral cortex were upregulated, which contrasts with small and balanced changes in the expression of GABAergic genes. The VPA rat model, useful in studying the molecular basis of ASD, may be suitable for testing experimental therapies in these disabilities.


Assuntos
Transtorno Autístico/induzido quimicamente , Transtorno Autístico/genética , Ácido Glutâmico/genética , Ácido Valproico , Ácido gama-Aminobutírico/genética , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Feminino , GABAérgicos , Perfilação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/biossíntese , Receptores de Glutamato Metabotrópico/genética , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/biossíntese , Proteína Vesicular 1 de Transporte de Glutamato/genética
8.
Mol Pharmacol ; 97(5): 336-350, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32111699

RESUMO

Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in preclinical and clinical studies. Their therapeutic window is narrow, however, and clinical application will likely only occur if greater discrimination in activity is achieved. Toward that end, we compared the modulatory activity of two PAMs recently considered as clinical candidates, LY451395 (mibampator) and PF-04958242/BIIB104, on recombinant and native AMPA receptors (AMPARs). We found that the principle molecular determinant that shaped modulatory activity of both PAMs on deactivation (recombinant) and decay (synaptic) of AMPARs was the auxiliary protein incorporated into the receptor complexes. AMPARs containing the stargazin/γ2 transmembrane AMPAR regulatory protein (TARP) were slowed to a >10-fold degree by both PAMs as compared with those incorporating γ8 TARP. Neither subunit composition nor flip/flop splice variation had substantive effect. Similarly, stargazin/γ2-containing mossy fiber EPSCs in cerebellar granule neurons were slowed to a ∼5-fold greater degree than EPSCs in hippocampal CA1 pyramidal cell neurons, which express the γ8 TARP. LY451395 exhibited greater efficacy than BIIB104 at both synapses. These studies provide insight into the receptor constituents that determine efficacy of sulfonamide PAMs. We conclude that compounds that discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/γ2 efficacy such as BIIB104, could act as viable procognitive therapeutics. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of AMPA receptors enhance cognitive function in a variety of preclinical models. A clearer understanding of the critical determinants of PAM activity could yield critical insight into pathways to maximize their therapeutic index. Here we show that auxiliary proteins for AMPARs play a major, but thus far underappreciated, role in shaping recombinant and neuronal AMPAR modulation by two clinical candidate PAMs. These data will inform both clinical outcomes as well as future rational development of new modulators.


Assuntos
Proteínas de Membrana/metabolismo , Receptores de AMPA/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Compostos de Bifenilo/farmacologia , Cerebelo/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Resultado do Tratamento , Triazóis/farmacologia
9.
Nucleic Acids Res ; 48(7): 3678-3691, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123907

RESUMO

Genomic instability resulting from defective DNA damage responses or repair causes several abnormalities, including progressive cerebellar ataxia, for which the molecular mechanisms are not well understood. Here, we report a new murine model of cerebellar ataxia resulting from concomitant inactivation of POLB and ATM. POLB is one of key enzymes for the repair of damaged or chemically modified bases, including methylated cytosine, but selective inactivation of Polb during neurogenesis affects only a subpopulation of cortical interneurons despite the accumulation of DNA damage throughout the brain. However, dual inactivation of Polb and Atm resulted in ataxia without significant neuropathological defects in the cerebellum. ATM is a protein kinase that responds to DNA strand breaks, and mutations in ATM are responsible for Ataxia Telangiectasia, which is characterized by progressive cerebellar ataxia. In the cerebella of mice deficient for both Polb and Atm, the most downregulated gene was Itpr1, likely because of misregulated DNA methylation cycle. ITPR1 is known to mediate calcium homeostasis, and ITPR1 mutations result in genetic diseases with cerebellar ataxia. Our data suggest that dysregulation of ITPR1 in the cerebellum could be one of contributing factors to progressive ataxia observed in human genomic instability syndromes.


Assuntos
Ataxia Cerebelar/genética , Cerebelo/metabolismo , Metilação de DNA , DNA Polimerase beta/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo/embriologia , Encéfalo/patologia , Cerebelo/anormalidades , Cerebelo/patologia , Citosina/metabolismo , Dano ao DNA , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Knockout , Neurogênese/genética
10.
J Neurosci ; 40(14): 2882-2894, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32111698

