**1. Introduction**

356 Amyotrophic Lateral Sclerosis

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The kynurenine pathway represents a major route for the catabolism of tryptophan (TRP). In the body, TRP is transported around the periphery either bound to albumin (90%) or in free form (10%), the two states existing in equilibrium (McMenamy 1965). However, only free form TRP can be transported across the blood-brain barrier (BBB) by the competitive and nonspecific L-type amino acid transporter (Hargreaves and Pardridge 1988). Once in the central nervous system (CNS), TRP acts as a precursor to several metabolic pathways, such as for the synthesis of kynurenine (KYN), serotonin, melatonin and protein (Fig. 1) (Ruddick *et al.* 2006).

Fig. 1. TRP in the CNS. Only free TRP can cross the BBB and act as precursor for protein, serotonin, tryptamine, and kynurenine and kynuramine synthesis. The kynurenine pathway is a major pathway for TRP catabolism. Adapted from (Ruddick *et al.* 2006).

The Kynurenine Pathway 359

Fig. 2. The kynurenine pathway. Via the kynurenine pathway, TRP is converted to nicotinamide adenine dinucleotide (NAD) in a series of biochemical steps. In the process, neuroactive intermediates are produced. The neuroprotectants include kynurenic acid and

picolinic, and the neurotoxin, QUIN.

In the CNS, the kynurenine pathway is present to varying extents in most cell types, including astrocytes (Guillemin *et al.* 2000), neurons (Guillemin *et al.* 2007), infiltrating macrophages and microglia (Guillemin *et al.* 2003), oligodendrocytes (Lim *et al.* 2007), and endothelial cells (Owe-Young *et al.* 2008). Infiltrating macrophages, activated microglia and neurons have the complete repertoire of kynurenine pathway enzymes. On the other hand, neuroprotective astrocytes and oligodendrocytes lack the enzyme, kynurenine 3-monooxygenase (KMO) and indoleamine 2,3-dioxygenase 1 (IDO-1) respectively, and are incapable of synthesizing the excitotoxin, quinolinic acid (QUIN) (Guillemin *et al.* 2000; Lim *et al.* 2007).

The oxidation of TRP, initiating the kynurenine pathway (Fig. 2), may be catalyzed by one of three enzymes - TRP 2,3-dioxygenase (TDO), IDO-1 or IDO-2, a newly discovered IDO related enzyme (Salter and Pogson 1985; Takikawa *et al.* 1986; Ball *et al.* 2007; Metz *et al.* 2007). TDO resides primarily in the liver, although it is also expressed in low quantities in the brain, and is induced by TRP or corticosteroids (Salter and Pogson 1985; Miller *et al.* 2004). In contrast, IDO-1 is the predominant enzyme extra-hepatically and is found in numerous cells, including macrophages, microglia, neurons and astrocytes (Guillemin *et al.* 2001; Guillemin *et al.* 2003; Guillemin *et al.* 2005; Guillemin *et al.* 2007). IDO-1 is up regulated by certain cytokines and inflammatory molecules, such as lipopolysaccharides, amyloid peptides and human immunodeficiency virus (HIV) proteins (Fujigaki *et al.* 1998; Guillemin *et al.* 2003; Takikawa 2005), and its most potent stimulant is interferon gamma (IFN-γ) (Hayaishi and Yoshida 1978; Werner-Felmayer *et al.* 1989). IFN-γ induces both the gene expression and enzymatic activity of IDO-1 (Yasui *et al.* 1986; Dai and Gupta 1990). IDO-2 possesses similar structural and enzymatic activities as IDO-1. However, the two enzymes differ in their expression pattern and signalling pathway, and IDO-2 is preferentially inhibited by D-1-methyl-tryptophan (D-1-MT) (Ball *et al.* 2007; Metz *et al.* 2007).

The first stable intermediate from the kynurenine pathway is KYN. Subsequently, several neuroactive intermediates are generated. They include the free-radical generator, 3 hydroxyanthranilic acid (3HAA) (Goldstein *et al.* 2000), the excitotoxin and *N*-methyl *D*aspartate (NMDA) receptor agonist, QUIN (Stone and Perkins 1981), the NMDA antagonist, kynurenic acid (KYNA) (Perkins and Stone 1982), and the neuroprotectant, picolinic acid (PIC) (Jhamandas *et al.* 1990).

