Farber disease is a fatal inherited disorder of lipid metabolism. It is characterized by a deficiency of the lysosomal enzyme acid ceramidase (AC), resulting in the buildup of the bio-effector molecule ceramide. A previous attempt to generate a Farber mouse model by Asah1 knockout was unsuccessful owing to embryonic lethality in homozygous embryos. Here, we successfully generated viable homozygotes that developed Farber disease by introducing a single-nucleotide human patient mutation into a conserved region of murine Asah1 (P361R). Indeed, homozygotes manifested Farber disease symptoms and died within 7-13 weeks. Furthermore, treatment of neonatal pups with intravenous injections of AC lentiviral vectors (LVs) yielded homozygotes with intermediate Farber phenotypes highlighted by reduced symptoms and increased longevity. Therefore, this first model of Farber disease can facilitate the advancement of experimental therapies and offer mechanistic insights into the integral roles of acid ceramidase, ceramide, and related sphingolipids in cell signaling, growth, and oncogenesis.

Farber disease is a fatal inherited disorder of lipid metabolism. It is characterized by a deficiency of the lysosomal enzyme acid ceramidase (AC), resulting in the buildup of the bio-effector molecule ceramide. A previous attempt to generate a Farber mouse model by Asah1 knockout was unsuccessful owing to embryonic lethality in homozygous embryos. Here, we successfully generated viable homozygotes that developed Farber disease by introducing a single-nucleotide human patient mutation into a conserved region of murine Asah1 (P361R). Indeed, homozygotes manifested Farber disease symptoms and died within 7-13 weeks. Furthermore, treatment of neonatal pups with intravenous injections of AC lentiviral vectors (LVs) yielded homozygotes with intermediate Farber phenotypes highlighted by reduced symptoms and increased longevity. Therefore, this first model of Farber disease can facilitate the advancement of experimental therapies and offer mechanistic insights into the integral roles of acid ceramidase, ceramide, and related sphingolipids in cell signaling, growth, and oncogenesis.

Lysosomal storage disorders (LSDs) are caused by defective lysosomal degradation of macromolecules resulting in accumulation of substrates in various tissues. This gradually leads to organ dysfunction and the classical clinical presentation with multisystem involvement. Historically the management of LSDs was confined to symptomatic treatment only. More recently other therapies have become available. Treatment options include cellular therapy in the form of Haemopoietic Stem Cell Transplant (HSCT), Enzyme Replacement Therapy (ERT), Substrate Reduction Therapy (SRT), Chaperone Mediated Therapy (CMT) and gene therapy. Whilst HSCT and ERT are established strategies in clinical practice for some LSDs, others are still in the development phase. The easy accessibility of ERT in the developed world (despite a high cost burden of approximately £144,000 per patient per annum in the UK), fewer risks associated with its administration and good metabolic and clinical outcome, have made ERT the treatment of choice for a number of LSDs. In recent years immune response has been identified as a significant factor in attenuating or nullifying the response to ERT. Despite recognition of this problem, there is a lack of reliable diagnostic tools to test and evaluate the antibody responses in the centres delivering ERT and far too little attention has been focused on development, optimisation and standardization of immune assays. In this project, IgG ELISA and two different functional enzyme inhibition assays (catalytic inhibition and cellular uptake inhibition) were developed and optimized. The immune response to ERT was then studied in recipients of ERT in MPSI, MPSVI and Pome disease. Our practice of delivering ERT in recipients of allogeneic HSCT prior to transplant provided us with an opportunity to study the immune response in MPSIH patients during ERT and following HSCT. We demonstrated functionally active antibodies in long term recipients of ERT in MPSI and Pompe disease. Allo-immune response in MPSVI did not inhibit the delivered enzyme therapy. A high titre inhibitory immune response was detected in the majority of MPSIH patients after exposure to ERT. This immune response was abrogated by allogeneic HSCT rendering these patients tolerant to replaced enzyme, confirming HSCT as an effective immune tolerance induction mechanism.

