A novel homozygous KCNQ3 loss-of-function variant causes non-syndromic intellectual disability and neonatal-onset pharmacodependent epilepsy

OBJECTIVE: Heterozygous variants in KCNQ2 or, more rarely, KCNQ3 genes are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical presentation and course, genetic transmission, and prognosis. While familial forms mostly include benign epilepsies with seizures starting in the neonatal or early-infantile period, de novo variants in KCNQ2 or KCNQ3 have been described in sporadic cases of early-onset encephalopathy (EOEE) with pharmacoresistant seizures, various age-related pathological EEG patterns, and moderate/severe developmental impairment. All pathogenic variants in KCNQ2 or KCNQ3 occur in heterozygosity. The aim of this work was to report the clinical, molecular, and functional properties of a new KCNQ3 variant found in homozygous configuration in a 9-year-old girl with pharmacodependent neonatal-onset epilepsy and non-syndromic intellectual disability. METHODS: Exome sequencing was used for genetic investigation. KCNQ3 transcript and subunit expression in fibroblasts was analyzed with quantitative real-time PCR and Western blotting or immunofluorescence, respectively. Whole-cell patch-clamp electrophysiology was used for functional characterization of mutant subunits. RESULTS: A novel single-base duplication in exon 12 of KCNQ3 (NM_004519.3:c.1599dup) was found in homozygous configuration in the proband born to consanguineous healthy parents; this frameshift variant introduced a premature termination codon (PTC), thus deleting a large part of the C-terminal region. Mutant KCNQ3 transcript and protein abundance was markedly reduced in primary fibroblasts from the proband, consistent with nonsense-mediated mRNA decay. The variant fully abolished the ability of KCNQ3 subunits to assemble into functional homomeric or heteromeric channels with KCNQ2 subunits. SIGNIFICANCE: The present results indicate that a homozygous KCNQ3 loss-of-function variant is responsible for a severe phenotype characterized by neonatal-onset pharmacodependent seizures, with developmental delay and intellectual disability. They also reveal difference in genetic and pathogenetic mechanisms between KCNQ2- and KCNQ3-related epilepsies, a crucial observation for patients affected with EOEE and/or developmental disabilities

The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea

Seagrasses colonized the sea(1) on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet(2). Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes(3), genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae(4) and that is important for ion homoeostasis, nutrient uptake and O-2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming(5,6), to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants(7)

Exploring the role of Kv7.3 in excitability control: novel insights from human mutations and a mice model

The KCNQ3 gene encodes for voltage-gated potassium channel subunits known as Kv7.3 which, together with subunits encoded by other members of the KCNQ gene subfamily, provide a critical contribution to the M-current. This current is a major controller of neuronal excitability at distinct brain areas and neuronal subtypes, as also revealed by the fact that variants in Kv7.3 cause mostly neonatal-onset epilepsies with wide phenotypic heterogeneity. However, despite these genetic evidences, several questions regarding the role of Kv7.3 subunits in controlling the excitability of specific brain regions and neuronal subtypes as well as the molecular pathophysiology of the human phenotypes associated to Kv7.3 variants are still poorly understood. In the present study, I have addressed some of these pressing issues in KCNQ3 neurobiology using tools and models ranging from kcnq3 KO mice to individuals/families carrying Kv7.3 mutations. In particular, using electrophysiological recordings in brain slices from kcnq3 KO mice, I have evaluated the role of Kv7.3 channel subunits in controlling the excitability of neurons located in the subiculum, the main output of the hippocampal circuit, and in the hippocampal CA1 area; notably, both the subuculum and the hippocampal formation are two cortical brain regions critically involved in seizure onset and propagation. In addition, I reported the functional consequences of mutations in Kv7.3 found associated with distinct clinical phenotypes in humans. In particular, I have investigated ex vivo and in vitro consequences of a new Kv7.3 variant (Kv7.3 F534Ifs*15), found in homozygous configuration in a 9‐year‐old girl with pharmacodependent neonatal‐onset epilepsy and non‐syndromic intellectual disability. This specific variant represents a unique opportunity to investigate the consequence of a complete deletion of Kv7.3 in humans given that all previously-found Kv7.3 mutations, except one, are found in heterozygosity. Finally, I have carried out electrophysiological and modeling studies to evaluated the functional consequences on channel properties determined by four de novo variants (Kv7.3-R227Q, -R230C, -R230S, and -R230C) found in patients with global neurodevelopmental disability (NDD), autism spectrum disorder (ASD), and Sleep-Activated Near-Continuous Multifocal Spikes. These pathogenic variants have been of great relevance given that were responsible of a unique phenotype, not associated to neonatal-onset seizure, which allowed a further expansion of the phenotypic spectrum of diseases associated to Kv7.3 gene variants

KCNQ3 is the principal target of retigabine in CA1 and subicular excitatory neurons

Retigabine is a first-in-class potassium channel opener approved for patients with epilepsy. Unfortunately, several side effects have limited its use in clinical practice, overshadowing its beneficial effects. Multiple studies have shown that retigabine acts by enhancing the activity of members of the voltage-gated KCNQ (Kv7) potassium channel family, particularly the neuronal KCNQ channels KCNQ2-KCNQ5. However, it is currently unknown whether retigabine's action in neurons is mediated by all KCNQ neuronal channels or by only a subset. This knowledge is necessary to elucidate retigabine's mechanism of action in the central nervous system and its adverse effects and to design more effective and selective retigabine analogs. Here, we show that the action of retigabine in excitatory neurons strongly depends on the presence of KCNQ3 channels. Deletion of Kcnq3 severely limited the ability of retigabine to reduce neuronal excitability in mouse CA1 and subiculum excitatory neurons. Additionally, we report that in the absence of KCNQ3 channels, retigabine can enhance CA1 pyramidal neuron activity, leading to a greater number of action potentials and reduced spike frequency adaptation; this finding further supports a key role of KCNQ3 channels in mediating the action of retigabine. Our work provides new insight into the action of retigabine in forebrain neurons, clarifying retigabine's action in the nervous system

