Glutaminase-deficient mice display hippocampal hypoactivity, insensitivity to pro-psychotic drugs and potentiated latent inhibition: relevance to schizophrenia.
ABSTRACT: Dysregulated glutamatergic neurotransmission has been strongly implicated in the pathophysiology of schizophrenia (SCZ). Recently, presynaptic modulation of glutamate transmission has been shown to have therapeutic promise. We asked whether genetic knockdown of glutaminase (gene GLS1) to reduce glutamatergic transmission presynaptically by slowing the recycling of glutamine to glutamate, would produce a phenotype relevant to SCZ and its treatment. GLS1 heterozygous (GLS1 het) mice showed about a 50% global reduction in glutaminase activity, and a modest reduction in glutamate levels in brain regions relevant to SCZ pathophysiology, but displayed neither general behavioral abnormalities nor SCZ-associated phenotypes. Functional imaging, measuring regional cerebral blood volume, showed hippocampal hypometabolism mainly in the CA1 subregion and subiculum, the inverse of recent clinical imaging findings in prodromal and SCZ patients. GLS1 het mice were less sensitive to the behavioral stimulating effects of amphetamine, showed a reduction in amphetamine-induced striatal dopamine release and in ketamine-induced frontal cortical activation, suggesting that GLS1 het mice are resistant to the effects of these pro-psychotic challenges. Moreover, GLS1 het mice showed clozapine-like potentiation of latent inhibition, suggesting that reduction in glutaminase has antipsychotic-like properties. These observations provide further support for the pivotal role of altered glutamatergic synaptic transmission in the pathophysiology of SCZ, and suggest that presynaptic modulation of the glutamine-glutamate pathway through glutaminase inhibition may provide a new direction for the pharmacotherapy of SCZ.
Project description:Dopamine neurons in the ventral tegmental area use glutamate as a cotransmitter. To elucidate the behavioral role of the cotransmission, we targeted the glutamate-recycling enzyme glutaminase (gene Gls1). In mice with a dopamine transporter (Slc6a3)-driven conditional heterozygous (cHET) reduction of Gls1 in their dopamine neurons, dopamine neuron survival and transmission were unaffected, while glutamate cotransmission at phasic firing frequencies was reduced, enabling a selective focus on the cotransmission. The mice showed normal emotional and motor behaviors, and an unaffected response to acute amphetamine. Strikingly, amphetamine sensitization was reduced and latent inhibition potentiated. These behavioral effects, also seen in global GLS1 HETs with a schizophrenia resilience phenotype, were not seen in mice with an Emx1-driven forebrain reduction affecting most brain glutamatergic neurons. Thus, a reduction in dopamine neuron glutamate cotransmission appears to mediate significant components of the GLS1 HET schizophrenia resilience phenotype, and glutamate cotransmission appears to be important in attribution of motivational salience.
Project description:Brain imaging has revealed that the CA1 subregion of the hippocampus is hyperactive in prodromal and diagnosed patients with schizophrenia (SCZ), and that glutamate is a driver of this hyperactivity. Strikingly, mice deficient in the glutamate synthetic enzyme glutaminase have CA1 hypoactivity and a SCZ-resilience profile, implicating glutamate-metabolizing enzymes. To address this further, we examined mice with a brain-wide deficit in the glutamate-metabolizing enzyme glutamate dehydrogenase (GDH), encoded by Glud1, which should lead to glutamate excess due to reduced glutamate metabolism in astrocytes. We found that Glud1-deficient mice have behavioral abnormalities in the 3 SCZ symptom domains, with increased baseline and amphetamine-induced hyperlocomotion as a positive symptom proxy, nest building and social preference as a negative symptom proxy, and reversal/extradimensional set shifting in the water T-maze and contextual fear conditioning as a cognitive symptom proxy. Neuroimaging of cerebral blood volume revealed hippocampal hyperactivity in CA1, which was associated with volume reduction. Parameters of hippocampal synaptic function revealed excess glutamate release and an elevated excitatory/inhibitory balance in CA1. Finally, in a direct clinical correlation using imaging-guided microarray, we found a significant SCZ-associated postmortem reduction in GLUD1 expression in CA1. These findings advance GLUD1 deficiency as a driver of excess hippocampal excitatory transmission and SCZ symptoms, and identify GDH as a target for glutamate modulation pharmacotherapy for SCZ. More broadly, these findings point to the likely involvement of alterations in glutamate metabolism in the pathophysiology of SCZ.
