glutaminase and Alzheimer-Disease

Despite numerous clinical trials and decades of endeavour, there is still no effective cure for Alzheimer's disease. Computational drug repositioning approaches may be employed for the development of new treatment strategies for Alzheimer's patients since an extensive amount of omics data has been generated during pre-clinical and clinical studies. However, targeting the most critical pathophysiological mechanisms and determining drugs with proper pharmacodynamics and good efficacy are equally crucial in drug repurposing and often imbalanced in Alzheimer's studies.. Here, we investigated central co-expressed genes upregulated in Alzheimer's disease to determine a proper therapeutic target. We backed our reasoning by checking the target gene's estimated non-essentiality for survival in multiple human tissues. We screened transcriptome profiles of various human cell lines perturbed by drug induction (for 6798 compounds) and gene knockout using data available in the Connectivity Map database. Then, we applied a profile-based drug repositioning approach to discover drugs targeting the target gene based on the correlations between these transcriptome profiles. We evaluated the bioavailability, functional enrichment profiles and drug-protein interactions of these repurposed agents and evidenced their cellular viability and efficacy in glial cell culture by experimental assays and Western blotting. Finally, we evaluated their pharmacokinetics to anticipate to which degree their efficacy can be improved.. We identified glutaminase as a promising drug target. Glutaminase overexpression may fuel the glutamate excitotoxicity in neurons, leading to mitochondrial dysfunction and other neurodegeneration hallmark processes. The computational drug repurposing revealed eight drugs: mitoxantrone, bortezomib, parbendazole, crizotinib, withaferin-a, SA-25547 and two unstudied compounds. We demonstrated that the proposed drugs could effectively suppress glutaminase and reduce glutamate production in the diseased brain through multiple neurodegeneration-associated mechanisms, including cytoskeleton and proteostasis. We also estimated the human blood-brain barrier permeability of parbendazole and SA-25547 using the SwissADME tool.. This study method effectively identified an Alzheimer's disease marker and compounds targeting the marker and interconnected biological processes by use of multiple computational approaches. Our results highlight the importance of synaptic glutamate signalling in Alzheimer's disease progression. We suggest repurposable drugs (like parbendazole) with well-evidenced activities that we linked to glutamate synthesis hereby and novel molecules (SA-25547) with estimated mechanisms for the treatment of Alzheimer's patients.

Topics: Alzheimer Disease; Drug Repositioning; Glutamates; Glutaminase; Humans; Transcriptome

Topics: Alzheimer Disease; Amino Acid Transport System A; Amyloid beta-Protein Precursor; Animals; Anti-Bacterial Agents; Ceftriaxone; Disease Models, Animal; Excitatory Amino Acid Transporter 2; Gene Knockdown Techniques; Glutamic Acid; Glutaminase; Mice; Mice, Transgenic; Presenilin-1; Presynaptic Terminals; Synaptic Vesicles; Vesicular Glutamate Transport Protein 1; Vesicular Glutamate Transport Protein 2

Given the emergent aging population, the identification of effective treatments for Alzheimer's disease (AD) is critical.. We investigated the therapeutic efficacy of JHU-083, a brain-penetrable glutamine antagonist, in treating AD using the humanized APOE4 knock-in mouse model.. Cell culture studies were performed using BV2 cells and primary microglia isolated from hippocampi of adult APOE4 knock-in mice to evaluate the effect of JHU-083 treatment on LPS-induced glutaminase (GLS) activity and inflammatory markers. Six-month-old APOE4 knock-in mice were administered JHU-083 or vehicle via oral gavage 3x/week for 4-5 months and cognitive performance was assessed using the Barnes maze. Target engagement in the brain was confirmed using a radiolabeled GLS enzymatic activity assay, and electrophysiology, gastrointestinal histology, blood chemistry, and CBC analyses were conducted to evaluate the tolerability of JHU-083.. JHU-083 inhibited the LPS-mediated increases in GLS activity, nitic oxide release, and pro-inflammatory cytokine production in cultured BV2 cells and primary microglia isolated from APOE4 knock-in AD mice. Chronic treatment with JHU-083 in APOE4 mice improved hippocampal-dependent Barnes maze performance. Consistent with the cell culture findings,postmortem analyses of APOE4 mice showed increased GLS activity in hippocampal CD11b+ enriched cells versus age-matched controls, which was completely normalized by JHU-083 treatment. JHU-083 was well-tolerated, showing no weight loss effect or overt behavioral changes. Peripheral nerve function, gastrointestinal histopathology, and CBC/clinical chemistry parameters were all unaffected by chronic JHU-083 treatment.. These results suggest that the attenuation of upregulated hippocampal glutaminase by JHU-083 represents a new therapeutic strategy for AD.

