GBA1-Associated Parkinson's Disease Is a Distinct Entity.

GBA1-associated Parkinson's disease (GBA1-PD) is increasingly recognized as a distinct entity within the spectrum of parkinsonian disorders. This review explores the unique pathophysiological features, clinical progression, and genetic underpinnings that differentiate GBA1-PD from idiopathic Parkinson's disease (iPD). GBA1-PD typically presents with earlier onset and more rapid progression, with a poor response to standard PD medications. It is marked by pronounced cognitive impairment and a higher burden of non-motor symptoms compared to iPD. Additionally, patients with GBA1-PD often exhibit a broader distribution of Lewy bodies within the brain, accentuating neurodegenerative processes. The pathogenesis of GBA1-PD is closely associated with mutations in the GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase). In this review, we discuss two mechanisms by which GBA1 mutations contribute to disease development: 'haploinsufficiency,' where a single functional gene copy fails to produce a sufficient amount of GCase, and 'gain of function,' where the mutated GCase acquires harmful properties that directly impact cellular mechanisms for alpha-synuclein degradation, leading to alpha-synuclein aggregation and neuronal cell damage. Continued research is advancing our understanding of how these mechanisms contribute to the development and progression of GBA1-PD, with the 'gain of function' mechanism appearing to be the most plausible. This review also explores the implications of GBA1 mutations for therapeutic strategies, highlighting the need for early diagnosis and targeted interventions. Currently, small molecular chaperones have shown the most promising clinical results compared to other agents. This synthesis of clinical, pathological, and molecular aspects underscores the assertion that GBA1-PD is a distinct clinical and pathobiological PD phenotype, necessitating specific management and research approaches to better understand and treat this debilitating condition.

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons.

BACKGROUND: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). OBJECTIVE: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD. METHODS: In our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. RESULTS: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. CONCLUSIONS: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

State-of-the-Art Molecular Genetics and Genomics in Germany.

The Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany focuses on German researchers and their international peers, covering their recent advances in genetics, genomics, epigenetics, and cytogenetics/cytogenomics in relation to prokaryotic and eukaryotic multicellular to mammalian organisms in arras ranging from basic to medical research [...].

Curcumin Has Beneficial Effects on Lysosomal Alpha-Galactosidase: Potential Implications for the Cure of Fabry Disease.

Fabry disease is a lysosomal storage disease caused by mutations in the GLA gene that encodes alpha-galactosidase (AGAL). The disease causes abnormal globotriaosylceramide (Gb3) storage in the lysosomes. Variants responsible for the genotypic spectrum of Fabry disease include mutations that abolish enzymatic activity and those that cause protein instability. The latter can be successfully treated with small molecules that either bind and stabilize AGAL or indirectly improve its cellular activity. This paper describes the first attempt to reposition curcumin, a nutraceutical, to treat Fabry disease. We tested the efficacy of curcumin in a cell model and found an improvement in AGAL activity for 80% of the tested mutant genotypes (four out of five tested). The fold-increase was dependent on the mutant and ranged from 1.4 to 2.2. We produced evidence that supports a co-chaperone role for curcumin when administered with AGAL pharmacological chaperones (1-deoxygalactonojirimycin and galactose). The combined treatment with curcumin and either pharmacological chaperone was beneficial for four out of five tested mutants and showed fold-increases ranging from 1.1 to 2.3 for DGJ and from 1.1 to 2.8 for galactose. Finally, we tested a long-term treatment on one mutant (L300F) and detected an improvement in Gb3 clearance and lysosomal markers (LAMP-1 and GAA). Altogether, our findings confirmed the necessity of personalized therapies for Fabry patients and paved the way to further studies and trials of treatments for Fabry disease.

Abnormal Pre-mRNA Splicing in Exonic Fabry Disease-Causing GLA Mutations.

