The identification of BCL-XL and MCL-1 as key anti-apoptotic proteins in medulloblastoma that mediate distinct roles in chemotherapy resistance.

Medulloblastoma is the most common malignant paediatric brain tumour, representing 20% of all paediatric intercranial tumours. Current aggressive treatment protocols and the use of radiation therapy in particular are associated with high levels of toxicity and significant adverse effects, and long-term sequelae can be severe. Therefore, improving chemotherapy efficacy could reduce the current reliance on radiation therapy. Here, we demonstrated that systems-level analysis of basal apoptosis protein expression and their signalling interactions can differentiate between medulloblastoma cell lines that undergo apoptosis in response to chemotherapy, and those that do not. Combining computational predictions with experimental BH3 profiling, we identified a therapeutically-exploitable dependence of medulloblastoma cells on BCL-XL, and experimentally validated that BCL-XL targeting, and not targeting of BCL-2 or MCL-1, can potentiate cisplatin-induced cytotoxicity in medulloblastoma cell lines with low sensitivity to cisplatin treatment. Finally, we identified MCL-1 as an anti-apoptotic mediator whose targeting is required for BCL-XL inhibitor-induced apoptosis. Collectively, our study identifies that BCL-XL and MCL-1 are the key anti-apoptotic proteins in medulloblastoma, which mediate distinct protective roles. While BCL-XL has a first-line role in protecting cells from apoptosis basally, MCL-1 represents a second line of defence that compensates for BCL-XL upon its inhibition. We provide rationale for the further evaluation of BCL-XL and MCL-1 inhibitors in the treatment of medulloblastoma, and together with current efforts to improve the cancer-specificity of BCL-2 family inhibitors, these novel treatment strategies have the potential to improve the future clinical management of medulloblastoma.

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.

Targeting the apoptosis pathway to treat tumours of the paediatric nervous system.

New, more effective therapeutics are required for the treatment of paediatric cancers. Current treatment protocols of cytotoxic treatments including chemotherapy trigger cancer-cell death by engaging the apoptosis pathway, and chemotherapy efficacy is frequently impeded by apoptosis dysregulation. Apoptosis dysregulation, through genetic or epigenetic mechanisms, is a feature of many cancer types, and contributes to reduced treatment response, disease progression and ultimately treatment resistance. Novel approaches are required to overcome dysregulated apoptosis signalling, increase the efficacy of cancer treatment and improve patient outcomes. Here, we provide an insight into current knowledge of how the apoptosis pathway is dysregulated in paediatric nervous system tumours, with a focus on TRAIL receptors, the BCL-2 proteins and the IAP family, and highlight preclinical evidence demonstrating that pharmacological manipulation of the apoptosis pathway can restore apoptosis signalling and sensitise cancer cells to treatment. Finally, we discuss the potential clinical implications of these findings.

PD-1 and TIGIT blockade differentially affect tumour cell survival under hypoxia and glucose deprived conditions in oesophageal adenocarcinoma; implications for overcoming resistance to PD-1 blockade in hypoxic tumours.

Recent studies have demontrated that immune checkpoint receptors are expressed on the surface of oesophageal adenocarcinoma (OAC) cells and might confer a survival advantage. This study explores the role of PD-1 and TIGIT signalling in OAC cells in either promoting or inhibiting the survival of OAC cells under characteristic features of the tumour microenvironment including nutrient-deprivation and hypoxia. PD-1 and TIGIT are expressed in normal and pre-malignant oesophageal epithelial cells and this expression significantly decreases along the normal- Barrett's Oesophagus- OAC disease sequence. However, glucose-deprivation and hypoxia significantly upregulated PD-1 and TIGIT on the surface of OAC cells in vitro. PD-1 blockade decreased OAC cell proliferation under normoxia but enhanced proliferation and decreased cell death in OAC cells under hypoxia and glucose-deprivation. TIGIT blockade decreased proliferation and induced OAC cell death, an effect that was maintained under nutrient-deprivation and hypoxia. Basal respiration and glycolytic reserve were enhanced and GLUT1 was upregulated on the surface of a subpopulation of OAC cells following PD-1 blockade. In contrast, TIGIT blockade enhanced a glycolytic phenotype in OAC cells, yet decreased other metabolic parameters including oxidative phosphorylation and basal respiration. Interestingly, inhibition of oxidative phosphorylation significantly upregulated TIGIT expression and inhibition of oxidative phosphorylation and glycolysis significantly decreased PD-1 on the surface of a subpopulation of OAC cells in vitro. These findings suggest an immune-independent mechanism for PD-1 inhibitor resistance in hypoxic tumours and suggest that TIGIT might be a more effective therapeutic target in OAC compared with PD-1 for treating hypoxic tumours.

