Gregory A. Grabowski

Gaucher Disease Glucocerebrosidase and α-Synuclein Form a Bidirectional Pathogenic Loop in Synucleinopathies

Parkinsons disease (PD), an adult neurodegenerative disorder, has been clinically linked to the lysosomal storage disorder Gaucher disease (GD), but the mechanistic connection is not known. Here, we show that functional loss of GD-linked glucocerebrosidase (GCase) in primary cultures or human iPS neurons compromises lysosomal protein degradation, causes accumulation of α-synuclein (α-syn), and results in neurotoxicity through aggregation-dependent mechanisms. Glucosylceramide (GlcCer), the GCase substrate, directly influenced amyloid formation of purified α-syn by stabilizing soluble oligomeric intermediates. We further demonstrate that α-syn inhibits the lysosomal activity of normal GCase in neurons and idiopathic PD brain, suggesting that GCase depletion contributes to the pathogenesis of sporadic synucleinopathies. These findings suggest that the bidirectional effect of α-syn and GCase forms a positive feedback loop that may lead to a self-propagating disease. Therefore, improved targeting of GCase to lysosomes may represent a specific therapeutic approach for PD and other synucleinopathies.

Fabry Disease, an Under-Recognized Multisystemic Disorder: Expert Recommendations for Diagnosis, Management, and Enzyme Replacement Therapy

