Investigation of Potential Drug Targets for Cholesterol Regulation to Treat Alzheimer's Disease

Abstract

Despite extensive research and seven approved drugs, the complex interplay of genes, proteins, and pathways in Alzheimer's disease remains a challenge. This implies the intricacies of the mechanism for Alzheimer's disease, which involves the interaction of hundreds of genes, proteins, and pathways. While the major hallmarks of Alzheimer's disease are the accumulation of amyloid plaques and tau protein tangles, excessive accumulation of cholesterol is reportedly correlated with Alzheimer's disease patients. In this work, protein-protein interaction analysis was conducted based upon the genes from a clinical database to identify the top protein targets with most data-indicated involvement in Alzheimer's disease, which include ABCA1, CYP46A1, BACE1, TREM2, GSK3B, and SREBP2. The reactions and pathways associated with these genes were thoroughly studied for their roles in regulating brain cholesterol biosynthesis, amyloid beta accumulation, and tau protein tangle formation. Existing clinical trials for each protein target were also investigated. The research indicated that the inhibition of SREBP2, BACE1, or GSK3B is beneficial to reduce cholesterol and amyloid beta accumulation, while the activation of ABCA1, CYP46A1, or TREM2 has similar effects. In this study, Sterol Regulatory Element-Binding Protein 2 (SREBP2) emerged as the primary protein target. SREBP2 serves a pivotal role in maintaining cholesterol balance, acting as a transcription factor that controls the expression of several enzymes pivotal for cholesterol biosynthesis. Novel studies suggest that SREBP2 performs a multifaceted role in Alzheimer's disease. The hyperactivity of SREBP2 may lead to heightened cholesterol biosynthesis, which suggested association with the pathogenesis of Alzheimer's disease. Lowering SREBP2 levels in an Alzheimer's disease mouse model results in reduced production of amyloid-beta, a major contributor to Alzheimer's disease progression. Moreover, its thoroughly analyzed crystal structure allows for computer-aided screening of potential inhibitors; SREBP2 is thus selected as a prospective drug target. While more protein targets can be added onto the list in the future, this work provides an overview of key proteins involved in the regulation of brain cholesterol biosynthesis that may be further investigated for Alzheimer's disease intervention.

Keywords: Alzheimer’s disease; amyloid beta; cholesterol biosynthesis; drug discovery; ligand-protein docking; protein target; protein-protein interaction.

Figure 1

Overview of the main pathways involved in cholesterol transport involved in Alzheimer’s Disease. Although not directly illustrated, SREBP2 is activated by GSK3B through the Akt pathway. De novo synthesis is indicated by Pathway 1, where SREBP2 and SCAP bind to each other and transport cholesterol from the ER to the Golgi apparatus. The SREBP2/SCAP complex activates SREBP2, which is involved in cholesterol synthesis in Pathway 2. Through Pathway 3, ABCA1 transports cholesterol to ApoE, which then transports the cholesterol to LRP1 to become synthesized in the neuron by Pathway 4. Pathways 5 and 6 demonstrate CYP46A1 converting excess cholesterol into 24S-OHC and then being exported from the neuron, which then crosses the Blood Brain Barrier through Pathway 7. Through Pathway 8, BACE1 cleaves APP to produce amyloid-beta plaques. When cholesterol is transported to the microglia via Pathway 9, TREM2 aids in cholesterol efflux out of the brain.

Figure 2

Overview of network between top genes associated with brain cholesterol metabolism and Alzheimer’s Disease (produced by the STRING program). Each colored line represents an individual known or predicted interaction between the gene pair. More lines indicate more interactions between the two genes they are connected to. Filled nodes indicate some known or predicted crystal structure that exists for the gene. Genes with a greater filled node indicate that more crystal structures exist for that specific gene.

Figure 3

The major pathways and interactions regulated by GSK3B: (A) Wnt/B-catenin pathway and (B)PI3K/Akt/mTor pathway. In the Wnt/B-catenin pathway, the binding of Wnt to LRP causes the GSK3B complex with other proteins to be inhibited. This same complex phosphorylates the LRP/Wnt/ligand complex. The inhibited GSK3B complex stabilizes this pathway and causes the accumulation of B-catenin outside of the plasma membrane. B-catenin then translocates into the nucleus and forms a complex with the TCF/LEF transcription factors, which reduces transcription and DNA binding. In the PI3K/Akt/mTor pathway, the binding of the growth factor to the receptor tyrosine kinase stimulates PI3K and PIP3 activity, which then stimulates Akt activity. This activation leads to the phosphorylation of GSK3B, which inactivates the protein. Once GSK3B is inhibited, the assembly of B-catenin is regulated for the Wnt signaling that is important to maintain synaptic signaling.

