David R. Wekstein

Alzheimer Neuropathologic Alterations in Aged Cognitively Normal Subjects

The histopathologic changes distinguishing early Alzheimer disease (AD) from normal or pathologic aging are not clearly defined. This report describes the autopsy findings of 59 elderly, well-educated, volunteers. They were examined longitudinally with mental status testing, some for up to 8 years, as part of our normal aging study. This study reveals that (1) the brains of many subjects who did not show cognitive impairment on neuropsychologic testing contain abundant senile plaques (SP) and/or neurofibrillary tangles (NFT); (2) 29 subjects met Khachaturian criteria for AD, 15 met CERAD and 7 met National Institute on Aging-Reagan Institute guidelines; (3) Braak and Braak staging method included 9 in stage IV subjects, 4 in stage V, and 1 in stage VI; (4) there was a progression of NFT from entorhinal cortex to hippocampus and amygdala as a function of age; (5) 2 subjects met criteria for a diagnosis of dementia with Lewy bodies but were not demented; (6) cerebral amyloid angiopathy was present in leptomeningeal vessels in 75% of subjects and in parenchymal vessels in 62% of subjects; (7) only 10 of 59 subjects (17%) had no or few degenerative brain changes. Our study demonstrates that the brains of a large percentage of cognitively normal, relatively well-educated individuals contain numerous degenerative changes and only a small percentage are relatively free of these changes.

“Preclinical” AD revisited Neuropathology of cognitively normal older adults

Objective: To classify neuropathologic alterations in the brains of nondemented older adults using current sets of criteria for AD. Background: AD neuropathologic alterations are found in the brains of some nondemented elderly subjects and suggest the possibility of presymptomatic AD. Three sets of guidelines have been developed to classify AD using senile plaques, neuritic plaques, and neurofibrillary tangles (NFT). Methods: Neuropathologic changes in 59 older adults followed longitudinally with a standard battery of mental status measures were investigated using Khachaturian, Consortium to Establish a Registry for Alzheimer’s Disease (CERAD), and National Institute on Aging–Reagan Institute (NIA-RI) guidelines. AD neuropathologic markers were evaluated in neocortical and allocortical regions. Cases were categorized as neuropathologically “normal” or “AD-like” and compared for possible mental status differences. Results: Between 11 and 49% of cases met one or more of the three classifications of AD. With adjustments for multiple comparisons, only NFT in hippocampal CA1 region were associated with autopsy age, suggesting that this may represent a pathologic process associated with normal brain aging. Using the NIA-RI guidelines, subjects in the AD-like group performed less well on the immediate paragraph recall and word-list delayed recall than their counterparts who did not meet these guidelines. Conclusions: These data indicate that the prevalence of “preclinical” AD in our population is relatively low based on the NIA-RI classification. Although many subjects had AD-like changes based on CERAD and Khachaturian guidelines, they exhibited no differences in mental performance, suggesting that the aging brain may be able to withstand such structural changes without meaningful impact on mental functioning.

Age and gender effects on human brain anatomy: A voxel-based morphometric study in healthy elderly

The adult human brain shrinks slowly with age, but the regional specificity and tissue class specificity of this loss is unclear. Subjects (n=122) were healthy aged participants in a longitudinal cohort who undergo periodic standardized cognitive and clinical examination. Multi-spectral segmentation of magnetic resonance images into grey matter (GM), white matter (WM) and CSF was performed on cross-sectional image data using a custom template and calculated prior probability maps. Global differences were evaluated by fitting a regression model for absolute and normalized subject GM, WM, and CSF values. Global and regional patterns of GM, WM and CSF differences were assessed using optimized voxel-based morphometry (VBM). GM volume decreased with age at a rate of 2.4 cm(3)/year (-0.18%/year); CSF increased by 2.5 cm(3)/year (0.20%/year). Regression analyses showed no significant decrease in WM volume, but a focal WM decrease with age was detected in the anterior corpus callosum using VBM. Diffuse reductions of GM volume were seen with age in the frontal, parietal, and temporal cortex, cerebellum and basal ganglia. Relative regional differences in cortical GM volume with age occurred in the frontal, parietal and temporal lobes, but not in medial temporal lobe or in posterior cingulate. We did not observe significant gender effects. These findings establish a baseline for comparison with pathologic changes in human brain volume between ages 58 and 95 years.