Transcriptional CDK inhibitors, CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation.

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

Transcriptional CDK Inhibitors CYC065 and THZ1 Induce Apoptosis in Glioma Stem Cells Derived from Recurrent GBM.

Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin-dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA-GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non-tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in gliomaspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non-stem tumour cells resulted in sphere disruption. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.

Marizomib sensitizes primary glioma cells to apoptosis induced by a latest-generation TRAIL receptor agonist.

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood-brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.

Phenotypic selection through cell death: stochastic modelling of O-6-methylguanine-DNA methyltransferase dynamics.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumour with a median overall survival of 15 months. To treat GBM, patients currently undergo a surgical resection followed by exposure to radiotherapy and concurrent and adjuvant temozolomide (TMZ) chemotherapy. However, this protocol often leads to treatment failure, with drug resistance being the main reason behind this. To date, many studies highlight the role of O-6-methylguanine-DNA methyltransferase (MGMT) in conferring drug resistance. The mechanism through which MGMT confers resistance is not well studied-particularly in terms of computational models. With only a few reasonable biological assumptions, we were able to show that even a minimal model of MGMT expression could robustly explain TMZ-mediated drug resistance. In particular, we showed that for a wide range of parameter values constrained by novel cell growth and viability assays, a model accounting for only stochastic gene expression of MGMT coupled with cell growth, division, partitioning and death was able to exhibit phenotypic selection of GBM cells expressing MGMT in response to TMZ. Furthermore, we found this selection allowed the cells to pass their acquired phenotypic resistance onto daughter cells in a stable manner (as long as TMZ is provided). This suggests that stochastic gene expression alone is enough to explain the development of chemotherapeutic resistance.

Cyclin-dependent kinase inhibitors in brain cancer: current state and future directions.

Cyclin-dependent kinases (CDKs) are important regulatory enzymes in the normal physiological processes that drive cell-cycle transitions and regulate transcription. Virtually all cancers harbour genomic alterations that lead to the constitutive activation of CDKs, resulting in the proliferation of cancer cells. CDK inhibitors (CKIs) are currently in clinical use for the treatment of breast cancer, combined with endocrine therapy. In this review, we describe the potential of CKIs for the treatment of cancer with specific focus on glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. Despite intense effort to combat GBM with surgery, radiation and temozolomide chemotherapy, the median survival for patients is 15 months and the majority of patients experience disease recurrence within 6-8 months of treatment onset. Novel therapeutic approaches are urgently needed for both newly diagnosed and recurrent GBM patients. In this review, we summarise the current preclinical and clinical findings emphasising that CKIs could represent an exciting novel approach for GBM treatment.

Implementing Patient-Derived Xenografts to Assess the Effectiveness of Cyclin-Dependent Kinase Inhibitors in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor with no available cure. As previously described, seliciclib, a first-generation cyclin-dependent kinase (CDK) inhibitor, down-regulates the anti-apoptotic protein, Mcl-1, in GBM, thereby sensitizing GBM cells to the apoptosis-inducing effects of the death receptor ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we have assessed the efficacy of seliciclib when delivered in combination with the antibody against human death receptor 5, drozitumab, in clinically relevant patient-derived xenograft (PDX) models of GBM. A reduction in viability and significant levels of apoptosis were observed in vitro in human GBM neurospheres following treatment with seliciclib plus drozitumab. While the co-treatment strategy induced a similar effect in PDX models, the dosing regimen required to observe seliciclib-targeted responses in the brain, resulted in lethal toxicity in 45% of animals. Additional studies showed that the second-generation CDK inhibitor, CYC065, with improved potency in comparison to seliciclib, induced a significant decrease in the size of human GBM neurospheres in vitro and was well tolerated in vivo, upon administration at clinically relevant doses. This study highlights the continued need for robust pre-clinical assessment of promising treatment approaches using clinically relevant models.

Ni(II), Pd(II), and Pt(II) Complexes of the Hedgehog Pathway Inhibitor GANT61-D.

