biomolecules Article Kynurenic Acid Levels are Increased in the CSF of Alzheimer’s Disease Patients Marta González-Sánchez 1,2 , Javier Jiménez 3 , Arantzazu Narváez 3 , Desiree Antequera 1,2 , Sara Llamas-Velasco 1,2 , Alejandro Herrero-San Martín 1,2 , Jose Antonio Molina Arjona 1,2 , Adolfo López de Munain 2,4 , Alberto Lleó Bisa 2,5 , M.-Pilar Marco 6,7 , Montserrat Rodríguez-Núñez 6,7 , David Andrés Pérez-Martínez 1,2 , Alberto Villarejo-Galende 1,2 , Fernando Bartolome 1,2, * , Elena Domínguez 3, * and Eva Carro 1,2, * 1 Group of Neurodegenerative Diseases, Hospital 12 de Octubre Research Institute (imas12), 28041 Madrid, Spain; martags.86@gmail.com (M.G.-S.); eeara@yahoo.es (D.A.); laisset@hotmail.com (S.L.-V.); alexportalrubio@hotmail.com (A.H.-S.M.); cvillaiza@telefonica.net (J.A.M.A.); daperezm@salud.madrid.org (D.A.P.-M.); avgalende@yahoo.es (A.V.-G.) 2 Biomedical Research Networking Center in Neurodegenerative Diseases (CIBERNED), 28031 Madrid, Spain; adolfojose.lopezdemunainarregui@osakidetza.eus (A.L.d.M.); alleo@santpau.cat (A.L.B.) 3 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, 28871 Alcalá de Henares, Spain; javier.jimenez@uah.es (J.J.); arantzazu.narvaez@uah.es (A.N.) 4 Neurosciences Research Area, Biodonostia Health Research Institute, 20014 San Sebastian, Spain 5 Neurology Department, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain 6 Nanobiotechnology for Diagnostics (Nb4D) Group, Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), 08034 Barcelona, Spain; pilar.marco@iqac.csic.es (M.-P.M.); montse.rodriguez@iqac.csic.es (M.R.-N.) 7 Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08034 Barcelona, Spain * Correspondence: fbartolome.imas12@h12o.es (F.B.); elena.dominguez@uah.es (E.D.); carroeva@h12o.es (E.C.); Tel.: +34-913-908-765 (F.B. & E.C.); +34-918-854-666 (E.D.); Fax: +34-913-908-544 (F.B. & E.C.); +34-918-854-666 (E.D.)  Received: 2 March 2020; Accepted: 2 April 2020; Published: 8 April 2020  Abstract: Kynurenic acid (KYNA) is a product of the tryptophan (TRP) metabolism via the kynurenine pathway (KP). This pathway is activated in neurodegenerative disorders, such as Alzheimer´s disease (AD). KYNA is primarily produced by astrocytes and is considered neuroprotective. Thus, altered KYNA levels may suggest an inflammatory response. Very recently, significant increases in KYNA levels were reported in cerebrospinal fluid (CSF) from AD patients compared with normal controls. In this study, we assessed the accuracy of KYNA in CSF for the classification of patients with AD, cognitively healthy controls, and patients with a variety of other neurodegenerative diseases, including frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP). Averaged KYNA concentration in CSF was higher in patients with AD when compared with healthy subjects and with all the other differentially diagnosed groups. There were no significant differences in KYNA levels in CSF between any other neurodegenerative groups and controls. These results suggest a specific increase in KYNA concentration in CSF from AD patients not seen in other neurodegenerative diseases. Keywords: Alzheimer´s disease; kynurenine pathway; cerebrospinal fluid; biomarkers; amyloid-β; tau protein Biomolecules 2020, 10, 571; doi:10.3390/biom10040571 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 571 2 of 15 1. Introduction Alzheimer’s disease (AD) is a complex and progressive neurodegenerative disorder that represents one of the major health problems in the world [1]. The main pathophysiological features of AD are the in-brain accumulation of amyloid-β peptide (Aβ) in neuritic extracellular plaques, the hyperphosphorylated