RESUMEN
BACKGROUND AND OBJECTIVES: Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. METHODS: We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. RESULTS: LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. DISCUSSION: Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.
Asunto(s)
Potenciales de Acción , Autoanticuerpos , Péptidos y Proteínas de Señalización Intracelular , Terminales Presinápticos , Transmisión Sináptica , Autoanticuerpos/inmunología , Autoanticuerpos/farmacología , Humanos , Animales , Transmisión Sináptica/fisiología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Terminales Presinápticos/metabolismo , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de los fármacos , Hipocampo/metabolismo , Ratas , Canal de Potasio Kv.1.1/inmunología , Proteínas/inmunología , Proteínas/metabolismo , Masculino , Células CultivadasRESUMEN
Currently, there is an unmet clinical need for novel immunosuppressive agents for long-term prevention of kidney transplant rejection as alternatives to the nephrotoxic calcineurin inhibitor cyclosporine (CsA). Recent studies have shown that K(+) channels have a crucial role in T-lymphocyte activity. We investigated whether combined blockade of the T-cell K(+) channels K(Ca)3.1 and K(v)1.3, both of which regulate calcium signaling during lymphocyte activation, is effective in prevention of rejection of kidney allografts from Fisher rats to Lewis rats. All recipients were initially treated with CsA (5 mg/kg d) for 7 days. In rats with intact allograft function, treatment was continued for 10 days with either CsA (5 mg/kg d), or a combination of TRAM-34 (K(Ca)3.1 inhibitor; 120 mg/kg d) plus Stichodactyla helianthus toxin (ShK, K(v)1.3 inhibitor; 80 microg/kg 3 times daily), or vehicle alone. Kidney sections were stained with periodic acid-Schiff or hematoxylin-eosin and histochemically for markers of macrophages (CD68), T-lymphocytes (CD43), or cytotoxic T-cells (CD8). Our results showed that treatment with TRAM-34 and ShK reduced total interstitial mononuclear cell infiltration (-42%) and the number of CD43+ T-cells (-32%), cytotoxic CD8+ T-cells (-32%), and CD68+ macrophages (-26%) in allografts when compared to vehicle treatment alone. Efficacy of TRAM-34/ShK treatment was comparable with that of CsA. In addition, no visible organ damage or other discernible adverse effects were observed with this treatment. Thus, selective blockade of T-lymphocyte K(Ca)3.1 and K(v)1.3 channels may represent a novel alternative therapy for prevention of kidney allograft rejection.
Asunto(s)
Rechazo de Injerto/prevención & control , Terapia de Inmunosupresión/métodos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Trasplante de Riñón/inmunología , Canal de Potasio Kv.1.1/inmunología , Linfocitos T/inmunología , Animales , Venenos de Cnidarios/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canal de Potasio Kv.1.1/antagonistas & inhibidores , Prueba de Cultivo Mixto de Linfocitos , Masculino , Pirazoles/farmacología , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
Potassium channels play an important role in microglial activation but their involvement in main functions of microglia including secretion of proinflammatory cytokines has remained uncertain. This study has revealed the specific expression of Kv1.1 in microglia both in vivo and in vitro. Kv1.1 immunoreactivity was localized in the amoeboid microglia in the rat brain between postnatal (P) day 1 (P1) and day 10 (P10); it was, however, progressively reduced with age and was hardly detected at P14 and P21 in ramified microglia, a derivative cell of amoeboid microglia. Following hypoxic exposure, Kv1.1 expression in amoeboid microglia was enhanced or induced in ramified microglia in more mature brain at P21 when compared with their matching controls. RT-PCR and Western blot analysis confirmed Kv1.1 mRNA and protein expression in murine BV-2 cells which was up-regulated by hypoxia or lipopolysaccharide (LPS) treatment; it was reduced significantly by dexamethasone. Neutralization with Kv1.1 antibody suppressed the expression and release of tumor necrosis factor-alpha, interleukin-1beta, endothelins and nitric oxide (NO) in LPS-activated BV-2 cells. It is concluded that Kv1.1, constitutively expressed by microglia, is elicited by hypoxia and LPS and this may be linked to production of proinflammatory cytokines, endothelins and NO.
Asunto(s)
Encéfalo/citología , Citocinas/metabolismo , Endotelinas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Canal de Potasio Kv.1.1/metabolismo , Microglía/metabolismo , Óxido Nítrico/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Línea Celular Transformada , Colorimetría/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hipoxia/metabolismo , Hipoxia/patología , Canal de Potasio Kv.1.1/genética , Canal de Potasio Kv.1.1/inmunología , Lipopolisacáridos/farmacología , Ratones , Microglía/efectos de los fármacos , Óxido Nítrico/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To document neurologic, oncologic, and serologic associations of patients in whom voltage-gated potassium channel (VGKC) autoantibodies were detected in the course of serologic evaluation for neuronal, glial, and muscle autoantibodies. METHODS: Indirect immunofluorescence screening of sera from 130,000 patients performed on a service basis for markers of paraneoplastic neurologic autoimmunity identified 80 patients whose IgG bound to the synapse-rich molecular layer of mouse cerebellar cortex in a pattern consistent with VGKC immunoreactivity. Antibody specificity was confirmed in all cases by immunoprecipitation of detergent-solubilized brain synaptic proteins complexed with (125)I-alpha-dendrotoxin. RESULTS: Clinical information was available for 72 patients: 51% women, median age at symptom onset 65 years, and median follow-up period 14 months. Neurologic manifestations were acute to subacute in onset in 71% and multifocal in 46%; 71% had cognitive impairment, 58% seizures, 33% dysautonomia, 29% myoclonus, 26% dyssomnia, 25% peripheral nerve dysfunction, 21% extrapyramidal dysfunction, and 19% brainstem/cranial nerve dysfunction. Creutzfeldt-Jakob disease was a common misdiagnosis (14%). Neoplasms encountered (confirmed histologically in 33%) included 18 carcinomas, 5 adenomas, 1 thymoma, and 3 hematologic malignancies. Hyponatremia was documented in 36%, other organ-specific autoantibodies in 49%, and a co-existing autoimmune disorder in 33% (including thyroiditis 21%, type 1 diabetes mellitus 11%). Benefit was reported for 34 of 38 patients (89%) receiving immunotherapy and was marked in 50%. CONCLUSIONS: The spectrum of neurologic manifestations and neoplasms associated with voltage-gated potassium channel (VGKC) autoimmunity is broader than previously recognized. Evaluation for VGKC antibodies is recommended in the comprehensive autoimmune serologic testing of subacute idiopathic neurologic disorders.
