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Journal of Neuroimmune Pharmacology :... Mar 2015We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require...
We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-β-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.
Topics: Animals; Aspartic Acid; Axons; Cerebellar Nuclei; Diazooxonorleucine; Enzyme Inhibitors; Female; Glutamic Acid; Glutaminase; Hypothalamic Area, Lateral; Hypothalamus; Immunity; Injections; Killer Cells, Natural; Male; Rats; Rats, Sprague-Dawley; Sympathetic Nervous System; T-Lymphocytes; Thalamus
PubMed: 25649846
DOI: 10.1007/s11481-014-9572-y -
Journal of Neurovirology Apr 2015Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits....
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.
Topics: Alphavirus Infections; Animals; Antimetabolites, Antineoplastic; Behavior, Animal; Diazooxonorleucine; Disease Models, Animal; Encephalitis, Viral; Enzyme-Linked Immunosorbent Assay; Glutamine; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Motor Activity; Sindbis Virus
PubMed: 25645378
DOI: 10.1007/s13365-015-0314-6 -
PloS One 2015Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated...
Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become "addicted" to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists.
Topics: Aniline Compounds; Animals; Antimetabolites, Antineoplastic; Apoptosis; Bone Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Drug Synergism; Glutamine; Humans; Mice; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Sarcoma, Ewing; Sulfonamides; Xenograft Model Antitumor Assays
PubMed: 25615615
DOI: 10.1371/journal.pone.0116998 -
Analytical Biochemistry Apr 2015Glutamine is an abundant amino acid that plays pivotal roles in cell growth, cell metabolism, and neurotransmission. Dysregulation of glutamine-using pathways has been...
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.
Topics: 1-Butanol; Animals; Brain; Chlorine; Chromatography, High Pressure Liquid; Diazooxonorleucine; Esters; Male; Mice, Inbred C57BL; Reference Standards; Tandem Mass Spectrometry; Time Factors
PubMed: 25584882
DOI: 10.1016/j.ab.2015.01.001