CaMKII and MEK1/2 inhibition time-dependently modify inflammatory signaling in rat cerebral arteries during organ culture.

BACKGROUND: Cerebral ischemia induces transcriptional upregulation of inflammatory genes in the brain parenchyma and in cerebral arteries, thereby contributing to the infarct development. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CaMKII) II and extracellular signal-regulated kinase1/2 (ERK1/2) on inflammatory mediators in rat cerebral arteries using organ culture as a method for inducing ischemic-like vascular wall changes. METHODS: Rat basilar arteries were cultured in serum-free medium for 0, 3, 6 or 24 hours in the presence or absence of the CaMKII inhibitor KN93 or the MEK1/2 inhibitor U0126. Protein expression of activated CaMKII, ERK1/2, and inflammatory-associated protein kinases and mediators were examined with western blot and immunohistochemistry. Caspase-3 mRNA levels in basilar arteries were studied with real-time PCR. RESULTS: Western blot evaluation showed that organ culture induced a significant increase in phosphorylated ERK1/2 at 3, 6 and 24 hours, while CaMKII was found to be already activated in fresh non-incubated arteries and to decrease with incubation time. The addition of U0126 or KN93 decreased levels of phosphorylated c-Jun N-terminal kinase and p-p38, as evaluated by immunohistochemistry. KN93 affected the increase in caspase-3 mRNA expression only when given at the start of incubation, while U0126 had an inhibitory effect when given up to six hours later. Tumor necrosis factor receptor 1 was elevated after organ culture. This inflammatory marker was reduced by both of the two different protein kinase inhibitors. CONCLUSIONS: The novel findings of the present study are that the cross-talk between the two protein kinases and the inhibition of CaMKII or MEK1/2 in a time-dependent manner attenuates inflammatory-associated protein kinases and mediators, suggesting that they play a role in cerebrovascular inflammation.

Retinal adaptation to changing glycemic levels in a rat model of type 2 diabetes.

PURPOSE: Glucose concentrations are elevated in retinal cells in undiagnosed and in undertreated diabetes. Studies of diabetic patients suggest that retinal function adapts, to some extent, to this increased supply of glucose. The aim of the present study was to examine such adaptation in a model of type 2 diabetes and assess how the retina responds to the subsequent institution of glycemic control. METHODS: Electroretinography (ERG) was conducted on untreated Zucker diabetic fatty (ZDF) rats and congenic controls from 8-22 weeks of age and on ZDFs treated with daily insulin from 16-22 weeks of age. Retinal sections from various ages were prepared and compared histologically and by immunocytochemistry. PRINCIPAL FINDINGS/CONCLUSIONS: Acute hyperglycemia did not have an effect on control rats while chronic hyperglycemia in the ZDF was associated with scotopic ERG amplitudes which were up to 20% higher than those of age-matched controls. This change followed the onset of hyperglycemia with a delay of over one month, supporting that habituation to hyperglycemia is a slow process. When glycemia was lowered, an immediate decrease in ZDF photoreceptoral activity was induced as seen by a reduction in a-wave amplitudes and maximum slopes of about 30%. A direct effect of insulin on the ERG was unlikely since the expression of phosphorylated Akt kinase was not affected by treatment. The electrophysiological differences between untreated ZDFs and controls preceded an activation of Müller cells in the ZDFs (up-regulation of glial fibrillary acidic protein), which was attenuated by insulin treatment. There were otherwise no signs of cell death or morphological alterations in any of the experimental groups. These data show that under chronic hyperglycemia, the ZDF retina became abnormally sensitive to variations in substrate supply. In diabetes, a similar inability to cope with intensive glucose lowering could render the retina susceptible to damage.

rd1 Mouse retina shows an imbalance in the activity of cysteine protease cathepsins and their endogenous inhibitor cystatin C.

