Master of Science / Department of Biology / Stella Y. Lee / CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Stella Yu-Chien Lee / Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative lysosomal storage disorders which is the most frequent group of inherited neurodegenerative disorders that affect children leading to severe pathological conditions such as progressive loss of motor neuron functions, loss of vision, mental retardation, epilepsy, ataxia and atrophy in cerebral, cerebella cortex and retina and eventually premature death. Among the many genes that cause NCL, mutations in CLN5 leads to different forms of NCL (infantile, late infantile, juvenile and adult) and mutations in CLN8 leads to progressive epilepsy with mental retardation (EPMR) and a variant late infantile form of NCL. The function(s) of both CLN5 and CLN8 proteins remain elusive. CLN5 is a glycosylated soluble protein that resides in the lysosome. We observed that endogenous CLN5 protein exist in two forms and identified a previously unknown C-terminal proteolytic processing event of CLN5. Using a cycloheximide chase experiment we demonstrated that the proteolytic processing of CLN5 is a post-translational modification. Furthermore treatment with chloroquine showed the processing occurs in low pH cellular compartments. After treatment with different protease inhibitors our results suggested the protease involved in the processing of CLN5 could be a cysteine protease. Using two glycosylation mutants of CLN5, retained in the endoplasmic reticulum (ER) or the Golgi we showed the proteolytic processing occurs in an organelle beyond the ER. This study contributes to understanding the characteristics of the CLN5 protein. CLN8 is an ER resident transmembrane protein that shuttles between the ER and the ER-Golgi intermediate compartment (ERGIC). In our study we identified a potential interaction between CLN8 and a PP2A holoenzyme complex consisting regulatory subunit A α isoform and regulatory subunit B α isoform. Using two CLN8 patient derived fibroblast cell lines we were able to show that the phosphorylated levels of PP2A target kinase Akt was reduced at both of its regulatory sites Ser473 and Thr308 and the activity of PP2A was increased. A delay of ceramide transport from ER to Golgi in CLN8 deficient patient cell lines was observed using BODIPY FL C5-Ceramide staining. Our results provide evidence for CLN8 protein being involved in the regulation of PP2A activity and trafficking of ceramide from ER to Golgi.

La thérapie chaperon représente une approche thérapeutique stratégique et innovante, en particulier dans le traitement des maladies lysosomales. Ces maladies génétiques rares ont une gravité variable, qui peut aller de la létalité avant la naissance jusqu’à la nécessité d‟une prise en charge permanente ; elles apparaissent à tous les stades de la vie. Des mimes du substrat appelé iminosucres, vont agir en allant au coeur du site actif de l’enzyme, stabiliser l’enzyme mutée qui est instable mais non inactive. Paradoxalement, la plupart des chaperons pharmacologiques sont des inhibiteurs de l’enzyme visée mais leur administration à faible concentration leur permet de réaliser leur mission de sauvetage de l’enzyme mutée. Dans cette optique, des recherches effectuées au sein de notre laboratoire ont fait état de la synthèse d’iminosucres, tels que les α-1-C-alkyl iminoxylitols qui sont de très bons inhibiteurs de la β-glucocérébrosidase, l’enzyme défaillante dans la maladie de Gaucher, mais aussi qui doublent l’activité enzymatique résiduelle. Une nouvelle voie de synthèse plus efficace a été réalisée afin d’obtenir plus efficacement ce type d’iminosucres et d’autres dérivés. Ces travaux ont également été l’occasion de développer des iminoxylitols structurellement simplifiés qui agissent comme chaperons pharmacologiques toujours pour le traitement de la maladie de Gaucher. Une partie de ces travaux a aussi été consacrée à la recherche d‟inhibiteurs de la β-galactocérébrosidase, l’enzyme impliquée dans la maladie de Krabbé, et qui pourront agir comme chaperons pharmacologiques. Différentes évaluations pharmacologiques ont été réalisées, notamment des tests d’inhibition et la détermination des effets chaperons. / Chaperone Mediated Therapy represents an innovative and strategic approach to treat lysosomal storage disorders which a class of rare genetic diseases. Competitive inhibitors for some of these lysosomal enzymes can, at sub inhibitory concentrations, act as chaperones and rescue the mutant proteins. In fact, enzymes carrying some mutations are still catalytically active. α-1-C-alkyl iminoxylitols represent a class of iminosugars which mimic the “gluco” configuration of the substrate and give powerful inhibitors of β-glucocerebrosidase, the enzyme involved in Gaucher disease. Moreover, this class of iminosugars, synthesized by our group, act as pharmacological chaperones and are able to double the residual activity of the N370S mutant. In order to synthesize more efficiently these iminosugars, the synthetic strategy was improved and optimized. Moreover, we focused our investigations on structural variations on our lead compound (α-1-C9 iminoxylitol) and draw important conclusions on structure-activity relationship. Then, we extended our expertise on iminosugars as pharmacological chaperones to another lysosomal glycosidase. In paricular, we targeted β-galactocerebrosidase, the enzyme responsible for Krabbe disease, and synthesized a series of iminosugars which mimic the “galacto” configuration. Biological assays were performed on our compounds to determine their activity as inhibitors and for some of them, their chaperone effects.

