Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.368
Filtrar
1.
Nat Commun ; 12(1): 2153, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846328

RESUMO

The signals in cerebellar Purkinje cells sufficient to instruct motor learning have not been systematically determined. Therefore, we applied optogenetics in mice to autonomously excite Purkinje cells and measured the effect of this activity on plasticity induction and adaptive behavior. Ex vivo, excitation of channelrhodopsin-2-expressing Purkinje cells elicits dendritic Ca2+ transients with high-intensity stimuli initiating dendritic spiking that additionally contributes to the Ca2+ response. Channelrhodopsin-2-evoked Ca2+ transients potentiate co-active parallel fiber synapses; depression occurs when Ca2+ responses were enhanced by dendritic spiking. In vivo, optogenetic Purkinje cell activation drives an adaptive decrease in vestibulo-ocular reflex gain when vestibular stimuli are paired with relatively small-magnitude Purkinje cell Ca2+ responses. In contrast, pairing with large-magnitude Ca2+ responses increases vestibulo-ocular reflex gain. Optogenetically induced plasticity and motor adaptation are dependent on endocannabinoid signaling, indicating engagement of this pathway downstream of Purkinje cell Ca2+ elevation. Our results establish a causal relationship among Purkinje cell Ca2+ signal size, opposite-polarity plasticity induction, and bidirectional motor learning.


Assuntos
Sinalização do Cálcio , Dendritos/metabolismo , Atividade Motora , Células de Purkinje/metabolismo , Potenciais de Ação , Animais , Channelrhodopsins/metabolismo , Endocanabinoides/metabolismo , Camundongos Transgênicos , Plasticidade Neuronal , Optogenética , Piperidinas/farmacologia , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Reflexo Vestíbulo-Ocular , Vigília
2.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804256

RESUMO

Lysosomal acid phosphatase 2 (Acp2) mutant mice (naked-ataxia, nax) have a severe cerebellar cortex defect with a striking reduction in the number of granule cells. Using a combination of in vivo and in vitro immunohistochemistry, Western blotting, BrdU assays, and RT-qPCR, we show downregulation of MYCN and dysregulation of the SHH signaling pathway in the nax cerebellum. MYCN protein expression is significantly reduced at P10, but not at the peak of proliferation at around P6 when the number of granule cells is strikingly reduced in the nax cerebellum. Despite the significant role of the SHH-MycN pathway in granule cell proliferation, our study suggests that a broader molecular pathway and additional mechanisms regulating granule cell development during the clonal expansion period are impaired in the nax cerebellum. In particular, our results indicate that downregulation of the protein synthesis machinery may contribute to the reduced number of granule cells in the nax cerebellum.


Assuntos
Fosfatase Ácida/genética , Ataxia Cerebelar/genética , Córtex Cerebelar/metabolismo , Proteínas Hedgehog/genética , Proteína Proto-Oncogênica N-Myc/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Córtex Cerebelar/anormalidades , Córtex Cerebelar/patologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lisossomos/genética , Lisossomos/patologia , Camundongos , Mutação , Neurônios/metabolismo , Neurônios/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Transdução de Sinais/genética
3.
Neurosci Lett ; 751: 135807, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33705934

RESUMO

Reduced cerebellar volume and motor dysfunction have previously been observed in the GFAP-IL6 murine model of chronic neuroinflammation. This study aims to extend these findings by investigating the effect of microglial activation and ageing on the total number of Purkinje cells and the morphology of their dendritic arborization. Through comparison of transgenic GFAP-IL6 mice and their wild-type counterparts at the ages of 12 and 24-months, we were able to investigate the effects of ageing and chronic microglial activation on Purkinje cells. Unbiased stereology was used to estimate the number of microglia in Iba1+ stained tissue and Purkinje cells in calbindin stained tissue. Morphological analyses were made using 3D reconstructions of images acquired from the Golgi-stained cerebellar tissue. We found that the total number of microglia increased by approximately 5 times in the cerebellum of GFAP-IL6 mice compared to their WT littermates. The number of Purkinje cells decreased by as much as 50 % in aged wild type mice and 83 % in aged GFAP-IL6 mice. The remaining Purkinje cells in these cohorts were found to have significant reductions in their total dendritic length and number of branching points, indicating how the complexity of the Purkinje cell dendritic arbor reduces through age and inflammation. GFAP-IL6 mice, when compared to WT mice, had higher levels of microglial activation and more profound neurodegenerative changes in the cerebellum. The presence of constitutive IL6 production, driving chronic neuroinflammation, may account for these neurodegenerative changes in GFAP-IL6 mice.