Project description:Glutaminase is the enzyme that converts glutamine into glutamate, which serves as a key excitatory neurotransmitter and one of the energy providers for cellular metabolism. Previous studies have revealed that mice lacking glutaminase 1 (GLS1), the dominant isoform in the brain and kidney, died shortly after birth due to disrupted glutamatergic transmission, suggesting the critical role of GLS1 in the physiological functions of synaptic network. However, whether GLS1 regulates neurogenesis, a process by which neurons are generated from neural progenitor cells (NPCs), is unknown. Using a human NPC model, we found that both GLS1 isotypes, kidney-type glutaminase and glutaminase C, were upregulated during neuronal differentiation, which were correlated with the expression of neuronal marker microtubule-associated protein 2 (MAP-2). To study the functional impact of GLS1 on neurogenesis, we used small interference RNA targeting GLS1 and determined the expressions of neuronal genes by western blot, real-time polymerase chain reaction, and immunocytochemistry. siRNA silencing of GLS1 significantly reduced the expression of MAP-2, indicating that GLS1 is essential for neurogenesis. To unravel the specific process(es) of neurogenesis being affected, we further studied the proliferation and survival of NPCs in vitro. siRNA silencing of GLS1 significantly reduced the Ki67(+) and increased the TUNEL(+) cells, suggesting critical roles of GLS1 for the proliferation and survival of NPCs. Together, these data suggest that GLS1 is critical for proper functions of NPCs, including neuronal differentiation, proliferation, and survival.
Project description:Abnormalities in glutamatergic neurotransmission are implicated in several psychiatric disorders, but in vivo neurochemical studies of the glutamate (Glu) system have been hampered by a lack of adequate probes. By contrast, glutamine (Gln) and Glu can be quantified separately in proton magnetic resonance spectroscopy studies in vivo. Accumulating evidence suggests that the Gln/Glu ratio is a putative index of glutamatergic neurotransmission but interpretation of changes in the Gln/Glu ratio depends on the conditions of the system, including ammonia levels.Here, we explored whether variation in GLS1 (the gene encoding the brain isoform of glutaminase, which catalyzes Gln-to-Glu conversion) is associated with Gln/Glu measured in vivo in two brain regions (anterior cingulate cortex, parieto-occipital cortex).A specific haplotype of four single nucleotide polymorphisms within GLS1 was significantly associated with Gln/Glu in the parieto-occipital cortex in an magnetic resonance spectroscopy-genetics dataset optimized for Gln/Glu detection (n = 42). This finding was replicated in a second magnetic resonance spectroscopy dataset that was optimized for ?-aminobutyric acid detection where Gln and Glu measurements could still be extracted (n = 40).These findings suggest that genetic variation in a key component of glutamatergic machinery is associated with a putative in vivo index of glutamatergic neurotransmission. Thus, GLS1 genotype might provide insight into normal brain function and into the pathophysiology of many psychiatric conditions where glutamatergic neurotransmission has been implicated. It might also serve as a biomarker for predicting response to existing and novel therapeutic interventions in psychiatry that target glutamatergic neurotransmission.
Project description:The targeting of glutamine metabolism specifically via pharmacological inhibition of glutaminase 1 (GLS1) has been translated into clinical trials as a novel therapy for several cancers. The results, though encouraging, show room for improvement in terms of tumor reduction. In this study, the glutaminase II pathway is found to be upregulated for glutamate production upon GLS1 inhibition in pancreatic tumors. Moreover, genetic suppression of glutamine transaminase K (GTK), a key enzyme of the glutaminase II pathway, leads to the complete inhibition of pancreatic tumorigenesis in vivo unveiling GTK as a new metabolic target for cancer therapy. These results suggest that current trials using GLS1 inhibition as a therapeutic approach targeting glutamine metabolism in cancer should take into account the upregulation of other metabolic pathways that can lead to glutamate production; one such pathway is the glutaminase II pathway via GTK.
Project description:Glutaminase mediates the recycling of neurotransmitter glutamate, supporting most excitatory neurotransmission in the mammalian central nervous system. A constitutive heterozygous reduction in GLS1 engenders in mice a model of schizophrenia resilience and associated increases in Gln, reductions in Glu and activity-dependent attenuation of excitatory synaptic transmission. Hippocampal brain slices from GLS1 heterozygous mice metabolize less Gln to Glu. Whether glutaminase activity is diminished in the intact brain in GLS1 heterozygous mice has not been assessed, nor the regional impact. Moreover, it is not known whether pharmacological inhibition would mimic the genetic reduction. We addressed this using magnetic resonance spectroscopy to assess amino acid content and 13C-acetate loading to assess glutaminase activity, in multiple brain regions. Glutaminase activity was reduced significantly in the hippocampus of GLS1 heterozygous mice, while acute treatment with the putative glutaminase inhibitor ebselen did not impact glutaminase activity, but did significantly increase GABA. This approach identifies a molecular imaging strategy for testing target engagement by comparing genetic and pharmacological inhibition, across brain regions.
