Isabel Gonçalves

Neuroprotective and neuroregenerative properties of metallothioneins

Metallothioneins (MTs) are low‐molecular weight cysteine‐ and metal‐rich proteins with unquestionable metal binding capacity, antioxidant and anti‐inflammatory properties, and a clear involvement in diverse physiological actions as inhibition of proapoptotic mechanisms, enhancement of cell survival, and tissue regeneration. Concurrent with this wide array of functions, MT‐1/2 have been implicated in neuroprotection and neuroregeneration. The zinc binding capacity and antioxidant properties of MTs may account for most of their physiological features in the brain. However, some receptor‐mediated actions of MT‐1/2 have also been reported recently, a subject to be fully elucidated. This review analyses and updates the current knowledge on the actions of MTs related to neuroprotection and neuroregeneration in an effort to distinguish receptor‐mediated actions of MTs from those arising from its zinc binding capacity and its antioxidant properties.

Transthyretin interacts with metallothionein 2.

Transthyretin (TTR) is a 55 kDa homotetrameric protein known for the transport of thyroxine and the indirect transportation of retinol. Within the central nervous system, TTR is primary synthesized and secreted into the cerebral spinal fluid by the choroid plexus (CP), whereas most TTR in the systemic circulation is produced and secreted by the liver. TTR is involved in two types of amyloid disease, the senile systemic amyloidosis and the familial amyloidotic polyneuropathy. TTR has also been implicated in the sequestration of amyloid beta peptide (Abeta), preventing its deposition. To explore other biological roles for TTR, we searched for protein-protein interactions using the yeast two-hybrid system with the full-length human TTR cDNA as bait. We found a novel interaction between TTR and metallothionein 2 (MT2) in human liver. This interaction was confirmed by competition binding assays, co-immunoprecipitation, cross-linking, and Western blotting experiments. Binding studies using MT1 showed a saturable specific interaction with TTR with a Kd of 244.8 +/- 44.1 nM. Western blotting experiments revealed a TTR-MT1/2 protein complex present in rat CP and kidney tissue extracts. Immunofluorescence experiments, in CP primary cell cultures and in CP paraffin sections, showed co-localization of TTR and MT1/2 in the cytoplasm of epithelial CP cells and localization of MT1/2 in the endoplasmic reticulum. Moreover, dot blot immunoassays of rat CSF provided the first evidence, to our knowledge, of circulating metallothionein in CSF. Taken together, we suggest that TTR-MT1/2 complexes may be functionally significant not only in healthy conditions but also in Abeta deposition in Alzheimer disease, thereby providing a novel potential therapeutic target.

5α-dihydrotestosterone up-regulates transthyretin levels in mice and rat choroid plexus via an androgen receptor independent pathway

Transthyretin (TTR) is a 55 kDa plasma homotetrameric protein mainly synthesized in the liver and choroid plexuses (CPs) of the brain that, functions as a carrier for thyroxin and retinol binding protein. It sequesters amyloid beta (Abeta) peptide, and TTR levels in the cerebrospinal fluid (CSF) appear to be inversely correlated with Alzheimers disease (AD) onset and progression. Androgen deprivation increases plasma Abeta levels, which indicate that androgens may reduce the levels of soluble Abeta, the peptide widely implicated in the initiation of AD pathogenesis; however, the underlying mechanisms are still poorly understood. In this study we examined the effects of 5alpha-dihydrotestosterone (DHT) on TTR protein and mRNA levels, in primary cultures of rat CPs epithelial cells (CPEC) by Western blot, and real time PCR, respectively. Moreover, TTR concentrations were measured in the CSF of castrated wild-type, and transgenic mice expressing human TTR subjected to DHT treatment, by radioimmunoassay and ELISA, respectively. TTR mRNA expression was also compared in the CPs, of the animals from each experimental group by real time PCR. DHT treatment increased TTR protein levels in CPEC, and induced TTR transcription in these cells. The combination of flutamide with DHT in the treatment of CPEC did not abrogate DHT-induced TTR levels, suggesting that TTR is up-regulated via an androgen receptor independent pathway. In the CPs of both mice strains, DHT also increased TTR mRNA levels, but no significant differences in TTR protein levels were detected in the CSF of these animals. These findings open a wide range of possibilities for future studies on Abeta deposition and cognitive function, in response to androgen induction of TTR in animal models of AD.

