Conference Agenda

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective death of motor neurons (MNs). No effective therapies exist which results in the death of the patient 3 to 5 years after the detection of the first signs. Although our knowledge about the neuropathology and genetic causes has made significant progress, our level of understanding based on currently existing cellular and animal models is insufficient. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different mutations, as well as from healthy controls. Patient-derived MNs showed progressive axonal transport defects of mitochondria. These axonal transport defect seems to be a general phenotype and also other cargoes, including lysosomes, were affected. We could rescue the axonal transport defects by CRISPR/Cas9-mediated genetic correction of the ALS-causing mutations. Moreover, pharmacological inhibition of histone deacetylase 6 (HDAC6) increased not only the α-tubulin acetylation within the microtubules, but also corrected the axonal transport defects in our patient-derived MNs. In conclusion, we are developing and studying completely new in vitro models of ALS. This will hopefully provide better insights into the ALS disease mechanism(s) and could allow us to identify new therapeutic targets for this dreadful disease. Our ultimate aim is to translate these strategies towards the clinic.

Spinal and bulbar muscular atrophy (SBMA) is a fatal motor neuron (MN) disease affecting lower MN and skeletal muscle cells. It is due to an expansion of a CAG triplet repeat of the androgen receptor (AR) gene, which is translated into a poly-glutamine (polyQ) tract in the N-terminus of the AR protein. AR functions are triggered by androgens, that lead to ARpolyQ misfolding and aggregation. Our strategy was aimed at reducing AR activation and nuclear ARpolyQ toxicity and at enhancing its cytoplasmic autophagic clearance. Using a knock-in SBMA mouse model, we tested the effects of bicalutamide, a type 1 pure antiandrogen blocking ARpolyQ activation, and trehalose a natural disaccharide with a pro-autophagic activity. Obtained data demonstrate that the compounds extend the survival of the KI mice, improve their motor behaviour, and partially recover the morphology of muscle fibres. At an early symptomatic stage, in KI mouse muscle the autophagic pathway is strongly activated. Despite this, SQSTM1 accumulates in muscle fibres indicating a blockage of the autophagic flux. Bicalutamide, alone or in combination with trehalose completely reverse the formation of AR insoluble forms leading to the removal of the autophagic flux blockage. Furthermore, we show that apoptosis is induced in KI AR113Q mouse muscles, and that trehalose and bicalutamide prevent apoptosis activation. Finally, reporting a decrease of mtDNA and OXPHOS enzymes content that are reverted by trehalose, our data indicate the importance of mitochondrial dysfunction in SBMA muscle. Altogether these results reveal modifications in muscle morphology and function at an early symptomatic stage of the disease suggesting the importance of developing muscle-targeted therapeutic intervention. Trehalose and bicalutamide counteracting ARpolyQ toxicity in skeletal muscle may be considered possible candidates for future clinical trials to be performed in SBMA patients.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in survival motor neuron (SMN) 1 gene, resulting in a truncated SMN protein, responsible for degeneration of brain stem and spinal motor neurons.

The recent availability of disease-modifying therapies for SMA represents a unique opportunity in the field of motor neuron diseases to identify reliable molecular factors that provide insight into disease progression and reveal biologic or pharmacologic phenomena, with potential complementary therapeutic implications.

SMN1 has ubiquitous expression in the organism, where it critically regulates several developmental and housekeeping cellular pathways, like RNA metabolism and biogenesis of microRNAs (miRNAs), key gene expression modulators, whose dysregulation contributes to neuromuscular diseases. MiRNAs are stable in body fluids and reflect distinct pathophysiological states, acting as promising biomarkers.

In this respect, we found that the expression of muscle-specific miR-133a, -133b, -206 and -1 (myomiRs), was increased in serum of SMA patients and reduced over disease course upon treatment. Notably, miR-133a reduction predicted patients’ response to therapy, both in children and in adults, in line with the different response timing.

Not least, myomiRs participate with inflammatory cytokines in regulating muscle tissue regeneration, and an immune system dysregulation has been previously reported in SMA preclinical models and patients. Accordingly, we demonstrated an inflammatory peripheral signature, that changes upon treatment, and the presence of inflammatory mediators in the cerebrospinal fluid of SMA patients, thus supporting an inflammatory/immunological contribution to SMA. Further, we provided evidence of a possible role for serum IL-23 as predictive biomarker of response to therapy, and of serum IL-10 as a potential on-treatment monitoring biomarker.

The molecules here identified could represent novel potential therapeutic targets, as well as reliable biomarkers to stratify patients, predict disease progression and monitor response to therapies, for a better management of SMA patients.

Spinal Muscular Atrophy (SMA) is a neurodegenerative disease affecting children, characterized by motor neuron (MN) impairment, skeletal muscle atrophy and premature death. It is caused by the mutation of the survival motor neuron 1 (SMN1) gene. Its homologous gene SMN2 is unaffected, but due to an alternative splicing generates only 10% of functional SMN protein. Fundamental limitations of current therapies still drive the need for new approaches aimed at increasing functional SMN production. Drug repositioning for SMA treatment represents a reliable tool to address significant unmet therapeutic needs. A screening of FDA-approved drugs identified two SMN2 splicing enhancers in SMA models (Drosophila-based reporter system and patient´s fibroblasts): Moxifloxacin and GT5 (code name). We already demonstrated that Moxifloxacin can be potentially repositioned for the SMA treatment (Januel et al., Cell Mol Life Sci. 2022). We now report the GT5 therapeutic efficacy, both in vitro (patient’s iPSCs-derived-MNs and primary SMA myoblasts) and in vivo (delta7 mice, murine model of severe SMA). In details, daily subcutaneous administration of GT5 in delta7 mice increased the SMN levels in spinal cord (≥50%), quadriceps and gastrocnemius (≥1 fold), compared to controls, also leading to improved motor skills. The analysis of the spinal cord ventral horns (lumbar tract) of GT5 mice confirmed: i) delayed MN degeneration (≤90%); ii) reduced levels of cleaved-caspase-3 (apoptotic marker) (≤63%), iii) lower neuroinflammation with reduced astrogliosis (GFAP signaling) (≤37%) and different degree of microglia ramification/activation compared with controls. Furthermore, GT5 mice skeletal muscles showed improved trophism and neuromuscular junction phenotypes. Notably, in vitro GT5 treatment prevented MN degeneration more efficiently than Risdiplam (a current SMA therapy) and rescued the impaired formation of myotubes in a MN-myoblast co-culture. Overall these results support the GT5 repositioning for the SMA treatment and strengthen the value of this strategy for discovering new therapies for rare diseases.