Quinolinic acid toxicity on oligodendroglial cells: relevance for multiple sclerosis and therapeutic strategies.

The excitotoxin quinolinic acid, a by-product of the kynurenine pathway, is known to be involved in several neurological diseases including multiple sclerosis (MS). Quinolinic acid levels are elevated in experimental autoimmune encephalomyelitis rodents, the widely used animal model of MS. Our group has also found pathophysiological concentrations of quinolinic acid in MS patients. This led us to investigate the effect of quinolinic acid on oligodendrocytes; the main cell type targeted by the autoimmune response in MS. We have examined the kynurenine pathway (KP) profile of two oligodendrocyte cell lines and show that these cells have a limited threshold to catabolize exogenous quinolinic acid. We further propose and demonstrate two strategies to limit quinolinic acid gliotoxicity: 1) by neutralizing quinolinic acid's effects with anti-quinolinic acid monoclonal antibodies and 2) directly inhibiting quinolinic acid production from activated monocytic cells using specific KP enzyme inhibitors. The outcome of this study provides a new insight into therapeutic strategies for limiting quinolinic acid-induced neurodegeneration, especially in neurological disorders that target oligodendrocytes, such as MS.

p38 MAPK inhibitors attenuate pro-inflammatory cytokine production and the invasiveness of human U251 glioblastoma cells.

Increasing evidence suggests that an inflammatory microenvironment promotes invasion by glioblastoma (GBM) cells. Together with p38 mitogen-activated protein kinase (MAPK) activation being regarded as promoting inflammation, we hypothesized that elevated inflammatory cytokine secretion and p38 MAPK activity contribute to expansion of GBMs. Here we report that IL-1ß, IL-6, and IL-8 levels and p38 MAPK activity are elevated in human glioblastoma specimens and that p38 MAPK inhibitors attenuate the secretion of pro-inflammatory cytokines by microglia and glioblastoma cells. RNAi knockdown and immunoprecipitation experiments suggest that the p38α MAPK isoform drives inflammation in GBM cells. Importantly, p38 MAPK inhibition strongly reduced invasion of U251 glioblastoma cells in an inflammatory microenvironment, providing evidence for a p38 MAPK-regulated link between inflammation and invasiveness in GBM pathophysiology.

Kynurenine 3-Monooxygenase Activity in Human Primary Neurons and Effect on Cellular Bioenergetics Identifies New Neurotoxic Mechanisms.

Upregulation of the kynurenine pathway (KP) of tryptophan metabolism is commonly observed in neurodegenerative disease. When activated, L-kynurenine (KYN) increases in the periphery and central nervous system where it is further metabolised to other neuroactive metabolites including 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA) and quinolinic acid (QUIN). Particularly biologically relevant metabolites are 3-HK and QUIN, formed downstream of the enzyme kynurenine 3-monooxygenase (KMO) which plays a pivotal role in maintaining KP homeostasis. Indeed, excessive production of 3-HK and QUIN has been described in neurodegenerative disease including Alzheimer's disease and Huntington's disease. In this study, we characterise KMO activity in human primary neurons and identified new mechanisms by which KMO activation mediates neurotoxicity. We show that while transient activation of the KP promotes synthesis of the essential co-enzyme nicotinamide adenine dinucleotide (NAD+), allowing cells to meet short-term increased energy demands, chronic KMO activation induces production of reactive oxygen species (ROS), mitochondrial damage and decreases spare-respiratory capacity (SRC). We further found that these events generate a vicious-cycle, as mitochondrial dysfunction further shunts the KP towards the KMO branch of the KP to presumably enhance QUIN production. These mechanisms may be especially relevant in neurodegenerative disease as neurons are highly sensitive to oxidative stress and mitochondrial impairment.

Involvement of the kynurenine pathway in human glioma pathophysiology.

The kynurenine pathway (KP) is the principal route of L-tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), the excitotoxin, quinolinic acid (QUIN) and the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2,3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. IDO-1 and TDO-2 induction in tumors are crucial mechanisms implicated to play pivotal roles in suppressing anti-tumor immunity. Here, we report the first comprehensive characterisation of the KP in 1) cultured human glioma cells and 2) plasma from patients with glioblastoma (GBM). Our data revealed that interferon-gamma (IFN-γ) stimulation significantly potentiated the expression of the KP enzymes, IDO-1 IDO-2, kynureninase (KYNU), kynurenine hydroxylase (KMO) and significantly down-regulated 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) and kynurenine aminotransferase-I (KAT-I) expression in cultured human glioma cells. This significantly increased KP activity but significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. KP activation (KYN/TRP) was significantly higher, whereas the concentrations of the neuroreactive KP metabolites TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlight a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumors, aimed at restoring anti-tumor immunity and reducing the capacity for malignant cells to produce NAD(+), which is necessary for energy production and DNA repair.