The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.

Iminosugars are known to behave as carbohydrate mimics in biological systems by virtue of their similar structures. However as the ring nitrogen prevents metabolism it means that iminosugars have the potential to become inhibitors of these systems. It is known, for example, that iminosugars can behave as mimics in the hydrolysis mechanism. This leads to possible medicinal applications of iminosugars. One such case is lysosomal storage disorders which arise as a result of a genetic defect which causes missense mutations coding for the N-acetylhexosaminidase enzymatic protein. N-Acetylhexosaminidases are a sub-member of the class of glycosidase enzymes. They are responsible for the cleavage of N-acetylhexosamine residues from glycoconjugates in the lysosome. Mutations in the gene coding for this protein lead to a deficiency in the enzymatic activity resulting in accumulation of unhydrolysed substrate in the lysosome. Lysosomal storage disorders have a phenotype of poor motor development and neurological problems. The infantile form usually leads to death before the age of five. An iminosugar mimic could give rise to a possible treatment for lysosomal storage disorders by acting as a molecular chaperone during protein folding, promoting correct folding by its intrinsic affinity for the native fold of the enzyme. Likewise in the treatment of cancer, the inhibitory ability of iminosugars has potential applications. In cancer, extracellular hydrolysis occurs which favours cancer cell survival. Macrophages, which attack and eliminate cancer cells, can be activated by macrophage activating factor (MAF) which displays an α-N-acetylgalactosamine residue that appears essential for the activation cascade. Cancer cells secrete an α-N-acetylgalactosaminidase enzyme that acts to decrease the potency of MAF, thus promoting cancer cell survival. Inhibition of cancer cell α-N-acetylgalactosaminidase may restore macrophage activation and generate potential therapeutics. Chapter 1 of this thesis contains extended discussion of the aforementioned, and related, diseases and the therapeutic applications of iminosugars. Some historically and biologically important iminosugars are described along with some current iminosugar drugs. Chapter 2 describes the synthetic strategies explored in an attempt to synthesise all the members of the 2-acetamido pyrrolidine iminosugars. An overview of the compounds synthesised towards this end by a past group member is given along with the work performed as part of this thesis to complete this goal. Both enantiomers with arabino- and ribo- stereochemistry and D-lyxo- were previously synthesised. The syntheses of both enantiomers with xylo- stereochemistry along with the L-lyxo- compound were completed as part of this thesis, from either D- or L-glucuronolactone and D-ribose, respectively. Chapter 3 details the synthetic strategy adopted to synthesise the enantiomer of D-DNJNAc, the first potent α-N-acetylgalactosaminidase inhibitor to be found. The synthesis towards another piperidine iminosugar, 6-deoxy DGJNAc, is presented in the second half of this chapter, along with two related compounds.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1(-/-) mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1(nmf164) for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1(nmf164)/Npc1(nmf164) pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1(nmf164) homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-beta-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs. These findings indicate that in Npc1(nmf164) homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases predominantly affecting infants and children, which in some cases can present into adulthood. There are fourteen genes comprising the 13 known subtypes of NCLs (CLN1-CLN8, CLN10-CLN14; CLN9 has been reclassified as CLN4). The NCL diseases share common molecular and clinical features, including cellular accumulation of autofluorescent storage material, characteristic histological findings (curvilinear inclusions, fingerprint profiles, and granular osmophilic deposits), markedly low brain weight, seizures, blindness, motor dysfunction and behavioral disabilities. Though the functions of the CLN proteins are not fully understood, they are mainly localized to the lysosomal compartment and autophagic pathway. Previous works have focused on understanding the individual functions of the CLN proteins. However, there is little research examining the interactions between CLN proteins and their involvement in neurogenesis. The CLN proteins also show involvement in various other signaling pathways, notably the mTOR and p53 pathways, and may therefore have implication as important signaling molecules during development and aging. In this thesis, I outline a variety of interactions between CLN proteins, as well as their role in lysosome formation and autophagy. I further examine the involvement of these proteins in lysosomes of microglia, and potential functions of microglia during neurogenesis in childhood and adulthood. I hypothesize that the CLN proteins are likely involved in a common pathway which is highly regulated during neurogenesis through microglial release of pro-inflammatory molecules. Though these diseases are incurable, enzyme replacement shows promise as a treatment for NCL; cerliponase alpha (BioMarin Pharmaceuticals) is the first and only FDA-approved enzyme replacement treatment for CLN2 disease. Future in-depth investigation of protein-protein interactions as well as their involvement in signaling pathways during development is necessary in order to find a cure for these devastating diseases.

