Interneuronal In Vivo Transfer of Synaptic Proteins.

Neuron-to-neuron transfer of pathogenic α-synuclein species is a mechanism of likely relevance to Parkinson's disease development. Experimentally, interneuronal α-synuclein spreading from the low brainstem toward higher brain regions can be reproduced by the administration of AAV vectors encoding for α-synuclein into the mouse vagus nerve. The aim of this study was to determine whether α-synuclein's spreading ability is shared by other proteins. Given α-synuclein synaptic localization, experiments involved intravagal injections of AAVs encoding for other synaptic proteins, ß-synuclein, VAMP2, or SNAP25. Administration of AAV-VAMP2 or AAV-SNAP25 caused robust transduction of either of the proteins in the dorsal medulla oblongata but was not followed by interneuronal VAMP2 or SNAP25 transfer and caudo-rostral spreading. In contrast, AAV-mediated ß-synuclein overexpression triggered its spreading to more frontal brain regions. The aggregate formation was investigated as a potential mechanism involved in protein spreading, and consistent with this hypothesis, results showed that overexpression of ß-synuclein, but not VAMP2 or SNAP25, in the dorsal medulla oblongata was associated with pronounced protein aggregation. Data indicate that interneuronal protein transfer is not a mere consequence of increased expression or synaptic localization. It is rather promoted by structural/functional characteristics of synuclein proteins that likely include their tendency to form aggregate species.

Rare coding variants in genes encoding GABAA receptors in genetic generalised epilepsies: an exome-based case-control study.

BACKGROUND: Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. METHODS: For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABAA receptors and was compared to the respective GABAA receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. FINDINGS: Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABAA receptors in cases (odds ratio [OR] 2·40 [95% CI 1·41-4·10]; pNonsyn=0·0014, adjusted pNonsyn=0·019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1·46 [95% CI 1·05-2·03]; pNonsyn=0·0081, adjusted pNonsyn=0·016). Comparison of genes encoding GABAA receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABAA receptor genes in cases compared with controls (OR 1·46 [95% CI 1·02-2·08]; pNonsyn=0·013, adjusted pNonsyn=0·027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. INTERPRETATION: Functionally relevant variants in genes encoding GABAA receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. FUNDING: EuroEPINOMICS (European Science Foundation through national funding organisations), Epicure and EpiPGX (Sixth Framework Programme and Seventh Framework Programme of the European Commission), Research Unit FOR2715 (German Research Foundation and Luxembourg National Research Fund).

Long-lasting pathological consequences of overexpression-induced α-synuclein spreading in the rat brain.

Increased expression of α-synuclein can initiate its long-distance brain transfer, representing a potential mechanism for pathology spreading in age-related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpression-induced α-synuclein transfer were assessed over a 1-year period after injection of viral vectors carrying human α-synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long-lasting pathological changes. Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression-induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by α-synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpression-induced spreading, even if temporary, causes long-lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder α-synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation.

The L444P Gba1 mutation enhances alpha-synuclein induced loss of nigral dopaminergic neurons in mice.

Mutations in glucocerebrosidase 1 (GBA1) represent the most prevalent risk factor for Parkinson's disease. The molecular mechanisms underlying the link between GBA1 mutations and Parkinson's disease are incompletely understood. We analysed two aged (24-month-old) Gba1 mouse models, one carrying a knock-out mutation and the other a L444P knock-in mutation. A significant reduction of glucocerebrosidase activity was associated with increased total alpha-synuclein accumulation in both these models. Gba1 mutations alone did not alter the number of nigral dopaminergic neurons nor striatal dopamine levels. We then investigated the effect of overexpression of human alpha-synuclein in the substantia nigra of aged (18 to 21-month-old) L444P Gba1 mice. Following intraparenchymal injections of human alpha-synuclein carrying viral vectors, pathological accumulation of phosphorylated alpha-synuclein occurred within the transduced neurons. Stereological counts of nigral dopaminergic neurons revealed a significantly greater cell loss in Gba1-mutant than wild-type mice. These results indicate that Gba1 deficiency enhances neuronal vulnerability to neurodegenerative processes triggered by increased alpha-synuclein expression.

Early left atrial tissue features in patients with chronic mitral regurgitation and sinus rhythm: Alterations of not remodeled left atria.

