d-serine levels in Alzheimer's disease: implications for novel biomarker development

Abstract

Alzheimer's disease (AD) is a severe neurodegenerative disorder still in search of effective methods of diagnosis. Altered levels of the NMDA receptor co-agonist, d-serine, have been associated with neurological disorders, including schizophrenia and epilepsy. However, whether d-serine levels are deregulated in AD remains elusive. Here, we first measured D-serine levels in post-mortem hippocampal and cortical samples from nondemented subjects (n=8) and AD patients (n=14). We next determined d-serine levels in experimental models of AD, including wild-type rats and mice that received intracerebroventricular injections of amyloid-β oligomers, and APP/PS1 transgenic mice. Finally, we assessed d-serine levels in the cerebrospinal fluid (CSF) of 21 patients with a diagnosis of probable AD, as compared with patients with normal pressure hydrocephalus (n=9), major depression (n=9) and healthy controls (n=10), and results were contrasted with CSF amyloid-β/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis.

Figure 1

d-serine levels are increased in Alzheimer's disease (AD) post-mortem brain tissue. d-serine levels in the hippocampus (a), parietal cortex (b) and occipital cortex (c) of post-mortem samples from control (Ctrl) and AD subjects. Values are presented as nmol of d-serine per g of wet tissue (WT). Horizontal lines represent mean values for each diagnostic group. Data points correspond to individual values. Statistical significance is given by the Student's t-test (*P<0.05; NS, not significant).

Figure 2

Amyloid-β oligomers (AβOs) increase d-serine and serine racemase (SR) levels in hippocampal cultures. Primary rat hippocampal neuronal cultures were exposed to 500 nM AβOs or vehicle (2% dimethyl sulfoxide in phosphate-buffered saline) for 24 h. (a) AβOs increased extracellular levels of d-serine. (b and c) AβOs increased total levels of SR messenger RNA (mRNA) (b) and protein (c). d-serine was measured by high-performance liquid chromatography and its values were corrected by total protein content in the analyzed samples. SR protein levels were detected by western blotting, using β-actin as a loading control. *P<0.05 (Student's t-test), statistical significance was assessed in comparison with control. Results are expressed as means±s.e.m. of three independent experiments (each carried out in triplicate wells) with different neuronal cultures.

Figure 3

Amyloid-β oligomers (AβOs) increase hippocampal d-serine and serine racemase (SR) levels in vivo. (a) AβOs increased d-serine levels in hippocampal homogenates of rats that received intrahippocampal injections of AβOs (1 μg) or vehicle (2% dimethyl sulfoxide in phosphate-buffered saline) once a week for 5 weeks (n=8 veh; n=7 AβOs), as analyzed 3 days after the last injection. (b) d-serine content is increased in the hippocampi of mice that received a single intracerebroventricular injection of AβOs (10 pmol, or 45 ng). d-serine levels were measured 8 days post injection (n=10 per group). (c and d) Thirteen- to fourteen-month-old APPSwe/PS1ΔE9 (APP/PS1) transgenic mice showed increased hippocampal levels of d-serine (c) and SR (d) compared with wild-type (WT) mice (n=8 per group). d-serine was measured by high-performance liquid chromatography and its values were corrected by total protein (ptn) content in the analyzed samples. SR protein levels were detected by western blotting using β-actin as the loading control. *P<0.05; **P<0.01 (Student's t-test), statistical significances were assessed in comparison with controls. Results are presented as means±s.e.m. of individuals.

Figure 4

Increased cerebrospinal fluid (CSF) levels of d-serine in patients with probable Alzheimer's disease (AD). (a) CSF levels of d-serine in healthy controls (Ctrl) and in patients with probable AD, major depressive disorder (MDD) or hydrocephalus (Hydro). Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Bonferroni adjustment for multiple comparisons. (b) CSF d-serine levels (μmol l⁻¹) in non-AD (white circles for INNOTEST amyloid/tau index (IATI)-based classification; blue circles for neuropsychological assessment) and AD cases (black circles for IATI-based classification; red circles for neuropsychological assessment) using different IATI cutoffs (0.83; 0.96; 1.24). Horizontal bars represent mean values for each group. ***P<0.001; Student's t-test. (c) CSF levels of d-serine as a function of the mini-mental state exam (MMSE). (d) CSF levels of d-serine as a function of CDR scores. Horizontal lines represent mean values for each CDR group. Data points correspond to individual values. Statistical significance is given by one-way ANOVA followed by Bonferroni adjustment for selected groups: CDR 0.5, 1, 2 and 3 versus CDR 0. (e) CSF levels of d-serine as a function of IATI across subject groups. (f) Receiver-operating characteristic curves for diagnostic based on IATI alone (blue line) or IATI/D-ser levels (red line), showing increased sensitivity and specificity when d-serine is added to the calculation. *P<0.05; ***P<0.001. CDR, clinical dementia rating score; NPA, neuropsychological assessment.