Effect of Transthyretin in the brain vasculature - Implications in physiology and pathology

FMUP/IBMC-I3S 9 Abstract The blood-brain barrier (BBB) is an important barrier that separates and protects the brain from the peripheral circulation. This BBB is formed by special tight junctions, a feature of the brain endothelium that is responsible for the BBB integrity and its low permeability. In several diseases, including Alzheimer’s disease (AD), BBB is impaired and changes in some important proteins are found. AD, the most common type of dementia, is characterized by an abnormal extracellular deposition of amyloid-β peptide (Aβ), which is believed to be a consequence of a failure in the balance between its production and clearance. Several molecules have been proposed as Aβ carriers and among them transthyretin (TTR) has been shown to have the capacity to bind and cleave Aβ, preventing its deposition and promoting its clearance, being considered as a neuroprotective protein. Moreover, previous reports highlighted the importance of TTR in the regulation of the expression of central nervous system (CNS) genes, pro-angiogenic genes in the eye and in the umbilical cord and bloodbrain barrier genes. In this project, we aimed at further investigating the participation of TTR in the brain vasculature, in biologic as in pathologic environments. In order to achieve our objectives, we began by evaluating the effect of TTR in the expression of tight junctions (TJs) related genes, at the transcript and protein levels, using hCMEC/D3 cells. Our initial results obtained by qRT-PCR, using a PCR array kit, suggested that TTR was modifying the expression of several genes, but further individual qRT-PCR for the most affected genes did not confirm the PCR array results. Moreover, three other genes not evaluated in the PCR array, were assayed by qRT-PCR and no effect of TTR was detected. Next, we decided to advance to the analysis of gene expression at the protein level of two important TJs proteins, occludin (OCLN) and Claudin-3 (CLDN3), by immunocytochemistry. Here, we observed that also protein levels of these TJs were not altered by TTR, as seen at the transcript level. Altogether, our results suggest that TTR is not capable to modulate the gene expression of TJs-related genes. Furthermore, to evaluate the role of TTR in brain vessel angiogenesis, we examined the expression of genes known to be imported in angiogenesis, using two different endothelial cell lines, hCMEC/D3 and bEnd.3 cells. The qRT-PCR results showed that both cell lines presented similar transcript levels in the absence or presence of TTR variants, indicating that TTR presence also does not influence the expression of angiogenesis-related FMUP/IBMC-I3S 10 Abstract genes. Additionally, using an in vitro wound healing assay, we investigated if the endothelial cell lines presented a different migration response, an important process of angiogenesis, when incubated in the presence or absence of TTR variants. However, the migratory response of the endothelial cells was similar in both conditions, as no significant differences were detected in the recovery of the wounded area in cells treated with TTR compared to control. These results supported our qRT-PCR results, demonstrating that TTR variants do not have a modulatory role on angiogenesis in the brain. Finally, we intended to understand if TTR is involved in the regulation of collagen IV levels in brain vessels. Using AD mice with different TTR backgrounds (TTR+/+, TTR+/and TTR-/-), by immunohistochemistry, we showed that the collagen IV layer was increased in AD/TTR+/and in AD/TTR-/-, as compared to AD/TTR+/+ animals. To further understand if TTR is directly responsible for this collagen IV alteration, we measured the collagen IV levels in non-transgenic mice with different TTR backgrounds. Here, we observed that collagen IV levels were similar in all animals, indicating that TTR is not directly responsible for the differences observed in the AD mice. Thus, we hypothesized that Aβ was the responsible for the increase of collagen IV levels in AD animals and, since TTR is capable of binding Aβ promoting its clearance, the differences in collagen IV measured in AD animals may result from an indirect action of TTR. In fact, incubation of bEnd.3 cells with Aβ species induced increased collagen IV which was counteracted by TTR, when coincubated with Aβ. TTR alone did not alter collagen IV in bEnd.3 cells, further corroborating our observations in the non-transgenic animals, and supporting the explanation that in AD mice, Aβ is responsible for the alterations in collagen IV layer and that TTR, indirectly, delays or partially avoids this alteration by sequestering the peptide, avoiding its aggregation and accumulation, and by enhancing its elimination from the brain. Previous results from our group also indicated that the protective effect of TTR on Aβ can be improved by stabilizing the tetrameric structure of this protein. With this in mind, also in a previous project, iododiflunisal (IDIF), a potent TTR stabilizer, was administrated to AD mice, resulting in cognitive improvement, as well as in decreased Aβ brain levels. To further investigate the relation between collagen IV levels and the protective role of TTR in AD, we now evaluated its levels in IDIF-treated and in control animals. Our results showed FMUP/IBMC-I3S 11 Abstract that levels of collagen IV were decreased upon IDIF treatment, strengthening the stability hypothesis. Altogether, our results support a neuroprotective role for TTR in AD, mainly by acting directly on Aβ and avoiding, at least some of, the downstream events caused by this peptide. We showed that alterations at the vascular level, such as the thickness of the basement membrane, i.e. collagen type IV layer, can be prevented by TTR. Given that the vascular alterations are thought to be part of the initial alterations in AD, it is of the outmost importance to explore therapeutic strategies aiming at increasing TTR levels, known to be decreased in this disorder. Key concepts: Blood-brain barrier; Tight-Junctions, Angiogenesis, Alzheimer’s disease, amyloid-β peptide, Collagen IV, Transthyretin.