Cellular and animal models for high-throughput screening of therapeutic agents for the treatment of the diseases of the elderly in general and Alzheimer's disease in particular(†).

It is currently thought that the dementia of Alzheimer's disease is due to the neurotoxicity of the deposits or aggregates of amyloid-ß (Aß) in the extracellular space of the cerebral cortex. This model has been widely criticized because there is a poor correlation between deposits and dementia. Others have questioned whether Aß is truly neurotoxic. Yet, in spite of these concerns, the search for therapeutic agents has been based on the development of mouse models transfected with mutant genes associated in humans with early onset Alzheimer's disease. A major limitation of these models is that although they exhibit many of the pathological and clinical manifestation of the human disease, the bulk of individuals who develop the dementia of Alzheimer's disease have none of these mutant genes. Furthermore, nine clinical trials of drugs that were effective in transgenic mice failed to show any benefit in patients. Finally, a major unresolved issue with the Aß model is that since Aß is produced in everyone, why are deposits only seen in the elderly? This issue must be resolved if we are to understand the etiology of the disease and develop test systems for both diagnosis and drug discovery. Published studies from my laboratory demonstrate that in human cerebrospinal fluid immunoreactive Aß is only present as a complex with two chaperones, ERp57 and calreticulin and is N-glycosylated. This complex formation is catalyzed by the posttranslational protein processing system of the endoplasmic reticulum (ER). Others have reported that in plaque Aß is present only as the naked peptide. Together these results suggest that both plaque and dementia are secondary to an age related decline in the capacity of the ER to catalyze protein, posttranslational processing. Since the synaptic membrane proteins necessary for a functioning memory are also processed in the ER, these findings would suggest that the loss of cognition is due to a decline in the capacity of the neuron to produce and maintain functioning synapses. Work from my laboratory and from others further indicate that the components of the ER, posttranslational, protein processing pathway do dramatically decline with age. These data suggest that this decline may be found in all cells and could account not only for the dementia of Alzheimer's disease, but also for many of the other manifestations of the aging process. These observations also suggest that declining ER function has a role in two well-recognized phenomena associated with aging: a loss of mitochondrial function and a decrease in myelin. Finally, based on this paradigm I propose new cellular and animals models for high-throughput screening for drug discovery.

Low serum glutathione peroxidase activity is associated with increased cardiovascular mortality in individuals with low HDLc's.

BACKGROUND: Since oxidized LDL is thought to initiate atherosclerosis and the serum glutathione peroxidase (GPx3) reduces oxidized lipids, we investigated whether high GPx3 activity reduces cardiovascular disease (CVD) mortality. METHODS: We determined GPx3 in stored samples from the Minnesota Heart Survey of 130 participants who after 5 to 12 years of follow-up had died of CVD and 240 controls. Participants were 26 to 85 years old and predominantly white. In a nested case-control, study we performed logistic regressions to calculate odds ratios (OR) adjusted for age, sex, baseline year, body mass index, smoking, alcohol intake, physical activity, total and HDL cholesterols, systolic blood pressure, serum glucose and gamma glutamyltransferase (GTT) activity. The referent was the quartile with the highest GPx3 activity (quartile 4). RESULTS: OR's for CVD mortality for increasing quartiles of GPx3 were 2.37, 2.14, 1.83 and 1.00 (P for trend 0.02). This inverse correlation was confined to those with HDLc's below the median (P for interaction, 0.006). The OR's for increasing quartiles of GPx3 in this group were 6.08, 5.00, 3.64 and 1.00 (P for trend, 0.002). CONCLUSIONS: Individuals with both low HDLc and GPx3 activity are at markedly increased risk for death from CVD.

The effect of aging on the chaperone concentrations in the hepatic, endoplasmic reticulum of male rats: the possible role of protein misfolding due to the loss of chaperones in the decline in physiological function seen with age.

The endoplasmic reticulum (ER) chaperones are highly conserved proteins that catalyze the posttranslational processing of all secretory and membrane proteins. Our studies suggest that chaperone declines are one of the two central defects in Alzheimer's disease. We propose that similar declines in other organ systems underlie the physiological deficits of aging. Rats were maintained in a colony from age 21 days to death. Animals were killed at regular intervals, and hepatic, ER chaperone contents were determined by immunoblotting. ERp55, ERp57, ERp72, BiP, and calnexin constitutive levels declined 30%-50% with age. Calreticulin was unaffected. BiP (also known as GRP78), ERp55, and ERp57 showed marked swings with peaks occurring in midwinter and midsummer. This cyclics declined 73% with age. Considering the role of the ER chaperones in membrane and secretory protein posttranslational processing, these data support the concept that their loss could lead to many of the physiological declines associated with aging.

In cerebrospinal fluid ER chaperones ERp57 and calreticulin bind beta-amyloid.

The beta-amyloids (abetas) are the major components of the plaque observed in the brains of patients with Alzheimer's disease. The conundrum is that although they are produced in everyone during the posttranslational processing in the endoplasmic reticulum (ER) of the amyloid precursor protein (APP), deposits are only observed in the elderly. Our work suggests that normals have a carrier protein(s) keeping them in solution. Based on immunoblotting studies of cerebrospinal fluid (CSF) from normals, we find that the bulk of the abetas are bound to the ER chaperones, ERp57 and calreticulin, suggesting that these may be carrier proteins which prevent aggregation of the abetas and that the deposits are due to faulty ER posttranslational processing of APP with the failure to form this complex. If membrane protein synthesis is similarly affected, it could explain the neuronal dysfunction characteristic of Alzheimer's disease.

Within-person variability of urinary 6beta-hydroxycortisol to urinaryl ratios in Caucasian women.

Cortisol is metabolized to 6beta-hydroxycortisol by human cytochrome p450-3A4 (CYP3A4), an important enzyme involved in the metabolism of a variety of exogenous and endogenous compounds. Both cortisol and 6beta-hydroxycortisol are excreted in urine, and the ratio of these steroids has been proposed as an indicator of CYP3A4 activity. We evaluated within-person variability of this biomarker in 10 healthy Caucasian women, aged 23-58 years. Each study participant was asked to provide a fasting morning urine sample once a week consecutively for 8 weeks. Urinary cortisol and 6beta-hydroxycortisol were determined by immunoassay kits purchased from the DiaSorin (Stillwater, MN) and the Stabiligen (Nancy, France), respectively. The coefficients of variation (CV) of urinary 6beta-hydroxycortisol to cortisol ratios from study participants ranged from 16.7 to 51.4% (mean, 31.1%) over the study period. The level of the ratio measured in any single urine sample was correlated reasonably well with the average of the ratios over the 8-week study period from the same woman, with the mean correlation coefficient of 0.79. These results indicated that urinary 6beta-hydroxycortisol to cortisol ratios measured in a spot urine sample may reflect the level of this biomarker over a relatively longer time period in Caucasian women, and thus, it can be used in epidemiologic studies as a biomarker to evaluate the association between CYP3A4 activity and disease risk.