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1.
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
2.
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
3.
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
4.
PLoS Comput Biol ; 16(2): e1007601, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040505

RESUMO

Recent experimental findings indicate that Purkinje cells in the cerebellum represent time intervals by mechanisms other than conventional synaptic weights. These findings add to the theoretical and experimental observations suggesting the presence of intra-cellular mechanisms for adaptation and processing. To account for these experimental results we propose a new biophysical model for time interval learning in a Purkinje cell. The numerical model focuses on a classical delay conditioning task (e.g. eyeblink conditioning) and relies on a few computational steps. In particular, the model posits the activation by the parallel fiber input of a local intra-cellular calcium store which can be modulated by intra-cellular pathways. The reciprocal interaction of the calcium signal with several proteins forming negative and positive feedback loops ensures that the timing of inhibition in the Purkinje cell anticipates the interval between parallel and climbing fiber inputs during training. We systematically test the model ability to learn time intervals at the 150-1000 ms time scale, while observing that learning can also extend to the multiple seconds scale. In agreement with experimental observations we also show that the number of pairings required to learn increases with inter-stimulus interval. Finally, we discuss how this model would allow the cerebellum to detect and generate specific spatio-temporal patterns, a classical theory for cerebellar function.


Assuntos
Células de Purkinje/fisiologia , Potenciais de Ação , Animais , Cálcio/metabolismo , Condicionamento Clássico , Humanos , Células de Purkinje/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia
5.
Nat Commun ; 11(1): 1101, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107387

RESUMO

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP3) levels and ameliorates Purkinje cell degeneration in SCA17 knock-in mice. Our findings demonstrate the important contribution of a tissue-specific protein to the polyQ protein-mediated selective neuropathology.


Assuntos
Inositol Polifosfato 5-Fosfatases/genética , Células de Purkinje/patologia , Ataxias Espinocerebelares/patologia , Proteína de Ligação a TATA-Box/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Inositol Polifosfato 5-Fosfatases/metabolismo , Camundongos , Camundongos Transgênicos , Peptídeos/genética , Peptídeos/metabolismo , Células de Purkinje/metabolismo , Fator de Transcrição Sp1/metabolismo , Ataxias Espinocerebelares/genética , Proteína de Ligação a TATA-Box/metabolismo , Expansão das Repetições de Trinucleotídeos
6.
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
7.
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
8.
Nat Commun ; 10(1): 5560, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804475

RESUMO

Ecomorphological studies evaluating the impact of environmental and biological factors on the brain have so far focused on morphology or size measurements, and the ecological relevance of potential multi-level variations in brain architecture remains unclear in vertebrates. Here, we exploit the extraordinary ecomorphological diversity of squamates to assess brain phenotypic diversification with respect to locomotor specialization, by integrating single-cell distribution and transcriptomic data along with geometric morphometric, phylogenetic, and volumetric analysis of high-definition 3D models. We reveal significant changes in cerebellar shape and size as well as alternative spatial layouts of cortical neurons and dynamic gene expression that all correlate with locomotor behaviours. These findings show that locomotor mode is a strong predictor of cerebellar structure and pattern, suggesting that major behavioural transitions in squamates are evolutionarily correlated with mosaic brain changes. Furthermore, our study amplifies the concept of 'cerebrotype', initially proposed for vertebrate brain proportions, towards additional shape characters.


Assuntos
Encéfalo/metabolismo , Córtex Cerebelar/metabolismo , Perfilação da Expressão Gênica , Locomoção/fisiologia , Répteis/genética , Animais , Evolução Biológica , Encéfalo/anatomia & histologia , Encéfalo/citologia , Córtex Cerebelar/citologia , Ecologia , Locomoção/genética , Filogenia , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Répteis/anatomia & histologia , Répteis/classificação
9.
Physiol Rep ; 7(24): e14296, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31872561

