Role of the c-Jun N-terminal kinase pathway in retinal excitotoxicity, and neuroprotection by its inhibition.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06705.x Abstract Retinal excitotoxicity is associated with retinal ischemia, and with glaucomatous and traumatic optic neuropathy. The present study investigates the role of c-Jun N-terminal kinase (JNK) activation in NMDA-mediated retinal excitotoxicity and determines whether neuroprotection can be obtained with the JNK pathway inhibitor, d-form of JNK-inhibitor 1 (d-JNKI-1). Young adult rats received intravitreal injections of 20 nmol NMDA, which caused extensive neuronal death in the inner nuclear and ganglion cell layers. This excitotoxicity was associated with strong activation of calpain, as revealed by fodrin cleavage, and of JNK. The cell-permeable peptide d-JNKI-1 was used to inhibit JNK. Within 40 min of its intravitreal injection, FITC-labeled d-JNKI-1 spread through the retinal ganglion cell layer into the inner nuclear layer and interfered with the NMDA-induced phosphorylation of JNK. Injections of unlabeled d-JNKI-1 gave unprecedentedly strong neuroprotection against cell death in both layers, lasting for at least 10 days. The NMDA-induced calpain-specific fodrin cleavage was likewise strongly inhibited by d-JNKI-1. Moreover the electroretinogram was partially preserved by d-JNKI-1. Thus, the JNK pathway is involved in NMDA-mediated retinal excitotoxicity and JNK inhibition by d-JNKI-1 provides strong neuroprotection as shown morphologically, biochemically and physiologically.

Critical roles of PPAR beta/delta in keratinocyte response to inflammation.

The immediate response to skin injury is the release of inflammatory signals. It is shown here, by use of cultures of primary keratinocytes from wild-type and PPAR beta/delta(-/-) mice, that such signals including TNF-alpha and IFN-gamma, induce keratinocyte differentiation. This cytokine-dependent cell differentiation pathway requires up-regulation of the PPAR beta/delta gene via the stress-associated kinase cascade, which targets an AP-1 site in the PPAR beta/delta promoter. In addition, the pro-inflammatory cytokines also initiate the production of endogenous PPAR beta/delta ligands, which are essential for PPAR beta/delta activation and action. Activated PPAR beta/delta regulates the expression of genes associated with apoptosis resulting in an increased resistance of cultured keratinocytes to cell death. This effect is also observed in vivo during wound healing after an injury, as shown in dorsal skin of PPAR beta/delta(+/+) and PPAR beta/delta(+/-) mice.

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.

Caspase-3 and RasGAP: a stress-sensing survival/demise switch.

The final decision on cell fate, survival versus cell death, relies on complex and tightly regulated checkpoint mechanisms. The caspase-3 protease is a predominant player in the execution of apoptosis. However, recent progress has shown that this protease paradoxically can also protect cells from death. Here, we discuss the underappreciated, protective, and prosurvival role of caspase-3 and detail the evidence showing that caspase-3, through differential processing of p120 Ras GTPase-activating protein (RasGAP), can modulate a given set of proteins to generate, depending on the intensity of the input signals, opposite outcomes (survival vs death).

Viral superantigen-induced negative selection of TCR transgenic CD4+ CD8+ thymocytes depends on activation, but not proliferation.

T-cell negative selection, a process by which intrathymic immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different experimental approaches suggest that this process is triggered as the result of activation-mediated cell death, the signal transduction pathways underlying this process is not fully understood. In the present report we have used an in vitro system to analyze the cell activation and proliferation requirements for the deletion of viral superantigen (SAg)-reactive Vbeta8.1 T-cell receptor (TCR) transgenic (TG) thymocytes. Our results indicate that in vitro negative selection of viral SAg-reactive CD4+ CD8+ thymocytes is dependent on thymocyte activation but does not require the proliferation of the negatively signaled thymocytes.

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10 % of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

IRF4 and BATF are critical for CD8(+) T-cell function following infection with LCMV.

CD8(+) T-cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses, beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T-cell function, which are still incompletely understood. Here, we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B-cell-activating transcription factor (BATF) are necessary for sustained CD8(+) T-cell effector function. Although Irf4(-/-) CD8(+) T cells were initially capable of proliferation, IRF4 deficiency resulted in limited CD8(+) T-cell responses after infection with the lymphocytic choriomeningitis virus. Consequently, Irf4(-/-) mice established chronic infections, but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T-cell function, limited immunopathology, and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T-cell immunity.

Involvement of microRNAs in the cytotoxic effects exerted by proinflammatory cytokines on pancreatic beta-cells.

OBJECTIVE: Pancreatic beta-cells exposed to proinflammatory cytokines display alterations in gene expression resulting in defective insulin secretion and apoptosis. MicroRNAs are small noncoding RNAs emerging as key regulators of gene expression. Here, we evaluated the contribution of microRNAs to cytokine-mediated beta-cell cytotoxicity. RESEARCH DESIGN AND METHODS: We used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in beta-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated beta-cell failure by modifying their expression in insulin-secreting MIN6 cells. RESULTS: We found that IL-1beta and TNF-alpha induce the expression of miR-21, miR-34a, and miR-146a both in MIN6 cells and human pancreatic islets. We further show an increase of these microRNAs in islets of NOD mice during development of pre-diabetic insulitis. Blocking miR-21, miR-34a, or miR-146a function using antisense molecules did not restore insulin-promoter activity but prevented the reduction in glucose-induced insulin secretion observed upon IL-1beta exposure. Moreover, anti-miR-34a and anti-miR-146a treatment protected MIN6 cells from cytokine-triggered cell death. CONCLUSIONS: Our data identify miR-21, miR-34a, and miR-146a as novel players in beta-cell failure elicited in vitro and in vivo by proinflammatory cytokines, notably during the development of peri-insulitis that precedes overt diabetes in NOD mice.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Motility tests for drugs preventing PolyQ aggregation in recombinant Caenorhabditis elegans.