Project description:BACKGROUND: Accumulating evidence suggests that dysfunction in the glutamatergic system may underlie the pathophysiology of autism. The anterior cingulate cortex (ACC) has been implicated in autism as well as in glutamatergic neurotransmission. We hypothesized that alterations in the glutamate-glutamine cycle in the ACC might play a role in the pathophysiology of autism. METHODS: We performed Western blot analyses for the protein expression levels of enzymes in the glutamate-glutamine cycle, including glutamine synthetase, kidney-type glutaminase, liver-type glutaminase, and glutamate dehydrogenases 1 and 2, in the ACC of postmortem brain of individuals with autism (n?=?7) and control subjects (n?=?13). RESULTS: We found that the protein levels of kidney-type glutaminase, but not those of the other enzymes measured, in the ACC were significantly lower in subjects with autism than in controls. CONCLUSION: The results suggest that reduced expression of kidney-type glutaminase may account for putative alterations in glutamatergic neurotransmission in the ACC in autism.
Project description:HIV-1 associated neurocognitive disorders (HAND) develop during progressive HIV-1 infection and affect up to 50% of infected individuals. Activated microglia and macrophages are critical cell populations that are involved in the pathogenesis of HAND, which is specifically related to the production and release of various soluble neurotoxic factors including glutamate. In the central nervous system (CNS), glutamate is typically derived from glutamine by mitochondrial enzyme glutaminase. Our previous study has shown that glutaminase is upregulated in HIV-1 infected monocyte-derived-macrophages (MDM) and microglia. However, how HIV-1 leads to glutaminase upregulation, or how glutaminase expression is regulated in general, remains unclear. In this study, using a dual-luciferase reporter assay system, we demonstrated that interferon (IFN) ? specifically activated the glutaminase 1 (GLS1) promoter. Furthermore, IFN-? treatment increased signal transducer and activator of transcription 1 (STAT1) phosphorylation and glutaminase mRNA and protein levels. IFN-? stimulation of GLS1 promoter activity correlated to STAT1 phosphorylation and was reduced by fludarabine, a chemical that inhibits STAT1 phosphorylation. Interestingly, STAT1 was found to directly bind to the GLS1 promoter in MDM, an effect that was dependent on STAT1 phosphorylation and significantly enhanced by IFN-? treatment. More importantly, HIV-1 infection increased STAT1 phosphorylation and STAT1 binding to the GLS1 promoter, which was associated with increased glutamate levels. The clinical relevance of these findings was further corroborated with investigation of post-mortem brain tissues. The glutaminase C (GAC, one isoform of GLS1) mRNA levels in HIV associated-dementia (HAD) individuals correlate with STAT1 (p<0.01), IFN-? (p<0.05) and IFN-? (p<0.01). Together, these data indicate that both HIV-1 infection and IFN-? treatment increase glutaminase expression through STAT1 phosphorylation and by binding to the GLS1 promoter. Since glutaminase is a potential component of elevated glutamate production during the pathogenesis of HAND, our data will help to identify additional therapeutic targets for the treatment of HAND.
Project description:Glutaminase, which converts glutamine to glutamate, is involved in Warburg effect in cancer cells. Two human glutaminase genes have been identified, GLS (GLS1) and GLS2. Two alternative transcripts arise from each glutaminase gene: first, the kidney isoform (KGA) and glutaminase C (GAC) for GLS; and, second, the liver isoform (LGA) and glutaminase B (GAB) for GLS2. While GLS1 is considered as a cancer therapeutic target, the potential role of GLS2 in cancer remains unclear. Here, we discovered a series of alkyl benzoquinones that preferentially inhibit glutaminase B isoform (GAB, GLS2) rather than the kidney isoform of glutaminase (KGA, GLS1). We identified amino acid residues in an allosteric binding pocket responsible for the selectivity. Treatment with the alkyl benzoquinones decreased intracellular glutaminase activity and glutamate levels. GLS2 inhibition by either alkyl benzoquinones or GLS2 siRNA reduced carcinoma cell proliferation and anchorage-independent colony formation, and induced autophagy via AMPK mediated mTORC1 inhibition. Our findings demonstrate amino acid sequences for selective inhibition of glutaminase isozymes and validate GLS2 as a potential anti-cancer target.
Project description:Glutaminase 1 (GLS1) expression is increased in non-small cell lung cancer (NSCLC). GLS1 knockdown using siRNA or inhibition using bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) induced cell cycle arrest with significant reduction of ATP level while levels of reactive oxygen species or glutathione were not affected in NSCLC cell lines. Recently we found that NSCLC significantly depends on cytosol NADH for ATP production. GLS1 remarkably contributes to ATP production through transferring cytosolic NADH into mitochondria via malate-aspartate shuttle by supply of glutamate in NSCLC. Regulation of malate-aspartate shuttle by knockdown or inhibition of glutamic-oxaloacetic transaminase 2 or malate dehydrogenase 2 mimicked GLS1 knockdown, which induced cell death with ATP reduction in NSCLC. Therefore, GLS1 inhibition induced cell cycle arrest with ATP depletion by glutamate reduction. Dual inhibition with BPTES and thymidylate synthase inhibitor, 5-fluorouracil (5-FU), elicits cell death synergistically through cell cycle arrest in NSCLC. A preclinical xenograft model of NSCLC showed remarkable anti-tumour effect synergistically in the BPTES and 5-FU dual therapy group.