Shamim Shaikh

Advanced glycation end products induce in vitro cross-linking of α-synuclein and accelerate the process of intracellular inclusion body formation

Cross‐linking of α‐synuclein and Lewy body formation have been implicated in the dopaminergic neuronal cell death observed in Parkinsons disease (PD); the mechanisms responsible, however, are not clear. Reactive oxygen species and advanced glycation end products (AGEs) have been found in the intracellular, α‐synuclein‐positive Lewy bodies in the brains of both PD as well as incidental Lewy body disease patients, suggesting a role for AGEs in α‐synuclein cross‐linking and Lewy body formation. The aims of the present study were to determine 1) whether AGEs can induce cross‐linking of α‐synuclein peptides, 2) the progressive and time‐dependent intracellular accumulation of AGEs and inclusion body formation, and 3) the effects of extracellular or exogenous AGEs on intracellular inclusion formation. We first investigated the time‐dependent cross‐linking of recombinant human α‐synuclein in the presence of AGEs in vitro, then used a cell culture model based on chronic rotenone treatment of human dopaminergic neuroblastoma cells (SH‐SY5Y) over a period of 1–4 weeks, in the presence of different doses of AGEs. Cells (grown on coverslips) and cell lysates, collected at the end of every week, were analyzed for the presence of intracellular reactive oxygen species, AGEs, α‐synuclein proteins, and intracellular α‐synuclein‐ and AGE‐positive inclusion bodies by using immunocytochemical, biochemical, and Western blot techniques. Our results show that AGEs promote in vitro cross‐linking of α‐synuclein, that intracellular accumulation of AGEs precedes α‐synuclein‐positive inclusion body formation, and that extracellular AGEs accelerate the process of intracellular α‐synuclein‐positive inclusion body formation.

Effects of chronic low dose rotenone treatment on human microglial cells.

BackgroundExposure to toxins/chemicals is considered to be a significant risk factor in the pathogenesis of Parkinsons disease (PD); one putative chemical is the naturally occurring herbicide rotenone that is now used widely in establishing PD models. We, and others, have shown that chronic low dose rotenone treatment induces excessive accumulation of Reactive Oxygen Species (ROS), inclusion body formation and apoptosis in dopaminergic neurons of animal and human origin. Some studies have also suggested that microglia enhance the rotenone induced neurotoxicity. While the effects of rotenone on neurons are well established, there is little or no information available on the effect of rotenone on microglial cells, and especially cells of human origin. The aim of the present study was to investigate the effects of chronic low dose rotenone treatment on human microglial CHME-5 cells.MethodsWe have shown previously that rotenone induced inclusion body formation in human dopaminergic SH-SY5Y cells and therefore used these cells as a control for inclusion body formation in this study. SH-SY5Y and CHME-5 cells were treated with 5 nM rotenone for four weeks. At the end of week 4, both cell types were analysed for the presence of inclusion bodies, superoxide dismutases and cell activation (only in CHME-5 cells) using Haematoxylin and Eosin staining, immunocytochemical and western blotting methods. Levels of active caspases and ROS (both extra and intra cellular) were measured using biochemical methods.ConclusionThe results suggest that chronic low dose rotenone treatment activates human microglia (cell line) in a manner similar to microglia of animal origin as shown by others. However human microglia release excessive amounts of ROS extracellularly, do not show excessive amounts of intracellular ROS and active caspases and most importantly do not show any protein aggregation or inclusion body formation. Human microglia appear to be resistant to rotenone (chronic, low dose) induced damage.

AGEs-RAGE mediated up-regulation of connexin43 in activated human microglial CHME-5 cells.

Microglial activation is a significant contributor to the pathogenesis of many neurodegenerative diseases. Microglia respond to a range of stimuli including pathogenic protein deposits such as advanced glycation endproducts (AGEs). AGEs are prominent inflammatory stimuli that accumulate in the ageing brain. AGEs can activate microglia, leading to the production of excessive amounts of inflammatory cytokines and coupling via gap junction proteins especially connexin43 (Cx43). The literature on the expression of microglial Cx43 during inflammation is controversial. Many cellular effects of AGEs are thought to be mediated by the receptor RAGE. There is however, no evidence suggesting Cx43 is a downstream effector of AGEs-RAGE interaction in microglia. In addition, most of the AGEs-related studies have been undertaken using rodent microglia; the information on human microglia is sparse. Microglia of human and rodent origin respond differently to certain stimuli. The aims of this study were to investigate the AGEs-RAGE-mediated activation of human microglia and establish if Cx43 is one of the downstream effectors of AGEs-RAGE interaction in these cells. Human microglial CHME-5 cells were treated with different doses of AGEs for a selected time-period and microglial activation studied using specific markers. The protein expression of RAGE, Cx43 and TNF-α-receptors (RI and RII) was analysed in response to AGEs in the absence/presence of various doses of anti-RAGE Fabs. TNF-α levels in media were measured using ELISA. TNF-α-induced opening of gap junctional channels was assessed by dye uptake assays and the effect of neutralising TNFRII on Cx43 levels was also studied. CHME-5 cells showed an up-regulation of RAGE, TNF-α, TNFRs (especially TNFRII) and Cx43 upon AGEs treatment and a significant dose-dependent drop in the levels of TNF-α, TNFRII and Cx43 in the presence of anti-RAGE Fabs. TNF-α induced gap junctional/hemichannel opening whereas blocking TNFRII inhibited TNF-α-induced increase in Cx43 levels. Results suggested that TNF-α, TNFRII and Cx43 are downstream effectors of the AGEs-RAGE interaction in human microglial CHME-5 cells.

