The immune system continuously modulates the balance between responsiveness to pathogens and tolerance to non‐harmful antigens. The mechanisms that mediate tolerance are not well understood, but recent findings have implicated tryptophan catabolism through the kynurenine metabolic pathway as one of many mechanisms involved. The enzymes that break down tryptophan through this pathway are found in numerous cell types, including cells of the immune system. Some of these enzymes are induced by immune activation, including the rate limiting enzyme present in macrophages and dendritic cells, indoleamine 2,3‐dioxygenase (IDO). It has recently been found that inhibition of IDO can result in the rejection of allogenic fetuses, suggesting that tryptophan breakdown is necessary for maintaining aspects of immune tolerance. Two theories have been proposed to explain how tryptophan catabolism facilitates tolerance. One theory posits that tryptophan breakdown suppresses T cell proliferation by dramatically reducing the supply of this critical amino acid. The other theory postulates that the downstream metabolites of tryptophan catabolism act to suppress certain immune cells, probably by pro‐apoptotic mechanisms. Reconciling these disparate views is crucial to understanding immune‐related tryptophan catabolism and the roles it plays in immune tolerance. In this review we examine the issue in detail, and offer additional insight provided by studies with antibodies to quinolinate, a tryptophan catabolite which is also necessary for nicotinamide adenine dinucleotide (NAD +) production. In addition to the immunomodulatory actions of tryptophan catabolites, we discuss the possible involvement of quinolinate as a means of replenishing NAD + in leucocytes, which is depleted by oxidative stress during an immune response.
Abstract Glioma, the most common primary brain tumor of the adult central nervous system, is associated with a poor prognosis due, in part, to the presence of chemoradiotherapy-resistant glioma stem-like cells responsible for inevitable post-surgical recurrence. N-acetyl-L-aspartate (NAA), one of the most concentrated metabolic sources of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors. NAA-derived acetate is converted to acetyl coenzyme A via acetyl-CoA synthetase (AceCS) for use in lipogenesis, protein/histone acetylation, and the TCA cycle. We propose that glyceryltriacetate (GTA), a FDA approved food additive with “generally regarded as safe” status, may be an effective means of reducing glioma growth via restoration of acetate levels. The effect of GTA on the growth of both established (Hs683, HOG) and stem-like (grade II OG33, grade III OG35) oligodendroglioma cell lines was assessed. In vitro, GTA induced growth arrest in all cells examined (i.e., increased proportion of cells in G0/G1 and reduced S phase cells by flow cytometry of propidium iodide labeled cells 24 hours after treatment and unbiased trypan blue exclusion based cytometry up to 5 days post-treatment). Growth arrest was not associated with apoptosis (lack of cleaved poly ADP-ribose polymerase immunolabeling), but differentiation (increased CNPase expression). ASPA expression was greater in stem-like cells when grown in stem cell media than differentiation media and was decreased in GTA-treated OG35 cells. Interestingly, GTA did not decrease ASPA expression in OG33 cells, but induced a novel 26 kDa ASPA isoform. ASPA and AceCS1 were co-localized within the nucleus. Nuclear, but not cytosolic, ASPA expression was decreased upon GTA addition in stem cell media, but not differentiation media. Finally, the effect of GTA on orthotopically grafted luciferase expressing OG33 and OG35 cells was assessed. Bioluminescence and tumor volume were reduced in GTA treated mice. These data suggest that the nuclear ASPA/AceCS1 co-localization provides acetate for histone acetylation to maintain cells in a progenitor/stem-like state and that decreased ASPA promotes gliomagenesis. Inasmuch as infants with Canavan Disease, a leukodystrophy due to ASPA mutation, treated with high dose GTA showed no significant side effects, GTA may prove an effective therapy to prevent recurrence by inducing growth arrest/differentiation of glioma stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3481. doi:1538-7445.AM2012-3481
Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite in the kynurenine pathway of tryptophan catabolism leading to the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As a precursor for NAD+, Quin can direct a portion of tryptophan catabolism toward replenishing cellular NAD+ levels in response to inflammation and infection. Intracellular Quin levels increase dramatically in response to immune stimulation (e.g., lipopolysaccharide; LPS or pokeweed mitogen; PWM) in macrophages, microglia, dendritic cells and other cells of the immune system. NAD+ serves numerous functions including energy production, the poly ADP ribose polymerization (PARP) reaction involved in DNA repair and the activity of various enzymes such as the NAD+-dependent deacetylases known as sirtuins. We used highly specific antibodies to protein-coupled Quin to delineate cells that accumulate Quin as a key aspect of the response to immune stimulation and infection. Here we describe Quin staining in the brain, spleen and liver after LPS administration to the brain or systemic PWM administration. Quin expression was strong in immune cells in the periphery after both treatments, whereas very limited Quin expression was observed in the brain even after direct LPS injection. Immunoreactive cells exhibited diverse morphology ranging from foam-cells to cells with membrane extensions related to cell motility. We also examined protein expression changes in the spleen after kynurenine administration. Acute (8 hr) and chronic (48 hr) kynurenine administration led to significant changes in protein expression in the spleen, including multiple changes involved with cytoskeletal rearrangements associated with cell motility and phagocytosis. Kynurenine administration resulted in several expression level changes in proteins associated with heat shock protein 90 (HSP90), a chaperone for the aryl-hydrocarbon receptor (AHR), which is the primary kynurenine metabolite receptor. We propose that cells with high levels of Quin are those that are currently releasing kynurenine pathway metabolites as well as accumulating Quin for sustained NAD+ synthesis from tryptophan. Further, the kynurenine pathway may be linked to the regulation of cell motility in immune and cancer cells.
Evidence has been presented in recent years that support the hypothesis thatN-acetylaspartylglutamate (NAAG) may be involved in synaptic transmission in the optic tract of mammals. Using a modified fixation protocol, we have determined the detailed distribution of NAAG immunoreactivity (NAAG-IR) in retinal ganglion cells and optic projections of the rat. Following optic nerve transection, dramatic losses of NAAG-IR were observed in the neuropil of all retinal target zones including the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, the dorsal and medial terminal nuclei and suprachiasmatic nucleus. Brain regions were microdissected and NAAG levels measured by a radioimmunoassay (RIA) (IC50:NAAG= 2.5nM,NAA= 100 μM;smallest detectable amount= 1–2pg/assay). decreases (50–60%) in NAAG levels were detected in the lateral geniculate, superior colliculus and suprachiasmatic nucleus. Moderate losses (25–45%) were noted in the pretectal nucleus and the nucleus of the optic tract. Smaller changes (15–20%) were detected in the paraventricular nucleus and the pretectal area. These results are consistent with a synaptic communication role for NAAG in the visual system.
Canavan disease (CD) is a fatal genetic neurodegenerative disorder caused by mutations in the gene for aspartoacylase, an enzyme that hydrolyzes N-acetylaspartate (NAA) into l-aspartate and acetate. Because aspartoacylase is localized in oligodendrocytes, and NAA-derived acetate is incorporated into myelin lipids, we hypothesize that an acetate deficiency in oligodendrocytes is responsible for the pathology in CD, and we propose acetate supplementation as a possible therapy. In our preclinical efforts toward this goal, we studied the effectiveness of orally administered glyceryl triacetate (GTA) and calcium acetate for increasing acetate levels in the murine brain. The concentrations of brain acetate and NAA were determined simultaneously after intragastric administration of GTA. We found that the acetate levels in brain were increased in a dose- and time-dependent manner, with a 17-fold increase observed at 1 to 2 h in 20- to 21-day-old mice at a dose of 5.8 g/kg GTA. NAA levels in the brain were not significantly increased under these conditions. Studies using mice at varying stages of development showed that the dose of GTA required to maintain similarly elevated acetate levels in the brain increased with age. Also, GTA was significantly more effective as an acetate source than calcium acetate. Chronic administration of GTA up to 25 days of age did not result in any overt pathology in the mice. Based on these results and the current Food and Drug Administration-approved use of GTA as a food additive, we propose that it is a potential candidate for use in acetate supplementation therapy for CD.
