Evaluation of Metabolic and Synaptic Dysfunction Hypotheses of Alzheimer's Disease (AD): A Meta-Analysis of CSF Markers

Abstract

Background: Alzheimer's disease (AD) is currently incurable and a majority of investigational drugs have failed clinical trials. One explanation for this failure may be the invalidity of hypotheses focusing on amyloid to explain AD pathogenesis. Recently, hypotheses which are centered on synaptic and metabolic dysfunction are increasingly implicated in AD.

Objective: Evaluate AD hypotheses by comparing neurotransmitter and metabolite marker concentrations in normal versus AD CSF.

Methods: Meta-analysis allows for statistical comparison of pooled, existing cerebrospinal fluid (CSF) marker data extracted from multiple publications, to obtain a more reliable estimate of concentrations. This method also provides a unique opportunity to rapidly validate AD hypotheses using the resulting CSF concentration data. Hubmed, Pubmed and Google Scholar were comprehensively searched for published English articles, without date restrictions, for the keywords "AD", "CSF", and "human" plus markers selected for synaptic and metabolic pathways. Synaptic markers were acetylcholine, gamma-aminobutyric acid (GABA), glutamine, and glycine. Metabolic markers were glutathione, glucose, lactate, pyruvate, and 8 other amino acids. Only studies that measured markers in AD and controls (Ctl), provided means, standard errors/deviation, and subject numbers were included. Data were extracted by six authors and reviewed by two others for accuracy. Data were pooled using ratio of means (RoM of AD/Ctl) and random effects meta-analysis using Cochrane Collaboration's Review Manager software.

Results: Of the 435 identified publications, after exclusion and removal of duplicates, 35 articles were included comprising a total of 605 AD patients and 585 controls. The following markers of synaptic and metabolic pathways were significantly changed in AD/controls: acetylcholine (RoM 0.36, 95% CI 0.24-0.53, p<0.00001), GABA (0.74, 0.58-0.94, p<0.01), pyruvate (0.48, 0.24-0.94, p=0.03), glutathione (1.11, 1.01- 1.21, p=0.03), alanine (1.10, 0.98-1.23, p=0.09), and lower levels of significance for lactate (1.2, 1.00-1.47, p=0.05). Of note, CSF glucose and glutamate levels in AD were not significantly different than that of the controls.

Conclusion: This study provides proof of concept for the use of meta-analysis validation of AD hypotheses, specifically via robust evidence for the cholinergic hypothesis of AD. Our data disagree with the other synaptic hypotheses of glutamate excitotoxicity and GABAergic resistance to neurodegeneration, given observed unchanged glutamate levels and decreased GABA levels. With regards to metabolic hypotheses, the data supported upregulation of anaerobic glycolysis, pentose phosphate pathway (glutathione), and anaplerosis of the tricarboxylic acid cycle using glutamate. Future applications of meta-analysis indicate the possibility of further in silico evaluation and generation of novel hypotheses in the AD field.

Keywords: Anaplerosis; CSF; GABA resistance; anaerobic glycolysis; cholinergic hypothesis; glutamate excitotoxicity; glutaminolysis; pentose phosphate pathway.

Fig. (1)

PRISMA Flow diagram showing meta-analysis selection of publications for all biomarkers, using PubMed database search as an example. Data represents pooled publications collected for 15 different markers for both space limitations and clarity. The duplicates were removed later in the work flow diagram, because this more accurately reflects the work flow which involved several authors independently searching for publications. Furthermore, several publications were common for several compounds, especially when amino acid analyses were performed. Additional publications from other sources highlight creativity of medical students in their searches. The workflow diagram is modified from Moher et al. [34]. Details of the individual markers investigated in this study are in Tables 1-4.

Fig. (2)

Meta-analysis of AD-specific changes in CSF markers. Bubble plot of the meta-analysis data plotting 3 values: effect size, z-score, and a number of subjects. The effect size, or the Ration of the Means (RoM), on the y-axis is the ratio of the pooled means (AD/Ctl concentrations). A ratio greater than unity indicates that CSF concentrations in AD are greater than the control, while a lower ratio indicates decreased CSF concentrations in AD vs Ctrl. The Z-score, shown on the x-axis, is a statistic indicating the deviation of the mean from the standard error. For example, any Z-score > 1.645 relates to significant p-value < 0.10. The bubble size reflects the total number of subjects in each meta-analysis (e.g. the smallest and largest bubbles represent pooled subject sizes of 67 and 323, respectively).

Fig. (3)

Mapping of meta-analysis data onto biochemical pathways. Several metabolic and synaptic pathways are depicted. Metabolic pathways include anaplerosis, glycolysis, and the pentose phosphate pathway (PPP), all shown as boxes. The synaptic pathways include the cholinergic (boxed), glutamatergic (boxed) and GABA. The relevant metabolic enzymes and CSF metabolites and amino acids were mapped to these biochemical pathways, with increases (green arrows) or decreases (red arrows), and significant changes denoted with their p-values. Note: for simplicity, several additional sources of glutamate are not depicted e.g. from the TCA cycle. For enzyme data lacking standard deviation, these values were imputed. The GDH fold change based on 1 publication [80].