Differential Stability of miR-9-5p and miR-9-3p in the Brain Is Determined by Their Unique Cis- and Trans-Acting Elements.

microRNAs (miRs) are fundamental regulators of protein coding genes. In the CNS, miR-9 is highly enriched and critical for neuronal development and function. Mature miRs are derived from a duplex precursor, and the -5p strand ("guide") is preferentially incorporated into an RNA-induced silencing complex (RISC) to exert its regulatory functions, while the complementary -3p strand ("passenger") is thought to be rapidly degraded. By contrast, both strands of the miR-9 duplex have unique functions critical for neuronal physiology, yet their respective degradation rates and mechanisms governing degradation are not well understood. Therefore, we determined the degradation kinetics of miR-9-5p and miR-9-3p and investigated the cis and trans elements that affected their stability in the brain. Using a combination of homogeneous neuronal/astrocyte cell models and heterogeneous brain tissue lysate, we demonstrate the novel finding that miR-9-3p was more stable than the miR-9-5p guide strand in all models tested. Moreover, the degradation kinetics of both miR-9-5p and miR-9-3p were brain-region specific, suggesting that each brain region was differentially enriched for specific degradation factors. We also determined that the 3' nucleotides harbor important cis elements required to not only maintain stability, but also to recruit potential protein degradation factors. We used mass spectrometry to assess the miR-9 interacting proteins and found that the -5p and -3p strands were associated with functionally distinct proteins. Overall, these studies revealed unique miR-9-5p and miR-9-3p degradation kinetics in the brain and proposed critical nucleotide sequences and protein partners that could contribute to this differential stability.

Structural and functional characteristics of oestrogen receptor ß splice variants: Implications for the ageing brain.

Oestrogen receptor (ER)ß is a multifunctional nuclear receptor that mediates the actions of oestrogenic compounds. Despite its well defined role in mediating the actions of oestrogens, a substantial body of evidence demonstrates that ERß has a broad range of physiological functions independent of those normally attributed to oestrogen signalling. These functions can partly be achieved by the activity of several alternatively spliced isoforms that have been identified for ERß. This short review describes structural differences between the ERß splice variants that are known to be translated into proteins. Moreover, we discuss how these alternative structures contribute to functional differences in the context of both healthy and pathological conditions. Our review also describes the principal factors that regulate alternative RNA splicing. The alternatively spliced isoforms of ERß are differentially expressed according to brain region, age and hormonal milieu, emphasising the likelihood that there are precise cell-specific mechanisms regulating ERß alternative splicing. However, despite these correlative data, the molecular factors regulating alternative ERß splicing in the brain remain unknown. We also review the basic mechanisms that regulate alternative RNA splicing and use that framework to make logical predictions about ERß alternative splicing in the brain.

Phosphorylation Alters Oestrogen Receptor ß-Mediated Transcription in Neurones.

Nuclear steroid hormone receptors are ubiquitously expressed transcription factors whose activity can be altered by post-translational modifications, such as phosphorylation. The consequences of post-translational modifications have been described for several members of the nuclear steroid hormone receptor superfamily; however, little is known about the effects of oestrogen receptor (ER)ß phosphorylation in the brain. Moreover, to our knowledge, the presence of phosphorylated ERß has not been detected in the brain of any species to date. Oestrogen receptor ß is highly expressed in several regions of the brain and in vitro studies have demonstrated that it can be phosphorylated at two serine residues (S87 and S105) in the N-terminal AF-1 region. The present study aimed to determine whether phosphorylated ERß is detectable in the hippocampus of aged female rats, as well as the functional consequences of ERß S87 and S105 phosphorylation on transcriptional activity in neuronal cells. First, we used a novel PhosTag(™) approach to detect phosphorylated forms of ERß in the dorsal hippocampus of aged female rats. The data obtained demonstrated abundant forms of phosphorylated ERß in the dorsal hippocampus, suggesting that this post-translational modification might be an important regulator of ERß function. To assess the functional consequences of ERß phosphorylation in neuronal cells, we created phospho-mimetic (S87E, S105E) and phospho-null (S87A, S105A) ERß receptors that were transiently transfected in a hippocampal-derived cell line. Collectively, our results showed that phosphorylation of S87 and S105 altered both ligand-independent and ligand-dependent ERß transcriptional regulation. Overall, these data demonstrate that phosphorylated forms of ERß are present in the brain of aged female rats and that phosphorylation of ERß could differentially alter ERß-mediated gene expression.

Characterisation of human oestrogen receptor beta (ERß) splice variants in neuronal cells.

Oestrogen receptor (ER)α and ERß are members of the ligand-activated superfamily of nuclear receptors and mediate most facets of oestrogen signalling. Several naturally occurring splice variants of each ER have been identified in the human brain, yet the biological significance of these splice variants in the brain remains unknown. In the present study, we exploit the unique structural differences of the human ERß splice variants to determine the functional significance of individual ER domains in the brain. We previously established that full-length rodent ERß (i.e. rERß1) has constitutive transcriptional activity in neuronal cells in the absence of ligand. By contrast to the rodent splice variants, the human ERß splice variants used in the present study contain varying length truncations of exon 8, which encodes for the E/F domains. Our results reveal that, in neuronal cells, each human-specific ERß splice variant constitutively activated promoters mediated by a canonical oestrogen response element and repressed promoters mediated by activator protein-1 sites via p38 activity. From these data, we conclude that the C-terminus, encoding the AF-2 region and F domain, is not essential for the constitutive properties of human ERß. Taken together, these studies show that human-specific ERß variants are constitutively active and also provide novel insight into the contributions of the functional domains of ERß towards mediating constitutive transcription at various promoters in neuronal cells.

Testosterone and estrogen act via different pathways to inhibit puberty in the male Siberian hamster (Phodopus sungorus).

The peripubertal transition in male mammals is accompanied by a gradual decrease in sensitivity to the inhibitory effects exerted by gonadal hormones, such as T and E2. Here, we investigated the effects of chronic T and its metabolites, 5alpha-dihydrotestosterone and E2 on the hypothalamo-pituitary-gonadal axis at puberty. We also examined if T effects are distinct or mediated through its conversion to 5alpha-dihydrotestosterone or E2. Twenty-day-old male Siberian hamsters were sc implanted with a SILASTIC brand capsule containing varying doses of T, 5alpha-dihydrotestosterone, or E2. Several functional parameters of the hypothalamo-pituitary-gonadal axis were evaluated including hypothalamic GnRH concentration, pituitary and plasma FSH levels, pituitary FSH and LH mRNA, and testicular status. Our results showed that gonadal steroids inhibited puberty in a dose-dependent manner as evaluated by testes mass (undiluted steroid: T, 27 +/- 3 mg; 5alpha-dihydrotestosterone, 18 +/- 1 mg; and E2, 62 +/- 4 mg relative to cholesterol-implanted controls, 510 +/- 42 mg). Also, T decreased plasma FSH below detectable levels, but pituitary FSH concentration was unaffected (1.37 +/- 0.16 ng/microg protein) while E2-treated hamsters had normal plasma FSH levels (3.5 +/- 0.98 ng/ml) yet significantly lower pituitary FSH concentration (0.09 +/- 0.04 ng/microg protein). These results showed that the pathways of T and E2 action on the hypothalamo-pituitary-gonadal axis are distinct.