Joseph A. Erhardt

Caspase 3 activation is essential for neuroprotection in preconditioning

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an in vivo model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an in vitro model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against N-methyl-d-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 up-regulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival.

Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia

Mitogen‐activated protein kinases (MAPKs) are involved in many cellular processes. The stress‐activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke‐induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre‐ and post‐stroke and intravenously post‐stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28–41%, and neurological deficits by 25–35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke‐induced expression of IL‐1β and TNFα (i.e., cytokines known to contribute to stroke‐induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063‐induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.

SB 239063, a novel p38 inhibitor, attenuates early neuronal injury following ischemia.

The aim of the present study was to evaluate p38 MAPK activation following focal stroke and determine whether SB 239063, a novel second generation p38 inhibitor, would directly attenuate early neuronal injury. Following permanent middle cerebral artery occlusion (MCAO), brains were dissected into ischemic and non-ischemic cortices and Western blots were employed to measure p38 MAPK activation. Neurologic deficit and MR imaging were utilized at various time points following MCAO to monitor the development and resolution of brain injury. Following MCAO, there was an early (15 min) activation of p38 MAPK (2.3-fold) which remained elevated up to 1 h (1.8-fold) post injury compared to non-ischemic and sham operated tissue. Oral SB 239063 (5, 15, 30, 60 mg/kg) administered to each animal 1 h pre- and 6 h post MCAO provided significant (P<0.05) dose-related neuroprotection reducing infarct size by 42, 48, 29 and 14%, respectively. The most effective dose (15 mg/kg) was further evaluated in detail and SB 239063 significantly (P<0.05) reduced neurologic deficit and infarct size by at least 30% from 24 h through at least 1 week. Early (i.e. observed within 2 h) reductions in diffusion weighted imaging (DWI) intensity following treatment with SB 239063 correlated (r=0.74, P<0.01) to neuroprotection seen up to 7 days post stroke. Since increased protein levels for various pro-inflammatory cytokines cannot be detected prior to 2 h in this stroke model, the early improvements due to p38 inhibition, observed using DWI, demonstrate that p38 inhibition can be neuroprotective through direct effects on ischemic brain cells, in addition to effects on inflammation.

Quantitative changes in interleukin proteins following focal stroke in the rat.

The aim of the present study was to quantitate the temporal changes in protein concentration for interleukin (IL)-1alpha, IL-1beta, IL-1ra, and IL-6 from 1 h to 15 days following focal ischemia. Protein expression was evaluated by enzyme-linked immunosorbent assay utilizing newly available rat antibodies. There were no detectable basal levels of IL-1alpha, 1L-1beta, or IL-6 in the sham-operated or non-ischemic control cortex. IL-1beta (increased significantly (P<0.05) as early as 4 h and peaked at 3 to 5 days. IL-1alpha (increased significantly (P<0.05) at 3 days. IL-6 increased early and peaked at 24 h (P<0.05). IL-1ra increased significantly (P<0.05) over basal levels from 12 h to 5 days. The present study provides the first quantitative determination of interleukin protein concentrations in the rat brain following focal stroke and demonstrates that this technology is now available for mechanistic studies in neuroprotection.

The novel β-blocker, carvedilol, provides neuroprotection in transient focal stroke

Increasing evidence supports a role for oxidative stress, proinflammatory cytokines, and apoptosis in the pathophysiology of focal ischemic stroke. Previous studies have found that the multi-action drug, carvedilol, is a mixed adrenergic antagonist, and that it behaves as an antioxidant and inhibits apoptosis. In the current study, the authors investigated whether carvedilol provides protection in focal cerebral ischemia and whether this protection is associated with reduced apoptosis and the downregulation of the inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) by an intraluminal filament technique. Carvedilol (1, 3, and 10 mg/kg) was injected daily subcutaneously 2 or 4 days before the induction of ischemia. Neurologic scores, infarct volumes, TUNEL staining, and mRNA levels of TNF-α and IL-Iβ were assessed at 24 hours reperfusion. The effect of carvedilol on microvascular cortical perfusion was studied with continuous laser—Doppler flowmetry. Twenty-four hours after MCAO, carvedilol at all three doses reduced infarct volumes by at least 40% and reduced neurologic deficits on average by 40% compared with vehicle-treated controls when given 2 or 4 days before the induction of ischemia. This protection was not mediated by changes in temperature or blood flow. Treatment with all three dose regimens resulted in fewer TUNEL positive cells compared with controls. At 24 hours reperfusion, carvedilol decreased TNF-α and IL-1β expression by 40% to 50% in the ipsilateral ischemic cortex compared with the contralateral controls. The results of the current study indicate that carvedilol is neuroprotective in focal cerebral ischemia and may protect the ischemic brain by inhibiting apoptosis and attenuating the expression of TNF-α and IL-1β.

Dabrafenib; Preclinical Characterization, Increased Efficacy when Combined with Trametinib, while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Comparative analyses of the small molecule thrombopoietin receptor agonist eltrombopag and thrombopoietin on in vitro platelet function

OBJECTIVE The thrombopoietin receptor (TPOR) is a therapeutic target for treatment of thrombocytopenia because stimulation of this receptor results in enhanced megakaryocyte proliferation, differentiation, and ultimately platelet production. In addition to effects on megakaryocytes, TPOR stimulation also impacts platelet function. The present study examined platelet function following stimulation with the small molecule TPOR agonist eltrombopag. MATERIALS AND METHODS Platelets were obtained from healthy volunteers, and signal transduction pathway activation was examined in washed platelet preparations. Platelet aggregation was examined in both washed platelet preparations and platelet-rich plasma. Platelet alpha-granule release was determined via fluorescein-activated cell sorting measurement of CD62P. RESULTS In signal transduction studies of washed human platelets, eltrombopag induced the phosphorylation signal transducers and activators of transcription (STAT) proteins with no phosphorylation of Akt, whereas recombinant human TPO (rhTPO) induced the phosphorylation of Akt as well as STAT-1, -3, and -5. In studies conducted at subthreshold/submaximal concentrations of adenosine diphosphate (ADP) or collagen, eltrombopag pretreatment did not result in platelet aggregation. In contrast, rhTPO acted in synergy with submaximal concentrations of ADP or collagen to induce maximal aggregation under all conditions examined. Similarly, platelet activation as examined via surface expression of CD62P was not enhanced by eltrombopag pretreatment as compared to rhTPO. CONCLUSIONS These results demonstrate that the nonpeptidyl TPOR agonist eltrombopag stimulates platelet signal transduction with little or no effect on overall platelet function, in contrast to TPO, which significantly primes platelet activation. These data demonstrate that effects of TPOR ligands on platelet function can vary depending on the specific mechanism utilized to stimulate the TPOR.

The selective p38 inhibitor SB-239063 protects primary neurons from mild to moderate excitotoxic injury

Inhibition of the p38 mitogen-activated protein kinase (MAP Kinase) pathway reduces acute ischemic injury in vivo, suggesting a direct role for this signaling pathway in a number of neurodegenerative processes. The present study was designed to evaluate further the role of p38 MAP Kinase in acute excitotoxic neuronal injury using the selective p38 inhibitor SB-239063 (trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl) imidazole). Unlike the widely used p38 inhibitor, SB-203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), this second generation p38 inhibitor more selectively inhibits p38 MAP Kinase without affecting the activity of other MAP Kinase signaling pathways and provides a more accurate means to selectively assess the role of p38 in excitotoxicity that has not been previously possible. SB-239063 provided substantial protection against cell death induced by either oxygen glucose deprivation (OGD) or magnesium deprivation in cultured neurons. The ability of this compound to block excitotoxicity was not due to direct inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated currents as SB-239063 did not alter NMDA electrophysiological responses. SB-239063 did not protect against a severe excitotoxic insult induced by 60-min exposure to NMDA. However, when tested against a less severe, brief (5 min) NMDA exposure, p38 inhibition provided substantial protection. These data demonstrate that inhibition of p38 MAP Kinase can confer neuroprotection in vitro against mild but not severe excitotoxic exposure, and suggests that other additional pathways/mechanism(s) may be involved in severe excitotoxic cell death.

Detection of Tumor Necrosis Factor-α mRNA Induction in Ischemic Brain Tolerance by Means of Real-Time Polymerase Chain Reaction

A short duration of ischemia (i.e., ischemic preconditioning) results in significant brain protection to subsequent severe ischemic insult. Because previous studies suggest that tumor necrosis factor-α (TNF-α) plays a role in both promoting ischemic damage and neuroprotection, the present work aimed to evaluate the expression of TNF-α mRNA in an established model of ischemic preconditioning using a transient 10-minute occlusion of the middle cerebral artery. Because the level of TNF-α mRNA expression in the brain was too low to be consistently detected by Northern technique, a real-time polymerase chain reaction method was applied to quantitate the absolute copy number of TNF-α transcript in rat brain after the preconditioning procedure. TNF-α mRNA was induced in the ipsilateral cortex as early as 1 hour (27 ± 1 copies of mRNA per microgram of tissue compared to 11 ± 3 copies in sham-operated samples) after preconditioning, reached a peak level at 6 hours (49 ± 10 copies of transcript, n = 4, P < 0.01), and persisted up to 2 days. These data not only demonstrate the utility of real-time polymerase chain reaction for sensitive and accurate measurement of mRNA expression in normal and injured tissues but also suggest a potential role of TNF-α in the phenomenon of ischemic preconditioning.

Expression of PEP-19 inhibits apoptosis in PC12 cells.

PEP-19 is a calmodulin-regulatory protein found specifically within neurons, though cellular functions of this protein have not been determined. In an effort to define potential effects of PEP-19, PC12 cell lines expressing this protein were generated and subjected to apoptotic stimuli. As measured by LDH release, cell death in PEP-19 expressing cells was 2-to 5-fold less following u.v. irradiation, and 2- to 4-fold less following staurosporine treatment than controls. Additionally, PEP-19-expressing cells displayed decreased DNA ladder formation, chromatin condensation, and caspase activation following staurosporine treatment. Overall, these results demonstrate that PEP-19 can inhibit apoptotic processes in PC12 cells, suggesting a potential regulatory mechanism for pathways leading to cell death.