Lysosomes are acidic, hydrolase-rich vesicles capable of degrading most biological macromolecules. During the past several decades, much has been learned about different aspects of lysosome biogenesis. The selective phosphorylation of mannose residues on lysosomal enzymes, in conjunction with specific receptors for the mannose-6-phosphate recognition marker, has been found to be largely responsible for the targeting of newly synthesized lysosomal enzymes to lyzosomes. It is known that lysosomes receive input from both the endocytotic and biosynthetic pathways. Nevertheless the exact molecular mechanisms responsible for sorting of the biosynthetic imput involved in the lysosome biogenesis is still a matter of debate. Because osteoclast precursors do not secrete their lysosomal enzymes and osteoclasts do, the observation of modifications occuring during osteoclastogenesis is a good model to observe mechanisms responsible for lysosomal enzymes traffic. Osteoclasts are bone-degrading cells. To perform this specific task they have to reorganise the sorting of their lysosomal enzymes to be able to target them toward the bone surface in mature cells. Since few years, the differentiation of osteoclasts in vitro did help to study these cells. Osteoclast morphology has been therefore already well studied, and the nature of their specific membrane domains is now established. Sensing the proximity of a bone-like surface the cell reorganises its cytoskeleton, and creates specific membrane domains: an actin-rich ring-like zone (named actin ring) surrounded by highly ruffled membrane (named the ruffled border) where enzymes are secreted, while subsequent bone degradation products are endocytosed. Endocytosed material is then transported through the cell inside transcytotic vesicles and released at the top of the cell in an area named the functional secretory domain. Several molecular machineries are thought to control these different phenomena. The main purpose of this thesis was to identify the major regulators of lysosomal enzymes secretion and therefore to identify the molecular switches responsible for such a membrane traffic re-organisation.

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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In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...

Alzheimers sjukdom är den vanligaste orsaken till demens och i samband med att befolkningen blir större och allt äldre ökar även antalet patienter. Vid sjukdomen sker en hjärnatrofi och de mikroskopiska fynd man ser är extracellulära plack av β-amyloid, intracellulära neurofibriller av fosforylerat tau och förlust av nervcellsutskott, axoner, synapser och dendriter. Några av de tidiga patologiska förändringarna man kan se är störningar i nervcellernas lysosomala system som fyller en viktig roll vid nedbrytning av makromolekyler. I en tidigare studie har man påvisat förhöjda nivåer av proteiner från det lysosomala systemet i cerebrospinalvätska. Syftet med den här studien var att mäta nivåer av det lysosomala systemets proteiner i human hjärnvävnad från patienter med Alzheimer och jämföra dessa med kontrollprover. De sex proteiner som analyserades med Western blot var EEA1, PICALM, LAMP-1, LAMP-2, LC3 och TFEB. Resultaten visar på signifikant ökning i temporala cortex av LAMP-1 och LAMP-2 och en signifikant minskning av LC3 och EEA1 hos patienter med Alzheimers sjukdom. För att kunna dra riktiga slutsatser kring hur de ökade nivåerna i cerebrospinalvätska speglar de olika sjukdomsmekanismerna i hjärnan krävs vidare analyser av fler patientprover samt prover från andra områden i hjärnan. / Alzheimer's disease is the most common cause of dementia, and when the population becomes larger and older also the number of patients increase. A cerebral atrophy and microscopic findings of extracellular plaques of β-amyloid, intracellular neurofibrillary of phosphorylated tau and loss of nerve cell protrusions, axons, synapses and dendrites are seen during the disease. One of the early pathological changes is the disruption of the neuronal lysosomal network that plays an important role in the degradation of macromolecules. In a previous study elevated levels of proteins of the lysosomal network in cerebrospinal fluid from Alzheimer’s disease patients was demonstrated. The purpose of this study was to measure levels of the lysosomal network system in the brain. The six proteins EEA1, PICALM, LAMP-1, LAMP -2, LC3 and TFEB were analyzed in human brain tissue from five Alzheimer's disease cases and five control cases by Western blot. The results show a significant increase in the temporal cortex of LAMP-1 and LAMP -2 and a significant decrease of LC3 and EEA1 in patients with Alzheimer's disease. In order to draw proper conclusions about how the increased levels in cerebrospinal fluid reflect the different disease mechanisms in the brain it requires further analysis of more patient samples and from other areas of the brain.

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...

Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...

Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...