RESUMO

Sensorimotor integration in the cerebellum is essential for refining motor output, and the first stage of this processing occurs in the granule cell layer. Recent evidence suggests that granule cell layer synaptic integration can be contextually modified, although the circuit mechanisms that could mediate such modulation remain largely unknown. Here we investigate the role of ACh in regulating granule cell layer synaptic integration in male rats and mice of both sexes. We find that Golgi cells, interneurons that provide the sole source of inhibition to the granule cell layer, express both nicotinic and muscarinic cholinergic receptors. While acute ACh application can modestly depolarize some Golgi cells, the net effect of longer, optogenetically induced ACh release is to strongly hyperpolarize Golgi cells. Golgi cell hyperpolarization by ACh leads to a significant reduction in both tonic and evoked granule cell synaptic inhibition. ACh also reduces glutamate release from mossy fibers by acting on presynaptic muscarinic receptors. Surprisingly, despite these consistent effects on Golgi cells and mossy fibers, ACh can either increase or decrease the spike probability of granule cells as measured by noninvasive cell-attached recordings. By constructing an integrate-and-fire model of granule cell layer population activity, we find that the direction of spike rate modulation can be accounted for predominately by the initial balance of excitation and inhibition onto individual granule cells. Together, these experiments demonstrate that ACh can modulate population-level granule cell responses by altering the ratios of excitation and inhibition at the first stage of cerebellar processing.SIGNIFICANCE STATEMENT The cerebellum plays a key role in motor control and motor learning. While it is known that behavioral context can modify motor learning, the circuit basis of such modulation has remained unclear. Here we find that a key neuromodulator, ACh, can alter the balance of excitation and inhibition at the first stage of cerebellar processing. These results suggest that ACh could play a key role in altering cerebellar learning by modifying how sensorimotor input is represented at the input layer of the cerebellum.


Assuntos
Acetilcolina/metabolismo , Cerebelo/metabolismo , Modelos Neurológicos , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Animais , Feminino , Masculino , Camundongos , Inibição Neural/fisiologia , Ratos , Ratos Sprague-Dawley
11.
J Neurosci ; 40(14): 2943-2959, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32122952

RESUMO

Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3). We have characterized recently generated Piccolo KO (Pclogt/gt ) rats. Analysis of rats of both sexes revealed a dramatic reduction in brain size compared with WT (Pclowt/wt ) animals, attributed to a decrease in the size of the cerebral cortical, cerebellar, and pontine regions. Analysis of the cerebellum and brainstem revealed a reduced granule cell layer and a reduction in size of pontine nuclei. Moreover, the maturation of mossy fiber afferents from pontine neurons and the expression of the α6 GABAA receptor subunit at the mossy fiber-granule cell synapse are perturbed, as well as the innervation of Purkinje cells by cerebellar climbing fibers. Ultrastructural and functional studies revealed a reduced size of mossy fiber boutons, with fewer synaptic vesicles and altered synaptic transmission. These data imply that Piccolo is required for the normal development, maturation, and function of neuronal networks formed between the brainstem and cerebellum. Consistently, behavioral studies demonstrated that adult Pclogt/gt rats display impaired motor coordination, despite adequate performance in tasks that reflect muscle strength and locomotion. Together, these data suggest that loss of Piccolo function in patients with PCH3 could be involved in many of the observed anatomical and behavioral symptoms, and that the further analysis of these animals could provide fundamental mechanistic insights into this devastating disorder.SIGNIFICANCE STATEMENT Pontocerebellar Hypoplasia Type 3 is a devastating developmental disorder associated with severe developmental delay, progressive microcephaly with brachycephaly, optic atrophy, seizures, and hypertonia with hyperreflexia. Recent genetic studies have identified non-sense mutations in the coding region of the PCLO gene, suggesting a functional link between this disorder and the presynaptic active zone. Our analysis of Piccolo KO rats supports this hypothesis, formally demonstrating that anatomical and behavioral phenotypes seen in patients with Pontocerebellar Hypoplasia Type 3 are also exhibited by these Piccolo deficient animals.


Assuntos
Cerebelo/metabolismo , Cerebelo/patologia , Cerebelo/fisiopatologia , Proteínas do Citoesqueleto/metabolismo , Neuropeptídeos/metabolismo , Atrofias Olivopontocerebelares , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Masculino , Fenótipo , Ratos
12.
J Neurosci ; 40(17): 3348-3359, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32169968

