Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

A novel de novo HCN2 loss-of-function variant causing developmental and epileptic encephalopathy treated with a ketogenic diet.

Missense variants of hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels cause variable phenotypes, ranging from mild generalized epilepsy to developmental and epileptic encephalopathy (DEE). Although variants of HCN1 are an established cause of DEE, those of HCN2 have been reported in generalized epilepsies. Here we describe the first case of DEE caused by the novel de novo heterozygous missense variant c.1379G>A (p.G460D) of HCN2. Functional characterization in transfected HEK293 cells and neonatal rat cortical neurons revealed that HCN2 p.G460D currents were strongly reduced compared to wild-type, consistent with a dominant negative loss-of-function effect. Immunofluorescence staining showed that mutant channels are retained within the cell and do not reach the membrane. Moreover, mutant HCN2 also affect HCN1 channels, by reducing the Ih current expressed by the HCN1-HCN2 heteromers. Due to the persistence of frequent seizures despite pharmacological polytherapy, the patient was treated with a ketogenic diet, with a significant and long-lasting reduction of episodes. In vitro experiments conducted in a ketogenic environment demonstrated that the clinical improvement observed with this dietary regimen was not mediated by a direct action on HCN2 activity. These results expand the clinical spectrum related to HCN2 channelopathies, further broadening our understanding of the pathogenesis of DEE.

Effect of the ketogenic diet in excitable tissues.

In the past decade, ketogenic diet (KD) has gained some popularity as a potential treatment for a wide range of diseases, including neurological and metabolic disorders, thanks to a beneficial role mainly related to its anti-inflammatory properties. The high-fat and carbohydrate-restricted regimen causes changes in the metabolism, leading, through the ß-oxidation of fatty acids, to the hepatic production of ketone bodies (KBs), which are used by many extrahepatic tissues as energy fuels. Once synthetized, KBs are delivered through the systemic circulation to all the tissues of the organism, where they play pleiotropic roles acting directly and indirectly on various targets, and among them ion channels and neurotransmitters. Moreover, they can operate as signaling metabolites and epigenetic modulators. Therefore, it is inappropriate to consider that the KD regimen can improve the patients' clinical condition simply by means of specific and localized effects; rather, it is more correct to think that KBs affect the organism as a whole. In this review, we tried to summarize the recent knowledge of the effects of KBs on various tissues, with a particular attention on the excitable ones, namely the nervous system, heart, and muscles.

Gabapentin treatment in a patient with KCNQ2 developmental epileptic encephalopathy.

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations.

Innovative Therapies and Nanomedicine Applications for the Treatment of Alzheimer's Disease: A State-of-the-Art (2017-2020).

The field of nanomedicine is constantly expanding. Since the first work dated in 1999, almost 28 thousand articles have been published, and more and more are published every year: just think that only in the last five years 20,855 have come out (source PUBMED) including original research and reviews. The goal of this review is to present the current knowledge about nanomedicine in Alzheimer's disease, a widespread neurodegenerative disorder in the over 60 population that deeply affects memory and cognition. Thus, after a brief introduction on the pathology and on the state-of-the-art research for NPs passing the BBB, special attention is placed to new targets that can enter the interest of nanoparticle designers and to new promising therapies. The authors performed a literature review limited to the last three years (2017-2020) of available studies with the intention to present only novel formulations or approaches where at least in vitro studies have been performed. This choice was made because, while limiting the sector to nanotechnology applied to Alzheimer, an organic census of all the relevant news is difficult to obtain.

Modulation of the intrinsic neuronal excitability by multifunctional liposomes tailored for the treatment of Alzheimer's disease.

PURPOSE: Nanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved. MATERIALS AND METHODS: We utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood-brain barrier and to be effective on mouse models of Alzheimer's disease (AD), both in the absence and in the presence of ß-amyloid peptide oligomers. RESULTS: Data show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86 MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3 pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with ß-amyloid peptide oligomers suggest a retention of liposome efficacy. CONCLUSION: These data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene.

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table.