17β-Estradiol Induces Transthyretin Expression in Murine Choroid Plexus via an Oestrogen Receptor Dependent Pathway

Oestrogen protects against AD by multiple mechanisms, including the enhancement of Aβ clearance. Transthyretin (TTR) is a homotetrameric protein mainly synthesized by the liver and choroid plexus (CP) of the brain that sequesters the amyloid beta (Aβ) peptide. In this study we examined the effects of 17β-estradiol (E2) on TTR protein and mRNA levels, in primary cultures of rat CP epithelial cells (CPEC) by Western blot and Real Time PCR, respectively. Moreover, the localization of oestrogen receptors alpha (ERα) and beta (ERβ) in response to E2 treatment was analysed by confocal microscopy in these cells. The expression of TTR, ERα and ERβ was also compared in the CP of castrated female mice treated with E2 to vehicle-treated animals by Real Time PCR. TTR concentration in the CSF of all these animals was measured by radioimmunoassay. E2 treatment induced TTR transcription and increased TTR protein content in CPEC. Pre-treatment with ICI 182,780 (ICI) abrogated E2-induced TTR expression suggesting that, TTR is up-regulated via an ER-dependent pathway. Confocal microscopy demonstrated extranuclear ERα and ERβ localization in untreated CPEC. Upon E2 treatment, translocation of ERα to the nucleus occurred, while ERβ remained in the cytosol. These data was concurrent with the up-regulation of TTR expression detected in the CP of castrated female mice subjected to E2 treatment. Our results highlight the importance of E2 on the regulation of TTR, which may participate in the oestrogen-induced decrease in Aβ levels and deposition described in the literature.

Human metallothioneins 2 and 3 differentially affect amyloid-beta binding by transthyretin.

Transthyretin (TTR), an amyloid‐beta (Aβ) scavenger protein, and metallothioneins 2 and 3 (MT2 and MT3), low molecular weight metal‐binding proteins, have recognized impacts in Aβ metabolism. Because TTR binds MT2, an ubiquitous isoform of the MTs, we investigated whether it also interacts with MT3, an isoform of the MTs predominantly expressed in the brain, and studied the role of MT2 and MT3 in human TTR–Aβ binding. The TTR–MT3 interaction was characterized by yeast two‐hybrid assays, saturation‐binding assays, co‐immunolocalization and co‐immunoprecipitation. The effect of MT2 and MT3 on TTR–Aβ binding was assessed by competition‐binding assays. The results obtained clearly demonstrate that TTR interacts with MT3 with a Kd of 373.7 ± 60.2 nm. Competition‐binding assays demonstrated that MT2 diminishes TTR–Aβ binding, whereas MT3 has the opposite effect. In addition to identifying a novel ligand for TTR that improves human TTR–Aβ binding, the present study highlights the need to clarify whether the effects of MT2 and MT3 in human TTR–Aβ binding observed in vitro have a relevant impact on Aβ deposition in animal models of Alzheimer’s disease.

Stress and Glucocorticoids Increase Transthyretin Expression in Rat Choroid Plexus via Mineralocorticoid and Glucocorticoid Receptors

Transthyretin (TTR) is a carrier for thyroid hormones and retinol binding protein. Several mutated forms of TTR cause familial amyloidotic polyneuropathy, an inheritable lethal disease. On the other hand, wild-type TTR has a protective role against Alzheimer’s disease. Despite its overall importance in normal animal physiology and in disease, few studies have focused on its regulation. An in silico analysis of the rat TTR gene revealed a glucocorticoid responsive element in the 3′ region of the first intron. Thus, we hypothesised that TTR could be regulated by glucocorticoid hormones and investigated the regulation of TTR expression in response to hydrocortisone in a rat choroid plexus cell line (RCP) and in primary cultures of choroid plexus epithelial cells (CPEC). In addition, the effect of psychosocial stress on TTR expression was analysed in rat liver, choroid plexus (CP) and cerebrospinal fluid (CSF). In RCP and CPEC cultures hydrocortisone upregulated TTR expression, an effect suppressed by glucocorticoid receptor and mineralocorticoid receptor antagonists. Moreover, induction of psychosocial stress increased TTR expression in liver, CP and CSF of animals subjected to acute and chronic stress conditions. Overall, we conclude that stress upregulates TTR expression in CP.