RESUMO

Cardiac Purkinje cells (PCs) are more susceptible to action potential abnormalities as compared to ventricular myocytes (VMs), which could be associated with their distinct intracellular calcium handling. We developed a detailed biophysical model of a mouse cardiac PC, which importantly reproduces the experimentally observed biphasic cytosolic calcium waves. The model includes a stochastic gating formulation for the opening and closing of ryanodine receptor (RyR) channels, simulated with a Monte Carlo method, to accurately reproduce cytosolic calcium wave propagation and the effects of spontaneous calcium release events. Simulations predict that during an action potential, smaller cytosolic calcium wavelets propagated from the sarcolemma towards the center of the cell and initiated larger magnitude cell-wide calcium waves via a calcium-induced-calcium release mechanism. In the presence of RyR mutations, frequent spontaneous calcium leaks from sarcoplasmic reticulum (SR) initiated calcium waves, which upon reaching the cell periphery produced delayed afterdepolarizations (DADs) via sodium-calcium exchanger (NCX) and T-type calcium (ICaT ) channel activation. In the presence of isoproterenol-mediated effects, DADs induced triggered activity by reactivation of fast sodium channels. Based on our model, we found that the activation of either L-type calcium channels (ICaL ), ICaT , sodium-potassium exchanger (INaK ) or NCX is sufficient for occurrence of triggered activity; however, a partial blockade of ICaT or INaK is essential for its successful termination. Our modeling study highlights valuable insights into the mechanisms of DAD-induced triggered activity mediated via cytosolic calcium waves in cardiac PCs and may elucidate the increased arrhythmogeneity in PCs.


Assuntos
Potenciais de Ação , Sinalização do Cálcio , Modelos Teóricos , Células de Purkinje/fisiologia , Animais , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Camundongos , Células de Purkinje/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
10.
Int J Mol Sci ; 20(24)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847086

RESUMO

Niemann-Pick Disease Type C1 (NPC1) is a rare hereditary neurodegenerative disease belonging to the family of lysosomal storage disorders. NPC1-patients suffer from, amongst other symptoms, ataxia, based on the dysfunction and loss of cerebellar Purkinje cells. Alterations in synaptic transmission are believed to contribute to a pathological mechanism leading to the progressive loss of Purkinje cells observed in NPC1-deficient mice. With regard to inhibitory synaptic transmission, alterations of GABAergic synapses are described but functional data are missing. For this reason, we have examined here the inhibitory GABAergic synaptic transmission of Purkinje cells of NPC1-deficient mice (NPC1-/-). Patch clamp recordings of inhibitory post-synaptic currents (IPSCs) of Purkinje cells revealed an increased frequency of GABAergic IPSCs in NPC1-/- mice. In addition, Purkinje cells of NPC1-/- mice were less amenable for modulation of synaptic transmission via the activation of excitatory NMDA-receptors (NMDA-Rs). Western blot testing disclosed a reduced protein level of phosphorylated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) subunit GluA2 in the cerebella of NPC1-/- mice, indicating a disturbance in the internalization of GluA2-containing AMPA-Rs. Since this is triggered by the activation of NMDA-Rs, we conclude that a disturbance in the synaptic turnover of AMPA-Rs underlies the defective inhibitory GABAergic synaptic transmission. While these alterations precede obvious signs of neurodegeneration of Purkinje cells, we propose a contribution of synaptic malfunction to the initiation of the loss of Purkinje cells in NPC1. Thus, a prevention of the disturbance of synaptic transmission in early stages of the disease might display a target with which to avert progressive neurodegeneration in NPC1.


Assuntos
Neurônios GABAérgicos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Doenças Neurodegenerativas/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Células de Purkinje/metabolismo , Sinapses/metabolismo , Animais , Neurônios GABAérgicos/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Células de Purkinje/patologia , Sinapses/genética , Sinapses/patologia
11.
Int J Mol Sci ; 20(20)2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635400

