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Progressive multiple sclerosis (MS) is a chronic disease with a unique pattern, which is histologically classified into the subpial type 3 lesions in the autopsy. The lesion is also homologous to that of cuprizone (CPZ) toxin-induced animal models of demyelination. Aberration of the tryptophan (TRP)-kynurenine (KYN) metabolic system has been observed in patients with MS; nevertheless, the KYN metabolite profile of progressive MS remains inconclusive. In this study, C57Bl/6J male mice were treated with 0.2% CPZ toxin for 5 weeks and then underwent 4 weeks of recovery. We measured the levels of serotonin, TRP, and KYN metabolites in the plasma and the brain samples of mice at weeks 1, 3, and 5 of demyelination, and at weeks 7 and 9 of remyelination periods by ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after body weight measurement and immunohistochemical analysis to confirm the development of demyelination. The UHPLC-MS/MS measurements demonstrated a significant reduction of kynurenic acid, 3-hydoxykynurenine (3-HK), and xanthurenic acid in the plasma and a significant reduction of 3-HK, and anthranilic acid in the brain samples at week 5. Here, we show the profile of KYN metabolites in the CPZ-induced mouse model of demyelination. Thus, the KYN metabolite profile potentially serves as a biomarker of progressive MS and thus opens a new path toward planning personalized treatment, which is frequently obscured with immunologic components in MS deterioration.

Background: Altered glutamatergic neurotransmission and neuropeptide levels play a central role in migraine pathomechanism. Previously, we confirmed that kynurenic acid, an endogenous glutamatergic antagonist, was able to decrease the expression of pituitary adenylate cyclase-activating polypeptide 1-38, a neuropeptide with known migraine-inducing properties. Hence, our aim was to reveal the role of the peripheral kynurenine pathway (KP) in episodic migraineurs. We focused on the complete tryptophan (Trp) catabolism, which comprises the serotonin and melatonin routes in addition to kynurenine metabolites. We investigated the relationship between metabolic alterations and clinical characteristics of migraine patients., Methods: Female migraine patients aged between 25 and 50 years (n = 50) and healthy control subjects (n = 34) participated in this study. Blood samples were collected from the cubital veins of subjects (during both the interictal/ictal periods in migraineurs, n = 47/12, respectively). 12 metabolites of Trp pathway were determined by neurochemical measurements (UHPLC-MS/MS)., Results: Plasma concentrations of the most Trp metabolites were remarkably decreased in the interictal period of migraineurs compared to healthy control subjects, especially in the migraine without aura (MWoA) subgroup: Trp (p < 0.025), L-kynurenine (p < 0.001), kynurenic acid (p < 0.016), anthranilic acid (p < 0.007), picolinic acid (p < 0.03), 5-hydroxy-indoleaceticacid (p < 0.025) and melatonin (p < 0.023). Several metabolites showed a tendency to elevate during the ictal phase, but this was significant only in the cases of anthranilic acid, 5-hydroxy-indoleaceticacid and melatonin in MWoA patients. In the same subgroup, higher interictal kynurenic acid levels were identified in patients whose headache was severe and not related to their menstruation cycle. Negative linear correlation was detected between the interictal levels of xanthurenic acid/melatonin and attack frequency. Positive associations were found between the ictal 3-hydroxykynurenine levels and the beginning of attacks, just as between ictal picolinic acid levels and last attack before ictal sampling., Conclusions: Our results suggest that there is a widespread metabolic imbalance in migraineurs, which manifests in a completely depressed peripheral Trp catabolism during the interictal period. It might act as trigger for the migraine attack, contributing to glutamate excess induced neurotoxicity and generalised hyperexcitability. This data can draw attention to the clinical relevance of KP in migraine.

Background: The kynurenine (KYN) pathway (KP) of the tryptophan (TRP) metabolism seems to play a role in the pathomechanism of multiple sclerosis (MS). Cuprizone (CPZ) treated animals develop both demyelination (DEM) and remyelination (REM) in lack of peripheral immune response, such as the lesion pattern type III and IV in MS, representing primary oligodendrogliopathy., Objective: To measure the metabolites of the KP in the CPZ treated animals, including TRP, KYN and kynurenic acid (KYNA). We proposed that KYNA levels might be decreased in the CPZ-induced demyelinating phase of the animal model of MS, which model represents the progressive phase of the disease., Methods: A total of 64 C57Bl/6J animals were used for the study. Immunohistochemical (IHC) measurements were performed to prove the effect of CPZ, whereas high-performance liquid chromatography (HPLC) was used to quantify the metabolites of the KP (n = 10/4 groups; DEM, CO1, REM, CO2)., Results: IHC measurements proved the detrimental effects of CPZ. HPLC measurements demonstrated a decrease of KYNA in the hippocampus (p < 0.05), somatosensory cortex (p < 0.01) and in plasma (p < 0.001)., Conclusion: This is the first evidence of marked reduction in KYNA levels in a non-immune mediated model of MS. Our results suggest an involvement of the KP in the pathomechanism of MS, which needs to be further elucidated., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