Fabry disease is an X-linked recessive lysosomal storage disorder that is caused by the deficient activity of -galactosidase A (-Gal A, also termed ceramide trihexosidase) (1, 2) and the resultant accumulation of globotriaosylceramide (also termed ceramide trihexoside) and related glycosphingolipids (3-5). In patients with the classic phenotype, levels of -Gal A activity are very low or undetectable. Patients with detectable -Gal A activity have a milder, variant phenotype (6-8). In classically affected males, the progressive glycosphingolipid accumulation, particularly in the vascular endothelium (Figure 1), leads to renal, cardiac, and cerebrovascular manifestations and early death. The disease is panethnic, and estimates of incidence range from about 1 in 40 000 to 60 000 males (5, 9). Fabry disease predominantly affects males, although carrier (heterozygous) females also can be affected to a mild or severe degree because of random X-chromosomal inactivation (5). Figure 1. Distinctive laboratory findings in Fabry disease. A B In the absence of a family member who has already received a diagnosis of the disorder, many cases are not diagnosed until adulthood (average age, 29 years) (9, 10), when the pathology of the disorder may already be advanced. Although clinical onset occurs in childhood, disease presentation may be subtle, and its signs and symptoms are often discounted as malingering or are mistakenly attributed to other disorders, such as rheumatic fever, erythromelalgia, neurosis, the Raynaud syndrome, multiple sclerosis, chronic intermittent demyelinating polyneuropathy, lupus, acute appendicitis, growing pains, or petechiae (5, 11). Fabry disease was first identified a century ago, but until now, no disease-specific treatment has been available. Patients have been managed with supportive, nonspecific treatment for pain management, cardiac and cerebrovascular complications, and end-stage renal disease. These interventions may prolong life, but their utility is limited because they do not address the underlying cause of the disease, that is, the lack of -Gal A and the progressive accumulation of globotriaosylceramide. Recently, enzyme replacement with human -Gal A has been shown to safely reverse the pathogenesis of the major clinical manifestations, to decrease pain, and to stabilize renal function in patients with Fabry disease (12, 13). The European Agency for the Evaluation of Medicinal Products has approved the treatment, and the U.S. Food and Drug Administration is currently reviewing it. Thus, recommendations for the diagnosis and treatment of Fabry disease are timely. Formation of Expert Panel and Basis of Recommendations In June and July of 2001, two groups of investigators published randomized, placebo-controlled trials that demonstrated that enzyme replacement therapy in Fabry disease can reverse the major pathologic consequences and improve outcomes (12, 13). With a disease-specific therapy finally available, the need for prompt and accurate diagnosis of this devastating, progressive disease became paramount so that patients could be identified and treated before incurring irreversible organ damage. Recognizing the need for initial guidelines for diagnosis, management, and the use of enzyme replacement therapy, Dr. Robert Desnick and Dr. Roscoe Brady (senior authors of the enzyme replacement trials) assembled an international panel of experts with extensive clinical experience and diverse subspecialty expertise in Fabry disease and lysosomal storage disorders. Panelists met face-to-face to identify and discuss salient issues. An independent coordinator conducted numerous global and specific searches of the MEDLINE database (19912001), including a global search of the recent literature on Fabry disease. The coordinator then interviewed each panelist in detail and, with the first author, prepared a draft statement. In a second face-to-face session, the draft was reviewed, revised, and finalized by the panel. A teleconference was convened to revise the manuscript after journal review. Support for the expert panel process was obtained from the Genzyme Corporation (Cambridge, Massachusetts), which had no formative role in the literature review, the formulation of recommendations, or the drafting and revising of the manuscript. As would be expected for a rare, under-recognized disease, the literature on Fabry disease mostly consists of single or small case studies and reviews in addition to book chapters written by Fabry experts. The few larger studies focus on disease manifestations and mechanisms of disease rather than the effectiveness of interventions or disease management. The literature on enzyme replacement therapy is limited to the clinical trials published in the last 2 years. Thus, clinical experience and expertise played an important role in the formulation of these recommendations. Disease Pathophysiology and Clinical Manifestations The major debilitating manifestations of Fabry