Kynurenine pathway and Parkinson’s disease

by Dr Jia-he Bai, Prof. Yong-peng Yu and Dr Ya-li Zheng
The diagnosis of early-stage Parkinson’s disease (PD) is still a worldwide clinical problem: it is necessary to identify a biomarker to aid the diagnosis of early-stage PD. This article discusses the current worldwide research progress of kynurenine and PD, in order to provide new directions and ideas for clinical and scientific research.

Background

Parkinson’s disease (PD) is a common neurodegenerative disease with progressive degeneration of dopaminergic neurons in the substantia nigra. It has become one of the major diseases that threaten the health of elderly people. Typical motor symptoms of PD are bradykinesia, rigidity and tremor. Besides motor symptoms, in the early stages of the disease, there are also some non-motor symptoms such as sleep disorders, constipation and cognitive decline, which increase with disease progression. The diagnosis of early-stage PD is still a worldwide clinical problem; therefore, it is necessary to identify a biomarker to aid the diagnosis of early-stage PD. Studies have shown that excitotoxicity, neuroinflammation, oxidative stress, misfolded protein accumulation, neuronal apoptosis and mitochondrial dysfunction affect the onset of PD and the evolution of disease [1,2]. The kynurenine pathway (KP) has also been found to correlate with PD and may play an important role in the pathogenesis of PD [3]. In addition, the kynurenine (KYN)/tryptophan (Trp) ratio have been found to be increased in the blood and cerebrospinal fluid (CSF) of PD patients [4]; interestingly, a report indicated that Trp levels were significantly higher in patients with PD than healthy controls [5]. Hence, this review discusses the current worldwide research progress on the KP and its metabolites and PD, in order to provide new directions a nd ideas for clinical and scientific research.

Different methods of measuring urine kynurenine

At present, the quantitative detection of KYN in body fluids mainly includes enzyme-linked immunoassay, high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS), and highperformance liquid chromatography with fluorescence (HPLC-FLD) and high-performance liquid chromatography ultraviolet method (HPLC-UV). Although the HPLC-MS/MS method has high sensitivity and selectivity in most cases, because of the small molecular structure of KYN, the fragments produced after proton impact are also small, which affects the selectivity and sensitivity of detection. In addition, because the equipment is expensive and the detection cost is high, it is not suitable for wide application in hospitals.
The HPLC-FLD method has to use an online derivatization method, as KYN itself cannot produce fluorescence, and so is more complicated and leads to poorer reproducibility. HPLC-UV equipment requirements are relatively low, so this can be attempted in the hospital. Although enzyme-linked immunoassay has cumbersome operating steps and takes a long time, it is more suitable for small-scale experimental research and testing in hospitals.

Kynurenine pathway

The metabolism of L-tryptophan is divided into two distinct pathways, the serotonin and the kynurenine pathway (KP) (Fig. 1). Indoleamine 2,3-dioxygenase 1 and 2 and tryptophan 2,3-dioxygenase convert L-tryptophan to N-formyl-L-kynurenine in the first step of the KP. N-formyl-L-kynurenine is further processed by kynurenine formamidase to L-kynurenine (L-KYN), the central metabolite of the KP. From L-KYN, three different enzymes produce the next metabolites, forming three branches of the metabolism. The first branch is the kynurenic acid (KNYA) branch, where kynurenine aminotransferases (KATs) produce KNYA from L-KYN. Production of anthranilic acid (AA) is on the second branch. On the third branch, kynurenine monooxygenase produces 3-hydroxykynurenine (3-HK), which is further metabolized by kynureninase to 3-hydroxyanthranilic acid (3-HA). 3-HK can be also metabolized by KATs to form xanthurenic acid or be autooxidized. 3-HA is converted by 3-hydro-xyanthranilic acid oxygenase to 2-amino-3-carboxymuconic-6-semialdehyde (ACMS) or suffers auto-oxidation to form cinnabarinic acid. ACMS can be converted by picolinic carboxylase to picolinic acid or can be converted by non-enzymatic cyclisation to quinolinic acid (QUIN), which, through conversion by quinolinic acid phosphoribosyltrans-ferase, results in the formation of nicotinamide adenine dinucleotide (NAD+).

