Role of TTR in A β peptide brain efflux - Impact in Alzheimer's disease

FCUP/ICBAS Abstract 11 Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder characterized by two main pathological features: neurofibrillary tangles (NFTs) composed of hyper-phosphorylated Tau protein and extracellular deposition of amyloid β peptide (Aβ) into plaques, found mainly in cortex and hippocampus. Brain levels of Aβ peptide, which is derived from proteolytic hydrolysis of amyloid-beta precursor protein (APP), are regulated by its production, its degradation and clearance through the brain, namely across the blood-brain barrier (BBB). Indeed, increasing evidence demonstrated that increased Aβ levels are a result of its faulty elimination across the BBB. Several molecules have been proposed as Aβ carriers and among them transthyretin (TTR) has been shown to be the major Aβ-binding protein in the cerebrospinal fluid (CSF), leading to decreased peptide aggregation and toxicity. Supporting the importance of TTR in AD is the observation of its decreased levels in CSF and plasma. It has been also suggested that TTR tetrameric stability is a key factor in TTR/Aβ interaction. Moreover, in vivo administration of iododiflunisal (IDIF), known to be a potent TTR tetrameric stabilizer, to an AD mice model resulted not only in decreased brain Aβ levels and deposition but also in improved cognitive function associated to the AD-like neuropathology in this AD model. Aβ levels were reduced in plasma and presented a trend for reduction in the CSF, suggesting TTR promoted Aβ clearance from the brain and the periphery. Furthermore, TTR expression in neurons has been recently reported, as well as the co-localization of extracellular TTR with Aβ in plaques, although it is still very controversial. Taking all these data into account, we hypothesize that TTR might also be involved in Aβ clearance at BBB. This project aims at investigating the influence of TTR in Aβ transport across the BBB, using an in vitro BBB cellular model and an in vivo study, and also at investigating the mechanisms leading to neuronal TTR expression using a neuronal cell line. In order to achieve our main goals, we started by evaluating the effect of TTR in Aβ plasma levels in AD/TTR transgenic mice with different ages and distinct genotypes for TTR. Our results showed that, Aβ1-42/Aβ1-40 ratios were elevated in plasma of AD/TTR mice compared with AD/TTR animals, at the age of 6 months at which senile plaque formation starts, in this model. This indicates that TTR contributes to the maintenance of normal plasma Aβ levels, supporting the idea that TTR promotes its elimination from the brain and from the periphery. After setting up and optimizing our cellular model, the hCMEC/D3 cell line, we performed internalization studies of labelled (FAM-Aβ1-42) or non-labelled Aβ peptide 12 FCUP/ICBAS Abstract (Aβ1-42) in the presence and absence of TTR, using flow cytometry and fluorescence microscopy. Our FACS results showed less internalization in the presence of TTR while fluorescence microscopy assays revealed no significant TTR effect in Aβ uptake. Further tests performed, using short periods of incubation, showed however, that TTR promoted Aβ internalization and possibly its degradation, as assessed by a fluorometric analysis of cellular lysates and supernatants. These results also indicated that in the initial analysis by FACS and fluorescence microscopy, and because cells were incubated for 3 hours, Aβ was internalized and then flowed out of cells, explaining the lower levels. We also conducted permeability studies to assess the effect of TTR in Aβ efflux, using cell monolayers established in transwells filters and measured Aβ transport from the basolateral (brain) to the apical (blood) side. The results showed that only TTR added to the basolateral chamber is able to promote Aβ efflux, when comparing to TTR added to the apical side. The permeability of TTR across the cell monolayers was also evaluated, and our results demonstrated that TTR was capable to cross the hCMEC/D3 cell monolayer, mostly from brain-to-blood, which was lower in the presence of Aβ. We suggest that TTR, while transporting Aβ, also crosses the monolayer or that Aβ causes impaired BBB integrity. Additionally, we performed a preliminary in vivo study, using mice with two copies of TTR (TTR), carrying only one copy of the TTR gene (TTR) and animals without TTR (TTR). We injected FAMAβ1-42 intracranially and our results indicated that, 30 minutes post injection, brains from TTR mice retained less peptide than TTR animals, further supporting a TTR role in Aβ brain efflux, corroborating our in vitro permeability results. However, we could not conclude on FAM-Aβ1-42 brain levels in TTR mice, due to technical issues. To further understand which mechanisms or stimulus are behind the induction of TTR gene expression in neurons, we decided to investigate if high levels of soluble, oligomeric and fibrillar forms of Aβ could induce such expression in a SH-SY5Y neuroblastoma cell line. We also studied the expression pattern of APP isoforms. Realtime polymerase chain reaction (RT-PCR) analysis showed that oligomers and fibrils were capable to induce TTR expression and to increase the expression of APP isoforms (i.e. total APP), whereas soluble Aβ did not cause a significant effect. We assessed also if 695APPswe gene produce TTR expression using the same cell line and our results showed no TTR expression. Finally, we evaluated if exogenous TTR could induce an alteration in APP isoforms expression, and no differences were observed in cells in presence or absence of TTR. FCUP/ICBAS Abstract 13 In summary, our results strengthen the importance of TTR in AD, indicating this protein as a promoter of Aβ clearance across the BBB. Our research also provided evidence that neuronal TTR production is a reaction to toxic Aβ species, further supporting TTR as a protective protein in the central nervous system (CNS).