OBJECTIVE: Left atrial (LA) enlargement, a compensatory mechanism in chronic mitral regurgitation (MR) increasing the risk of atrial fibrillation (AF) and predictive of cardiac events, involves structural alterations. We characterized LA features in patients in sinus rhythm with severe degree of MR, similar degrees of left ventricular remodeling but divergent LA size. METHODS: Among a consecutive series of 163 patients in stable sinus rhythm undergoing isolated mitral valve surgery for severe non-rheumatic MR, two groups were arbitrarily selected according to their LA size (antero-posterior): NRLA group (non-remodeled LA) included 8 patients with LA≤40mm, RLA group (remodeled LA) included 8 patients with LA>55mm. LA biopsies were processed for paraffin inclusion and sectioning. Fibrosis, cardiomyocytes morphology, capillaries density, cytochrome c and F-actin expression were evaluated by microscopy. RESULTS: Histology and immunohistochemistry demonstrated alteration of moderate entity: higher amounts of endomysial fibrosis (not of collagen type III) and of hypertrophic cardiomyocytes in RLA than in NRLA. Confocal microscopy displayed focally disorganized F-actin and no nuclear fragmentation in both groups, but more intra-cytoplasm cytochrome c in RLA vs. NRLA, possibly indicative of more successful escape to apoptosis by NRLA cardiomyocytes. CONCLUSIONS: Our study shows the presence of early cellular and interstitial alterations in LA tissue in patients with chronic MR and sinus rhythm. These features were analogous to those of patients with AF, and suggest that macroscopic remodeling LA in the settings of MR is preceded by structural changes, paving the way to further investigation on the preventive role of early mitral valve repair.

Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

Aggregation and neuron-to-neuron transmission are attributes of α-synuclein relevant to its pathogenetic role in human synucleinopathies such as Parkinson's disease. Intraparenchymal injections of fibrillar α-synuclein trigger widespread propagation of amyloidogenic protein species via mechanisms that require expression of endogenous α-synuclein and, possibly, its structural corruption by misfolded conformers acting as pathological seeds. Here we describe another paradigm of long-distance brain diffusion of α-synuclein that involves inter-neuronal transfer of monomeric and/or oligomeric species and is independent of recruitment of the endogenous protein. Targeted expression of human α-synuclein was induced in the mouse medulla oblongata through an injection of viral vectors into the vagus nerve. Enhanced levels of intra-neuronal α-synuclein were sufficient to initiate its caudo-rostral diffusion that likely involved at least one synaptic transfer and progressively reached specific brain regions such as the locus coeruleus, dorsal raphae and amygdala in the pons, midbrain and forebrain. Transfer of human α-synuclein was compared in two separate lines of α-synuclein-deficient mice versus their respective wild-type controls and, interestingly, lack of endogenous α-synuclein expression did not counteract diffusion but actually resulted in a more pronounced and advanced propagation of exogenous α-synuclein. Self-interaction of adjacent molecules of human α-synuclein was detected in both wild-type and mutant mice. In the former, interaction of human α-synuclein with mouse α-synuclein was also observed and might have contributed to differences in protein transmission. In wild-type and α-synuclein-deficient mice, accumulation of human α-synuclein within recipient axons in the pons, midbrain and forebrain caused morphological evidence of neuritic pathology. Tissue sections from the medulla oblongata and pons were stained with different antibodies recognizing oligomeric, fibrillar and/or total (monomeric and aggregated) α-synuclein. Following viral vector transduction, monomeric, oligomeric and fibrillar protein was detected within donor neurons in the medulla oblongata. In contrast, recipient axons in the pons were devoid of immunoreactivity for fibrillar α-synuclein, indicating that non-fibrillar forms of α-synuclein were primarily transferred from one neuron to the other, diffused within the brain and led to initial neuronal injury. This study elucidates a paradigm of α-synuclein propagation that may play a particularly important role under pathophysiological conditions associated with enhanced α-synuclein expression. Rapid long-distance diffusion and accumulation of monomeric and oligomeric α-synuclein does not necessarily involve pathological seeding but could still result in a significant neuronal burden during the pathogenesis of neurodegenerative diseases.

Neuron-to-neuron α-synuclein propagation in vivo is independent of neuronal injury.

