Effector Repertoire of the Sweetpotato Black Rot Fungal Pathogen Ceratocystis fimbriata.

In 2015, sweetpotato producers in the United States experienced one of the worst outbreaks of black rot recorded in history, with up to 60% losses reported in the field and packing houses and at shipping ports. Host resistance remains the ideal management tool to decrease crop losses. Lack of knowledge of Ceratocystis fimbriata biology represents a critical barrier for the deployment of resistance to black rot in sweetpotato. In this study, we scanned the recent near chromosomal-level assembly for putative secreted effectors in the sweetpotato C. fimbriata isolate AS236 using a custom fungal effector annotation pipeline. We identified a set of 188 putative effectors on the basis of secretion signal and in silico prediction in EffectorP. We conducted a deep RNA time-course sequencing experiment to determine whether C. fimbriata modulates effectors in planta and to define a candidate list of effectors expressed during infection. We examined the expression profile of two C. fimbriata isolates, a pre-epidemic (1990s) isolate and a post-epidemic (2015) isolate. Our in planta expression profiling revealed clusters of co-expressed secreted effector candidates. Based on fold-change differences of putative effectors in both isolates and over the course of infection, we suggested prioritization of 31 effectors for functional characterization. Among this set, we identified several effectors that provide evidence for a marked biotrophic phase in C. fimbriata during infection of sweetpotato storage roots. Our study revealed a catalog of effector proteins that provide insight into C. fimbriata infection mechanisms and represent a core catalog to implement effector-assisted breeding in sweetpotato. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Syngap1 regulates experience-dependent cortical ensemble plasticity by promoting in vivo excitatory synapse strengthening.

A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing-dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.

Phytophthora capsici, 100 Years Later: Research Mile Markers from 1922 to 2022.

In 1922, Phytophthora capsici was described by Leon Hatching Leonian as a new pathogen infecting pepper (Capsicum annuum), with disease symptoms of root rot, stem and fruit blight, seed rot, and plant wilting and death. Extensive research has been conducted on P. capsici over the last 100 years. This review succinctly describes the salient mile markers of research on P. capsici with current perspectives on the pathogen's distribution, economic importance, epidemiology, genetics and genomics, fungicide resistance, host susceptibility, pathogenicity mechanisms, and management.

Phytophthora capsici Populations Are Structured by Host, Geography, and Fluopicolide Sensitivity.

Phytophthora capsici epidemics are propelled by warm temperatures and wet conditions. With temperatures and inland flooding in many locations worldwide expected to rise as a result of global climate change, understanding of population structure can help to inform management of P. capsici in the field and prevent devastating epidemics. Thus, we investigated the effect of host crop, geographical origin, fungicide sensitivity, and mating type on shaping the population structure of P. capsici in the eastern United States. Our fungicide in vitro assays identified the emergence of insensitive isolates for fluopicolide and mefenoxam. A set of 12 microsatellite markers proved informative to assign 157 P. capsici isolates to five distinct genetic clusters. Implementation of Bayesian structure, population differentiation, genetic diversity statistics, and index of association analysis, allowed us to identify population structure by host with some correspondence with genetic clusters for cucumber and squash isolates. We found weak population structure by state for geographically close isolates. In this study, we discovered that North Carolina populations stratify by fluopicolide sensitivity with insensitive isolates experiencing nonrandom mating. Our findings highlight the need for careful monitoring of local field populations, improved selection of relevant isolates for breeding efforts, and hypervigilant surveillance of resistance to different fungicides.

SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.

SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.

Allosteric kidney-type glutaminase (GLS) inhibitors with a mercaptoethyl linker.

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl (SCH2CH2) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC50 value of 50 nM. Interestingly, the corresponding derivative with an n-propyl (CH2CH2CH2) linker showed substantially lower inhibitory potency (IC50 = 2.3 µM) while the derivative with a dimethylsulfide (CH2SCH2) linker showed no inhibitory activity at concentrations up to 100 µM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety.

Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors.

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders.