Fabry disease (FD) is a rare X-linked disease due to a multiverse of disrupting mutations within the GLA gene encoding lysosomal α-galactosidase A (AGAL). Absent AGAL activity causes the accumulation of complex glycosphingolipids inside of lysosomes in a variety of cell types and results in a progressive multisystem disease. Known disease-associated point mutations in protein-coding gene regions usually cause translational perturbations and result in premature chain termination, punctual amino acid sequence alterations or overall altered sequence alterations downstream of the mutation site. However, nucleotide exchanges at the border between introns and exons can affect splicing behavior and lead to abnormal pre-mRNA processing. Prediction with the Human Splicing Finder (HSF) revealed an indication of a significant change in splicing-relevant information for some known FD-associated GLA mutations. To experimentally determine the extent of the change, we made use of a minigene reporter assay and verified alternative splicing events for the exonic mutations c.194G>T and c.358C>G, which led to the usage of alternative donor splice sites at exon 1 and exon 2, respectively. In addition, the mutations c.548G>T and c.638A>T led to significant exon 4 skipping. We conclude that splicing phenotype analysis should be employed in the in vitro analysis of exonic GLA gene mutations, since abnormal splicing may result in a reduction of enzyme activity and alter the amenability for treatment with pharmacological chaperone (PC).

Drug Repositioning for Fabry Disease: Acetylsalicylic Acid Potentiates the Stabilization of Lysosomal Alpha-Galactosidase by Pharmacological Chaperones.

Fabry disease is caused by a deficiency of lysosomal alpha galactosidase and has a very large genotypic and phenotypic spectrum. Some patients who carry hypomorphic mutations can benefit from oral therapy with a pharmacological chaperone. The drug requires a very precise regimen because it is a reversible inhibitor of alpha-galactosidase. We looked for molecules that can potentiate this pharmacological chaperone, among drugs that have already been approved for other diseases. We tested candidate molecules in fibroblasts derived from a patient carrying a large deletion in the gene GLA, which were stably transfected with a plasmid expressing hypomorphic mutants. In our cell model, three drugs were able to potentiate the action of the pharmacological chaperone. We focused our attention on one of them, acetylsalicylic acid. We expect that acetylsalicylic acid can be used in synergy with the Fabry disease pharmacological chaperone and prolong its stabilizing effect on alpha-galactosidase.

Mechanistic Insight into the Mode of Action of Acid ß-Glucosidase Enhancer Ambroxol.

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid ß-glucosidase (ß-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on ß-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits ß-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant ß-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated ß-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant ß-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional ß-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize ß-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.

Proteostasis regulators modulate proteasomal activity and gene expression to attenuate multiple phenotypes in Fabry disease.

The lysosomal storage disorder Fabry disease is characterized by a deficiency of the lysosomal enzyme α-Galactosidase A. The observation that missense variants in the encoding GLA gene often lead to structural destabilization, endoplasmic reticulum retention and proteasomal degradation of the misfolded, but otherwise catalytically functional enzyme has resulted in the exploration of alternative therapeutic approaches. In this context, we have investigated proteostasis regulators (PRs) for their potential to increase cellular enzyme activity, and to reduce the disease-specific accumulation of the biomarker globotriaosylsphingosine in patient-derived cell culture. The PRs also acted synergistically with the clinically approved 1-deoxygalactonojirimycine, demonstrating the potential of combination treatment in a therapeutic application. Extensive characterization of the effective PRs revealed inhibition of the proteasome and elevation of GLA gene expression as paramount effects. Further analysis of transcriptional patterns of the PRs exposed a variety of genes involved in proteostasis as potential modulators. We propose that addressing proteostasis is an effective approach to discover new therapeutic targets for diseases involving folding and trafficking-deficient protein mutants.

Misfolding of Lysosomal α-Galactosidase a in a Fly Model and Its Alleviation by the Pharmacological Chaperone Migalastat.

Fabry disease, an X-linked recessive lysosomal disease, results from mutations in the GLA gene encoding lysosomal α-galactosidase A (α-Gal A). Due to these mutations, there is accumulation of globotriaosylceramide (GL-3) in plasma and in a wide range of cells throughout the body. Like other lysosomal enzymes, α-Gal A is synthesized on endoplasmic reticulum (ER) bound polyribosomes, and upon entry into the ER it undergoes glycosylation and folding. It was previously suggested that α-Gal A variants are recognized as misfolded in the ER and undergo ER-associated degradation (ERAD). In the present study, we used Drosophila melanogaster to model misfolding of α-Gal A mutants. We did so by creating transgenic flies expressing mutant α-Gal A variants and assessing development of ER stress, activation of the ER stress response and their relief with a known α-Gal A chaperone, migalastat. Our results showed that the A156V and the A285D α-Gal A mutants underwent ER retention, which led to activation of unfolded protein response (UPR) and ERAD. UPR could be alleviated by migalastat. When expressed in the fly's dopaminergic cells, misfolding of α-Gal A and UPR activation led to death of these cells and to a shorter life span, which could be improved, in a mutation-dependent manner, by migalastat.

Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells.

Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer's disease and Parkinson's disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

Pharmacological Chaperones: A Therapeutic Approach for Diseases Caused by Destabilizing Missense Mutations.

The term "pharmacological chaperone" was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.

Assessment of Gene Variant Amenability for Pharmacological Chaperone Therapy with 1-Deoxygalactonojirimycin in Fabry Disease.

Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal α-galactosidase A (α-Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the α-galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of ≥3% of wild type enzyme activity and a relative increase in enzyme activity of ≥1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own α-galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.

Dose-Dependent Effect of Enzyme Replacement Therapy on Neutralizing Antidrug Antibody Titers and Clinical Outcome in Patients with Fabry Disease.

BACKGROUND: Use of enzyme replacement therapy (ERT) to treat Fabry disease, caused by deficient lysosomal α-galactosidase A activity, can lead to formation of neutralizing antidrug antibodies (ADAs). These antibodies are associated with increased accumulation of plasma globotriaosylceramide (Gb3) and disease progression. Because agalsidase ERT can saturate ADA-binding sites during infusions (achieving agalsidase/antibody equilibrium), we investigated in this open cohort study whether saturated patients (who have excess agalsidase after infusions) experience better clinical outcomes compared with not saturated patients (who have excess ADAs after infusions). METHODS: We isolated ADAs from sera of 26 men with Fabry disease receiving ERT (for a median of 94 months) and determined the amount of agalsidase necessary for antibody saturation. Clinical and biochemical outcomes included measurements of eGFR, interventricular septum thickness, and lyso-Gb3. RESULTS: ADA titers decreased significantly in all patients during infusion. Agalsidase-α and agalsidase-ß had similar ADA-binding capacity and comparable ADA saturation frequency. Fourteen patients with saturated ADAs presented with mild (but significant) loss of eGFR, stable septum thickness, and significantly decreased lyso-Gb3 levels. The 12 not saturated patients had a more pronounced and significant loss of eGFR, increased septum thickness, and a smaller, nonsignificant reduction in lyso-Gb3, over time. In three patients, dose escalation resulted in partially elevated ADA titers, but importantly, also in reduced lyso-Gb3 levels. CONCLUSIONS: A not saturated ADA status during infusion is associated with progressive loss of eGFR and ongoing cardiac hypertrophy. Dose escalation can result in saturation of ADAs and decreasing lyso-Gb3 levels, but may lead to increased ADA titers.

Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test.

Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.

Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen-substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH-EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.-Bohl, C., Pomorski, A., Seemann, S., Knospe, A.-M., Zheng, C., Krezel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

ADAM23 promotes neuronal differentiation of human neural progenitor cells.

BACKGROUND: ADAM23 is widely expressed in the embryonic central nervous system and plays an important role in tissue formation. RESULTS: In this study, we showed that ADAM23 contributes to cell survival and is involved in neuronal differentiation during the differentiation of human neural progenitor cells (hNPCs). Upregulation of ADAM23 in hNPCs was found to increase the number of neurons and the length of neurite, while its downregulation decreases them and triggers cell apoptosis. RNA microarray analysis revealed mechanistic insights into genes and pathways that may become involved in multiple cellular processes upon up- or downregulation of ADAM23. CONCLUSIONS: Our results suggest that ADAM23 regulates neuronal differentiation by triggering specific signaling pathways during hNPC differentiation.

Glucosylsphingosine Causes Hematological and Visceral Changes in Mice-Evidence for a Pathophysiological Role in Gaucher Disease.