Topics: Alzheimer Disease; Animals; Apolipoprotein E4; Azo Compounds; Caproates; Cell Line; Cells, Cultured; Cognition; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Glutaminase; Glutamine; Hippocampus; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia

Ammonia is a toxic by-product of protein catabolism and is involved in changes in glutamate metabolism. Therefore, ammonia metabolism genes may link a range of diseases involving glutamate signaling such as Alzheimer's disease (AD), major depressive disorder (MDD), and type 2 diabetes (T2D). We analyzed data from a National Institute on Aging study with a family-based design to determine if 45 single nucleotide polymorphisms (SNPs) in glutaminase (GLS), carbamoyl phosphate synthetase 1 (CPS1), or glutamate-ammonia ligase (GLUL) genes were associated with AD, MDD, or T2D using PLINK software. HAPLOVIEW software was used to calculate linkage disequilibrium measures for the SNPs. Next, we analyzed the associated variations for potential effects on transcriptional control sites to identify possible functional effects of the SNPs. Of the SNPs that passed the quality control tests, four SNPs in the GLS gene were significantly associated with AD, two SNPs in the GLS gene were associated with T2D, and one SNP in the GLUL gene and three SNPs in the CPS1 gene were associated with MDD before Bonferroni correction. The in silico bioinformatic analysis suggested probable functional roles for six associated SNPs. Glutamate signaling pathways have been implicated in all these diseases, and other studies have detected similar brain pathologies such as cortical thinning in AD, MDD, and T2D. Taken together, these data potentially link GLS with AD, GLS with T2D, and CPS1 and GLUL with MDD and stimulate the generation of testable hypotheses that may help explain the molecular basis of pathologies shared by these disorders.

Topics: Alzheimer Disease; Ammonia; Carbamoyl-Phosphate Synthase (Ammonia); Depressive Disorder, Major; Diabetes Mellitus, Type 2; Glutamate-Ammonia Ligase; Glutaminase; Humans; Polymorphism, Single Nucleotide

Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amide Synthases; Cerebellum; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Glutamate Dehydrogenase; Glutamic Acid; Glutaminase; Humans; Middle Aged; Nerve Tissue Proteins

Phosphate activated glutaminase (PAG) was quantified in human cerebellar cortex extracts in 13 patients with Alzheimer's disease (AD) and 13 controls by Western immunoblotting using antibody to C-terminus of PAG kidney isoform. The majority of samples from the AD group contained less amount of PAG in comparison with control samples. Although medians in these groups were slightly different (21 and 28 arbitrary units in AD patients and controls, respectively), the Mann Whitney U-test demonstrated a significant between-group difference (U= 28.5, Z= -2.87, p=0.004). Since the both groups were matched for gender, age and postmortem interval, the difference in the PAG level was probably due to the presence of AD. The alteration in the PAG level observed in the cerebellum of patients with AD results in the disturbance of probably not only glutamate metabolism but also many other pathways involving PAG and leads to crucial consequences, particularly, to neurodegeneration.

Topics: Alzheimer Disease; Case-Control Studies; Cerebellum; Glutaminase; Humans

Amounts of glutamate metabolizing enzymes such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), GS-like protein (GSLP), and phosphate-activated glutaminase (PAG) were compared in prefrontal cortex of control subjects and patients with Alzheimer disease (AD). The target proteins were quantified by ECL-Western immunoblotting in extracts from brain tissue prepared by two different techniques separating enzymes preferentially associated with cytoplasm (GDH I and II isoenzymes, GS, and partially GSLP) and membrane (GDH III, PAG, and partially GSLP) fractions. Amounts of all listed enzymes were found significantly increased in the patient group compared with controls. Some links between the measured values were observed in the control, but not in the AD patient group. The results may suggest for the pathological interruption of regulatory relations between distinct enzymes of glutamate metabolism in brain of AD patients.

Topics: Aged; Alzheimer Disease; Amide Synthases; Female; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamic Acid; Glutaminase; Humans; Isoenzymes; Male; Middle Aged; Nerve Tissue Proteins; Prefrontal Cortex; Statistics as Topic; Tissue Extracts

There is strong evidence for the involvement of the neurotransmitter glutamate system in the pathogenesis of Alzheimer's disease and schizophrenia. In these mental diseases, the brain shows changes in the levels of glutamate and in the function and expression of its transporters and receptors. Since the levels of glutamate are largely determined by the rate of its metabolism, the changes of its concentrations may be associated with dysfunctions of appropriate enzymes. Actually, disturbances of glutamate metabolic enzymes, such as glutaminase, glutamate decarboxylase, and glutamine synthetase were detected in the brain of patients with Alzheimer's disease. The alterations in the expression of glutamine synthetase, glutamine synthetase-like protein, and three isoenzymes of glutamate dehydrogenase in the frontal cortex of patients with schizophrenia suggest that there are impaired glutamate metabolism in this mental disease and Alzheimer's disease.

