Hypoxia-induced gene expression is a critical determinant of neuron survival after stroke. Understanding the cell autonomous genetic program controlling adaptive and pathological transcription could have important therapeutic implications. To identify the factors that modulate delayed neuronal apoptosis after hypoxic injury, we developed an in vitro culture model that recapitulates these divergent responses and characterized the sequence of gene expression changes using microarrays. Hypoxia induced a disproportionate number of bZIP transcription factors and related targets involved in the endoplasmic reticulum stress response. Although the temporal and spatial aspects of ATF4 expression correlated with neuron loss, our results did not support the anticipated pathological role for delayed CHOP expression. Rather, CHOP deletion enhanced neuronal susceptibility to both hypoxic and thapsigargin-mediated injury and attenuated brain-derived neurotrophic factorinduced neuroprotection. Also, enforced expression of CHOP prior to the onset of hypoxia protected wild-type cultures against subsequent injury. Collectively, these findings indicate CHOP serves a more complex role in the neuronal response to hypoxic stress with involvement in both ischemic preconditioning and delayed neuroprotection.
The dual specificity phosphatase MAPK phosphatase-1 (MKP-1) feeds back on MAP kinase signaling to regulate metabolic, inflammatory and survival responses. MKP-1 is widely expressed in the central nervous system (CNS) and induced after ischemic stress, although its function in these contexts remains unclear. Here we report that MKP-1 activated several cell death factors, including BCL2 and adenovirus E1B 19 kDa interacting protein 3, and caspases 3 and 12 culminating in apoptotic cell death in vitro. MKP-1 also exerted inhibitory effects on the bZIP transcription factor CCAAT/enhancer-binding protein (C/EBPb), previously shown to have neuroprotective properties. These effects included reduced expression of the full-length C/EBPb variant and hypo-phosphorylation at the MEK-ERK1/2-sensitive Thr 188 site. Notably, enforced expression C/EBPb rescued cells from MKP-1-induced toxicity. Studies performed in knock-out mice indicate that the MKP-1 activity is required to exclude C/EBPb from the nucleus basally, and that MKP-1 antagonizes C/EBPb expression after global forebrain ischemia, particularly within the vulnerable CA1 sector of the hippocampus. Overall, MKP-1 appears to lower the cellular apoptotic threshold by inhibiting C/EBPb and enhancing both BH3 protein expression and cellular caspase activity. Thus, although manipulation of the MKP-1-C/EBPb axis could have therapeutic value in ischemic disorders, our observations using MKP-1 catalytic mutants suggest that approaches geared towards inhibiting MKP-1's phosphatase activity alone may be ineffective.
To facilitate genetic studies in primary neurons, we analyzed the efficiency of cationic lipid-mediated plasmid DNA transfection using adherent and acutely dissociated neuronal suspensions derived from embryonic mouse cortical tissue. Compared to transfections using adherent cultures, the in-tube procedure enhanced the delivery of a GFP reporter plasmid between four-to eight-fold depending on the age of the harvested embryo. The procedure required relatively brief complex incubation times, and supported the transfection of cells expressing the neuronal markers NeuN and TuJ1 with improved uniformity in transfection events across the well surface. To demonstrate the utility of this approach in studying the genetic mechanisms controlling neuron development, we provide data regarding the role of the bZIP transcription factor c/EBP-β in regulating neurite outgrowth. It is anticipated that this in vitro protocol will facilitate the identification of novel genes involved in both developmental and disease-relevant signaling pathways.Keywords primary neuron; transfection; liposome; gene expression; high-throughput screening; in vitro assay IntroductionIt is estimated that the human genome expresses between 20,000 and 25,000 unique mRNAs that are ultimately translated into proteins. However, this estimate does not account for the range of expressed non-coding transcripts, which include tRNAs, rRNAs, snoRNAs and microRNAs (HGSC, 2004, Wright, et al., 2001. Moreover, only a fraction of the protein coding transcripts have been functionally annotated. This complexity is of particular relevance in the central nervous system given the range of neuron subtypes present in the various specialized brain regions (Muotri and Gage, 2006). Further transcript diversity is supported through alternative splicing and RNA editing, both of which can undergo dynamic regulation in response to physiologic and pathological perturbations (Blencowe, 2005). With the availability of comprehensive commercial cDNA and RNAi libraries, understanding the function of these unique transcripts in the context of disease-relevant models has become a tenable goal. However, the lack of cost-effective methods that support efficient, high-throughput gene delivery to primary neuronal cultures remains a rate limiting step in studying genetic responses within post-mitotic neurons. Lipid mediated gene transfer is one of the most widely used techniques in basic cell biological research. In the neurosciences, transfection is often used to perturb gene function in tumor cell lines derived from neural or glial lineages. While these systems recapitulate various aspects of neuronal physiology, results obtained using transformed cell lines are an approximation of the signaling events occurring in the post-mitotic neuron. Unfortunately, the genetic manipulation of primary neuronal cultures remains a challenge given their resistance to lipid-mediated gene transfer using standard techniques. Although efficiencies as high as 25% have been reported using rat cortical neu...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.