The endogenous tryptophan metabolite and NAD+ precursor quinolinic acid confers resistance of gliomas to oxidative stress.

Quinolinic acid is a product of tryptophan degradation and may serve as a precursor for NAD(+), an important enzymatic cofactor for enzymes such as the DNA repair protein PARP. Pathologic accumulation of quinolinic acid has been found in neurodegenerative disorders including Alzheimer and Huntington disease, where it is thought to be toxic for neurons by activating the N-methyl-D-aspartate (NMDA) receptor and inducing excitotoxicity. Although many tumors including gliomas constitutively catabolize tryptophan, it is unclear whether quinolinic acid is produced in gliomas and whether it is involved in tumor progression. Here, we show that quinolinic acid accumulated in human gliomas and was associated with a malignant phenotype. Quinolinic acid was produced by microglial cells, as expression of the quinolinic acid-producing enzyme 3-hydroxyanthranilate oxygenase (3-HAO) was confined to microglia in glioma tissue. Human malignant glioma cells, but not nonneoplastic astrocytes, expressed quinolinic acid phosphoribosyltransferase (QPRT) to use quinolinic acid for NAD(+) synthesis and prevent apoptosis when de novo NAD(+) synthesis was blocked. Oxidative stress, temozolomide, and irradiation induced QPRT in glioma cells. QPRT expression increased with malignancy. In recurrent glioblastomas after radiochemotherapy, QPRT expression was associated with a poor prognosis in two independent datasets. Our data indicate that neoplastic transformation in astrocytes is associated with a QPRT-mediated switch in NAD(+) metabolism by exploiting microglia-derived quinolinic acid as an alternative source of replenishing intracellular NAD(+) pools. The elevated levels of QPRT expression increase resistance to oxidative stress induced by radiochemotherapy, conferring a poorer prognosis. These findings have implications for therapeutic approaches inducing intracellular NAD(+) depletion, such as alkylating agents or direct NAD(+) synthesis inhibitors, and identify QPRT as a potential therapeutic target in malignant gliomas.

The kynurenine pathway in brain tumor pathogenesis.

Brain tumors are among the most common and most chemoresistant tumors. Despite treatment with aggressive treatment strategies, the prognosis for patients harboring malignant gliomas remains dismal. The kynurenine pathway (KP) is the principal route of L-tryptophan catabolism leading to the formation of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)), and important neuroactive metabolites, including the neurotoxin, quinolinic acid (QUIN), the neuroprotective agent, picolinic acid (PIC), the T(H)17/Treg balance modulator, 3-hydroxyanthranilic acid (3-HAA), and the immunosuppressive agent, L-kynurenine (KYN). This review provides a new perspective on KP dysregulation in defeating antitumor immune responses, specifically bringing light to the lower segment of the KP, particularly QUIN-induced neurotoxicity and downregulation of the enzyme α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) as a potential mechanism of tumor progression. Given its immunosuppressive effects, 3-HAA produced from the KP may also play a role in suppressing antitumor immunity in human tumors. The enzyme indoleamine 2, 3-dioxygenase (IDO-1) initiates and regulates the first step of the KP in most cells. Mounting evidence directly implicates that the induction and overexpression of IDO-1 in various tumors is a crucial mechanism facilitating tumor immune evasion and persistence. Tryptophan 2, 3-dioxygenase (TDO-2), which initiates the same first step of the KP as IDO-1, has likewise recently been shown to be a mechanism of tumoral immune resistance. Further, it was also recently shown that TDO-2-dependent production of KYN by brain tumors might be a novel mechanism for suppressing antitumor immunity and supporting tumor growth through the activation of the Aryl hydrocarbon receptor (AhR). This newly identified TDO-2-KYN-AhR signaling pathway opens up exciting future research opportunities and may represent a novel therapeutic target in cancer therapy. Our discussion points to a number of KP components, namely TDO-2, IDO-1, and ACMSD, as important therapeutic targets for the treatment of brain cancer. Targeting the KP in brain tumors may represent a viable strategy likely to prevent QUIN-induced neurotoxicity and KYN and 3-HAA-mediated immune suppression.

Neuroprotective effects of naturally occurring polyphenols on quinolinic acid-induced excitotoxicity in human neurons.