The pathological formation of proteinaceous aggregates that accumulate into the brain cells of patients are hallmarks of neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis and the heterogeneous group of polyglutamine (polyQ) diseases. In the polyQ diseases, the most upstream events of the pathogenic cascade are the misfolding and aggregation of proteins, such as huntingtin in Huntington's disease, that contain expanded stretch of glutamine residues above 35--‐40 repeats. This expanded polyQ stretch triggers the misfolding and aggregation of cytotoxic polyQ proteins in the neurons that cause cell death through different processes, like apoptosis, excessive inflammation, formation of free radicals, eventually leading to neuronal loss and neurodegeneration. This study focuses on the cellular network of chaperone proteins that can prevent protein aggregation by binding misfolding intermediates and may, as in the case of HSP70, actively unfold misfolded proteins into refoldable non--‐toxic ones (Hinault et al., 2010; Sharma et al., 2011). The chaperones can also collaborate with the proteasome to convert stable harmful proteins into harmless amino acids. Thus, the chaperone proteins that are the most important cellular factors of prevention and curing of protein misfolding, are negatively affected by aging (Morley et al., 2002) and fail to act properly in the neurons of aged persons, which eventually may lead to neurodegenerative pathologies. The general aim of this research was to identify least toxic drugs that can upregulate the expression of chaperone genes in cells suffering from polyQ--‐ mediated protein aggregation and degeneration. The specific aim of this study was to observe the effect of ten drugs on polyQ aggregation in a recombinant nematode Caenorhabditis elegans expressing a chimeric protein containing a sequence of 35 glutamines (Q35) fused to the green fluorescent protein in muscle cells, which causes an age--‐ and temperature--‐ dependent phenotype of accelerated paralysis. The drugs were selected after having proven their causing the overexpression of chaperone proteins in a previous wide screening of 2000 drugs on the moss plant Physcomitrella patens. The screening that we performed in this study was on these ten drugs. It suggested that piroxicam and anisindione were good reducers of polyglutamine disease mediated paralysis. A hypothesis can be made that they may act as good enhancers of the heat shock response, which causes the overexpression of many HSP chaperones and thus reduce motility impairment of polyQ disease expressing nematodes. Piroxicam was found to have the greatest effect on reducing polyQ35 proteins aggregates mediated paralysis in a dose--‐dependent manner but was also found to either have a toxic effect on wild type C.elegans, either to change its natural motility behavior, eventually reducing its motility in both cases. Chloroform should be preferred over DMSO as a drug solvent as it appears to be less toxic to C.elegans.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.

Deleterious Actions Of Vegf On The Blood Retinal Barrier And Photoreceptor Survival In The Light-damage Model

Purpose: The retinal balance between pro- and anti-angiogenic factors is critical for angiogenesis control, but is also involved in cell survival. We previously reported upregulation of VEGF and photoreceptor (PR) cell death in the Light-damage (LD) model. Preliminary results showed that anti-VEGF can rescue PR from cell death. Thus, we investigated the role of VEGF on the retina and we herein described the effect of anti-VEGF antibody delivered by lentiviral gene transfer in this model.Methods: To characterize the action of VEGF during the LD, we exposed Balb/c mice subretinally injected with LV-anti-VEGF, or not, to 5'000 lux for 1h. We next evaluated the retinal function, PR survival and protein expression (VEGF, VEGFR1/2, Src, PEDF, p38MAPK, Akt, Peripherin, SWL-opsin) after LD. We analyzed Blood retinal barrier (BRB) integrity on flat-mounted RPE and cryosections stained with β-catenin, ZO-1, N-cadherin and albumin.Results: Results indicate that the VEGF pathway is modulated after LD. LD leads to extravascular albumin leakage and BRB breakdown: β-catenin, ZO-1 and N-cadherin translocate to the cytoplasm of RPE cells showing loss of cell cohesion. This phenomenon is in adequacy with the VEGF time-course expression. Assessment of the retinal function reveals that PR rescue correlates with the level of LV-anti-VEGF expression. Rhodopsin content was higher in the LV-anti-VEGF group than in controls and measures of the ONL thickness indicate that LV-anti-VEGF preserves by 82% the outer nuclear layer from degeneration. Outer segments (OS) appeared well organized with an appropriate length in the LV-anti-VEGF group compared to controls, and the expression of SWL-opsin is maintained in the OS without being mislocalized as in the LV-GFP group. Finally, LV-anti-VEGF treatment prevents BRB breakdown and maintained RPE cell integrity.Conclusions: This study involves VEGF in LD and highlights the prime importance of the BRB integrity for PR survival. Taken together, these results show that anti-VEGF is neuroprotective in this model and maintains functional PR layer in LD-treated mice.

Controlled delivery of 5-chlorouracil using poly(ortho esters) in filtering surgery for glaucoma.

PURPOSE: To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). METHODS: Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. RESULTS: In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. CONCLUSIONS: The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.