tau protein in intracellular neurofibrillary tangles (NFT), and neuronal loss. The consequence of the brain accumulation of Aβ and tau can be detected by analyzing the levels of Aβ42 peptide (the most neurotoxic isoform of Aβ) and total tau (t-tau) protein in the cerebrospinal fluid (CSF) of AD patients. This analysis made it possible to establish these parameters as clinical biomarkers indicating that reduced Aβ levels and increased t-tau levels in CSF are distinctive of AD [2]. The inverse correlation between Aβ42 levels in the CSF and the brain is explained because Aβ42 in the AD brain is sequestered in the cerebral neuritic plaques, as it is their main component, with less Aβ being available to diffuse into the CSF, and therefore reduced levels of Aβ42 in CSF from AD patients (reviewed in [3]). In the case of t-tau protein, CSF level probably reflects the intensity of neuronal damage and neurodegeneration (reviewed in [3]). Although highly studied, the exact initiating and pathophysiological AD-initiating factors remain unknown, but the majority of AD-related research is based on the amyloid cascade and tau hypotheses. In addition to these theories, different pathological mechanisms, such as mitochondrial dysfunction, glutamate excitotoxicity, and neuroinflammation have been implicated in AD [2–8]. Neuroinflammation in AD is associated with the activation of microglia and astrocytes along with increased levels of neuroinflammatory mediators and modulators such as proinflammatory cytokines and chemokines [8,9]. One consequence of microglial activation is the overproduction of quinolinic acid (QA). This excitotoxin is a downstream-derived product from the kynurenine pathway (KP) and strongly contributes to the neuroinflammatory environment [10]. KP is the main route that metabolizes tryptophan (TRP), and particularly, this pathway has also been implicated in AD [11]. There is a bidirectional connection between inflammatory cytokines and the KP, and an imbalance in this association may possibly lead to neurological or psychiatric disorders [12,13]. One study suggested that interleukin (IL)-6 activates the KP in patients with schizophrenia, leading to an increased production of kynurenic acid (KYNA) [14]. It was shown that the KP regulates both innate and adaptive immune responses, and that TRP metabolism reflects a crucial interface between immune and central nervous system (CNS) [15]. Therefore, any imbalance in the KP and subsequently in the production of TRP metabolites may cause neuronal damage, giving rise to multiple physiological and neurological impairments [16,17]. TRP is an essential amino acid necessary for protein biosynthesis, and a precursor of several compounds with important biological functions, such as serotonin, melatonin, and kynurenines [18]. TRP enters the brain by a carrier-mediated transport across the blood-brain barrier, where it is lately degraded. KYNA is a product of the TRP metabolism via the KP, and it is synthesized in and released by astrocytes in the brain [19,20]. KYNA is an N-methyl-D-aspartate (NMDA) and α7 nicotinic acetylcholine (α7nACh) receptor antagonist and is considered to be neuroprotective [18,21–23]. It counteracts the excitotoxic activity of other KP metabolites, such as QA [24,25]. Increased levels of KYNA were found in AD brains and AD animal models, and authors explained that by exerting its antagonist abilities on NMDA and α7nACh receptors, KYNA may disrupt the learning and cognitive abilities [26,27]. Additionally, increased levels of KYNA in the developing brain resulted in biochemical impairments and behavioral abnormalities [28]. However, several reports