Assuntos
Envelhecimento/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Interleucina-6/metabolismo , Microglia/metabolismo , Células de Purkinje/citologia , Envelhecimento/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Proteína Glial Fibrilar Ácida/genética , Inflamação/metabolismo , Interleucina-6/genética , Camundongos , Microglia/citologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia
4.
J Physiol ; 599(7): 2085-2102, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33527421

RESUMO

KEY POINTS: Recent studies have repeatedly demonstrated the cross-talk of heterogeneous signals between neuronal and glial circuits. Here, we investigated the mechanism and the influence of physiological interactions between neurons and glia in the cerebellum. We found that the cerebellar astrocytes, Bergmann glial cells, react to exogenously applied glutamate, glutamate transporter substrate (d-aspartate) and synaptically released glutamate. In response, the Bergmann glial cells release glutamate through volume-regulated anion channels. It is generally assumed that all of the postsynaptic current is mediated by presynaptically released glutamate. However, we showed that a part of the postsynaptic current is mediated by glutamate released from Bergmann glial cells. Optogenetic manipulation of Bergmann glial state with archaerhodpsin-T or channelrhodopsin-2 reduced or augmented the amount of glial glutamate release, respectively. Our data indicate that glutamate-induced glutamate release in Bergmann glia serves as an effective amplifier of excitatory information processing in the brain. ABSTRACT: Transmitter released from presynaptic neurons has been considered to be the sole generator of postsynaptic excitatory signals. However, astrocytes of the glial cell population have also been shown to release transmitter that can react on postsynaptic receptors. Therefore, we investigated whether astrocytes take part in generation of at least a part of the synaptic current. In this study, mice cerebellar acute slices were prepared and whole cell patch clamp recordings were performed. We found that Bergmann glial cells (BGs), a type of astrocyte in the cerebellum, reacts to a glutamate transporter substrate, d-aspartate (d-Asp) and an anion conductance is generated and glutamate is released from the BGs. Glutamate release was attenuated or augmented by modulating the state of BGs with activation of light-sensitive proteins, archaerhodopsin-T (ArchT) or channelrhodopsin-2 (ChR2) expressed on BGs, respectively. Glutamate release appears to be mediated by anion channels that can be blocked by a volume-regulated anion channel-specific blocker. Synaptic response to a train of parallel fibre stimulation was recorded from Purkinje cells. The latter part of the response was also attenuated or augmented by glial modulation with ArchT or ChR2, respectively. Thus, BGs effectively function as an excitatory signal amplifier, and a part of the 'synaptic' current is actually mediated by glutamate released from BGs. These data show that the state of BGs have potential for having direct and fundamental consequences on the functioning of information processing in the brain.


Assuntos
Neuroglia , Células de Purkinje , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Cerebelo/metabolismo , Ácido Glutâmico , Camundongos , Neuroglia/metabolismo , Células de Purkinje/metabolismo
5.
JCI Insight ; 6(3)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33554954

RESUMO

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder characterized by motor incoordination, mild cognitive decline, respiratory dysfunction, and early lethality. It is caused by the expansion of the polyglutamine (polyQ) tract in Ataxin-1 (ATXN1), which stabilizes the protein, leading to its toxic accumulation in neurons. Previously, we showed that serine 776 (S776) phosphorylation is critical for ATXN1 stability and contributes to its toxicity in cerebellar Purkinje cells. Still, the therapeutic potential of disrupting S776 phosphorylation on noncerebellar SCA1 phenotypes remains unstudied. Here, we report that abolishing S776 phosphorylation specifically on the polyQ-expanded ATXN1 of SCA1-knockin mice reduces ATXN1 throughout the brain and not only rescues the cerebellar motor incoordination but also improves respiratory function and extends survival while not affecting the hippocampal learning and memory deficits. As therapeutic approaches are likely to decrease S776 phosphorylation on polyQ-expanded and WT ATXN1, we further disrupted S776 phosphorylation on both alleles and observed an attenuated rescue, demonstrating a potential protective role of WT allele. This study not only highlights the role of S776 phosphorylation to regulate ATXN1 levels throughout the brain but also suggests distinct brain region-specific disease mechanisms and demonstrates the importance of developing allele-specific therapies for maximal benefits in SCA1.


