RESUMEN
OBJECTIVE: Family members carrying the same SCN1A variant often exhibit differences in the clinical severity of epilepsy. This variable expressivity suggests that other factors aside from the primary sodium channel variant influence the clinical manifestation. However, identifying such factors has proven challenging in humans. METHODS: We perform whole exome sequencing (WES) in a large family in which an SCN1A variant (p.K1372E) is segregating that is associated with a broad spectrum of phenotypes ranging from lack of epilepsy, to febrile seizures and absence seizures, to Dravet syndrome. We assessed the hypothesis that the severity of the SCN1A-related phenotype was affected by alternate alleles at a modifier locus (or loci). RESULTS: One of our top candidates identified by WES was a second variant in the SCN1A gene (p.L375S) that was shared exclusively by unaffected carriers of the K1372E allele. To test the hypothesized that L375S variant nullifies the loss-of-function effect of K1372E, we transiently expressed Nav1.1 carrying the two variants in HEK293T cells and compared their biophysical properties with the wild-type (WT) variant, and then co-expressed WT with K1372E or L375S with K1372E in equal quantity and tested the functional consequence. The data demonstrated that co-expression of the L375S and K1372E alleles reversed the loss-of-function property brought by the K1372E variant, whereas WT-K1372E co-expression remained partial loss-of-function. SIGNIFICANCE: These results support the hypothesis that L375S counteracts the loss-of-function effect of K1372E such that individuals carrying both alleles in trans do not present epilepsy-related symptoms. We demonstrate that monogenic epilepsies with wide expressivity can be modified by additional variants in the disease gene, providing a novel framework for the gene-phenotype relationship in genetic epilepsies.
Asunto(s)
Epilepsias Mioclónicas , Epilepsia , Convulsiones Febriles , Epilepsias Mioclónicas/genética , Epilepsia/complicaciones , Epilepsia/genética , Células HEK293 , Humanos , Mutación , Canal de Sodio Activado por Voltaje NAV1.1/genética , Fenotipo , Convulsiones Febriles/complicaciones , Convulsiones Febriles/genética , Virulencia , Secuenciación del ExomaRESUMEN
Amyloid beta (Aß), the hallmark of Alzheimer's Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aß fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aß aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aß aggregates, and current therapeutics are summarized in this review.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Humanos , Microdominios de Membrana/metabolismoRESUMEN
Adenylate cyclase (EC 4.6.1.1) type 9 (AC9) activity has been shown to be inhibited by PMA activation of novel protein kinase C (nPKC) isoforms. In the current study the effect on AC9 activity of activating PKC in physiological relevant manner was examined. Contrary to the anticipated inhibitory effect of activating PKCs through Gq-coupled receptors, activation of transiently expressed Gq-coupled serotonin 5-HT2A or muscarinic M5 receptors resulted in the potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK293 cells stably expressing AC9 (HEK-AC9). Consistent with Gq-mediated activation of PKC, the addition of the PKC inhibitor bisindolylmaleimide further potentiated isoproterenol-stimulated cyclic AMP accumulation. Expression of a constitutively active mutant of Galphaq in HEK-AC9 cells also produced an enhancement in basal and isoproterenol-stimulated cyclic AMP accumulation. We also examined the role of Galphaq-mediated release of intracellular Ca2+ on the observed potentiation of AC9 activity, by depleting intracellular Ca2+ stores with thapsigargin. In Ca2+-depleted HEK-AC9 cells, activation of transiently expressed M5 receptors resulted in inhibition of isoproterenol-stimulated cyclic AMP accumulation that was blocked by bisindolylmaleimide, indicating that M5 potentiation of AC9 activity requires Ca2+. This prompted us to examine the effects of the calmodulin antagonist W7 and the Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor KN-93. Pretreating cells with W7 and KN-93 significantly inhibited M5-mediated potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK-AC9 cells, suggesting that Galphaq potentiation of AC9 activity involves Ca2+/calmodulin and CaMK II. This data provides evidence for Ca2+-mediated potentiation of AC9 activity.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , AMP Cíclico/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Bencilaminas/farmacología , Línea Celular , AMP Cíclico/análisis , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Indoles/farmacología , Isoproterenol/farmacología , Maleimidas/farmacología , Mutación , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/metabolismo , Receptores de Serotonina 5-HT2/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Tapsigargina/farmacologíaRESUMEN