disease result from the progressive accumulation of globotriaosylceramide in the vascular endothelium (Figure 1), leading to ischemia and infarction, especially in the kidney, heart, and brain. The ischemia and infarction of small vessels are primarily due to vascular occlusion (5); however, evidence for a prothrombotic state has recently been published (14). In addition, early and substantial deposition of globotriaosylceramide occurs in podocytes, leading to proteinuria and, with age, in cardiomyocytes, causing cardiac hypertrophy and conduction abnormalities (Figure 1). Patients are generally divided into two major groups on the basis of the absence or presence of residual -Gal A activity: classic disease and milder, later-onset, atypical variants (5). Presentation and clinical course can vary within these phenotypes, and an intermediate phenotype has also been described (15-17). The Classic Phenotype Males with classic disease have no or very low -Gal A activity, resulting in severe renal, cardiac, and cerebrovascular disease manifestations. Before treatment of uremia became available, the average lifespan of affected males was about 40 years (18). With the advent of renal dialysis or transplantation, the median survival was about 50 years (19). Clinical manifestations (Figures 1 and 2), which usually begin in childhood or adolescence, include intermittent pain in the extremities (acroparesthesias); episodic Fabry crises of acute pain lasting hours to days; characteristic skin lesions (angiokeratomas); a corneal opacity that does not affect vision; hypohidrosis; heat, cold, and exercise intolerance; mild proteinuria; and gastrointestinal problems. By adulthood, the renal involvement inevitably results in end-stage renal disease, which requires dialysis or transplantation (20, 21). Cardiac manifestations include left ventricular hypertrophy, valvular disease (especially mitral insufficiency), ascending aortic dilatation, coronary artery disease, and conduction abnormalities (Figure 1), leading to congestive heart failure, arrhythmias, and myocardial infarction (5, 22-24). Cerebrovascular manifestations include early stroke, transient ischemic attacks, white matter lesions, hemiparesis, vertigo or dizziness, and complications of vascular disease (such as diplopia, dysarthria, nystagmus, tinnitus, hemiataxia, memory loss, and hearing loss) (5, 25). Figure 2. Distinctive clinical features of Fabry disease. A B C Clinical manifestations in carrier females range from asymptomatic to full-blown disease as severe as that in affected males (5, 26-28). Although many carriers will be relatively asymptomatic and have a normal lifespan, carriers may experience symptoms in childhood and adolescence (such as pain and proteinuria) and adulthood (such as cardiac or, more rarely, renal manifestations). In late adulthood, some carriers develop left ventricular hypertrophy and substantial cardiomyopathy. Data on carriers are limited. A recent study of obligate carrier females found significant disease manifestations in 20 of 60 women, including 17 of whom who had experienced transient ischemic attacks or cerebrovascular accidents (28). Atypical Variants Atypical male variants have a milder, later-onset phenotype (5-7, 17, 29). Because of low residual -Gal A levels, these patients do not have the early major clinical manifestations of classic Fabry disease. For example, cardiac variants present with cardiomegaly and mild proteinuria usually after 40 years of age, when patients with classic Fabry disease would be severely affected or would have died (6, 7, 29). Two recent studies have suggested that the cardiac variant of Fabry disease may be an important cause of idiopathic left ventricular hypertrophy (7) or late-onset hypertrophic cardiomyopathy (30). Tissue biopsies or autopsy studies of cardiac variants reveal globotriaosylceramide accumulation in the myocardium and not in the vascular endothelium throughout the body (5, 6, 29). These findings suggest that even low levels of -Gal A can prevent globotriaosylceramide accumulation in the microvasculature and that this lack of accumulation is associated with the absence or attenuation of disease manifestations. Thus, reversal of the underlying vascular endothelial pathology by enzyme replacement therapy will probably be clinically therapeutic in patients with classic Fabry disease. Enzymatic and Molecular Diagnosis In affected males with the classic or variant phenotype, the disease is readily diagnosed by determining the -Gal A activity in plasma or peripheral leukocytes. In contrast, female carriers can have normal to very low -Gal A activity; therefore, their specific family mutation in the -Gal A gene must be demonstrated. Most kindreds have family-specific or private mutations; to date, more than 300 mutations have been identified, of which most are missense (amino acid substitutions) or nonsense (causing premature truncation of the amino acid sequence) mutations. Spli