RESUMO

S-equol is a major bacterial metabolite of the soy isoflavone daidzein. It is known to be a phytoestrogen that acts by binding to the nuclear estrogen receptors (ERs) that are expressed in various brain regions, including the cerebellum. However, the effects of S-equol on cerebellar development and function have not yet been extensively studied. In this study, the effects of S-equol were evaluated using a mouse primary cerebellar culture, Neuro-2A clonal cells, and an astrocyte-enriched culture. S-equol augmented the dendrite arborization of Purkinje cells induced by triiodothyronine (T3) and the neurite growth of Neuro-2A cell differentiation. Such augmentation was suppressed by G15, a selective G-protein coupled ER (GPR30) antagonist, and ICI 182,780, an antagonist for ERs in both cultures. On the other hand, in astrocytes, S-equol induced cell proliferation and cell migration with an increase in the phosphorylated extracellular-signal-regulated kinase 1/2 and F-actin rearrangements. Such effects were suppressed by G15, but not by ICI. These findings indicated that S-equol may enhanced cerebellar development by affecting both neurons and astrocytes through several signaling pathways, including GPR30 and ERs. We here report a novel mechanism of S-equol in cerebellar development that may provide a novel possibility to use S-equol supplementation during development.


Assuntos
Astrócitos/metabolismo , Equol/metabolismo , Neurônios/metabolismo , Actinas/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Equol/farmacologia , Feminino , Humanos , Camundongos , Modelos Biológicos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosforilação , Gravidez , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Nat Neurosci ; 22(10): 1731-1742, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501572

RESUMO

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.


Assuntos
Encéfalo/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Proteômica , Células de Purkinje/metabolismo
13.
Cerebellum ; 18(6): 999-1010, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31273610

RESUMO

The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Cerebelo/embriologia , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sobrevivência Celular/fisiologia , Cerebelo/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células de Purkinje/metabolismo
14.
Brain Struct Funct ; 224(7): 2421-2436, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31256239

RESUMO

In the standard model for the development of climbing and mossy fiber afferent pathways to the cerebellum, the ingrowing axons target the embryonic Purkinje cell somata (around embryonic ages (E13-E16 in mice). In this report, we describe a novel earlier stage in afferent development. Immunostaining for a neurofilament-associated antigen (NAA) reveals the early axon distributions with remarkable clarity. Using a combination of DiI axon tract tracing, analysis of neurogenin1 null mice, which do not develop trigeminal ganglia, and mouse embryos maintained in vitro, we show that the first axons to innervate the cerebellar primordium as early as E9 arise from the trigeminal ganglion. Therefore, early trigeminal axons are in situ before the Purkinje cells are born. Double immunostaining for NAA and markers of the different domains in the cerebellar primordium reveal that afferents first target the nuclear transitory zone (E9-E10), and only later (E10-E11) are the axons, either collaterals from the trigeminal ganglion or a new afferent source (e.g., vestibular ganglia), seen in the Purkinje cell plate. The finding that the earliest axons to the cerebellum derive from the trigeminal ganglion and enter the cerebellar primordium before the Purkinje cells are born, where they seem to target the cerebellar nuclei, reveals a novel stage in the development of the cerebellar afferents.


Assuntos
Cerebelo/metabolismo , Neurônios/metabolismo , Células de Purkinje/metabolismo , Gânglio Trigeminal/metabolismo , Vias Aferentes/metabolismo , Animais , Axônios/metabolismo , Núcleos Cerebelares/metabolismo , Nervo Vestibular/metabolismo
15.
Exp Neurol ; 320: 112983, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31251935

RESUMO

Although multiple reports using animal models have confirmed that melatonin appears to promote neuroprotective effects following ischemia/reperfusion-induced brain injury, the relationship between its protective effects and activation of autophagy in Purkinje cells following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) remains unclear. Rats used in this study were randomly assigned to 6 groups as follows; vehicle-treated sham operated group, vehicle-treated asphyxial CA/CPR operated group, melatonin-treated sham operated group, melatonin-treated asphyxial CA/CPR operated group, PDOT (a MT2 melatonin receptor antagonist) plus (+) melatonin-treated sham operated group and PDOT+melatonin-treated asphyxial CA/CPR operated group. Melatonin (20 mg/kg, i.p., 4 times before CA and 3 times after CA) treatment significantly improved survival rate and neurological deficit compared with the vehicle-treated asphyxial CA/CPR rats (survival rates ≥40% vs 10%), showing that melatonin treatment exhibited protective effect against asphyxial CA/CPR-induced Purkinje cell death. The protective effect of melatonin against CA/CPR-induced Purkinje cell death paralleled a remarkable attenuation of autophagy-like processes (Beclin-1, Atg7 and LC3), as well as a dramatic reduction in superoxide anion radical (O2·-), intense enhancements of CuZn superoxide dismutase (SOD1) and MnSOD (SOD2) expressions. Furthermore, the protective effect was notably reversed by treatment with PDOT, which is a selective MT2 antagonist. In brief, melatonin conferred neuroprotection against asphyxial CA/CPR-induced Purkinje cell death via inhibiting autophagic activation by reducing expressions of O2·- and increasing expressions of antioxidant enzymes, and suggests that MT2 is involved in neuroprotective effect of melatonin against Purkinje cell death caused by asphyxial CA/CPR.


