Cell-imaging based analyses to identify new prognostic and predictive biomarker for Hereditary Spastic Paraplegia

Hereditary Spastic Paraplegia type 4 (HSP-SPG4) is a neurodegenerative disorder characterized by great heterogeneity, both in terms of clinical manifestations and genetic causes. HSP-SPG4 is caused by mutations in the SPG4 gene, which encodes spastin, a microtubule (MT)-severing ATPase. Spastin plays a crucial role in regulating MT dynamics to facilitate cytoskeletal reorganization, intracellular transport, and mitotic functions. Additionally, spastin is involved in the control of lipid droplet (LD) homeostasis. The primary aim of this thesis is to identify novel biomarkers for HSP-SPG4, by using an advanced imaging-based approach for the analysis of peripheral blood mononuclear cells (PBMCs) from patients. The first part of the study is dedicated to establishing the parameter “distance between the centroid of the nucleus and that of the cell (dcnc)” as a MT based biomarker, distinguishing SPG4 cells from healthy donor (HD) cells (Sardina et al., 2023). To generalize these results, we expanded the study to a well-characterized cohort of 48 SPG4 patients and 21 HD. The dcnc-method resulted able to distinguish SPG4 from HD cells independently of mutation type Pag. 7 and of disease severity, with a good diagnostic performance and accuracy. A detailed clinical characterisation was conducted for the cohort of 48 SPG4 patients, and correlation analyses were performed between dcnc and the aforementioned parameters. The strongest correlation was observed between dcnc and the disease progression (DP). Furthermore, the study explored the effects of spastin-elevating drugs, such as MLN4924, NSC1892, and valproic acid (VPA). These drugs significantly restore spastin levels in cells carrying truncating mutations associated with lack of spastin mutant expression and induce a recovery of MT organization defects. Notably, dcnc was shown able to monitor drug response, providing a potential tool to evaluate the effects of spastin-elevating drug in non-neuronal cells. Additionally, the study investigated whether different classes of spastin mutations, including missense mutations, respond differently to spastin-elevating drugs, providing preliminary evidence on the diverse responses to this type of therapeutic strategy. By cell-imaging, we also assessed whether other cellular components in addition to MT are affected by spastin mutations, such as LD. Despite the high variability observed, the parameter “number of LD in Pag. 8 each cell (nLD)” shows an enrichment of cells with a higher number of LD in HSP-SPG4 cells compared to HD cells. A multi-parametric analysis including parameters based on MT features (dcnc, fluorescence intensity, texture and shape) and on LD features (fluorescence intensity and nLD) confirms that dcnc is the best discriminative parameter for SPG4 cells among those analyzed. The second part of this thesis was conducted during my 6-month research period abroad in the laboratory of Dr. Barbara Ciani (Department of Chemistry) at the University of Sheffield, UK. We investigated how post-translational modifications regulate the ATPase activity of AAA-ATPase family proteins such as spastin. This hypothesis is based on evidence that post-translational modifications in the disordered linker between the N-terminal domains and the ATPase domain of AAA-ATPase family enzymes could control their protein levels and activity. Using in vitro biochemical approaches, the study analyzed the impact of phosphorylation in S268 on spastin’s enzymatic function, suggesting that phosphorylation in the linker stabilises the active form of the enzyme. The findings suggest that phosphorylation at key residues modulates spastin’s ATP hydrolysis, Pag. 9 providing new insights into the regulation of AAA ATPase family proteins. Overall, this thesis provides insights into spastin’s regulation and offers potential biomarkers, which could inform future approaches for HSP-SPG4.