PURPOSE: To compare in vivo levels, spatial localization, and in vitro secretion of cysteine protease cathepsins and cystatin C (cysC) in the retinal degeneration 1 (rd1) mouse model of retinitis pigmentosa and control (wt) mouse retinas. METHODS: The spatial localization, protein contents, cysC levels and cathepsin-B, -S, and -L activities in wt and rd1 retinas at postnatal (PN) days 2, 7, 14, 21, and 28 were analyzed by immunostaining, spectrophotometry, ELISA, and fluorescence spectrophotometry. The in vitro secretion of cysC and cysteine proteases by PN7 retinal explants into the conditioned medium (RCM) was quantified. RESULTS: The pigment epithelium, photoreceptors, and inner retinal and ganglion cell layers of both wt and rd1 retinas showed cysC and cathepsin-B labeling. CysC immunostaining was extensive in the optic nerve head fibers. The rd1 explants secreted higher amounts of cysteine protease into the RCM. The protein content in wt and rd1 retinal extracts increased up to PN14, then decreased in rd1 but not in wt. In rd1 extracts at PN14 to -28, cathepsin activity was higher and increased with age, but the cysC level was higher and constant. The ratios of cathepsin activity to cysC (cathepsin-L at PN2 and total, -B, and -L at PN14 to -28) were higher in rd1 extracts. CONCLUSIONS: Similar localization of both cathepsin-B and cysC in wt and rd1 retinas along with lower proteins and higher cathepsin activity in rd1 retinal extracts and RCM are consistent with their localization in extracellular matrix and a role in physiopathologic remodeling in wt and rd1 retinas.

Excessive activation of poly(ADP-ribose) polymerase contributes to inherited photoreceptor degeneration in the retinal degeneration 1 mouse.

Retinitis pigmentosa (RP) is an inherited blinding disease for which there is no treatment available. It is characterized by a progressive and neurodegenerative loss of photoreceptors but the underlying mechanisms are poorly understood. Excessive activation of the enzyme poly(ADP-ribose) polymerase (PARP) has recently been shown to be involved in several neuropathologies. To investigate the possible role of PARP in retinal photoreceptor degeneration, we used the retinal degeneration 1 (rd1) mouse RP model to study PARP expression, PARP activity, and to test the effects of PARP inhibition on photoreceptor viability. PARP expression was found to be equal between rd1 and wild-type counterpart retinas. In contrast to this, a dramatic increase in both PARP activity per se and PARP product formation was detected by in situ assays in rd1 photoreceptors actively undergoing cell death. Furthermore, PARP activity colabeled with oxidatively damaged DNA and nuclear translocation of AIF (apoptosis-inducing factor), suggesting activation of PARP as a bridge between these events in the degenerating photoreceptors. The PARP-specific inhibitor PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide x HCl[ reduced the number of cells exhibiting death markers in a short-term retinal culture paradigm, a protective effect that was translated into an increased number of surviving photoreceptors when the inhibitor was used in a long-term culture setting. Our results thus demonstrate an involvement of PARP activity in rd1 photoreceptor cell death, which could have a bearing on the understanding of neurodegenerations as such. The findings also suggest that the therapeutical possibilities of PARP inhibition should include retinal diseases like RP.

CNTF+BDNF treatment and neuroprotective pathways in the rd1 mouse retina.

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and cAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants.

Differential Akt activation in the photoreceptors of normal and rd1 mice.

Retinitis pigmentosa is a blinding disease in which unknown mechanisms cause the degeneration of retinal photoreceptors. The retinal degeneration (rd1) mouse is a relevant model for this condition, since it carries a mutation also found in some forms of retinitis pigmentosa. To understand the degenerative process in the rd1 mouse, we must identify the survival and apoptosis-related signaling pathways in its photoreceptors and determine whether signaling differs from that in normal mice. The phosphatidylinositol 3-kinase/Akt kinase pathway promotes survival in several different cell types. The purpose of the present study has been to compare Akt activity in retinal cells of normal and rd1 mice. We have found that, in normal mice, Akt becomes activated in the retina in a developmentally regulated and cell-type-specific fashion, encompassing essentially all retinal cells. In most cell types, once Akt activation has begun, it remains in this state throughout life. An exception is seen in the rod photoreceptors, in which Akt is activated only transiently during their development. The rd1 retina behaves identically in all but one respect, namely that the activation of Akt in rod photoreceptors persists until these cells undergo apoptosis. Thus, Akt may participate in constitutive survival processes in retinal neurons, except in rod photoreceptors in which the role of this pathway may be restricted to the developmental period. However, Akt activation in the rods may be part of a defense mechanism initiated in response to insults, such as the retinal degeneration seen in the rd1 mouse.