GANT61-D is an important hedgehog pathway inhibitor and an interesting ligand candidate for metal coordination. The first examples of metal complexes of the potent hedgehog pathway inhibitor GANT61-D are described. The reaction of Ni(II), Pd(II), and Pt(II) precursors with the hedgehog pathway inhibitor GANT61-D gave [NiII(GANT61-D)(OH2)3(µ2-SO4)(µ3-SO4)] (1), [PdII(Cl)(GANT61-D)]Cl (2), [PtII(Cl)(GANT61-D)]Cl, and [PtII(CBDCA-2H)(GANT61-D)]. X-ray crystal structure analysis revealed that GANT61-D is a versatile N-donor ligand that can act as a bidentate ligand via the diaminopropane (DAP) N atoms or a tridentate ligand via the DAP N atoms and one dimethylaniline N atom. Protonation constants of the GANT61-D ligand in water and in a 60:40 (w/w) dimethyl sulfoxide-water solvent mixture were determined. Potentiometric and spectroscopic data on the NiII(GANT61-D) system indicate the formation of octahedral 1:1 species with medium stability in solution. 1 and 2 exhibited noteworthy in vitro cytotoxicity against medulloblastoma cancer cells.

System-based approaches as prognostic tools for glioblastoma.

BACKGROUND: The evasion of apoptosis is a hallmark of cancer. Understanding this process holistically and overcoming apoptosis resistance is a goal of many research teams in order to develop better treatment options for cancer patients. Efforts are also ongoing to personalize the treatment of patients. Strategies to confirm the therapeutic efficacy of current treatments or indeed to identify potential novel additional options would be extremely beneficial to both clinicians and patients. In the past few years, system medicine approaches have been developed that model the biochemical pathways of apoptosis. These systems tools incorporate and analyse the complex biological networks involved. For their successful integration into clinical practice, it is mandatory to integrate systems approaches with routine clinical and histopathological practice to deliver personalized care for patients. RESULTS: We review here the development of system medicine approaches that model apoptosis for the treatment of cancer with a specific emphasis on the aggressive brain cancer, glioblastoma. CONCLUSIONS: We discuss the current understanding in the field and present new approaches that highlight the potential of system medicine approaches to influence how glioblastoma is diagnosed and treated in the future.

Sensitization of glioblastoma cells to TRAIL-induced apoptosis by IAP- and Bcl-2 antagonism.

Due to the lack of effective treatments for glioblastoma (GBM), we here studied the responsiveness of GBM cell lines to the combination of death ligand, TRAIL and the IAP antagonist, TL32711 (Birinapant). Responses were highly heterogeneous, with synergistic apoptosis as well as treatment resistance observed. Caspase-8 and Bid, together with caspase-3, form a nonlinear signalling hub that efficiently induced apoptosis in responder cell lines. Cells resistant to TRAIL/TL32711 expressed low amounts of procaspase-8 and Bid and poorly activated caspase-3. We therefore hypothesised that improving caspase-8 activation or sensitising mitochondria to truncated Bid (tBid) could convert non-responder GBM cell lines to responders. Mathematical simulations of both strategies predicted mitochondrial sensitization to tBid would outperform enhancing caspase-8 activation. Indeed, antagonising Bcl-2 by ABT-199 allowed TRAIL/TL32711 response synergies to manifest in otherwise TRAIL resistant cell lines. These findings were further corroborated in experiments with a translationally relevant hexavalent TRAIL variant. Our study therefore demonstrates that a high caspase-8/Bid signature is associated with synergistic TRAIL/TL32711-induced apoptosis in GBM cells and outlines Bcl-2 antagonism as a highly potent intervention to sensitize highly TRAIL-resistant GBM cells to TRAIL/TL32711 combination treatment.

Low cleaved caspase-7 levels indicate unfavourable outcome across all breast cancers.

