SummaryDysfunctions in mitochondrial dynamics and metabolism are common pathological processes associated with Parkinson’s disease (PD). It was recently shown that an inherited form of PD and dementia is caused by mutations in the OPA1 gene, which encodes for a key player in mitochondrial fusion and structure. iPSC-derived neural cells from these patients exhibited severe mitochondrial fragmentation, respiration impairment, ATP deficits, and heightened oxidative stress. Reconstitution of normal levels of OPA1 in PD-derived neural cells normalized mitochondria morphology and function. OPA1-mutated neuronal cultures showed reduced survival in vitro. Intriguingly, selective inhibition of necroptosis effectively rescued this survival deficit. Additionally, dampening necroptosis in MPTP-treated mice protected from DA neuronal cell loss. This human iPSC-based model captures both early pathological events in OPA1 mutant neural cells and the beneficial effects of blocking necroptosis, highlighting this cell death process as a potential therapeutic target for PD.
An original multivariate multi-scale methodology for assessing the complexity of physiological signals is proposed. The technique is able to incorporate the simultaneous analysis of multi-channel data as a unique block within a multi-scale framework. The basic complexity measure is done by using Permutation Entropy, a methodology for time series processing based on ordinal analysis. Permutation Entropy is conceptually simple, structurally robust to noise and artifacts, computationally very fast, which is relevant for designing portable diagnostics. Since time series derived from biological systems show structures on multiple spatial-temporal scales, the proposed technique can be useful for other types of biomedical signal analysis. In this work, the possibility of distinguish among the brain states related to Alzheimer's disease patients and Mild Cognitive Impaired subjects from normal healthy elderly is checked on a real, although quite limited, experimental database.
Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here, we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Na v 1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Na v 1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, thus rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recover their firing ability and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach in DS and other disorders resulting from altered gene dosage.
The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.
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