The incidence of neurodegenerative diseases has increased greatly worldwide due to the rise in life expectancy. In spite of notable development in the understanding of these disorders, there has been limited success in the development of neuroprotective agents that can slow the progression of the disease and prevent neuronal death. Some natural products and molecules are very promising neuroprotective agents because of their structural diversity and wide variety of biological activities. In addition to their neuroprotective effect, they are known for their antioxidant, anti-inflammatory and antiapoptotic effects and often serve as a starting point for drug discovery. In this review, the following natural molecules are discussed: firstly, kynurenic acid, the main neuroprotective agent formed via the kynurenine pathway of tryptophan metabolism, as it is known mainly for its role in glutamate excitotoxicity, secondly, the dietary supplement pantethine, that is many sided, well tolerated and safe, and the third molecule, α-lipoic acid is a universal antioxidant. As a conclusion, because of their beneficial properties, these molecules are potential candidates for neuroprotective therapies suitable in managing neurodegenerative diseases.

Kynurenic acid (KYNA) is one of the most significant metabolite of the kynurenine pathway both in terms of functional and potential therapeutic value. It is an N-methyl-D-aspartate (NMDA) receptor antagonist, but it can also activate the G-protein coupled receptor 35 (GPR35), which shares several structural and functional properties with cannabinoid receptors. Previously our group demonstrated that systemic chronic KYNA treatment altered opioid receptor G-protein activity. Opioid receptors also overlap in many features with cannabinoid receptors. Thus, our aim was to examine the direct in vitro and systemic, chronic in vivo effect of KYNA on type 1 cannabinoid receptor (CB 1 R) binding and G-protein activity. Based on competition and [ 35 S]GTPγS G-protein binding assays in rat brain, KYNA alone did not show significant binding towards the CB 1 R, nor did it alter CB 1 R ligand binding and agonist activity in vitro. When rats were chronically treated with KYNA (single daily, i.p., 128 mg/kg for 9 days), the KYNA plasma and cerebrospinal fluid levels significantly increased compared to vehicle treated group. Furthermore, in G-protein binding assays, in the whole brain the amount of G-proteins in basal and in maximum activity coupled to the CB 1 R also increased due to the treatment. At the same time, the overall stimulatory properties of the receptor remained unaltered in vehicle and KYNA treated samples. Similar observations were made in rat hippocampus, but not in the cortex and brainstem. In saturation binding assays the density of CB 1 Rs in rat whole brain and hippocampus were also significantly enhanced after the same treatment, without significantly affecting ligand binding affinity. Thus, KYNA indirectly and brain region specifically increases the abundance of functional CB 1 Rs, without modifying the overall binding and activity of the receptor. Supposedly, this can be a compensatory mechanism on the part of the endocannabinoid system induced by the long-term KYNA exposure., Competing Interests: Declaration of competing interest There are none., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Several studies supported an increased vulnerability of males regarding Parkinson's disease (PD) and its animal models, the background of which has not been exactly revealed, yet. In addition to hormonal differences, another possible factor behind that may be a female-predominant increase in endogenous striatal alpha-tocopherol (αT) level with aging, even significant at 16 weeks of age, previously demonstrated by the authors. Accordingly, the aim of the current study was the assessment whether this difference in striatal αT concentration may contribute to the above-mentioned distinct vulnerability of genders to nigrostriatal injury. Female and male C57Bl/6 mice at the age of 16 weeks were injected with 12 mg/kg body weight 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 5 times at 2 h intervals or with saline. The levels of some biogenic amines (striatum) and αT (striatum and plasma) were determined by validated high performance liquid chromatography methods. Although the results proved previous findings, i.e., striatal dopamine decrease was less pronounced in females following MPTP treatment, and striatal αT level was significantly higher in female mice, the correlation between these 2 variables was not significant. Surprisingly, MPTP treatment did not affect striatal αT concentrations, but significantly decreased plasma αT levels without differences between genders. The current study, examining the possible role of elevated αT in female C57Bl/6 mice behind their decreased sensitivity to MPTP intoxication for the first time, was unable to demonstrate any remarkable connection between these 2 variables. These findings may further confirm that αT does not play a major role against neurotoxicity induced by MPTP., (© 2020 The Author(s).)