No description available.

Les maladies lysosomales forment un groupe hétérogène d’une cinquantaine d’affections qualifiées de « rares ». Actuellement, seulement 9 maladies lysosomales disposent d’un traitement spécifique, principalement par enzymothérapie substitutive, mais les effets bénéfiques sont souvent limités. Le manque de ciblage pour le Récepteur du Mannose 6-Phosphate (RM6P), responsable de l’internalisation dans les lysosomes, expliquerait en partie cette efficacité modérée des enzymes thérapeutiques. Dans ce contexte, nous avons développé une approche de ciblage innovante basée sur des Analogues synthétiques du Mannose 6-Phosphate fonctionnalisés sur l’Aglycone (appelés AMFA) afin de répondre aux besoins non satisfaits par les traitements actuels.Les travaux de cette thèse portent principalement sur la maladie de Pompe, myopathie causée par la déficience d’une enzyme lysosomale, l’Alpha Glucosidase Acide (GAA), responsable de la conversion du glycogène en glucose. Afin d’améliorer l’adressage de l’enzyme thérapeutique aux lysosomes via le RM6P, nous avons fonctionnalisé la GAA recombinante humaine (rhGAA) avec les AMFA. Nos études sur la forme adulte de la maladie ont démontré une augmentation significative de l’internalisation et pour la première fois, chez des souris âgées modèles de la maladie, une restauration de la santé musculaire et une amélioration significative de la fonction motrice ont été observées (article 1). Nous nous sommes ensuite intéressés aux propriétés de la rhGAA-AMFA. Nous avons démontré que l’efficacité de la rhGAA-AMFA n’était pas uniquement due à une meilleure internalisation mais également à une meilleure maturation intracellulaire de l’enzyme (article 2). En effet, nos résultats ont démontré que chez les patients atteints de la maladie de Pompe, il existe une surexpression des phosphatases acides ACP2 et ACP5. Ces phosphatases peuvent détruire le signal mannose 6-phosphate (M6P) naturellement présent sur l’enzyme, ce qui interrompt sa maturation en forme active. L’AMFA, contrairement au M6P, est insensible à cette dégradation et assure donc la stabilité de l’adressage de l’enzyme in vitro, mais également in vivo.L’ensemble de ces résultats suggèrent que le greffage des AMFA sur des enzymes recombinantes représente une nouvelle solution thérapeutique pour le traitement de la maladie de Pompe et potentiellement pour le traitement d’autres maladies lysosomales. / Lysosomal diseases form a heterogeneous group of about fifty rare diseases. At present, only 9 lysosomal diseases have a specific treatment, mainly by enzyme replacement therapy but the beneficial effects appear often limited. The lack of targeting for the Mannose 6-Phosphate Receptor (M6PR), responsible for internalization into the lysosomes, would partly explain this moderate efficiency of the therapeutic enzymes. In this context, we have developed an innovative targeting approach based on Mannose 6-Phosphate Synthetic Analogues Functionalized at the Aglycone position (called AMFAs) to address the unmet needs of current treatments.The work of this thesis focuses mainly on Pompe disease which is a myopathy caused by the deficiency of a lysosomal enzyme, Acid Alpha Glucosidase (GAA), responsible for the conversion of glycogen into glucose. In order to improve the targeting of the therapeutic enzyme to lysosomes via the M6PR, we have functionalized the human recombinant GAA (rhGAA) with the AMFAs. Our studies on aged mice model of the adult form of the disease have demonstrated a significant increase of the enzyme internalization and for the first time, the restoration of muscle health and the significant improvement in motor function (article 1). We then investigated the properties of rhGAA-AMFA. We have proved that the effectiveness of rhGAA-AMFA is not only due to a better cell uptake but also to a more complete intracellular processing of the enzyme (article 2). Indeed, our results demonstrated that in myoblasts of patients affected by Pompe disease there is an overexpression of ACP2 and ACP5 acid phosphatases. These phosphatases can destroy the mannose 6-phosphate signal (M6P) naturally present on the enzyme, therefore possibly interrupting its processing into the active form. AMFA, unlike M6P, is insensitive to this degradation and thus ensures the stability of enzyme addressing in vitro, but also in vivo.All together, these results suggest that the grafting of the AMFAs on recombinant enzymes represents a new therapeutic solution for the treatment of Pompe disease and potentially for other lysosomal diseases.

