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.

In vitro evaluation of the activity of teriflunomide against SARS-CoV-2 and the human coronaviruses 229E and OC43.

Previous data have suggested an antiviral effect of teriflunomide, including against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the agent underlying the ongoing COVID-19 pandemic. We undertook an in vitro investigation to evaluate the inhibitory activity of teriflunomide against SARS-CoV-2 in a cell-based assay. Teriflunomide was added to Vero (kidney epithelial) cells that had been infected with SARS-CoV-2. A nucleocapsid immunofluorescence assay was performed to examine viral inhibition with teriflunomide and any potential cytotoxic effect. The 50% effective concentration (EC50) for teriflunomide against SARS-CoV-2 was 15.22 µM. No cytotoxicity was evident for teriflunomide in the Vero cells (i.e., the 50% cytotoxic concentration [CC50] was greater than the highest test concentration of 100 µM). The data were supported by additional experiments using other coronaviruses and human cell lines. In the SARS-CoV-2-infected Vero cells, the prodrug leflunomide had an EC50 of 16.49 µM and a CC50 of 54.80 µM. Our finding of teriflunomide-mediated inhibition of SARS-CoV-2 infection at double-digit micromolar potency adds to a growing body of evidence for a broad-ranging antiviral effect of teriflunomide.

Glutamine antagonist-mediated immune suppression decreases pathology but delays virus clearance in mice during nonfatal alphavirus encephalomyelitis.

Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.

Glutamate carboxypeptidase II inhibition behaviorally and physiologically improves pyridoxine-induced neuropathy in rats.

Pyridoxine is used as a supplement for treating conditions such as vitamin deficiency as well as neurological disorders such as depression, epilepsy and autism. A significant neurologic complication of pyridoxine therapy is peripheral neuropathy thought to be a result of long-term and high dose usage. Although pyridoxine-induced neuropathy is transient and can remit after its withdrawal, the process of complete recovery can be slow. Glutamate carboxypeptidase II (GCP II) inhibition has been shown to improve symptoms of both chemotherapy- and diabetic-induced neuropathy. This study evaluated if GCP II inhibition could behaviorally and physiologically improve pyridoxine-induced neuropathy. In the current study, high doses of pyridoxine (400 mg/kg, twice a day for seven days) were used to induce neuropathy in rats. An orally bioavailable GCP II inhibitor, 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), was administered daily at a dose of 30 mg/kg starting from the onset of pyridoxine injections. Body weight, motor coordination, heat sensitivity, electromyographical (EMG) parameters and nerve morphological features were monitored. The results show beneficial effects of GCP II inhibition including normalization of hot plate reaction time, foot fault improvements and increased open field distance travelled. H wave frequency, amplitude and latency as well as sensory nerve conduction velocity (SNCV) were also significantly improved by 2-MPPA. Lastly, GCP II inhibition resulted in morphological protection in the spinal cord and sensory fibers in the lumbar region dorsal root ganglia (DRG). In conclusion, inhibition of GCP II may be beneficial against the peripheral sensory neuropathy caused by pyridoxine.

Histone deacetylase inhibition rescues structural and functional brain deficits in a mouse model of Kabuki syndrome.

Kabuki syndrome is caused by haploinsufficiency for either of two genes that promote the opening of chromatin. If an imbalance between open and closed chromatin is central to the pathogenesis of Kabuki syndrome, agents that promote chromatin opening might have therapeutic potential. We have characterized a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), leading to impairment of methyltransferase function. In vitro reporter alleles demonstrated a reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) activity in mouse embryonic fibroblasts from Kmt2d(+/ßGeo) mice. These activities were normalized in response to AR-42, a histone deacetylase inhibitor. In vivo, deficiency of H3K4me3 in the dentate gyrus granule cell layer of Kmt2d(+/ßGeo) mice correlated with reduced neurogenesis and hippocampal memory defects. These abnormalities improved upon postnatal treatment with AR-42. Our work suggests that a reversible deficiency in postnatal neurogenesis underlies intellectual disability in Kabuki syndrome.

All about running: synaptic plasticity, growth factors and adult hippocampal neurogenesis.