INTRODUCTION: Interneuronal propagation of α-synuclein has been demonstrated in a variety of experimental models and may be involved in disease progression during the course of human synucleinopathies. The aim of this study was to assess the role that neuronal injury or, vice versa, cell integrity could have in facilitating interneuronal α-synuclein transfer and consequent protein spreading in an in vivo animal model. RESULTS: Viral vectors carrying the DNA for human α-synuclein were injected into the rat vagus nerve to trigger protein overexpression in the medulla oblongata and consequent spreading of human α-synuclein toward pons, midbrain and forebrain. Two vector preparations sharing the same viral construct were manufactured using identical procedures with the exception of methods for their purification. They were also injected at concentrations that induced comparable levels of α-synuclein transduction/overexpression in the medulla oblongata. α-Synuclein load was associated with damage (at 6 weeks post injection) and death (at 12 weeks) of medullary neurons after treatment with only one of the two vector preparations. Of note, neuronal injury and degeneration was accompanied by a substantial reduction of caudo-rostral propagation of human α-synuclein. CONCLUSIONS: Interneuronal α-synuclein transfer, which underlies protein spreading from the medulla oblongata to more rostral brain regions in this rat model, is not a mere consequence of passive release from damaged or dead neurons. Neuronal injury and degeneration did not exacerbate α-synuclein propagation. In fact, data suggest that cell-to-cell passage of α-synuclein may be particularly efficient between intact, relatively healthy neurons.

Rescuable folding defective NaV1.1 (SCN1A) mutants in epilepsy: properties, occurrence, and novel rescuing strategy with peptides targeted to the endoplasmic reticulum.

Mutations of the voltage gated Na(+) channel Na(V)1.1 (SCN1A) are important causes of different genetic epilepsies and can also cause familial hemiplegic migraine (FHM-III). In previous studies, some rescuable epileptogenic folding defective mutants located in domain IV of Na(V)1.1 have been identified, showing partial loss of function also with maximal rescue. Variable rescue may be one of the causes of phenotypic variability, and rescue might be exploited for therapeutic approaches. Recently, we have identified a folding defective FHM-III Na(V)1.1 mutant that showed overall gain of function when rescued, consistent with a differential pathomechanism. Here, we have evaluated functional properties and cell surface expression of six Na(V)1.1 epileptogenic missense mutations in different rescuing conditions, including a novel one that we have developed expressing a selective sodium channel toxin (CsEI) targeted to the endoplasmic reticulum (ER). All the mutants showed loss of function and reduced cell surface expression, consistently with possibility of rescue. Four of them were rescuable by incubation at low temperature and interactions with different co-expressed proteins or a pharmacological chaperone (phenytoin). Notably, CsEI was able to rescue four mutants. Thus, Na(V)1.1 folding defective mutants can be relatively common and mutations inducing rescuable folding defects are spread in all Na(V)1.1 domains. Importantly, epileptogenic mutants showed overall loss of function even upon rescue, differently than FHM-III ones. The effectiveness of CsEI demonstrates that interactions in the ER are sufficient for inducing rescue, and provides a proof of concept for developing possible therapeutic approaches that may overcome some limitations of pharmacological chaperones.

Nonfunctional NaV1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects.

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura. Mutations causing FHM type 3 have been identified in SCN1A, the gene encoding the Nav1.1 Na(+) channel, which is also a major target of epileptogenic mutations and is particularly important for the excitability of GABAergic neurons. However, functional studies of NaV1.1 FHM mutations have generated controversial results. In particular, it has been shown that the NaV1.1-L1649Q mutant is nonfunctional when expressed in a human cell line because of impaired plasma membrane expression, similarly to NaV1.1 mutants that cause severe epilepsy, but we have observed gain-of-function effects for other NaV1.1 FHM mutants. Here we show that NaV1.1-L1649Q is nonfunctional because of folding defects that are rescuable by incubation at lower temperatures or coexpression of interacting proteins, and that a partial rescue is sufficient for inducing an overall gain of function because of the modifications in gating properties. Strikingly, when expressed in neurons, the mutant was partially rescued and was a constitutive gain of function. A computational model showed that 35% rescue can be sufficient for inducing gain of function. Interestingly, previously described folding-defective epileptogenic NaV1.1 mutants show loss of function also when rescued. Our results are consistent with gain of function as the functional effect of NaV1.1 FHM mutations and hyperexcitability of GABAergic neurons as the pathomechanism of FHM type 3.

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.