Topics: Alzheimer Disease; Autopsy; Brain; Frontal Lobe; Glutamate Decarboxylase; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutaminase; Humans; Schizophrenia

Much is known about modular organization in the cerebral cortex, but this knowledge is skewed markedly toward primary sensory areas, and in fact, it has been difficult to demonstrate elsewhere. In this report, we test the hypothesis that a unique form of modules exists in the entorhinal area of the human cortex (Brodmann's area 28). We examined this issue using classic cyto- and myeloarchitectonic stains, immunolabeling for various neurochemicals, and histochemistry for certain enzymes. The findings reveal that the entorhinal cortex in the human is formed by a mosaic of cellular aggregates whose most conspicuous elements are the cell islands of layer II and myelinated fibers around the cell islands, the disposition of glutamic acid decarboxylase-positive neurons and processes, cytochrome oxidase staining, and the pattern of cholinergic afferent fibers. The neuropathology of Alzheimer's disease cases highlights the modules, but inversely so, by destroying their features. The findings are of interest because 1) anatomically defined modules are shown to be present in areas other than the sensory and motor cortices, 2) the modules are morphological entities likely to reflect functions of the entorhinal cortex, and 3) the destruction of entorhinal cortex modules may account disproportionately for the severity of memory impairments in Alzheimer's disease.

Topics: Acetylcholinesterase; Adolescent; Adult; Aged; Aged, 80 and over; Alzheimer Disease; Child; Child, Preschool; Dementia; Electron Transport Complex IV; Glutamate Decarboxylase; Glutaminase; Hippocampus; Histocytochemistry; Humans; Immunohistochemistry; Middle Aged; Neurons; Neuropeptide Y; Parvalbumins; Reference Values; Somatostatin

We used immunoblotting and radioligand binding techniques to compare levels of the calcium-mobilizing receptor for the phosphoinositide hydrolysis-derived intracellular second messenger inositol (1,4,5)-trisphosphate (IP3) in post mortem samples from the temporal, frontal and parietal cortices of eight Alzheimer's disease (AD) and eight matched control cases. Immunoblotting with an antibody directed against the C-terminal end of the rat type I IP3-receptor showed that IP3-receptor protein levels were significantly reduced in the temporal (to 59 +/- 6% of controls, P = 0.0002) and frontal (to 62 +/- 10% of controls, P = 0.04), but not in the parietal cortices (to 63 +/- 13% of controls, P = 0.1) of the AD cases, compared to controls. The number of [3H]IP3 radioligand binding sites was significantly decreased in the temporal cortex, but not frontal and parietal cortices, of the AD brains. The decreased levels of both immunoreactive IP3-receptor protein and [3H]IP3 binding in the temporal cortex correlated with a semi-quantitative score for the severity of AD neuropathology. No significant changes were seen in the levels of glial fibrillary acidic protein, synaptophysin or phosphate-activated glutaminase, as markers for astrocytes, neuronal vesicles and mitochondria, respectively. It is concluded that in affected AD brain regions, the IP3-receptor may represent a sensitive target for proteolysis, possibly mediated by activation of the Ca(2+)-activated neutral protease calpain. These degenerative changes may in part be responsible for the disruption of Ca2+ homeostasis in AD-sensitive neurons.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Autopsy; Biomarkers; Calcium Channels; Cell Membrane; Cerebral Cortex; Glial Fibrillary Acidic Protein; Glutaminase; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Rats; Receptors, Cytoplasmic and Nuclear; Reference Values; Regression Analysis; Second Messenger Systems; Statistics, Nonparametric; Synaptophysin; Temporal Lobe

Neurologic and psychologic tests without brain tissue biopsy do not establish the diagnosis of Alzheimer's disease. This pilot study demonstrates significant increases in the activity of plasma glutamate dehydrogenase and the plasma concentrations of aspartate, glutamate, and alpha-ketoglutarate in nursing home residents with previously diagnosed Alzheimer's disease when compared with that in other nursing home residents without Alzheimer's disease who had no complicating conditions. Plasma concentrations of gamma-aminobutyric acid, glutamine, and activities of plasma glutamate decarboxylase, glutaminase, and glutamine synthetase were not significantly different in the two groups. A discriminant analysis number, based on the four significantly different compounds, is obtained that may be used as the basis for an inexpensive, non-invasive, and accurate screening test for Alzheimer's disease.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Aminobutyrates; Aspartic Acid; Discriminant Analysis; Female; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutamic Acid; Glutaminase; Humans; Ketoglutaric Acids; Male; Mass Screening; Middle Aged; Pilot Projects; Sensitivity and Specificity

