Spinal muscular atrophy (SMA), the leading genetic cause of infant death, is a motor neuron disease (MND) caused by mutations or deletion of the survival motor neuron 1 (SMN1) gene, which codes for SMN protein. While SMN protein is ubiquitously expressed and crucial for the survival of all types of cells, motor neuron (MN) degeneration is the primary pathological result of SMN protein reduction. The origin of this selective vulnerability in SMA remains unsolved. In agreement with the large number of identified SMN binding partners, SMN has been linked to a vast number of cellular functions (e.g. splicing, transport and local translation of messenger ribonucleic acid (mRNA), endocytosis or autophagy), many of which impact protein homeostasis. The correct functionality of the mentioned housekeeping processes is critical for all cellular types, and thus it is puzzling why MNs are especially vulnerable to the reduction of SMN protein. The role that SMN plays in the regulation of the autophagy-lysosomal pathway (ALP), a major cellular degradative system, is not well studied. Recent studies have shown that SMN deficient cells display defects in the catabolic endosomal-autophagy pathway, leading to accumulation of autophagosomes (APs) and their undegraded cargo. The fact that APs form properly in SMN deficient cells, but are not correctly cleared from the cell, suggests a failure in the final step of the ALP, the AP degradation mediated by lysosomes. The main goal of this thesis was therefore to investigate the molecular mechanisms underlying the regulation of ALP by SMN, and whether and how alterations in this axis can result in the selective degeneration of MNs in SMA. To this end, MNs, derived from human induced pluripotent stem cell (hiPSC) lines, generated from patients affected by SMA and healthy individuals, have been used to uncover specific alterations in the ALP upon SMN reduction and the underlying molecular factors. Utilizing image-based experiments, I was able to discover that SMA MNs display a reduced number of lysosomes, compared to healthy MNs and isogenic controls, which leads to a defective AP-lysosome fusion. Interestingly, the remaining reduced pool of lysosomes in these SMA MNs exhibits an increased acidity, protease activity and axonal transport, none of which, seems to be sufficient to prevent MN loss. These findings demonstrate that SMN loss leads to a dysregulation of several key elements of the ALP, ultimately resulting in a reduced capacity of SMA MNs to degrade superfluous and potentially harmful material and to obtain essential building blocks from its recycling. To assess if the observed alterations in the ALP are specific to MNs or shared among other neuronal types that are typically not affected upon SMN reduction, I generated cortical neurons (CxNs) from the same hiPSCs and performed similar studies. These SMA CxNs did not show a reduction in the number of lysosomes or a change in their acidification status. Therefore, these findings indicate that the defective ALP upon SMN protein deficiency seems to be specific to spinal MNs and does not occur in all neuronal types. To explore the potential origin of the observed ALP abnormalities in SMA MNs, I focused on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy functionality. The main inhibitor of TFEB activity, the mammalian target of rapamycin (mTOR), has been previously shown to be over-activated in SMA, but whether SMN protein exerts any regulation over TFEB had not been explored before. During this thesis, I found that TFEB, and several of its downstream targets essential for autophagy function, are indeed dysregulated in SMA. The decline in the expression levels of several TFEB target genes that I have discovered in SMA MNs confirmed the reduced activity of TFEB in these cells. In addition, overexpression of TFEB in SMA MNs and in an in vivo SMA zebrafish model did ameliorate the reduced survival of MNs and axonal dystrophy characteristic of these models, respectively, further confirming TFEB as a potential key protein in the loss of protein homeostasis of SMA MNs. These results also strengthened the observed over-activation of mTOR as potential key link between SMN reduction, defective ALP and MN vulnerability, but the mechanistic origin of this abnormally active mTOR in SMA MNs is unknown. I was able to find a potential candidate for this link in a previously published RNA-sequencing dataset, namely the mTOR activating tumor protein, translationally-controlled 1 (TPT1). A previous study showed that TPT1 acts as a negative regulator of basal autophagy, through activation of mTOR. Intriguingly, TPT1 was over-represented in the mentioned dataset. Upon TPT1 knock down in SMN deficient cells, autophagy flux and MN survival was ameliorated, which suggests TPT1 as a promising candidate downstream of SMN loss to revert the lysosomal and autophagic defects identified in SMA MNs. The dysregulation of the ALP, including alterations in TFEB levels, has been linked to the appearance of toxic protein aggregates in many neurodegenerative diseases (NDs). I therefore wondered if the observed reduction in ALP functionality in SMA MNs could result in an overlooked aberrant protein aggregation phenotype, similar to other NDs. Indeed, I showed in this study that three commonly used markers for protein aggregation, p62 protein - an autophagy cargo that accumulates when autophagy does not function properly -, vimentin - an integral component of the aggresome structure - and Proteostat - a fluorescent dye that binds protein aggregates - were increased in SMA hMNs compared to healthy controls. Together, these findings show that SMA MNs selectively display an accumulation of undegraded material, including APs, likely due to a dysregulation of TFEB, which additionally leads to a reduction in the number of lysosomes per MN and therefore to a decreased proteostasis capacity. Additionally, clear signs of intracellular protein aggregation were observed in SMA MNs, which could further increase the vulnerability of these neurons. These phenomena seem to be specific to MNs as no similar decrease in survival or lysosomal defects were observed in SMA CxNs and could at least partially explain the observed selective vulnerability of spinal MNs in SMA patients. Collectively, the presented Ph.D. thesis demonstrates that SMN protein regulates the correct activity of the ALP, and that low SMN levels result in the dysfunction of this critical pathway, specifically in MNs. This study highlights the importance of this axis in the survival of MNs, and places it in the spotlight for further research aiming to improve MN health, not only in SMA but potentially as well for other MNDs.

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Objective: Activated astroglia is involved in the pathophysiology of neurodegenerative diseases and has also been described in animal models of spinal muscular atrophy (SMA). Given the urgent need of biomarkers for treatment monitoring of new RNA-modifying and gene replacement therapies in SMA, we examined glial fibrillary acidic protein concentrations in cerebrospinal fluid (cGFAP) as a marker of astrogliosis in SMA. - Methods: 58 adult patients and 21 children with genetically confirmed 5q-associated SMA from four German motor neuron disease specialist care centers and 30 age- and sex-matched controls were prospectively included in this study. cGFAP was measured and correlated to motor performance and disease severity. Additionally, we compared fL). - Results: cGFAP concentrations did not differ from controls but showed higher levels in more severely affected patients after adjustment for patients’ age. Normalized cNfL values were associated with disease severity. Within 14 months of nusinersen treatment, cGFAP concentrations did not change, while cNfL decreased significantly. - Interpretation: cGFAP is not an outstanding biomarker in SMA, but might support the hypothesis that glial activation is involved in SMA pathology. Unlike previously suggested, cNfL may be a promising biomarker also in adult patients with SMA, which should be subject to further investigations.

No description available.