Genetic Pharmacotherapy as an Early CNS Drug Development Strategy: Testing Glutaminase Inhibition for Schizophrenia Treatment in Adult Mice

Genetic pharmacotherapy is an early drug development strategy for the identification of novel CNS targets in mouse models prior to the development of specific ligands. Here for the first time, we have implemented this strategy to address the potential therapeutic value of a glutamate-based pharmacotherapy for schizophrenia involving inhibition of the glutamate recycling enzyme phosphate-activated glutaminase. Mice constitutively heterozygous for GLS1, the gene encoding glutaminase, manifest a schizophrenia resilience phenotype, a key dimension of which is an attenuated locomotor response to propsychotic amphetamine challenge. If resilience is due to glutaminase deficiency in adulthood, then glutaminase inhibitors should have therapeutic potential. However, this has been difficult to test given the dearth of neuroactive glutaminase inhibitors. So, we used genetic pharmacotherapy to ask whether adult induction of GLS1 heterozygosity would attenuate amphetamine responsiveness. We generated conditional floxGLS1 mice and crossed them with global CAGERT2cre∕+ mice to produce GLS1 iHET mice, susceptible to tamoxifen induction of GLS1 heterozygosity. One month after tamoxifen treatment of adult GLS1 iHET mice, we found a 50% reduction in GLS1 allelic abundance and glutaminase mRNA levels in the brain. While GLS1 iHET mice showed some recombination prior to tamoxifen, there was no impact on mRNA levels. We then asked whether induction of GLS heterozygosity would attenuate the locomotor response to propsychotic amphetamine challenge. Before tamoxifen, control and GLS1 iHET mice did not differ in their response to amphetamine. One month after tamoxifen treatment, amphetamine-induced hyperlocomotion was blocked in GLS1 iHET mice. The block was largely maintained after 5 months. Thus, a genetically induced glutaminase reduction—mimicking pharmacological inhibition—strongly attenuated the response to a propsychotic challenge, suggesting that glutaminase may be a novel target for the pharmacotherapy of schizophrenia. These results demonstrate how genetic pharmacotherapy can be implemented to test a CNS target in advance of the development of specific neuroactive inhibitors. We discuss further the advantages, limitations, and feasibility of the wider application of genetic pharmacotherapy for neuropsychiatric drug development

Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

Neuronal epigenomes, including chromosomal loopings moving distal cis-regulatory elements into proximity of target genes, could serve as molecular proxy linking present-day-behaviour to past exposures. However, longitudinal assessment of chromatin state is challenging, because conventional chromosome conformation capture assays essentially provide single snapshots at a given time point, thus reflecting genome organization at the time of brain harvest and therefore are non-informative about the past. Here we introduce ‘NeuroDam’ to assess epigenome status retrospectively. Short-term expression of the bacterial DNA adenine methyltransferase Dam, tethered to the Gad1 gene promoter in mouse prefrontal cortex neurons, results in stable G[superscriptmethyl]ATC tags at Gad1-bound chromosomal contacts. We show by NeuroDam that mice with defective cognition 4 months after pharmacological NMDA receptor blockade already were affected by disrupted chromosomal conformations shortly after drug exposure. Retrospective profiling of neuronal epigenomes is likely to illuminate epigenetic determinants of normal and diseased brain development in longitudinal context.United States. National Institutes of Healt

Beyond NMDA Receptors: Homeostasis at the Glutamate Tripartite Synapse and Its Contributions to Cognitive Dysfunction in Schizophrenia

Cognitive deficits are core symptoms of schizophrenia but remain poorly addressed by dopamine-based antipsychotic medications. Glutamate abnormalities are implicated in schizophrenia-related cognitive deficits. While the role of the NMDA receptor has been extensively studied, less attention was given to other components that control glutamate homeostasis. Glutamate dynamics at the tripartite synapse include presynaptic and postsynaptic components and are tightly regulated by neuron–astrocyte crosstalk. Here, we delineate the role of glutamate homeostasis at the tripartite synapse in schizophrenia-related cognitive dysfunction. We focus on cognitive domains that can be readily measured in humans and rodents, i.e., working memory, recognition memory, cognitive flexibility, and response inhibition. We describe tasks used to measure cognitive function in these domains in humans and rodents, and the relevance of glutamate alterations in these domains. Next, we delve into glutamate tripartite synaptic components and summarize findings that implicate the relevance of these components to specific cognitive domains. These collective findings indicate that neuron–astrocyte crosstalk at the tripartite synapse is essential for cognition, and that pre- and postsynaptic components play a critical role in maintaining glutamate homeostasis and cognitive well-being. The contribution of these components to cognitive function should be considered in order to better understand the role played by glutamate signaling in cognition and develop efficient pharmacological treatment avenues for schizophrenia treatment-resistant symptoms

Hippocampal glutamate NMDA receptor loss tracks progression in Alzheimer's disease: quantitative autoradiography in postmortem human brain.

