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.