Assuntos
Ataxina-1/química , Ataxina-1/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Alelos , Animais , Ataxina-1/genética , Comportamento Animal , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Modelos Neurológicos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Estabilidade Proteica , Células de Purkinje/metabolismo , Serina/química , Ataxias Espinocerebelares/terapia , Expansão das Repetições de Trinucleotídeos
6.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466390

RESUMO

Niemann-Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-ß-CD in Npc1 gene-deficient (Npc1-/-) mice. Intracerebroventricular HP-ß-CD inhibited cerebellar Purkinje cell damage in Npc1-/- mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1-/- mice. Repeated doses of intracerebroventricular HP-ß-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1-/- mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-ß-CD treatment.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/administração & dosagem , Cerebelo/efeitos dos fármacos , Proteínas do Olho/metabolismo , Fígado/efeitos dos fármacos , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/metabolismo , Animais , Biomarcadores/metabolismo , Cerebelo/metabolismo , Colesterol/metabolismo , Modelos Animais de Doenças , Feminino , Glicoproteínas/metabolismo , Infusões Intraventriculares , Fígado/metabolismo , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo
7.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33443203

RESUMO

Activity of sensory and motor cortices is essential for sensorimotor integration. In particular, coherence between these areas may indicate binding of critical functions like perception, motor planning, action, or sleep. Evidence is accumulating that cerebellar output modulates cortical activity and coherence, but how, when, and where it does so is unclear. We studied activity in and coherence between S1 and M1 cortices during whisker stimulation in the absence and presence of optogenetic Purkinje cell stimulation in crus 1 and 2 of awake mice, eliciting strong simple spike rate modulation. Without Purkinje cell stimulation, whisker stimulation triggers fast responses in S1 and M1 involving transient coherence in a broad spectrum. Simultaneous stimulation of Purkinje cells and whiskers affects amplitude and kinetics of sensory responses in S1 and M1 and alters the estimated S1-M1 coherence in theta and gamma bands, allowing bidirectional control dependent on behavioral context. These effects are absent when Purkinje cell activation is delayed by 20 ms. Focal stimulation of Purkinje cells revealed site specificity, with cells in medial crus 2 showing the most prominent and selective impact on estimated coherence, i.e., a strong suppression in the gamma but not the theta band. Granger causality analyses and computational modeling of the involved networks suggest that Purkinje cells control S1-M1 phase consistency predominantly via ventrolateral thalamus and M1. Our results indicate that activity of sensorimotor cortices can be dynamically and functionally modulated by specific cerebellar inputs, highlighting a widespread role of the cerebellum in coordinating sensorimotor behavior.


Assuntos
Córtex Motor/metabolismo , Células de Purkinje/metabolismo , Córtex Somatossensorial/metabolismo , Animais , Córtex Cerebelar , Cerebelo/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Optogenética , Córtex Sensório-Motor , Núcleos Ventrais do Tálamo , Vibrissas/fisiologia
8.
J Neurosci ; 41(9): 2053-2068, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33478986