Accumulating evidence from animal and human research shows exercise benefits learning and memory, which may reduce the risk of neurodegenerative diseases, and could delay age-related cognitive decline. Exercise-induced improvements in learning and memory are correlated with enhanced adult hippocampal neurogenesis and increased activity-dependent synaptic plasticity. In this present chapter we will highlight the effects of physical activity on cognition in rodents, as well as on dentate gyrus (DG) neurogenesis, synaptic plasticity, spine density, neurotransmission and growth factors, in particular brain-derived nerve growth factor (BDNF).

Prolonged running, not fluoxetine treatment, increases neurogenesis, but does not alter neuropathology, in the 3xTg mouse model of Alzheimer's disease.

Reductions in adult neurogenesis have been documented in the original 3xTg mouse model of Alzheimer's disease (AD), notably occurring at the same age when spatial memory deficits and amyloid plaque pathology appeared. As this suggested reduced neurogenesis was associated with behavioral deficits, we tested whether activity and pharmacological stimulation could prevent memory deficits and modify neurogenesis and/or neuropathology in the 3xTg model backcrossed to the C57Bl/6 strain. We chronically administered the antidepressant fluoxetine to one group of mice, allowed access to a running wheel in another, and combined both treatments in a third cohort. All treatments lasted for 11 months. The female 3xTg mice failed to exhibit any deficits in spatial learning and memory as measured in the Morris water maze, indicating that when backcrossed to the C57Bl/6 strain, the 3xTg mice lost the behavioral phenotype that was present in the original 3xTg mouse maintained on a hybrid background. Despite this, the backcrossed 3xTg mice expressed prominent intraneuronal amyloid beta (Aß) levels in the cortex and amygdala, with lower levels in the CA1 area of the hippocampus. In the combined cohort, fluoxetine treatment interfered with exercise and reduced the total distance run. The extent of Aß neuropathology, the tau accumulations, or BDNF levels, were not altered by prolonged exercise. Thus, neuropathology was present but not paralleled by spatial memory deficits in the backcrossed 3xTg mouse model of AD. Prolonged exercise for 11 months did improve the long-term survival of newborn neurons generated during middle-age, whereas fluoxetine had no effect. We further review and discuss the relevant literature in this respect.

Targeting the glutamatergic system for the treatment of HIV-associated neurocognitive disorders.

The accumulation of excess glutamate in the extracellular space as a consequence of CNS trauma, neurodegenerative diseases, infection, or deregulation of glutamate clearance results in neuronal damage by excessive excitatory neurotransmission. Glutamate excitotoxicity is thought to be one of several mechanisms by which HIV exerts neurotoxicity that culminates in HIV-associated neurocognitive disorders (HAND). Excess glutamate is released upon HIV infection of macrophage/microglial cells and has been associated with neurotoxicity mediated by gp120, transactivator of transcription (Tat) and other HIV proteins. Several strategies have been used over the years to try to prevent glutamate excitotoxicity. Since the main toxic effects of excess glutamate are thought to be due to excitotoxicity from over activation of glutamate receptors, antagonists of these receptors have been popular therapeutic targets. Early work to ameliorate the effects of excess extracellular glutamate focused on NMDA receptor antagonism, but unfortunately, potent blockade of this receptor has been fraught with side effects. One alternative to direct receptor blockade has been the inhibition of enzymes responsible for the production of glutamate such as glutaminase and glutamate carboxypeptidase II. Another approach has been to regulate the transporters responsible for modulation of extracellular glutamate such as excitatory amino acid transporters and the glutamate-cystine antiporter. There is preliminary experimental evidence that these approaches have potential therapeutic utility for the treatment of HAND. These efforts however, are at an early stage where the next steps are dependent on the identification of drug-like inhibitors as well as the development of predictive neuroAIDS animal models.

Running throughout middle-age improves memory function, hippocampal neurogenesis, and BDNF levels in female C57BL/6J mice.