Enzyme Therapy in Type 1 Gaucher Disease: Comparative Efficacy of Mannose-Terminated Glucocerebrosidase from Natural and Recombinant Sources

Gaucher disease, an inborn error of glycosphingolipid metabolism, is the most frequent lysosomal storage disease [1]. Non-neuronopathic or type 1 disease is the most common variant and the most prevalent genetic disease among Ashkenazi Jews [2, 3]. Various point mutations, deletions, and insertions within the glucocerebrosidase (acid -glucosidase, EC 3.2.1.45) locus at chromosome 1q21 result in a deficiency of this lysosomal enzyme [4, 5]. The subsequent accumulation of glucocerebroside (glucosylceramide) in cells of monocyte-macrophage lineage leads to the visceral manifestations of anemia, thrombocytopenia, hepatosplenomegaly, skeletal disease, and, less frequently, primary lung involvement [1]. Gaucher disease has become a prototype genetic disease for the development of prenatal diagnosis [6], genotype-phenotype correlations [2, 7, 8], and effective therapy [9-13]. On the basis of the clear efficacy of targeted enzyme therapy [10], recent studies [9, 12, 14, 15] in more than 90 patients have established that regular infusions of enzyme purified from placenta (alglucerase [Ceredase; Genzyme Corp., Cambridge, Massachusetts]) lead to regression of the clinical manifestations of Gaucher disease. In addition, antibody-mediated and non-antibody-mediated adverse events have occurred in only 5% to 7% of treated patients [15, 16]. Ceredase is a commercial form of placenta glucocerebrosidase that has been modified for targeting mannose receptor sites on macrophages and other cells [17, 18]. A theoretical limitation to the use of Ceredase is the remote possibility of infective contaminants in the preparation from human placenta. A practical limitation is the finite availability of acceptable placentae. For each patient, approximately 10 to 12 tons of placentae or about 50 000 per year are needed as source material for Ceredase. Enzyme produced by heterologous expression of human complementary DNA (cDNA) for glucocerebrosidase in eukaryotic cells could eliminate both of these limitations. To determine the efficacy of recombinant glucocerebrosidase, we did a randomized, double-blind, parallel trial with mannose-terminated glucocerebrosidase (alglucerase, Ceredase) from human placenta and the human enzyme that is produced in Chinese hamster ovary cells and deglycosylated to expose mannose residues in the oligosaccharide chains (imiglucerase, Cerezyme [Genzyme Corp.]). Methods Thirty patients with non-neuronopathic type 1 Gaucher disease were entered into the trial after consent was obtained. Deficiency of glucocerebrosidase (5% to 15% of mean normal activities) was shown by natural or artificial substrates in peripheral blood mononuclear cells, cultured skill fibroblasts, or lymphoblastoid cell lines obtained from each patient [19, 20]. Inclusion criteria for the study were as follows: 1) enzymatically confirmed glucocerebrosidase deficiency; 2) patient age between 2 and 75 years; 3) an intact, enlarged spleen; 4) a hemoglobin level at least 10 g/L less than the lower limit of normal; and 5) in women, a willingness to avoid pregnancy during the trial. Exclusion criteria were as follows: 1) inability to comply with study requirements; 2) previous receipt of any form of glucocerebrosidase; 3) total splenectomy; 4) a concurrent major medical disorder, such as active infectious disease or substance abuse; and 5) positive serologic response to hepatitis B surface antigen or human immunodeficiency virus (HIV) type 1, or both. The patients had moderate to severe Gaucher disease. The study was a double-blind, parallel trial with random assignment to Ceredase or Cerezyme. We categorized randomization into three groups according to patient age: 1) younger than 12 years; 2) 12 to 17 years; and 3) older than 17 years. In each study center, patients were independently randomly assigned in blocks by age and were assigned study numbers. All study personnel except the pharmacist at each institution were blinded to the allocated treatment. No children younger than 12 years were enrolled in the study. Of the 30 patients enrolled, 17 were male and 13 were female (age range, 12 to 69 years). Of the 7 patients between 12 and 17 years of age, 4 received Ceredase and 3 received Cerezyme. Twenty-three patients older than 17 years were enrolled; 11 of them received Ceredase and 12 received Cerezyme. The groups of patients treated with Ceredase or Cerezyme did not differ in sex distribution, age, weight, or height. All patients received Cerezyme or Ceredase at a dose of 60 U/kg body weight once every 2 weeks for 9 months. Complete hematologic and clinical chemistry data were available for this period. At this dose, complete data for hepatic and splenic volumes were available for all patients for the first 6 months. Hepatic and splenic volumes were evaluated in the 16 patients from the Mt. Sinai School of Medicine at 9 months, just before a dose-reduction study began. The