HEPATIC LIPOPROTEIN RECEPTOR RELATED PROTEIN MODULATORS AS POTENTIAL THERAPEUTICS FOR ALZHEIMER'S DISEASE

Several risk factors for Alzheimer's disease (AD), such as amyloid precursor protein, presenilins, insulin degrading enzyme and apolipoprotein E, are all associated with amyloid beta. Therefore, targeting Aβ production, aggregation and clearance has been at the forefront of research on therapeutic strategies for AD. Lipoprotein receptor related protein 1 mediates transport of Aβ across the blood brain barrier and also binds Aβ circulating in the blood in its soluble form (soluble LRP or sLRP). Hence, upregulating hepatic LRP expression in periphery results in increased clearance of Aβ from the brain. In agreement with this, semi-purified extract of root of Withania somnifera completely reverses behavioural deficits and plaque pathology in nine months old APPSwe/PS1dE9 mice via upregulation of hepatic LRP. The main objectives of this study were to identify the compounds in the crude extract of WS that upregulate hepatic LRP and to understand transcriptional upregulation of hepatic LRP. Identification of these compounds and proteins involved in the regulation of hepatic LRP expression will help in designing new drugs that target Aβ clearance via the periphery. The extract was fractionated using flash chromatography. Luciferase-based reporter assay was used to screen the fractions in vitro and also to identify the site of action of the active principle(s) in the extract within the LRP promoter region. The efficacy of fractions was validated in vivo in 9 months old APPSwe/PS1dE9 mice based on performance of animals on radial arm maze, cortical amyloid load and upregulation of hepatic LRP. We found that the site of action of the compounds in the crude extract is most probably within the first 400 base pairs upstream sequence of LRP promoter. An in silico analysis of this region revealed transcription factor binding sites for AP-2, Sp-1, NF- kappa β and Cox-2. We also found that one of the fractions, fraction 4.4, reversed behavioural deficits and plaque pathology in transgenic mice at a substantially lower dose as compared to the crude extract. The active principle(s) is present in fraction 4.4. Mass spectrometry and high-pressure liquid chromatography helped in identification of four potential compounds in fraction 4.4.