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Background: Apolipoprotein E4 (apoE4) is associated with perturbed lipid metabolism and elevated risk for CVD and Alzheimer’s disease (AD). Data suggests impact of both non-brain and brain apoE on risk to develop AD. Homozygosity for apoE4 increases AD risk approximately 10-fold. Brain apoE4particles are smaller and less lipidated than brain apoE2 or apoE3. ApoE is lipidated by ATP Binding Cassette A1 (ABCA1) and both proteins are synthesized predominantly in astrocytes. We hypothesized that enhancing apoE4 lipidation by an ABCA1 agonist, CS6253, would have AD therapeutic actions. Methods and Results: Astrocyte cholesterol efflux is highly responsive to stimulation by exogenous CS6253 peptide. Time-course showed that basal [3H]cholesterol efflux from cells to serum-free medium was compromised in apoE4 astrocytes. Incubating with peptide CS6253 (10 μg/mL) showed 6-fold improvement in [3H] cholesterol efflux to serum-free medium containing either apoE4 or E3 astrocytes. CS6253 crossed the BBB and was found to co-localize with astrocytes in hippocampus. CS6253 i.p. injection (20 mg/kg/48h for 6 weeks) in apoE4 Targeted Replacement mice significantly lowered apoJ in plasma and the brain. The distribution of serum apoE4 on HPLC to smaller lipoproteins was seen rendering similar profile to apoE3. Plasma apoA-I levels, cholesterol and triglyceride levels were unchanged. Lipidation of apoE in brain was compromised in apoE4 compared to apoE3 mice and CS6253 treatment increased apoE4 lipidation. CS6253 treatment prevented cognition decline and development of the brain AD pathological phenotype. CS6253 treatment decreased Aβ42 and P-tau and increased levels of VGluT1 and apoER2. Cognitive decline was fully prevented in apoE4 mice by CS6253 treatment as assessed by novel object recognition and Morris water maze. Conclusion: ABCA1 agonist treatment by CS653 resulted in improved apoE4 lipidation in cells and in mice. Six weeks treatment counteracted apoE4 driven AD with regard to phenotype and cognition. The data point to the potential therapeutic utility of CS6253 in apoE4 AD.

Background: Type 2 Diabetes Mellitus (T2DM) is associated with high cardiovascular disease (CVD) risk. Addressing the underlying atherogenesis and diabetes causing CVD in T2DM is important. CS6253 is an ABCA1 agonist peptide derived from the C-terminal of apoE that has shown macrophage specific reverse cholesterol transport, anti-atherosclerosis and anti-diabetic properties. Further studies were carried out to characterize metabolic effects. Methods: CS6253 was incubated with a) INS-1 823/13 cells to assess effects on insulin secretion and b) with L6-Glut4myc rat myoblasts to assess glucose uptake properties. Diet Induced Obesity (DIO) mice, i.e. C57BL/6 mice that had been fed 60% high-fat diet for 6 weeks, were treated with CS6253 and Glucose Tolerance Tests (GTT) performed after overnights fasting administering glucose 1g/kg ip. Results: CS6253 1mg/mL incubated for 2 hours under standard conditions with 3mM glucose showed a 3-fold increase in insulin secretion compared to control, i.e. 232(32) vs. 79(7) ng/M cells, p3 H-glucose uptake by CS6253 peptide in L6-Glut4myc rat myoblasts increased insulin’s glucose uptake capacity from 3800 to 4619 DPM/well, p3 H-glucose uptake compared to control. DIO mice were treated with CS6253 30mg/kg sc alternate days or PBS control for 16 weeks. GTTs were performed after 2, 6 and 15 weeks treatment showing 39%, 45% and 57% reductions in the glucose-AUC compared to control, respectively, p Discussion: CS6253 shows potent, sustained and increased anti-diabetic actions over the 16 weeks treatment period in DIO mice. In vivo and in vitro studies show improved pancreas β-cell function with increased glucose-mediated insulin secretion and also insulin sensitizing properties. CS6253’s combined anti-diabetic and anti-atherosclerosis properties suggest utility in the treatment of CVD and T2DM.

The physiological functions of microparticles (MPs) released from cells remain poorly defined. Here, we study MPs released from various cell lines; baby hamster kidney (BHK) cells, human THP-1 and hepG2 cells also expressing ABCA1. To assess the role of MPs in cholesterol homeostasis we incubated cells with apoA-I or CS-6253 (CS) a novel apoE derived mimetic peptide and potential cholesterol efflux and HDL modulator. When incubated with BHK cells expressing ABCA1 under mifepristone and Mock cells in time dependent manner (0,5 to 24h) we found that MPs are ABCA1 dependent. Accordingly the dependence of MPs release on ABCA1 activity is confirmed in human THP-1 cells and hepG2 cells after blocking ABCA1 with probucol (10 μM, 24h). Given the rate-limiting step of ABCA1 in cholesterol homeostasis, we hypothesized that MPs may have a role in cholesterol transport from cells. To address this point we isolated MPs by FPLC from media collected from 3 H-cholesterol labelled cells that were exposed or not to (1 μM) apoA-I, or CS in time dependent manner. We demonstrate that MPs accounted for ~ 1/3 of the total cholesterol released to the medium. In addition, we found that cholesterol carried by MPs is redistributed among resident lipoproteins under conditions when 3 H-MPs were incubated in plasma (1h, 37°C), as shown by FPLC profiles. In addition, the cholesterol regulatory role in cellular release of MPs was tested after depletion of plasma membrane cholesterol by methyl-β-cyclodextrin (CDX, 10 mM, for 30 min). There was a significant decrease in cholesterol counts from MPs released in CDX treated macrophages THP-1 cells when compared to cells not treated relative to each condition: cells alone (16%, p

Microparticles (MPS) are lipoprotein-sized structures created by the ABCA1 transporter. Their biological roles in health and in disease remain unknown. Here, we study MPS released from baby hamster kidney (BHK) cells stably expressing ABCA1 and human THP-1 cells also expressing ABCA1. Media cell culture was first collected from BHK-ABCA1 expressing cells after (45min, 2, 4, 8, and 24h) incubation. After centrifugation (4000xg for 15min and 10000xg for 30min) to remove cell debris, the supernatant was passed through a 10kDa cutoff filter and subsequently subjected to analytical FPLC. FPLC analysis shows creation of a single peak in the presence of ABCA1 but not in mock-transfected BHK cells. In a time-course study, the estimated hydrodynamic diameter remained stable (≥20nm). After 8h incubation of BHK cell with apoA-I or an apo-E mimetic peptide, CS-6253 (1μM), ABCA1 mediated formation of MPS of similar size with a significant increase in 3[H]-FC content than those generated by ABCA1 alone, (373±23 % cpm, P