<p>GM2 gangliosidoses encompass a group of chronic neurodegenerative disorders characterized by metabolic defects in ganglioside catabolism and marked intralysosomal accumulation of GM2 in central nervous system (CNS)-resident neurons. Included in this group are Tay-Sachs and Sandhoff disease. Human cases of Tay-Sachs and Sandhoff disease present with devastating neurological deterioration; however, murine models display drastically divergent phenotypes. Tay-Sachs mice avoid pathology via a sialidase-mediated bypass of β-hexosaminidase A (HEXA) deficiency, though the precise mechanism of avoidance is not fully elucidated. The following work aimed to: i) determine if the murine sialidase-mediated bypass could be potentiated in human cells, and ii) help clarify the mechanism of disease avoidance in Tay-Sachs animals.</p> <p>Adenoviral overexpression of truncated CCAAT displacement protein (CDP^831-1505) in human Tay-Sachs neuroglia augmented neuraminidase 1/lysosomal sialidase (NEU1) protein levels, which reduced intralysosomal GM2 accumulations. Chromatin immunoprecipitation revealed binding of CDP^831-1505 to the human <em>NEU1</em> promoter in Tay-Sachs neuroglia. These results provide mechanistic and functional evidence supporting therapeutic exploitation of <em>NEU1</em> for Tay-Sachs disease.</p> <p>Comparison of immunological responses of bone marrow-derived macrophages (BMDMs) to pathogen associated molecular patterns (PAMPs) or GM2 demonstrated that Sandhoff macrophages secrete increased TNF and reduced IL-10 following lipopolysaccharide stimulation. GM2 treatment failed to stimulate an immune response. Such behaviour occurred in the absence of clearly observable intralysosomal ganglioside accumulations. Altered LAMP2 protein size, potentially due to aberrant glycosylation, is hypothesized to disrupt autophagosomal/lysosomal fusion. Subsequent autophagosomal accumulation could result in inherent macrophage hypersensitivity and immunologic irritability. Downstream interleukin-10 (IL-10)/signal transducer and activator of transcription 3 (Stat3) axis, mitogen activated protein kinase (MAPK), and glycogen synthase kinase 3-beta (GSK3β) signaling pathways were affected in Sandhoff BMDMs. These data indicate inherent differences in immunological responses of BMDMs from Sandhoff mice, presumably related to their β-hexosaminidase B (HEXB) deficiency.</p> <p>Data presented here provides evidence to suggest a paradigm shift in the neurodegenerative model of Tay-Sachs and Sandhoff Diseases towards one that places immune cells as an initiating factor for widespread neuroinflammation.</p> / Master of Science (MSc)

Les glycosidases sont des enzymes impliquées dans de nombreux processus biologiques. Entre autres, elles sont responsables de la dégradation des déchets polysaccharidiques de nos cellules. Lorsqu’une modification génétique touche un gène qui code pour une de ces enzymes, des pathologies graves regroupées sous l’appellation de « maladies lysosomales » peuvent être déclenchées. L'objectif de ce projet a été de proposer une méthode de synthèse efficace de molécules potentiellement actives spécifiquement sur l'une ou l'autre de ces maladies. Les molécules ciblées sont des inhibiteurs de glycosidases de la famille des aminocyclitols, utilisés dans une stratégie thérapeutique émergente « par molécules chaperonnes ». La méthode de synthèse développée s’appuie sur une étape enzymatique clé utilisant les aldolases comme catalyseurs et répondant aux contraintes environnementales actuelles de la chimie verte. Nous avons atteint nos objectifs grâce à l’utilisation de trois aldolases différentes, produites et purifiées pour la première fois au sein de notre laboratoire. Il s’agit de la fuculose-1-phosphate aldolase F1PA, de la rhamnulose-1-phosphate aldolase R1PA et de la nouvellement découverte fructose-6-phosphate aldolase FSA. La formation d’une quarantaine de nitrocyclitols, de stéréochimies définies, précurseurs des aminocyclitols correspondant, a ainsi été réalisée avec de très bons rendements de synthèse. / Glycosidases are enzymes involved in many biological processes. For example, they are responsible for breaking up polysaccharide waste materials of our cells. When a genetic mutation concerns a gene encoding for one of theses enzymes, acute pathologies named lysosomal storage disorders can appear. Aim of this work was to find an effective synthesis method of molecules potentially active specifically on one or others diseases. Target molecules are glycosidases inhibitors from the aminocyclitols family, used in an emergent strategy “by molecular chaperones”. The method of synthesis developed in the course of this work is based on an enzymatic key step using aldolases as catalyst, and follows current environment constraints of the green chemistry concept. Goals were reached thanks to the use of three different aldolases, produced and purified for the first time in our lab. It consists in fuculose-1-phosphate aldolase F1PA, rhamnulose-1-phosphate aldolase R1PA and the newly discovered fructose-6-phosphate aldolase FSA. Formation of around forty nitrocyclitols (aminocyclitols precursors) with a defined stereochemistry was realised with very good yields of synthesis.

