Daniela Alberati-Giani

Regulation of the Kynurenine Metabolic Pathway by Interferon‐γ in Murine Cloned Macrophages and Microglial Cells

Abstract: Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon‐γ (IFN‐γ) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN‐γ induced the expression of indoleamine 2,3‐dioxygenase (IDO) activity. Whereas tumor necrosis factor‐α did not affect enzyme induction by IFN‐γ, lipopolysaccharide modulated IDO activity differently in the two IFN‐γ‐activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3‐hydroxylase, and 3‐hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3‐hydroxylase activity was stimulated by IFN‐γ. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN‐γ markedly stimulated the activity of this enzyme only in MT2 cells. IFN‐γ‐treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3‐hydroxyanthranilic acid quinolinic acids from l‐[5‐3H]tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.

Identification of a mitochondrial form of kynurenine aminotransferase/glutamine transaminase K from rat brain

A soluble aminotransferase with kynurenine aminotransferase (KAT) activity has been recently isolated from rat brain. This enzyme corresponds to a cytosolic form of glutamine transaminase K (GTK). In addition to the cytosolic enzyme, a mitochondrial‐associated form of this KAT/GTK also exists. In the present work we have isolated a rat brain cDNA clone encoding a KAT/GTK enzyme identical to the soluble form but carrying an additional stretch of 32 amino acids at its NH2‐terminus. Several structural features of this sequence resemble those of leader peptides for mitochondrial import. Evidence that the isolated cDNA encoded for mitochondrial KAT/GTK was obtained after transfection of HEK‐293 cells with the cDNA coding for this new KAT/GTK isoenzyme. In fact, a significant enrichment of both KAT and GTK enzymatic activities was found in the crude mitochondrial fraction of the transfected cells.

Cloning and Characterization of a Soluble Kynurenine Aminotransferase from Rat Brain: Identity with Kidney Cysteine Conjugate β-Lyase

Abstract: In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate β‐lyase, indicating that rat kidney KAT and β‐lyase represent the same molecular entity. Oligonucleotide probes derived from the β‐lyase cDNA were then used as primers for PCR of reverse‐transcribed rat brain poly(A)+ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the β‐lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK‐293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 mM pyruvate and 2‐oxoglutarate, the Km values for l‐kynurenine were 1.2 mM and 86.3 µM, respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/β‐lyase mRNA of ∼2.1 kb.

Cloning and functional expression of human kynurenine 3-monooxygenase

Kynurenine 3‐monooxygenase, an NADPH‐dependent flavin monooxygenase, catalyses the hydroxylation of l‐kynurenine to l‐3‐hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3‐monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55 762 Da. Transfection of the human cDNA in HEK‐293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of ∼2.0 kb in liver, placenta and kidney.

Regulation of the Kynurenine Pathway by IFN-γ in Murine Cloned Macrophages and Microglial Cells

Several pieces of evidence indicate that, in neurological disorders associated with immune activation, brain infiltrating macrophages and microglial cells secrete various neurotoxic factors, such as glutamate and reactive oxygen species, which may greatly influence the survival of neurons (Mallat and Chamak, 1994; Giulian et al., 1993; Stone, 1993). Among the putative endogenous neurotoxins released by phagocytic cells during inflammatory processes, particular attention has been devoted to quinolinic acid (QUIN) (Heyes et al., 1993; Heyes, 1993). This tryptophan metabolite, formed along the kynurenine pathway, exerts its effect through activation of glutamate NMDA receptors and its accumulation within the CNS, observed in brain inflammatory disorders, has been speculatively linked to neuronal dysfunctions (Heyes et al., 1993). The overproduction of QUIN is mainly attributed to the induction of indoleamine-2,3 dioxygenase (IDO), the enzyme converting L-tryptophan into L-kynurenine (Takikawa et al., 1988; Taylor and Feng, 1991), by cytokines, such as IFN-γ. It has been found that IFN-γ activated human macrophages are able to produce QUIN directly from L-tryptophan (Heyes et al., 1992), whereas neurons and astroglial cells, even if they express inducible IDO activity (Saito et al., 1993a), do not appear to synthesise this neurotoxin directly from L-tryptophan. Whereas most of the available findings suggest that the large increase in QUIN cerebral levels in brain inflammatory conditions is mainly due to infiltrating activated macrophages, the relevance of activated microglial cells in the production of neuroactive kynurenines, has been only partially investigated (Saito et al., 1993a).