thapsigargin and Retinal-Degeneration

Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.

Topics: Animals; Apoptosis; Boronic Acids; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Retina; Retinal Degeneration; Retinal Ganglion Cells; Thapsigargin; Tunicamycin

Although the photoreceptors cell death is the main cause of some retinopathies diseases, the mechanisms involved in this process are poorly understood. The neuroprotective effects of interleukin-4 (IL-4) have been shown in several tissues, including retina. We demonstrate that treatment of rat retinal explants with IL-4 completely inhibited the thapsigargin-induced rod photoreceptor cell death after 24 hr in culture. We also showed that IL-4 receptor alpha subunit (IL-4Ralpha) is abundantly present in retina. Colocalization of IL-4Ralpha and rhodopsin indicate a direct effect of this cytokine in rod photoreceptor cells. Moreover, IL-4 increased the intracellular levels of cAMP in 7.4-fold, indicating that the neuroprotective effect of this cytokine was completely blocked by RpcAMP, an inhibitor of protein kinase (PKA). Our data demonstrate, for the first time, the neuroprotective effect of IL-4 through cAMP/PKA pathway in thapsigargin-induced photoreceptor cell death.

Topics: Animals; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Enzyme Inhibitors; Interleukin-4; Nerve Degeneration; Neuroprotective Agents; Organ Culture Techniques; Rats; Receptors, Interleukin-4; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Rhodopsin; Signal Transduction; Thapsigargin; Up-Regulation

The authors investigated intercellular communication among cultured rat retinal pigment epithelial (RPE) cells isolated from dystrophic Royal College of Surgeons (RCS) rats by studying the conduction of the [Ca2+]i wave elicited by mechanical stimulation. The effect of protein phosphorylation was measured by modulating the protein kinase C (PKC), protein kinase A (PKA), and tyrosine kinase activity.. Cultured RPE cells isolated from neonatal control Long-Evans (LE) and dystrophic RCS rats were analyzed using the fluorescent dye fluo-3 to measure the Ca2+-wave propagation on mechanical stimulation to investigate the intercellular communication.. Mechanical stimulation in LE-RPE cells resulted in a centrifugally spreading Ca2+ wave through the neighboring cells. When a mechanical stimulus was applied on RCS-RPE cells, a significantly reduced Ca2+-response was found in the neighboring cells compared with that of control RPE cells. Activation of PKC almost completed blocked the mechanically induced Ca2+ rise in the neighboring RCS-RPE cells. In contrast to LE-RPE cells, an activation of PKA also significantly decreased the Ca2+-wave propagation in RCS-RPE cells. Inhibition of PKA had no effect on the intercellular communication in LE- or RCS-RPE cells. In addition, when protein phosphatase activity or tyrosine kinase activity was inhibited, an increased Ca2+ rise in the neighboring cells on mechanical stimulation was measured, reaching levels currently found for LE-RPE cells.. In dystrophic RCS-RPE cells, a decreased intercellular Ca2+-wave propagation is found. This intercellular communication can be mediated by protein phosphorylation.

Topics: Aniline Compounds; Animals; Calcium; Calcium-Transporting ATPases; Cell Communication; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Fluorescent Dyes; Okadaic Acid; Phosphorylation; Pigment Epithelium of Eye; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Rats, Mutant Strains; Retinal Degeneration; Stress, Mechanical; Thapsigargin; Xanthenes