Introdução: As Mucopolissacaridoses (MPS) são doenças lisossômicas causadas pela deficiência de enzimas envolvidas na degradação dos glicoaminoglicanos. O acúmulo anormal dessa molécula compromete a função celular e orgânica, levando a um espectro de manifestações clínicas, de caráter multissistêmico e progressivo. Manifestações gastrointestinais, tais como episódios frequentes de fezes amolecidas e diarréia, têm sido frequentemente evidenciadas e relatadas por pacientes com MPS. Ainda que os sintomas gastrointestinais sejam frequentemente ofuscados pelos fenótipos neurológicos graves, eles podem afetar a qualidade de vida dos pacientes e de seus familiares. Objetivo: Avaliar as manifestações gastrointestinais em pacientes com MPS que estavam ou não recebendo Terapia de Reposição Enzimática (TRE). Adicionalmente, para melhor compreender o assunto, avaliamos a histologia da mucosa intestinal em camundongos com MPS I. Métodos: Estudo transversal com amostragem de conveniência. Foram incluídos no estudo pacientes com diagnóstico de qualquer tipo de MPS que estavam ou não em TRE. Os sujeitos foram avaliados através de inquérito alimentar, questionamentos quanto ao aparecimento de sintomas gastrointestinais e realização de uma série de exames bioquímicos. Adicionalmente, foram realizados testes de bioimpedância elétrica (para avaliação da composição corporal) e teste molecular para hipolactasia primária a partir de amostras de DNA dos pacientes, assim como análise da histologia da mucosa intestinal em um modelo de camundongo com MPS I. Resultados: Foram incluídos 27 pacientes com diferentes tipos de MPS, sendo 15 (55,6%) do sexo feminino e 12 (44,4%) do sexo masculino, com mediana de idade de 12 (1-28) anos. Os sintomas gastrointestinais mais prevalentes foram flatulência, distensão abdominal, dor abdominal e fezes amolecidas. Houve diferença significativa na prevalência de flatulência entre os tipos de MPS (p=0,004). A prevalência de flatulência e de distensão abdominal foi significativamente maior no grupo dos que não recebiam TRE, em comparação ao grupo dos que recebia TRE (p = 0,04 e 0,03, respectivamente). A maioria dos exames bioquímicos realizados para investigação e/ou exclusão de alguma patologia específica apresentaram resultados normais, sugerindo que o aparecimento de sintomas gastrointestinais nesses pacientes possa ser decorrente da própria MPS através de mecanismo fisiopatológico independente. A análise da histologia do intestino delgado de camundongos MPS I identificou células aumentadas de volume, sugerindo algum tipo de acúmulo intracelular. Em relação ao Teste Molecular para Hipolactasia, 58,8% dos pacientes apresentaram genótipo CC, o qual é compatível com intolerância à lactose. Os exames de Bioimpedância Elétrica sugerem que, os pacientes em TRE apresentam menor proporção de massa gorda (MG) em relação aos pacientes que não estavam em TRE. Conclusão: Este foi o primeiro estudo sobre manifestações gastrointestinais de pacientes brasileiros com MPS realizado em nosso meio. Inúmeros sinais e sintomas foram observados, com maior prevalência relativa nas MPS I, II, III e IV do que na MPS VI. O sintoma mais frequentemente relatado foi flatulência. Embora não tenha sido estatisticamente significativo a diferença, a proporção de pacientes com sintomas gastrointestinais foi menor nos pacientes que estavam em TRE. A Intolerância à lactose observada em maior proporção nos pacientes com MPS com manifestações gastrointestinais, a avaliação da composição corporal e as alterações observadas na mucosa intestinal do camundongo com MPS I devem ser levadas em consideração na interpretação dos resultados. Acreditamos que mais estudos, dirigidos para