Early Alzheimer's disease (AD) is characterized by memory loss and hippocampal atrophy with relative sparing of basal ganglia. Activation of glutamate NMDA receptors in the hippocampus is an important step in memory formation. We measured the density of NMDA receptors in samples of hippocampus, entorhinal cortex and basal ganglia obtained from subjects who died with pathologically confirmed AD and age- and sex- matched non-demented controls. We found significant decreases in NMDA receptor density in the hippocampus and entorhinal cortex but not in the basal ganglia. Loss of NMDA receptors was significantly correlated with neuropathological progression as assessed by Braak staging postmortem. The same samples were probed for neuroinflammation by measuring the density and gene expression of translocator protein 18 kDA (TSPO), an established marker of microglial activation. Unlike NMDA receptor loss, increased densities of TSPO were found in all of the brain regions sampled. However hippocampal, but not striatal TSPO density and gene expression were inversely correlated with NMDA receptor density and positively correlated with Braak stage, suggesting NMDA receptors exacerbate neuroniflammatory damage. The high correlation between hippocampal NMDA receptor loss and disease progression supports the use of non invasive imaging with NMDA receptor tracers and positron emission tomography as a superior method for diagnosis, staging and treatment monitoring of AD in vivo

Regional NMDA receptor density in AD and control subjects measured with [³H]MK801 autoradiography.

<div><p>A. Representative autoradiographic and anatomical images. </p> <p>Top –Hippocampal anatomy is shown on the left followed by a representative pseudocolored NMDAR autoradiogram from hippocampus of a control subject and a representative autoradiogram from hippocampus of a subject with AD. The bottom row depicts striatal anatomy on the left followed by a representative NMDAR autoradiogram from a control striatum and a representative autoradiogram from a subject with AD. Autoradiograms were pseudocolored using the rainbow spectrum (bar on the right).</p> <p>B. Quantitative autoradiographic measurements of regional NMDAR density (expressed as specific binding of [³H]MK801 in nCi/mg tissue).</p> <p>Abbreviations: CA1-4 cornu ammoni fields of the hippocampus, DG=gentate gyrus, SubP=subpyramidal layers of CA1(including stratum radiatum, lacunosum, and moleculare),, Sub=subiculum, ECx=entorhinal cortex, Str=striatum. </p> <p>*p<0.05, **p<0.005 AD relative to control; ANOVA followed by Fisher’s PLSD posthoc test. </p> <p>C. NMDAR density in the CA1 pyramidal layer as a function of Braak stage (left) and genotype (right). </p> <p>*p<0.05 relative to Braak stages 0-II, one way ANOVA followed by Fisher’s PLSD posthoc test.</p> <p>**p<0.005, student’s t test. </p></div

Regional neuroinflammation in AD subjects and controls measured with [³H]PK11195 autoradiography.

<div><p>A. Representative autoradiographic and anatomical images. </p> <p>Top –Hippocampal anatomy is shown on the left followed by a representative pseudocolored TSPO autoradiogram from hippocampus of a control subject and a representative autoradiogram from hippocampus of a subject with AD. The bottom row depicts striatal anatomy on the left followed by a representative TSPO autoradiogram from a control striatum and a representative autoradiogram from a subject with AD. </p> <p>Autoradiograms were pseudocolored using the rainbow spectrum (bar on the right).</p> <p>B. Quantitative autoradiographic measurements of regional TSPO density (expressed as specific binding of [3H]PK11195 in nCi/mg tissue).</p> <p>Abbreviations: CA1-4 cornu ammoni fields of the hippocampus, DG=gentate gyrus, SubP=subpyramidal layers of CA1, Sub=subiculum, ECx=entorhinal cortex, Str=striatum. </p> <p>*p<0.05, **p<0.005 AD relative to control; ANOVA followed by Fisher’s PLSD posthoc test. </p> <p>C. TSPO density in CA1 pyramidal cell body layer as a function of Braak stage (left) and genotype (right). </p> <p>*p<0.05 relative to Braak stages 0-II, one way ANOVA followed by Fisher’s PLSD posthoc test.</p> <p>**p<0.005, student’s t test. </p></div

Regional TSPO mRNA gene expression in relation to AD pathology and ApoE genotype.

<div><p>A. TSPO gene expression by region and disease state. TSPO mRNA gene expression was measured by RT-PCR and expressed as fold change over expression in the reference sample. **p<0.005.</p> <p>B. TSPO gene expression as a function of Braak stage in hippocampus (left) and striatum (right).</p> <p>*p<0.05 relative to Braak stages 0-II, one way ANOVA followed by Fisher’s PLSD posthoc test.</p> <p>C. Effect of genotype on TSPO gene expression in hippocampus (left) and striatum (right).</p> <p>*p<0.05, students t-test 2 tailed.</p></div

Region dependent correlation between TSPO and NMDAR density.

<div><p>Quantitative analyses of [³H]MK801 binding to NMDAR and [³H]PK11195 binding to TSPO were performed by autoradiography. The correlation between NMDAR and TSPO density was negative in the CA1 hippocampal field (left, <i>n</i> = 38, Spearman’s <i>p</i> < 0.001) and positive in the striatum (right, <i>n</i> = 37, Spearman’s <i>p</i> < 0.001).</p> <p>SB=specific binding.</p></div