Kumar Sambamurti - US grants

The amyloid beta protein is an approximately 4 kD secreted protein normally found in human plasma and cerebrospinal fluid. Abeta is invariably deposited as insoluble amyloid fibrils in the brains of patients with Alzheimer's disease (AD), and there is increasing evidence that Abeta deposition plays an important role in AD pathogenesis. Abeta is released from the larger amyloid beta protein precursor (betaAPP) through cleavage on the amino and carboxyl side of Abeta by proteolytic activities referred to as beta and gamma secretase, respectively. betaAPP is also cleaved at Abeta 16 by a third secretase, a secretase, which may serve to prevent amyloid deposition by bisecting the Abeta peptide. Despite their important role in Alzheimer's disease, none of the betaAPP secretases has yet been isolated or cloned. Here, I propose to isolate betaAPP secretase cDNAs using a system in which the fission yeast Schizosaccharomyces pombe expresses a chimeric cDNA construct consisting of a secreted derivative of the human placental alkaline phosphatase (SEAP) linked to the C-terminal 105 amino acids of betaAPP (SEAP-APP). I have shown that the secretases in mammalian cells readily process SEAP-APP so that Abeta and secreted SEAP-containing derivatives (sSEAP-APP) are efficiently secreted. In contrast, S. pombe expressing SEAP-APP does not produce secreted derivatives indicating that it lacks endogenous secretase activity. In my initial studies, S. pombe cells expressing SEAP-APP were transformed with a human cDNA (HeLa cell) expression library, and 80,000 transformants, each of which could contain several cDNAs, were screened for their ability to produce secreted derivatives. Five transformants contained putative secretase cDNAs that resulted in the production of sSEAP-APP. Thus complementation in S. pombe appears to be an effective and convenient method to isolate putative human secretase cDNAs. In this application, I propose to employ this system and modifications of it to isolate as many independent candidate betaAPP-secretase cDNAs as possible. I will then determine which of these cDNAs encode authentic betaAPP secretases by determining whether coexpression with authentic betaAPP in S. pombe results in cleavage at the predicted site(s).

DESCRIPTION (provided by applicant): Recent studies have shown that increased levels of Ab peptides are among the earliest detectable abnormalities in Alzheimer's disease and may mediate a chain of downstream events leading to neuronal degeneration and cognitive decline. There is increasing evidence from clinical, epidemiological and laboratory studies that cholesterol plays a role in the pathogenesis of Alzheimer's disease. This body of evidence includes in vitro studies indicating that cellular cholesterol levels modulate Ab production and the enzymatic processing of APP, animal studies demonstrating that cholesterol levels modulate Ab accumulation in the brain (preliminary data) and several observational, clinical studies suggesting that the prevalence and incidence of probable Alzheimer's disease was significantly lower in patients taking cholesterol-lowering drugs. Taken together the studies support the hypothesis that Alzheimer's disease may be a disease in which cholesterol homeostasis is altered and that cholesterol may participate in a chain of events that modulate the disease neuropathology. The application proposes to test the following hypotheses: 1-that in the human brain increased cholesterol content contributes to amyloid accumulation by changing APP processing in a more amyloidogenic manner. 2-that there are correlative interactions between levels of apoE expression, cholesterolemia and amyloid pathology. 3-that certain apoE promoter polymorphisms act in concert with cholesterol levels influencing the extent of apoE expression and amyloid accumulation. Preliminary and recently published data from our laboratory suggest that cholesterol content in plasma and brain of Alzheimer's transgenic mice is strongly correlated with rate of development of amyloid pathology and with apoE expression. These hypotheses are amenable to testing as outlined in the corresponding sections of the proposal and their study will advance our understanding of the pathogenesis of Alzheimer's disease.

PROJECT SUMMARY/ABSTRACT Our hypothesis is that overnutrition coupled with impaired dietary amino acid turnover may play a role in Alzheimer disease (AD) pathogenesis. goals are to manage amyloid and tangle homeostasis by nutrition to prevent and treat AD. Restriction of protein, Leu, Met, branched-chain amino acids (BCAA) can improve longevity in rodents without calorie restriction. Failure of Met or BCAA degradation are caused by inherited diseases -- homocystinuria and maple syrup urine disease -- that are fatal when untreated and cause mental retardation. Treatments include restricting Met or BCAA or Leu. Therefore, our hypothesis is that MR and BCAA- R can help in managing and preventing AD by limiting metabolic load and epigenetic changes. The primary purpose of this study is to treat amyloidosis and tauopathy in an AD mouse model by restricting Met levels and to compare the treatments with other dietary restriction (DR) approaches. We propose that by restricting Met and other nutrients, the reduced metabolic load will lead to a fasting condition and reduce amyloid ? (A?) protein precursor (APP) levels due to its rapid turnover (20 min half-life). The studies will use behavior as an endpoint to measure overall health of the mice. Although behavior is a complex change that may not linearly correlate with degeneration and pathology, it provides a go/no go decision for mental health improvement. We will use tissues from control and restricted mice to follow AD-like neuropathology and neuronal integrity to test the hypothesis that the interventions can prevent AD and related dementias. Most studies on AD have focused on A? and senile plaques. A salient feature of the current study is that we will determine the effects of DR on A?, Microtubule-associated protein Tau (MAPT) and a combination of both lesions in littermates. Finally, it has recently been recognized that the number of microbes rival human cells particularly in the alimentary canal. Moreover, studies have shown that the gut microbiota can affect nutrition, and vice versa, in both positive and negative ways and impact immune, metabolic and neurological functions. Hence, it is important to understand the associations between gut microbiota composition, dietary modification and disease outcome to realize the contribution of gut microbes to dietary intervention of AD associated neuropathology. We will therefore examine the gut microbiome in all the treatments. Since a 1.5 x increase in APP can induce FAD, the small reduction over time in its expression should prevent or delay AD. Dietary restriction and exercise may provide useful paradigms that may be readily implemented for prevention of AD.