Induced pluripotent stem cell model recapitulates pathologic hallmarks of Gaucher disease.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ß-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.

Alpha-synuclein interacts with Glucocerebrosidase providing a molecular link between Parkinson and Gaucher diseases.

The presynaptic protein α-synuclein (α-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between α-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that α-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the α-syn C-terminal residues, 118-137. This α-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 µm in the presence of 25 to 100 mm NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for α-syn, as does the inhibitor conduritol-ß-epoxide-bound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the α-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.

Beta-glucosidase 1 (GBA1) is a second bile acid ß-glucosidase in addition to ß-glucosidase 2 (GBA2). Study in ß-glucosidase deficient mice and humans.

Beta-glucosidase 1 (GBA1; lysosomal glucocerebrosidase) and ß-glucosidase 2 (GBA2, non-lysosomal glucocerebrosidase) both have glucosylceramide as a main natural substrate. The enzyme-deficient conditions with glucosylceramide accumulation are Gaucher disease (GBA-/- in humans), modelled by the Gba-/- mouse, and the syndrome with male infertility in the Gba2-/- mouse, respectively. Before the leading role of glucosylceramide was recognised for both deficient conditions, bile acid-3-O-ß-glucoside (BG), another natural substrate, was viewed as the main substrate of GBA2. Given that GBA2 hydrolyses both BG and glucosylceramide, it was asked whether vice versa GBA1 hydrolyses both glucosylceramide and BG. Here we show that GBA1 also hydrolyses BG. We compared the residual BG hydrolysing activities in the GBA1-/-, Gba1-/- conditions (where GBA2 is the almost only active ß-glucosidase) and those in the Gba2-/- condition (GBA1 active), with wild-type activities, but we used also the GBA1 inhibitor isofagomine. GBA1 and GBA2 activities had characteristic differences between the studied fibroblast, liver and brain samples. Independently, the hydrolysis of BG by pure recombinant GBA1 was shown. The fact that both GBA1 and GBA2 are glucocerebrosidases as well as bile acid ß-glucosidases raises the question, why lysosomal accumulation of glucosylceramide in GBA1 deficiency, and extra-lysosomal accumulation in GBA2 deficiency, are not associated with an accumulation of BG in either condition.

The role of saposin C in Gaucher disease.

Saposin C is one of four homologous proteins derived from sequential cleavage of the saposin precursor protein, prosaposin. It is an essential activator for glucocerebrosidase, the enzyme deficient in Gaucher disease. Gaucher disease is a rare autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene that exhibits vast phenotypic heterogeneity, despite its designation as a "simple" Mendelian disorder. The observed phenotypic variability has led to a search for disease modifiers that can alter the Gaucher phenotype. The PSAP gene encoding saposin C is a prime candidate modifier for Gaucher disease. In humans, saposin C deficiency due to mutations in PSAP results in a Gaucher-like phenotype, despite normal in vitro glucocerebrosidase activity. Saposin C deficiency has also been shown to modify phenotype in one mouse model of Gaucher disease. The role of saposin C as an activator required for normal glucocerebrosidase function, and the consequences of saposin C deficiency are described, and are being explored as potential modifying factors in patients with Gaucher disease.

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide.

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.

A mutation in SCARB2 is a modifier in Gaucher disease.

Lysosomal integral membrane protein type 2 (LIMP-2) is responsible for proper sorting and lysosomal targeting of glucocerebrosidase, the enzyme deficient in Gaucher disease (GD). Mutations in the gene for LIMP-2, SCARB2, are implicated in inherited forms of myoclonic epilepsy, and myoclonic epilepsy is part of the phenotypic spectrum associated with GD. We investigated whether SCARB2 mutations impact the Gaucher phenotype focusing on patients with myoclonic epilepsy, including a pair of siblings with GD who were discordant for myoclonic seizures. Sequencing of SCARB2 genomic and cDNA identified a heterozygous, maternally inherited novel mutation, c.1412A>G (p.Glu471Gly), in the brother with GD and myoclonic epilepsy, absent from his sibling and controls. Glucocerebrosidase activity, Western blots, real-time PCR, and immunofluorescence studies demonstrated markedly decreased LIMP-2 and glucocerebrosidase in cells from the sibling with (p.Glu471Gly) LIMP-2, and diminished glucocerebrosidase in lysosomes. The cells secreted highly glycosylated enzyme and showed mistrafficking of glucocerebrosidase. Sequencing of SCARB2 in 13 other subjects with GD and myoclonic epilepsy and 40 controls failed to identify additional mutations. The study provides further evidence for the association of LIMP-2 and myoclonic epilepsy, explains the drastically different phenotypes encountered in the siblings, and demonstrates that LIMP-2 can serve as a modifier in GD.

Mucolipidosis type IV: an update.

