Compelling studies have shown that changes in the cerebrospinal fluid (CSF) and blood levels of
amino acids acting on the glutamatergic N-methyl-D-aspartate receptor (NMDAR) represent a neurochemical signature in various neuropathologies. These include psychiatric conditions such as schizophrenia39,40 and major depression41, and a wide spectrum of neurological diseases, including Alzheimer’s disease (AD)42–44, frontotemporal dementia45, Parkinson’s disease (PD)46–49, amyotrophic lateral sclerosis50,51, mild cognitive impairment52,53, multiple sclerosis54,55 and traumatic brain injury56. Surprisingly, no investigation so far specifically addressed the relationship between these neuroactive molecules and frailty phenotypes, including those related to cognitive decline and depression. Here, we sought to fill this gap by investigating the endogenous levels of D-serine, glycine and the other amino acids acting on glutamatergic neurotransmission in a well-characterized cohort of older subjects encompassing the entire continuum existing between fit and frail aging. Overall, our biochemical determinations suggest that disrupted systemic D-serine homeostasis may represent a potential predictive biomarker of frailty, while increased serum glycine and D-/Total serine ratio could be specifically associated with cognitive decline and depression in frail elderly individuals.
Previous blood metabolomics studies identified several metabolites associated with frailty, belonging to redox homeostasis, inflammation, amino acids, purine metabolism, urea and tricarboxylic acid cycles and sugar metabolism pathways14. Among the amino acids identified as dysregulated, glutamate metabolism was found to be affected in frail compared to non-frail subjects16–18,21,25. In light of this finding, and given the close relationship linking frailty with cognitive decline12,27,57, we investigated whether the serum levels of amino acids acting on glutamatergic NMDAR and their precursors could predict frailty status, and specifically its cognitive domain, in elderly adults. Interestingly, we found that serum D-serine is an independent predictor of the EFS score. D-serine is synthetized by serine racemase (SR)58 starting from its L-enantiomer and then degraded through D-amino acid oxidase (DAO) activity59,60. Once released in the forebrain, D-serine act as an obligatory co-agonist at the glycine modulatory site on GluN1 subunit of NMDAR, a ionotropic glutamatergic receptor playing a key role in sensorimotor gating, synaptic plasticity and cognitive functions61. Despite a few reports suggested that circulating blood D-serine concentration decrease61 or remains unchanged49,62,63 during healthy aging, recent studies found a positive correlation between serum D-serine and age in patients affected by AD and PD49,63.
Our observations showing that D-serine and D-/Total serine ratio significantly increase with ageing in frail but not in non-frail controls suggests that a dysregulation of blood D-serine homeostasis may represent a common ageing-related metabolic variation across different neuropathologies.
While EFS was conceived to evaluate frailty through a multidimensional approach, the Fried’s frailty phenotype is a widely used tool able to capture the physical domain of frailty8. Notably, we failed to find any association between D-serine or the other amino acids levels and frailty phenotype. Therefore, we argue that D-serine may not mirror all the components of frailty syndrome, but could instead represent a specific biochemical fingerprint of its cognitive domain. Consistent with this view, D-serine levels have recently been proposed as an early gender-related biomarker of AD since their serum concentrations correlated with cognitive deterioration in female patients44,63. However, other Authors failed to confirm significant changes of CSF and blood D-Ser levels in the whole AD clinical spectrum42,64. Interestingly, a recent clinical-pathological study showed that Aβ and tau brain deposition and frailty have a synergistic impact in determining the onset of dementia57. This finding, considered together with (i) the previous studies linking increased D-serine with AD-related pathology and cognitive decline43,65,66 and (ii) the ability of D-serine to diffuse across the blood-brain barrier67, suggest that blood levels of this D-amino acid could be adopted as a metabolic signature to identify older adults at higher risk of conversion to dementia.
Notably, the stratification of our elderly cohort by sex disclosed that the correlation between serum D-serine, EFS score and global cognition was mainly driven by females. In agreement with this view, recent investigations showed increased D-/Total Ser ratio in the human post-mortem hippocampus and serum of AD female patients compared to healthy females66,68. Similarly, we recently found a significant increase of serum D-serine in PD female, but not in male patients, compared to healthy controls49. These findings suggest that a dysregulation of blood D-serine may reflect the occurrence of different neuropathologies in a sex-dependent manner. Considering the neuroprotective roles played by estrogens and the compelling evidence that estrogens loss after menopause can accelerate the effect of aging on cognitive functions69, we speculate that the link between increased systemic D-serine levels and cognitive decline may be mediated, at least in part, by the reduced estrogens levels which characterize females aging. However, further studies on larger elderly cohorts are needed to address this outstanding issue.
We also found that higher serum glycine concentrations correlated with worse cognitive function and depressive symptoms in the frail but not in the non-frail group. Similarly to D-serine, glycine binds the GluN1 subunit of NMDAR and acts as a major obligatory co-agonist70. However, in other central nervous system (CNS) regions, glycine also regulates inhibitory neurotransmission via glycine receptors (GlyR)71. Considering that glycinergic transmission has been implicated in the physiopathology of cognitive decline and depression70,71, the correlation between blood glycine levels, cognitive performance and depressive symptoms may mirror a dysregulation of this amino acid in the CNS of frail subjects.