showing KYNA and other KP metabolite levels in CSF and blood from AD patients and healthy subjects reached inconclusive results [29–35]. Using high-performance liquid chromatography (HPLC), recent studies found increased KYNA concentration in CSF in different cohorts of AD patients [36,37]. However, it was almost simultaneously published that KYNA levels were strongly reduced in CSF from AD patients when compared with control subjects [38]. In this work, we aimed to clarify KP metabolite concentrations measured in CSF and serum and to determine their specificity in AD. Therefore, we analyzed TRP and KYNA levels in CSF and plasma from a well-characterized cohort of patients who had mild cognitive impairment (MCI), AD, or one of a range of other neurodegenerative disorders, including Biomolecules 2020, 10, 571 3 of 15 frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP), using a sensitive enzyme-linked immunosorbent assay (ELISA). 2. Materials and Methods 2.1. Cohorts In total, 132 subjects were included in this study: (i) elderly non-demented subjects classified as controls (n = 23); (ii) patients with mild cognitive impairment due to AD (MCI) (n = 24); (iii) probable mild AD (n = 41); (iv) moderate–severe AD (n = 20); (v) frontotemporal dementia (FTD) (n = 8); (vi) amyotrophic lateral sclerosis (ALS) (n = 8); and (vii) progressive supranuclear palsy (PSP) (n = 8). Subjects were recruited from the Neurology Service of three different geographical areas in Spain: 12 de Octubre University Hospital (Madrid); Donostia-Osakidetza University Hospital (San Sebastian); and Santa Creu i Sant Pau Hospital (Barcelona). Diagnoses were based on detailed clinical assessments, neuropsychological studies, neuroimaging, electromyography, and CSF Aβ42 and t-tau levels. An AD- indicative biomarker profile (Aβ+ ) was defined as CSF Aβ42 <550 pg/µL and t-tau/Aβ42 ratio >0.52, according to ELISA manufacture instructions and reported cut-offs [39]. Those cases with CSF Aβ42 ≥550 pg/µL and t-tau/Aβ42 ratio ≤0.52 were defined as non-AD biomarker profiles (Aβ− ). Diagnosis of MCI and dementia due to AD were established according to the National Institute on Aging and the Alzheimer’s Association guidelines [2,40], and these criteria were used in the present study. The majority of MCI patients were amnesic MCI but some of them presented an atypical non-amnesic MCI (visual variant or language variant). These atypical cases were, however, then confirmed as MCI due to AD within their Aβ+ CSF biomarker profiles. Global cognition was assessed using the Mini Mental State Examination (MMSE) [41] and disease severity using the Clinical Dementia Rating (CDR) score [42]. All FTD patients met the clinical diagnosis criteria of behavioral variant FTD [43], or the nonfluent/semantic variant primary progressive aphasia (PPA) [44]. ALS and PSP diagnosis were established according to revised El Escorial criteria [45] and Boxer criteria [46] respectively. All MCI and AD dementia patients had an Aβ+ CSF biomarker profile, while it was Aβ− in patients with other neurodegenerative diseases and healthy controls. Controls were usually recruited from among the patients’ partners when they fulfilled the age and health criteria. Inclusion criteria for old, cognitively-normal individuals were age over 50, no history or clinical signs of neurological or psychiatric disease, and absence of AD CSF biomarker profile. Exclusion criteria for patients were evidence of concomitant cerebrovascular disease or any neurological, psychiatric, or non-neurological medical conditions; or medications that could affect cognition or motor function. Consent was in accordance with the Declaration of Helsinki, and approval was obtained from the research ethics committee of the responsible institution (Research Institute Hospital 12 de Octubre (imas12: 16/079, Date: 26 April 2016); Biodonostia Health Research Institute (22/2016 Date: 19 July 2017); Sant Pau Biomedical Research Institute (16/2013 Date: 14 January 2014)). Written informed consent was signed by all participants or representatives. 