The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the "best" reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC-MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The neurochemical background of the evolution of headache disorders, still remains partially undiscovered. Accordingly, our aim was to further explore the neurochemical profile of Complete Freund's adjuvant (CFA)-induced orofacial pain, involving finding the shift point regarding small molecule neurotransmitter concentrations changes vs. that of the previously characterized headache-related neuropeptides. The investigated neurotransmitters consisted of glutamate, γ-aminobutyric acid, noradrenalin and serotonin. Furthermore, in light of its influence on glutamatergic neurotransmission, we measured the level of kynurenic acid (KYNA) and its precursors in the kynurenine (KYN) pathway (KP) of tryptophan metabolism., Methods: The effect of CFA was evaluated in male Sprague Dawley rats. Animals were injected with CFA (1 mg/ml, 50 μl/animal) into the right whisker pad. We applied high-performance liquid chromatography to determine the concentrations of the above-mentioned compounds from the trigeminal nucleus caudalis (TNC) and somatosensory cortex (ssCX) of rats. Furthermore, we measured some of these metabolites from the cerebrospinal fluid and plasma as well. Afterwards, we carried out permutation t-tests as post hoc analysis for pairwise comparison., Results: Our results demonstrated that 24 h after CFA treatment, the level of glutamate, KYNA and that of its precursor, KYN was still elevated in the TNC, all diminishing by 48 h. In the ssCX, significant concentration increases of KYNA and serotonin were found., Conclusion: This is the first study assessing neurotransmitter changes in the TNC and ssCX following CFA treatment, confirming the dominant role of glutamate in early pain processing and a compensatory elevation of KYNA with anti-glutamatergic properties. Furthermore, the current findings draw attention to the limited time interval where medications can target the glutamatergic pathways.

The development of a validated method, applicable for the measurement of tryptophan (TRP) and serotonin (5-HT), and that of the neuroprotective branch of the kynurenine pathway from several different biological matrices, including mouse brain, is described. Following the spectral analysis of the metabolites, they were quantified with reversed-phase high-performance liquid chromatography (HPLC), using separate internal standards (ISs) for UV (3-nitro-L-tyrosine) and fluorescent (the newly utilized 4-hydroxyquinazoline-2-carboxylic acid) detectors. With regard to validation parameters, selectivity, linearity, limit of detection, limit of quantification, precision and recovery were determined. Although the linearity ranges were different for the assessed matrices, the correlation coefficient was >0.999 in each case. Furthermore, good intra- and inter-day precision values were obtained with coefficient of variation <5%, and bias <6.5% (except the 5-HT level in brain samples), respectively. The recoveries varied between 82.5% and 116%. The currently developed methods yield opportunities for the assessment of concentration changes in the TRP metabolism from a wide range of biological matrices, therefore they may well be utilized in future clinical and preclinical studies, especially in view that so many metabolites with the application of ISs have not been detected from mouse brain with such a simple HPLC method before., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Manipulation of kynurenic acid (KYNA) level through kynurenine aminotransferase-2 (KAT-2) inhibition with the aim of therapy in neuro-psychiatric diseses has been the subject of extensive recent research. Although mouse models are of particular importance, neither the basic mechanism of KYNA production and release nor the relevance of KAT-2 in the mouse brain has yet been clarified. Using acute mouse brain slice preparations, we investigated the basal and L-kynurenine (L-KYN) induced KYNA production and distribution between the extracellular and intracellular compartments. Furthermore, we evaluated the effect of specific KAT-2 inhibition with the irreversible inhibitor PF-04859989. To ascertain that the observed KYNA release is not a simple consequence of general cell degradation, we examined the structural and functional integrity of the brain tissue with biochemical, histological and electrophysiological tools. We did not find relevant change in the viability of the brain tissue after several hours incubation time. HPLC measurements proved that mouse brain slices intensively produce and liberate KYNA to the extracellular compartment, while only a small proportion retained in the tissue both in the basal and L-KYN supplemented state. Finally, specific KAT-2 inhibition significantly reduced the extracellular KYNA content. Taken together, these results provide important data about KYNA production and release, and in vitro evidence for the first time of the function of KAT-2 in the adult mouse brain. Our study extends investigations of KAT-2 manipulation to mice in a bid to fully understand the function; the final, future aim is to assign therapeutical kynurenergic manipulation strategies to humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Since the definite diagnosis of Creutzfeldt-Jakob disease (CJD) can currently only be provided by autopsy, there is a special need for fine diagnostic tools in live patients to achieve accurate diagnosis as early as possible. The aim of this study was to perform a preliminary retrospective analysis on the utility of the measurement of total Tau (tTau) and some other biomarkers from the cerebrospinal fluid (CSF) of patients with rapidly progressive dementia in the diagnostic work up of CJD. Beside the assessment of relevant clinical data and the findings of electroencephalography and brain magnetic resonance imaging, the presence of 14-3-3 protein and the levels of tTau were determined by Western blot technique and enzyme-linked immunosorbent assay from the CSF of 19 patients diagnosed with rapidly progressive dementia between the period of 2004-2017 at the Department of Neurology, University of Szeged. This preliminary study provided 100% sensitivity for 14-3-3, and interestingly, only 40% specificity to support the clinical diagnosis of CJD. Regarding tTau, the sensitivity values were calculated to be 100% or 83%, whereas the specificity values were 71% or 86%, depending on the applied cut-off levels. The poor specificity of 14-3-3 is not in line with literature data and may be the result of the small number of patients in the cohort with non-prion disease, predominantly consisting of disorders with considerable tissue damage, whereas tTau presented good sensitivity and specificity values. The combined application of these and novel chemical biomarkers may increase both sensitivity and specificity to a desired level.