RESUMO

Spinocerebellar ataxias (SCAs) are diseases characterized by cerebellar atrophy and loss of Purkinje neurons caused by mutations in diverse genes. In SCA14, the disease is caused by point mutations or small deletions in protein kinase C γ (PKCγ), a crucial signaling protein in Purkinje cells. It is still unclear whether increased or decreased PKCγ activity may be involved in the SCA14 pathogenesis. In this study, we present a new knock-in mouse model related to SCA14 with a point mutation in the pseudosubstrate domain, PKCγ-A24E, known to induce a constitutive PKCγ activation. In this protein conformation, the kinase domain of PKCγ is activated, but at the same time the protein is subject to dephosphorylation and protein degradation. As a result, we find a dramatic reduction of PKCγ protein expression in PKCγ-A24E mice of either sex. Despite this reduction, there is clear evidence for an increased PKC activity in Purkinje cells from PKCγ-A24E mice. Purkinje cells derived from PKCγ-A24E have short thickened dendrites typical for PKC activation. These mice also develop a marked ataxia and signs of Purkinje cell dysfunction making them an interesting new mouse model related to SCA. Recently, a similar mutation in a human patient was discovered and found to be associated with overt SCA14. RNA profiling of PKCγ-A24E mice showed a dysregulation of related signaling pathways, such as mGluR1 or mTOR. Our results show that the induction of PKCγ activation in Purkinje cells results in the SCA-like phenotype indicating PKC activation as one pathogenetic avenue leading to a SCA.SIGNIFICANCE STATEMENT Spinocerebellar ataxias (SCAs) are hereditary diseases affecting cerebellar Purkinje cells and are a one of neurodegenerative diseases. While mutation in several genes have been identified as causing SCAs, it is unclear how these mutations cause the disease phenotype. Mutations in PKCγ cause one subtype of SCAs, SCA14. In this study, we have generated a knock-in mouse with a mutation in the pseudosubstrate domain of PKCγ, which keeps PKCγ in the constitutive active open conformation. We show that this mutation leading to a constant activation of PKCγ results in a SCA-like phenotype in these mice. Our findings establish the constant activation of PKC signaling as one pathogenetic avenue leading to an SCA phenotype and a mechanism causing a neurodegenerative disease.


Assuntos
Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Células de Purkinje/metabolismo , Ataxias Espinocerebelares/genética , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Atividade Motora/fisiologia , Mutação , Células de Purkinje/patologia , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
9.
Neuron ; 109(4): 629-644.e8, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33352118

RESUMO

The synaptotrophic hypothesis posits that synapse formation stabilizes dendritic branches, but this hypothesis has not been causally tested in vivo in the mammalian brain. The presynaptic ligand cerebellin-1 (Cbln1) and postsynaptic receptor GluD2 mediate synaptogenesis between granule cells and Purkinje cells in the molecular layer of the cerebellar cortex. Here we show that sparse but not global knockout of GluD2 causes under-elaboration of Purkinje cell dendrites in the deep molecular layer and overelaboration in the superficial molecular layer. Developmental, overexpression, structure-function, and genetic epistasis analyses indicate that these dendrite morphogenesis defects result from a deficit in Cbln1/GluD2-dependent competitive interactions. A generative model of dendrite growth based on competitive synaptogenesis largely recapitulates GluD2 sparse and global knockout phenotypes. Our results support the synaptotrophic hypothesis at initial stages of dendrite development, suggest a second mode in which cumulative synapse formation inhibits further dendrite growth, and highlight the importance of competition in dendrite morphogenesis.


Assuntos
Cerebelo/citologia , Cerebelo/metabolismo , Dendritos/metabolismo , Proteínas do Tecido Nervoso/deficiência , Precursores de Proteínas/deficiência , Células de Purkinje/metabolismo , Receptores de Glutamato/deficiência , Animais , Dendritos/genética , Feminino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Gravidez , Ligação Proteica/fisiologia , Precursores de Proteínas/genética , Receptores de Glutamato/genética
10.
FASEB J ; 35(1): e21092, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33378124

RESUMO

Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is ß-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here, we show that myosin 18A⍺ concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⍺ results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A⍺. Importantly, ß-Pix co-localizes with myosin 18A⍺ in spines, and its spine localization is lost upon myosin 18A⍺ knockdown or when its myosin 18A⍺ binding site is deleted. Finally, we show that the spines of myosin 18A⍺ knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⍺ form a complex with ß-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of ß-Pix's GEF activity.


Assuntos
Espinhas Dendríticas/metabolismo , Miosinas/metabolismo , Células de Purkinje/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Espinhas Dendríticas/genética , Deleção de Genes , Camundongos , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Miosinas/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética
11.
Nat Commun ; 11(1): 6407, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335094

RESUMO

Endocannabinoids retrogradely regulate synaptic transmission and their abundance is controlled by the fine balance between endocannabinoid synthesis and degradation. While the common assumption is that "on-demand" release determines endocannabinoid signaling, their rapid degradation is expected to control the temporal profile of endocannabinoid action and may impact neuronal signaling. Here we show that memory formation through fear conditioning selectively accelerates the degradation of endocannabinoids in the cerebellum. Learning induced a lasting increase in GABA release and this was responsible for driving the change in endocannabinoid degradation. Conversely, Gq-DREADD activation of cerebellar Purkinje cells enhanced endocannabinoid signaling and impaired memory consolidation. Our findings identify a previously unappreciated reciprocal interaction between GABA and the endocannabinoid system in which GABA signaling accelerates endocannabinoid degradation, and triggers a form of learning-induced metaplasticity.