2.2. Sample Collection CSF samples were collected from all subjects and processed according to standardized procedures by lumbar puncture in 15-mL sterile polypropylene tubes. Samples were then centrifuged at 3000 rpm at 4 ◦ C for 10 min. Supernatant aliquots were stored at −80 ◦ C into 0.5 mL polypropylene tubes with Protease Inhibitor Cocktail (Roche Applied Science, Basel, Switzerland). Blood samples were obtained through antecubital vein puncture from patients and healthy subjects. Plasma was isolated from whole blood and collected in 7-mL-EDTA-2Na tubes. Whole blood was centrifuged at 2000 rpm for 10 min at room temperature. Supernatants were then collected in tubes with Protease Inhibitor Cocktail (Roche Applied Science, Basel, Switzerland) and stored at −80 ◦ C. 2.3. DNA Purification and Apolipoprotein E (APOE) Genotyping Genomic DNA was extracted from peripheral blood using QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer instructions. Human APOE C112R and R158C Biomolecules 2020, 10, 571 4 of 15 polymorphisms were detected to identify the APOE ε2, ε3, and ε4 alleles, using LightCycler 480 II Instruments Kit (Roche Diagnostics, Basel, Switzerland) following manufacturer instructions. 2.4. Aβ42 and t-tau Quantification Endogenous Aβ42 and t-tau levels in CSF samples were quantified using INNOTEST ELISAs (Fujirebio Europe NV, Gent, Belgium), according to manufacturer’s instructions. 2.5. KYNA and TRP Quantification KYNA and 5-hydroxy-L-tryptophan (L-5-HTP; from now on TRP) levels in CSF and plasma samples were quantified using an in-house competitive ELISA in triplo developed by the Bioelectrochemistry and Biosensors Group from the University of Alcalá in collaboration with the Nanobiotechnology for Diagnostics (Nb4D) group from IQAC-CSIC. Analytical parameters found in CSF were a limit of detection (LOD) of 0.26 µg/L with an IC50 of 23.6 µg/L and a linear range (LR) from 2.8 to 100 µg/L for KYNA; and a LOD of 2.5 µg/L with an IC50 of 250 µg/L and a LR from 40–912 µg/L for TRP. The immunoreagents used for KYNA detection (the polyclonal antibody (As301) and the enzyme tracer (HRP-SIA-IHSH)) were developed by the Nb4D group with the support of the ICTS “NANBIOSIS”; more specifically, by the Custom Antibody Service (CAbS, CIBER-BBN, IQAC-CSIC). Their preparation and characterization will be described elsewhere. The tryptophan hydroxylase polyclonal antibody (ref. PA5-19749) was purchased from Thermo Fisher Scientific, (Thermo Fisher Scientific, MA, USA). A 96-well ELISA microplate was coated overnight at 4 ◦ C with anti-KYNA and anti-TRP primary antibodies. Then, plates were washed with phosphate-buffered saline containing 0.05% Tween-20 (PBST). After washing, following standards and CSF samples (50 µL), 50 µL of HRP-bioconjugates (HRP-SIA-IHSH or HRP-L-5-HTP) were added to each well and the plate was incubated for 30 min at room temperature. Next, wells were washed and 100 µL of substrate solution were added. After 30 min incubation, absorbances were measured at 450 nm using a Fisher Multiskan Go microplate reader (Thermo Fisher Scientific, MA, USA). Inter-assay variability for KYNA and L-5-HTP assays was estimated at 21.4% (n = 17) and 21% (n = 4), respectively. Intra-assay variability was estimated at 13.4% (n = 18) and 10.6% (n = 6) for KYNA and L-5-HTP, respectively. 