Glucosylceramide and glucosylsphingosine are the two major storage products in Gaucher disease (GD), an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The build-up of glucosylceramide in the endoplasmic reticulum and prominent accumulation in cell lysosomes of tissue macrophages results in decreased blood cell and platelet counts, and skeletal abnormalities. The pathological role of the deacylated form of glucosylceramide, glucosylsphingosine (lyso-Gb1), a recently identified sensitive and specific biomarker for GD, is not well investigated. We established a long-term infusion model in C57BL/6JRj mice to examine the effect of lyso-Gb1 on representative hallmark parameters of GD. Mice received lyso-Gb1 at a dosage of 10 mg·kg-1 per day as a continuous subcutaneous administration, and were routinely checked for blood lyso-Gb1 levels using liquid chromatography-multiple reaction monitoring mass spectrometry (LC/MRM-MS) measurements at four-weekly intervals throughout treatment. The C57BL/6JRj mice showed a stable increase of lyso-Gb1 up to->500-fold greater than the normal reflecting concentrations seen in moderately to severely affected patients. Furthermore, lyso-Gb1 accumulated in peripheral tissues. The mice developed hematological symptoms such as reduced hemoglobin and hematocrit, increased spleen weights and a slight inflammatory tissue response after eight weeks of treatment. The above findings indicate a measurable visceral and hematological response in treated mice that suggests a role for lyso-Gb1 in the development of peripheral signs of GD.

Functional and Clinical Consequences of Novel α-Galactosidase A Mutations in Fabry Disease.

Fabry disease (FD) is a rare metabolic disorder of glycosphingolipid storage caused by mutations in the GLA gene encoding lysosomal hydrolase α-galactosidase A (α-gal A). Recently, the diagnostic procedure for FD has advanced in several ways, through the development of a specific biomarker (lyso-Gb3) and the implementation of newborn screenings, which acted as a catalyst to augment general awareness of the disease. Heterologous over-expression of α-gal A variants and subsequent in vitro measurement of enzyme activity provided molecular data to elucidate the relationship between mutation, enzyme damage, lyso-Gb3 biomarker levels, and clinical phenotype. This knowledge is the foundation for improved counseling with regard to prognosis and therapeutic decisions. Herein, we resume the approach of in vitro characterization, with a further 73 mainly novel GLA gene mutations. Patient lyso-Gb3 data were available for most of the mutations. All mutations were tested for responsiveness to pharmacological chaperone treatment and phenotypic data for 61 hemizygous male and 116 heterozygous female patients carrying a mutation associated with ≥ 20% residual activity, formerly classified as "mild" variant, were collected in order to evaluate the pathogenicity. We conclude that a mild GLA variant is typically characterized by high residual enzyme activity and normal biomarker levels. We found evidence that these variants can still be classified as a distinctive, but milder, sub-type of FD.

Enzyme enhancers for the treatment of Fabry and Pompe disease.

Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies.

Functional characterisation of alpha-galactosidase a mutations as a basis for a new classification system in fabry disease.

Fabry disease (FD) is an X-linked hereditary defect of glycosphingolipid storage caused by mutations in the gene encoding the lysosomal hydrolase α-galactosidase A (GLA, α-gal A). To date, over 400 mutations causing amino acid substitutions have been described. Most of these mutations are related to the classical Fabry phenotype. Generally in lysosomal storage disorders a reliable genotype/phenotype correlation is difficult to achieve, especially in FD with its X-linked mode of inheritance. In order to predict the metabolic consequence of a given mutation, we combined in vitro enzyme activity with in vivo biomarker data. Furthermore, we used the pharmacological chaperone (PC) 1-deoxygalactonojirimycin (DGJ) as a tool to analyse the influence of individual mutations on subcellular organelle-trafficking and stability. We analysed a significant number of mutations and correlated the obtained properties to the clinical manifestation related to the mutation in order to improve our knowledge of the identity of functional relevant amino acids. Additionally, we illustrate the consequences of different mutations on plasma lyso-globotriaosylsphingosine (lyso-Gb3) accumulation in the patients' plasma, a biomarker proven to reflect the impaired substrate clearance caused by specific mutations. The established system enables us to provide information for the clinical relevance of PC therapy for a given mutant. Finally, in order to generate reliable predictions of mutant GLA defects we compared the different data sets to reveal the most coherent system to reflect the clinical situation.