Browsing by Subject "blood brain barrier"
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Item Biochemical and Phylogenetic Analysis of Organic Anion Transporting Polypeptides(2017-11) DeVoll, KaylaOrganic Anion Transporting Polypeptides (Human: OATPs, all others: Oatps), are sodium-independent membrane transporters predicted to have 12-transmembrane domains. Oatps/OATPs mediate the transport of a broad range of endo- and xenobiotics including bile salts, hormones and their conjugates, statins, thyroid hormones, prostaglandins, and anticancer agents. Oatps have varying tissue expression profiles with some Oatps having a very discrete tissue-specific expression pattern while other Oatps are ubiquitously expressed throughout multiple tissues. The functional diversity displayed by Oatps/OATPs has made it difficult to understand their overall physiological role. The research presented here takes a two-pronged approach to elucidate the evolved physiological role of Oatps. The first approach investigates the active site structure of a specific Oatp, Oatp1c1, through transport inhibition and the second approach involves a comprehensive phylogenetic analysis of the Oatp family. Oatp1c1 is expressed predominantly in the blood-brain barrier and the blood-cerebrospinal fluid barrier cells with minimal expression in specialized cells of the eye and gonads. Oatp1c1 has a narrower range of recognizable substrates compared to other Oatps. However, Oatp1c1 is a high affinity thyroxine transporter with the lowest Km¬ yet identified (90nM-340nM) of all transporters, indicating an important role in thyroid hormone homeostasis. Previous research suggests Oatp1c1 contains multiple binding sites based on the biphasic uptake curves of T4 and E2-17β-G as well as T4 and E2-17β-G cis-inhibition values differing from their Km¬s. Unpublished data from the Rumbley Laboratory was used to characterize the Oatp1c1 T4 uptake site by calculating IC50 values for multiple inhibitors of T4 uptake. However, the Oatp1c1 E2-17β-G uptake site is not fully characterized. In this study, HEK293 cells were transiently transfected with a Gateway expression vector, pEF-DEST 51, containing the Oatp1c1 gene sequence and uptake of 3H-E2-17β-G was assessed. Oatp1c1 expressing cells were found to uptake 3H-E2-17β-G 4-fold over empty vector transfected cells and uptake was shown in a time dependent manner. Next, multiple inhibitors were used to test their ability to inhibit the uptake of 3H-E2-17β-G and preliminary IC50 values were calculated and compared to the IC50 values calculated for the same inhibitors against the uptake of T4. Data presented here supports the idea of Oatp1c1 possessing a single flexible binding site capable of binding multiple substrate molecules simultaneously. However, the presence of multiple substrate binding sites capable of communication was not ruled out. Oatps/OATPs are promiscuous membrane transporters and are highly conserved proteins present in all metazoans sequenced to date but are absent in all other organisms including plants, bacteria and yeast. Sequence alignments of Homo sapiens OATPs with that of primitive organisms have shown Oatps/OATPs have a high level of evolutionary conservation. Understanding why such primitive organisms like Monosiga, Trichoplax and Nematostella possess Oatps may allow clarification of the evolved physiological role of Oatps/OATPs. Due to the high level of Oatps/OATPs conservation among all metazoans, understanding the physiological purpose these transport proteins serve is important, especially since Oatps/OATPs are present in such a diverse range of organisms. For comprehensive phylogenetic analysis of the Oatp family, H. sapiens OATP4A1 protein sequence was used as the query sequence for BLASTp searches completed against organisms with complete or nearly complete genomes. Multiple sequence alignments of all 1264 sequences were completed using MAFFT, QuickProbs and Clustal Omega. These alignments were compared to one another and subsequently submitted to FastTree 2.0 and MrBayes 3.2 for phylogenetic analysis. Amphimedon queenslandica was identified as the first metazoan to possess Oatp protein sequences more closely related to the Oatp4 family than to any other indicating the Oatp4 family was likely the first to evolve. Oatps/OATPs where shown to cluster into seven divergent families, with the seventh labeled as the Uncharacterized Oatp cluster. The Uncharacterized Oatp cluster emerges with the Bilaterian split of Protostomia and Deuterostomia but is lost somewhere