Age-related memory loss is considered to commence at middle-age and coincides with reduced adult hippocampal neurogenesis and neurotrophin levels. Consistent physical activity at midlife may preserve brain-derived neurotrophic factor (BDNF) levels, new cell genesis, and learning. In the present study, 9-month-old female C57Bl/6J mice were housed with or without a running wheel and injected with bromodeoxyuridine (BrdU) to label newborn cells. Morris water maze learning, open field activity and rotarod behavior were tested 1 and 6 months after exercise onset. Here we show that long-term running improved retention of spatial memory and modestly enhanced rotarod performance at 15 months of age. Both hippocampal neurogenesis and mature BDNF peptide levels were elevated after long-term running. Thus, regular exercise from the onset and during middle-age may maintain brain function.

Monosynaptic inputs to new neurons in the dentate gyrus.

Adult hippocampal neurogenesis is considered important for cognition. The integration of newborn dentate gyrus granule cells into the existing network is regulated by afferent neuronal activity of unspecified origin. Here we combine rabies virus-mediated retrograde tracing with retroviral labelling of new granule cells (21, 30, 60, 90 days after injection) to selectively identify and quantify their monosynaptic inputs in vivo. Our results show that newborn granule cells receive afferents from intra-hippocampal cells (interneurons, mossy cells, area CA3 and transiently, mature granule cells) and septal cholinergic cells. Input from distal cortex (perirhinal (PRH) and lateral entorhinal cortex (LEC)) is sparse 21 days after injection and increases over time. Patch-clamp recordings support innervation by the LEC rather than from the medial entorhinal cortex. Mice with excitotoxic PRH/LEC lesions exhibit deficits in pattern separation but not in water maze learning. Thus, PRH/LEC input is an important functional component of new dentate gyrus neuron circuitry.

Fluctuations in endogenous kynurenic acid control hippocampal glutamate and memory.

Kynurenic acid (KYNA), an astrocyte-derived metabolite, antagonizes the α7 nicotinic acetylcholine receptor (α7nAChR) and, possibly, the glycine co-agonist site of the NMDA receptor at endogenous brain concentrations. As both receptors are involved in cognitive processes, KYNA elevations may aggravate, whereas reductions may improve, cognitive functions. We tested this hypothesis in rats by examining the effects of acute up- or downregulation of endogenous KYNA on extracellular glutamate in the hippocampus and on performance in the Morris water maze (MWM). Applied directly by reverse dialysis, KYNA (30-300 nM) reduced, whereas the specific kynurenine aminotransferase-II inhibitor (S)-4-(ethylsulfonyl)benzoylalanine (ESBA; 0.3-3 mM) raised, extracellular glutamate levels in the hippocampus. Co-application of KYNA (100 nM) with ESBA (1 mM) prevented the ESBA-induced glutamate increase. Comparable effects on hippocampal glutamate levels were seen after intra-cerebroventricular (i.c.v.) application of the KYNA precursor kynurenine (1 mM, 10 µl) or ESBA (10 mM, 10 µl), respectively. In separate animals, i.c.v. treatment with kynurenine impaired, whereas i.c.v. ESBA improved, performance in the MWM. I.c.v. co-application of KYNA (10 µM) eliminated the pro-cognitive effects of ESBA. Collectively, these studies show that KYNA serves as an endogenous modulator of extracellular glutamate in the hippocampus and regulates hippocampus-related cognitive function. Our results suggest that pharmacological interventions leading to acute reductions in hippocampal KYNA constitute an effective strategy for cognitive improvement. This approach might be especially useful in the treatment of cognitive deficits in neurological and psychiatric diseases that are associated with increased brain KYNA levels.

Exercise is not beneficial and may accelerate symptom onset in a mouse model of Huntington's disease.

