Supplement: Multi-Modal MRI to Assess Alzheimer's Disease Prevention in an APOE4 Mouse Model

Apolipoprotein E4 (APOE4) allele is the strongest genetic risk factor for Alzheimer’s disease. APOE4 carriers accumulate beta-amyloid and neurofibrillary tau tangles earlier and with more extensive pathology compared to non-carriers. However, decades before the aggregation of beta-amyloid and tau, cognitively normal APOE4 carriers have developed neurometabolic and neurovascular deficits, including reduced glucose metabolism, cerebral blood flow (CBF), and blood-brain barrier (BBB) function. Emerging evidence shows that gut microbiota plays a critical role in determining brain vascular and metabolic integrity. Brainiste et al. recently showed that BBB permeability is increased in germ free mice due to lack of butyrate, a short chain fatty acids (SCFAs) produced by microbiota, such as Clostridium tyrobutyricum. BBB dysfunction will further lead to impaired clearance of beta-amyloid, increased inflammation, and reduced CBF. Metabolically, scyllo-inositol produced by microbiota Bacillus subtilis has shown to play a major role for alleviating beta-amyloid burden in AD patients. In line with this, we found significantly changed microbiome composition, impaired BBB function, and reduced CBF in mice that overexpresses human beta-amyloid and expresses human APOE4 (E4FAD), compared to their APOE3 (E3FAD) littermates (see preliminary data). Our preliminary findings suggest that modifying gut microbiome of the E4FAD mice may be critical to preserve brain functions and potentially prevent the development of AD-related neuropathology for the APOE4 carriers. As diet is an important modulator of the gut microbiome, in this Supplement, we will extend from our parent R01 to include a nutritional intervention as well as gut microbiome analyses. We have formulated a diet containing prebiotic Inulin, and established a new collaboration with Dr. Stefan Green of University of Illinois at Chicago for microbiome sequencing and bacteria taxonomy analysis. We will use the MRI and magnetic resonance spectroscopy (MRS) techniques proposed in the parent R01 to determine brain vascular and metabolic integrity. In this study, our goal is to determine if our prebiotic Inulin can restore the gut microbiome of the E4FAD mice; and if the microbiota-induced neurovascular and neurometabolic changes can be detected by MRI and MRS. Inulin is a non-digestible fiber of carbohydrates that is known to be fermented in the gastrointestinal tract and increase beneficial bacterial populations and SCFA. Inulin also has been demonstrated to reduce inflammation and enhance neuroprotection. The central hypothesis is that Inulin is protective to brain vascular and metabolic functions in E4FAD mice by modulating the gut microbiome; and multimodal MRI/MRS can be used as surrogate markers to assess the efficacy of Inulin. We will test the hypothesis by pursuing the following two Specific Aims: Aim 1. Identify effects of the prebiotic Inulin on gut microbiome. We will feed asymptomatic E4FAD and E3FAD mice (2 months of age) with either Inulin or vehicle control diet for 16 weeks. We will collect pre- and post-treatment fecal samples, extract fecal DNA, analyze gut microbiome, and generate data of bacteria taxonomy and diversity. Hypothesis: E4FAD mice fed with prebiotic Inulin will have restored microbiome composition and diversity (including Clostridium tyrobutyricum and Bacillus subtilis) to the level of E3FAD mice, compared with the E4FAD mice fed with control diet. Aim 2. Determine effects of the prebiotic Inulin on brain vascular and metabolic functions. After collecting fecal samples, we will image BBB leakage, CBF, and scyllo-inositol level using MRI and MRS. We will compare pre- and post-treatment efficacy of Inulin. In the end of the study, we will euthanize the mice, and quantify the number of beta-amyloid transporter and tight junction protein on BBB. We will send the blood samples to Metabolon Inc. for analyzing SCFAs. Hypothesis: E4FAD mice with Inulin diet will have restored CBF, BBB integrity, and scyllo-inositol level, compared with the E4FAD mice fed with control diet. Inulin will also enhance beta-amyloid transport and SCFAs, and reduce BBB permeability (by preserving tight junctions) of the E4FAD mice. The parent R01 is focused on Rapamycin, a pharmaceutical intervention, to directly restore brain vascular and metabolic functions of the E4FAD mice. The purpose of the Supplement is to use dietary intervention and gut microbiome manipulation for preserve brain physiology of the mice. Our project will for the first time use multimodal MRI/MRS to determine the potential effectiveness of prebiotic diet in preventing AD-related deficits using an APOE4 mouse model. If the results of the proposed study support our hypothesis, a future R01 grant application will seek to more rigorously investigate the underlying mechanisms that link brain-gut axis. More broadly, this new collaboration will enable us to combine analyses of neuroimaging, brain physiology, and gut microbiome with verity of dietary supplements to begin to understand the interplay between brain and gut that may prevent or even reverse processes of AD for the APOE4 carriers. As our approaches are highly translational, we would be able to rapidly move to clinical studies for asymptomatic APOE4 carriers in the future.