Brain structural alterations before mild cognitive impairment

Objective: To determine whether alterations of brain structure in normal aged individuals precede the development of mild cognitive impairment (MCI) or Alzheimer disease (AD). Background: Persons with MCI and AD demonstrate cortical volume losses vs asymptomatic aged individuals, particularly in the hippocampus, amygdala, and entorhinal cortex. It is unknown whether these losses or other volumetric changes are present, and to what degree, in cognitively normal individuals before the clinical diagnosis of MCI. Methods: Structural MRI was performed on a cross-section of 136 longitudinally examined normal aged subjects. All subjects were cognitively normal at the time of their scan, but 23 later developed MCI, and 9 of these 23 went on to an AD diagnosis. Extracted volumes from voxel-based morphometric analysis were combined with clinical data to compare the 23 subjects who eventually developed MCI to 113 subjects who remained cognitively normal over an average follow-up of 5.4 years. Results: Initially normal subjects who eventually developed MCI demonstrated decreased gray matter volumes in the anteromedial temporal lobes bilaterally and left angular gyrus while still cognitively normal. Conclusion: Structural brain changes in anatomic areas involved in higher cognitive processes precede clinical signs and symptoms in longitudinally followed normal subjects destined to develop mild cognitive impairment.

N-terminal Heterogeneity of Parenchymal and Cerebrovascular Aβ Deposits

The goals of this study were twofold: to determine whether species differences in Aβ N-terminal heterogeneity explain the absence of neuritic plaques in the aged dog and aged bear in contrast to the human; and to compare Aβ N-terminal isoforms in parenchymal vs cerebrovascular Aβ (CVA) deposits in each of the species, and in individuals with Alzheimer disease (AD) vs nondemented individuals. N-terminal heterogeneity can affect the aggregation, toxicity, and stability of Aβ. The human, polar bear, and dog brain share an identical Aβ amino acid sequence. Tissues were immunostained using affinity-purified polyclonal antibodies specific for the L-aspartate residue of Aβ at position one (AβN1[D]), D-aspartate at N1 (AβN1[rD]), and pyroglutamate at N3 (AβN3[pE]) and p3, a peptide beginning with leucine at N17 (AβN17[L]). The results demonstrate that each Aβ N-terminal isoform can be present in diffuse plaques and CVA deposits in AD brain, nondemented human, and the examined aged animal models. Though each Aβ N-terminal isoform was present in diffuse plaques, the average amyloid burden of each isoform was highest in AD vs polar bear and dog (beagle) brain. Moreover, the ratio of AβN3(pE) (an isoform that is resistant to degradation by most aminopeptidases) vs AβSN17(L)-x (the potentially nonamyloidogenic p3 fragment) was greatest in the human brain when compared with aged dog or polar bear. Neuritic plaques in AD brain typically immunostained with antibodies against AβN1(D) and AβN3(pE), but not AβN17(L) or AβN1(rD). Neuritic deposits in nondemented individuals with atherosclerotic and vascular hypertensive changes could be identified with ApNl(D), ApN3(pE), and AβN1(rD). The presence of AβN1(rD) in neuritic plaques in nondemented individuals with atherosclerosis or hypertension, but not in AD, suggests a different evolution of the plaques in the two conditions. AβN1(rD) was usually absent in human CVA, except in AD cases with atherosclerotic and vascular hypertensive changes. Together, the results demonstrate that diffuse plaques, neuritic plaques, and CVA deposits are each associated with distinct profiles of Aβ N-terminal isoforms.

Autoantibodies to amyloid β-peptide (Aβ) are increased in Alzheimer’s disease patients and Aβ antibodies can enhance Aβ neurotoxicity

Studies of amyloid precursor protein transgenic mice suggest that immune responses to amyloid β peptide (Aβ) may be instrumental in the removal of plaques from the brain, but the initial clinical trial of an Aβ vaccine in patients with Alzheimer’s disease (AD) was halted as the result of serious neurological complications in some patients. We now provide evidence that AD patients exhibit an enhanced immune response to Aβ and that, contrary to expectations, Aβ antibodies enhance the neurotoxic activity of the peptide. Serum titers to Aβ were significantly elevated in AD patients and Aβ antibodies were found in association with amyloid plaques in their brains, but there was no evidence of cell-mediated immune responses to Aβ in the patients. Aβ antibodies were detected in the serum of old APP mutant transgenic mice with plaque-like Aβ deposits, but not in the serum of younger transgenic or nontransgenic mice. Serum from APP mutant mice potentiated the neurotoxicity of Aβ. Our data suggest that a humoral immune response to Aβ in AD patients may promote neuronal degeneration, a process with important implications for the future of vaccine-based therapies for AD.

