MORPHOLOGICAL AND SYNAPTIC CHANGES OF THE CORTICAL GABAERGIC INHIBITORY SYSTEM IN SPINAL MUSCULAR ATROPHY

Menduti G^1,2, Ferrini F^3,4, Caretto A^1,2, Hassan A^5,6, di Vito R^6,7, Beltrando G^1,2, Marnetto D², Usiello A^6,7, Di Cunto F^1,2, Boido M^1,2, and Vercelli A^1,2 | ¹Neuroscience Institute Cavalieri Ottolenghi, Orbassano, Turin, Italy; ²Department of Neuroscience «Rita Levi Montalcini», University of Turin, Italy; ³Department of Veterinary Sciences, University of Turin, Grugliasco, Turin, Italy; ⁴Department of Psychiatry and Neuroscience, Université Laval, Québec, Canada; ⁵European School of Molecular medicine, University of Milan, Italy; ⁶Laboratory of Translational Neuroscience, Ceinge Biotecnologie Avanzate, Naples, Italy; ⁷Department of Environmental, Biological and Pharmaceutical Science and Technologies, Università degli Studi della Campania Luigi Vanvitelli, Caserta, Italy

Spinal Muscular Atrophy (SMA) is a neuromuscular disease due to the lack of Survival Motor Neuron (SMN) protein, characterized by lower motor neuron (MN) degeneration and muscle atrophy. Moreover, evidence shows motor cortex (CRTX) morphofunctional abnormalities in SMA patients, suggesting altered maturation and maladaptive plasticity. Given limited access to patient samples, preclinical models are mandatory to clarify motor cortical involvement and underlying mechanisms in SMA pathogenesis. Building on our previous observation of upper MN vulnerability in SMA mice, we dissected cortical inhibitory GABAergic signalling, metabolism, and interneuron (IN) function in the sensorimotor (SM) CRTX and in primary neuron-astrocyte co-cultures from a severe SMA mouse model, compared to WT controls. Bioinformatic and biochemical analyses revealed stage-specific alterations in GABAergic pathways and metabolite profiles, particularly at late stage (P12), indicating a critical window of cortical network vulnerability. Imaging and molecular studies showed GABA+ neuron loss (-38%) and impaired GABA synthesis (GAD65/67). Moreover, confocal analysis revealed marked morphological alterations of parvalbumin (PV)+ INs in the SM CRTX of SMA mice, characterized by smaller somas and reduced dendritic dendritic complexity compared to controls. To determine whether these changes affected specific cortical layers, we quantified PV+ IN distribution and found a pronounced reduction in layer 5 of the motor CRTX (M1: -33.1%; S1: -25.7%). Consistently, the density of inhibitory synapses, assessed by gephyrin/GAD puncta, was markedly decreased in the SMA motor CRTX, particularly in layer 5 compared to WT controls (≤50%). Electrophysiological characterizations further confirmed decreased inhibitory neurotransmission onto layer 5 pyramidal neurons. Subsequent analyses showed that SMN loss impairs GABA release and reuptake by altering astrocytic transporter expression (SNAT5: -45%; GAT3: +29%), leading to reduced neuronal GABA and its accumulation in astrocytes. This imbalance leads to cortical GABAergic dysfunction, contributing to SMA pathology and highlighting SMN’s critical role in neurotransmitter regulation. Our data support a mechanistic model in which SMN deficiency impairs cortical GABAergic networks, expanding our understanding of cortical mechanisms in SMA and providing a framework for cortical-targeted therapeutic strategies.