RATIONALE: There is substantial evidence that lisuride can produce effects linked to 5-HT(1A) receptor occupancy. Nevertheless, this action has generally been ignored in the mechanism of action of lisuride, in favor of an exclusive role for dopamine receptors in considering its antiparkinsonian effects, or an exclusive role of 5-HT(2A/2C) receptor activation in hallucinogenesis. These conclusions are surprising when one considers that the potent interaction of lisuride with 5-HT(1A) receptors has been demonstrated in several different laboratories and that activation of 5-HT(1A) and 5-HT(1B) receptors can modulate dopaminergically mediated responses. OBJECTIVE: The lack of full substitution of lisuride for lysergic acid diethylamide (LSD) in drug discrimination experiments and induction of a pronounced 5-HT syndrome by this compound at relatively low doses convinced us to execute two series of experiments that might explain the primary mechanism responsible for lisuride-mediated biological effects and its paradoxical classification as a dopamine agonist in the literature. RESULTS: In drug discrimination studies, lisuride fully mimicked the 5-HT(1A) agonist LY 293284, only partially substituted for LSD and DOI, and failed to substitute for (+)-amphetamine. Lisuride produced a significant dose-related increase in flat body posture, forepaw treading, and lower-lip retraction which reflect a modulation of behavior by action at central 5-HT(1A) receptors. Only pMPPI [4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridynyl-benzamide hydrochloride], a selective 5-HT(1A) antagonist, was effective in inhibiting all 5-HT syndrome behaviors produced by lisuride, whereas pMPPI was without effect on any behavior induced by LSD. Lisuride dose dependently decreased body temperature in rats with a potency similar to that of the selective 5-HT(1A) agonist LY 293284. The hypothermic effect of lisuride was prevented by pre-injection of pMPPI, but not by ketanserin or haloperidol. CONCLUSION: We have demonstrated that the behavioral effects of low doses of lisuride are clearly mediated by stimulation of 5-HT(1A) receptors.
Asunto(s)
Temperatura Corporal/efectos de los fármacos , Discriminación en Psicología/efectos de los fármacos , Lisurida/farmacología , Receptores de Serotonina/fisiología , Animales , Temperatura Corporal/fisiología , Células CHO , Cricetinae , Discriminación en Psicología/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Lisurida/metabolismo , Dietilamida del Ácido Lisérgico/metabolismo , Dietilamida del Ácido Lisérgico/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Agonistas de Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
Nine membrane-bound members of the mammalian adenylate cyclase family have been identified. The least characterized and most divergent in sequence of the nine adenylate cyclase isoforms is AC9. Stimulation by Galpha(s) and inhibition by Ca2+/calcineurin are two modes of regulation that have been reported for AC9. We explored the possibility of additional modes of regulation of human AC9. We now report that quinpirole activation of the inhibitory G protein-coupled D2L dopamine receptor inhibits Galpha(s) stimulation of AC9 by approximately 50%. The effects of quinpirole were reversed by the D2 antagonist spiperone and by pertussis toxin pretreatment. We also report the first evidence for regulation of AC9 by protein kinase C (PKC). Specifically, phorbol ester activation of PKC significantly attenuated (approximately 50%) Galpha(s)-stimulated AC9 activity. The effect of PKC activation on AC9 was reversed by the PKC inhibitor bisindolylmaleimide. Galpha(s)-stimulated cyclic accumulation was reduced more by simultaneous addition of both quinpirole and phorbol 12-myristate 13-acetate than by either drug alone. Additional studies investigated the role of glycosylation on AC9 activity. The results show that blocking glycosylation of AC9 significantly attenuates Galpha(s) stimulation. In contrast, the ability of PKC and Galpha(i/o) to negatively regulate AC9 did not seem to be affected by the glycosylation state of AC9. These observations demonstrate the diverse regulatory features of AC9 and the ability of AC9 to integrate multiple signals.