Assuntos
Endocanabinoides/metabolismo , Consolidação da Memória/fisiologia , Transmissão Sináptica/fisiologia , Animais , Cerebelo/metabolismo , Condicionamento Clássico , Medo , Masculino , Camundongos Endogâmicos C57BL , Monoacilglicerol Lipases/metabolismo , Células de Purkinje/metabolismo , Células de Renshaw/metabolismo , Ácido gama-Aminobutírico/metabolismo
12.
Nat Commun ; 11(1): 5304, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082323

RESUMO

A missense mutation, S85C, in the MATR3 gene is a genetic cause for amyotrophic lateral sclerosis (ALS). It is unclear how the S85C mutation affects MATR3 function and contributes to disease. Here, we develop a mouse model that harbors the S85C mutation in the endogenous Matr3 locus using the CRISPR/Cas9 system. MATR3 S85C knock-in mice recapitulate behavioral and neuropathological features of early-stage ALS including motor impairment, muscle atrophy, neuromuscular junction defects, Purkinje cell degeneration and neuroinflammation in the cerebellum and spinal cord. Our neuropathology data reveals a loss of MATR3 S85C protein in the cell bodies of Purkinje cells and motor neurons, suggesting that a decrease in functional MATR3 levels or loss of MATR3 function contributes to neuronal defects. Our findings demonstrate that the MATR3 S85C mouse model mimics aspects of early-stage ALS and would be a promising tool for future basic and preclinical research.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Neurônios Motores/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Mutação de Sentido Incorreto , Células de Purkinje/metabolismo
13.
Parasitol Res ; 119(10): 3433-3441, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32789733

RESUMO

Angiostrongylus cantonensis causes a human central nervous system (CNS) infection characterized by eosinophilic meningitis or meningoencephalitis. Individuals infected with A. cantonensis exhibit unbalanced walking. The mechanism of extensive neurological impairments of hosts caused by A. cantonensis larvae remains unclear. Tight junction proteins (e.g., claudin-5 and zonula occludens-1) are the most important regulators of paracellular permeability and cellular adhesion. In a previous study, we found that increased matrix metalloproteinase-9 (MMP-9) activity may be associated with blood-CNS barrier disruption and/or the degeneration of Purkinje cells in eosinophilic meningitis caused by A. cantonensis. In the present study, the co-localization of MMP-9 and tight junction proteins on the degeneration of Purkinje cells was measured via confocal laser scanning immunofluorescence microscopy. The statistical evidence indicated that MMP-9 correlated between tight junction protein disruption and Purkinje cell degeneration at 20 days post-infection with A. cantonensis. In conclusion, Purkinje cell degeneration is highly correlated with tight junction protein disruption via the MMP-9 activation pathway.


Assuntos
Angiostrongylus cantonensis/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Células de Purkinje/patologia , Infecções por Strongylida/parasitologia , Proteínas de Junções Íntimas/metabolismo , Animais , Modelos Animais de Doenças , Larva/fisiologia , Camundongos , Células de Purkinje/metabolismo , Células de Purkinje/parasitologia , Infecções por Strongylida/metabolismo , Infecções por Strongylida/patologia
14.
Proc Natl Acad Sci U S A ; 117(29): 17348-17358, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636261

RESUMO

The cerebellar posterior vermis generates an estimation of our motion (translation) and orientation (tilt) in space using cues originating from semicircular canals and otolith organs. Theoretical work has laid out the basic computations necessary for this signal transformation, but details on the cellular loci and mechanisms responsible are lacking. Using a multicomponent modeling approach, we show that canal and otolith information are spatially and temporally matched in mouse posterior vermis Purkinje cells and that Purkinje cell responses combine translation and tilt information. Purkinje cell-specific inhibition of protein kinase C decreased and phase-shifted the translation component of Purkinje cell responses, but did not affect the tilt component. Our findings suggest that translation and tilt signals reach Purkinje cells via separate information pathways and that protein kinase C-dependent mechanisms regulate translation information processing in cerebellar cortex output neurons.