Despite our biochemical data are highly intriguing, the findings of the present study should be interpreted cautiously, bearing in mind that (i) the assessment of AD and other dementia-related biomarkers was not included in this study, thus preventing any inference linking the serum amino acid levels and the presence of concomitant neurodegenerative diseases; (ii) frailty is characterized by a decline in the function of multiple organ systems, which may directly influence the serum concentration of D-serine, glycine and the other amino acids. Indeed, recent studies showed that blood D-serine levels correlate positively with biochemical renal parameters62,72–74, while various L-amino acids correlated with metabolic parameters such as liver enzymes, lipids and blood glucose62. In the kidney and liver, glycine is rapidly interconverted with L-serine, which may then enter the glycolytic or gluconeogenic pathways through its conversion in pyruvate, or be employed in phospholipids biogenesis or mitochondrial metabolism75. Dietary intake and D-amino acids produced by the gut microbiota may also affect serine enantiomers metabolism76,77.
Interestingly, studies in animal models showed that D-serine is detectable in multiple organs, including heart, pancreas, spleen, liver, kidney, lung and muscles78,79, and glutamatergic receptors play relevant functions in the modulation of physiological processes in several peripheral tissues80. Of note, recent studies showed that serine racemase and NMDAR are highly expressed in human pancreatic islet β cells81, and systemic D-serine administration modulates insulin secretion in a dose-dependent manner82,83. Despite we found similar serum D-serine levels between diabetic and non-diabetic subjects, L-serine and L-glutamate were increased in diabetic compared to non-diabetic group, consistently with previous blood metabolomics evidence17,25. In line with other studies84, we also observed a positive correlation between serum L-glutamate concentration, BMI and visceral adiposity in both non-frail and frail participants. Although the biological mechanisms responsible for this association are still unclear, considering that glutamate signalling modulates the immune system85 and that increased VAT promotes systemic inflammation86, elevated blood L-glutamate levels could represent a metabolic signature underpinning the abnormal increase in oxidative stress and inflammation associated to obesity. Concurrently, our data suggest a correlation between serum L-glutamate concentration and SMI. This is in line with previous investigations showing that glutamate is crucial in maintaining the homeostasis of energy metabolism in skeletal muscle87. Surprisingly, this relationship was observed in the non-frail but not in frail group, suggesting that different biological pathways may modulate the maintenance of skeletal muscle mass across healthy and frail aging. However, our results may be affected by the very low prevalence of subjects with SMI scores below the proposed cut-off to define sarcopenia34 and therefore require validation in larger cohorts.
Besides its neuroactive role, glycine primarily influences anti-oxidative reactions and immune system75. In agreement with this knowledge, glycine has been used to prevent tissue injury, enhance anti-oxidative capacity, improve immunity, and treat metabolic disorders in obesity, diabetes and various inflammatory diseases88. Thus, consistent with the multiple beneficial effects of glycine, we cannot rule out that the negative correlation between this amino acid and cognitive functions in frail older individuals might represent an epiphenomenon triggered by inflammation and metabolic dysfunctions, rather than being causally linked to memory impairments. In the same way, we cannot rule out that systemic D-serine metabolism variation in frailty may represent a biochemical adaptation to CNS and multi-system deteriorations. In line with this hypothesis, D-serine supplementation or treatment with DAO inhibitors significantly improved cognitive functions in healthy subjects, PD and schizophrenia patients89–92. Future investigations are warranted to clarify these important issues.
The strengths of our work include (i) the novelty of investigating NMDAR-related amino acids and their precursors in the serum of a well-characterized elderly cohort, including the entire clinical spectrum existing from fit to frail condition; (ii) the correlation of serum amino acids with multiple potential confounding factors, such as diabetes, body composition and cigarette smoking93; (iii) the assessment of frailty with two different but complementary tools8,9.
However, we also acknowledge some limitations. First, the cross-sectional design and the clinical-biochemical correlations observed in the present study did not allow to draw any causal relationship between the serum changes in amino acids levels and the clinical phenotypes. Future longitudinal studies on larger elderly cohorts adopting a multidimensional approach, including the measurement of blood biomarkers mirroring brain (e.g. neurofilament light chain, Aβ42/Aβ40 ratio, phosphorylated tau94) and peripheral organs damage, as well as inflammation, are warranted to elucidate this issue. Second, the sex ratio was unbalanced with an higher prevalence of females, potentially biasing the analyses conducted after stratifying the cohort by sex. Third, the assessment of biochemical parameters of kidney and liver function was not included in the study protocol, thus preventing the adjustment of the analyses for the serum levels of creatinine, aspartate transaminase and alanine transaminase, which correlate with the blood levels of D-Ser and several L-amino acids, respectively62. However, the history of any kidney or liver disease or altered parameters of renal and hepatic function was strictly considered as an exclusion criteria at the time of participants enrolment.
In conclusion, this study highlights serum D-/serine and glycine as putative biochemical signatures of cognitive decline and depression in frail older subjects. The observation that D-serine correlates with frailty scores and global cognition in females but not in males suggest that this effect may also be modulated by sex-related biological factors.