Objectives: Alzheimer's disease (AD) is a growing global health crisis exacerbated by increasing life span and an aging demographic. Convergent lines of evidence, including genome-wide association studies, strongly implicate neuroinflammation in the pathogenesis of AD. Several dietary agents, including phenolic-rich foods, show promise for the prevention and/or management of AD, which in large part, has been attributed to their anti-inflammatory effects. We previously reported that a food-grade phenolic-enriched maple syrup extract (MSX) inhibited neuroinflammation in vitro but whether these effects are translatable in vivo remain unknown. Herein, we assessed MSX's ability to attenuate early neuroinflammation in a transgenic mouse model of AD. Methods: The effects of MSX on AD-related neuroinflammation was evaluated by orally administering MSX (100 and 200 mg/kg/day for 30 days) to the 3xTg-AD mouse model of AD. The expression of inflammatory markers in mouse brains were analyzed with LC-MS/MS with SWATH acquisition. Results: 3xTg-AD mice dosed orally with MSX have decreased expression of several inflammatory proteins, including, most notably, the AD risk-associated protein ‘triggering receptor expressed on myeloid cells-2’ (TREM2), and stimulator of interferon genes TMEM173, and suppressor of cytokine signaling-6 (SOCS6). However, this decrease in inflammation did not coincide with a decrease in pathogenic amyloid generation or lipid peroxidation. Discussion: These data demonstrate that oral administration of this maple syrup derived natural product reduces key neuroinflammatory indices of AD in the 3xTg-AD model of AD. Therefore, further studies to investigate MSX's potential as a dietary intervention strategy for AD prevention and/or management are warranted.

Proteins that regulate the coagulation cascade, including thrombin, are elevated in the brains of Alzheimer's disease (AD) patients. While studies using amyloid-based AD transgenic mouse models have implicated thrombin as a protein of interest, the role of thrombin in tau-based animal models has not been explored. The current study aims to determine how inhibiting thrombin could alter oxidative stress, inflammation, and AD-related proteins in a tau-based mouse model, the Tg4510. Aged Tg4510 mice were treated with the direct thrombin inhibitor dabigatran or vehicle for 7 days, brains collected, and western blot and data-independent proteomics using mass spectrometry with SWATH-MS acquisition performed to evaluate proteins related to oxidative stress, intracellular signaling, inflammation, and AD pathology. Dabigatran reduced iNOS, NOX4, and phosphorylation of tau (S396, S416). Additionally, dabigatran treatment increased expression of several signaling proteins related to cell survival and synaptic function. Increasing evidence supports a chronic procoagulant state in AD, highlighting a possible pathogenic role for thrombin. Our data demonstrate that inhibiting thrombin produces alterations in the expression of proteins involved in oxidative stress, inflammation, and AD-related pathology, suggesting that thrombin-mediated signaling affects multiple AD-related pathways providing a potential future therapeutic target.

The field of Alzheimer’s disease (AD) has witnessed recent breakthroughs in the development of disease-modifying biologics and diagnostic markers. While immunotherapeutic interventions have provided much-awaited solutions, nucleic acid-based tools represent other avenues of intervention; however, these approaches are costly and invasive, and they have serious side effects. Previously, we have shown in AD animal models that tolfenamic acid (TA) can lower the expression of AD-related genes and their products and subsequently reduce pathological burden and improve cognition. Using TA as a scaffold and the zinc finger domain of SP1 as a pharmacophore, we developed safer and more potent brain-penetrating analogs that interfere with sequence-specific DNA binding at transcription start sites and predominantly modulate the expression of SP1 target genes. More importantly, the proteome of treated cells displayed ~75% of the downregulated products as SP1 targets. Specific levels of SP1-driven genes and AD biomarkers such as amyloid precursor protein (APP) and Tau proteins were also decreased as part of this targeted systemic response. These small molecules, therefore, offer a viable alternative to achieving desired therapeutic outcomes by interfering with both amyloid and Tau pathways with limited off-target systemic changes.

Nonalcoholic fatty liver disease (NAFLD) is growing in prevalence across the world and is associated with a significant economic cost. In addition to life style modification, no pharmacotherapy method is available to treat NAFLD. As the histology of the liver changes over the course of the disease, it can be expected that the liver proteome will change too. Included in the liver proteome are drug disposition proteins comprised of drug metabolizing enzymes and transporters. These proteins are responsible for the removal of drugs and endogenous compounds from the body. The goals of this dissertation were: (1) Develop methodology to accurately and reproducibly measure drug metabolizing enzymes and transporters and (2) determine the alterations in these proteins as a result of the progression of NAFLD and diabetes. Manuscripts I and II are used to develop the methodology required to measure the proteins of interest and determine other variables that may be affecting protein expression changes besides disease. Manuscripts III and IV bring biological effect into the equation to determine how NAFLD and diabetes change the proteome and compare the results to a mouse model of NASH to assess its validity. The manuscripts are briefly described below: Manuscript I: Hepatic xenobiotic transporters in the human liver play an important role in the elimination of drugs or toxins and significantly contribute to variability in drug response. We have developed a label-free mass spectrometry-based method to study the protein expression of 13 of 25 clinically relevant transporter proteins (6 ABC and 7 SLC) in liver tissue from 20 donors (9 female, 11 male). Whole tissue homogenate was used, and in-solution trypsin digestion was performed using pressure- cycling technology. Data was acquired in data-dependent and sequential window acquisition of all theoretical mass spectra (SWATH-MS) mode. Bovine serum albumin (BSA) was spiked in each sample before digestion and monitored for batch to batch variability. Spectronaut™ was used for peptide identification and data extraction from SWATH files. Na+/K+ transporting subunit alpha 1 (ATP1A1), was quantified as a cell membrane marker and its coefficient of variation was 16.6% across different liver samples. The work highlights the suitability of SWATH-MS for large-scale simultaneous quantification of several xenobiotic transporters important in drug disposition. We found that average differential expression of transporters proteins was similar and much smaller than the inter-individual variability observed between the genders. Manuscript II: Human liver tissue utilized in drug metabolism (DM) studies is retrieved from brain-dead