Effects of glucosamine and nucleotide association on fibroblast: extracellular matrix gene expression

Glucosamine (Gluc) is a drug used as an anti-inflammatory in moderate forms of knee arthrosis. A further off label use of Gluc is in the anti-aging treatments associated with Polideoxirybonucleotide (PDRN) through intra-dermal injection for a procedure called bio-stimulation. An unexpected effect on cultured dermal fibroblasts, during an experimental study on the gene activation in aesthetic bio-stimulation, was observed. The results have potential application in orthopaedic medical therapy. Fibroblast primary cultures were carried out, seeding cells on a layer of Gluc or PDRN alone or in combination for 24 h. Real Time-PCR was performed to investigate several gene expressions. The MMP13 and the IGF-I gene expression in fibroblast cultures were strongly inhibited after 24 h of incubation with the association of Gluc and PDRN, whereas Gluc and PDRN alone produced a modest inhibition of IGF-I and an activation of MMP13. MMP13 is present in osteoarthritic cartilage and this enzyme plays a significant role in cartilage collagen degradation. IGF1 is involved in growth and development and is successfully used in tissue-engineering for cartilage repair. Based on the reported data, we infer that the association of Gluc and PDRN has a potential application in cartilage therapy. Additional basic science and clinical studies are needed to confirm this preliminary report

An Orally Active Cannabis Extract with High Content in Cannabidiol attenuates Chemically-induced Intestinal Inflammation and Hypermotility in the Mouse.

Anecdotal and scientific evidence suggests that Cannabis use may be beneficial in inflammatory bowel disease (IBD) patients. Here, we have investigated the effect of a standardized Cannabis sativa extract with high content of cannabidiol (CBD), here named CBD BDS for "CBD botanical drug substance," on mucosal inflammation and hypermotility in mouse models of intestinal inflammation. Colitis was induced in mice by intracolonic administration of dinitrobenzenesulfonic acid (DNBS). Motility was evaluated in the experimental model of intestinal hypermotility induced by irritant croton oil. CBD BDS or pure CBD were given - either intraperitoneally or by oral gavage - after the inflammatory insult (curative protocol). The amounts of CBD in the colon, brain, and liver after the oral treatments were measured by high-performance liquid chromatography coupled to ion trap-time of flight mass spectrometry. CBD BDS, both when given intraperitoneally and by oral gavage, decreased the extent of the damage (as revealed by the decrease in the colon weight/length ratio and myeloperoxidase activity) in the DNBS model of colitis. It also reduced intestinal hypermotility (at doses lower than those required to affect transit in healthy mice) in the croton oil model of intestinal hypermotility. Under the same experimental conditions, pure CBD did not ameliorate colitis while it normalized croton oil-induced hypermotility when given intraperitoneally (in a dose-related fashion) or orally (only at one dose). In conclusion, CBD BDS, given after the inflammatory insult, attenuates injury and motility in intestinal models of inflammation. These findings sustain the rationale of combining CBD with other minor Cannabis constituents and support the clinical development of CBD BDS for IBD treatment

Preclinical investigation in FAAH inhibition as a neuroprotective therapy for frontotemporal dementia using TDP-43 transgenic male mice

Abstract Background Frontotemporal dementia (FTD) is a heterogeneous group of early onset and progressive neurodegenerative disorders, characterized by degeneration in the frontal and temporal lobes, which causes deterioration in cognition, personality, social behavior and language. Around 45% of the cases are characterized by the presence of aggregates of the RNA-binding protein TDP-43. Methods In this study, we have used a murine model of FTD that overexpresses this protein exclusively in the forebrain (under the control of the CaMKIIα promoter) for several biochemical, histological and pharmacological studies focused on the endocannabinoid system. Results These mice exhibited at postnatal day 90 (PND90) important cognitive deficits, signs of emotional impairment and disinhibited social behaviour, which were, in most of cases, maintained during the first year of life of these animals. Motor activity was apparently normal, but FTD mice exhibited higher mortality. Their MRI imaging analysis and their ex-vivo histopathological evaluation proved changes compatible with atrophy (loss of specific groups of pyramidal neurons: Ctip2- and NeuN-positive cells) and inflammatory events (astroglial and microglial reactivities) in both cortical (medial prefrontal cortex) and subcortical (hippocampus) structures at PND90 and also at PND365. The analysis of the endocannabinoid system in these mice proved a decrease in the hydrolysing enzyme FAAH in the prefrontal cortex and the hippocampus, with an increase in the synthesizing enzyme NAPE-PLD only in the hippocampus, responses that were accompanied by modest elevations in anandamide and related N-acylethanolamines. The potentiation of these elevated levels of anandamide after the pharmacological inactivation of FAAH with URB597 resulted in a general improvement in behaviour, in particular in cognitive deterioration, associated with the preservation of pyramidal neurons of the medial prefrontal cortex and the CA1 layer of the hippocampus, and with the reduction of gliosis in both structures. Conclusions Our data confirmed the potential of elevating the endocannabinoid tone as a therapy against TDP-43-induced neuropathology in FTD, limiting glial reactivity, preserving neuronal integrity and improving cognitive, emotional and social deficits