The role of receptor for advanced glycation end products (RAGE) in neuronal differentiation

The receptor for advanced glycation end products (RAGE) is a multiligand receptor protein thought to play an important role in neuronal differentiation. RAGE can bind a number of ligands and activate a variety of signalling pathways that lead to diverse downstream effects. Amphoterin and S100B are endogenous ligands, the interaction of which with RAGE is known to be involved in defined physiological processes. The present study investigated the spatiotemporal pattern of the expression for RAGE and its ligands, amphoterin and S100B, during neuronal differentiation of NT2/D1 cells. In this study, all three proteins were shown to increase with progression of neuronal differentiation as determined by Western blotting, raising the possibility that both amphoterin and S100B may interact with RAGE and have important functions during the process of cell differentiation. Moreover, blocking the activation of RAGE with neutralizing antibody in the presence of retinoic acid disrupted the progression of normal neuronal differentiation. Immunocytochemistry (ICC) studies showed that amphoterin partially colocalized with RAGE within differentiating NT2 cells, whereas S100B showed a high degree of colocalization. This result suggests that S100B is more likely to be the principal ligand for RAGE during the differentiation process and that RAGE and amphoterin might have both independent and combined roles. Moreover, RAGE was expressed only in cells that were committed to a neuronal phenotype, suggesting direct involvement of RAGE in mediating cellular changes within differentiating neuronal cells. Further detailed studies are now required to characterize fully the role of RAGE during the neuronal differentiation period.

Recovery of Neurological Functions in Non-Human Primate Model of Parkinson's Disease by Transplantation of Encapsulated Neonatal Porcine Choroid Plexus Cells

Parkinsons disease (PD) is a neurodegenerative disease that is primarily characterized by degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) and a loss of their fibre projections in the striatum. We utilized the neonatal porcine choroid plexus (CP), an organ that secretes cerebrospinal fluid containing various types of neurotrophic and neuroprotective factors, to ameliorate the Parkinsonian symptoms in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated rhesus monkeys without requiring immunosuppression. We demonstrate that transplanted encapsulated CP clusters (eCPs) significantly improved neurological functions in MPTP-treated monkeys during the course of six months after transplantation (p < 0.001) when compared with monkeys implanted with empty capsules or subjected to sham surgery. The improvement in neurological scores was accompanied by a corresponding improvement in apomorphine-induced circling behaviour (p < 0.001) as well as increased tyrosine hydroxylase (TH) staining in the striatum. Our results suggest that eCPs are a promising cell therapeutic agent to treat Parkinsons disease.

Comparison of bidirectional and bicistronic inducible systems for coexpression of connexin genes and fluorescent reporters.

Gene expression studies often require inducible coexpression of both a gene of interest and a reporter gene. Fusion of fluorescent reporters can, however, modify protein structure and function. We have generated inducible expression systems for two connexin genes: Cx30 and Cx43. It has been reported recently that reporter fusion to connexins can modify their function. Therefore, we compared two methods of independent reporter coexpression and examined colocalization with induced connexin expression. Identical levels of connexin expression were observed for both the bidirectional and bicistronic expression systems. In contrast, however, reporter gene expression by the bidirectional promoter provided brighter average fluorescent pixel intensity than expression of a reporter gene in a bicistronic transcript. Moreover, as a result of this difference in reporter expression, bidirectional expression systems provided equal or better colocalization between the connexins and reporter gene fluorescence. The results of our study indicate that bidirectional reporter expression provides a robust indicator of transfection and gene expression and, therefore, may favor the use of bidirectional over bicistronic reporters in the design of expression systems where the gene of interest, such as a connexin gene, contains translational motifs or long intronic regions.

The effects of methylation and histone deacetylation on prostaglandin H synthase (PGHS)-1 and PGHS-2 expression in human gestational tissue explants

WithdrawnCONTEXT 9alpha,11beta-Prostaglandin F(2) (9alpha,11beta-PGF(2)) can contract uterine smooth muscle with a potency equal to PGF(2alpha). Its presence in the human uterus and production by human gestational tissues is unknown. OBJECTIVE These studies were performed to determine whether the PGD(2)-derived 9alpha,11beta-PGF(2) is both present in human amniotic fluid and synthesized by human gestational tissues and if so, whether labor-related substances could regulate its production. RESULTS Detectable concentrations of 9alpha,11beta-PGF(2) were found in amniotic fluid samples and appeared to increase in late gestation. All gestational tissues studied synthesized 9alpha,11beta-PGF(2), with the placenta having the highest basal production rate, followed by the amnion and then the choriodecidua. IL-1beta and TNFalpha caused concentration-dependent increases in 9alpha,11beta-PGF(2) production in human amnion and choriodecidual explants. Moreover, treatment of choriodecidual and placental explants with lipopolysaccharide resulted in a significant increase in 9alpha,11beta-PGF(2) production rates, reaching a maximum of 13-fold in the choriodecidua. Studies examining the effects of the addition of exogenous PGD(2) strongly indicated that the choriodecidua has significant ability to convert PGD(2) to 9alpha,11beta-PGF(2), whereas the amnion has little. CONCLUSIONS These results demonstrate for the first time that 9alpha,11beta-PGF(2) is present in human amniotic fluid and that it is produced by human gestational tissues and up-regulated by bacterial cell wall components and proinflammatory cytokines. We suggest that this prostaglandin may play a part in the mechanisms of human labor at term and preterm.