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Autoanticuerpos/sangre , Síndromes Paraneoplásicos/inmunología , Enfermedades del Sistema Nervioso Periférico/inmunología , Canales de Potasio de la Superfamilia Shaker/inmunología , Adenoma/complicaciones , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades del Sistema Nervioso Autónomo/etiología , Enfermedades del Sistema Nervioso Autónomo/inmunología , Enfermedades de los Ganglios Basales/etiología , Enfermedades de los Ganglios Basales/inmunología , Niño , Enfermedades de los Nervios Craneales/etiología , Enfermedades de los Nervios Craneales/inmunología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Neoplasias Hematológicas/complicaciones , Humanos , Canal de Potasio Kv.1.1/inmunología , Canal de Potasio Kv.1.2/inmunología , Canal de Potasio Kv1.6 , Masculino , Persona de Mediana Edad , Mioclonía/etiología , Mioclonía/inmunología , Síndromes Paraneoplásicos/complicaciones , Enfermedades del Sistema Nervioso Periférico/etiología , Timoma/complicaciones , Neoplasias del Timo/complicacionesRESUMEN
Autoantibodies to Shaker-type (Kv1) K+ channels are now known to be associated with three syndromes. Peripheral nerve hyperexcitability is the chief manifestation of acquired neuromyotonia; the combination of neuromyotonia with autonomic and CNS involvement is called Morvan's syndrome (MoS); and CNS manifestations without peripheral involvement is called limbic encephalitis (LE). To determine the cellular basis of these clinical manifestations, we immunostained mouse neural tissues with sera from patients with neuromyotonia (n = 10), MoS (n = 2) or LE (n = 5), comparing with specific antibodies to relevant K+ channel subunits. Fourteen of 17 patients' sera were positive for Kv1.1, Kv1.2 or Kv1.6 antibodies by immunoprecipitation of 125I-alpha-dendrotoxin-labelled rabbit brain K+ channels. Most sera (11 out of 17) labelled juxtaparanodes of peripheral myelinated axons, co-localizing with Kv1.1 and Kv1.2. In the CNS, all sera tested (n = 12) co-localized with one or more areas of high Kv1.1, Kv1.2 or Kv1.6 channel expression: 10 out of 12 sera co-localized with Kv1.1 and Kv1.2 at spinal cord juxtaparanodes or cerebellar layers, while 3 out of 12 sera co-localized additionally (n = 2) or exclusively (n = 1) with Kv1.6 subunits in Purkinje cells, motor and hippocampal neurons. However, only sera from LE patients labelled the hippocampal areas that are enriched in excitatory, Kv1.1-positive axon terminals. All sera (17 out of 17) labelled one or more of these Kv1 subunits when expressed at the cell membrane of transfected HeLa cells, but not when they were retained in the endoplasmic reticulum. Again, LE sera labelled Kv1.1 subunits more prominently than did MoS or neuromyotonia sera, suggesting an association between higher Kv1.1 specificity and limbic manifestations. In contrast, neuromyotonia sera bound more strongly to Kv1.2 subunits than to Kv1.1 or Kv1.6. These studies support the hypothesis that antibodies to mature surface membrane-expressed Shaker-type K+ channels cause acquired neuromyotonia, MoS and LE, and suggest that future assays based on immunofluorescence of cells expressing individual Kv1 subunits will prove more sensitive than the immunoprecipitation assay. Although more than one type of antibody is often detectable in individual sera, higher affinity for certain subunits or subunit combinations may determine the range of clinical manifestations.
Asunto(s)
Autoanticuerpos/sangre , Enfermedades Autoinmunes/inmunología , Síndrome de Isaacs/inmunología , Encefalitis Límbica/inmunología , Canales de Potasio de la Superfamilia Shaker/inmunología , Adolescente , Adulto , Anciano , Animales , Especificidad de Anticuerpos , Biomarcadores/sangre , Encéfalo/inmunología , Femenino , Células HeLa , Hipocampo/inmunología , Humanos , Canal de Potasio Kv.1.1/inmunología , Canal de Potasio Kv.1.2/inmunología , Canal de Potasio Kv1.6 , Masculino , Ratones , Persona de Mediana Edad , Nervios Periféricos/inmunología , Terminales Presinápticos/inmunología , Médula Espinal/inmunología , Siringomielia/inmunología , TransfecciónRESUMEN
BACKGROUND: The megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice. RESULTS: We found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi) glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER). Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons. CONCLUSION: The megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.