Immunoelectron microscope localization of androgen receptors and proliferating cell nuclear antigen in the epithelial cells of albino rat ventral prostate

AbstractAndrogen receptor (AR) and proliferating cell nuclear antigen (PCNA) play a crucial role in development and progression of various prostatic diseases including prostatic carcinoma that is a leading cause of death in males. Previous studies have evaluated the expression pattern of AR and PCNA in prostate epithelial cells using immunohistochemistry (IHC). However, this technique has limited ability to identify their precise subcellular localization. Therefore, the aim of this study was to localize, subcellularly, AR and PCNA in the secretory epithelial cells of rat ventral prostate using post embedding immunogold-electron microscopy. The ventral lobes were dissected from six adult male albino rats after being perfused with paraformaldehyde. Some specimens were immuno-labeled with AR or PCNA and others were processed for immunoelectron microscope of AR and PCNA using 15-nm gold conjugated secondary antibodies. The results showed that, by immunoperoxidase reaction, AR and PCNA were localized diffusely throughout the nuclei of the epithelial cells of prostatic acini without visible cytoplasmic expression. However, the higher resolution immuno-electron microscopy was able to detect AR and PCNA in the nucleus and some cytoplasmic organelles. In conclusion, this study emphasizes the importance of immuoelectron microscopy in precise localization of AR and PCNA at the subcelullar levels in the secretory epithelial cells of the rat prostatic acini. These findings will help to further understand the mechanism of action of these receptors under normal and pathological conditions that could have future clinical application after careful human investigation

Reduced axonal diameter of peripheral nerve fibres in a mouse model of Rett syndrome

Rett syndrome (RTT) is a neurological disorder characterized by motor and cognitive impairment, autonomic dysfunction and a loss of purposeful hand skills. In the majority of cases, typical RTT is caused by de novo mutations in the X-linked gene, MECP2. Alterations in the structure and function of neurons within the central nervous system of RTT patients and Mecp2-null mouse models are well established. In contrast, few studies have investigated the effects of MeCP2-deficiency on peripheral nerves. In this study, we conducted detailed morphometric as well as functional analysis of the sciatic nerves of symptomatic adult female Mecp2+/- mice. We observed a significant reduction in the mean diameter of myelinated nerve fibers in Mecp2+/- mice. In myelinated fibers, mitochondrial densities per unit area of axoplasm were significantly altered in Mecp2+/- mice. However, conduction properties of the sciatic nerve of Mecp2 knockout mice were not different from control. These subtle changes in myelinated peripheral nerve fibers in heterozygous Mecp2 knockout mice could potentially explain some RTT phenotypes

Development of a novel AAV gene therapy cassette with improved safety features and efficacy in a mouse model of Rett syndrome

Rett syndrome (RTT), caused by loss-of-function mutations in the MECP2 gene, is a neurological disorder characterized by severe impairment of motor and cognitive functions. The aim of this study was to investigate the impact of vector design, dosage and delivery route on the efficacy and safety of gene augmentation therapy in mouse models of RTT. Our results show that AAV-mediated delivery of MECP2 to Mecp2-null mice by systemic administration, and utilizing a minimal endogenous promoter, was associated with a narrow therapeutic window and resulted in liver toxicity at higher doses. Lower doses of this vector significantly extended survival of mice lacking MeCP2 or expressing a mutant T158M allele but had no impact on RTT-like neurological phenotypes. Modifying vector design by incorporating an extended Mecp2 promoter and additional regulatory 3’-UTR elements significantly reduced hepatic toxicity after systemic administration. Moreover, direct cerebroventricular injection of this vector into neonatal Mecp2-null mice resulted in high brain transduction efficiency, increased survival and bodyweight and an amelioration of RTT-like phenotypes. Our results show that controlling levels of MeCP2 expression in the liver is achievable through modification of the expression cassette. However, it also highlights the importance of achieving high brain transduction to impact the RTT-like phenotypes

Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes

This is a pre-copyedited, author-produced version of an article accepted for publication in Human Molecular Genetics following peer review. The version of record Ross, P. D., et al. (2016). "Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes." Human Molecular Genetics 25(20): 4389-4404.] is available online at: https://doi.org/10.1093/hmg/ddw269Work in SC’s laboratory was supported by the Biotechnology and Biological Sciences Research Council (PhD studentship for PDR), a consortium grant from the Rett Syndrome Research Trust, the Chief Scientist Office (Scottish Executive Health Department) [grant ETM/334], RS Macdonald Charitable Trust, Rosetrees Trust [grant M530], and the Rett Syndrome Association Scotland. Work in AB’s laboratory was supported by a Consortium Grant from the Rett Syndrome Research Trust, by Wellcome Trust programme grant [091580] and by Wellcome Trust Centre Core Grant [092076]. Funding to pay the Open Access publication charges for this article was provided by the Scottish Executive Health Department

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