Our research aims at understanding the roles of seminolipid (sulfogalactosylglycerolipid or SGG) and its associated membrane domains in male reproduction. SGG is a sulfoglycolipid present selectively and abundantly in mammalian male germ cells. Therefore, information on its properties would be relevant towards the development of male fertility biomarkers and spermicide-based contraceptives. We have shown that SGG has direct affinity for zona pellucida (ZP, egg extracellular matrix) and plays a role in the formation of sperm lipid rafts, the ZP-binding platforms on the sperm anterior head plasma membrane (APM), the initial ZP binding site. For a better understanding of mechanisms underlying sperm-ZP interaction, I performed proteomic characterization of APM vesicles (SGG-associated membrane domains with ZP affinity) isolated from sperm before and after capacitation, a process through which sperm gain maximal ZP affinity. Proteomic results revealed that capacitated APM vesicles contained high-molecular-weight protein complexes, with higher ZP affinity and levels of ZP-binding proteins as compared with those of the non-capacitated samples. ZP-binding proteins known to exist in the acrosome (i.e., zonadhesin, proacrosin/acrosin) were found in these APM protein complexes. Immunofluorescence suggested that a fraction of these proteins trafficked from the acrosome to APM during capacitation. These findings provided a new mechanism on how sperm gain full ZP-binding ability during capacitation. Since SGG is a major component of APM, proper SGG levels at this site would be important for male fertility. Levels of sperm SGG are regulated through the synthesis and degradation. In fact, lack of SGG-synthesis enzymes causes a spermatogenesis disruption, resulting in male infertility. However, significance of SGG degradation remains unknown. SGG can be desulfated in vitro by arylsulfatase A (ARSA), an enzyme existing in the acrosomes of sperm/spermatids and lysosomes of Sertoli cells, testicular somatic cells that nurture developing germ cells. Sertoli cells also phagocytose ~50% of germ cells that become apoptotic during spermatogenesis. To understand physiological importance of SGG degradation, the fertility status and SGG levels of Arsa-/- male mice were determined. We found that Arsa-/- males became subfertile when they were older than 5 months, and when they were 8-month-old (~40-year-old men) they produced sperm at 50% wild type rate. Arsa-/- sperm had minimal in vitro fertilizing ability and a number of them showed abnormal morphology. Quantitative mass spectrometry revealed that SGG levels in Sertoli cells of 8-month-old Arsa-/- mice were increased to ~250% of the wild type level; this SGG accumulation may lead to a decrease in Sertoli cell ability to support spermatogenesis. However, SGG levels in sperm of 8-month-old Arsa-/- mice were ~50% of the wild type value, a result that partly explained the decreased fertilizing ability of these sperm. The reduced SGG level of Arsa-/- sperm was likely due to a lack of SGG’s building-block lipid (palmitylpalmitoylglycerol) putatively generated in Arsa-/- Sertoli cells and recycled to the next generation of primary spermatocytes for SGG synthesis. Hence, levels of sperm SGG are a promising bioindex for male fertility. Since Sertoli cells also regulate SGG homeostasis, their functionality should be now included in male fertility/subfertility diagnosis.