2.6. Data and Statistical Analysis Numerical data are shown as means ± standard deviations (SD) and categorical data as a percentages. To compare demographic, clinical, and plasma and CSF biomarker data between groups, we used the non-parametric Kruskal–Wallis rank test and non-parametric pairwise comparisons adjusted by Bonferroni. Fisher’s exact test was used to compare the distribution of categorical data across groups. Correlations between biomarkers were assessed using Spearman rank correlation. Receiver operating characteristic (ROC) curves were used to determine the diagnostic accuracy of KYNA from CSF to differentiate between the diagnostic groups. The optimal cut-off point, sensitivity, specificity, and area under the curve (AUC) were calculated in all cases. Statistical significance was set out at p < 0.05. Statistical analysis was performed using STATA/IC 14.2 for Windows, Texas, USA. 3. Results 3.1. Demographic and Clinical Characteristics Clinical, demographic, and genetic profiles from all subjects are shown in Table 1. No differences in sex were found between groups. Regarding age, healthy donors and ALS patients were younger than AD dementia patients. MMSE showed a progressive decreased score according to cognitive severity in the MCI and AD dementia groups. APOE genotype was more prevalent in the MCI/AD patients than in controls, according to previous publications [47]. Aβ42 and t-tau levels in CSF are also shown in Table 1. Biomolecules 2020, 10, 571 5 of 15 Table 1. Demographic and biomarker characteristics of the study participants. Control MCI Mild AD Mod AD FTD ALS PSP p Characteristics (n = 23) (n = 24) (n = 41) (n = 20) (n = 8) (n = 8) (n = 8) # Age, mean (SD), y 64.7 (10.8) 72.0 (7.1) 71.9 (8.1) a 73.3 (7.2) a,b 66.4 (5.2) 58.0 (14.1) 74.0 (6.0) <0.001 Female sex, n (%) 8 (34.8) 14 (58.4) 22 (53.7) 13 (65.0) 3 (37.4) 3 (37.5) 4 (50.0) ns Disease duration, mean (SD), y - 2.4 (1.4) 2.6 (1.7) 4.0 (1.6) c,d,e 3.3 (1.2) 1.8 (0.9) 4.1 (2.2) <0.01 MMSE score, mean (SD) - 25.4 (3.2) 21.6 (3.0) e 16.0 (4.3) f,g - - - <0.0001 CDR, mean (SD) 0 0.5 1,0 2.2 (0.4) - - - NA APOE ε4 carrier, No./Total (%) 0/15 (0.0%) 9/15 (60.0%) h 9/18 (50.0%) h 2/9 (22.2%) - - - <0.0001 CSF biomarkers Aβ42 , mean (SD), pg/mL 1026.1 (278.9) 380.0 (89.3) 355.9 (103.8) 360.9 (88.0) 832.2 (188.5) 701.2 (224.1) 1058.7 (219.3) NA T-tau, mean (SD), pg/mL 303.9 (110.7) 928.8 (545.0) 836.4 (444.0) 871.2 (389.7) 305.4 (162.0) 137.0 (56.5) 252.3 (88.7) NA CSF KYNA, mean (SD), µg/L 3.9 (2.9) 9.8 (6.9) a 11.1 (7.2) i 10.5 (7.4) h 5.6 (2.5) 4.0 (1.4) 4.7 (3.7) <0.0001 CSF TRP, mean (SD), µg/L 73.7 (19.7) 75.5 (20.6) 77.1 (25.3) 79.7 (27.0) 78.4 (18.6) 67.5 (23.6) - ns Ratio KYNA/TRP, mean (SD) 0.06 (0.06) 0.13 (0.09) 0.16 (0.13) h 0.17 (0.17) 0.08 (0.05) 0.07 (0.04) - <0.05 Plasma biomarkers (n = 20) (n = 11) (n = 17) (n = 9) Plasma KYNA, mean (SD), µg/L 164.0 (118.5) 179.4 (126.9) 167.4 (126.4) 176.9 (82.8) - - - ns Plasma TRP, mean (SD), µg/L 2333.3 (1216.3) 2022.7 (1335.2) 2104.7 (822.7) 1956.0 (1259.3) - - - ns Ratio KYNA/TRP, mean (SD) 0.08 (0.07) 0.10 (0.07) 0.08 (0.09) 0.11 (0.08) - - - ns Abbreviations: MCI, mild cognitive impairment; Mild AD, mild Alzheimer’s disease; Mod AD, moderate–severe Alzheimer’s disease; FTD, frontotemporal dementia; ALS, amyotrophic lateral sclerosis; PSP, progressive supranuclear palsy; MMSE, Mini-Mental State Examination; CDR, Clinical Dementia Rating; Aβ42 , β-amyloid 42; t-tau, total tau; KYNA, kynurenic acid; TRP, tryptophan; NA, not applicable; ns, non-significant. # p-value indicates statistical difference between all