Caratterizzazione delle alterazioni cellulari e strutturali di motoneuroni affetti da Atrofia Muscolare Spinale

Spinal muscular atrophy (SMA) is a paediatric genetic disease characterized by the loss of motor neurons (MNs) in brainstem and spinal cord, leading to progressive muscular weakness. SMA is caused by mutations of the SMN1 (Survival Motor Neuron 1) gene, that becomes ineffective in producing functional SMN protein, and is partially compensated by its homologous, the SMN2 gene. The pathology exists with different degrees of severity, from type I to type IV, related mainly with SMN2 copy number. Currently three SMN-dependent therapies are available for SMA patients. However, not all SMA patients take advantages from SMN-dependent therapies: in fact, they are particularly effective when administered in the earliest phase of the disease to severe SMA patients. Therefore, other strategies are required to face also the SMN-independent features of SMA pathology. In the last years, increasing evidence indicates that SMN protein deficiency, among other pathological features, could be related to the mitochondria impairment. Dysregulation in mitochondrial dynamics affects cellular functions and can be crucial in heritable diseases such as SMA. For these reasons, we decided to study mitochondria in vitro in cultured mouse embryonic fibroblasts (MEFs) from SMA mouse model (SMN2+/+; SMNΔ7+/+; Smn-/-), and ex vivo in lumbar spinal cord MNs, in order to deeply investigate the cellular alterations due to SMN lack, paying particular attention to mitochondrial impairment in early (postnatal day 5, P5) and late symptomatic (P10) disease stages. The present study has morphologically characterized MNs affected by SMA, pointing out subcellular alterations through ultrastructural transmission electron microscope (TEM) and following imaging analysis. SMA MNs revealed cytoplasm shrinkage and autophagic features. Intriguingly, electron-dense and clogging material into degenerating neurites was evident at P5 and no more detectable at P10; on the contrary, in this later stage autophagosome and autolysosome accumulation was seen at the MN soma level. This observations suggest that neurite degeneration may be the initial pathological mechanism that progressively leads to dysfunction in the cell soma. Concerning mitochondria alterations, our findings from MEF analysis underlined an increased mitochondrial network fragmentation in SMA compared to WT cells, together with a larger mitochondrial footprint, which suggested the presence of giant and swollen mitochondria. These preliminary results were further confirmed by ultrastructural analysis from spinal cord MNs: statistically significant discrepancies, regarding size, amount, and structural alterations, in mitochondria from SMA affected MNs compared to healthy controls were detected. Mitochondria in SMA models showed greater dimensions and increased density in MNs cytoplasm. Defects in their morphology and cristae organization, such as edema and cristae fragmentation, were also identified and quantified. These preliminary results suggest that mitochondria could represent a promising therapeutic target to weaken SMA symptoms, in combination with the currently available SMN-based therapies, or even a potential biomarker to follow disease progression.