A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death

Pro-apoptotic Bax is essential for RGC (retinal ganglion cell) death. Gene dosage experiments in mice, yielding a single wild-type Bax allele, indicated that genetic background was able to influence the cell death phenotype. DBA/2JBax+/− mice exhibited complete resistance to nerve damage after 2 weeks (similar to Bax−/− mice), but 129B6Bax+/− mice exhibited significant cell loss (similar to wild-type mice). The different cell death phenotype was associated with the level of Bax expression, where 129B6 neurons had twice the level of endogenous Bax mRNA and protein as DBA/2J neurons. Sequence analysis of the Bax promoters between these strains revealed a single nucleotide polymorphism (T129B6 to CDBA/2J) at position −515. A 1.5- to 2.5-fold increase in transcriptional activity was observed from the 129B6 promoter in transient transfection assays in a variety of cell types, including RGC5 cells derived from rat RGCs. Since this polymorphism occurred in a p53 half-site, we investigated the requirement of p53 for the differential transcriptional activity. Differential transcriptional activity from either 129B6 or DBA/2J Bax promoters were unaffected in p53−/− cells, and addition of exogenous p53 had no further effect on this difference, thus a role for p53 was excluded. Competitive electrophoretic mobility-shift assays identified two DNA–protein complexes that interacted with the polymorphic region. Those forming Complex 1 bound with higher affinity to the 129B6 polymorphic site, suggesting that these proteins probably comprised a transcriptional activator complex. These studies implicated quantitative expression of the Bax gene as playing a possible role in neuronal susceptibility to damaging stimuli

The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

Abstract Background Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Methods Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116 BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. Results HCT116 BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Conclusion Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the translocation step of BAX activation may be impaired

Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways

BACKGROUND: Retinal ganglion cell (RGC) soma death is a consequence of optic nerve damage, including in optic neuropathies like glaucoma. The activation of the innate immune network in the retina after nerve damage has been linked to RGC pathology. Since the eye is immune privileged, innate immune functions are the responsibility of the glia, specifically the microglia, astrocytes, and Müller cells that populate the retina. Glial activation, leading to the production of inflammatory cytokines, is a hallmark feature of retinal injury resulting from optic nerve damage and purported to elicit secondary degeneration of RGC somas. METHODS: A mouse model of optic nerve crush (ONC) was used to study retinal glial activation responses. RGC apoptosis was blocked using Bax-deficient mice. Glial activation responses were monitored by quantitative PCR and immunofluorescent labeling in retinal sections of activation markers. ATP signaling pathways were interrogated using P2X receptor agonists and antagonists and Pannexin 1 (Panx1)-deficient mice with RGC-specific deletion. RESULTS: ONC induced activation of both macroglia and microglia in the retina, and both these responses were dramatically muted if RGC death was blocked by deletion of the Bax gene. Macroglial, but not microglial, activation was modulated by purinergic receptor activation. Release of ATP after optic nerve damage was not mediated by PANX1 channels in RGCs. CONCLUSIONS: RGC death in response to ONC plays a principal stimulatory role in the retinal glial activation response. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-016-0558-y) contains supplementary material, which is available to authorized users