KP is involved in the development of PD

Several neuroactive compounds produced in KP have different function, for example: KYNA is an antagonist of α7 nicotinic acetylcholine receptors (α7nAchRs), and is also antagonist of N-methyl-D-aspartate receptors (NMDARs) when KYNA at a high concentration level [6], while some ex vivo studies disputed the actions of KYNA on α7nAChRs, several in vivo and electrophysiological studies suggested that α7nAChRs were the preferential target of endogenous KYNA in the brain [7]. Overall, it is possible that KYNA blocked NMDARs and α7nAChRs with comparable potency. So, KYNA can inhibit the excitatory amino acid toxicity caused by NMDARs. In addition, KYNA can also be used as a potential endogenous antioxidant and exert neuroprotective effects by inhibiting mitochondrial apoptosis and inflammatory actions [8]. In addition to KYNA, AA and 3-HA also seem to be anti-inflammatory factors. QUIN can produce neurotoxic effects by selectively activating NMDARs, generating reactive oxygen intermediates, consuming endogenous antioxidants and causing mitochondrial dysfunction [9]. In fact, QUIN can also act as an initiator and promoter of inflammation. 3-HK produces free radicals that lead to neuronal toxicity in PD. The inflammatory factors in turn induce the activating enzymes of the KP process which means inflammation leads to a concomitant activation of the KP. This raises the possibility of a positive feedback loop.
In PD, production of QUIN is so high that there is insufficient KYNA to block QUIN production. Moreover, KYNA is decreased in PD patients. In a word, the rising levels of KYN are shifted to feed into the QUIN path rather than the KYNA path, thus leading to PD progression (Fig. 2). Similarly, the urine KYN levels in the PD group were significantly higher than those in the control group in our previous research (Table 1), which indicated that urine KYN might be used as a valuable biomarker for early-stage PD [10].
A recent study found that the gene encoding 2-amino-3-carboxymuconate- semialdehyde decarboxylase (ACMSD) is closely related to the risk of PD. As mentioned above, ACMSD is located at a key branch-point in the KP and converts ACMS into (eventually) picolinic acid, and hence limits the availability of ACMS for conversion into neurotoxic QUIN. Therefore, mutations that affect the expression and/or function of ACMSD will affect the formation of QUIN, indicating that the gene encoding ACMSD may be involved in the development of PD [11]. It is currently believed that the disorder of the brain-gut-microbial axis may be one of the mechanisms of the pathogenesis of PD, but the specific mechanism of the disorder is still unclear. Some scientists have found that KYN may affect the microbial community of the gastrointestinal tract [12], which may affect the intestine (causing changes in the homeostasis and permeability of the tract), and the occurrence of inflammation and the activation of immune cells lead to harmful substances entering the brain through the blood–brain barrier, triggering the death of dopaminergic neurons. The microbiota can also consume Trp, the precursor of KYN, and subsequently affect KYN levels. As an essential amino acid, Trp can only be obtained from the diet. There may be a loop mechanism involving KYN and the gastrointestinal microbiota. When KP is disturbed, it also leads to the destruction of the loop, leading to the onset of PD.

