Computational identification and experimental validation of early glycinergic impairments in spinal muscular atrophy

Spinal Muscular Atrophy (SMA) is a progressive neurodegenerative disorder characterized by lower motor neuron (MN) loss, due to mutations or deletions in the Survival Motor Neuron 1 (SMN1) gene. Increasing evidence implies mitochondrial dysfunctions in SMA, suggesting these organelles are potential therapeutic targets. Through bioinformatic analysis of microarray-based gene expression data, obtained from a wide range of mouse tissues and cell lines (not specifically mutated in Smn), we identified eight mitochondrial genes significantly anticorrelated with Smn expression, which may be potentially associated with SMN-related pathways.

Among them, Gcsh (glycine cleavage system protein H), encoding a subunit of the glycine cleavage system (GCS), was confirmed by experimental validation as actually deregulated after SMN loss of function. In the severe SMNΔ7 mouse model, we revealed by quantitative Real Time-PCR a strong upregulation of Gcsh in the lumbar spinal cord at an early symptomatic stage (postnatal day 5, P5) compared to wild type (WT), although by western blot protein levels were not significantly increased in total lysate. Moreover, RNA-Scope in-situ hybridization showed significantly increased Gcsh expression in the MN soma, and immunofluorescence corroborated these findings, revealing elevated GCSH protein in both MNs and astrocytes.

We also demonstrated a significant downregulation of the astrocyte-associated glycine transporter GlyT1, alongside a slight, non-significant increase in the neuronal presynaptic transporter GlyT2. These findings were further explored at the late symptomatic stage (P12) confirming the persistence of glycinergic alterations during disease progression. Finally, morphological studies of Renshaw cells (glycinergic interneurons crucial for recurrent inhibition) revealed early morphological alterations at P5, that became more evident at the late symptomatic stage (P12).

Together, these findings suggest an association between SMA and early glycinergic system dysfunction already at the initial stages of the disease, potentially affecting excitation/inhibition balance and contributing to MN degeneration. In conclusion, our results highlight the need to further investigate GCS expression/activity and glycinergic pathways to elucidate mitochondrial contributions to SMA pathogenesis and to uncover novel therapeutic opportunities. DnaJ heat shock protein family member C5 Glyceraldehyde-3-phosphate dehydrogenase Quantitative reverse transcription polymerase chain reaction Sodium dodecyl sulfate–polyacrylamide gel electrophoresis Non-phosphorylated neurofilament H antibody marker Ubiquinol-cytochrome c reductase core protein 2 We are deeply grateful to Prof.

Alain Prochiantz (Collège de France, Paris) for his insightful discussions and constructive suggestions, as well as for hosting AC for a short internship during her PhD. This work was supported by Girotondo Onlus and Smarathon Foundation to AV and MB, and by the Italian Ministry of University and Research (MUR) national project “Dipartimenti di Eccellenza 2023–2027”, awarded to the Department of Neuroscience “Rita Levi Montalcini” (University of Turin). This work was in partial fulfilment of the requirements for the doctorate degree of AC.

Department of Neuroscience “Rita Levi Montalcini”, University of Turin, Turin, Italy Anna Caretto, Ferdinando Di Cunto, Giovanna Menduti, Marina Boido & Alessandro Vercelli Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Orbassano, TO, Italy Center for Interdisciplinary Research in Biology, Collège de France, CNRS, INSERM, Université PSL, Paris, France European School of Molecular Medicine, University of Milan, Milan, Italy Laboratory of Translational Neuroscience, Ceinge Biotecnologie Avanzate, Naples, Italy Amber Hassan, Raffaella di Vito & Alessandro Usiello Department of Environmental, Biological and Pharmaceutical Science and Technologies, Università degli Studi della Campania “Luigi Vanvitelli”, Caserta, Italy Dept. Neuroscience, Neuroscience Institute Cavalieri Ottolenghi (NICO), Univ. Turin, Regione Gonzole 10, Orbassano, 10043, TO, Italy The authors declare no competing interests.