Analysis of the effects of sex hormone background on the rat choroid plexus transcriptome by cDNA microarrays.

The choroid plexus (CP) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF), the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis.

Progesterone Enhances Transthyretin Expression in the Rat Choroid Plexus In Vitro and In Vivo via Progesterone Receptor

Depletion of ovarian hormones 17β-estradiol (E2) and progesterone (P) after menopause may contribute to the decline in cognitive performance and increases the risk of Alzheimer’s disease (AD) in women, striking the importance of understanding the regulation of pivotal proteins involved in AD pathogenesis by ovarian hormones. Transthyretin (TTR) is now recognized as one of such proteins due to its ability to sequester and degrade amyloid β (Aβ) into less harmful peptides and preventing their aggregation. We have previously demonstrated that E2 enhances TTR expression. In this study, we investigate the effects of P on TTR expression in primary cultures of rat choroid plexus epithelial cells and in adult ovariectomized female rats. The results obtained demonstrate that, in vitro and in vivo, TTR is up-regulated by P. In addition, the mechanism underlying the response of TTR to P was investigated, and we provide evidence that this response is achieved through a progesterone receptor-mediated mechanism. Our results reinforce the importance of ovarian hormones on the regulation of TTR, which may reflect on the processing of Aβ peptides and consequently on AD onset and progression.

Androgen Receptor is Expressed in Murine Choroid Plexus and Downregulated by 5α-Dihydrotestosterone in Male and Female Mice

The choroid plexuses (CPs) of the brain form a unique interface between the peripheral blood and the cerebrospinal fluid (CSF). CPs produce several neuroprotective peptides, which are secreted into the CSF. Despite their importance in neuroprotection, the mechanisms underlying the regulation of most of these peptides in CPs remain unknown. Androgens regulate the expression of neuroprotective peptides in several tissues where the androgen receptor (AR) is coexpressed, including the brain. The presence of AR in CPs has never been investigated, but recent studies in our laboratory show that the CP is an androgen-responsive tissue. In order to fulfill this gap, we investigated and characterized AR distribution and expression in male and female rat CPs and in primary cultures from rat CP epithelial cells. In addition, the response of AR to 5α-dihydrotestosterone (DHT) in castrated male and female mice subjected to DHT replacement was analyzed. We show that rat CP epithelial cells contain AR mRNA and protein. Moreover, we demonstrate that AR is downregulated by DHT in mice CPs.

Gender associated circadian oscillations of the clock genes in rat choroid plexus

It is well-documented that circadian rhythms are controlled by the circadian master clock of the mammalian brain, located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN clockwork is a cell autonomous mechanism consisting of a series of interlocked transcriptional/post-translational feedback loops. In turn, the SCN controls the seasonal rhythmicity of various biological processes, in particular the secretion pattern of hormones. Although the effects of gonadal hormones on circadian rhythmicity are clearly established, how the SCN integrates and regulates these hormonal stimuli remains unknown. We have previously found that clock genes are expressed in the choroid plexus (CP). Therefore, we compared the circadian expression of these genes in female and male rat CP. We show that there is a 24-h rhythm in the expression of Per2 and Cry2 in males and females. Bmal1 and Per1 expression also varied along the day, but only in females. Bmal1, Clock and Per1 mRNA did not show any significant differences in the CP of males. Moreover, data from cultured CP cells collected at different timepoints revealed significant circadian rhythms in mRNA abundance of Bmal1, Clock and Per2. In conclusion, our data show that the rat CP expresses all canonical clock genes and that their circadian expression differs between genders suggesting that hormones can regulate circadian rhythmicity in CP.