RESUMO
Stable and entrainable physiological circadian rhythms are crucial for overall health and well-being. The suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals, consists of diverse neuron types that collectively generate a circadian profile of electrical activity. However, the mechanisms underlying the regulation of endogenous neuronal excitability in the SCN remain unclear. Two-pore domain potassium channels (K2P), including TASK-3, are known to play a significant role in maintaining SCN diurnal homeostasis by inhibiting neuronal activity at night. In this study, we investigated the role of TASK-3 in SCN circadian neuronal regulation and behavioural photoentrainment using a TASK-3 global knockout mouse model. Our findings demonstrate the importance of TASK-3 in maintaining SCN hyperpolarization during the night and establishing SCN sensitivity to glutamate. Specifically, we observed that TASK-3 knockout mice lacked diurnal variation in resting membrane potential and exhibited altered glutamate sensitivity both in vivo and in vitro. Interestingly, despite these changes, the mice lacking TASK-3 were still able to maintain relatively normal circadian behaviour.
Assuntos
Ritmo Circadiano , Camundongos Knockout , Canais de Potássio de Domínios Poros em Tandem , Núcleo Supraquiasmático , Animais , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/fisiologia , Núcleo Supraquiasmático/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Comportamento Animal/fisiologia , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Neurônios/metabolismo , Potenciais da Membrana/fisiologia , Canais de PotássioRESUMO
Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.
Assuntos
Barreira Hematoencefálica , Transportador 1 de Aminoácidos Catiônicos , Animais , Humanos , Camundongos , Barreira Hematoencefálica/metabolismo , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Transportador 1 de Aminoácidos Catiônicos/genética , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Establishing preclinical models of Alzheimer's disease that predict clinical outcomes remains a critically important, yet to date not fully realized, goal. Models derived from human cells offer considerable advantages over non-human models, including the potential to reflect some of the inter-individual differences that are apparent in patients. Here we report an approach using induced pluripotent stem cell-derived cortical neurons from people with early symptomatic Alzheimer's disease where we sought a match between individual disease characteristics in the cells with analogous characteristics in the people from whom they were derived. We show that the response to amyloid-ß burden in life, as measured by cognitive decline and brain activity levels, varies between individuals and this vulnerability rating correlates with the individual cellular vulnerability to extrinsic amyloid-ß in vitro as measured by synapse loss and function. Our findings indicate that patient-induced pluripotent stem cell-derived cortical neurons not only present key aspects of Alzheimer's disease pathology but also reflect key aspects of the clinical phenotypes of the same patients. Cellular models that reflect an individual's in-life clinical vulnerability thus represent a tractable method of Alzheimer's disease modelling using clinical data in combination with cellular phenotypes.
RESUMO
A loss of function mutation in the TRESK K2P potassium channel (KCNK18), has recently been linked with typical familial migraine with aura. We now report the functional characterisation of additional TRESK channel missense variants identified in unrelated patients. Several variants either had no apparent functional effect, or they caused a reduction in channel activity. However, the C110R variant was found to cause a complete loss of TRESK function, yet is present in both sporadic migraine and control cohorts, and no variation in KCNK18 copy number was found. Thus despite the previously identified association between loss of TRESK channel activity and migraine in a large multigenerational pedigree, this finding indicates that a single non-functional TRESK variant is not alone sufficient to cause typical migraine and highlights the genetic complexity of this disorder.
Assuntos
Coreia/imunologia , Coreia/terapia , Encefalite/imunologia , Encefalite/metabolismo , Proteínas/metabolismo , Idoso , Sítios de Ligação de Anticorpos/imunologia , Coreia/metabolismo , Encefalite/complicações , Humanos , Imunoterapia/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismoRESUMO
Multiple sclerosis (MS) is a complex trait with a sibling relative risk (lambda(sibs)) between 18 and 36. We report a multistage genome scan of 552 sibling pairs from 442 families, the largest MS family sample assessed for linkage. The first stage consisted of a genome scan for linkage with 498 microsatellite markers at an average spacing of 7 cM in 219 sibling pairs. The second stage involved further genotyping of markers from positive regions in an independent sample of 333 affected sibling pairs. The global distribution of allele sharing for all markers showed a shift towards greater sharing within the affected sibling pair group but not in the discordant sibling pair group. This shift indicates that the number of contributing genetic factors is likely to be moderate to large. Only markers at chromosome 6p showed significant evidence for linkage (MLOD=4.40), while other regions were only suggestive (1p, 2q, 5p, 9q, 11p, 12q, 18p, 18q and 21q) with MLODs greater than 1.0. The replication analysis involving all 552 affected sibling pairs confirmed suggestive evidence for five locations, namely, 2q27 (MLOD=2.27), 5p15 (MLOD=2.09), 18p11 (MLOD=1.68), 9q21 (MLOD=1.58) and 1p31 (MLOD=1.33). Suggestive linkage evidence for a previously reported location on chromosome 17q (MLOD=1.67) and a prior association with marker D17S789 was replicated. We showed that the overall excess allele sharing we observed for the entire sample was due to increased allele sharing within the DRB1*15 negative subgroup alone. This observation is most consistent with a model of genetic heterogeneity between HLA and other genetic loci. These findings offer guidance for future genetic studies including dense SNP linkage disequilibrium analysis.
Assuntos
Genoma Humano , Esclerose Múltipla/genética , Canadá/etnologia , Cromossomos Humanos Par 6/genética , Frequência do Gene , Genes MHC da Classe II/genética , Marcadores Genéticos , Testes Genéticos , Humanos , Desequilíbrio de Ligação , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único , População/genética , IrmãosRESUMO
Migraine with aura (MA) is a prevalent neurological condition with strong evidence for a genetic basis. Familial hemiplegic migraine, a rare Mendelian form of MA, can be caused by mutations in the calcium channel gene, CACNA1A or in the ATP1A2 gene, a Na+/K+ pump. Susceptibility genes for the more prevalent forms of migraine have yet to be identified despite several reports of linkage including loci on 4q24, 1q31, 19p13 and Xq24-28. We have undertaken a genome-wide screen of 43 Canadian families, segregating MA with families chosen for an apparent autosomal dominant pattern of transmission. Diagnosis was based upon International Headache Society Criteria. Parametric linkage analysis revealed a novel locus on 11q24 with a two-point LOD score of 4.2 and a multi-point parametric LOD score of 5.6. We did not find any support for linkage at previously reported loci. The lack of consensus amongst linkage studies, including this study, is probably an indication of the heterogeneity that is inherent for MA. Nevertheless, the finding of a highly significant locus with a LOD score of 5.6 is powerful evidence that a gene increasing susceptibility to MA resides on 11q24. Several candidate genes map to this region of the genome including a number of ion channel genes such as GRIK4, SCNB2, KCNJ5 and KCNJ1.