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Introduction Lafora disease (LD) is an ultrarare fatal progressive myoclonic epilepsy, causing drug-resistant epilepsy, myoclonus and psychomotor deterioration. LD is caused by mutations in EPM2A or NHLRC1, which lead to the accumulation of polyglucosans in the brain and neurodegeneration. There are no approved treatments for LD. VAL-1221 is a fusion protein comprising the Fab portion of a cell-penetrating antibody and recombinant human acid alpha glucosidase, and has demonstrated an ability to clear polyglucosans. We hypothesise that intravenous infusion of VAL-1221 might be able to degrade cerebral polyglucosans and stabilise or improve disease outcomes. The aim of this study is to assess the safety and preliminary efficacy of VAL-1221 in patients with LD.Methods and analysis The study is a phase 2, single-arm, open-label, baseline-controlled clinical trial which will be conducted in a single investigational study centre in Italy, namely the sponsor ‘IRCCS Istituto delle Scienze Neurologiche di Bologna—Azienda USL di Bologna’. The study will enrol six genetically confirmed patients with mid- to late-stage LD. The global duration of the study for each participant will be 18 months, including screening period, open-label treatment (12 months) and follow-up period. VAL-1221 20 mg/kg will be administered as an intravenous infusion every week for 3 weeks, then every other week. Patients will undergo full clinical assessments at baseline, at an intermediate and at the end-of-treatment visit. The primary objective is to evaluate the safety. The exploratory efficacy endpoints will be related to epilepsy, neuropsychological and motor functions, global assessment and disease burden, in addition to biomarkers. Statistical analyses will be primarily descriptive.Ethics and dissemination The study protocol was approved by the local ethics committee (number 232-2023-FARM-AUSLBO-23020, 22 March 2023). The results of this study will be disseminated by the investigators through presentations at international scientific conferences and reported in peer-reviewed scientific journals.Trial registration number European Union Clinical Trials Register (EudraCT 2023-000185-34).

Introduction: During aging, sarcopenia and decline in physiological processes lead to partial loss of muscle strength, atrophy, and increased fatigability. Muscle changes may be related to a reduced intake of essential amino acids playing a role in proteostasis. We have recently shown that branched-chain amino acid (BCAA) supplements improve atrophy and weakness in models of muscle disuse and aging. Considering the key roles that the alteration of Ca2+-related homeostasis and store-operated calcium entry (SOCE) play in several muscle dysfunctions, this study has been aimed at gaining insight into the potential ability of BCAA-based dietary formulations in aged mice on various players of Ca2+ dyshomeostasis.Methods: Seventeen-month-old male C57BL/6J mice received a 12-week supplementation with BCAAs alone or boosted with two equivalents of L-alanine (2-Ala) or with dipeptide L-alanyl-L-alanine (Di-Ala) in drinking water. Outcomes were evaluated on ex vivo skeletal muscles indices vs. adult 3-month-old male C57BL/6J mice.Results: Ca2+ imaging confirmed a decrease in SOCE and an increase of resting Ca2+ concentration in aged vs. adult mice without alteration in the canonical components of SOCE. Aged muscles vs. adult muscles were characterized by a decrease in the expression of ryanodine receptor 1 (RyR1), the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) pump, and sarcalumenin together with an alteration of the expression of mitsugumin 29 and mitsugumin 53, two recently recognized players in the SOCE mechanism. BCAAs, particularly the formulation BCAAs+2-Ala, were able to ameliorate all these alterations.Discussion: These results provide evidence that Ca2+ homeostasis dysfunction plays a role in the functional deficit observed in aged muscle and supports the interest of dietary BCAA supplementation in counteracting sarcopenia-related SOCE dysregulation.

Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse model for DMD is the C57BL/10ScSn-Dmdmdx/J (BL10-mdx), but this model exhibits mild pathology and does not replicate key features of human disease. The D2.B10-Dmdmdx/J (D2-mdx) mouse is a more recent model which seems to better mimics the complex human DMD phenotype. However, the D2-mdx mouse remains less extensively characterised than its BL10-mdx counterpart. Quantitative PCR analysis of gene expression is an important tool to monitor disease progression and evaluate therapeutic efficacy, but measurements must be normalised to stably expressed reference genes, which should ideally be determined and validated empirically. We examined gene expression in the gastrocnemius (GC), diaphragm (DIA) and heart in the D2-mdx mouse, the BL10-mdx mouse, and appropriate strain-matched wild-type controls (D2-wt and BL10-wt), from 4 to 52 weeks of age, using a large panel of candidate references (ACTB, AP3D1, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13A, SDHA, and in the heart, also HTATSF1 and HMBS). Data was analyzed using GeNorm, Bestkeeper, deltaCt and Normfinder algorithms to identify stable references under multiple possible scenarios. We show that CSNK2A2, AP3D1 and ACTB represent strong universal reference genes in both GC and DIA, regardless of age, muscle type, strain and genotype, while HTATSF1 and SDHA are optimal for the heart. GAPDH, HPRT1 and RPL13A were conversely revealed to be poor references, showing tissue-, age- or disease-specific changes in expression. Our results illustrate the importance of determining appropriate reference genes for specific comparative scenarios, but also reconfirm that universal panels can nevertheless be identified for normalising gene expression studies in even complex pathological states.

IntroductionGrowth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD).MethodsHere, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.).ResultsIn vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-β1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD.DiscussionOur results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.