The syntheses of prostaglandin (PG) F2 alpha, E2 and D2, and thromboxane (TX) B2 from [14C]arachidonic acid were studied in frontal cortex of human control and Alzheimer's disease (AD) brains using the microsomal fractions. Under the assay conditions employed, it was found that the major metabolite of [14C]arachidonic acid was PGE2 accounting for 63% of total prostanoid production; PGF2 alpha accounted for 21.5%, TXB2 for 9%, and PGD2 for 6.5%. When AD samples were compared to control samples, microsomal PG synthesis was significantly decreased, with reduced production of PGE2, PGF2 alpha and PGD2. Such decreases in AD brain seem unrelated to age, sex, postmortem delay and, as far as could be determined, antemortem state. In both control and Alzheimer groups, a history of anti-inflammatory therapy seemed to correlate with increased PG synthesis.

Topics: Aged; Alzheimer Disease; Animals; Arachidonic Acid; Carbon Radioisotopes; Cerebral Cortex; Choline O-Acetyltransferase; Dinoprost; Dinoprostone; Female; Glutaminase; Humans; Kinetics; Male; Microsomes; Postmortem Changes; Prostaglandin D2; Prostaglandins; Rats; Rats, Sprague-Dawley; Reference Values; Thromboxane B2

Although formation of neurofibrillary tangles is a major pathological feature of Alzheimer's disease (AD), the neurotransmitter content of neurofibrillary tangle-bearing neurons has not been well characterized. We studied the hippocampus of 6 patients with pathologically verified AD and 6 control subjects using a monoclonal antibody to glutamyl-glutamate and polyclonal antisera against glutaminase and taurine. In normal hippocampus, glutamate and glutaminase stained pyramidal neurons in the cornu ammonis (CA) fields and the subiculum, as well as the dentate granule cells. Fiber staining was better seen with glutamate antisera, which in AD specimens showed reduced numbers of glutamate-immunoreactive fibers in the molecular layer of the dentate gyrus. In AD specimens, glutamate- and glutaminase-immunoreactive pyramidal neurons in the hippocampal CA fields were decreased in number and remaining neurons showed irregular shortened and disorganized dendritic fields. Taurine immunoreactivity was localized to a subset of hippocampal pyramidal neurons, which showed similar degenerative changes in AD specimens. Glutamate-, glutaminase-, and taurine-stained neurons were found to contain neurofibrillary tangles using either double immunofluorescence with tau antisera, double immunoperoxidase stains, or silver and thioflavine S counterstains. These studies show that two distinct neurochemically defined populations of pyramidal neurons in allocortex frequently show degenerative changes and develop neurofibrillary tangles in AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Female; Glutamates; Glutamic Acid; Glutaminase; Hippocampus; Humans; Immunologic Techniques; Male; Middle Aged; Neurofibrils; Neurons; Taurine

Phosphate activated glutaminase (PAG) was evaluated in the neocortex of Alzheimer and control cases. Consistent with previously reported results in rat cerebral cortex, pyramidal cells were stained immunohistochemically by a PAG specific polyclonal rabbit antibody, especially in layers II, III and V. An Alzheimer's case showed drastic depletion of PAG-positive pyramidal neurons, especially in layers II and III. Cortical PAG levels by biochemical assay were reduced to 18% of control in a small series of Alzheimer's cases (n = 3), while choline acetyltransferase (ChAT) was reduced to 28% of control in the same tissue samples. PAG staining was also observed in large neurons of the rat neostriatum. Double immunostaining for PAG and ChAT established that these large neurons also contained both enzymes.

Topics: Alzheimer Disease; Animals; Cerebral Cortex; Glutaminase; Humans; Immunohistochemistry; Rats

