Dibutyryl cAMP- or Interleukin-6-induced astrocytic differentiation enhances mannose binding lectin (MBL)-associated serine protease (MASP)-1/3 expression in C6 glioma cells.

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells.

BACKGROUND: The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. METHODS: Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription-PCR, viability assays, and single cell Ca(2+) imaging. RESULTS: ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca(2+), activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca(2+). Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca(2+) permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. CONCLUSIONS: Excessive ER Ca(2+) loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies.

Stressed to death: targeting endoplasmic reticulum stress response induced apoptosis in gliomas.

Glial tumors are the main primary adult brain tumor. Even with the most advanced treatments, which include stereotactic microscope aided surgical resection, internal and external radiation therapy and local and systemic chemotherapy, median survival time for patients diagnosed with these malignancies is about 12 months. We explore here the possibility that the endoplasmic reticulum stress response (ERSR) could be a possible target to develop chemotherapeutic agents to induce toxicity in glioma cells. ERSR has the dual capacity of activating repair and/or cytotoxic mechanisms. ERSR is triggered by the accumulation of unfolded proteins in the ER. The presence of unfolded proteins in the ER regulates, via a complex biochemical cascade, the upregulation of molecular chaperones, inhibition of protein synthesis, and an increase of proteasome mediated unfolded protein degradation. ERSR in particular conditions can also contribute to cell death via activation of programmed cell death. Apoptosis activation during ERSR is usually caused by the activation of one or a combination of three biochemical cascades. Induction of these pathways ultimately leads to caspase 3 activation culminating in apoptosis. Glioma cells are in a condition of constant low grade ERSR, which possibly contributes to their resistance to treatment protocols. It is conceivable that small molecules that interact with this phenomenon ultimately could be used to modulate the system to activate apoptosis and cause gliotoxicity. We will discuss here ERSR biochemically relevant features to death mechanisms and already identified small molecules that by modulating ERSR are able to activate glioma cell death.

Identification of novel small molecule activators of nuclear factor-κB with neuroprotective action via high-throughput screening.

Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.

Inactivation of hnRNP K by expanded intronic AUUCU repeat induces apoptosis via translocation of PKCdelta to mitochondria in spinocerebellar ataxia 10.

We have identified a large expansion of an ATTCT repeat within intron 9 of ATXN10 on chromosome 22q13.31 as the genetic mutation of spinocerebellar ataxia type 10 (SCA10). Our subsequent studies indicated that neither a gain nor a loss of function of ataxin 10 is likely the major pathogenic mechanism of SCA10. Here, using SCA10 cells, and transfected cells and transgenic mouse brain expressing expanded intronic AUUCU repeats as disease models, we show evidence for a key pathogenic molecular mechanism of SCA10. First, we studied the fate of the mutant repeat RNA by in situ hybridization. A Cy3-(AGAAU)(10) riboprobe detected expanded AUUCU repeats aggregated in foci in SCA10 cells. Pull-down and co-immunoprecipitation data suggested that expanded AUUCU repeats within the spliced intronic sequence strongly bind to hnRNP K. Co-localization of hnRNP K and the AUUCU repeat aggregates in the transgenic mouse brain and transfected cells confirmed this interaction. To examine the impact of this interaction on hnRNP K function, we performed RT-PCR analysis of a splicing-regulatory target of hnRNP K, and found diminished hnRNP K activity in SCA10 cells. Cells expressing expanded AUUCU repeats underwent apoptosis, which accompanied massive translocation of PKCdelta to mitochondria and activation of caspase 3. Importantly, siRNA-mediated hnRNP K deficiency also caused the same apoptotic event in otherwise normal cells, and over-expression of hnRNP K rescued cells expressing expanded AUUCU repeats from apoptosis, suggesting that the loss of function of hnRNP K plays a key role in cell death of SCA10. These results suggest that the expanded AUUCU-repeat in the intronic RNA undergoes normal transcription and splicing, but causes apoptosis via an activation cascade involving a loss of hnRNP K activities, massive translocation of PKCdelta to mitochondria, and caspase 3 activation.

Reduced penetrance in a Brazilian family with spinocerebellar ataxia type 10.

OBJECTIVE: To describe reduced penetrance associated with early onset in a Brazilian family with spinocerebellar ataxia type 10. DESIGN: Clinical examination and molecular analysis for the ATTCT repeat responsible for spinocerebellar ataxia type 10 in a patient and family members through 3 generations. SETTING: Ambulatory care. Patients A 28-year-old female Brazilian patient who presented with early-onset cerebellar ataxia and epilepsy, and her 9 asymptomatic relatives. Main Outcome Measure Genotype-phenotype correlation. RESULTS: Molecular testing on this patient showed an expansion of approximately 850 ATTCT repeats at the SCA10 locus. Similar SCA10 expansions of approximately 850 repeats were identified in 6 of 8 asymptomatic paternal relatives examined. CONCLUSION: The stably transmitted pentanucleotide expansion of approximately 850 repeats may represent a mutant SCA10 allele with reduced penetrance that may express an early-onset, severe phenotype.