RESUMEN
Staggerer mutant mice have functional loss of a transcription factor, retinoid-related orphan receptor α (RORα), which is abundantly expressed in Purkinje cells (PCs) of the cerebellum.Homozygous staggerer (sg/sg)mice show cerebellar hypoplasia and congenital ataxia. Sg/sg mice serve as an important extreme mouse model of the hereditary spinocerebellar ataxia type 1 (SCA1), since it has been shown that RORα dysfunction is strongly correlated with SCA1 pathogenesis. However, synaptic abnormalities, especially at parallel fibre (PF)-PC synapses, in SCA1-related sg/sg mice have not been examined in detail electrophysiologically. In this study, we report that PFs can still establish functional synapses onto PCs in sg/sg mice in spite of reduction in the number of PF-PC synapses. Compared with PF-evoked EPSCs in the wild-type or heterozygotes, the success rate of the EPSC recordings in sg/sg was quite low (â¼40%) and the EPSCs showed faster kinetics and slightly decreased paired pulse facilitation at short intervals. The prominent synaptic dysfunction is that sg/sg mice lack metabotropic glutamate receptor (mGluR)-mediated slow EPSCs completely. Neither intense PF stimulation nor an exogenously applied mGluR agonist, DHPG, could elicit mGluR-mediated responses.Western blot analysis in the sg/sg cerebellum revealed low-level expression of mGluR1 and TRPC3, both of which underlie mGluR-mediated slow currents in PCs. Immunohistochemical data demonstrated marked mislocalization of mGluR1 on sg/sg PCs.We found that mGluR-mediated retrograde suppression of PF-PC EPSCs by endocannabinoid is also impaired completely in sg/sg mice. These results suggest that disruption of mGluR signalling at PF-PC synapses is one of the major synaptic defects in sg/sg mice and may manifest itself in SCA1 pathology.
Asunto(s)
Potenciales Postsinápticos Excitadores/genética , Células de Purkinje/patología , Receptores de Glutamato Metabotrópico/deficiencia , Transducción de Señal/genética , Ataxias Espinocerebelosas/genética , Sinapsis/genética , Animales , Cerebelo/metabolismo , Cerebelo/patología , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Mutantes Neurológicos , Células de Purkinje/metabolismo , Distribución Aleatoria , Receptores de Glutamato Metabotrópico/fisiología , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/patología , Sinapsis/patologíaRESUMEN
Hotfoot5J mice are spontaneously occurring ataxic mice that lack delta2 glutamate receptor (GluRdelta2) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GluRdelta2 in Purkinje cells. Lentiviral vectors expressing GluRdelta2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluRdelta2 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GluRdelta2 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders.
Asunto(s)
Ataxia/terapia , Cerebelo/virología , Terapia Genética , Vectores Genéticos , Lentivirus/genética , Receptores de Glutamato/genética , Animales , Ataxia/fisiopatología , Cerebelo/fisiopatología , Espinas Dendríticas/fisiología , Espinas Dendríticas/virología , Femenino , Ácido Glutámico/metabolismo , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microglía/fisiología , Microglía/virología , Actividad Motora , Neuronas/fisiología , Neuronas/virología , Células de Purkinje/fisiología , Células de Purkinje/virología , Receptores de Glutamato/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinapsis/fisiología , Sinapsis/virologíaRESUMEN
The delta2 glutamate receptor (GluRdelta2) is expressed predominantly in cerebellar Purkinje cells. GluRdelta2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluRdelta2 function, we produced various GluRdelta2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluRdelta2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluRdelta2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluRdelta2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluRdelta2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluRdelta2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluRdelta2, which functions substantially without conventional ligand binding and ion channel structures.