Large pyramidal neurons of rat and human neocortex stain immunohistochemically for phosphate-activated glutaminase (PAG). In a limited number of postmortem brains, we find large reductions in cortical PAG activity in Alzheimer's disease (AD). This finding is consistent with histological evidence that pyramidal neurons are affected in AD. The reductions are greater than those found in the same samples in choline acetyltransferase (ChAT) but the possible deleterious effects of coma and similar premortem factors on human PAG activity have yet to be assessed. The activity of beta-glucuronidase, a lysosomal enzyme which occurs in reactive astrocytes, is elevated in the same samples. Positron emission tomography (PET) studies, using 18F-fluorodeoxyglucose (FDG), have demonstrated significant deficiencies in glucose metabolism in the cortex in AD, with the parietal, temporal and some frontal areas being particularly affected. We found in serial scans of 13 AD cases, including one relatively young (44-46 year old) familial case, an exacerbation of the defect over time in most cases. We have found a negative correlation between the regional metabolic rates for glucose (LCMR(s] measured premortem and the beta-glucuronidase activities measured postmortem on a few AD cases that have come to autopsy. The correlations between LCMR(s) and PAG and ChAT activities tend to be positive. The results are consistent with previous suggestions that decreased LCMR(s) in AD reflect local neuronal loss and gliosis.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Brain; Deoxyglucose; Fluorodeoxyglucose F18; Glucose; Glucuronidase; Glutaminase; Humans; Middle Aged; Tomography, Emission-Computed

Biochemical indices of cortical nerve cells affected in Alzheimer's disease have been proposed (excitatory dicarboxylic amino acid, EDAA, sodium-dependent carrier; phosphate-activated glutaminase activity; serotonin type 2 recognition site; somatostatin-like immunoreactivity). These and the content of EDAAs and two related amino acids, and choline acetyltransferase (ChAT) activity have been measured in up to 13 areas of cerebral cortex and the cerebellar cortex from 16 patients with Alzheimer's disease and 17 controls. Reduction of the index of the serotonin recognition site, somatostatin content and another biochemical index of interneurones coincide and indicate a rather unexpected focal loss of such neurones from the parietal lobe. No unequivocal measure of the integrity of pyramidal neurones could be established as the content of no amino acid was reduced, the index of the EDAA carrier showed evidence of change in few brain regions and glutaminase activity was subject to unexplained variability. ChAT activity alone closely paralleled a previous report of the distribution of morphological degeneration. The results are discussed in relation to therapy and positron emission tomography.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amino Acids, Dicarboxylic; Asparagine; Aspartic Acid; Cerebellar Cortex; Cerebral Cortex; Choline O-Acetyltransferase; Glutamates; Glutaminase; Glutamine; Humans; Middle Aged; Somatostatin

Excitatory dicarboxylic amino acids previously have been ascribed several functions in the brain. Here their total concentration and proposed neurochemical markers of neurotransmitter function have been measured in brain from patients with Alzheimer's disease (AD) and controls. Specimens were obtained antemortem (biopsy) approximately 3 years after emergence of symptoms and promptly (less than 3 h) postmortem some 10 years after onset. Early in the disease a slight elevation in aspartic acid concentration of cerebral cortex was observed in the patients with AD. A reduction in glutamic acid concentration of a similar magnitude was found. It is argued that this, together with a decrease in CSF glutamine content and lack of change in the phosphate-activated brain glutaminase activity of tissue, reflects an early metabolic abnormality. Later in the disease evidence of glutamatergic neurone loss is provided by the finding that in many regions of the cerebral cortex the Na+-dependent uptake of D-[3H]aspartic acid was almost always lowest in AD subjects compared with control when assessed by a method designed to minimise artifacts and epiphenomena. Release of endogenous neurotransmitters from human brain tissue postmortem did not appear to have the characteristics of that from human tissue antemortem and rat brain.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amino Acids; Brain; Glutaminase; Glutamine; Humans; Phosphates; Time Factors

One of the microtubule associated proteins, tau factor, that appears associated to the paired helical filaments of Alzheimer's disease presents, by itself, after urea treatment, the ability of polymerizing in vitro as tested by immunoelectronmicroscopy. These polymers resemble in their width and appearance those of the paired helical filaments. The conditions required for this assembly have been studied and determined the protein concentration needed, the influence of salt concentration and pH as well as the possible modifications (deamination, acylation) which may be implied in such in vitro polymerization.

Topics: Alzheimer Disease; Animals; Glutaminase; Gold; Humans; Hydrogen-Ion Concentration; Immunologic Tests; Microscopy, Electron; Microtubule-Associated Proteins; Nerve Tissue Proteins; Polymers; Protein Conformation; Staphylococcal Protein A; Swine; tau Proteins; Urea

Microtubule associated protein tau factor self-assembles into filamentous structures resembling the paired helical filaments found in Alzheimer disease. Tau polymerization requires of a previous modification; conversion of glutamine into glutamic acid by deamination.

Topics: Alzheimer Disease; Animals; Glutamates; Glutaminase; Glutamine; Macromolecular Substances; Microtubule-Associated Proteins; Neurofibrils; Peptide Fragments; Polymers; Protein Binding; Swine; tau Proteins