Mucopolysaccharidosis type III (Sanfilippo) is comprised of four phenotypically similar lysosomal storage disorders (MPS IIIA-D) caused by the deficiency of enzymes that catabolise heparan sulphate (HS). Progressive accumulation of HS results in abnormal behaviour, progressive cognitive and motor impairment and death in mid-teens. There are currently no treatments for MPS III. To assess the effect of novel therapeutics in the mouse models of MPS III it is necessary to examine the effect on primary storage of HS, secondary storage and behaviour. The reported behaviour of MPS IIIA and B mice is conflicting therefore we developed a one-hour open field test, performed at the same time of day during a period of hyperactivity observed in a previous circadian rhythm study of MPS IIIB mice. At 8 months of age MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and a reduction in immobility time. The MPS IIIA mice presented with the same behavioural phenotype as the MPS IIIB mice and were significantly hyperactive at 4 and 6 months of age and also displayed a reduced sense of danger. The hyperactivity and reduced sense of danger observed in the mice is consistent with the patient phenotype. Whilst haematopoietic stem cell transplant (HSCT) is the standard therapy used to treat the similar HS storage disorder MPS I Hurler, it is ineffectual in MPS IIIA. We hypothesise that HSCT failure in MPS IIIA is due to insufficient enzyme production in the brain by donor-derived microglial cells. By increasing expression of N-sulphoglucosamine sulphohydrolase (SGSH) we may be able to treat MPS IIIA. Therefore we compared the effect of HSCT using normal haematopoietic stem cells (WT-HSCT) to lentiviral overexpression of SGSH in normal cells (LV-WT-HSCT) or MPS IIIA cells (LV-IIIA-HSCT) in MPS IIIA mice, using the behavioural tests developed.SGSH activity in the brain of MPS IIIA recipients was not significantly increased by WT-HSCT, but was significantly increased by LV-IIIA-HSCT and LV-WT-HSCT. HS was significantly reduced by all transplants but the best treatment was LV-WT-HSCT. Neuroinflammation, indicated by the number of microglia in the brain, was significantly reduced by all treatments but remains significantly elevated. GM2 gangliosides were significantly reduced by WT-HSCT and LV-WT-HSCT and were no longer significantly elevated, but LV-IIIA-HSCT had no significant effect. Critically LV-WT-HSCT corrected the behaviour at 4 and 6 months of age whilst the other treatments had no significant effect. LV-WT-HSCT and WT-HSCT reduced GM2 gangliosides and neuroinflammation equally but only LV-WT-HSCT corrected behaviour and primary HS storage, suggesting they are the important factors in MPS IIIA pathology. LV-WT-HSCT corrects the neurological phenotype in MPS IIIA mice and is a clinically viable approach to treat MPS IIIA and other neuropathic lysosomal storage disorders.

<p>  </p> <p>Niemann-Pick Type C (NPC) disease is a rare lysosomal storage disorder that occurs in about 1/89,000 to 1/120,000 live births and is characterized by an aberrant accumulation of cholesterol within the late endosome/lysosome of cells. Symptoms of this disease include splenomegaly, neurological deterioration, and often death before adulthood. Mutations in the membrane bound NPC1 or luminal NPC2 proteins lead to a decrease in cholesterol efflux within the lysosomes by which excess cholesterol crystallizes within membranes resulting in cell death. It has been demonstrated that increasing the amount of the lysosomal specific phospholipid Bis(monoacylglycerol)phosphate (BMP), also known as Lysobisphosphatidic acid (LBPA), in cells increases the rate of cholesterol transport in <em>npc1</em>-/- cells, but not in <em>npc</em>2-/- cells, indicating a strong synergistic relationship between the NPC2 protein and the lysosomal membranes. Increasing the amount of phosphatidyl glycerol (PG), a hypothesized precursor to BMP, has also shown an increase in cholesterol egress. While it is hypothesized that the increase in cholesterol clearance in the latter is due to the biosynthesis of LBPA from PG, there is no study to directly confirm this phenomenon. Therefore, we set out to synthesize diastereochemically pure PG containing isotopically labeled oleyl acyl chains to examine LBPA levels using lipidomic analysis of <em>npc1-/-</em> cells post treatment with PG. </p> <p>Initially, efforts centered around the use of phosphoramidite methodology commonly encountered in DNA oligonucleotide synthesis. While this route proved to be successful in making PG in modest yield (52%), reproducibility of this route with consistent yields was hindered due to the use of tetrabutylammonium fluoride (TBAF) in the final global deprotection step. Thus, we set out to discover a phosphorylated intermediate that did not require TBAF in the final step or contain easily hydrolysable protecting groups. It was discovered that H-phosphonate methodology using diphenyl phosphite for phosphorylation of the glycerol headgroup and backbone proved to be robust enough for PG synthesis. In this strategy, PG can be isolated in two steps from the final protected intermediate by first oxidizing the H-phosphonate from PIII to PV followed by deprotection of the glycerol head group under acidic conditions. Additionally, the H-phosphonate strategy also allowed us to omit headgroup modification prior to phosphorylation which reduced the number of synthetic steps from 11 steps to 7 steps. As a result, we were able to synthesize diastereochemically pure PG more consistently than the previous route in 75% yield. The route was further modified further to incorporate asymmetric acyl chains allowing the selective installation of a labeled acyl chain on the <em>sn</em>-1 or <em>sn</em>-2 positions of the phosphoglycerol backbone. The results from the lipidomic experiments indicate that increased LBPA concentrations in cells rise upon incubation with labeled PG. Additionally, increases in lyso-PG and acyl-PG are also observed leading to several hypotheses on how LBPA might be synthesized from PG. Future directions on this effort include identification of phospholipid species made from PG containing asymmetrically labeled acyl chains.  Synthesis of photoaffinity labeled PG is also underway to determine the protein partners involved in PG metabolism.</p>