This thesis focusses on the further unravelling of one of the mechanisms involved in developing Parkinson's disease: the GBA1 gene, encoding the lysosomal enzyme GCase. Several questions are... Show moreThis thesis focusses on the further unravelling of one of the mechanisms involved in developing Parkinson's disease: the GBA1 gene, encoding the lysosomal enzyme GCase. Several questions are addressed: How prevalent are mutations in this gene in the Netherlands and does it affect disease onset (chapter 2 and 5)? What methodological challenges accompany the sequencing of this gene (chapter 3 and 4)? What biomarkers may be used in clinical trials targeting GCase (chapter 6)? And what are the effects of the novel GCase activator LTI-291, when first administered to healthy volunteers (chapter 7) and to GBA-PD patients (chapter 8)? Show less
In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph... Show moreIn Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson's disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1-/- fish) and without GlcSph (gba1-/-:asah1b-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1-/-:asah1b-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons. Show less
Lysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A... Show moreLysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A common LSD is Gaucher disease, which is characterized by a defect in glucocerebrosidase (GCase) degrading glucosylceramide (GlcCer) in lysosomes. In this thesis, the zebrafish is evaluated as vertebrate animal model for the investigation of lysosomal storage disorders, in particular Gaucher disease. Zebrafish are an appealing model organism to study genetic disorders with a high evolutionary conservation of genes and proteins compared to humans, easy maintenance and simple genetic and pharmacological manipulation. Zebrafish larvae are of particular use as zebrafish can generate hundreds of off-spring which have a rapid embryonal development, are transparent and fit in a 96-wells plate. In this thesis several biochemical and genetic techniques have been developed in order to 1) compare the catalytic features of zebrafish GCase with human GCase, 2) investigate the consequences of its defect in zebrafish larvae and adults as well as a concomitant defect in non-lysosomal GBA2 and 3) study the potential toxicity of excessive glucosylsphingosine during GCase deficiency as consequence of a defect in lysosomal acid ceramidase. GCase-deficient zebrafish showed similar symptoms and affected molecular mechanisms as patients and mouse models. Therefore the zebrafish offers exciting new possibilities to study molecular mechanisms underlying pathological processes during lysosomal hydrolase deficiencies. Show less
This Thesis describes the design, synthesis and evaluation as glycoprocessing enzyme inhibitors of focused libraries of iminosugars. In the studies described, 1-deoxynojirimycin (DNJ), and its... Show moreThis Thesis describes the design, synthesis and evaluation as glycoprocessing enzyme inhibitors of focused libraries of iminosugars. In the studies described, 1-deoxynojirimycin (DNJ), and its known N-alkylated derivatives, served as starting points. DNJ modifications presented here include alteration of the substitution pattern of the piperidine core structure; variation in the N substituent, or a combination of the two. Biological evaluation of the synthesized compounds focused on the glycoprocessing enzymes involved in glucosylceramide metabolism: glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA1) and neutral glucosylceramidase (GBA2), and in all examples presented the inhibitory potency of newly synthesized compounds are compared with that of literature compounds. Show less
This thesis describes biochemical investigations of glucocerebrosidase (GBA), the lysosomal β- glucosidase that is deficient in Gaucher disease (GD). Central in the performed research was the... Show moreThis thesis describes biochemical investigations of glucocerebrosidase (GBA), the lysosomal β- glucosidase that is deficient in Gaucher disease (GD). Central in the performed research was the examination of factors influencing the intralysosomal stability and half-life of GBA. The investigations made use of new chemical biology tools such as activity based probes (ABPs) and photo-activatable and clickable (PAC) lipids.The Discussion reviews the present insights into GBA in health and disease. In this connection, the molecular basis and clinical manifestation of Gaucher disease and Action Myoclonus Renal Failure syndrome are discussed, including the metabolic adaptations to GBA deficiency. Particular attention is paid to the lysosomal structural stability of GBA and associated resistance against proteolytic degradation by cysteine cathepsins. Literature findings and novel own results on this topic are discussed. New technology to study GBA by labeling with GlcCer and cyclophellitol derived probes is introduced and the application is described. Unresolved research questions on GBA and related disease conditions are identified. As future research objective the translation of fundamental knowledge on GBA to effective therapy of neuronopathic Gaucher disease and other disease conditions caused by enzyme reduction are discussed. Show less
Moraitou, M.; Dermentzaki, G.; Dimitriou, E.; Monopolis, I.; Dekker, N.; Aerts, H.; ... ; Michelakakis, H. 2016
The exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective... Show moreThe exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective group in the solution-phase synthesis of (phosphorothioate) oligonucleotides. Pioneered by de Koning et al. a Solution-Phase Extraction Method (SPEM) amendable to scale-up was developed for the preparation of DNA and phosphorthioate oligonucleotide fragments. This approach utilizes extractive work-up procedures as the only tool in the isolation of oligonucleotides. Next, the design and synthesis of diamoniod decorated iminosugars as potential inhibitors of glycoside proccesing enzymes are described. Several libraries of natural iminosugars such as deoxynojirimycin and castanospermine decorated with an N-pentyloxy spacer were prepared. These iminosugar derivatives were evaluated as inhibitors of the enzymes involved in glucosylcramide metabolism, namely glucosylceramide synthase, beta-glucocerebrosidase and beta-glucosidase. Show less