SYNGAP1 loss-of-function variants are causally associated with intellectual disability, severe epilepsy, autism spectrum disorder and schizophrenia. While there are hundreds of genetic risk factors for neurodevelopmental disorders (NDDs), this gene is somewhat unique because of the frequency and penetrance of loss-of-function variants found in patients combined with the range of brain disorders associated with SYNGAP1 pathogenicity. These clinical findings indicate that SYNGAP1 regulates fundamental neurodevelopmental processes that are necessary for brain development. Here, we describe four phenotypic domains that are controlled by Syngap1 expression across vertebrate species. Two domains, the maturation of cognitive functions and maintenance of excitatory-inhibitory balance, are defined exclusively through a review of the current literature. Two additional domains are defined by integrating the current literature with new data indicating that SYNGAP1/Syngap1 regulates innate survival behaviors and brain structure. These four phenotypic domains are commonly disrupted in NDDs, suggesting that a deeper understanding of developmental Syngap1 functions will be generalizable to other NDDs of known or unknown etiology. Therefore, we discuss the known molecular and cellular functions of Syngap1 and consider how these functions may contribute to the emergence of disease-relevant phenotypes. Finally, we identify major unexplored areas of Syngap1 neurobiology and discuss how a deeper understanding of this gene may uncover general principles of NDD pathobiology.

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Enhanced Brain Delivery of 2-(Phosphonomethyl)pentanedioic Acid Following Intranasal Administration of Its γ-Substituted Ester Prodrugs.

2-(Phosphonomethyl)pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II (GCPII) with efficacy in multiple neurological and psychiatric disease models, but its clinical utility is hampered by low brain penetration due to the inclusion of multiple acidic functionalities. We recently reported an improvement in the brain-to-plasma ratio of 2-PMPA after intranasal (IN) dosing in both rodents and primates. Herein, we describe the synthesis of several 2-PMPA prodrugs with further improved brain delivery of 2-PMPA after IN administration by masking of the γ-carboxylate. When compared to IN 2-PMPA in rats at 1 h post dose, γ-(4-acetoxybenzyl)-2-PMPA (compound 1) resulted in significantly higher 2-PMPA delivery to both plasma (4.1-fold) and brain (11-fold). Subsequent time-dependent evaluation of 1 also showed high brain as well as plasma 2-PMPA exposures with brain-to-plasma ratios of 2.2, 0.48, and 0.26 for olfactory bulb, cortex, and cerebellum, respectively, as well as an improved sciatic nerve to plasma ratio of 0.84. In contrast, IV administration of compound 1 resulted in similar plasma exposure of 2-PMPA versus the IN route (AUCIV: 76 ± 9 h·nmol/mL versus AUCIN: 99 ± 24 h·nmol/mL); but significantly lower nerve and brain tissue exposures with tissue-to-plasma ratios of 0.21, 0.03, 0.04, and 0.04 in nerve, olfactory bulb, cortex, and cerebellum, respectively. In primates, IN administration of 1 more than doubled 2-PMPA concentrations in the cerebrospinal fluid relative to previously reported levels following IN 2-PMPA. The results of these experiments provide a promising strategy for testing GCPII inhibition in neurological and psychiatric disorders.

Maternal inflammation leads to impaired glutamate homeostasis and up-regulation of glutamate carboxypeptidase II in activated microglia in the fetal/newborn rabbit brain.

Astrocyte dysfunction and excessive activation of glutamatergic systems have been implicated in a number of neurologic disorders, including periventricular leukomalacia (PVL) and cerebral palsy (CP). However, the role of chorioamnionitis on glutamate homeostasis in the fetal and neonatal brains is not clearly understood. We have previously shown that intrauterine endotoxin administration results in intense microglial 'activation' and increased pro-inflammatory cytokines in the periventricular region (PVR) of the neonatal rabbit brain. In this study, we assessed the effect of maternal inflammation on key components of the glutamate pathway and its relationship to astrocyte and microglial activation in the fetal and neonatal New Zealand white rabbit brain. We found that intrauterine endotoxin exposure at gestational day 28 (G28) induced acute and prolonged glutamate elevation in the PVR of fetal (G29, 1day post-injury) and postnatal day 1 (PND1, 3days post-injury) brains along with prominent morphological changes in the astrocytes (soma hypertrophy and retracted processes) in the white matter tracts. There was a significant increase in glutaminase and N-Methyl-d-Aspartate receptor (NMDAR) NR2 subunit expression along with decreased glial L-glutamate transporter 1 (GLT-1) in the PVR at G29, that would promote acute dysregulation of glutamate homeostasis. This was accompanied with significantly decreased TGF-ß1 at PND1 in CP kits indicating ongoing neuroinflammation. We also show for the first time that glutamate carboxypeptidase II (GCPII) was significantly increased in the activated microglia at the periventricular white matter area in both G29 and PND1 CP kits. This was confirmed by in vitro studies demonstrating that LPS activated primary microglia markedly upregulate GCPII enzymatic activity. These results suggest that maternal intrauterine endotoxin exposure results in early onset and long-lasting dysregulation of glutamate homeostasis, which may be mediated by impaired astrocyte function and GCPII upregulation in activated microglia.

Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

D-Amino acid oxidase inhibitors based on the 5-hydroxy-1,2,4-triazin-6(1H)-one scaffold.

A series of 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one derivatives were designed and synthesized as a new class of d-amino acid oxidase (DAAO) inhibitors. Some of the newly synthesized derivatives showed potent inhibitory activity against human DAAO with IC50 values in the nanomolar range. Among them, 6-hydroxy-3-phenethyl-1,2,4-triazin-5(2H)-one 6b and 3-((6-fluoronaphthalen-2-yl)methylthio)-6-hydroxy-1,2,4-triazin-5(2H)-one 6m were found to be metabolically stable in mouse liver microsomes. In addition, compound 6b was found to be orally available in mice and able to enhance plasma d-serine levels following its co-administration with d-serine compared to the oral administration of d-serine alone.

Input-specific regulation of hippocampal circuit maturation by non-muscle myosin IIB.

Myh9 and Myh10, which encode two major isoforms of non-muscle myosin II expressed in the brain, have emerged as risk factors for developmental brain disorders. Myosin II motors regulate neuronal cytoskeletal dynamics leading to optimization of synaptic plasticity and memory formation. However, the role of these motor complexes in brain development remains poorly understood. Here, we disrupted the in vivo expression of Myh9 and/or Myh10 in developing hippocampal neurons to determine how these motors contribute to circuit maturation in this brain area important for cognition. We found that Myh10 ablation in early postnatal, but not mature, CA1 pyramidal neurons reduced excitatory synaptic function in the Schaffer collateral pathway, whereas more distal inputs to CA1 neurons were relatively unaffected. Myh10 ablation in young neurons also selectively impaired the elongation of oblique dendrites that receive Schaffer collateral inputs, whereas the structure of distal dendrites was normal. We observed normal spine density and spontaneous excitatory currents in these neurons, indicating that Myh10 KO impaired proximal pathway synaptic maturation through disruptions to dendritic development rather than post-synaptic strength or spine morphogenesis. To address possible redundancy and/or compensation by other Myosin II motors expressed in neurons, we performed similar experiments in Myh9 null neurons. In contrast to findings in Myh10 mutants, evoked synaptic function in young Myh9 KO hippocampal neurons was normal. Data obtained from double Myh9/Myh10 KO neurons largely resembled the MyH10 KO synaptic phenotype. These data indicate that Myosin IIB is a key molecular factor that guides input-specific circuit maturation in the developing hippocampus. Non-muscle myosin II is an actin binding protein with three isoforms in the brain (IIA, IIB and IIC) encoded by the myh9, myh10, and myh14 genes in mice, respectively. We have studied the structure and the function of hippocampal CA1 neurons missing NMIIB and/or NMIIA proteins at different times during development. We have discovered that NMIIB is the major isoform regulating Schaffer collateral inputs, and that this regulation is restricted to early postnatal development.

Neurological sequelae induced by alphavirus infection of the CNS are attenuated by treatment with the glutamine antagonist 6-diazo-5-oxo-l-norleucine.

Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits. Infection of mice with the prototypic alphavirus, Sindbis virus, provides an animal model with which to study disease mechanisms and examine potential therapeutics. Infectious virus is cleared from the brain within a week after infection, but viral RNA is cleared slowly and persists for the life of the animal. However, no studies have examined the effect of infection on neurocognitive function over time. In the present study, we examined neurocognitive function at different phases of infection in 5-week-old C57BL/6 mice intranasally inoculated with Sindbis virus. At the peak of active virus infection, mice demonstrated hyperactivity, decreased anxiety, and marked hippocampal-dependent memory deficits, the latter of which persisted beyond clearance of infectious virus and resolution of clinical signs of disease. Previous studies indicate that neuronal damage during alphavirus encephalomyelitis is primarily due to inflammatory cell infiltration and glutamate excitotoxicity rather than directly by virus infection. Therefore, mice were treated with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist that can suppress both the immune response and excitotoxicity. Treatment with DON decreased inflammatory cell infiltration and cell death in the hippocampus and partially prevented development of clinical signs and neurocognitive impairment despite the presence of infectious virus and high viral RNA levels. This study presents the first report of neurocognitive sequelae in mice with alphavirus encephalomyelitis and provides a model system for further elucidation of the pathogenesis of virus infection and assessment of potential therapies.

Bioanalysis of 6-diazo-5-oxo-l-norleucine in plasma and brain by ultra-performance liquid chromatography mass spectrometry.

Glutamine is an abundant amino acid that plays pivotal roles in cell growth, cell metabolism, and neurotransmission. Dysregulation of glutamine-using pathways has been associated with pathological conditions such as cancer and neurodegenerative diseases. 6-Diazo-5-oxo-l-norleucine (DON) is a reactive glutamine analog that inhibits enzymes affecting glutamine metabolism such as glutaminase, 2-N-amidotransferase, l-asparaginase, and several enzymes involved in pyrimidine and purine de novo synthesis. As a result, DON is actively used in preclinical models of cancer and neurodegenerative disease. Moreover, there have been several clinical trials using DON to treat a variety of cancers. Considerations of dose and exposure are especially important with DON treatment due to its narrow therapeutic window and significant side effects. Consequently, a robust quantification bioassay is of interest. DON is a polar unstable molecule that has made quantification challenging. Here we report on the characterization of a bioanalytical method to quantify DON in tissue samples involving DON derivatization with 3 N HCl in butanol. The derivatized product is lipophilic and stable. Detection of this analyte by mass spectrometry is fast and specific and can be used to quantify DON in plasma and brain tissue with a limit of detection at the low nanomolar level.

Inhibition of glutamate carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis.

Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylglutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated glutamate carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function.

Small molecule glutaminase inhibitors block glutamate release from stimulated microglia.

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.

Mutant disrupted-in-schizophrenia 1 in astrocytes: focus on glutamate metabolism.

Disrupted-in-schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase to regulate production of D-serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, reuptake, or secretion of GLU remains unexplored. Therefore, we studied the effects of dominant-negative mutant DISC1 on various aspects of GLU metabolism by using primary astrocyte cultures and hippocampal tissue from transgenic mice with astrocyte-restricted expression of mutant DISC1. Although mutant DISC1 had no significant effects on astrocyte proliferation, GLU reuptake, glutaminase, or glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine-serine-cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus, and elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of N-methyl-D-aspartate (NMDA) receptors in the hippocampus, at postnatal day 21. Our findings indicate that decreased D-serine production by astrocytic mutant DISC1 might lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse.

Regional brain distribution of translocator protein using [(11)C]DPA-713 PET in individuals infected with HIV.

Imaging the brain distribution of translocator protein (TSPO), a putative biomarker for glial cell activation and neuroinflammation, may inform management of individuals infected with HIV by uncovering regional abnormalities related to neurocognitive deficits and enable non-invasive therapeutic monitoring. Using the second-generation TSPO-targeted radiotracer, [(11)C]DPA-713, we conducted a positron emission tomography (PET) study to compare the brains of 12 healthy human subjects to those of 23 individuals with HIV who were effectively treated with combination antiretroviral therapy (cART). Compared to PET data from age-matched healthy control subjects, [(11)C]DPA-713 PET of individuals infected with HIV demonstrated significantly higher volume-of-distribution (VT) ratios in white matter, cingulate cortex, and supramarginal gyrus, relative to overall gray matter VT, suggesting localized glial cell activation in susceptible regions. Regional TSPO abnormalities were evident within a sub-cohort of neuro-asymptomatic HIV subjects, and an increase in the VT ratio within frontal cortex was specifically linked to individuals affected with HIV-associated dementia. These findings were enabled by employing a gray matter normalization approach for PET data quantification, which improved test-retest reproducibility, intra-class correlation within the healthy control cohort, and sensitivity of uncovering abnormal regional findings.