Figure 4

The major pathways and interactions regulated by BACE1: (A) Notch signaling pathway, (B) Amyloidogenic pathway, and (C) NF$\kappa$B pathway. In the Notch signaling pathway, the binding of Notch1 to the Jag1 ligand causes a conformational change in Notch1 and results in cleavage. This cleavage releases NCID, which then relocates into the nucleus. NCID’s relocation activates downstream signaling. This signaling can be regulated by binding Jag1 to the surface of the cell. BACE1 then cleaves Jag1, which regulates the activity of Jag1 in the sending cell and stimulates Notch1 signaling in the receiving cell. In the amyloidogenic pathway, APP is cleaved by BACE1 to generate amyloid-beta. The two cleavage fragments, C99 and sAPP-$\beta$, are released. The C99 complex with amyloid-beta is cleaved by $\gamma$ -secretase, which generates AICD and releases amyloid-beta. The BACE1 cleavage is the rate-limiting step in amyloid-beta formation, therefore the liberation of amyloid-beta leads to an accumulation of amyloid-beta. In the NFκB pathway, GSK3B activates NFκB, which causes the phosphorylation of IκB. This phosphorylation causes IκB to relocate to the nucleus and bind to DNA. This activates multiple target genes associated with amyloid-beta production including BACE1, which can reactivate NFκB.

Figure 5

Major signaling pathways that regulate SREBP2 activation during cholesterol synthesis: (A) P53 pathway, and (B) Akt pathway. In the p53 pathway, mutant p53 binds to SREBP2 and subsequently activates the Ras, RhoA, YAP/TAZ pathways, while p53 blocks the activation of SREBP2 through ABCA1. Once SREBP2 is activated, it signals the mevalonate pathway to synthesize acetyl-CoA into cholesterol. In the Akt pathway, growth factors bind to receptor tyrosine kinases to activate PI3K, which then activates Akt. Akt phosphorylates and inhibits TSC2, which inhibits RHEB and activates mTORC1. MTORC1 works with other targets to stimulate and activate SREBP2. However, the activation of Akt may directly phosphorylate GSK3B, which will mediate phosphorylation and inhibition of transcription factors including SREBP2. This promotes proteasomal degradation.

Figure 6

Major pathways in which ABCA1 plays an important role for cholesterol transport in brains: (A) Reverse cholesterol transport pathway, (B) LXR/RXR pathway, and (C) SREBP2 pathway. In the reverse cholesterol transport pathway, free cholesterol is transported out of the macrophage to ApoA-1. ApoA-1 then becomes pre-B-HDL or nascent HDL. Nascent HDL then becomes mature HDL which is mediated by LCAT. LCAT then mediates cholesterol esterification of HDL into cholesterol ester (CE). The cholesterol esters are then taken up by SCARB1 receptors and taken up into hepatocytes. Cholesterol esters are then hydrolyzed to generate free cholesterol (FCh) in hepatocytes. In the LXR/RXR pathway, ligand LDL binds to receptor CD36, which oxidizes LDL into cholesterol esters. The cholesterol esters are converted into cholesterol, which is then converted into oxysterols. The oxysterols activate LXR/RXR, which then promotes cholesterol efflux from the nucleus, which then activates ABCA1 to transfer cholesterol out of the macrophage to Apo acceptors. In the SREBP2 pathway, SCAP transports SREBP2 from the ER to the Golgi apparatus. SREBP2 is then translocated to the sterol regulatory element in the nucleus. Then miR-33a is co-transcribed with SREBP2, which inhibits ABCA1 activity. By inhibiting ABCA1, cholesterol efflux is hindered, therefore cholesterol accumulates. However, inhibiting miR-33a would activate ABCA1 and promote cholesterol efflux.

Figure 7

The TREM2/DAP12 pathway. In this pathway, various ligands bind to TREM2 which creates an electrostatic interaction between TREM2 and DAP12. This interaction generates tyrosine phosphorylation of DAP12 with ITAM. This recruits the Syk kinase to activate signaling molecules such as PIP3, which then activates Akt. Through the Akt pathway, GSK3B is inhibited and therefore B-catenin is stabilized. The activation of Akt also activates NF$\kappa$B, and NF$\kappa$ B then relocates into the nucleus and binds to DNA. This increases the release of proinflammatory cytokines into the cytoplasm.