RESUMO

Nitric oxide (NO) is an important signaling molecule that fulfills diverse functional roles as a neurotransmitter or diffusible second messenger in the developing and adult CNS. Although the impact of NO on different behaviors such as movement, sleep, learning, and memory has been well documented, the identity of its molecular and cellular targets is still an area of ongoing investigation. Here, we identify a novel role for NO in strengthening inhibitory GABAA receptor-mediated transmission in molecular layer interneurons of the mouse cerebellum. NO levels are elevated by the activity of neuronal NO synthase (nNOS) following Ca2+ entry through extrasynaptic NMDA-type ionotropic glutamate receptors (NMDARs). NO activates protein kinase G with the subsequent production of cGMP, which prompts the stimulation of NADPH oxidase and protein kinase C (PKC). The activation of PKC promotes the selective strengthening of α3-containing GABAARs synapses through a GΑΒΑ receptor-associated protein-dependent mechanism. Given the widespread but cell type-specific expression of the NMDAR/nNOS complex in the mammalian brain, our data suggest that NMDARs may uniquely strengthen inhibitory GABAergic transmission in these cells through a novel NO-mediated pathway.SIGNIFICANCE STATEMENT Long-term changes in the efficacy of GABAergic transmission is mediated by multiple presynaptic and postsynaptic mechanisms. A prominent pathway involves crosstalk between excitatory and inhibitory synapses whereby Ca2+-entering through postsynaptic NMDARs promotes the recruitment and strengthening of GABAA receptor synapses via Ca2+/calmodulin-dependent protein kinase II. Although Ca2+ transport by NMDARs is also tightly coupled to nNOS activity and NO production, it has yet to be determined whether this pathway affects inhibitory synapses. Here, we show that activation of NMDARs trigger a NO-dependent pathway that strengthens inhibitory GABAergic synapses of cerebellar molecular layer interneurons. Given the widespread expression of NMDARs and nNOS in the mammalian brain, we speculate that NO control of GABAergic synapse efficacy may be more widespread than has been appreciated.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cerebelo/metabolismo , Interneurônios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Cerebelo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Interneurônios/efeitos dos fármacos , Masculino , Camundongos , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Técnicas de Patch-Clamp , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
13.
Toxicol Lett ; 322: 87-97, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935479

RESUMO

1,2-Dichloroethane (1,2-DCE) is a widely used chlorinated organic toxicant, but little is known about the cerebellar dysfunction induced by excessive exposure to it. To uncover 1,2-DCE-induced neurotoxicity in cerebellar granular cells (CGCs), and to investigate the underlying mechanisms, we explored this, both in vitro and in vivo. Our findings showed significant cell viability inhibition in human CGCs (HCGCs) treated with 1,2-DCE. Flow cytometry and mitochondrial membrane potential analyses discovered an increase in apoptotic-mediated cell death in HCGCs after 1,2-DCE treatment. This HCGC apoptosis was involved in the increases of protein expression in Cytochrome c, Caspase-3, Bad, Bim, transformation related protein 53, Caspase-8, tumor necrosis factor-α, and Survivin. Quantitative real-time PCR (qPCR) and western blot confirmed the increases in Cytochrome c, Caspase-3, cleaved Caspase-3, and Bad in HCGCs after 1,2-DCE treatment. Bax inhibitor peptide V5 rescued 1,2-DCE-induced HCGC apoptosis. Furthermore, 80 CD-1 male mice were exposed to 1,2-DCE by inhalation at 0, 100, 350, and 700 mg/m3 for 6 h/day for 4 weeks. An open field test found abnormal neurobehavioral changes in the mice exposed to 1,2-DCE. Histopathological examination showed significantly shrunken and hypereosinophilic cytoplasm with nuclear pyknosis in mouse CGCs from the 700 mg/m3 1,2-DCE group. TdT-mediated dUTP nick-end labeling assay verified significant increases in apoptotic positive cells in the mouse CGCs after 1,2-DCE exposure. We confirmed the increases in the expressions of Cytochrome c, Caspase-3, cleaved Caspase-3 and Bad in the mice exposed to 1,2-DCE. These findings suggest that 1,2-DCE exposure can induce CGC apoptosis and cerebellar dysfunction, at least in part, through mitochondrial pathway.


Assuntos
Apoptose/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Dicloretos de Etileno/toxicidade , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/patologia , Cerebelo/fisiopatologia , Humanos , Locomoção/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Medição de Risco , Transdução de Sinais
14.
PLoS One ; 15(1): e0227077, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929594