in the chordate lineage before the emergence of vertebrates. Combined, both research studies will aid in the understanding of the evolved biological role of Oatps/OATPs and potentially lead to rational drug design of specifically targeted pharmaceutics.Item Influence of active efflux transport on the distribution of targeted agents to brain tumors(2014-07) Oberoi, Rajneet KaurGlioblastoma multiforme (GBM) is a lethal disease of the whole brain. Despite complete surgical resection of the tumor, recurrence is inevitable and leads to patient death. Several molecularly-targeted agents have shown promising results preclinically, although clinical results have been disappointing. One plausible explanation for clinical failure of drugs is their inability to effectively target the invasive glioma cells that reside in areas away from the tumor core. These regions of the brain have an intact blood brain barrier (BBB), which through a combination of endothelial tight junctions and active efflux transporters restricts brain penetration of several drugs. The objective of this thesis was to investigate the influence of active efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) on the brain distribution of molecularly targeted agents. To enhance our understanding of brain distribution with statistical certainty, we proposed a population-based analysis method to estimate variability around the brain partition coefficient (Kp). We showed that the brain distribution of sunitinib (a tyrosine-kinase inhibitor) is limited by active efflux mediated by P-gp and Bcrp at the BBB. We further demonstrated that brain distribution could be enhanced by administration of a dual P-gp/Bcrp inhibitor. To statistically ascertain the variability associated with Kp in a serial sacrifice design, we developed a pharmacokinetic model to simultaneously describe plasma and brain concentration time profile data. We further evaluated the influence of study design features such as between subject variability (BSV) and sample size at each time point on bias and precision of estimation of Kp. Our results show that bias is unaffected by the assumptions regarding the magnitude of BSV and sample size, however, precision improves with sample size.We further examined the influence of BBB on delivery and efficacy of dual PI3K/mTOR inhibitors, GNE-317 (higher BBB permeability) and GDC-0980 (restricted BBB penetration) using three glioma mouse models, GL261-luc-GFP model, GBM10 and U87. Disruption of the BBB in the tumor core resulted in higher drug concentrations for GDC-0980. However, intact BBB in the areas adjacent to the core restricted the brain concentrations of GDC-0980. Contrary to this, the brain concentrations of GNE-317 were similar in all the three regions of the brain. Furthermore, results from survival studies indicated that although both GNE-317 and GDC-0980 significantly improved survival compared to placebo, the treatment groups were not significantly different from each other. Therefore, it is important to consider that effective treatment of GBM relies not only on effective delivery across the BBB to invasive glioma cells but also on an effective drug.Item Investigation into the use of Pulsed Focused Ultrasound as a Method of Facilitating Homing of Umbilical Cord Blood Stem Cells after Systemic Administration in Ischemic Stroke Rat Models(2016-04) Hamborg, JoshuaStroke is a leading cause of mortality with no current therapies for chronic stroke victims. Our work investigates how to help chronic stroke patients regain function lost due to their stroke. This was accomplished by exploring how the use umbilical cord blood stem cells (UCBSCs), used in conjunction with pulsed focused ultrasound (pFUS), could provide a safe, efficient, and relatively noninvasive method for providing neuroregenerative therapy. Using rat stroke models that have undergone unilateral MCA occlusion, we propose that tail vein injections of UCBSCs, followed immediately afterwards by pFUS targeted to regions of ischemic damage, will result in functional improvements due to engraftment and neural differentiation of the stem cells. Initial immunohistochemical analysis of control rat brain tissue investigated how the local neuroenvironment may become therapeutically favorable after transcranial pFUS treatments. Results obtained so far are preliminary.Item Trafficking of Amyloid beta protein at the Blood Brain Barrier: Novel Insights in Alzheimer's Disease Pathogenesis(2016-12) Sharda, NidhiAlzheimer’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.