Assuntos
Vermis Cerebelar/fisiologia , Membrana dos Otólitos/fisiologia , Proteína Quinase C/metabolismo , Células de Purkinje/metabolismo , Animais , Córtex Cerebelar , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Plasticidade Neuronal
15.
Nat Commun ; 11(1): 3343, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620905

RESUMO

The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although known to form distinctive intranuclear bodies, the cellular pathways and processes that polyQ-ataxin-1 influences remain poorly understood. Here we identify the direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells, pathways analyses indicating a significant enrichment of essential nuclear transporters, pointing to disruptions in nuclear transport processes in the presence of elevated levels of ataxin-1. Our direct assessments of nuclear transporters and their cargoes confirm these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Analogous changes in importin-ß1, nucleoporin 98 and nucleoporin 62 nuclear rim staining are observed in Purkinje cells of ATXN1[82Q] mice. The results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQ-ataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.


Assuntos
Ataxina-1/metabolismo , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células de Purkinje/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Ataxina-1/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HeLa , Humanos , Camundongos , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Peptídeos/genética , Ligação Proteica , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
16.
Sci Rep ; 10(1): 9420, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523011

RESUMO

We investigated the role of nitric oxide synthase (NOS) in mediating blood-brain barrier (BBB) disruption and peripheral immune cell infiltration in the cerebellum following blast exposure. Repetitive, but not single blast exposure, induced delayed-onset BBB disruption (72 hours post-blast) in cerebellum. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) administered after blast blocked BBB disruption and prevented CD4+ T-cell infiltration into cerebellum. L-NAME also blocked blast-induced increases in intercellular adhesion molecule-1 (ICAM-1), a molecule that plays a critical role in regulating blood-to-brain immune cell trafficking. Blocking NOS-mediated BBB dysfunction during this acute/subacute post-blast interval (24-71 hours after the last blast) also prevented sensorimotor impairment on a rotarod task 30 days later, long after L-NAME cleared the body. In postmortem brains from Veterans/military Servicemembers with blast-related TBI, we found marked Purkinje cell dendritic arbor structural abnormalities, which were comparable to neuropathologic findings in the blast-exposed mice. Taken collectively, these results indicate that blast provokes delayed-onset of NOS-dependent pathogenic cascades that can later emerge as behavioral dysfunction. These results also further implicate the cerebellum as a brain region vulnerable to blast-induced mTBI.


Assuntos
Traumatismos por Explosões/metabolismo , Traumatismos por Explosões/fisiopatologia , Concussão Encefálica/fisiopatologia , Doenças Cerebelares/metabolismo , Doenças Cerebelares/fisiopatologia , Cerebelo/fisiopatologia , Óxido Nítrico Sintase/metabolismo , Animais , Traumatismos por Explosões/tratamento farmacológico , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Concussão Encefálica/tratamento farmacológico , Concussão Encefálica/metabolismo , Doenças Cerebelares/tratamento farmacológico , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Modelos Animais de Doenças , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NG-Nitroarginina Metil Éster/farmacologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Células de Purkinje/patologia
17.
Proc Natl Acad Sci U S A ; 117(26): 15085-15095, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32546527

RESUMO

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.


Assuntos
Diferenciação Celular , Células de Purkinje/metabolismo , Transcriptoma , Animais , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas/genética , Proteínas/metabolismo , Células de Purkinje/citologia
18.
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
20.
Sci Rep ; 10(1): 4452, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157179

RESUMO

The cerebellum coordinates voluntary movements for balanced motor activity in a normal gravity condition. It remains unknown how hypergravity is associated with cerebellum-dependent motor behaviors and Purkinje cell's activities. In order to investigate the relationship between gravity and cerebellar physiology, we measured AMPA-mediated fast currents and mGluR1-mediated slow currents of cerebellar Purkinje cells along with cerebellum-dependent behaviors such as the footprint and irregular ladder under a hypergravity condition. We found abnormal animal behaviors in the footprint and irregular ladder tests under hypergravity. They are correlated with decreased AMPA and mGluR1-mediated synaptic currents of Purkinje cells. These results indicate that gravity regulates the activity of Purkinje cells, thereby modulating cerebellum-dependent motor outputs.


Assuntos
Cerebelo/patologia , Hipergravidade , Atividade Motora , Células de Purkinje/patologia , Animais , Comportamento Animal , Cerebelo/metabolismo , Masculino , Células de Purkinje/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...