Gary E. Gibson

Consensus Report of the Working Group on : Molecular and Biochemical Markers of Alzheimer's Disease

The ideal biomarker for Alzheimers disease (AD) should detect a fundamental feature of neuropathology and be validated in neuropathologically-confirmed cases; it should have a sensitivity >80% for detecting AD and a specificity of >80% for distinguishing other dementias; it should be reliable, reproducible, non-invasive, simple to perform, and inexpensive. Recommended steps to establish a biomarker include confirmation by at least two independent studies conducted by qualified investigators with the results published in peer-reviewed journals. Our review of current candidate markers indicates that for suspected early-onset familial AD, it is appropriate to search for mutations in the presenilin 1, presenilin 2, and amyloid precursor protein genes. Individuals with these mutations typically have increased levels of the amyloid Abeta42 peptide in plasma and decreased levels of APPs in cerebrospinal fluid. In late-onset and sporadic AD, these measures are not useful, but detecting an apolipoprotein E e4 allele can add confidence to the clinical diagnosis. Among the other proposed molecular and biochemical markers for sporadic AD, cerebrospinal fluid assays showing low levels of Abeta42 and high levels of tau come closest to fulfilling criteria for a useful biomarker.The ideal biomarker for Alzheimers disease (AD) should detect a fundamental feature of neuropathology and be validated in neuropathologically-confirmed cases: it should have a sensitivity >80% for detecting AD and a specificity of >80% for distinguishing other dementias: it should be reliable, reproducible non-invasive, simple to perform, and inexpensive. Recommended steps to establish a biomarker include confirmation by at least two independent studies conducted by qualified investigators with the results published in peer-reviewed journals. Our review of current candidate markers indicates that for suspected early-onset familial AD. it is appropriate to search for mutations in the presenilin 1, presenilin 2, and amyloid precursor protein genes. Individuals with these mutations typically have increased levels of the amyloid Aβ 42 peptide in plasma and decreased levels of APPs in cerebrospinal fluid. In late-onset and sporadic AD. these measures are not useful. but detecting an apolipoprotein E e4 allele can add confidence to the clinical diagnosis. Among the other proposed molecular and biochemical markers for sporadic AD. cerebrospinal fluid assays showing low levels of Aβ 42 and high levels of tau come closest to fulfilling criteria for a useful biomarker.

Mitochondrial abnormalities in Alzheimer brain: Mechanistic implications

Reductions in cerebral metabolism sufficient to impair cognition in normal individuals also occur in Alzheimers disease (AD). The degree of clinical disability in AD correlates closely to the magnitude of the reduction in brain metabolism. Therefore, we tested whether impairments in tricarboxylic acid (TCA) cycle enzymes of mitochondria correlate with disability. Brains were from patients with autopsy‐confirmed AD and clinical dementia ratings (CDRs) before death. Significant (p < 0.01) decreases occurred in the activities of the pyruvate dehydrogenase complex (−41%), isocitrate dehydrogenase (−27%), and the α‐ketoglutarate dehydrogenase complex (−57%). Activities of succinate dehydrogenase (complex II) (+44%) and malate dehydrogenase (+54%) were increased (p < 0.01). Activities of the other four TCA cycle enzymes were unchanged. All of the changes in TCA cycle activities correlated with the clinical state (p < 0.01), suggesting a coordinated mitochondrial alteration. The highest correlation was with pyruvate dehydrogenase complex (r = 0.77, r2 = 0.59). Measures to improve TCA cycle metabolism might benefit AD patients. Ann Neurol 2005;57:695–703

Calcium and the aging nervous system

Many aspects of calcium homeostasis change with aging. Numerous calcium compartments complicate studies of altered calcium regulation. However, age-related decreases in calcium permeation across membranes and mobilization from organelles may be a common fundamental change. Deficits in ion movements appear to lead to altered coupling of calcium-dependent biochemical and neurophysiological processes and may lead to pathological and behavioral changes. The calcium-associated changes during aging probably do not occur with equal intensity in all cell types or in different parts of the same cell. Thus, cells or compartments with a high proportion of calcium activated processes would be more sensitive to diminished calcium availability. These age-related changes may predispose the brain to the development of age-related neurological disorders. The effects of decreased ion movement may be further aggravated by an age-related decline in other calcium-dependent processes. Depression of some of these calcium-dependent functions appears physiologically significant, since increasing calcium availability ameliorates age-related deficits in neurotransmission and behavior. A better understanding of the interactions between calcium homeostasis and calcium-dependent processes during aging will likely help in the design of more effective therapeutic strategies.

Protein-bound acrolein: a novel marker of oxidative stress in Alzheimer's disease.

Abstract : Several lines of evidence support the role of oxidative stress, including increased lipid peroxidation, in the pathogenesis of Alzheimers disease (AD). Lipid peroxidation generates various reactive aldehydes, such as 4‐hydroxynonenal (HNE), which have been detected immunochemically in AD, particularly in neurofibrillary tangels, one of the major diagnostic lesions in AD brains. A recent study demonstrated that acrolein, the most reactive among the α, β‐unsaturated aldehyde products of lipid peroxidation, could be rapidly incorporated into proteins, generating a carbonyl derivative, a marker of oxidative stress to proteins. The current studies used an antibody raised against acrolein‐modified keyhole limpet hemocyanin (KLH) to test whether acrolein modification of proteins occurs in AD. Double immunofluorescence revealed strong acrolein‐KLH immunoreactivity in more than half of all paired helical filament (PHF)‐1‐labeled neurofibrillary tangles in AD cases. Acrolein‐KLH immunoreactivity was also evident in a few neurons lacking PHF‐1‐positive neurofibrillary tangles. Light acrolein‐KLH immunoreactivity occurred in dystrophic neurites surrounding the amyloid‐β core, which itself lacked acrolein‐KLH staining. The pattern of acrolein‐KLH immunostaining was similar to that of HNE. Control brains did not contain any acrolein‐KLH‐immunoreactive structures. The current results suggest that protein‐bound acrolein is a powerful marker of oxidative damage to protein and support the hypothesis that lipid peroxidation and oxidative damage to protein may play a crucial role in the formation of neurofibrillary tangles and to neuronal death in AD.

Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease.

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range of biological effects. Since resveratrols properties seem ideal for treating neurodegenerative diseases, its ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for forty-five days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless, resveratrol diminished plaque formation in a region specific manner. The largest reductions in the percent area occupied by plaques were observed in medial cortex (-48%), striatum (-89%) and hypothalamus (-90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing. However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and decreased glutathione may be linked to the diminished plaque formation. This study supports the concept that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive agents that protect against beta-amyloid plaque formation and oxidative stress.

IMPAIRED SYNTHESIS OF ACETYLCHOLINE IN BRAIN ACCOMPANYING MILD HYPOXIA AND HYPOGLYCEMIA

Abstract— Lowering the concentration of oxygen or of glucose to which mouse and rat brains were exposed impaired the synthesis of acetylcholine from labelled precursors in vivo.

Abnormalities of mitochondrial enzymes in Alzheimer disease.

Summary. Abundant evidence, including critical information gathered by Prof. Siegfried Hoyer and his colleagues, indicates that abnormalities of cerebral metabolism are common in neurodegenerative diseases, including Alzheimers Disease (AD). Alterations in mitochondrial enzymes likely underlie these deficits. Replicable reductions in AD brain occur in the pyruvate dehydrogenase complex (the link of glycolysis to the Krebs cycle), the α-ketoglutarate dehydrogenase complex (KGDHC; the link of Krebs cycle to glutamate metabolism) and cytochrome oxidase (the link of the Krebs cycle to oxygen utilization). Available evidence suggests that deficiencies in KGDHC may be genetic in some cases, whereas evidence that the other two enzyme systems have a genetic component is lacking. Additional results indicate that the reductions can also be secondary to other causes including oxidative stress. A variety of data suggest that the mitochondrial insufficiencies contribute significantly to the pathophysiology of AD.

Dopaminergic cell death induced by MPP+, oxidant and specific neurotoxicants shares the common molecular mechanism

Recent etiological study in twins (Tanner et al. 1999) strongly suggests that environmental factors play an important role in typical, non‐familial Parkinsons disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP+ (the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Son et al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP+ and the other PD‐related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H2O2‐related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP+, the neurotoxicants and an oxidant, H2O2 equally induce the ROS‐dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase‐1 and Caspase‐3‐like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan‐caspase inhibitor Boc‐(Asp)‐fmk (BAF) exerted significant neuroprotection against ROS‐induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase‐1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.

Brain dysfunction in mild to moderate hypoxia

Hypoxia is commonly invoked to explain alterations in mental function, particularly in patients with cardiac pulmonary failure. The effects of acute graded hypoxia or higher integrative functions are well documented experimentally in man. Hypoxia in experimental animal models demonstrates that the pathophysiology is complex. In mild to moderate hypoxia, in contrast to severe hypoxia and to ischemia, the supply of energy for the brain is not impaired; cerebral levels of adenosine triphosphate (ATP) and adenylate energy charge are normal. In contrast, the turnover of several neurotransmitters is altered by mild hypoxia. For example, acetylcholine synthesis is reduced proportionally to the reduction in carbohydrate oxidation. This relationship holds in vitro and with several in vivo models of hypoxia. Pharmacologic and physiologic studies in man and experimental animals are consistent with acetylcholine having an important role in mediating the cerebral effects of mild hypoxia. These observations raise the possibility that treatments directed to cholinergic or other central neurotransmitter systems may benefit patients with cerebral syndromes secondary to chronic hypoxia.

Aging Decreases Oxidative Metabolism and the Release and Synthesis of Acetylcholine

Acetylcholine (ACh) synthesis in vivo is known to decrease during the aging process (senescence). To elucidate the molecular mechanism(s) of this age‐related decline, we studied brain slices from 3‐, 10‐, and 30‐month‐old mice of two strains (C57B1 and Balb/c). In low K+ media, oxidative metabolism as measured by 14CO2 production decreased with aging from 100% (3 months) to 85% (10 months) or 71% (30 months) whether [U−14C]glucose, [3,4‐14C]glucose, or [l‐14C]pyruvate was the substrate. In the aged brain (3 months) the increase in 14CO2 production with K+ stimulation was about twofold higher than in the young brain (3 months). Thus, in high K+ media, only slight decreases (<10%) in oxidative metabolism occurred with aging. Changes in ACh synthesis paralleled the decreases in 14CO2 production. Synthesis of [14C]ACh from [U‐14C]glucose in low K+ media declined from 100% (3 months) to 85% (10 months) or 66% (30 months), while in high K+ media only slight decreases (<10.5%) occurred with aging. The Ca2+‐dependent, K+‐stimulated release of [14C]ACh declined from 100% (3 months) to 58% (10 months) or 25% (30 months). Only the decrease in the release of ACh declined to the same extent as the reduced in vivo synthesis of ACh with aging. The results suggest that decreases in oxidative metabolism, ACh synthesis, and in the release of ACh contribute to a reduction in cholinergic function in the senescent brain.