The proapoptotic activities of Bax and Bak limit the size of the neural stem cell pool - PubMed (original) (raw)

The proapoptotic activities of Bax and Bak limit the size of the neural stem cell pool

Tullia Lindsten et al. J Neurosci. 2003.

Abstract

The proapoptotic Bcl-2 family members Bak and Bax play central and redundant roles in the regulation of apoptosis. In this study, we investigated the effect of loss of Bax and Bak in the CNS. The adult bax-/-bak-/- mice display masses of densely staining cells in the proliferative zones of the brain. These cells are shown to be a mix of neural progenitor cells and postmitotic cells at different stages of neural and glial differentiation. Both neural progenitor cells and mature neurons derived from bax-/-bak-/- mice were resistant to various apoptotic stimuli. Despite this resistance, postmitotic mature bax-/-bak-/- neurons remain as sensitive to excitoxic death as wild-type neurons. Thus, Bax and Bak play a critical role in regulating the number of neural progenitor cells in the adult brain but are not absolutely required for the initiation of neuronal cell death after neurotoxic injury.

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Figures

Figure 1.

Figure 1.

Abnormal cells in the brains of adult _bax_-/-_bak_-/- mice are found in multiple areas. Hematoxylin and eosin-stained coronal sections at the level of the basal ganglia (BG) and hippocampus (hipp) from bax+/-bak+/- (A, E), bax+/+_bak_-/- (C), _bax_-/-bak+/+ (D), and _bax_-/-_bak_-/- (B, F) mice. Representative sections are shown. The surface area of the SVZ (*) and cell counts in the corpus callosum (co.ca.) were determined as outlined in Material and Methods and are shown in Results. E and F also include white matter (wm) and cerebral cortex (cc). Scale bar, 150 μm.

Figure 2.

Figure 2.

Phenotypic features of excess cells seen in the _bax_-/-_bak_-/- compared with wild-type mouse brains. Cells in the SVZ were phenotyped by immunohistochemistry. In the _bax_-/-_bak_-/- SVZ, 16.9 ± 3.1% of the cells labeled with the neuronal marker NeuN, labeling immature and mature neurons, whereas 11.2 ± 3.1% of the cells expressed the low molecular weight neurofilament proteins (NFP). Among the small immature cells, 13.4 ± 1.3% cells showed astrocytic differentiation (GFAP labeling). A total of 13.7 ± 2.8% NeuN-positive, 7.3 ± 1.3% NFP-positive, and 3.1 ± 0.5% GFAP-positive cells were present in the SVZ of wild-type mice. Immunofluorescence was used to demonstrate the expression of the intermediate filament nestin found in immature neurons within the SVZ (red; nuclear counter stain with DAPI, blue). Scale bar, 75 μm.

Figure 3.

Figure 3.

Cell proliferation occurs in the SVZ of the adult _bax_-/-_bak_-/- brain. After 12 daily BrdU injections, mice were killed 1 d (acute), 12 d, or 4 weeks later. Histological sections were stained with anti-BrdU (green) and with an anti-histone-3 antibody (red). BrdU-incorporating cells were observed in the _bax_-/-_bak_-/- mice at increased numbers when compared with the wild-type mice (for quantitation, see Results). In addition, the _bax_-/-_bak_-/- mice show a relatively constant (over time) number of cells in the cell cycle as visualized by antibody to acetylated histone-3. Scale bar, 150 μm.

Figure 4.

Figure 4.

SVZ cells show BrdU incorporation and labeling with neuronal markers. Dual-color fluorescent labeling of sections from bax+/+bak+/+ and _bax_-/-_bak_-/- mice showed many cells with BrdU incorporation (red nuclear label) and β-tubulin (green cytoplasmic label). Double-labeled cells (arrows in β-tubulin/BrdU images) are seen in both the wild-type and _bax_-/-_bak_-/- mice. Although more double-labeled cells are present in the _bax_-/-_bak_-/- mice, similar percentages of double-labeled cells were seen in the bax+/+bak+/+ and _bax_-/-_bak_-/- brains (62.5 vs 61.3%, respectively). No GFAP/BrdU double-labeled cells were seen in the sections from the bax+/+bak+/+ mice, and only a few were seen in the _bax_-/-_bak_-/- mice (6 per 100 μm2) (arrowhead identified GFAP labeling in a BrdU-positive nucleus identified by the arrow). Scale bar, 25 μm.

Figure 5.

Figure 5.

_bax_-/-_bak_-/- NPC cultures are able to differentiate along neural and glial lineages. NPC cultures were grown in the presence of N2 supplement, 1% FCS and FGF-2, in N2 supplement and 1% FCS, or in the presence of 10% FCS and BDNF. After 2 weeks in differentiation medium, cells were stained with antibodies against O1 (oligodendral marker), MAP2 (neural marker), and GFAP (astrocyte marker) (A), and quantitation is shown in B. Culture in N2, 1% FCS, and FGF-2 resulted in small cells with relatively short blunt processes that occasionally labeled with O1 but not with GFAP or MAP2. The morphology and O1 staining indicate that some of these cells are immature oligodendrocytes. In N2 and 1% FCS media, the cells become large and elaborate extensive processes. These cells show differentiation along the lines of mature neurons (MAP2 labeling) and mature oligodendrocytes (compare the complex morphology of the O1 labeled cells with N2 alone vs those with N2 and FGF) (B). No astrocytes were seen with the N2 media alone. FCS and BDNF, conditions known to differentiate astrocytes from NPCs, exhibit the expected result with large flat cells showing strong GFAP immunoreactivity. Under these conditions, no neurons or oligodendrocytes were observed. For percentages of differentiated cells from wild-type mice, see Results. Scale bar, 25 μm.

Figure 6.

Figure 6.

NPC cultures from _bax_-/-_bak_-/- mice are resistant to apoptotic stimuli. NPC cultures from _bax_-/-_bak_-/- and control genotypes were grown without N2 supplement, 1% FCS, and FGF-2 for 3 d (A), treated with staurosporine for 24 hr (B), or treated with etoposide for 24 hr (C). Cells were harvested, and viability was determined by trypan blue exclusion. The mean ± SD of triplicate samples are shown. GF, Growth factor.

Figure 7.

Figure 7.

Effects of long-term growth factor deprivation in _bax_-/-_bak_-/- NPC cultures. _bax_-/-_bak_-/- (filled squares) and wild-type (open squares) NPC cultures were grown to confluency in standard NPC medium and then deprived of N2 supplement, 1% FCS, and FGF-2 for 31 d. At day 31, growth factors were readded. Relative cell number compared with the number of cells present at the onset of the experiment (A) and cell size (B) are shown at indicated time points. The majority of the wild-type cells died within 1 week and was not available for additional sampling. The viability of the _bax_-/-_bak_-/- cells was 91% at day 31. A representative experiment is shown.

Figure 8.

Figure 8.

_bax_-/-_bak_-/- cerebellar granule cells are sensitive to NMDA-induced excitotoxic death. Cerebellar granule cell cultures were established from _bax_-/-_bak_-/- and control mice. Cells were treated with NMDA at indicated concentrations in the absence or presence of MK-801 (A) and treated with NMDA with or without MK-801 or thapsigargin (B; Thap). Viablity was determined by calcein AM-ethidium bromide staining. The mean ± SD of triplicates are shown from a representative experiment. c, No treatment control.

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