Assuntos
Antioxidantes/farmacologia , Parada Cardíaca/patologia , Melatonina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células de Purkinje/efeitos dos fármacos , Animais , Asfixia/etiologia , Autofagia/efeitos dos fármacos , Parada Cardíaca/complicações , Masculino , Fármacos Neuroprotetores/farmacologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptor MT2 de Melatonina/metabolismo
16.
Neurobiol Dis ; 130: 104516, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31229688

RESUMO

Spinocerebellar ataxia 42 (SCA42) is a neurodegenerative disorder recently shown to be caused by c.5144G > A (p.Arg1715His) mutation in CACNA1G, which encodes the T-type voltage-gated calcium channel CaV3.1. Here, we describe a large Japanese family with SCA42. Postmortem pathological examination revealed severe cerebellar degeneration with prominent Purkinje cell loss without ubiquitin accumulation in an SCA42 patient. To determine whether this mutation causes ataxic symptoms and neurodegeneration, we generated knock-in mice harboring c.5168G > A (p.Arg1723His) mutation in Cacna1g, corresponding to the mutation identified in the SCA42 family. Both heterozygous and homozygous mutants developed an ataxic phenotype from the age of 11-20 weeks and showed Purkinje cell loss at 50 weeks old. Degenerative change of Purkinje cells and atrophic thinning of the molecular layer were conspicuous in homozygous knock-in mice. Electrophysiological analysis of Purkinje cells using acute cerebellar slices from young mice showed that the point mutation altered the voltage dependence of CaV3.1 channel activation and reduced the rebound action potentials after hyperpolarization, although it did not significantly affect the basic properties of synaptic transmission onto Purkinje cells. Finally, we revealed that the resonance of membrane potential of neurons in the inferior olivary nucleus was decreased in knock-in mice, which indicates that p.Arg1723His CaV3.1 mutation affects climbing fiber signaling to Purkinje cells. Altogether, our study shows not only that a point mutation in CACNA1G causes an ataxic phenotype and Purkinje cell degeneration in a mouse model, but also that the electrophysiological abnormalities at an early stage of SCA42 precede Purkinje cell loss.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Cerebelo/metabolismo , Fenótipo , Células de Purkinje/metabolismo , Ataxias Espinocerebelares/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Canais de Cálcio Tipo T/genética , Cerebelo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Células de Purkinje/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
17.
Bull Exp Biol Med ; 167(1): 39-42, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31177457

RESUMO

Voltage clamp and concentration-jump methods were employed to examine the effects of endogenous neuropeptide cycloprolylglycine on GABA-activated ionic currents in isolated cerebellar Purkinje cells. In the concentration range of 0.1-10.0 µM, short-term (600 msec) external application of cycloprolylglycine against the background of GABA-evoked current produced no effect on its amplitude. In contrast, application of 1 µM cycloprolylglycine increased current up to 177±15% control level. The development of potentiating effect and return to the control level of ionic current were slow, which was indicative of possible implication of second messenger systems in these processes. Functional augmentation of GABAA receptors under the action of cycloprolylglycine can underlie the established neuroprotective and anxiolytic effects of this endogenous dipeptide.