The aging process clearly increases the demand for antioxidant protection, especially in the brain, involving that provided by α-tocopherol (αT). However, little is known about the age-related changes in brain αT levels and the influencing effect of gender on it, in human or murine samples as well. Accordingly, the aim of the current study was to detect age-, gender- and region-specific changes in αT concentrations in mouse brain tissue and to assess the influencing effect of plasma αT levels on it. Female and male C57BL/6 mice at the ages of 6, 16 and 66 weeks (n = 9 in each group) were applied. αT levels were determined with high performance liquid chromatography (HPLC) from the striatum, cortex, hippocampus, cerebellum, brainstem and from plasma samples. A detailed validation process was carried out for the applied HPLC method as well. The results demonstrated that brain αT levels significantly increased in the striatum, cortex, and hippocampus with aging in both genders, but in a more pronounced way in females with an increasing magnitude of this difference. In case of the cerebellum, a moderate elevation could be detected only in females, whereas in case of the brainstem there was no significant change in αT level. With regard to plasma samples, no clear trend could be identified. The current study is the first to present age-dependent gender-specific changes in αT level in certain brain regions of the C57Bl/6 mouse strain, and may provide meaningful information for future therapeutic studies targeting aging-related processes.

Dysfunction of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α) has been linked to various neurodegenerative and neuropsychiatric disorders; however, reports on psychic behavioral alterations on PGC-1α-deficient animals are sparse. The present study revisited prior observations of anxiety-related, depression-related, and hippocampal memory-related observations having been made on different PGC-1α-deficient murine strains, in a large-scale analysis on whole-body full-length (FL-)PGC-1α-deficient mice. The examinations were performed on animals covering a wide age range enrolled from both sexes, and included paradigms such as the open-field, elevated plus maze, light-dark box, tail suspension test, and spatial recognition two-trial Y-maze. The findings revealed no signs of previously reported anxiety-like behavior, but revealed an unexpected phenotype with decreased anxiety behavior consistent throughout different paradigms, with slight male preponderance. This was associated with despair-like anhedonic behavior, consistent with that reported previously, but did not associate with either peripheral or brain alterations in kynurenic acid synthesis, which was previously proposed. Though male FL-PGC-1α-deficient mice tended to perform poorer in the hippocampus-based spatial learning paradigm, the genotype overall was not associated with impairment in spatial memory, contradicting with prior observations. None of the observed alterations deteriorated with age, similarly to motor alterations as reported previously. The most likely contributors of this peculiar phenotype are discussed, with clinicopathological correlations drawn. Being the first to address these behavioral domains within the same PGC-1α-deficient strain, our findings extend the knowledge about the complex in vivo effect of PGC-1α dysfunction and add important notes to research in the field of PGC-1α in connection with neuropsychiatric disorders.