Transthyretin and metallothioneins affect amyloid-beta metabolism and are regulated by glucocorticoids in choroid plexus

The choroid plexus (CP), localized within brain ventricles, constitutes the barrier between the blood and the cerebrospinal fluid (CSF) and is an essential structure for the maintenance of brain homeostasis. It participates in the synthesis, secretion and regulation of the CSF and various biologically active compounds, in the maintenance of central nervous system (CNS) metal bioavailability and in the removal of brain toxic compounds, protecting it against neurotoxic insults. Transthyretin (TTR) is a homotetrameric protein mostly produced and secreted by the liver to the peripheral circulation and by the CP epithelial cells (CPECs) to the CSF. The TTR major functions are the transport of thyroid hormones, principally thyroxin, and, indirectly, retinol. Furthermore, in the last decades, it has also been shown that TTR acts as an amyloid-beta (Aβ) peptide scavenger, a key molecule in Alzheimer’s disease (AD), preventing its aggregation and/or deposition and promoting its clearance. Actually, the non-mutated form of TTR has been identified as the main Aβ binding protein in the CSF. Metallothioneins (MTs) are multipurpose proteins with widely described metal binding, antioxidant and anti-inflammatory properties. In mammals, four distinct isoforms had been identified (MT-1 to MT-4). In addition, other physiological actions have also been attributed to MTs: inhibition of pro-apoptotic mechanisms, enhancement of cell survival and tissue regeneration. Previous studies showed that the expression of brain MTs is regulated in several neurodegenerative disorders, as AD, and MT-1/2, contrarily to MT-3, are highly inducible by physiological and psychological stress. TTR and MTs act in several metabolic pathways, especially within the CNS, where they play major roles in its homeostasis. Previously, our group showed that TTR interacts with MT-2, an ubiquitous isoform of the MTs, with unknown effects on the functions of each protein. Thus, we firstly investigated whether it also interacts with MT-3, an isoform predominantly expressed in the brain, and studied the role of MT-2 and MT-3 in human TTR–Aβ binding. The TTR–MT-3 interaction was characterized by yeast two-hybrid assays, saturation-binding assays, co-immunolocalization and co-immunoprecipitation assays. Moreover, the effect of MT-2 and MT-3 in TTR–Aβ binding was assessed by competition-binding assays. The results demonstrated that TTR interacts with MT-3 with a dissociation constant (Kd) of 373.7 ± 60.2 nM. Also, TTR-MT-2 interaction diminished the TTR–Aβ binding, whereas MT-3 enhanced the binding of TTR to Aβ, most likely promoting its degradation. Furthermore, both proteins co-localized with the endoplasmic reticulum of CPECs, indicating that, as TTR, MTs may also be secreted and interactions between TTR and MTs might occur inside and outside these cells. Stress is related with neurodegenerative disorders because it raises glucocorticoid levels, which generate adequate responses to stressors and regulate key molecules, as TTR and MT-1/2, in some brain regions, participating in neuroprotection and neuroregeneration. However, in CP, nothing is known concerning the regulation of TTR and MT-1/2 expressions by glucocorticoids. In silico analyses of TTR and MT-1/2 genes identified glucocorticoid responsive elements (GREs) in both genes. Also, CP expresses TTR, MTs (isoforms 1, 2 and 3) and glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) turning it into a likely glucocorticoid responsive tissue. Thus, we hypothesized that TTR and MT-1/2 could be regulated by glucocorticoids within the CP. We investigated the regulation of TTR expression in response to hydrocortisone in a rat choroid plexus (RCP) cell line and in primary cultures of CPECs. In addition, the effect of psychosocial stress induction in TTR expression was analyzed in rat liver, CP and CSF. The results showed that hydrocortisone up-regulated TTR expression in RCP and CPEC cultures, and, this effect was suppressed upon addition of GR and/or MR antagonists, suggesting the involvement of these receptors in this regulatory mechanism. Moreover, induction of psychosocial stress increased TTR expression in liver, CP and CSF of animals subjected to acute or chronic stress conditions, showing that stress up-regulates TTR expression, particularly in CP. To test the hypothesis that MT-1/2 expression in CP could also be regulated by glucocorticoids, with implications in apoptosis, we performed analogous experiments to those described above. Data obtained showed that hydrocortisone up-regulated MT-1/2 expression in RCP cells and CPECs and the incubation with GR and/or MR antagonists abrogated this effect. In addition, comparing to controls, the incubation of RCP cells with hydrocortisone diminished the ratio of apoptotic/late apoptotic cells from 17.8% to 9.7% and, this effect was abolished by the addition of an anti-MT-1/2 antibody. Thus, the up-regulation of MT-1/2 expression after incubation with glucocorticoids diminished apoptosis in CP and in this regard, glucocorticoids may beneficiate CP integrity and therefore become neuroprotective. In vivo studies showed that induction of chronic psychosocial stress increased MT-1/2 expression in liver and CP of male and female rats. A similar pattern was observed after acute stress in liver. Interestingly, in CP, induction of acute stress caused different effects between genders as, in females, it promoted an up-regulation of MT-1/2 expression, while a down-regulation was observed in males, indicating a distinct regulation gender-dependent and, suggesting distinct readjustment in response to stress between males and females in this structure. Generally, results showed that glucocorticoids regulate MT-1/2 expression in rat CP, time-, tissue- and gender-dependently, with implications in apoptosis. Numerous pathways and molecules and its complex interactions are involved in neurodegenerative disorders. In line with this, we suggest that the putative and isolated neuroprotective effects promoted by glucocorticoids in CP may not be sufficient per se to prevent the negative effects promoted by high levels of cortisol in other brain regions, other molecules or in the overall progression of a disease. Taken together, our results suggest that the up-regulation of TTR and MT-1/2 in CP, promoted by glucocorticoids, also result in a higher bioavailability of these proteins that may potentiate the occurrence of TTR-MT-2 interactions, which negatively affect the Aβ clearance and, consequently, AD. Future studies will be crucial for clarification of the role of these molecules and their interactions with Aβ metabolism and apoptosis, which may have far reaching effects in various neurodegenerative disorders, as AD.

Topic: Amyloidosis: Diagnosis, Treatment, Outcomes
Subfield: Molecular Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences