No abstract

SUMMARYPurpose: 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 a 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 wildtype 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.

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.

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.

Loss of ataxia telangiectasia mutated (ATM) kinase, a key factor of the DNA damage response (DDR) pathway, causes the cancer predisposing and neurodegenerative syndrome ataxia-telangiectasia (A-T). To investigate the mechanisms of neurodegeneration, we have reprogrammed fibroblasts from ATM-null A-T patients and normal controls to pluripotency (human-induced pluripotent stem cells), and derived from these neural precursor cells able to terminally differentiate into post-mitotic neurons positive to >90% for β-tubulin III+/microtubule-associated protein 2+. We show that A-T neurons display similar voltage-gated potassium and sodium currents and discharges of action potentials as control neurons, but defective expression of the maturation and synaptic markers SCG10, SYP and PSD95 (postsynaptic density protein 95). A-T neurons exhibited defective repair of DNA double-strand breaks (DSBs) and repressed phosphorylation of ATM substrates (e.g., γH2AX, Smc1-S966, Kap1-S824, Chk2-T68, p53-S15), but normal repair of single-strand breaks, and normal short- and long-patch base excision repair activities. Moreover, A-T neurons were resistant to apoptosis induced by the genotoxic agents camptothecin and trabectedin, but as sensitive as controls to the oxidative agents. Most notably, A-T neurons exhibited abnormal accumulation of topoisomerase 1-DNA covalent complexes (Top1-ccs). These findings reveal that ATM deficiency impairs neuronal maturation, suppresses the response and repair of DNA DSBs, and enhances Top1-cc accumulation. Top1-cc could be a risk factor for neurodegeneration as they may interfere with transcription elongation and promote transcriptional decline.