Alzheimer’s disease (AD) is the most common form of dementia in elderly population. Unfortunately, the current treatment approaches for AD are symptomatic and do not interfere with the disease progression at any stage. The risk of AD increases with age; from 12 %, above 70 years of age, to 50 % beyond 80 years of age. Due to an improvement in average life span in many regions across the globe, AD is being recognized as a major socio-economic health problem that is expected to worsen in the near future. Also, with 99.6 % of current clinical trials failing in AD due to insufficient knowledge of the disease targets and lack of early diagnostic methods; detailed investigations into AD pathogenesis is critically important. Development of amyloid beta (Aβ) plaques in the brain is one of the primary pathological hallmarks of AD. Impaired clearance and not overproduction of toxic Aβ proteins leads to their accumulation and subsequent plaque formation in sporadic AD that accounts for more than 90 % of total AD cases. Blood brain barrier (BBB) is expected to be the primary clearance portal of Aβ from the brain. Being at the interface between brain and plasma, the BBB also maintains the dynamic equilibrium of brain and plasma Aβ pools. This equilibrium is believed to be perturbed in AD due to BBB dysfunction. However, the manifestations of BBB dysfunction and the precise mechanisms that may trigger it, are not clearly understood. Two major Aβ isoforms, Aβ40 and Aβ42, play a predominant role in AD pathogenesis. Aβ42 is believed to be pathological whereas Aβ40 is constitutively expressed and believed to have protective properties against neurological anomalies. Also, Aβ40 expression is 10- and 1.5- fold higher than Aβ42 in cerebral spinal fluid (CSF) and plasma, respectively. This ratio of Aβ42:Aβ40 increases in brain and decreases in plasma, respectively, during disease progression. However, the pathophysiological mechanisms driving this clinical observation is not known yet. In my thesis, I demonstrate distinct age dependent changes in the BBB permeability and plasma exposure of Aβ40 and Aβ42 at the BBB endothelium in wild type (WT) and AD-transgenic (APP,PS1) mice. Further, to investigate the pathophysiological mechanisms driving these observations, I conducted in-vitro studies in polarized hCMEC/D3 monolayers, a widely used BBB model, and demonstrated that Aβ40 and Aβ42 are internalized at the BBB endothelium via clathrin- and lipid raft-mediated endocytosis, respectively. This mechanistic difference offers an opportunity to the BBB to independently handle and modulate the clearances of Aβ40 and Aβ42. Also, in-vivo investigations coupled with quantitative modeling, have indicated that Aβ40 may accumulate more than Aβ42 at the BBB endothelium. This may decrease Aβ40 transcytosis at the BBB and increase the exposure of the BBB endothelium to the vasculotropic Aβ40. To investigate the mechanisms driving this observation, I tested the hypothesis published by our lab previously that the impaired transcytosis of Aβ40 at the BBB endothelium is due to the perturbed vesicular exocytosis. Also, synaptic transmission involving SNARE exocytosis machinery is known to be impaired in AD. Thus, I investigated the ability of Aβ40 and Aβ42 to perturb the SNARE exocytosis machinery at the BBB endothelium and in neurons, in comparison with tetanus neurotoxin, a well-established disruptor of the VAMP-2 mediated exocytosis. My findings reveal that Aβ40 and Aβ42 isoforms use VAMP-2 (vesicular SNARE) to exocytose at the BBB endothelium and neurons. Further, using fluorescence resonance energy transfer (FRET) and lifetime microscopy (FLIM), I have demonstrated that Aβ40 and Aβ42 interfere with the functioning of SNARE fusion between VAMP-2 and SNAP-25. Moreover, my findings suggest that Aβ40 may be more efficient than Aβ42 in perturbing this process, and consequently may interfere with its own exocytosis. In summary, my work provides evidence for the presence of distinct clearance mechanisms of Aβ40 and Aβ42 isoforms at the BBB endothelium. This novel assertion provides a framework to explain the disrupted clearance of Aβ, perturbed Aβ42:Aβ40 ratios and dysregulated transport of Aβ and other endogenous proteins at the BBB endothelium observed in AD patients. These findings could be applied to identify new drug targets to ameliorate BBB dysfunction in AD.
University of Minnesota Ph.D. dissertation. December 2016. Major: Pharmaceutics. Advisor: Karunya Kandimalla. 1 computer file (PDF); xvi, 254 pages.
Trafficking of Amyloid beta protein at the Blood Brain Barrier: Novel Insights in Alzheimer's Disease Pathogenesis.
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