as manifestações gastrointestinais, devam ser realizados para comprovar esses achados e melhor compreender os mecanismos fisiopatológicos dos sintomas relacionados nos pacientes aferidos. / Introduction: The mucopolysaccharidoses (MPS) are lysosomal storage disorders characterized by a deficiency in enzymes involved in glycosaminoglycan degradation. Abnormal accumulation of this molecule compromises cellular and organic function, leading to a spectrum of progressive, multisystem clinical manifestations. Gastrointestinal manifestations, such as frequent episodes of loose stools and diarrhea, are often reported by patients with MPS. Although these gastrointestinal symptoms are often overshadowed by severe neurological phenotypes, they can have a negative impact on the quality of life of patients and their family members. Objective: To assess gastrointestinal manifestations in patients with MPS who were or not receiving enzyme replacement therapy (ERT). Furthermore, we sought to assess bowel mucosa histology in a mouse model of MPS I. Methods: Cross-sectional study with a convenience sampling strategy. The sample comprised patients with a diagnosis of MPS of any type and regardless of ERT status. Patients were assessed by means of a dietary recordatory and an interview focused on gastrointestinal symptoms, as well as a battery of biochemical tests. Bioelectrical Impedance was performed for body composition assessment.Patient DNA samples were also tested by molecular tests for primary lactase hypolactasia, and bowel mucosa specimens from MPS I mice underwent histological examination. Results: A total of 27 MPS patients were included, 15 (55.6%) female and 12 (44.4%) male, with a median age of 12 (1-28) years. The most prevalent gastrointestinal symptoms were flatulence, abdominal distension, abdominal pain, and loose stools. A significant difference in the prevalence of flatulence was observed among different MPS types (p=0.004). The prevalence of flatulence and abdominal distension was significantly higher in the non-ERT group than in the ERT group (p = 0.04 and 0.03 respectively). Most biochemical tests performed to work up and/or rule out specific conditions were within normal limits, which suggests that the development of gastrointestinal systems in these patients may be due to MPS itself through an independent pathophysiological mechanism. Histological analysis of smallbowel tissue from MPS I mice found increased cell volume indicative of some form of intracellular accumulation. On molecular testing for lactase deficiency, 58.8% of patients had the CC genotype, which is consistent with lactose intolerance. Bioelectrical Impedance analysis suggest that patients with TRE have lower proportion of fat mass (FM) compared to patients who were not on ERT. Conclusion: This was the first study to assess gastrointestinal manifestations in Brazilian patients with different MPS subtypes.Several signs and symptoms were observed with higher relative prevalence in MPS I, II, III and IV to that in MPS VI. The most frequently reported symptom was flatulence. Although was no statistical difference the proportion of patients with gastrointestinal symptoms was lower in patients on ERT.The lactose intolerance observed in a higher proportion of MPS patients with gastrointestinal manifestations, body composition assessment and observed changes in the bowel mucosa of MPS I mice should be taken into account when interpreting the results. Further studies focusing on the gastrointestinal manifestations of MPS are warranted to corroborate our findings and provide a better understanding of the pathophysiological mechanisms associated with these symptoms in affected patients.