RESUMO

Gaucher disease is caused by a deficiency in glucocerebrosidase that can result in non-neuronal as well as neuronal symptoms. Common visceral symptoms are an increased organ size, specifically of the spleen, and glucosylceramide as well as glucosylsphingosine substrate accumulations as a direct result of the glucocerebrosidase deficiency. Neuronal symptoms include motor deficits and strong alterations in the cerebellum. To evaluate the effect of new compounds for the treatment of this devastating disease, animal models are needed that closely mimic the human phenotype. The 4L/PS-NA mouse as model of Gaucher disease is shown to present reduced glucocerebrosidase activity similar to human cases but an in-depth characterization of the model was still not performed. We therefore analyzed 4L/PS-NA mice for visceral alterations, motor deficits and also neuronal changes like glucocerebrosidase activity, substrate levels and neuroinflammation. A special focus was set at pathological changes of the cerebellum. Our results show that 4L/PS-NA mice have strongly enlarged visceral organs that are infiltrated by enlarged leukocytes and macrophages. Furthermore, animals present strong motor deficits that are accompanied by increased glucosylceramide and glucosylsphingosine levels in the brain, astrocytosis and activated microglia in the cortex and hippocampus as well as reduced calbindin levels in the cerebellum. The latter was directly related to a strong Purkinje cell loss. Our results thus provide a detailed characterization of the 4L/PS-NA mouse model over age showing the translational value of the model and validating its usefulness for preclinical efficiency studies to evaluate new compounds against Gaucher disease.


Assuntos
Modelos Animais de Doenças , Doença de Gaucher/genética , Glucosilceramidase/genética , Fenótipo , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Feminino , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Glucosilceramidase/metabolismo , Leucócitos/patologia , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Movimento , Mutação , Neurônios/metabolismo , Neurônios/patologia , Baço/metabolismo , Baço/patologia , Timo/metabolismo , Timo/patologia
15.
PLoS Genet ; 16(1): e1008527, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999692

RESUMO

A form of hereditary cerebellar ataxia has recently been described in the Norwegian Buhund dog breed. This study aimed to identify the genetic cause of the disease. Whole-genome sequencing of two Norwegian Buhund siblings diagnosed with progressive cerebellar ataxia was carried out, and sequences compared with 405 whole genome sequences of dogs of other breeds to filter benign common variants. Nine variants predicted to be deleterious segregated among the genomes in concordance with an autosomal recessive mode of inheritance, only one of which segregated within the breed when genotyped in additional Norwegian Buhunds. In total this variant was assessed in 802 whole genome sequences, and genotyped in an additional 505 unaffected dogs (including 146 Buhunds), and only four affected Norwegian Buhunds were homozygous for the variant. The variant identified, a T to C single nucleotide polymorphism (SNP) (NC_006585.3:g.88890674T>C), is predicted to cause a tryptophan to arginine substitution in a highly conserved region of the potassium voltage-gated channel interacting protein KCNIP4. This gene has not been implicated previously in hereditary ataxia in any species. Evaluation of KCNIP4 protein expression through western blot and immunohistochemical analysis using cerebellum tissue of affected and control dogs demonstrated that the mutation causes a dramatic reduction of KCNIP4 protein expression. The expression of alternative KCNIP4 transcripts within the canine cerebellum, and regional differences in KCNIP4 protein expression, were characterised through RT-PCR and immunohistochemistry respectively. The voltage-gated potassium channel protein KCND3 has previously been implicated in spinocerebellar ataxia, and our findings suggest that the Kv4 channel complex KCNIP accessory subunits also have an essential role in voltage-gated potassium channel function in the cerebellum and should be investigated as potential candidate genes for cerebellar ataxia in future studies in other species.


Assuntos
Ataxia Cerebelar/genética , Doenças do Cão/genética , Proteínas Interatuantes com Canais de Kv/genética , Polimorfismo de Nucleotídeo Único , Animais , Ataxia Cerebelar/veterinária , Cerebelo/metabolismo , Cães , Proteínas Interatuantes com Canais de Kv/metabolismo , Mutação , Sequenciamento Completo do Genoma/veterinária
16.
PLoS One ; 15(1): e0227829, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999726

RESUMO

A hallmark of Niemann-Pick disease, type C (NPC) is the progressive degeneration of Purkinje neurons in the cerebellum caused by the accumulation of free cholesterol and glycosphingolipids in the lysosome. Recent studies suggest that the state of glycosylation of lysosomal membrane proteins may play a role in disease progression. Our study has identified the presence of a highly glycosylated form of Lysosome Associated Membrane Protein 1 (LAMP1) that correlated spatiotemporally with Purkinje neuron loss. This form of LAMP1 was predominantly localized to activated microglia; showing a ~5-fold increase in surface labeling by FACS analysis. This suggests a potential role for LAMP1 in the neuro-inflammatory process in these mice during disease progression. Analysis of other mouse models of neurodegeneration that exhibit neuro-inflammation showed little or no presence of this glycosylated form of LAMP1, suggesting this observation for LAMP1 is specific to NPC disease. Furthermore, early treatment of Npc1-/- mice with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), significantly prevented the appearance of the glycosylated LAMP1 in the cerebellum of Npc1-/- mice at 7 weeks, consistent with the prevention of neuro-inflammation in mice treated with this drug. Treatment of Npc1-/- mice with HPßCD at 7 weeks, after disease onset, did not reverse or prevent further appearance of the hyperglycosylated LAMP1, demonstrating that once this aspect of neuro-inflammation began, it continued despite the HPßCD treatment. Analysis of LAMP1 in cerebellar tissue of NPC1 patients showed a small level of hyperglycosylated LAMP1 in the tissue, however, this was not seen in the CSF of patients.