OBJECTIVE: Neuronal channelopathies cause brain disorders, including epilepsy, migraine, and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders remain uncertain. One particularly devastating channelopathy is Dravet syndrome (DS), a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Na(v) 1.1. Heterologous expression of mutant channels suggests loss of function, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Mouse model studies suggest that decreased Na(v) 1.1 function in interneurons causes disinhibition. We aim to determine how mutant SCN1A affects human neurons using the induced pluripotent stem cell (iPSC) method to generate patient-specific neurons. METHODS: Here we derive forebrain-like pyramidal- and bipolar-shaped neurons from 2 DS subjects and 3 human controls by iPSC reprogramming of fibroblasts. DS and control iPSC-derived neurons are compared using whole-cell patch clamp recordings. Sodium current density and intrinsic neuronal excitability are examined. RESULTS: Neural progenitors from DS and human control iPSCs display a forebrain identity and differentiate into bipolar- and pyramidal-shaped neurons. DS patient-derived neurons show increased sodium currents in both bipolar- and pyramidal-shaped neurons. Consistent with increased sodium currents, both types of patient-derived neurons show spontaneous bursting and other evidence of hyperexcitability. Sodium channel transcripts are not elevated, consistent with a post-translational mechanism. INTERPRETATION: These data demonstrate that epilepsy patient-specific iPSC-derived neurons are useful for modeling epileptic-like hyperactivity. Our findings reveal a previously unrecognized cell-autonomous epilepsy mechanism potentially underlying DS, and offer a platform for screening new antiepileptic therapies.

Caudo-rostral brain spreading of α-synuclein through vagal connections.

α-Synuclein accumulation and pathology in Parkinson's disease typically display a caudo-rostral pattern of progression, involving neuronal nuclei in the medulla oblongata at the earliest stages. In this study, selective expression and accumulation of human α-synuclein within medullary neurons was achieved via retrograde transport of adeno-associated viral vectors unilaterally injected into the vagus nerve in the rat neck. The exogenous protein progressively spread toward more rostral brain regions where it could be detected within axonal projections. Propagation to the pons, midbrain and forebrain followed a stereotypical pattern of topographical distribution. It affected areas such as the coeruleus-subcoeruleus complex, dorsal raphae, hypothalamus and amygdala ipsilateral and, to a lesser extent, contralateral to the injection side. Spreading was accompanied by evidence of neuritic pathology in the form of axonal varicosities intensely immunoreactive for human α-synuclein and containing Thioflavin-S-positive fibrils. Thus, overexpression of human α-synuclein in the lower brainstem is sufficient to induce its long-distance caudo-rostral propagation, recapitulating features of Parkinson's disease and mechanisms of disease progression.

Divergent effects of the T1174S SCN1A mutation associated with seizures and hemiplegic migraine.

PURPOSE: To report the identification of the T1174S SCN1A (NaV 1.1) mutation in a three-generation family with both epileptic and familial hemiplegic migraine (FHM) phenotypes and clarify the pathomechanism. METHODS: The five affected individuals underwent detailed clinical analyses. Mutation analyses was performed by direct sequencing of SCN1A; functional studies by expression in tsA-201 cells. A computational model was used to compare the effects of T1174S with those of a typical FHM mutation (Q1489K). KEY FINDINGS: The proband had benign occipital epilepsy (BOE); two relatives had simple febrile seizures (FS) and later developed BOE. Two additional relatives had FHM without epilepsy or FS. All affected members and one obliged carrier carried the T1174S mutation. Functional effects were divergent: positive shift of the activation curve and deceleration of recovery from fast inactivation, consistent with loss of function, and increase of persistent current (I(NaP)), consistent with gain of function. The I(NaP) increase was inhibited by dialysis of the cytoplasm, consistent with a modulation. Therefore, as shown by the computational model, T1174S could in some conditions induce overall loss of function, and in others gain of function; Q1489K induced gain of function in all the conditions. SIGNIFICANCE: Modulation of the properties of T1174S can lead to a switch between overall gain and loss of function, consistent with a switch between promigraine end epileptogenic effect and, thus, with coexistence of epileptic and FHM phenotypes in the same family. These findings may help to shed light on the complex genotype-phenotype relationship of SCN1A mutations.

Pure haploinsufficiency for Dravet syndrome Na(V)1.1 (SCN1A) sodium channel truncating mutations.

