Novel Therapeutic Targets for Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by impaired memory and other cognitive deficits. Even though the genes associated with familial AD were discovered more than three decades ago, no cure has been developed yet. The drugs that are prescribed for AD currently provide only symptomatic relief and do not cure or alter the progression of the disease. Hence, the need for better therapeutic strategies is greater than ever The study and the thesis titled ‘Novel therapeutic targets for Alzheimer’s disease’ is divided into two parts: Part 1 titled ‘Lipoprotein receptor related protein 1 as a therapeutic target for Alzheimer’s disease’ and Part 2 titled ‘Differential gene expression in entorhinal cortex of one-month old APPSwe/PS1dE9 animals’. The scope of both the studies is described below. One of the lesser studied mechanisms of Aβ clearance from brain is the clearance via periphery. It is known that lowering the amyloid beta levels in blood or periphery leads to increased clearance of amyloid beta from the brain (Zlokovic et al, 2000; Zlokovic et al, 2008). We demonstrated in our laboratory that a partially purified extract of root of Withania somnifera completely reverses plaque pathology and behavioural deficits in APPSwe/PS1dE9 animals via upregulation of hepatic lipoprotein receptor related protein 1 (LRP) (Sehgal et al, 2012). The soluble form of LRP (sLRP) circulates in blood and binds and clears Aβ42 from blood. Upregulation of hepatic LRP leads to increased levels of sLRP in blood and hence, increased clearance of Aβ42 from blood. This in turn results in increased efflux of Aβ42 monomers from brain into periphery which leads to breakdown of plaques in the brain. Inhibition of hepatic LRP completely diminished this therapeutic effect. Identification of the active principle(s) in the semi-purified extract of root of Withania somnifera will help in designing a novel class of therapeutics that target Aβ clearance from brain through periphery. Therefore, the objective of part 1 of this study was to identify the active principle(s) in the semi-purified extract of root of Withania somnifera. The extract was fractionated using flash chromatography. We obtained sixteen fractions from the crude extract. The effect of each fraction was first studied in vitro in mouse hepatoma cell line (H6). Our in vitro experiments were designed with two objectives: i) identification of region of LRP promoter associated with upregulation of LRP expression by WS extract and ii) identification of the fraction with active principle(s). We developed a luciferase-based reporter assay system such that the luciferase gene was expressed under a specific region of LRP promoter. We cloned three such promoter regions into the promoterless vector to screen the fractions (400 base pairs upstream sequence, 1100 base pairs upstream sequence and 2000 base pairs upstream sequence). Our results suggested that the site of action of the chemical compounds in WS extract possibly lies within the first 400 base pairs upstream sequence of LRP promoter. Five of the sixteen fractions demonstrated the highest efficiency in driving luciferase expression via the LRP promoter. The efficacy of these five fractions was then validated in vivo in nine months old APPSwe/PS1dE9 animals. The therapeutic efficiency of these fractions was determined based on the performance of animals on radial arm maze, upregulation of hepatic LRP, reduction in cortical Aβ42 load and plaque pathology. Treatment with fraction 4.4, one of the five fractions, led to complete reversal of behavioural deficits and plaque pathology in transgenic mice. A significant increase in hepatic LRP and decrease in cortical Aβ42 levels was also observed. The mass spectrometric analysis of fraction 4.4 helped in lowering the number of potential active principle(s) from over a hundred compounds to four compounds. Identification of these compounds and a comprehensive study of structure and effects of each of these compounds will help in developing a new class of drugs that acts through periphery. The second part of this study focuses on identification of genes that are differentially expressed early on in AD transgenic animals. It is known that the physiological changes associated with AD begin long before the symptoms of AD appear (Ahmad et al, 2017; Kommaddi et al, 2018). Hence, identification of the genes that are differentially expressed very early on in AD will provide an insight into the mechanisms associated with disease causality. This will also help in identification of new therapeutic targets and biomarkers which will help in better management of the disease and early diagnosis. Hence, we studied differential gene expression using RNA sequencing in entorhinal cortex of one-month old APPSwe/PS1dE9 animals. Age matched wildtype littermates were used as control. We found that of the total read counts, 37 genes were significantly upregulated, and 31 genes were significantly downregulated in APPSwe/PS1dE9 animals when compared to wildtype animals. The genes that were differentially expressed are associated with axon guidance, axonal growth cone organization, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and mitogen-activated protein kinase (MAPK) signalling, synaptic transmission and synaptic vesicle recycling. Differential expression of some of these genes in human AD post mortem brain has been shown but there are no studies on expression profiles of these gene in presymptomatic phase either in animals or humans. This study, hence, is the first RNA sequencing study on changes in gene expression profiles in one-month old AD transgenic animals. The study provides several new candidate genes that are differentially expressed in young transgenic mice. But comprehensive studies need to be done both in mice models and humans to ascertain the roles of these genes in the pathogenesis of AD.