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Abstract Objective Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by autosomal‐dominant pathogenic variants in either the TSC1 or TSC2 gene, and it is characterized by hamartomas in multiple organs, such as skin, kidney, lung, and brain. These changes can result in epilepsy, learning disabilities, and behavioral complications, among others. The mechanistic link between TSC and the mechanistic target of the rapamycin (mTOR) pathway is well established, thus mTOR inhibitors can potentially be used to treat the clinical manifestations of the disorder, including epilepsy. Methods In this study, we tested the efficacy of a novel mTOR catalytic inhibitor (here named Tool Compound 1 or TC1) previously reported to be more brain‐penetrant compared with other mTOR inhibitors. Using a well‐characterized hypomorphic Tsc2 mouse model, which displays a translationally relevant seizure phenotype, we tested the efficacy of TC1. Results Our results show that chronic treatment with this novel mTOR catalytic inhibitor (TC1), which affects both the mTORC1 and mTORC2 signaling complexes, reduces seizure burden, and extends the survival of Tsc2 hypomorphic mice, restoring species typical weight gain over development. Interpretation Novel mTOR catalytic inhibitor TC1 exhibits a promising therapeutic option in the treatment of TSC.

Abstract ERLIN2‐related disorders are rare conditions of the motor system and clinical details are limited to a small number of prior descriptions. We here presented clinical and genetic details in five individuals (four different families) where three subjects carried a common homozygous p.Asn292ArgfsX26, associated also with sensorineural hearing loss in one child. One further subject had a de novo p.Gln63Lys and one harbors the homozygous p.Val136Gly because of maternal isodisomy of chromosome 8. Overall, we expanded the clinical and genetic spectrum of ERLIN2‐related disorders and we reiterate that autosomal‐dominant transmission is a potential mode of inheritance. Future research will elucidate disease mechanisms.

Abstract Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP‐4). Using next‐generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP‐4 complex formation in patient‐derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP‐4 deficiency using morpholino‐mediated knockdown of ap4s1. In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability.

Biallelic loss-of-function variants in the subunits of the adaptor protein complex 4 lead to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), and SPG52 (AP4S1). Here, we describe the generation of induced pluripotent stem cells (iPSCs) from three AP-4-HSP patients with biallelic, loss-of-function variants in AP4M1 and their sex-matched parents (asymptomatic, heterozygous carriers). Following reprogramming using non-integrating Sendai virus, iPSCs were characterized following standard protocols including karyotyping, embryoid body formation, pluripotency marker expression and STR profiling. These first iPSC lines for SPG50 provide a valuable resource for studying this rare disease and related forms of hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) and hereditary ataxia (HA) are two groups of disorders characterized, respectively, by progressive dysfunction or degeneration of the pyramidal tracts (HSP) and of the Purkinje cells and spinocerebellar tracts (HA). Although HSP and HA are generally shown to have distinct clinical-genetic profiles, in several cases the clinical presentation, the causative genes, and the cellular pathways and mechanisms involved overlap between the two forms. Genetic analyses in humans in combination with in vitro and in vivo studies using model systems have greatly expanded our knowledge of spinocerebellar degenerative disorders. In this review, we focus on the zebrafish (Danio rerio), a vertebrate model widely used in biomedical research since its overall nervous system organization is similar to that of humans. A critical analysis of the literature suggests that zebrafish could serve as a powerful experimental tool for molecular and genetic dissection of both HA and HSP. The zebrafish, found to be very useful for demonstrating the causal relationship between defect and mutation, also offers a useful platform to exploit for the development of therapies.

Bi-allelic variants in the subunits of the adaptor protein complex 4 lead to childhood-onset, complex hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), and SPG52 (AP4S1). Here, we describe the generation of induced pluripotent stem cells (iPSCs) from three AP-4-HSP patients with compound-heterozygous, loss-of-function variants in AP4B1 and sex-matched parents. Fibroblasts were reprogrammed using non-integrating Sendai virus. iPSCs were characterized according to standard protocols including karyotyping, embryoid body formation, pluripotency marker expression and STR profiling. These first iPSC lines for SPG47 provide a valuable resource for studying this rare disease and related forms of hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity—there are over 80 potential disease-associated genes—and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy.

Glucocorticoid-induced osteoporosis (GIO) is a secondary cause of bone loss. Bisphosphonates approved for GIO, might induce jaw osteonecrosis; thus additional therapeutics are required. Adenosine receptor agonists are positive regulators of bone remodeling, thus the efficacy of adenosine receptor stimulation for treating GIO was tested. In a preventive study GIO was induced in Sprague-Dawley rats by methylprednisolone (MP) for 60 days. Animals were randomly assigned to receive polydeoxyribonucleotide (PDRN), an adenosine A2 receptor agonist, or PDRN and DMPX (3,7-dimethyl-1-propargylxanthine, an A2 antagonist), or vehicle (0.9% NaCl). Another set of animals was used for a treatment study, following the 60 days of MP-induction rats were randomized to receive (for additional 60 days) PDRN, or PDRN and DMPX (an adenosine A2 receptor antagonist), or zoledronate (as control for gold standard treatment), or vehicle. Control animals were administered with vehicle for either 60 or 120 days. Femurs were analyzed after treatments for histology, imaging, and breaking strength analysis. MP treatment induced severe bone loss, the concomitant use of PDRN prevented the developing of osteoporosis. In rats treated for 120 days, PDRN restored bone architecture and bone strength; increased b-ALP, osteocalcin, osteoprotegerin and stimulated the Wnt canonical and non-canonical pathway. Zoledronate reduced bone resorption and ameliorated the histological features, without significant effects on bone formation. Our results suggest that adenosine receptor stimulation might be useful for preventing and treating GIO.