Date of Degree
Alzheimer's, amyloid, therapeutics, repurposing, inflammation, multi-target
Alzheimer’s disease (AD) is multifactorial, and its hallmarks include the formation of amyloid-beta (Ab) plaques and neurofibrillary tau tangles, accompanied by an increase in glial cell activation, culminating in neurodegeneration, chronic neuroinflammation, and cognitive decline in human patients. AD will cost the United States over $300 million this year alone and is projected to cost over $1 trillion by 2050, AD is a serious concern for the aging population, and efforts need to be redirected towards more effective therapeutic intervention strategies. Drugs aimed at halting AD progression have so far proven unsuccessful due to the development of pharmaceuticals that target one aspect of the pathology while ignoring the others.
Investigators invested resources towards developing animal models that mimic human AD progression. However, most of the current animal models do not capture all hallmarks of AD. For example, the 5X FAD mouse model does not develop neurofibrillary tau tangles, while the 3X Tg-AD mouse model does not exhibit synaptic loss. Efforts should be focused to generating animal that more accurately reflect disease progression and include the most significant risk factor, which is aging, along with the other hallmarks. The goal would be to translate the results of obtained with animal models to human therapeutic interventions.
Another problem investigators face is the diagnosis and treatment timeline. Patients are diagnosed when they already exhibit cognitive impairment, and by this time they have moderate AD and significant neurodegeneration. At this stage it is too late for currently available pharmaceuticals to have significant effects on halting disease progression. Thus it is important, to identify early AD biomarkers that are significantly altered in AD patients prior to the development of the plaques, tangles, and cognitive impairment so that therapeutics act more efficiently.
Considering the aforementioned problems with current AD research approaches, the overall goal of my studies was to investigate the effects of using novel early therapeutic interventions that target neuroinflammation, or a multitarget approach to mitigate AD progression.
My hypothesis was that (1) targeting neuroinflammation with ibudilast (IBU) or (2) using a combined treatment with diazoxide (DZ) and dibenzoylmethane (DIB), will effectively slow down the progression of AD. IBU is a multi-target drug that is a non-selective phosphodiesterase inhibitor, a Toll-like receptor 4 (TLR4) antagonist, and is predicted (in-silico) to inhibit off-target kinases, such as interleukin-1 receptor associated kinase 1 (IRAK1). DZ is a potassium channel activator and DIB restores eIF2B activity, thus reversing stress-induced translational depression. To test my hypothesis in vivo, I used the transgenic Tg-AD Fisher 344 rat model of AD. These rats (Tg-AD) develop multiple hallmarks of AD including Ab plaques, tau tangles, neuronal loss, neuroinflammation, and cognitive deficits in an age-dependent manner that is comparable to human AD. My studies included mainly 11-month-old rats, because at this age, the Tg-AD rats are mainly in the moderate AD stage (goal 1). Some of my studies (goal 3) also used rats at 4-months of age to be able to compare the 4-month pre-pathology stage with the 11-month moderate pathology stage.
Goal 1: Determine the effects of ibudilast, which alters Toll-like receptor signaling, on AD pathology in a transgenic rat model of AD (Chapter 2).
I found that chronic ibudilast (IBU) treatment in Tg-AD rats slowed the development of AD pathology, such as Ab plaques and neurofibrillary tau tangles in the hippocampal dentate gyrus (DG) region. IBU also reduced the degree of microglia cell activation across the entire rat hippocampus and improved the cognitive performance of Tg-AD treated rats compared to age-matched untreated Tg-AD rats. IBU also significantly altered the expression of genes associated with the toll-like receptor signaling pathway and ubiquitin proteasome pathway. Based on my data, I conclude that IBU has potential as a therapeutic agent for AD.
Goal 2: Explore the Toll-like receptor variations between mice and human brain tissue as it could affect the efficacy of drugs predicted to treat neuroinflammation in AD (Chapter 3).
Prior investigations determined that the brains of AD patients had increased levels of microgliosis and pro-inflammatory cytokines. This suggests that Toll-like receptor signaling could be upregulated in AD brains, likely due to the buildup of extracellular Ab plaques and other neurotoxic factors that disrupt homeostasis. I performed a literature search to provide a review of the variation among TLR distribution and levels in human and mouse brain tissue, as it could impact the predicated efficacy of drugs developed to target these receptors to treat neuroinflammation in AD.
Goal 3: Determine the therapeutic potential on DZ/DIB co-treatment on AD pathology in a transgenic rat model of AD (Chapter 4)
I opted to use a combination treatment of DZ and DIB in Tg-AD rats to mitigate the progression of AD because previous studies examined each drugs’ therapeutic benefits on attenuating neurodegeneration and apoptosis in other animal model systems. However, their combined therapeutic potential was not addressed. I found that DZ / DIB treatment reduced the buildup of Ab plaques in the hippocampal hilar subregion of the DG, and tau tangle buildup in DG and cornu ammonis 3 (CA3) hippocampal regions. I did not detect drug treatment effects on microglia or neuronal loss. However, I found that DZ / DIB treatment decreased eIF2a levels in Tg-AD treated rats compared to untreated Tg-AD rats. The translation initiator factor eIF2-alpha is a DIB target, as DIB inhibits its activity, thus reversing stress-induced translational depression.
In collaboration with Charles Wallace, another graduate student in our lab, and using RNA sequencing analysis, we found that at 4 months of age (pre-pathology stage), gene expression for the early growth response factor 2 (EGR2) and histone cluster H1 H2AA (HIST1H2AA) was altered in female Tg-AD rats. Based on these results, I propose that alterations in EGR2 and HIST1H2AA levels could serve as early AD biomarkers, prior to the development of other conventional AD hallmarks.
My studies on the effects of the DZ/DIB combination treatment in Tg-AD rats was complemented by studies carried out by Charles Wallace. Charles established that the DZ/DIB treatment mitigated spatial memory defects of the Tg-AD rats and improved spatial memory of WT rats. Based on our results, I conclude that the combination DZ/DIB treatment is an effective strategy to mitigate AD pathology due to its multi-target approach that affects multiple signaling pathways.
Conclusions: I found that two novel therapeutic approaches (1) IBU and (2) DZ/DIB combination, tested on a transgenic rat model of AD, mitigated some aspects of AD pathology. Thus these multitarget drug approaches should be explored as novel treatments for AD.
Oliveros, Giovanni, "Novel Therapeutic Strategies for Alzheimer’s Disease: Targeting Toll-Like Receptor Signaling and a Multi-Target Approach" (2022). CUNY Academic Works.