Assuntos
Peptídeos Cíclicos/farmacologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Receptores de GABA-A/metabolismo , Animais , Células Cultivadas , Agonistas GABAérgicos/farmacologia , Humanos , Técnicas de Patch-Clamp , Peptídeos Cíclicos/metabolismo , Ratos
18.
Brain Struct Funct ; 224(6): 2121-2142, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31165301

RESUMO

Neurofibromatosis type 2 (NF2) patients are prone to develop glial-derived tumors in the peripheral and central nervous system (CNS). The Nf2 gene product -Merlin is not only expressed in glia, but also in neurons of the CNS, where its function still remains elusive. Here, we show that cerebellar Purkinje cells (PCs) of isoform-specific Merlin-deficient mice were innervated by smaller vGluT2-positive clusters at presynaptic terminals than those of wild-type mice. This was paralleled by a reduction in frequency and amplitude of miniature excitatory postsynaptic currents (mEPSC). On the contrary, in conditional transgenic mice in which Merlin expression was specifically ablated in PCs (L7Cre;Nf2fl/fl), we found enlarged vGluT2-positive clusters in their presynaptic buttons together with increased amplitudes of miniature postsynaptic currents. The presynaptic terminals of these PCs innervating neurons of the deep cerebellar nuclei were also enlarged. When exploring mice with Merlin-deficient granule cells (GCs) (Math1Cre;Nf2fl/fl), we found cerebellar extracts to contain higher amounts of vGluT1 present in parallel fiber terminals. In parallel, mEPSC frequency was increased in Math1Cre;Nf2fl/fl mice. On the contrary, VGluT2 clusters in cerebellar glomeruli composed of NF2-deficient presynaptic Mossy fiber terminals and NF2-deficient postsynaptic GC were reduced in size as shown for isoform-specific knockout mice. These changes in Math1Cre;Nf2fl/fl-deficient mice were paralleled by an increased activation of Rac1-Cofilin signaling which is known to impact on cytoskeletal reorganization and synapse formation. Consistent with the observed synaptic alterations in these transgenic mice, we observed altered ultrasonic vocalization, which is known to rely on proper cerebellar function. No gross morphological changes or motor coordination deficits were observed in any of these transgenic mice. We therefore conclude that Merlin does not regulate overall cerebellar development, but impacts on pre- and post-synaptic terminal organization.


Assuntos
Cerebelo/metabolismo , Neurofibromina 2/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Animais , Axônios/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Células de Purkinje/metabolismo
19.
J Biol Chem ; 294(30): 11498-11512, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31177092

RESUMO

Neurolastin is a dynamin family GTPase that also contains a RING domain and exhibits both GTPase and E3 ligase activities. It is specifically expressed in the brain and is important for synaptic transmission, as neurolastin knockout animals have fewer dendritic spines and exhibit a reduction in functional synapses. Our initial study of neurolastin revealed that it is membrane-associated and partially co-localizes with endosomes. Using various biochemical and cell-culture approaches, we now show that neurolastin also localizes to mitochondria in HeLa cells, cultured neurons, and brain tissue. We found that the mitochondrial localization of neurolastin depends upon an N-terminal mitochondrial targeting sequence and that neurolastin is imported into the mitochondrial intermembrane space. Although neurolastin was only partially mitochondrially localized at steady state, it displayed increased translocation to mitochondria in response to neuronal stress and mitochondrial fragmentation. Interestingly, inactivation or deletion of neurolastin's RING domain also increased its mitochondrial localization. Using EM, we observed that neurolastin knockout animals have smaller but more numerous mitochondria in cerebellar Purkinje neurons, indicating that neurolastin regulates mitochondrial morphology. We conclude that the brain-specific dynamin GTPase neurolastin exhibits stress-responsive localization to mitochondria and is required for proper mitochondrial morphology.


Assuntos
Dinaminas/metabolismo , Mitocôndrias/metabolismo , Células de Purkinje/metabolismo , Animais , Células Cultivadas , Dinaminas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Mutação , Transporte Proteico
20.
Mol Cell ; 75(1): 13-25.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31151856

RESUMO

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células de Purkinje/metabolismo , Sequência de Aminoácidos , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/metabolismo , Animais , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Células de Purkinje/citologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinapses/fisiologia , Transmissão Sináptica
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