hepatic and splenic volumes of the patients from the National Institutes of Health (NIH) were evaluated at 12 months. We did not include these data in our report. We did analyses of variance and other statistical analyses using Systat software (Systat Inc., Evanston, Illinois). In addition to physical examinations, clinical laboratory studies were done to monitor both therapeutic efficacy and potential toxic effects, including total serum acid phosphatase levels, angiotensin-converting enzyme levels, serum bilirubin levels, hemoglobin levels, platelet counts, peripheral blood leukocyte counts, and serum iron and clotting studies. At baseline and at study completion, hepatitis B surface antigen assay; HIV-1 serologic testing; serum protein electrophoresis; and complement C3, C4, and CH-50 studies were done. Hepatic and splenic volumes were estimated by computed tomography (at Mt. Sinai School of Medicine) [12, 15] or magnetic resonance imaging (at NIH) [21]. We calculated the increases over normal volumes by assuming that the hepatic and splenic masses (1 g/mL density) were 2.5% and 0.2% of body weight, respectively [22]. To avoid biasing the results because of the nearly universal weight gain in treated patients, we averaged the body weight of adults over the study period. In children (patients younger than 18 years), we calculated the hepatic and splenic volumes on the basis of body weight at each time point to allow for growth. We monitored the formation of antibodies to natural or recombinant glucocerebrosidases every 3 months by radioimmunoprecipitation assay [16]. Adverse events were monitored at each infusion. Before each infusion, we also obtained a history of intra-infusion adverse events. Ceredase was supplied as a clear liquid solution stored at 4 C, solubilized in the presence of albumin. Cerezyme was purified from culture media of Chinese hamster ovary cell clones that contained numerous copies of the human glucocerebrosidase cDNA. Carbohydrate removal to expose core mannose moieties was done by the sequential exoglycosidase treatment used to produce Ceredase. The amino acid sequences of glucocerebrosidase in Ceredase and Cerezyme were identical except for a single amino acid substitution of a histidine for a natural arginine at position 495 in the latter. The lack of effect of this amino acid change on the catalytic function of glucocerebrosidase has been documented [23]. Cerezyme was supplied as a lyophilized powder containing mannitol, sodium citrate, and polysorbate 80. Cerezyme as lyophilized powder was stored at 4 C until use. Immediately before administration, Cerezyme was reconstituted with sterile water to a concentration of 40 U/mL. Ceredase or Cerezyme stocks were diluted in 0.9% NaCl just before infusion. During the study, the patients were weighed at each infusion, and the total dose to be administered was adjusted to 60 U/kg on the basis of the current weight. A unit of enzyme activity (U) is defined as the amount of enzyme required to cleave 1 mol of p-nitrophenol--d-glucopyranoside per minute. Results Hematologic Findings The effects of Ceredase and Cerezyme infusions on hemoglobin levels by 6 and 9 months are shown in Tables 1 and 2. Neither the mean initial hemoglobin level (approximately 107 g/L) nor changes in hemoglobin levels differed between the two treatment groups. During the first 6 months of therapy, hemoglobin levels increased by a mean of 17.1 g/L. In patients with hemoglobin levels less than 120 g/L, 54% and 30% of the patients receiving Ceredase and Cerezyme, respectively, achieved this or a greater level by 6 months. Sixty-nine percent and 40%, respectively, of patients receiving Ceredase and Cerezyme achieved these levels by 9 months. The rates at which hemoglobin levels increased by 10 and 15 g/L were also similar. An average of 92 days was required for the hemoglobin level to increase 10 g/L in the Ceredase group, whereas an average of 77 days was needed in the Cerezyme group. For the Ceredase and Cerezyme groups, hemoglobin levels increased 15 g/L in 113 and 125 days, respectively. None of the above differences was significant (P > 0.2). In both treatment groups, we observed lesser degrees of response in patients with higher initial hemoglobin levels. However, this conclusion was strongly influenced by two patients who had initial hemoglobin levels less than 90 g/L and large responses to treatment, that is, an increase of more than 30 g/L during the first 6 months of therapy. Table 1. Clinical Findings from Patients with Type I Gaucher Disease Treated with Ceredase and Cerezyme* Table 2. Effects of Ceredase and Cerezyme on Hematologic Measurements All patients in the study had thrombocytopenia (mean platelet count, approximately 71.5 109/L). About half (7 of 15) of the patients in each group had increases in platelet counts of 20% and 40% or more during the 6- and 9-month treatment periods, respectively (Table 2). These responses to therapy did not differ between the Ceredase and Cerezyme groups. In each group and in the entire st