PURPOSE: Dravet syndrome (DS), a devastating epileptic encephalopathy, is mostly caused by mutations of the SCN1A gene, coding for the voltage-gated Na(+) channel Na(V)1.1 α subunit. About 50% of SCN1A DS mutations truncate Na(V)1.1, possibly causing complete loss of its function. However, it has not been investigated yet if Na(V)1.1 truncated mutants are dominant negative, if they impair expression or function of wild-type channels, as it has been shown for truncated mutants of other proteins (e.g., Ca(V) channels). We studied the effect of two DS truncated Na(V)1.1 mutants, R222* and R1234*, on coexpressed wild-type Na(+) channels. METHODS: We engineered R222* or R1234* in the human cDNA of Na(V)1.1 (hNa(V)1.1) and studied their effect on coexpressed wild-type hNa(V)1.1, hNa(V)1.2 or hNa(V)1.3 cotransfecting tsA-201 cells, and on hNa(V)1.6 transfecting an human embryonic kidney (HEK) cell line stably expressing this channel. We also studied hippocampal neurons dissociated from Na(V)1.1 knockout (KO) mice, an animal model of DS expressing a truncated Na(V)1.1 channel. KEY FINDINGS: We found no modifications of current amplitude coexpressing the truncated mutants with hNa(V)1.1, hNa(V)1.2, or hNa(V)1.3, but a 30% reduction coexpressing them with hNa(V)1.6. However, we showed that also coexpression of functional full-length hNa(V)1.1 caused a similar reduction. Therefore, this effect should not be involved in the pathomechanism of DS. Some gating properties of hNa(V)1.1, hNa(V)1.3, and hNa(V)1.6 were modified, but recordings of hippocampal neurons dissociated from Na(V)1.1 KO mice did not show any significant modifications of these properties. Therefore, Na(V)1.1 truncated mutants are not dominant negative, consistent with haploinsufficiency as the cause of DS. SIGNIFICANCE: We have better clarified the pathomechanism of DS, pointed out an important difference between pathogenic truncated Ca(V)2.1 mutants and hNa(V)1.1 ones, and shown that hNa(V)1.6 expression can be reduced in physiologic conditions by coexpression of hNa(V)1.1. Moreover, our data may provide useful information for the development of therapeutic approaches.

Epileptogenic ion channel mutations: from bedside to bench and, hopefully, back again.

Mutations of genes coding for ion channels cause several genetically determined human epileptic syndromes. The identification of a gene variant linked to a particular disease gives important information, but it is usually necessary to perform functional studies in order to completely disclose the pathogenic mechanisms. The functional consequences of epileptogenic mutations have been studied both in vitro and in vivo with several experimental systems, studies that have provided significant knowledge on the pathogenic mechanisms that leads to inherited human epilepsies, and possibly also on the pathogenic mechanisms of non-genetic human epilepsies due to "acquired channelopathies". However, several open issues remain and difficulties in the interpretation of the experimental data have arisen that limit translational applications. We will highlight the value and the limits of different approaches to the study of epileptogenic channelopathies, focussing on the importance of the experimental systems in the assessment of the functional effects of the mutations and on the possible applications of the obtained results to the clinical practice.

A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation.

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.

A rescuable folding defective Nav1.1 (SCN1A) sodium channel mutant causes GEFS+: common mechanism in Nav1.1 related epilepsies?

Mutations of voltage-gated Na(+) channels are the most common known cause of genetically determined epilepsy; Na(v)1.1 (SCN1A) is the most frequent target. They can cause both mild and severe forms, also in patients harboring the same mutation. We have recently characterized in a family with extreme phenotypes the first epileptogenic folding-defective Na(+) channel mutant (Na(v)1.1-M1841T), whose loss of function is attenuated by interactions with associated proteins and drugs. We hypothesized that in vivo variability of the interactions may modulate the functional effect and thus the phenotype (Rusconi et al., 2007). Here we characterize another Na(v)1.1 folding-defective mutant (Na(v)1.1-R1916G) that, however, has been identified in a GEFS+ family with relatively mild phenotypes. This novel mutant shows a number of specific characteristics, but, similarly to Na(v)1.1-M1841T, it can be rescued by interactions with associated proteins and drugs. Thus, loss of function caused by folding defects that can be attenuated by molecular interactions may be a common pathogenic mechanism for Na(v)1.1 epileptogenic mutants. Folding defects can be present also in families showing only mild phenotypes in which, however, severe phenotypes could emerge within a permissive genetic background.

Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels.

The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.

Self-limited hyperexcitability: functional effect of a familial hemiplegic migraine mutation of the Nav1.1 (SCN1A) Na+ channel.

