Accumulation of Glucosylceramide and Glucosylsphingosine (Psychosine) in Cerebrum and Cerebellum in Infantile and Juvenile Gaucher Disease

Abstract: Three major clinical variants of Gaucher disease have been defined: Type I, chronic nonneuronopathic; Type II, acute neuronopathic; and Type III, subacute neuronopathic. In a search for the underlying molecular basis of the neurological manifestations, the concentration and composition of cholesterol, phospholipids, neutral glycosphingolipids, and gangliosides were examined in cerebral and cerebellar cortices of five cases of Type II, eight cases of Type III, and one case of presumed Type I/III. In Type II the concentration of glucosylceramide was 140-530 μmol/kg in cerebral cortex and 51-450 μmol/kg in cerebellar cortex, the highest values found in the most fulminant cases. These concentrations were 20-80 times greater than normal in cerebral cortex and 5-40 times normal in cerebellar cortex. In Type III the concentration of glucosylceramide was 37-65 and 59-1750 μmol/kg in cerebral and cerebellar cortex, respectively. The highest concentrations were found in the cerebellum of patients who had survived splenectomy for several years. The ceramide composition of the accumulated glucosylceramide suggested that brain gangliosides were the major precursors of the glucosylceramide in brains of Type II but in cerebellar cortex in Type III was partly of extracerebral origin. The levels of lactosylceramide and oligohexaosylceramides were slightly raised in all brain specimens from the Gaucher cases. The ganglioside concentration was normal, whereas there was a certain increase in the proportion of GM2 and GM3 gangliosides. The brain glycosphingolipid changes in the Type I/III case were similar but slightly less than those in Type III cases of corresponding age. Glucosylsphingosine (psychosine), never detected in normal human brain, was demonstrated in brains from all the Gaucher cases. The psychosine concentration was highest in Type II cases, 3.8-8.8 and 3.9-12.3 μmol/kg in cerebral and cerebellar cortex, respectively, with the highest values found in the most fulminant cases. In Type III the psychosine concentration varied more widely, 0.8-4.6 and 1.4-6.3 μmol/kg in cerebral and cerebellar cortex, respectively. The lowest value, 0.7 μmol/kg, was found in the Type I/III case. Our method detected psychosine down to 0.01 μmol/kg, which means that the concentration of psychosine was increased at least 100- to 1000-fold in Gaucher grey matter. We suggest that the accumulation of the cell-toxic substance psychosine is the basis for the extensive neuronal cell loss in Gaucher disease, which is most striking in Type II disease.     

Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.     

Viable Neuronopathic Gaucher Disease Model in Medaka (Oryzias latipes) Displays Axonal Accumulation of Alpha-Synuclein

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson’s disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.     

Efficacy of Enzyme and Substrate Reduction Therapy with a Novel Antagonist of Glucosylceramide Synthase for Fabry Disease

Fabry disease, an X-linked glycosphingolipid storage disorder, is caused by the deficient activity of α-galactosidase A (α-Gal A). This results in the lysosomal accumulation in various cell types of its glycolipid substrates, including globotriaosylceramide (GL-3) and lysoglobotriaosylceramide (globotriaosyl lysosphingolipid, lyso-GL-3), leading to kidney, heart, and cerebrovascular disease. To complement and potentially augment the current standard of care, biweekly infusions of recombinant α-Gal A, the merits of substrate reduction therapy (SRT) by selectively inhibiting glucosylceramide synthase (GCS) were examined. Here, we report the development of a novel, orally available GCS inhibitor (Genz-682452) with pharmacological and safety profiles that have potential for treating Fabry disease. Treating Fabry mice with Genz-682452 resulted in reduced tissue levels of GL-3 and lyso-GL-3 and a delayed loss of the thermal nociceptive response. Greatest improvements were realized when the therapeutic intervention was administered to younger mice before they developed overt pathology. Importantly, as the pharmacologic profiles of α-Gal A and Genz-682452 are different, treating animals with both drugs conferred the greatest efficacy. For example, because Genz-682452, but not α-Gal A, can traverse the blood–brain barrier, levels of accumulated glycosphingolipids were reduced in the brain of Genz-682452–treated but not α-Gal A–treated mice. These results suggest that combining substrate reduction and enzyme replacement may confer both complementary and additive therapeutic benefits in Fabry disease.     

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.     