Mucolipidosis type IV (MLIV) is a neurodevelopmental as well as neurodegenerative disorder with severe psychomotor developmental delay, progressive visual impairment, and achlorydria. It is characterized by the presence of lysosomal inclusions in many cell types in patients. MLIV is an autosomal recessive disease caused by mutations in MCOLN1, which encodes for mucolipin-1, a member of the transient receptor potential (TRP) cation channel family. Although approximately 70-80% of patients identified are Ashkenazi Jewish, MLIV is a pan-ethnic disorder. Importantly, while MLIV is thought to be a rare disease, its frequency may be greater than currently appreciated, for its common presentation as a cerebral palsy-like encephalopathy can lead to misdiagnosis. Moreover, patients with milder variants are often not recognized as having MLIV. This review provides an update on the ethnic distribution, clinical manifestations, laboratory findings, methods of diagnosis, molecular genetics, differential diagnosis, and treatment of patients with MLIV. An enhanced awareness of the manifestations of this disorder may help to elucidate the true frequency and range of symptoms associated with MLIV, providing insight into the pathogenesis of this multi-system disease.

Aggregation of α-synuclein in brain samples from subjects with glucocerebrosidase mutations.

Recent studies show an increased frequency of mutations in the glucocerebrosidase gene (GBA1) in patients with α-synucleinopathies including Parkinson disease. Some patients with Gaucher disease (GD) develop parkinsonism with α-synuclein-positive inclusions post mortem. Proteins were extracted from the cerebral cortex of subjects with synucleinopathies with and without GBA1 mutations, controls and patients with GD. Patients with GBA1-associated synucleinopathies showed aggregation of oligomeric forms of α-synuclein in the SDS-soluble fraction, while only monomeric forms of α-synuclein were seen in subjects with GBA1 mutations without parkinsonism. Thus, brains from patients with GBA1-associated parkinsonism show biochemical characteristics typical of Lewy body disorders.

Gaucher disease and parkinsonism, a molecular link theory.

Mutant GBA was found recently to be the most prevalent risk factor for familial parkinsonism. The two diseases do not share common symptoms and there is no direct pathway to explain the mechanism by which GBA mutations can confer the risk. Increased burden on the degradative pathway caused by defective glucocerebrosidase, or toxic side effects of glycosylated lipids accumulation were proposed to explain brain damage. Both hypotheses are not sufficient to explain the linkage. In order to develop a more inclusive theory we introduced into the model the prion theory and the second hit. Other possibilities are also brought into consideration.

A new resorufin-based alpha-glucosidase assay for high-throughput screening.

Mutations in alpha-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, alpha-glucosidase is a therapeutic target for type II diabetes, and alpha-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the alpha-glucosidase enzyme assay, resorufin alpha-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-alpha-D-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK(a) of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the alpha-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.

Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase.

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in alpha-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl alpha-D-galactopyranoside, for a new alpha-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK(a) of resorufin (approximately 6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.

Apoptotic abnormalities in differential gene expression in peripheral blood mononuclear cells from children with Fabry disease.

AIM: This study was designed to examine the effect of enzyme replacement therapy (ERT) on differential gene expression in peripheral blood mononuclear cells (PBMCs) of children with Fabry disease who had not previously been exposed to ERT. METHODS: Thirteen children with Fabry disease (age range, 6.5-17.0 years) were studied as part of a 6-month, open-label study of ERT with agalsidase alfa. Paired blood samples were taken at the start of the study and after 6 months of ERT. Further blood samples were also taken from 16 age-matched control subjects. PBMCs were isolated and, following RNA extraction, differential gene expression analysis was performed using the Human Genome U133 Plus 2.0 microarray. RESULTS: Twenty-one genes were determined to be differentially expressed in PBMCs of ERT-naïve children with Fabry disease compared with healthy controls; neuronal apoptosis inhibitory protein ranked as the most significantly differentially expressed gene. Comparison of gene expression in children with Fabry disease prior to and after ERT showed that two genes were significantly differentially expressed (p < or = 0.05) following treatment; the expressed sequence tag (probe set ID, 243259_at) was downregulated, while expression of apoptosis-inducing factor was increased, possibly as an antioxidant counter-regulatory response. CONCLUSION: This study identifies a number of genes that are differentially expressed in a small cohort of children with Fabry disease relative to healthy controls. These genes may relate to the underlying biological abnormalities in Fabry disease.

Using peripheral blood mononuclear cells to determine a gene expression profile of acute ischemic stroke: a pilot investigation.