Elevated levels of the anti-apoptotic BCL2 protein associate with favourable outcome in breast cancer. We investigated whether executioner caspase activation downstream of mitochondrial apoptosis was associated with, or independent, of BCL2's prognostic signature in breast cancer. Levels of pro- and anti-apoptotic BCL2 family proteins were quantified in triple negative breast cancer (TNBC) samples and utilised to calculate BCL2 profiles of 845 breast cancer patients. Biomarkers including single apoptosis proteins and network-enriched apoptosis system signatures were evaluated using uni- and multi-variate Cox-models. In both TNBC and non-TNBC breast cancer, the anti-apoptotic BCL2 protein was particularly abundant when compared to other solid tumours. High BCL2 protein levels were prognostic of favourable outcome across all breast cancers (HR 0.4, 95% CI 0.2-0.6, Wald p < 0.0001). Although BCL2 and cleaved caspase-7 levels were negatively correlated, levels of cleaved caspase-7 were also associated with favourable outcome (HR 0.4, 95% CI 0.3-0.7, Wald p = 0.001). A combination of low BCL2 and low cleaved caspase-7 protein levels was highly prognostic of unfavourable outcome across all breast cancers (HR 11.29, 95% CI 2.20-58.23, Wald p = 0.01). A combination of BCL2 and cleaved caspase-7 levels is a promising prognostic biomarker in breast cancer patients. KEY MESSAGE: BCL2 levels are elevated in breast cancer where they are marker of good prognosis. BCL2 and active caspase levels correlate negatively; yet, active caspases indicate good outcome. Low BCL2 and low caspase-7 are highly prognostic of unfavourable outcome across all breast cancers. BCL2 levels indicate molecular subtype and tumour proliferation status in breast cancer.

Radiation-Induced Gliomas.

Radiation therapy has been a cornerstone of cancer management for many decades and is an integral part of the multi-modality care of patients with brain tumors. The known serious side effects of radiation therapy on the head or central nervous system are uncommon and include radiation necrosis, microangiopathy, and progressive leukencephalopathy. In addition, there have been descriptions of radiation-induced tumors including sarcomas, gliomas, lymphomas, and carcinomas of the thyroid. Patients who have received radiation therapy of the head or face may rarely develop radiation-induced tumors, a majority of which are meningiomas, followed by radiation-induced gliomas (RIGs) and sarcomas. The increased risk of RIGs is well described in both the pediatric and adult populations and after the use of both therapeutic and diagnostic radiations. The incidence of RIGs is estimated at approximately 0.5-2.7% at a latent period of approximately 15 years. The risk appears to be dependent on patient age at treatment, as well as radiation dose and treatment volume considerations. The scope of this review focuses on the etiology, clinical features, and management of RIGs as they relate to previous radiation therapy.

Targeting Autophagy in Glioblastoma.

The role of autophagy in cancer cell survival and cell death has received much attention in recent years; however, scientists are still trying to unravel the complex relationship that exists between autophagy as a tumor suppressor mechanism and as a promoter of tumor progression. In glioblastoma (GBM), the most fatal tumor of the central nervous system, mounting evidence suggests that autophagy processes are tightly intertwined with GBM tumorigenesis and the development of different molecular subtypes. This has led to exciting prospects that autophagy-targeted therapies may improve the efficacy of conventional therapies as well as therapies targeted at specific genetic alterations in individual GBM patients.

Predicting the cell death responsiveness and sensitization of glioma cells to TRAIL and temozolomide.

Genotoxic chemotherapy with temozolomide (TMZ) is a mainstay of treatment for glioblastoma (GBM); however, at best, TMZ provides only modest survival benefit to a subset of patients. Recent insight into the heterogeneous nature of GBM suggests a more personalized approach to treatment may be necessary to overcome cancer drug resistance and improve patient care. These include novel therapies that can be used both alone and with TMZ to selectively reactivate apoptosis within malignant cells. For this approach to work, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified first. Here, we describe the first proof-of-principle study that merges quantitative protein-based analysis of apoptosis signaling networks with data- and knowledge-driven mathematical systems modeling to predict treatment responsiveness of GBM cell lines to various apoptosis-inducing stimuli. These include monotherapies with TMZ and TRAIL, which activate the intrinsic and extrinsic apoptosis pathways, respectively, as well as combination therapies of TMZ+TRAIL. We also successfully employed this approach to predict whether individual GBM cell lines could be sensitized to TMZ or TRAIL via the selective targeting of Bcl-2/Bcl-xL proteins with ABT-737. Our findings suggest that systems biology-based approaches could assist in personalizing treatment decisions in GBM to optimize cell death induction.

Identification of a novel Bcl-2-interacting mediator of cell death (Bim) E3 ligase, tripartite motif-containing protein 2 (TRIM2), and its role in rapid ischemic tolerance-induced neuroprotection.