Familial hemiplegic migraine (FHM) is an autosomal dominant inherited subtype of severe migraine with aura. Mutations causing FHM (type 3) have been identified in SCN1A, the gene encoding neuronal voltage-gated Na(v)1.1 Na(+) channel alpha subunit, but functional studies have been done using the cardiac Na(v)1.5 isoform, and the observed effects were similar to those of some epileptogenic mutations. We studied the FHM mutation Q1489K by transfecting tsA-201 cells and cultured neurons with human Na(v)1.1. We show that the mutation has effects on the gating properties of the channel that can be consistent with both hyperexcitability and hypoexcitability. Simulation of neuronal firing and long depolarizing pulses mimicking promigraine conditions revealed that the effect of the mutation is a gain of function consistent with increased neuronal firing. However, during high-frequency discharges and long depolarizations, the effect became a loss of function. Recordings of firing of transfected neurons showed higher firing frequency at the beginning of long discharges. This self-limited capacity to induce neuronal hyperexcitability may be a specific characteristic of migraine mutations, able to both trigger the cascade of events that leads to migraine and counteract the development of extreme hyperexcitability typical of epileptic seizures. Thus, we found a possible difference in the functional effects of FHM and familial epilepsy mutations of Nav1.1.

Heme oxygenase-1 expression in the left atrial myocardium of patients with chronic atrial fibrillation related to mitral valve disease: its regional relationship with structural remodeling.

Atrial fibrillation becomes a self-perpetuating arrhythmia as a consequence of electrophysiologic and structural remodeling involving the atrium. Oxidative stress may be a link between this rhythm disturbance and electrophysiologic remodeling. The aim of this study was to evaluate whether the heme oxygenase-1 (HO-1) marker of oxidative stress was more expressed in left atrial sites with stronger structural remodeling in patients affected by chronic atrial fibrillation (CAF) and mitral valve disease (MD). Myocardial samples were taken from the left atrial posterior wall (LAPW) and left atrial appendage (LAA) of 24 patients with CAF-MD in addition to 10 autopsy controls. The levels of HO-1 messenger RNA (mRNA) and HO-1 protein in each pathologic LAPW and LAA were quantified using reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Furthermore, light microscopy was used to morphometrically evaluate the differential myocyte and interstitial changes in the same CAF-MD LAPW and LAA samples. In controls, HO-1 protein was quantified using enzyme-linked immunosorbent assay. Unlike controls, patients with CAF-MD had higher levels of HO-1 mRNA and its protein product, expressed as LAPW/LAA ratios, in the LAPW (2.18 +/- 1.18, P < .0001, and 1.55 +/- 0.67, P < .005), and their LAPW also showed greater histologic changes in myocytolytic myocytes (15.1% +/- 3.1% versus 6.9% +/- 3.3%, P < .0001), interstitial fibrosis (8.2% +/- 2.2% versus 2.8% +/- 1.2%, P < .0001), and capillary density (816 +/- 120 number/mm(2) versus 1114 +/- 188 number/mm(2); P < .05). In addition, markers of oxidative stress were immunohistochemically studied with antinitrotyrosine and anti-iNOS antibodies. In patients with CAF-MD, the inducible enzyme HO-1 is more expressed in the left atrial areas that show greater structural remodeling. This finding strongly suggests a pathogenetic relationship between oxidative stress and the degree of histologic change.

Modulatory proteins can rescue a trafficking defective epileptogenic Nav1.1 Na+ channel mutant.

Familial epilepsies are often caused by mutations of voltage-gated Na+ channels, but correlation genotype-phenotype is not yet clear. In particular, the cause of phenotypic variability observed in some epileptic families is unclear. We studied Na(v)1.1 (SCN1A) Na+ channel alpha subunit M1841T mutation, identified in a family characterized by a particularly large phenotypic spectrum. The mutant is a loss of function because when expressed alone, the current was no greater than background. Function was restored by incubation at temperature <30 degrees C, showing that the mutant is trafficking defective, thus far the first case among neuronal Na+ channels. Importantly, also molecular interactions with modulatory proteins or drugs were able to rescue the mutant. Protein-protein interactions may modulate the effect of the mutation in vivo and thus phenotype; variability in their strength may be one of the causes of phenotypic variability in familial epilepsy. Interacting drugs may be used to rescue the mutant in vivo.