Synergistic activation of AMPK prevents from polyglutamine-inducedtoxicity inCaenorhabditis elegans

11 páginas, 4 figuras. Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.phrs.2020.105105.Expression of abnormally long polyglutamine (polyQ) tracks is the source of a range of dominant neurodegenerative diseases, such as Huntington disease. Currently, there is no treatment for this devastating disease, although some chemicals, e.g., metformin, have been proposed as therapeutic solutions. In this work, we show that metformin, together with salicylate, can synergistically reduce the number of aggregates produced after polyQ expression in Caenorhabditis elegans. Moreover, we demonstrate that incubation polyQ-stressed worms with low doses of both chemicals restores neuronal functionality. Both substances are pleitotropic and may activate a range of different targets. However, we demonstrate in this report that the beneficial effect induced by the combination of these drugs depends entirely on the catalytic action of AMPK, since loss of function mutants of aak-2/AMPKα2 do not respond to the treatment. To further investigate the mechanism of the synergetic activity of metformin/salicylate, we used CRISPR to generate mutant alleles of the scaffolding subunit of AMPK, aakb-1/AMPKβ1. In addition, we used an RNAi strategy to silence the expression of the second AMPKβ subunit in worms, namely aakb-2/AMPKβ2. In this work, we demonstrated that both regulatory subunits of AMPK are modulators of protein homeostasis. Interestingly, only aakb-2/AMPKβ2 is required for the synergistic action of metformin/salicylate to reduce polyQ aggregation. Finally, we showed that autophagy acts downstream of metformin/salicylate-related AMPK activation to promote healthy protein homeostasis in worms.We thank the CGC, funded by the NIH Office of ResearchInfrastructure Programs (P40 OD010440), for worm strains. [...] RPVMis aMiguel Servet type IIresearcher (CPII16/00004) funded by Institutode Salud Carlos III (ISCIII, Madrid, Spain). Grants from the ISCIII wereused to perform this work (PI14/00949 and PI17/00011). All grantsfrom ISCIII are co-financed by the European Development RegionalFund”A way to achieve Europe”(ERDF). JBY holds a grant from theGeneralitat Valenciana and the European Social Fund (ACIF/2019/249). Some equipment used in this work has been funded in partnershipbetween the Generalitat Valenciana (Conselleria de Sanitat I SalutPública, Valencian Community, Spain) and European Funds (ERDF/FSE), through the call "Improvement of research infrastructures for rarediseases”CV FEDER 2014-2020. This work has been partially supportedby a grant from the Fundació Telemarató de la TV3 (Reference 559),which covered the work of MDS. The funds from the ISCIII are partiallysupported by the European Regional Development Fund. RPVM is also aMarie Curie fellow (CIG322034, EU). This work has been partiallysupported by a grant from the CIBERER (ACCI2016), a grant from theFundación Ramón Areces (CIVP19S8119) and anAyuda Miguel Gilgrantto RPVM (VII Convocatoria Ayudas a la Investigación MHER, 2019Peer reviewe

Study of USH1 Splicing Variants through Minigenes and Transcript Analysis from Nasal Epithelial Cells

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by minigene assays. These variants were selected according to bioinformatic analysis. The second aim was to analyze the USH1 transcripts, obtained from nasal epithelial cells samples of our patients, in order to corroborate the observed effect of mutations by minigenes in patient’s tissues. The last objective was to evaluate the nasal ciliary beat frequency in patients with USH1 and compare it with control subjects. In silico analysis were performed using four bioinformatic programs: NNSplice, Human Splicing Finder, NetGene2 and Spliceview. Afterward, minigenes based on the pSPL3 vector were used to investigate the implication of selected changes in the mRNA processing. To observe the effect of mutations in the patient’s tissues, RNA was extracted from nasal epithelial cells and RT-PCR analyses were performed. Four MYO7A (c.470G>A, c.1342_1343delAG, c.5856G>A and c.3652G>A), three CDH23 (c.2289+1G>A, c.6049G>A and c.8722+1delG) and one PCDH15 (c.3717+2dupTT) variants were observed to affect the splicing process by minigene assays and/or transcripts analysis obtained from nasal cells. Based on our results, minigenes are a good approach to determine the implication of identified variants in the mRNA processing, and the analysis of RNA obtained from nasal epithelial cells is an alternative method to discriminate neutral Usher variants from those with a pathogenic effect on the splicing process. In addition, we could observe that the nasal ciliated epithelium of USH1 patients shows a lower ciliary beat frequency than control subjects

Altered Antioxidant-Oxidant Status in the Aqueous Humor and Peripheral Blood of Patients with Retinitis Pigmentosa

Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa

USH2A Gene Editing Using the CRISPR System

Usher syndrome (USH) is a rare autosomal recessive disease and the most common inherited form of combined visual and hearing impairment. Up to 13 genes are associated with this disorder, with USH2A being the most prevalent, due partially to the recurrence rate of the c.2299delG mutation. Excluding hearing aids or cochlear implants for hearing impairment, there are no medical solutions available to treat USH patients. The repair of specific mutations by gene editing is, therefore, an interesting strategy that can be explored using the CRISPR/Cas9 system. In this study, this method of gene editing is used to target the c.2299delG mutation on fibroblasts from an USH patient carrying the mutation in homozygosis. Successful in vitro mutation repair was demonstrated using locus-specific RNA-Cas9 ribonucleoproteins with subsequent homologous recombination repair induced by an engineered template supply. Effects on predicted off-target sites in the CRISPR-treated cells were discarded after a targeted deep-sequencing screen. The proven effectiveness and specificity of these correction tools, applied to the c.2299delG pathogenic variant of USH2A, indicates that the CRISPR system should be considered to further explore a potential treatment of USH. Keywords: Usher syndrome, USH2A, c.2299delG, CRISPR, gene editing, RNP

Metformin intake associates with better cognitive function in patients with Huntington's disease

Huntington's disease (HD) is an inherited, dominant neurodegenerative disorder caused by an abnormal expansion of CAG triplets in the huntingtin gene (htt). Despite extensive efforts to modify the progression of HD thus far only symptomatic treatment is available. Recent work suggests that treating invertebrate and mice HD models with metformin, a well-known AMPK activator which is used worldwide to treat type 2-diabetes, reduces mutant huntingtin from cells and alleviates many of the phenotypes associated to HD. Herein we report statistical analyses of a sample population of participants in the Enroll-HD database, a world-wide observational study on HD, to assess the effect of metformin intake in HD patients respect to cognitive status using linear models. This cross-sectional study shows for the first time that the use of metformin associates with better cognitive function in HD patients