Phenotype, diagnosis, and treatment of Gaucher's disease.

Gauchers disease continues to be a model for applications of molecular medicine to clinical delineation, diagnosis, and treatment. Analyses of several thousand affected individuals have broadened the range of the pan-ethnic disease variants, provided initial genotype and phenotype correlations, and established the effectiveness of enzyme therapy. Large numbers of affected individuals worldwide have provided insight into the effect of disease variation related to ethnic origin, prognosis, and outcome. The ability to safely and effectively use enzyme therapy to inhibit or reverse visceral-disease progression and involvement has provided impetus for design of new enzyme therapies, and creation of substrate depletion and pharmacological chaperone strategies. Such innovations could provide interventions that are effective for neuronopathic variants and, potentially, could be more cost effective than other treatments. These developments are novel, clinically important, advancements for patients with other lysosomal storage diseases and genetic diseases.

Acid β‐glucosidase mutants linked to gaucher disease, parkinson disease, and lewy body dementia alter α‐synuclein processing

Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α‐synuclein (SNCA). A loss in lysosomal acid–β‐glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the genes association with increased synucleinopathy risk.

An in vivo model of human small intestine using pluripotent stem cells

Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.

Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA

Primary pulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by accumulation of surfactant in the lungs that is presumed to be mediated by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling based on studies in genetically modified mice. The effects of GM-CSF are mediated by heterologous receptors composed of GM-CSF binding (GM-CSF-Rα) and nonbinding affinity-enhancing (GM-CSF-Rβ) subunits. We describe PAP, failure to thrive, and increased GM-CSF levels in two sisters aged 6 and 8 yr with abnormalities of both GM-CSF-Rα–encoding alleles (CSF2RA). One was a 1.6-Mb deletion in the pseudoautosomal region of one maternal X chromosome encompassing CSF2RA. The other, a point mutation in the paternal X chromosome allele encoding a G174R substitution, altered an N-linked glycosylation site within the cytokine binding domain and glycosylation of GM-CSF-Rα, severely reducing GM-CSF binding, receptor signaling, and GM-CSF–dependent functions in primary myeloid cells. Transfection of cloned cDNAs faithfully reproduced the signaling defect at physiological GM-CSF concentrations. Interestingly, at high GM-CSF concentrations similar to those observed in the index patient, signaling was partially rescued, thereby providing a molecular explanation for the slow progression of disease in these children. These results establish that GM-CSF signaling is critical for surfactant homeostasis in humans and demonstrate that mutations in CSF2RA cause familial PAP.

CNS expression of glucocerebrosidase corrects α-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy

Emerging genetic and clinical evidence suggests a link between Gaucher disease and the synucleinopathies Parkinson disease and dementia with Lewy bodies. Here, we provide evidence that a mouse model of Gaucher disease (Gba1D409V/D409V) exhibits characteristics of synucleinopathies, including progressive accumulation of proteinase K-resistant α-synuclein/ubiquitin aggregates in hippocampal neurons and a coincident memory deficit. Analysis of homozygous (Gba1D409V/D409V) and heterozygous (Gba1D409V/+ and Gba1+/−) Gaucher mice indicated that these pathologies are a result of the combination of a loss of glucocerebrosidase activity and a toxic gain-of-function resulting from expression of the mutant enzyme. Importantly, adeno-associated virus-mediated expression of exogenous glucocerebrosidase injected into the hippocampus of Gba1D409V/D409V mice ameliorated both the histopathological and memory aberrations. The data support the contention that mutations in GBA1 can cause Parkinson disease-like α-synuclein pathology, and that rescuing brain glucocerebrosidase activity might represent a therapeutic strategy for GBA1-associated synucleinopathies.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK

Significance Cancer cells reprogram their metabolism for optimal growth and survival. AMPK-activated protein kinase (AMPK) is a key energy sensor that controls many metabolic pathways including metabolic reprogramming. However, its role in cancer is poorly understood. Some studies claim that it has a tumor suppressor role while others show its protumor role. Two AMPK-activating compounds (including metformin, now in many clinical trials) are widely used to suppress cancer cell proliferation. We found that AMPK is abundantly expressed in high-grade gliomas and, in contrast to popular belief, these two AMPK activators suppressed glioma cell proliferation through unique AMPK-independent mechanisms. The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis—two crucial arms of cancer growth—AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40’s association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

Viable Mouse Models of Acid β-Glucosidase Deficiency: The Defect in Gaucher Disease

Gaucher disease is an autosomal recessively inherited disease caused by mutations at the acid beta-glucosidase (GCase) locus (GBA). To develop viable models of Gaucher disease, point mutations (pmuts), encoding N370S, V394L, D409H, or D409V were introduced into the mouse GCase (gba) locus. DNA sequencing verified each unique pmut. Mutant GCase mRNAs were near wild-type (WT) levels. GCase activities were reduced to 2 to 25% of WT in liver, lung, spleen, and cultured fibroblasts from pmut/pmut or pmut/null mice. The corresponding brain GCase activities were approximately 25% of WT. N370S homozygosity was lethal in the neonatal period. For the other pmut mice, a few storage cells appeared in the spleen at > or =7 months (D409H or D409V homozygotes) or > or =1 year (V394L homozygotes). V394L/null, D409H/null, or D409V/null mice showed scattered storage cells in spleen at approximately 3 to 4 months. Occasional storage cells (sinusoidal cells) were present in liver. In D409V/null mice, large numbers of Mac-3-positive storage cells (ie, macrophages) accumulated in the lung. Glycosphingolipid analyses showed varying rates of progressive glucosylceramide accumulation in visceral organs of pmut/pmut or pmut/null mice, but not in brain. These GCase-deficient mice provide tools for gaining insight into the pathophysiology of Gaucher disease and developing improved therapies.