Multiplicity of Antioxidant Enzyme Catalase in Mouse Liver Cells

Multiplicity of catalase activity has been observed in crude homogenates from the tissue and cell lines of mouse liver by ethanol/Triton X-100/heat treatment. The five enzymatically active catalase bands were designated as CAT1, CAT2, CAT3, CAT4, and CAT5 with a nondenatured molecular mass of 270kDa, 258kDa, 229kDa, 2lOkDa, or 197kDa, respectively. Cultured mouse liver cell lines, mouse liver tissue homogenate, and pure mouse liver catalase showed only one catalase band (CAT1) after ethanol/Triton X-100 treatment at 4°C for 72 hr. The same treatment but incubated at 37°C for 72 hr yielded three bands (CAT2, CAT4, CAT5) in normal cell line, only one band (CAT5) in MNNG-transformed and SV40-transformed cells, two bands (CAT1, CAT4) in mouse liver tissue homogenates, and two bands (CAT1, CAT3) in pure mouse liver catalase. These five catalase bands were further biochemically characterized. The CATl, CAT2, and CAT3 are sensitive to heat (68°C, 1 min), while CAT4 and CAT5 are rather heat resistant. The sensitivity to catalase inhibitors, such as aminotriazole, azide, or cyanide varies among the isoforms. Protease inhibitors could prevent the formation of CAT3 and CAT4, but not CAT5. Treatment with protease, however, removed all forms of catalase except CAT5. We conclude from this study that the appearance of different catalase bands is likely due to epigenetic modification of the protein, particularly proteolysis. The lowered catalase activity in transformed cells might also be attributable to the loss of two catalase isoforms.     

Inducible Diabetes and Transgene Expression in a Single Mouse: A Tool to Elucidate Autoimmune Mechanisms

Transgenic Research -     

Acetylcholinesterase in Mouse Neuroblastoma Cells: Intracellular and Released Enzyme

Abstract: Mouse neuroblastoma cells in cultures release acetylcholinesterase into the growth medium. The released enzyme, as well as the intracellular activity, separate on a density gradient into two molecular forms, sedimenting as 4.5S and 10.5S entities. The relative amounts of these forms are different in the two cases: whereas the slower sedimenting form is the major one in the cellular extract, the 10.5S form predominates in the released activity. The cellular and released proteins were labelled by [³H]diisopropylphosphofluoridate and analysed on polyacrylamide-SDS gels. The results suggest that the intracellular as well as the extracellular molecules are oligomers of similar subunits.     

Modified Extracorporeal Photopheresis with Cells from a Healthy Donor for Acute Graft-versus-Host Disease in a Mouse Model

Background

Graft-versus-host disease (GvHD) is a major challenge after hematopoietic stem cell transplantation but treatment options for patients are still limited. In many cases first-line treatment with glucocorticoids is not successful. Among second-line therapies the extracorporeal photopheresis (ECP) is frequently performed, due to induction of selective tolerance instead of general immunosuppression. However, for some patients with severe acute GvHD the leukapheresis step of the ECP procedure is physically exhausting and limits the number of ECP cycles.

Methods

We hypothesized that leukocytes from healthy cell donors could be used as a replacement for ECP leukocytes gained from the GvHD patient. For this purpose we used a well established mouse model of acute GvHD. The ECP therapy was based on cells with the genetic background of the initial donor of the stem cell transplantation. As a precondition we developed a protocol representing conventional ECP in mice equivalent to clinical used ECP setup.

Results

We could demonstrate that conventional, clinically derived ECP setup is able to alleviate acute GvHD. By using leukocytes obtained from healthy mice with the bone marrow donor’s genetic background we could not observe a statistically significant therapeutic effect.

Conclusions

Conventional human ECP setup is effective in the mouse model of severe acute GvHD. In addition we could not prove that ECP cells from healthy mice with bone marrow donor’s genetic background are as effective as ECP cells derived from GvHD mice. Based on our findings, new questions arise for further studies, in which the cellular characteristics for ECP mediated immune tolerance are a matter of investigation.     

Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.     

Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.Gaucher disease (GD),³ caused by deficiency of a lysosomal enzyme glucocerebrosidase (GCase), an acidic β-glucosidase (EC 3.2.1.45), encompasses a continuum of clinical spectrum from a perinatal lethal disorder to an asymptomatic form. Three major clinical subtypes (1–3) have been broadly defined, primarily based on the degree of neurological involvement. These are useful in determining prognosis and management (1, 2). Type 1 (GD-1), known as the non-neuronopathic form, is characterized by the presence of bone disease, hepatosplenomegaly, anemia, and thrombocytopenia. Type 2 and 3 (GD-2 and GD-3), also known as neuronopathic forms, are characterized by the presence of primary neurological disease. In the past, GD-2 and -3 were distinguished by age of onset and rate of disease progression, but these distinctions are not absolute (3). Generally, patients with GD-2 have an age of onset from days to months with a rapidly progressive neurological course. Individuals with GD-3 may have onset before reaching 2 years of age, often have a more slowly progressive neurological course, and may live into the 3rd or 4th decade. Interestingly, carriers of GD are also considered to have an increased risk of developing Parkinson disease (4–6).Skin fibroblasts from GD-1 patients contain 10–25% of residual GCase activity. The level of 25–30% is believed to represent the “critical threshold” of GCase activity, under which symptoms associated with GD are observed (7). Over 250 mutations have been reported in the GBA gene (encoding GCase) to cause GD. Of these, 203 are missense mutations (8). The most prevalent mutations found are N370S in GD-1 patients and L444P in GD-3 patients (9). However, L444P can also be found in GD-2 patients (10). Most of these missense mutations are believed to affect the proper folding of GCase in the endoplasmic reticulum (ER), re-directing the misfolded protein to the ER-associated degradation pathway. The mutations that have been demonstrated to affect the early folding of GCase are G202R, N370S, and F213I (11–14). In GD patient fibroblasts, particularly those with an N370S or F213I allele, growth in media containing a sub-inhibitory concentration of N-nonyl-deoxynojirimycin, N-octyl-β-valienamine, or the iminosugar isofagomine (IFG), specific competitive inhibitors of GCase, has been demonstrated to increase levels of lysosomal GCase activity 2–4-fold (7, 11, 13, 15, 16). These compounds function as pharmacological chaperones (PCs). PCs act by stabilizing the native conformation of the mutant GCase in the ER, allowing more functional molecules to form and evade the ER-associated degradation pathway by instead being passed onto the ER transport machinery (17), ultimately resulting in increased amounts in the lysosome. It is believed that once the complex enters the lysosomes of cells containing stored substrate, the inhibitors will be displaced allowing GCase to hydrolyze the substrate (18). However, the ideal PC for any lysosomal storage disease (LSD) should bind maximally at the neutral pH of the ER and minimally at the acidic pH of lysosomes.The current enzyme replacement therapy (ERT) for GD is limited to the treatment of non-neurological symptoms, because of the inability of the enzyme to cross the blood-brain barrier (19). This therapeutic approach is also very expensive (∼$200,000/year/patient) and must be administered intravenously. Small molecules are more likely to cross the blood-brain barrier, are less expensive to manufacture, and can be taken orally. Most of the small molecules, currently identified as PCs for GCase, are chemical compounds that have never been tested in humans and therefore are not approved by the drug regulation authorities. The screen of chemical libraries of Food and Drug Administration (FDA)-approved compounds has already proven to be a practical and successful strategy to identify small molecules that can behave as EET agents for specific lysosomal enzymes deficient in LSDs (20). To date, all reported approaches to library screening have utilized an assay aimed at identifying novel inhibitors of the target enzyme (20–23). In this context, we describe here the identification of ambroxol (ABX) as an EET agent for GCase using a more general thermal denaturation assay to screen the NINDS, National Institutes of Health, library of 1,040 drugs that have been previously used to treat humans. We then characterize the biochemical effects of ABX treatment on three GD patient cell lines, L444P/L444P, N370S/N370S, and F213I/L444P. Only the latter two cell lines responded with enhanced lysosomal GCase activity and protein levels. Additionally, substrate storage was reduced as demonstrated by liquid chromatography-electrospray ionization-tandem mass spectrometry after 15 days of treatment of an N370S/N370S lymphoblast line with ABX.     

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.     

MAR提高重组CHO细胞中转基因表达的分子特征分析

分析核基质结合区（matrix attachment region,MAR）调控转基因表达的分子序列特征,鉴定能有效提高CHO细胞转基因表达的MAR特征性元件。将人β-珠蛋白MAR片段从5′到3′ 端分为6个分段（1～540,421～1 020,901～1 500,1 381～1 980,1 861～2 460,2 341～2 999位置）,分别采用PCR进行克隆,经测序证实正确后,分别连接到含有氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因的表达载体SV40启动子及上游,构建β-珠蛋白MAR渐次片段介导的表达载体,转染CHO细胞,G418筛选出稳定表达细胞株,ELISA分析CAT报告基因的表达水平,生物信息学分析MAR序列特征。结果表明,β-珠蛋白MAR全长能显著提高转基因的表达,6个渐次片段相比较,421～1 020位的第2个分段和 901～1 500位的第3个分段提高转基因表达作用显著。生物信息学分析结果显示,MAR-like motif有助于转基因表达提高。     

Brain Purines in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current-studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT^-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [¹⁴C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT^- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.     

Mechanism of Accumulation of DNA in Giant Cells of Mouse Trophoblast

IN rodents, the foetal part of the placenta contains giant trophoblast cells which are unique among mammalian cell types in that each nucleus contains several hundred times the haploid amount of DNA^1–4. We have investigated the mechanism by which this DNA is accumulated, in order to understand its relation to trophoblast function. Galassi⁵ suggested that engulfment of maternal cells might be responsible for the formation of the giant nuclei, while Avery and Hunt⁶ raised the possibility that diploid trophoblast cells fused. Recent studies^4,7 make both these possibilities seem unlikely. On the basis mainly of cytological observations, Zybina¹ has proposed that giant trophoblast nuclei arise in the rat by a series of endoreduplications, that is replication of the genome without subsequent mitosis and cell division. Her claim⁸ that polytene chromosomes⁹ could be seen in these nuclei was not supported by our studies on mouse trophoblast⁴.