BACKGROUND: Direct brain biopsy is rarely indicated during acute stroke. This study uses peripheral blood mononuclear cells (PBMCs) to determine whether a systemic gene expression profile could be demonstrated in patients with acute ischemic stroke. METHODS AND RESULTS: Using oligonucleotide microarrays, we compared the gene expression profile of an index cohort of 20 patients with confirmed ischemic stroke on neuroimaging studies with that of 20 referent subjects. Validation studies used quantitative real-time polymerase chain reaction to measure the levels of 9 upregulated genes in the index cohort, and an independent cohort of 9 patients and 10 referent subjects was prospectively studied to determine the accuracy of the Prediction Analysis for Microarrays list to classify stroke. After correction for multiple comparisons with the Bonferroni technique, 190 genes were significantly different between the stroke and referent groups. Broad classes of genes included white blood cell activation and differentiation (approximately 60%), genes associated with hypoxia and vascular repair, and genes potentially associated with an altered cerebral microenvironment. Real-time polymerase chain reaction confirmed increased mRNA expression in 9 of 9 upregulated stroke-associated genes in the index cohort. A panel of 22 genes derived from the Prediction Analysis for Microarrays algorithm in the index cohort classified stroke in the validation cohort with a sensitivity of 78% and a specificity of 80%. Control for the Framingham stroke risk score revealed only a partial dependence of the stroke gene expression profile in PBMCs on vascular risk. CONCLUSIONS: This study demonstrated an altered gene expression profile in PBMCs during acute ischemic stroke. Some genes with altered expression were consistent with an adaptive response to central nervous system ischemia.

Fabry disease and vascular risk factors: future strategies for patient-based studies and the knockout murine model.

UNLABELLED: Fabry disease is secondary to deficiency of the lysosomal enzyme alpha-galactosidase A, leading to altered glycosphingolipid metabolism and accumulation that is often associated with endothelial dysfunction. Current evidence suggests that there is impairment of the vascular nitric oxide pathway, with abnormalities evident in the cerebral circulation and in the dermal vasculature of patients with Fabry disease. Some of these findings have been confirmed in a mouse model of Fabry disease. The murine model, however, allows investigation of Fabry disease at a non-clinical level and a near complete investigation of biological processes within an affected tissue. This is of particular utility in allowing gene expression analysis of clinically inaccessible tissues such as the aorta. CONCLUSION: Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, may open a new chapter in our understanding of the biology of lysosomal storage disorders.

Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.

A patient with mucolipidosis-IV heterozygous for two mutations in MCOLN1 expressed only her father's cDNA mutation c.1207C>T predicting an R403C change in mucolipin. She inherited a 93bp segment from mitochondrial NADH dehydrogenase 5 (MTND5) from her mother that was inserted in-frame prior to the last nucleotide of exon 2 of MCOLN1 (c.236_237ins93). This alteration abolished proper splicing of MCOLN1. The splice site at the end of the exon was not used due to an inhibitory effect of the inserted segment, resulting in two aberrant splice products containing stop codons in the downstream intron. These products were eliminated via nonsense-mediated decay. This is the first report of an inherited transfer of mitochondrial nuclear DNA causing a genetic disease. The elimination of the splice site by the mitochondrial DNA requires a change in splicing prediction models.

Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans.

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. RESULTS: The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. CONCLUSIONS: Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

MS screening strategies: investigating the glycomes of knockout and myodystrophic mice and leukodystrophic human brains.

The implementation of highly sensitive and rapid mass spectrometric screening strategies for defining the glycosylation repertoires of organs in knockout mice is helping to reveal the roles that glycans play in health and disease. Thus novel glycosylation pathways have been uncovered in two such knockouts, namely alpha-mannosidase II null mice and UDP-N-acetylglucosamine: alpha 6-D-mannoside beta 1,2-N-acetylglucosaminyltransferase II null mice. This chapter documents the glycosylation profiles of a wide range of organs from the normal mouse which should facilitate future glycomics studies of knockout mice. Furthermore, we report applications of our screening technology in studies of the myodystrophy mouse and a human leukodystrophy.

Diffuse neuroaxonal involvement in mucolipidosis IV as assessed by proton magnetic resonance spectroscopic imaging.

Mucolipidosis IV is an autosomal recessive disorder caused by mutations in MCOLN1, which codes for mucolipin, a transient receptor potential protein. In order to investigate brain metabolic abnormalities in mucolipidosis IV, we studied 14 patients (11 children, 3 adults) by proton magnetic resonance spectroscopic imaging. The ratios of N-acetylaspartate/ creatine-phosphocreatine and N-acetylaspartate/choline-containing compounds in patients with mucolipidosis IV were significantly reduced in all regions of interest except the parietal gray matter and thalamus. The ratios of choline-containing compounds/creatine-phosphocreatine was not significantly reduced in patients compared with controls. The ratio of N-acetylaspartate/creatine-phosphocreatine were significantly lower (P = .005) in the more neurologically impaired patients compared with the least impaired. For every region of interest, except for parietal gray matter, the ratio of N-acetylaspartate/creatine-phosphocreatine was lower in the more motorically impaired patient group. There was no difference for the ratio of N-acetylaspartate/creatine-phosphocreatine between younger and older patients. These findings suggest that mucolipidosis IV is largely a static developmental encephalopathy associated with diffuse neuronal and axonal damage or dysfunction. Mucolipin deficiency impairs motor more than sensory central nervous system pathways.

Macrophage models of Gaucher disease for evaluating disease pathogenesis and candidate drugs.

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.

High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.