Mature neurons dynamically restrict apoptosis via redundant premitochondrial brakes

Abstract

Apoptotic cell death is critical for the early development of the nervous system, but once the nervous system is established, the apoptotic pathway becomes highly restricted in mature neurons. However, the mechanisms underlying this increased resistance to apoptosis in these mature neurons are not completely understood. We have previously found that members of the miR-29 family of microRNAs (miRNAs) are induced with neuronal maturation and that overexpression of miR-29 was sufficient to restrict apoptosis in neurons. To determine whether endogenous miR-29 alone was responsible for the inhibition of cytochrome c release in mature neurons, we examined the status of the apoptotic pathway in sympathetic neurons deficient for all three miR-29 family members. Unexpectedly, we found that the apoptotic pathway remained largely restricted in miR-29-deficient mature neurons. We therefore probed for additional mechanisms by which mature neurons resist apoptosis. We identify miR-24 as another miRNA that is upregulated in the maturing cerebellum and sympathetic neurons that can act redundantly with miR-29 by targeting a similar repertoire of prodeath BH3-only genes. Overall, our results reveal that mature neurons engage multiple redundant brakes to restrict the apoptotic pathway and ensure their long-term survival.

Keywords: apoptosis; maturation; miR-24; miR-29; neurons.

Figure 1

Neuronal maturation is associated with progressive resistance to neuronal apoptosis at both pre and post-mitochondrial checkpoints. A) Representative images of identical fields of 5 DIV and 11 DIV neurons imaged at 24 hour intervals after NGF deprivation. B) Quantification of sympathetic neuronal survival in response to NGF deprivation for 48 hours after maturing in culture for the indicated amount of time. Data represent mean ± SEM of 3 independent experiments. C) Neurons isolated from neonatal XIAP^−/− mice and cultured for the indicated time were injected with 10 mg/mL of either bovine cyt c to induce apoptosis or yeast cyt c as a negative control and survival was quantified 24 hours post-injection. Data are displayed as mean ± SEM of 3 independent experiments for 5, 10 and 15 DIV and 2 independent experiments for 20 and 25 DIV(**=P<0.01). D) Quantification of cyt c release from neurons cultured for the indicated time and deprived of NGF for 48 hours (**=P<.01, ***=P<.001). E) Representative images of cyt c and Tom20 staining in neurons matured for the indicated amount of time and maintained (+NGF) or deprived (-NGF) for 48 hours.

Figure 2

Mature miR-29 deficient neurons remain resistant to NGF-deprivation induced cyt c release. A) Timecourse of miR-29b induction in maturing neurons. Neurons were isolated from neonatal mice and cultured for the indicated amount of time and miR-29 levels were assessed by RT-qPCR. Values are expressed as mean fold change ± SEM relative to 5 DIV neurons from 3 independent experiments (**=p<0.01, ***=p<0.001, ns=not significant). B) Verification of miR-29 deficiency in mature mir-29 knockout neurons as assessed by RT-qPCR. Values are expressed as mean fold change ± SEM relative to WT 5 DIV neurons from 3 independent experiments. C) Representative images of cyt c staining in mature WT and miR-29 KO cells deprived of NGF for 48 hours. D) Quantification of cyt c release in young WT neurons and mature WT and mature miR-29 KO neurons deprived of NGF for 48 hours. Data represent the percentage of cells with mitochondrial cyt c and are presented as mean ± SEM of 3 independent experiments. E) RT-qPCR quantification of selected BH3-only genes in young, mature WT, and mature miR-29 KO neurons deprived of NGF for 48 hours. Fold changes were normalized to P5 +NGF neurons and represent mean ± SEM of 3 independent experiments.

Figure 3

Other miRNAs predicted to regulate the apoptotic pathway are also induced with neuronal maturation. A) Schematic showing candidate pools of miRNAs that may also regulate cell death in maturing neurons. B) Table of predicted targets for candidate miRNAs by TargetScan software. C–E) Relative expression levels of candidate miRNAs in maturing cerebellum. Values are expressed relative to young (P5) cerebellum and represent mean ± SEM of 3 independent experiments. F–H) Relative expression levels of candidate miRNAs in young (5 DIV) and mature (28 DIV) sympathetic neurons measured by RT-qPCR. Values are expressed as fold-change relative to expression in young neurons and represent mean ± SEM of 3 independent experiments (*=P<0.05 **=p<0.01 ***=P<0.001, ns=not significant).

Figure 4

Overexpression of miR-29 or miR-24 is sufficient to inhibit cyt c release and cell death in young, NGF deprived neurons. A) Representative images of sympathetic neurons injected with mimics to candidate miRNAs or negative control mimic. Injected cells (arrows) are marked with FITC-Dextran (green) and cyt c release (red) was assessed after 48 hours of NGF deprivation. B) Quantification of cyt c release in injected cells after 48 hours of NGF deprivation. Data are represented as mean ± SEM of 3 independent experiments (*=P<0.05, ***=P<0.001). C) Quantification of neuronal survival in neurons injected with mimics to miR-29, miR-24, or a negative control (ncmiR) after 48 hours of NGF deprivation. Survival is expressed as the percentage of cells remaining compared to the number alive pre-deprivation. Data represent mean ± SEM of at least 3 independent experiments.

Figure 5

Overexpression of miR-29 or miR-24 is sufficient to inhibit the induction of Bim and Puma in young sympathetic neurons. A) Representative images of Bim staining in neurons. Neurons were injected at 3 DIV with mimics to miR-29, miR-24, or a negative control, along with FITC-Dextran (Green) to mark injected cells. At 5 DIV neurons were deprived of NGF and after 48 hours of NGF deprivation, neurons were fixed and stained for Bim (red). B) Quantification of normalized Bim staining intensity. Bim staining intensity was measured, and values for injected cells were normalized to NGF-deprived, mock injected neurons. Data presented as mean intensity ± SEM of 3 independent experiments (*=P>0.05). C) Sequence and alignment of miR-24 seed sequence with two putative miR-24 target sites in Puma 3′UTR. D) Luciferase activity was measured 48 hours after transfection in HEK293T cells transfected with reporter plasmids containing the Puma 3′UTR fused to a firefly luciferase gene. Plasmids were transfected either alone or with 100 nM mimics of miR-24 or negative control. Expression was normalized by measuring the ratio of firefly to renilla luciferase. Values are plotted relative to vector alone and represent mean ± SEM of 3 independent experiments (*=P<0.05). E) Representative images of cyt c staining in neurons injected with mimics to miR-29, miR-24, or negative control miR (ncmiR) and treated with 20 μM etoposide. Green indicates injected cells (arrows). F) Quantification of cyt c release in young neurons injected with mimics to miR-29, miR-24, or negative control after 48 hours of etoposide treatment. Data are plotted as mean ± SEM of 3 independent experiments (***=P<0.001).