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.

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.

FDG-PET imaging in HIV-infected subjects: relation with therapy and immunovirological variables.

PURPOSE: To characterise tissue sites of immune activation and HIV replication we performed FDG-PET in ART-treated and ART-naive HIV-infected individuals. Specific aims were to establish whether HIV-infected patients can be differentiated on the basis of the detection of specific locations of viral replication, even in the presence of an apparently optimal immunovirological response to ART, and whether these FDG-PET findings can be related to immunovirological variables and AIDS history status. PATIENTS AND METHODS: Patients were divided into five groups as follows: subgroup A1 (full responders, n = 8): current ART treatment, CD4+ T lymphocytes >500/mL, viral load <50 copies/mL; subgroup A2 (full responders, n = 5): same criteria as A-1, but with a previous history of AIDS; subgroup A3 (immunological non responders, n = 5): current ART treatment, viral load <50 copies/mL, low CD4+ T lymphocytes (<200/mL); group B (virological non responders, n = 2): current ART treatment, CD4+ T lymphocytes around 500/mL, viral load >50,000 copies/mL; group C (ART-naïve, n = 5): no current or previous ART treatment, increased viral load. RESULTS: PET images revealed different patterns of FDG uptake. All ART-treated patients with either suppressed (<50 copies/mL; Group A) or high viremia (group B) showed a normal pattern of FDG uptake. On the contrary, the ART-naïve subjects with high viraemia (group C) displayed multiple foci of increased glucose metabolism in the lymph nodes. In the ART-naïve subjects, FDG uptake, apparently related to viraemia level, was observed in the upper torso mainly in the axillary nodes bilaterally in patients with viraemia below 100,000 copies/mL; in those with viraemia higher than 100,000 copies/mL, FDG uptake was also observed in the inguinal lymph nodes. CONCLUSIONS: The emergence, in our study, of a correlation between the percentage of CD8+/CD38+/RO+ T cells (well established markers of progression to AIDS independently of CD4+ T lymphocytes) and positive FDG-PET in ART-naive patients is a novel finding that seems to confer prognostic value on FDG uptake. FDG uptake is strongly associated with response to ART independently of a previous AIDS diagnosis. Notably, no differences were observed between ART-treated subjects classed as immunological responders and those classed as non responders. Data herewith indicate that FDG uptake and immunological variables are unrelated when ART is being administered. This is evidence of the complementarity of immunological and FDG measures. FDG uptake is a sensitive marker of disease state and its relation with CD8+/CD38+/CD45RO+ T cells indicates that it can be considered a marker of disease status. The lack of a correlation between FDG uptake and immunological variables in patients under ART warrants further investigation.

Hyperexcitability in Cultured Cortical Neuron Networks from the G93A-SOD1 Amyotrophic Lateral Sclerosis Model Mouse and its Molecular Correlates.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the corticospinal tract and leading to motor neuron death. According to a recent study, magnetic resonance imaging-visible changes suggestive of neurodegeneration seem absent in the motor cortex of G93A-SOD1 ALS mice. However, it has not yet been ascertained whether the cortical neural activity is intact, or alterations are present, perhaps even from an early stage. Here, cortical neurons from this model were isolated at post-natal day 1 and cultured on multielectrode arrays. Their activity was studied with a comprehensive pool of neurophysiological analyses probing excitability, criticality and network architecture, alongside immunocytochemistry and molecular investigations. Significant hyperexcitability was visible through increased network firing rate and bursting, whereas topological changes in the synchronization patterns were apparently absent. The number of dendritic spines was increased, accompanied by elevated transcriptional levels of the DLG4 gene, NMDA receptor 1 and the early pro-apoptotic APAF1 gene. The extracellular Na+, Ca2+, K+ and Cl- concentrations were elevated, pointing to perturbations in the culture micro-environment. Our findings highlight remarkable early changes in ALS cortical neuron activity and physiology. These changes suggest that the causative factors of hyperexcitability and associated toxicity could become established much earlier than the appearance of disease symptoms, with implications for the discovery of new hypothetical therapeutic targets.

Comparative neuronal differentiation of self-renewing neural progenitor cell lines obtained from human induced pluripotent stem cells.

Most human neuronal disorders are associated with genetic alterations that cause defects in neuronal development and induce precocious neurodegeneration. In order to fully characterize the molecular mechanisms underlying the onset of these devastating diseases, it is important to establish in vitro models able to recapitulate the human pathology as closely as possible. Here we compared three different differentiation protocols for obtaining functional neurons from human induced pluripotent stem cells (hiPSCs): human neural progenitors (hNPs) obtained from hiPSCs were differentiated by co-culturing them with rat primary neurons, glial cells or simply by culturing them on matrigel in neuronal differentiation medium, and the differentiation level was compared using immunofluorescence, biochemical and electrophysiological methods. We show that the differentiated neurons displayed distinct maturation properties depending on the protocol used and the faster morphological and functional maturation was obtained when hNPs were co-cultured with rat primary neurons.