The kynurenine pathway first aroused great interest when it was observed that an accelerated and sustained degradation of TRP occurred when activated T cells released IFNγ during an immune response (Pfefferkorn 1984). The significance was speculated to be a defence mechanism that starved tumour cells, pathogens and parasites of TRP (Pfefferkorn 1984; Brown *et al.* 1991). Further research soon discovered that IDO-1 activity was necessary for the preservation of allogeneic foetuses in mice, and that TRP depletion had an antiproliferative and apoptotic effect on T cells (Munn *et al.* 1998; Munn *et al.* 1999; Lee *et al.* 2002). Hence, the kynurenine pathway appeared to exert an immuno-regulatory effect. In particular, the general control non-derepressible-2 kinase (GCN2) was identified as a key mediator in IDO-1 induced TRP depletion immunosuppression (Munn *et al.* 2005). The activation of GCN2 triggered a stress-response program that resulted in cell-cycle arrest, differentiation, adaptation or apoptosis (de Haro *et al.* 1996; Rao *et al.* 2004; Bi *et al.* 2005). Furthermore, some of the kynurenines, such as QUIN and 3HAA, can selectively target immune cells undergoing activation, thus suppressing T cell proliferation (Frumento *et al.* 2002; Fallarino *et al.* 2003). They can also act in concert to produce an additive effect (Terness *et al.* 2002). Lastly, the production of the excitotoxic QUIN was often significantly increased following inflammation and resulting immune activation (Moffett *et al.* 1997).

In the CNS, the kynurenine pathway is present to varying extents in most cell types, including astrocytes (Guillemin *et al.* 2000), neurons (Guillemin *et al.* 2007), infiltrating macrophages and microglia (Guillemin *et al.* 2003), oligodendrocytes (Lim *et al.* 2007), and endothelial cells (Owe-Young *et al.* 2008). Infiltrating macrophages, activated microglia and neurons have the complete repertoire of kynurenine pathway enzymes. On the other hand, neuroprotective astrocytes and oligodendrocytes lack the enzyme, kynurenine 3-monooxygenase (KMO) and indoleamine 2,3-dioxygenase 1 (IDO-1) respectively, and are incapable of synthesizing the

The oxidation of TRP, initiating the kynurenine pathway (Fig. 2), may be catalyzed by one of three enzymes - TRP 2,3-dioxygenase (TDO), IDO-1 or IDO-2, a newly discovered IDO related enzyme (Salter and Pogson 1985; Takikawa *et al.* 1986; Ball *et al.* 2007; Metz *et al.* 2007). TDO resides primarily in the liver, although it is also expressed in low quantities in the brain, and is induced by TRP or corticosteroids (Salter and Pogson 1985; Miller *et al.* 2004). In contrast, IDO-1 is the predominant enzyme extra-hepatically and is found in numerous cells, including macrophages, microglia, neurons and astrocytes (Guillemin *et al.* 2001; Guillemin *et al.* 2003; Guillemin *et al.* 2005; Guillemin *et al.* 2007). IDO-1 is up regulated by certain cytokines and inflammatory molecules, such as lipopolysaccharides, amyloid peptides and human immunodeficiency virus (HIV) proteins (Fujigaki *et al.* 1998; Guillemin *et al.* 2003; Takikawa 2005), and its most potent stimulant is interferon gamma (IFN-γ) (Hayaishi and Yoshida 1978; Werner-Felmayer *et al.* 1989). IFN-γ induces both the gene expression and enzymatic activity of IDO-1 (Yasui *et al.* 1986; Dai and Gupta 1990). IDO-2 possesses similar structural and enzymatic activities as IDO-1. However, the two enzymes differ in their expression pattern and signalling pathway, and IDO-2 is preferentially

excitotoxin, quinolinic acid (QUIN) (Guillemin *et al.* 2000; Lim *et al.* 2007).

inhibited by D-1-methyl-tryptophan (D-1-MT) (Ball *et al.* 2007; Metz *et al.* 2007).

following inflammation and resulting immune activation (Moffett *et al.* 1997).

(PIC) (Jhamandas *et al.* 1990).