Assuntos
Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Feminino , Glicosilação , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/análise , Masculino , Camundongos Endogâmicos BALB C , Doença de Niemann-Pick Tipo C/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patologia
17.
PLoS Biol ; 18(1): e3000596, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905212

RESUMO

Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such "intrinsic plasticity" in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell-specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning.


Assuntos
Potenciais de Ação/genética , Aprendizagem/fisiologia , Memória/fisiologia , Atividade Motora/fisiologia , Células de Purkinje/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Reflexo Vestíbulo-Ocular , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
18.
PLoS One ; 15(1): e0227667, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978088

RESUMO

The apolipoprotein E gene (APOE) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), yet the expression of APOE is not clearly understood. For example, it is unclear whether AD patients have elevated or decreased APOE expression or why the correlation levels of APOE RNA and the ApoE protein differ across studies. Likewise, APOE has a single CpG island (CGI) that overlaps with its 3'-exon, and this CGI's effect is unknown. We previously reported that the APOE CGI is highly methylated in human postmortem brain (PMB) and that this methylation is altered in AD frontal lobe. In this study, we comprehensively characterized APOE RNA transcripts and correlated levels of RNA expression with DNA methylation levels across the APOE CGI. We discovered the presence of APOE circular RNA (circRNA) and found that circRNA and full-length mRNA each constitute approximately one third of the total APOE RNA, with truncated mRNAs likely constituting some of the missing fraction. All APOE RNA species demonstrated significantly higher expression in AD frontal lobe than in control frontal lobe. Furthermore, we observed a negative correlation between the levels of total APOE RNA and DNA methylation at the APOE CGI in the frontal lobe. When stratified by disease status, this correlation was strengthened in controls but not in AD. Our findings suggest a possible modified mechanism of gene action for APOE in AD that involves not only the protein isoforms but also an epigenetically regulated transcriptional program driven by DNA methylation in the APOE CGI.


Assuntos
Doença de Alzheimer/genética , Apolipoproteínas E/genética , Encéfalo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Autopsia , Estudos de Casos e Controles , Cerebelo/metabolismo , Ilhas de CpG , Metilação de DNA , Feminino , Lobo Frontal/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , RNA Circular/genética , RNA Circular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(4): 2194-2200, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932443

RESUMO

LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson's disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca2+-activated K+ (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a "functional knockdown" of these currents and may contribute to the tremor associated with increased LINGO1 levels.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cerebelo/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica
20.
J Neuropathol Exp Neurol ; 79(3): 266-276, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999342

RESUMO

For amyotrophic lateral sclerosis (ALS), achieving and maintaining effective drug levels in the brain is challenging due to the activity of ATP-binding cassette (ABC) transporters which efflux drugs that affect drug exposure and response in the brain. We investigated the expression and cellular distribution of the ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using immunohistochemistry in spinal cord (SC), motor cortex, and cerebellum from a large cohort of genetically well characterized ALS patients (n = 25) and controls (n = 14). The ALS group included 17 sporadic (sALS) and 8 familial (fALS) patients. Strong P-gp expression was observed in endothelial cells in both control and ALS specimens. Immunohistochemical analysis showed higher P-gp expression in reactive astroglial cells in both gray (ventral horn) and white matter of the SC, as well as in the motor cortex of all ALS patients, as compared with controls. BCRP expression was higher in glia in the SC and in blood vessels and glia in the motor cortex of ALS patients, as compared with controls. P-gp and BCRP immunoreactivity did not differ between sALS and fALS cases. The upregulation of both ABC transporters in the brain may explain multidrug resistance in ALS patients and has implications for the use of both approved and experimental therapeutics.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Cerebelo/metabolismo , Córtex Motor/metabolismo , Proteínas de Neoplasias/metabolismo , Medula Espinal/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/análise , Adulto , Idoso , Astrócitos/metabolismo , Astrócitos/patologia , Cerebelo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Motor/patologia , Proteínas de Neoplasias/análise , Medula Espinal/patologia
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