We have previously shown that the cell death-promoting protein Bcl-2-interacting mediator of cell death (Bim) is ubiquitinated and degraded following a neuroprotection-conferring episode of brief ischemia (preconditioning). Here, we identify the E3 ligase that ubiquitinates Bim in this model, using a proteomics approach. Using phosphorylated GST-Bim as bait, we precipitated and identified by mass spectrometry tripartite motif protein 2 (TRIM2), a RING (really interesting new gene) domain-containing protein. The reaction between TRIM2 and Bim was confirmed using co-immunoprecipitation followed by immunoblotting. We show that TRIM2 binds to Bim when it is phosphorylated by p42/p44 MAPK but does not interact with a nonphosphorylatable Bim mutant (3ABim). 12-O-tetradecanoylphorbol-13-acetate activation of p42/p44 MAPK drives Bim ubiquitination in mouse embryonic fibroblast cells and is associated with an increased interaction between TRIM2 and Bim. One hour following preconditioning ischemia, the binding of Bim to TRIM2 increased, consistent with the time window of enhanced Bim degradation. Blocking p42/p44 MAPK activation following preconditioning ischemia with U0126 or using the nonphosphorylatable 3ABim reduced the binding between Bim and TRIM2. Immunodepletion of TRIM2 from cell lysates prepared from preconditioned cells reduced Bim ubiquitination. Finally, suppression of TRIM2 expression, using lentivirus transduction of shRNAmir, stabilized Bim protein levels and blocked neuroprotection observed in rapid ischemic tolerance. Taken together, these data support a role for TRIM2 in mediating the p42/p44 MAPK-dependent ubiquitination of Bim in rapid ischemic tolerance.

Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.

The functional significance of neuronal death for pathogenesis of epilepsy and the underlying molecular mechanisms thereof remain incompletely understood. The p53 transcription factor has been implicated in seizure damage, but its target genes and the influence of cell death under its control on epilepsy development are unknown. In the present study, we report that status epilepticus (SE) triggered by intra-amygdala kainic acid in mice causes rapid p53 accumulation and subsequent hippocampal damage. Expression of p53-up-regulated mediator of apoptosis (Puma), a proapoptotic Bcl-2 homology domain 3-only protein under p53 control, was increased within a few hours of SE. Induction of Puma was blocked by pharmacologic inhibition of p53, and hippocampal damage was also reduced. Puma induction was also blocked in p53-deficient mice subject to SE. Compared to Puma-expressing mice, Puma-deficient mice had significantly smaller hippocampal lesions after SE. Long-term, continuous telemetric EEG monitoring revealed a approximately 60% reduction in the frequency of epileptic seizures in the Puma-deficient mice compared to Puma-expressing mice. These are the first data showing genetic deletion of a proapoptotic protein acting acutely to influence neuronal death subsequently alters the phenotype of epilepsy in the long-term, supporting the concept that apoptotic pathway activation is a trigger of epileptogenesis.-Engel, T., Murphy, B. M., Hatazaki, S., Jimenez-Mateos, E. M., Concannon, C. G., Woods, I., Prehn, J. H. M., Henshall, D. C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.

Depletion of 14-3-3 zeta elicits endoplasmic reticulum stress and cell death, and increases vulnerability to kainate-induced injury in mouse hippocampal cultures.

14-3-3 proteins are ubiquitous signalling molecules that regulate development and survival pathways in brain. Altered expression and cellular localization of 14-3-3 proteins has been implicated in neurodegenerative diseases and in neuronal death after acute neurological insults, including seizures. Presently, we examined expression and function of 14-3-3 isoforms in vitro using mouse organotypic hippocampal cultures. Treatment of cultures with the endoplasmic reticulum (ER) stressor tunicamycin caused an increase in levels of 14-3-3 zeta within the ER-containing microsomal fraction, along with up-regulation of Lys-Asp-Glu-Leu-containing proteins and calnexin, and the selective death of dentate granule cells. Depletion of 14-3-3 zeta levels using small interfering RNA induced both ER stress proteins and death of granule cells. Treatment of hippocampal cultures with the excitotoxin kainic acid increased levels of Lys-Asp-Glu-Leu-containing proteins and microsomal 14-3-3 zeta levels and caused cell death within the CA1, CA3 and dentate gyrus of the hippocampus. Kainic acid-induced damage was significantly increased in each hippocampal subfield of cultures treated with small interfering RNA targeting 14-3-3 zeta. The present data indicate a role for 14-3-3 zeta in survival responses following ER stress and possibly protection against seizure injury to the hippocampus.