Postnatal Changes in Rat Ventricular Function

Experiments were designed to assess the intrinsic changes in myocardial function with age and to determine the role of increasing left ventricular mass in this process. Isometric ventricular pressure-volume curves and length-tension curves were measured in isolated perfused rat hearts of different ages and masses. Incremental tension, peak active tension, and peak passive tension were less for 10-day-old hearts than they were for older hearts subjected to proportionately equal increases in length. At resting length, peak active tension developed by hearts at least 16 days old was 78% higher than that developed by 10-day-old hearts, and peak passive tension was 133% higher. There was no significant difference between 16-day-old rats and older rats. Thus, by the sixteenth postnatal day, the heart had developed functional maturity. These functional changes appeared to be related to age rather than to left ventricular mass. Histological studies of the left ventricle showed that, at birth, the myofibrils were disoriented with a high ratio of nonmyofibrillar elements to myofibrillar elements. Within 16–20 days after birth, the structural organization and the ratio of nonmyofibrillar elements to myofibrillar elements were similar to the organization and the ratio found in an adult. Thus a direct relationship existed between the functional properties and the anatomical development of the myocardium. Mass per se was not the sole factor that contributed to improved myocardial function with age.

The Immune Status of Healthy Centenarians

The immune status of 17 healthy individuals 100–103 years of age (centenarians) was investigated. Qualitative values for immunoglobulins IgG, IgM, IgA, and IgE were within normal ranges for subjects more than 60 years of age with the exception of elevated IgM in one individual. Cell marker studies employing a panel of 27 monoclonal antibodies delineating T and B lymphocytes, monocytes, natural killer cells, granulocytes, and functional and developmental subsets of each were performed to phenotype the peripheral blood leukocytes. Although the total lymphocyte count was normal in every subject, the numbers of T4‐positive helper–inducer T lymphocytes were profoundly depressed, as were responses to the mitogen phytohemagglutinin and interleukin‐2 production. Activated immature T lymphocytes and the number of cells bearing the phenotype of natural killer cells were increased, but natural killer cell activity was normal. Early B lymphocytes were also increased. The relative concentration of monocytes was normal. Taken together these findings indicate that the immune system in centenarians is similar to that in younger but still elderly individuals, i.e., discriminating T‐lymphoid functions are reduced in association with an apparent failure of some T, B, and natural killer cells to differentiate to functional maturity.

Methylenetetrahydrofolate reductase and angiotensin converting enzyme gene polymorphisms in two genetically and diagnostically distinct cohort of Alzheimer patients

The role of methylenetetrahydrofolate reductase (MTHFR) and angiotensin converting enzyme (ACE) gene polymorphisms as risk factors for the occurrence of Alzheimers disease (AD) is still controversial. In this study, we investigated the common MTHFR C677-->T and ACE insertion/deletion (I/D) gene polymorphisms as risk factors for AD in two genetically and diagnostically distinct cohort of Alzheimers patients. We analyzed a neuropathologically confirmed American cohort of 124 AD patients and 97 elderly controls, and a clinically diagnosed Italian cohort of 126 probable AD cases, 106 elderly controls, and a community-based sample of 1232 subjects aged under 65 years. No difference was found in polymorphism distribution between cases and controls in both study cohorts. We also tested a possible association between the polymorphisms investigated. No interaction was found between the MTHFR and ACE alleles. Moreover, no association was found for the ACE and MTHFR polymorphisms with age at onset, disease duration and MMSE score at observation. Thus, in our study, MTHFR C677-->T and ACE I/D polymorphisms do not appear to confer an added risk for AD.

Pharmacokinetics, Pharmacodynamics, and Safety of Metrifonate in Patients with Alzheimer's Disease

Metrifonate is converted nonenzymatically to 2,2, dimethyl dichlorovinyl phosphate (DDVP), an inhibitor of acetylcholinesterase (AChE). This 21‐day, randomized, double‐blind, placebo‐controlled trial of metrifonate in patients with Alzheimers disease (n = 27) evaluated four doses, each administered orally once daily. All patients received a loading dose (LD) for 6 days followed by a maintenance dose (MD) for 15 days. The treatment groups were: panel 1, LD = 1.5 mg/kg (75–135 mg), MD = 0.25 mg/kg (12.5–25 mg); panel 2, LD = 2.5 mg/kg (125–225 mg), MD = 0.40 mg/kg (20–35 mg); panel 3, LD = 4.0 mg/kg (200–335 mg), MD = 0.65 mg/kg (30–60 mg); and panel 4, LD = 4.0 mg/kg (200–335 mg), MD = 1.0 mg/kg (50–90 mg). All metrifonate doses were well tolerated. Most adverse events were mild to moderate in intensity, gastrointestinal in nature, and transient. Mean area under the concentration—time curve (AUC) and maximum concentration (Cmax for both metrifonate and DDVP increased in relation to dose. Metrifonate and DDVP had similar, largely dose‐independent mean values for time to Cmax (tmax) and half‐life (t1/2). There was little or no accumulation of either metrifonate or DDVP with long‐term administration. After 21 days of treatment, mean percent erythrocyte AChE inhibition was 14%, 35%, 66%, 77%, and 82% for placebo and panels 1 through 4, respectively. Cognitive improvement was observed with the two highest metrifonate doses. These results reflect favorable safety and pharmacokinetic profiles for the use of metrifonate in the treatment of Alzheimers disease.