groups; a p < 0.05 vs. control; b p < 0.05 vs. ALS; c p < 0.05 vs. MCI; d p < 0.05 vs. Mild AD; e p < 0.01 vs. ALS; f p < 0.001 vs. MCI; g p < 0.01 vs. Mild AD; h p < 0.01 vs. control; i p < 0.0001 vs. control. Biomolecules 2020, 10, 571 6 of 15 3.2. KYNA and TRP Levels in CSF from the Study Participants Biomolecules 2020, 9, x FOR PEER REVIEW 7 of 16 Averaged KYNA and TRP levels in CSF from all subjects are shown in Table 1, and each KYNA individual 3.2. KYNA value and TRPis Levels represented in CSF from in Figure the Study 1. Data show increased KYNA concentrations Participants in MCI (9.8 ± 6.9Averaged µg/L; pKYNA < 0.05), mild and TRP AD levels (11.1 in CSF ± all from 7.2subjects < 0.0001), µg/L;arep shown in Tableand 1, andmoderate–severe each KYNA AD (10.5 ± 7.4 µg/L; p < 0.01) individual value patients is represented in Figure 1.with compared Data show increased controls (3.9KYNA ± 2.9concentrations µg/L) (Figure in MCI 1a).(9.8Although ± the 6.9 µg/L; p < 0.05), mild AD (11.1 ± 7.2 µg/L; p < 0.0001), and moderate–severe AD (10.5 ± 7.4 µg/L; p < analysis of KYNA levels in other neurodegenerative diseases reported no significance when compared 0.01) patients compared with controls (3.9 ± 2.9 µg/L) (Figure 1A). Although the analysis of KYNA Commented [M6]: The writing shou to MCI, mildlevels AD,inand moderate other AD, a trend neurodegenerative diseasesof reduced reported levels was no significance whenobserved comparedintoFTD mild ± 2.5 µg/L), MCI, (5.6 picture, e.g., ‘Figure 1a’, the same as ALS (4.0 ± 1.4AD, and moderate µg/L), and PSP AD, a trend (4.7 of reduced ± 3.7 levels wasRegarding µg/L) groups. observed in FTD TRP (5.6 ± 2.5 µg/L), levels, ALS (4.0 ± 1.4 were found no differences between groupsµg/L),(Table and PSP 1).(4.7KYNA/TRP ± 3.7 µg/L) groups. ratioRegarding TRP levels, was increased innoCSFdifferences from AD werepatients found between compared with groups (Table 1). KYNA/TRP ratio was increased in CSF from AD patients compared with healthy healthy subjects (0.06 subjects ±±0.06), (0.06 but 0.06), but only only the mild the mild AD groupADreached groupstatistical reached statistical significance ± 0.13; p < 0.01;(0.16 ± 0.13; significance (0.16 p < 0.01; Table Table 1). 1). Figure 1. KYNAFigure 1. KYNA levels levelsfrom in CSF in CSF allfrom all differentially differentially diagnosed groups diagnosed groups ofofdonors. donors.(a) Scatter plots plots over (a) Scatter over box plots showing KYNA levels in CSF from all groups following the diagnosis. Boxes represent box plots showing KYNA levels in CSF from all groups following the diagnosis. Boxes represent the 25th, the 25th, 50th, and 75th data percentiles. KYNA levels were increased in MCI and AD dementia 50th, and 75thgroups data compared percentiles. KYNA to controls. (b)levels weredisplaying Scatter plots increased in MCI KYNA levelsand AD in CSF dementia from groups compared subjects grouped throughout to controls. (b) Scattertheir plotsCSFdisplaying biomarker profile: KYNA controls (n = 23), levels patients in CSF withsubjects from AD-indicative CSF biomarker grouped throughout their CSF biomarker profiles (n = 85), profile: and patients controls (n = without AD-indicative 23), patients CSF biomarker profiles with AD-indicative CSF (n = 24). Differences biomarker profiles (n = 85), between groups were assessed using Kruskal–Wallis test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < and patients without AD-indicative CSF biomarker profiles (n = 24). Differences between groups were 0.0001. MCI, mild cognitive impairment; Mild AD, mild Alzheimer’s disease; Mod AD, moderate– assessed using Kruskal–Wallis severe Alzheimer’s disease; * p