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Effects of p21 on adult hippocampal neuronal development after irradiation

Effects of p21 on adult hippocampal neuronal development after irradiation Inhibition of hippocampal neurogenesis is implicated in neurocognitive impairment after cranial irradiation. We recently demonstrated that disruption of neuronal development after DNA damage was regulated by p53. The cyclin- dependent kinase inhibitor 1 or p21, a downstream effector p53, mediates cell cycle arrest in response to DNA damage. There is evidence that p21 negatively regulates proliferation of neural progenitors (NPCs). Here we characterized the effects of p21 on disruption of neuronal development in the hippocampal dentate gyrus after irradiation. We irradiated young adult mice wild type (+/+) or knockout (−/−) of the Cdkn1a (p21) gene, and used different bromodeoxyuridine (BrdU) paradigms for cell fate mapping. The acute apoptotic response of NPCs in the subgranular zone of the dentate gyrus was independent of p21 after irradiation. In nonirradiated mice, p21 knockout resulted in an increase in neuroblast proliferation and neurogenesis. At 9 weeks after 5Gy, NPCs in the subgranular zone demonstrated increased p21 expression. Loss of newborn type-1 cells and disruption of hippocampal neurogenesis was evident at 9 weeks after irradiation, and these effects were independent of p21 genotype status. Within the developmental milestones of NPCs, irradiation resulted in loss of early intermediate NPCs (type-2a cells) in wild-type mice, whereas the principal effect of irradiation with p21 loss was culling of proliferating late intermediate (type-2b cells) and neuroblasts. These results suggest that p21 exerts differential effects on cell fate of NPCs after irradiation. p21 may serve to protect proliferating late NPCs but does not alter the ultimate inhibition of new neuron production after DNA damage. Introduction radiation . Neurogenesis is associated with hippocampal 5,6 Multipotent neural stem cells and/or neural progenitor function of learning and memory . Inhibition of neuro- cells (NPCs) are present in the adult mammalian central genesis is implicated in neurocognitive decline following nervous system. In the adult mammalian brain, the den- radiation treatment for brain tumors . How DNA damage tate gyrus of the hippocampus represents an area where following ionizing radiation leads to impaired neuronal NPCs continue to generate new neurons which become development in the adult hippocampus remains unclear . 1,2 integrated into the neuronal circuitry . In the commonly accepted model of hippocampal neu- Many physiologic conditions such as an enriched ronal development, radial glial-like cells or type-1 cells are environment and exercise have been reported to result in thought to be the neural stem cells .Theygiveriseto enhanced adult neurogenesis . Neuronal development in transient amplifying or intermediate NPCs (type-2a, type- the adult hippocampus is disrupted in various pathologic 2b, and type-3 cells) which differ by their potential for 1,2 8 conditions and brain injuries including after ionizing proliferation and increasing neuronal differentiation . NPCs in the adult mouse hippocampus are known to undergo apoptosis after irradiation , a response mediated by the 10,11 tumor suppressor p53 . Despite the absence of NPC Correspondence: C. S. Wong (shun.wong@sunnybrook.ca) Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, apoptosis, p53 loss resulted in increased ablation of new- Canada born type-1 cells and profound inhibition of adult neuro- Departments of Radiation Oncology and Medical Biophysics, University of genesis after irradiation . Activation of p53 after irradiation Toronto, Toronto, ON, Canada Edited by A. Rufini © 2018 The Author(s). 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Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Li and Wong Cell Death Discovery (2019) 5:15 Page 2 of 12 results in upregulation of its downstream effector, the Expression of p21 is increased in neural progenitors cyclin-dependent kinase inhibitor 1 or p21. There is evi- after irradiation dence that p21 negatively regulates NPC proliferation . Irradiation is known to result in upregulation of p21 in Here we asked whether p21 might play a role in disruption mouse huppocampus . In nonirradiated dentate gyrus, of hippocampal neuronal development after irradiation. there was no evidence of p21 immunoreactivity. At Using mice wild type (+/+)or knockout(−/−)of the p21 9 weeks after 5 Gy, we observed a generalized increase in gene, p21 was found to have differential effects on cell fate p21 nuclear immunoreactivity in the granular cell layer of of NPCs, and specifically on disruption of the intermediate dentate gyrus (Fig. 2a). Since cells in the subgranular zone NPC stages of neuronal development after irradiation. Loss also expressed p21 immunoreactivity, sections were of p21 however did not alter the extent of inhibition of stained for phenotypic markers of NPCs to determine if production of new neurons after irradiation. there was differential expression of p21 in NPCs after irradiation. Among the nestin+/GFAP+ type-1 cells Results (Fig. 2b), 10.1 ± 2.0% demonstrated p21 nuclear immu- Apoptosis of neural progenitors after irradiation is nostaining after 5 Gy, and 12.0 ± 3.8% of p21+ subganular independent of p21 cells were nestin+/GFAP+. Within hours after irradiation, there is a robust p53- We used immunohistochemistry for T-box transcription mediated apoptotic response of NPCs in the subgranular factor (Tbr2) and DCX to further sort intermediate NPCs zone of the dentate gyrus . Two apoptosis radiosensitive into type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+), and