The first stable intermediate from the kynurenine pathway is KYN. Subsequently, several neuroactive intermediates are generated. They include the free-radical generator, 3 hydroxyanthranilic acid (3HAA) (Goldstein *et al.* 2000), the excitotoxin and *N*-methyl *D*aspartate (NMDA) receptor agonist, QUIN (Stone and Perkins 1981), the NMDA antagonist, kynurenic acid (KYNA) (Perkins and Stone 1982), and the neuroprotectant, picolinic acid

The kynurenine pathway first aroused great interest when it was observed that an accelerated and sustained degradation of TRP occurred when activated T cells released IFNγ during an immune response (Pfefferkorn 1984). The significance was speculated to be a defence mechanism that starved tumour cells, pathogens and parasites of TRP (Pfefferkorn 1984; Brown *et al.* 1991). Further research soon discovered that IDO-1 activity was necessary for the preservation of allogeneic foetuses in mice, and that TRP depletion had an antiproliferative and apoptotic effect on T cells (Munn *et al.* 1998; Munn *et al.* 1999; Lee *et al.* 2002). Hence, the kynurenine pathway appeared to exert an immuno-regulatory effect. In particular, the general control non-derepressible-2 kinase (GCN2) was identified as a key mediator in IDO-1 induced TRP depletion immunosuppression (Munn *et al.* 2005). The activation of GCN2 triggered a stress-response program that resulted in cell-cycle arrest, differentiation, adaptation or apoptosis (de Haro *et al.* 1996; Rao *et al.* 2004; Bi *et al.* 2005). Furthermore, some of the kynurenines, such as QUIN and 3HAA, can selectively target immune cells undergoing activation, thus suppressing T cell proliferation (Frumento *et al.* 2002; Fallarino *et al.* 2003). They can also act in concert to produce an additive effect (Terness *et al.* 2002). Lastly, the production of the excitotoxic QUIN was often significantly increased

Fig. 2. The kynurenine pathway. Via the kynurenine pathway, TRP is converted to nicotinamide adenine dinucleotide (NAD) in a series of biochemical steps. In the process, neuroactive intermediates are produced. The neuroprotectants include kynurenic acid and picolinic, and the neurotoxin, QUIN.

The Kynurenine Pathway 361

Fig. 3. Hypothetical model of the involvement of QUIN in the pathogenesis of ALS. QUIN, released from activated microglia, can induce various effects in astrocytes and motor

neurons, including excitotoxicity, oxidative stress, apoptosis, mitochondrial dysfunction and the inflammatory cascade, all putatively thought to contribute to ALS disease pathogenesis

QUIN is an excitotoxin and can be linked to excitotoxicity in ALS in two ways: (1) through the activation of the NMDA receptor; and (2) its effect on glutamate levels. The heteromeric NMDA receptor (NR) has three families of subunits: NR1 (A and B), NR2 (A to D) and NR3 (A and B). In the ventral and dorsal horns of ALS spinal cord, up to 78% loss of NR2A has been detected (Samarasinghe *et al.* 1996). Interestingly, QUIN acts on the NR subtypes, NR1+NR2A and NR1+NR2B (Priestley *et al.* 1995), and the loss of NR2A in ALS patients

Glutamate induced toxicity has been implicated in the selective neuronal damage seen in ALS and counteracting glutamatergic toxicity, thus far, is the only treatment available for ALS. QUIN can potentiate its own toxicity and that of other excitatory amino acids, such as glutamate, under energy deprived conditions (Schurr and Rigor 1993). Moreover, QUIN

and progression. Adapted from (Guillemin *et al.* 2005).

may possibly reflect an excitotoxic mechanism involving QUIN.

**3.2 QUIN and excitotoxicity** 

To date, the kynurenine pathway has been implicated in a wide range of diseases and disorders, including infectious diseases (e.g. HIV), neurological disorders (e.g. Alzheimer's disease (AD), Huntington's disease (HD) and ALS), affective disorders (e.g. schizophrenia, depression and anxiety), autoimmune diseases (e.g. multiple sclerosis and rheumatoid arthritis), peripheral conditions (e.g. cardiovascular disease) and malignancy, and a key indicator is often the upregulation in IDO-1 resulting in an accelerated and sustained degradation in TRP.
