The kynurenine pathway in Alzheimer’s disease
Significantly lower serum tryptophan, kynurenine and xanthurenic acid were found in participants clinically diagnosed with AD compared with controls, whilst serum xanthurenic acid demonstrated a significant positive correlation with participant MMSE cognitive scores.
Previous literature regarding tryptophan pathway metaboltes in AD contains conflicting results. Lower levels of tryptophan, xanthurenic acid, 3-hydroxyanthranilic acid [18] and tryptophan [33] and tryptophan and kynurenic acid [9] have been reported in the plasma of AD patients in agreement with our findings. However, conversely there are conflicting reports of higher levels of serum 3-hydroxykynurenine in AD [19], higher levels of serum kynurenine and anthranilic acid in females with a high neocortical amyloid-β load [10] and positive correlations between serum kynurenine metabolites with plasma amyloid-β(1–42) and neurofilament light chain [11]. This reported discrepancy may be a consequence of the relatively small sample sizes typically used for analysis, emphasising the need for further investigation into the association between AD and tryptophan metabolism using larger cohorts.
In addition, the metabolites kynurenic acid and quinolinic acid have been reported to be significantly higher in the cerebrospinal fluid (CSF) of individuals clinically diagnosed with AD [33]. The same study reported no significant differences in plasma concentrations of these metabolites collected from the same study participants. This finding is in agreement with the serum data presented here, where we report no significant differences of the two metabolites when comparing between the three clinical groups. Our data did however demonstrate a trend that suggested that kynurenic acid was lower in the individuals diagnosed with AD. Although this finding was significant in the initial Kruskal-Wallis inter-group test, it did not pass the correction for multiple testing threshold for the study. This result may suggest that metabolic changes in the kynurenine pathway observed in CSF may differ to those seen in blood-based biofluids. A study consisting of 20 AD cases and 18 controls that analysed the kynurenine pathway in both plasma and CSF reported significant metabolite correlations across CSF and plasma for kynurenine, 3-hydroxykynurenine, anthranilic acid, picolinic acid and neopterin; however, kynurenic acid was not significantly correlated across the biofluids [12]. In future, a large cohort study with matched sample types including CSF, serum/plasma and urine collected at the same study visit would provide valuable information on the translation of metabolic biomarkers in AD across the different biofluid compartments.
Previous literature has reported associations between quinolinic acid and Alzheimer’s disease pathology [34, 35] with reports of β-amyloid inducing the production of quinolinic acid by macrophages and microglia in vitro [36]. However, our data reported no significant differences between the concentrations of quinolinic acid in control and AD participant groups (Table 3). Likewise, the concentrations of picolinic acid have also been previously reported to associate with AD pathology [12]. Again, our data did not show any significant differences in the serum concentrations of picolinic acid between the participant groups (Table 3). The reasons behind the disparity in previous literature reports and our data are unclear but may be because the previous literature has typically compared the metabolites with specific features of AD pathology rather than overall clinical classification. Future large-scale studies that are able to collect pathological data in tandem with the clinical, cognitive and metabolic data may reveal more valuable information about these apparent relationships.
In urine, we also found significantly lower levels of tryptophan, xanthurenic acid and kynurenic acid, and urine xanthurenic acid demonstrated a significant positive correlation with participant MMSE cognitive scores. To the best of the authors’ knowledge, comparisons of urinary kynurenines in clinical cases of AD, MCI and controls have not been previously reported. The lower levels observed of the three urinary metabolites are consistent with our findings in serum.
Mechanistically, the rate limiting enzyme in the kynurenine metabolic pathway is indoleamine 2,3-dioxygenase (IDO)—a critical enzyme in systemic inflammation expressed by key cells of the immune system, including microglia [37]. The activity of IDO can be monitored using the circulating kynurenine/tryptophan ratio [16]. Here, we showed a higher urinary kynurenine/tryptophan ratio in the AD group and a significant negative correlation of the ratio with participant MMSE score, suggesting increased conversion of tryptophan to kynurenine prior to renal excretion, perhaps as a result of systemic inflammation and IDO upregulation in cases of AD and cognitive decline [38].
Our data also found no significant differences when comparing metabolite concentrations between participant groups for NAD+ and its precursors nicotinic acid, nicotinic riboside and β-nicotinamide mononucleotide. NAD is a key functional metabolite in cellular metabolism and has been hypothesised as playing a role in the disrupted energy metabolism pathways that occur in AD [39]. To the authors’ knowledge, there are no literature references that directly compare differences in the concentration of blood- or urine-based nicotinamide pathway metabolites in AD. However, there have been many examples of in vivo murine model work that has investigated the potential use of nicotinic metabolites as a treatment to slow AD pathology [40,41,42]. Our data suggest that these metabolites are not present at different concentrations in circulatory serum when comparing between the participant groups; however, further investigation into alternative biofluids such as CSF or post-mortem brain would add useful information regarding the role of the nicotinic pathway in AD.
The serotonin pathway in Alzheimer’s disease
A consequence of lower tryptophan bioavailability and an increase IDO enzyme activity is a reduced capacity for serotonin biosynthesis. This is reflected in our results, with lower levels of serotonin and 5-hydroxyindole acetic acid reported in the AD group.
Despite reports of lower amounts of serotonin in cerebrospinal fluid [43] and post-mortem brains [44, 45] in AD, to the best of the authors’ knowledge, differences in blood or urine have not been previously published and are reported here for the first time.
However, serotonin and serotonergic signalling have previously been proposed to be disrupted in AD [3], including reports of an increase in serotonin-4 receptors in the brain in response to an increased amyloid burden [46]. Madsen et al. hypothesised that this may be a consequence of lower serotonin, thereby acting as a compensatory effect to improve cognitive function, to increase acetylcholine release or to counteract increased amyloid accumulation [46]. Subsequent studies in mice have reported that amyloid precursor protein processing is regulated by the serotonin-4 receptor and activation of serotonin-4 receptor upregulates α-secretase, resulting in the formation of soluble amyloid, rather than the insoluble amyloid-β otherwise produced by cleavage via the β- and γ-secretase route [5]. Receptor agonists of serotonin-4 receptor, serotonin-5 receptor and serotonin-6 receptor have also been shown to reduce brain interstitial fluid levels of amyloid-β in the brains of mouse models [47].
In addition, selective serotonin reuptake inhibitors (SSRIs) are under investigation as therapeutic agents in AD. SSRIs work by increasing free serotonin at the synapse or neuronal cells resulting in increased levels of free serotonin available to synaptic receptors [14]. In both mouse models and humans, SSRIs have been reported to reduce levels of interstitial brain amyloid-β [48].
SSRIs are currently licenced for use in depression, and therefore, the study contained samples collected from participants who reported prescription of SSRI medication across all of the participant groups (Table 1). The mechanism of SSRI action is not fully understood, and there are conflicting reports in the literature regarding the effect of SSRI medication on blood and urine levels of serotonin [49,50,51]. In our data, individuals who were prescribed SSRI medication had significantly lower levels of serotonin in their serum; however, this difference was not observed in urine (Fig. 7). Further longitudinal work would be required to interpret whether this observation is a direct result of the SSRI medication, or due to the underlying pathophysiology of the individual that leads to treatment for depressive symptoms, a condition frequently linked to lower levels of plasma and serum serotonin [49].
To assess the impact of SSRI on the overall result of the study, univariate analysis was repeated only with the participants who did not take SSRI medication. In the re-analysis, serum serotonin no longer reported significant differences between AD and control groups (Table S5). However, interestingly, results in urine mirrored the full cohort, including the continued observation of significantly lower levels in the AD group of urinary tryptophan, serotonin and 5-hydroxyindoleacetic acid suggesting altered tryptophan and serotonin metabolism and renal excretion in the AD group, regardless of SSRI intake status. The discrepancy between serum and urine is unexplained, nevertheless the results raise important questions about the serotonergic signalling system in AD. Future longitudinal phenotyping resulting in accurate patient stratification may enable greater insight into the impact of serotonin bioavailability and SSRI medication in AD.
The bioavailability of tryptophan in Alzheimer’s disease
Tryptophan is the parent metabolite in both the serotonin and kynurenine pathways, and therefore, its bioavailability may have a downstream effect on the resultant bioavailability of key neuroactive metabolites in the circulatory system (Fig. 9).
As tryptophan is an essential amino acid that cannot be synthesised in mammalian systems, the bioavailability of circulatory free tryptophan is primarily influenced by the consumption of protein in the diet combined with the rate of usage in protein synthesis and the ability to absorb amino acids through the intestinal wall. In AD, the impact on bioavailability of essential amino acids is highly complex and multifactorial. Changes in appetite are well documented in AD with many occurrences of eating disturbances reported varying between both the loss and increase of appetite, as well as changes in dietary preference [52]. In addition, faecal calprotectin (a marker of intestinal inflammation) has been reported to be negatively associated with serum essential amino acids in individuals with AD—suggesting a disturbed intestinal barrier function leading to the lowering of essential amino acid blood concentrations [53].
The bioavailability of tryptophan is also known to be controlled by the population and diversity of an individual’s gut microbiome [54], with manipulation of the microbial composition demonstrated to impact plasma concentrations of tryptophan [55].
Research investigating alterations in the composition of the gut microbiome of individuals with AD have suggested that they have differences in the prevalence of Firmicutes, Bifidobacteria and Bacteroidetes compared with controls [56], all of which have been reported to possess tryptophan decarboxylase enzymes [54, 57], suggesting that gut diversity could impact the bioavailability of tryptophan and its downstream metabolites. However, in our results, the metabolite indole-3-acetic acid, an indole molecule known to be produced from tryptophan by gut bacteria [54], remained unchanged between participant groups (Kruskal-Wallis p = 0.8518, Holm-adjusted p = 1.0000), suggesting that the differences observed here in tryptophan metabolite may not be attributable to alterations in the gut microciome. Future studies warrant further investigation of indole containing metabolites (e.g. indole, tryptamine, indole lactic acid, indole aldehyde and indole propionic acid), which when combined with microbiome sequencing in a sample from AD cohorts will establish if any associations exist between tryptophan bioavailability, indole metabolites and gut microbial diversity.