Asunto(s)
Células de Purkinje/fisiología , Receptores de Glutamato/química , Animales , Western Blotting , Ataxia Cerebelosa/genética , Inmunohistoquímica , Depresión Sináptica a Largo Plazo/fisiología , Ratones , Ratones Noqueados , Microscopía Confocal , Técnicas de Placa-Clamp , Fenotipo , Receptores de Glutamato/deficiencia , Receptores de Glutamato/genética , Sinapsis/fisiologíaRESUMEN
We recently produced transgenic mice that expressed an abnormally expanded polyglutamine (polyQ) specifically in cerebellar Purkinje cells (polyQ mice). The polyQ mice showed inclusion body formation, cerebellar atrophy and severe ataxia. Here we analyzed polyQ mice using immunohistochemistry, immunoelectronmicroscopy and electrophysiology. A diffuse form of polyQ was detected in the nucleus. Interestingly, ubiquitinated large inclusions were located close to, but apparently outside of the soma of Purkinje cells. Infusion of lucifer yellow into Purkinje cells clearly indicated the traffic between the periplasmic inclusions and soma of Purkinje cells. To examine whether the formation of periplasmic inclusions was an active process or a result of cell death, the polyQ mouse cerebellum was immunolabeled for cleaved caspase-3, a marker of apoptosis. Interestingly, no Purkinje cells in P80 polyQ mice immunoreacted with the antibody. The results were substantiated by electrophysiological assay, which showed that P80 Purkinje cells with large periplasmic inclusions were functionally active: excitatory postsynaptic currents (EPSCs) were reliably evoked upon electrical stimulation of parallel fibers (PFs) or climbing fibers (CFs), and current injection into Purkinje cells generated action potentials; however, the frequency of action potentials in response to various volumes of current injection was consistently lower in polyQ mice than in wild-type animals, and aberrant innervation by multiple CFs was detected in polyQ mouse Purkinje cells. These results suggest that Purkinje cells with periplasmic inclusions were not apoptotic, but their functions were substantially impaired, which could contribute to the severe ataxic phenotype.
Asunto(s)
Cerebelo/citología , Péptidos/genética , Péptidos/metabolismo , Células de Purkinje/fisiología , Células de Purkinje/ultraestructura , Animales , Caspasa 3/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cerebelo/metabolismo , Cerebelo/fisiología , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Inmunohistoquímica , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Ratones , Ratones Endogámicos , Ratones Transgénicos , Microscopía Electrónica , UbiquitinaciónRESUMEN
Antiepileptic drugs protect against seizures by modulating neuronal excitability. Ethosuximide is selectively used for the treatment of absence epilepsy, and has also been shown to have the potential for treating several other neuropsychiatric disorders in addition to several antiepileptic drugs. Although ethosuximide inhibits T-type Ca(2+), noninactivating Na(+), and Ca(2+)-activated K(+) channels, the molecular mechanisms underlying the effects of ethosuximide have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) channels (GIRK, or Kir3) play an important role in regulating neuronal excitability, heart rate and platelet aggregation. In the present study, the effects of various antiepileptic drugs on GIRK channels were examined first by using the Xenopus oocyte expression assay. Ethosuximide at clinically relevant concentrations inhibited GIRK channels expressed in Xenopus oocytes. The inhibition was concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. However, the other antiepileptic drugs tested: phenytoin, valproic acid, carbamazepine, phenobarbital, gabapentin, topiramate and zonisamide, had no significant effects on GIRK channels even at toxic concentrations. In contrast, Kir1.1 and Kir2.1 channels were insensitive to all of the drugs tested. Ethosuximide also attenuated ethanol-induced GIRK currents. These inhibitory effects of ethosuximide were not observed when ethosuximide was applied intracellularly. In granule cells of cerebellar slices, ethosuximide inhibited GTPgammaS-activated GIRK currents. Moreover, ADP- and epinephrine-induced platelet aggregation was inhibited by ethosuximide, but not by charybdotoxin, a platelet Ca(2+)-activated K(+) channel blocker. These results suggest that the inhibitory effects of ethosuximide on GIRK channels may affect some of brain, heart and platelet functions.
Asunto(s)
Anticonvulsivantes/farmacología , Etosuximida/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Potenciales de la Membrana/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Adenosina Difosfato/farmacología , Animales , Animales Recién Nacidos , Compuestos de Bario/farmacología , Depresores del Sistema Nervioso Central/farmacología , Cerebelo/citología , Cloruros/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Etanol/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Microinyecciones/métodos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , XenopusRESUMEN
Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin-proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases.