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

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.

GSK-3 and CK2 Kinases Converge on Timeless to Regulate the Master Clock.

The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, we identify a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro also delay nuclear accumulation in both tissue culture and in vivo and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity.

Posttranscriptional and posttranslational regulation of clock genes.

Circadian rhythms have been observed in diverse organisms, including plants, animals, bacteria, and fungi. In such organisms, the circadian clock is primarily composed of a cell-autonomous transcriptional feedback loop. In addition to transcriptional regulation, the modification of core clock transcripts and proteins can dramatically affect the circadian clock. In this review, the authors discuss some of the posttranscriptional and posttranslational modifications and their effects on the circadian clock. The combined outcome of these modifications is to adjust the timing of the clock to produce a circadian oscillator that takes approximately 24 h.

CK1 and GSK3 in the Drosophila and mammalian circadian clock.

Two kinases, DOUBLETIME and SHAGGY, have been shown to play a role in the circadian clock. DOUBLETIME, the Drosophila orthologue of casein kinase 1, can phosphorylate PERIOD in the cytoplasm and in the nucleus. This phosphorylation destabilizes PERIOD in both locations and sets patterns of both cytoplasmic accumulation and nuclear turnover. Cytoplasmic phosphorylation postpones accumulation of PERIOD and affects timing of nuclear accumulation of PERIOD/ TIMELESS complexes. SHAGGY, the Drosophila orthologue of glycogen synthase kinase 3, phosphorylates TIMELESS and promotes nuclear translocation of PERIOD/ TIMELESS complexes. Thus, the opposing effects of these two kinases in the cytoplasm are crucial for establishing the approximately 24 h period of circadian rhythmicity in Drosophila. Casein Kinase 1 has been shown to be a component of the circadian clock in mammals. Recent studies are also pointing to a role for glycogen synthase kinase 3 in the mammalian clock.