Exercise benefits both general health and brain function in rodents and humans. However, it is less clear whether physical activity prevents or ameliorates neurodegenerative diseases. The aim of the present study was to determine whether voluntary wheel running can delay the onset or reduce the severity of Huntington's disease (HD) in a mouse model. To investigate whether running may delay HD symptoms lifespan, disease onset, locomotor activity, glucose levels, weight, striatal volume, inclusions, cognition and hippocampal neurogenesis were studied in male N171-82Q transgenic HD mice. Running started in pre-symptomatic (44±1 days old) male HD mice, did not improve function and appeared to accelerate disease onset. In particular, HD runners had an earlier onset of disease symptoms (shaking, hunched back and poor grooming), reduced striatal volume and impaired motor behavior, including a shorter latency to fall from the rotarod compared to sedentary controls. Furthermore, weight loss, reduced lifespan, hyperglycemia, Morris water maze learning deficits, diminished hippocampal neurogenesis, deficits in immature neuronal morphology, intranuclear inclusions and decreased dentate gyrus volume were refractory to physical activity. Taken together our research indicates that exercise is not beneficial, and may be detrimental to a vulnerable nervous system.

Reduction of endogenous kynurenic acid formation enhances extracellular glutamate, hippocampal plasticity, and cognitive behavior.

At endogenous brain concentrations, the astrocyte-derived metabolite kynurenic acid (KYNA) antagonizes the alpha 7 nicotinic acetylcholine receptor and, possibly, the glycine co-agonist site of the NMDA receptor. The functions of these two receptors, which are intimately involved in synaptic plasticity and cognitive processes, may, therefore, be enhanced by reductions in brain KYNA levels. This concept was tested in mice with a targeted deletion of kynurenine aminotransferase II (KAT II), a major biosynthetic enzyme of brain KYNA. At 21 days of age, KAT II knock-out mice had reduced hippocampal KYNA levels (-71%) and showed significantly increased performance in three cognitive paradigms that rely in part on the integrity of hippocampal function, namely object exploration and recognition, passive avoidance, and spatial discrimination. Moreover, compared with wild-type controls, hippocampal slices from KAT II-deficient mice showed a significant increase in the amplitude of long-term potentiation in vitro. These functional changes were accompanied by reduced extracellular KYNA (-66%) and increased extracellular glutamate (+51%) concentrations, measured by hippocampal microdialysis in vivo. Taken together, a picture emerges in which a reduction in the astrocytic formation of KYNA increases glutamatergic tone in the hippocampus and enhances cognitive abilities and synaptic plasticity. Our studies raise the prospect that interventions aimed specifically at reducing KYNA formation in the brain may constitute a promising molecular strategy for cognitive improvement in health and disease.

Strain-specific nicotinic modulation of glutamatergic transmission in the CA1 field of the rat hippocampus: August Copenhagen Irish versus Sprague-Dawley.

Prepulse inhibition (PPI), a measure of sensorimotor gating impaired in patients with schizophrenia, is more sensitive to disruption by apomorphine in prepubertal August Copenhagen Irish (ACI) than Sprague-Dawley (SD) rats. In brain regions including the hippocampus, PPI is modulated by alpha7* nicotinic receptors (nAChRs) and kynurenic acid (KYNA), a kynurenine metabolite that blocks alpha7 nAChRs. Here, KYNA levels and nAChR activities were measured in the hippocampi of 10- to 23-day-old ACI and SD rats of both sexes. Hippocampal KYNA levels were not different between ACI and SD rats. In hippocampal slices from both rat strains, choline (10 mM) evoked alpha7* nAChR-mediated type IA currents in CA1 stratum radiatum (SR) interneurons. In the presence of alpha7 nAChR antagonists, acetylcholine (ACh, 1 mM) evoked alpha4beta2* nAChR-mediated type II currents. ACh also triggered excitatory postsynaptic currents (EPSCs) that resulted from alpha3beta4* nAChR activation in glutamatergic neurons/axons synapsing onto the interneurons. The magnitude of the nicotinic responses did not differ significantly between male and female rats. Only the magnitude of alpha3beta4* nAChR responses and the frequency of spontaneous EPSCs recorded from CA1 SR interneurons differed between the rat strains, being significantly larger in ACI than SD rats. These results indicate that the alpha3beta4* nAChR activity in glutamatergic neurons/axons and the number of glutamatergic terminals synapsing onto CA1 SR interneurons are larger in prepubertal ACI than SD rats. The differential sensitivity of these rats to PPI disruption by apomorphine may result from strain-specific levels of glutamatergic activity and its strain-specific modulation by alpha3beta4* nAChRs in the hippocampus.