The therapeutic effect of KP in PD

There are countless studies on the treatment of PD, the commonly used Madopar [containing levodopa (L-DOPA) and benserazide] and dopaminergic agonists can only relieve the clinical symptoms of PD, and cannot reverse the course of PD, and long-term use will cause dyskinesias, end-of-dose phenomena and affect the quality of life of PD patients. The role of KP in the pathogenesis of PD demonstrates its potential as a therapeutic target in the treatment of PD.
Using PD animal models, several therapeutic strategies have been tested to increase endogenous KYNA or decrease QUIN production to prevent or slow down the progression of PD and related disorders. Silva-Adaya showed that co-administration of the main KYNA precursor, L-KYN and an inhibitor of organic anion transporter, increased KYNA and resulted in the reversal of glutamate-induced excitotoxicity in 6-OHDA-induced PD rats. The increase in KYNA not only modulated glutamate release from cortical areas to striatum but also directly acted on the NMDA receptors as antagonist, thereby limiting glutamate excitotoxicity. To overcome the short half-life of these metabolites, analogues of L-KYN and KYNA [such as L-4- chlorokynurenine (4-Cl-KYN), 7-chlorokynurenic acid (7-Cl-KYNA) and more recently 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1Hquinolin- 4-one hydrochloride] have been designed to enhance the stability and pharmacological properties. 4-Cl-L-KYN crosses the blood–brain barrier and blocks QUIN toxicity at the glycine site on NMDA receptors. These KYNA analogues are blood–brain barrier permeable, capable of suppressing glutamate release and NMDA activation with therapeutic potential in PD that is yet to be assessed. KYNA analogues are at this time going into clinical trials for the treatment of probable PD as potential neuroprotective agents [13].
Zonisamide is a sulfonamide antiepileptic drug that has been used to treat PD and show improved motor symptom of PD patient with levodopa-induced dyskinesia. Although the mechanism remains unknown, zonisamide is able to enhance the production of KYNA. Among the KP rate-limiting enzymes, kynurenine monooxygenase (KMO), located in the middle reaches of the KP, is also important. KMO can regulate the production of downstream metabolites of 3-HK, and inhibiting KMO can turn KYN to the direction of KYNA production. Early work found that when the KMO inhibitor nicotinylalanine is injected into the brain ventricle, it can have a protective effect on QUIN-mediated neurotoxicity in the nigrostriatal dopamine system [14]. KMO inhibitors can significantly reduce the severity of hamster dystonia [15], and the most widely used KMO inhibitor 3,4-dimethoxy-N-[4-(3-nitrophenyl)-1,3- thiazol-2-yl]benzenesulfonamide (Ro 61-8048) can increase the level of KYNA and can also attenuate L-DOPA-induced dyskinesia in 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, and will not damage the therapeutic effect of L-DOPA [16].
FK506 (also known as tacrolimus), a neuroimmunophilin ligand that is being used as an immunosuppressant in PD therapy, not only enhances the formation of KYNA in the cortex but also abolishes the inhibition of KYNA synthesis induced by MPP+ (1-methyl-4-phenylpyridinium) and 3-nitropropionic acid. A recent study showed that treatment with FK506 could significantly increase the survival of dopaminergic neurons in a dose-dependent manner. However, the short-term treatment has only a minor effect on disease progression. Nevertheless, FK506 significantly lowered the infiltration of both T-helper and cytotoxic T cells and the number and subtype of microglia and macrophages [17].
These data suggest that the anti-inflammatory properties of FK506 significantly reduce neurodegeneration, and highlight a causal role of neuroinflammation in the pathobiology of PD.

Conclusions and future directions

Many studies have shown that the KP and its metabolites involved in PD, participate in the occurrence and development of PD through several mechanisms, such as affecting mitochondrial function, the brain-gut-microbial axis, excitotoxicity and neurotoxicity. It was demonstrated that specific KP metabolite analogues and inhibitors could increase the level of KYNA and reduce the production of QUIN and, consequently, the restoration and maintenance of the normal balance of the KP by addition of zonisamide and neuroimmunosuppressant FK506, which contributes to treat PD. In the future, experimental research on targeted drugs based on the characteristics of the KP and its metabolites for PD therapy should be carried out. The KP might become a new direction for the treatment of PD.

Acknowledgement

This research was supported by National Natural Science Foundation of China (81400957).
The authors
Jia-he Bai1,2 MM, Yong-peng Yu*2,3 MD and Ya-li Zheng2 MM
1 Department of Neurology, Heze Municipal Hospital affiliated to Heze
Medical College, Heze, People’s Republic of China
2 Department of Neurology, Weihai Central Hospital affiliated to Qingdao
University, Weihai, People’s Republic of China
3 Department of Neurology, Weihai Central Hospital affiliated to Weifang
Medical college, Weihai, People’s Republic of China

*Corresponding author:
E-mail: yypeng6688@126.com