Quinolinic acid (QUIN) excitotoxicity is mediated by elevated intracellular Ca(2+) levels, and nitric oxide-mediated oxidative stress, resulting in DNA damage, poly(ADP-ribose) polymerase (PARP) activation, NAD(+) depletion and cell death. We evaluated the effect of a series of polyphenolic compounds [i.e. epigallocatechin gallate (EPCG), catechin hydrate, curcumin, apigenin, naringenin and gallotannin] with antioxidant properties on QUIN-induced excitotoxicity on primary cultures of human neurons. We showed that the polyphenols, EPCG, catechin hydrate and curcumin can attenuate QUIN-induced excitotoxicity to a greater extent than apigenin, naringenin and gallotannin. Both EPCG and curcumin were able to attenuate QUIN-induced Ca(2+) influx and neuronal nitric oxide synthase (nNOS) activity to a greater extent compared with apigenin, naringenin and gallotannin. Although Ca(2+) influx was not attenuated by catechin hydrate, nNOS activity was reduced, probably through direct inhibition of the enzyme. All polyphenols reduced the oxidative effects of increased nitric oxide production, thereby reducing the formation of 3-nitrotyrosine and poly (ADP-ribose) polymerase activity and, hence, preventing NAD(+) depletion and cell death. In addition to the well-known antioxidant properties of these natural phytochemicals, the inhibitory effect of some of these compounds on specific excitotoxic processes, such as Ca(2+) influx, provides additional evidence for the beneficial health effects of polyphenols in excitable tissue, particularly within the central nervous system.

Characterisation of the expression of NMDA receptors in human astrocytes.

Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS). However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs) in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN). Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH) activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B) are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.

Mechanism for quinolinic acid cytotoxicity in human astrocytes and neurons.

There is growing evidence implicating the kynurenine pathway (KP) and particularly one of its metabolites, quinolinic acid (QUIN), as important contributors to neuroinflammation in several brain diseases. While QUIN has been shown to induce neuronal and astrocytic apoptosis, the exact mechanisms leading to cell death remain unclear. To determine the mechanism of QUIN-mediated excitotoxicity in human brain cells, we measured intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and poly(ADP-ribose) polymerase (PARP) and extracellular lactate dehydrogenase (LDH) activities in primary cultures of human neurons and astrocytes treated with QUIN. We found that QUIN acts as a substrate for NAD(+) synthesis at very low concentrations (<50 nM) in both neurons and astrocytes, but is cytotoxic at sub-physiological concentrations (>150 nM) in both the cell types. We have shown that the NMDA ion channel blockers, MK801 and memantine, and the nitric oxide synthase (NOS) inhibitor, L-NAME, significantly attenuate QUIN-mediated PARP activation, NAD(+) depletion, and LDH release in both neurons and astrocytes. An increased mRNA and protein expression of the inducible (iNOS) and neuronal (nNOS) forms of nitric oxide synthase was also observed following exposure of both cell types to QUIN. Taken together these results suggests that QUIN-induced cytotoxic effects on neurons and astrocytes are likely to be mediated by an over activation of an NMDA-like receptor with subsequent induction of NOS and excessive nitric oxide (NO(*))-mediated free radical damage. These results contribute significantly to our understanding of the pathophysiological mechanisms involved in QUIN neuro- and gliotoxicity and are relevant for the development of therapies for neuroinflammatory diseases.

Effects of Kynurenine Pathway Metabolites on Intracellular NAD Synthesis and Cell Death in Human Primary Astrocytes and Neurons.

The kynurenine pathway (KP) is a major route of L-tryptophan catabolism resulting in the production of the essential pyridine nucleotide nicotinamide adenine dinucleotide, (NAD(+)). Up-regulation of the KP during inflammation leads to the release of a number of biologically active metabolites into the brain. We hypothesised that while some of the extracellular KP metabolites may be beneficial for intracellular NAD(+) synthesis and cell survival at physiological concentrations, they may contribute to neuronal and astroglial dysfunction and cell death at pathophysiological concentrations. In this study, we found that treatment of human primary neurons and astrocytes with 3-hydroxyanthranilic acid (3-HAA), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and picolinic acid (PIC) at concentrations below 100 nM significantly increased intracellular NAD(+) levels compared to non-treated cells. However, a dose dependent decrease in intracellular NAD(+) levels and increased extracellular LDH activity was observed in human astrocytes and neurons treated with 3-HAA, 3-HK, QUIN and PIC at concentrations >100 nM and kynurenine (KYN), at concentrations above 1 µM. Intracellular NAD(+) levels were unchanged in the presence of the neuroprotectant, kynurenic acid (KYNA), and a dose dependent increase in intracellular NAD(+) levels was observed for TRP up to 1 mM. While anthranilic acid (AA) increased intracellular NAD(+) levels at concentration below 10 µM in astrocytes. NAD(+) depletion and cell death was observed in AA treated neurons at concentrations above 500 nM. Therefore, the differing responses of astrocytes and neurons to an increase in KP metabolites should be considered when assessing KP toxicity during neuroinflammation.