NPC subpopulations, proliferating type-2 cells and non- type-3 (Tbr2−/DCX+) cells. Of the p21+ subgranular cells, proliferating neuroblasts (type-3 cells) have been descri- 9.2 ± 1.6% were Tbr2+, and 10.4 ± 3.7% were DCX+.Of bed .We first determined whether p21, a downstream the type-2a, type-2b, and type-3 cells, 32.5 ± 10.3%, 56.7 ± effector of p53, plays a role in radiation-induced apopto- 3.3%, and 14.3 ± 6% respectively demonstrated p21 sis. Using nonbiased stereology, we compared the number immunoreactivity after irradiation (Fig. 2c). of apoptotic cells at 8 h, the peak apoptotic response after 5Gy in the dentate gyrus of p21−/− mice with p21+/+ Loss of p21 does not alter inhibition of newborn neurons mice. Apoptotic cells with characteristic morphology on after irradiation 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining To determine whether p21 plays a role in inhibition of and positive (+) for terminal deoxynucleotidyl transferase hippocampal neurogenesis after irradiation, we compared dUTP nick-end labeling (TUNEL) or caspase-3 (Fig. 1a, b) the number of neuroblasts and newborn neurons in p21 were readily observed in the subgranular zone of both +/+ mice and p21−/− mice after 0 or 5 Gy (Fig. 3) using irradiated p21+/+ mice and p21−/− mice. Using dou- stereologic estimates. BrdU (50 mg/kg) was given daily for blecortin (DCX) to label neuroblasts, we observed no 7 consecutive days at 4 weeks after cranial irradiation. The difference in the number of TUNEL+/DCX+ and caspase- number of DCX+ cells and BrdU-labeled NeuN+ cells 3+/DCX+ cells in p21−/− mice compared to p21+/+ were determined at 9 weeks after irradiation or at 4 weeks mice at 8 h after irradiation (Fig. 1c). after the last BrdU injection. Ki67 was used as a marker Since only a fraction of apoptotic cells expressed nestin, for proliferating cells. Compared to controls, there was an a marker of early NPCs , we determined loss of pro- apparent loss of DCX+ cells in both p21+/+ mice and liferating type-2 cells at 24 h to provide evidence for p21−/− mice after irradiation consistent with inhibition apoptosis in this apoptotic-sensitive NPC subpopula- of neurogenesis (Fig. 3a). Regardless of p21 genotype, tion . Bromodeoxyuridine (BrdU, 50 mg/kg) was given irradiation resulted in loss of total Ki67+ cells (irradia- every 2 h for four doses, and animals were given a single tion, p < 0.001; p21 genotype, p = not significant; two-way dose of 5 Gy immediately after the final BrdU injection. ANOVA; Fig. 3b, d) and BrdU+ cells (irradiation, p < 0.01; No change in the number of BrdU+/GFAP+/nestin+ p21 genotype, p = not significant; Fig. 3c, e). Consistent cells with characteristic radial glia morphology (type- with the negative effects of p21 on neurogenesis ,an 1 cells) in irradiated p21+/+ mice and p21−/− mice at increase in DCX+ cells and BrdU+/NeuN+ cells was 24 h was observed compared to their respective 0-Gy observed in control nonirradiated p21−/− mice com- controls (Fig. 1d). A marked loss of BrdU+/nestin+ but pared to wild-type mice. Irradiation resulted in loss of GFAP-negative (−) cells or proliferating type-2 cells was DCX cells (irradiation, p < 0.001; p21 genotype, p = not observed in irradiated p21+/+ mice and p21−/− mice significant; Fig. 3f), Ki67+/DCX+ cells (irradiation, p < after 5 Gy compared to 0-Gy genotype controls (irradia- 0.01; p21 genotype, p = not significant; Fig. 3g), and tion, p = 0.001; p21 genotype, p = 0.8; two-way ANOVA; BrdU+/NeuN+ cells (irradiation, p < 0.01; p21 genotype, Fig. 1e). These results are consistent with the lack of a p = not significant; two-way ANOVA; Fig. 3h) indepen- role for p21 in the apoptotic response of NPCs after dent of the p21 genotype. Taken together, although p21 irradiation. loss increases neurogenesis in the absence of irradiation, it Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 3 of 12 TUNEL DCX DAPI Merged 5 µm b Caspase-3 DCX DAPI Merged 10 µm p21+/+ p21−/− TUNEL Caspase-3 de 120 4000 0 Gy 0 Gy 5 Gy 5 Gy 0 0 p21+/+ p21−/− p21+/+ p21−/− Fig. 1 Irradiation induces apoptosis of neuronal progenitors in the subgranular zone of dentate gyrus independent of p21. a A TUNEL+ cell (arrow, green) is DCX+ (red, arrow) and demonstrates characteristic nuclear condensation (DAPI, blue). b A caspase-3+ cell (arrow, red) shows perinuclear DCX immunoreactivity (green) and nuclear condensation on DAPI (blue). c There is no difference in the number of DCX+ apoptotic cells labeled by TUNEL or caspase-3 at 8 h after 5 Gy between wild-type (+/+) mice and p21−/− mice. d At 24 h after 5 Gy, there is no loss of BrdU +/nestin+/GFAP+ cells in dentate gyrus of p21+/+ mice and p21−/− mice. e A marked reduction in BrdU+/nestin+/GFAP− cells is noted at 24 h after 5 Gy regardless of p21 genotype. BrdU (50 mg/kg) was given every 2 h for four doses, and animals were irradiated with a single dose of 0 or 5 Gy immediately after the final BrdU injection. Data are represented as mean ± SEM and analyzed using two-way ANOVA, †p < 0.01, §p < 0.001, post hoc Bonferroni comparison with 3−4 mice per experimental group does not have an effect on inhibition of neurogensis after estimated by stereology to determine the effects of p21 on irradiation. proliferating type-1 cells after irradiation. In nonirradiated mice, p21 loss did not appear to alter the total number of Loss of p21 does not alter loss of neural stem cells type-1 cells, number of BrdU+ type-1 cells, and number of after irradiation Ki67+ type-1 cells. A 5 Gy dose resulted in loss of BrdU+ We next asked if p21 loss might alter the ablation of type-1 and Ki67+ type-1 cells but the p21 genotype did type-1 cells after irradiation. The same 7-day BrdU para- not have an effect on loss of these neural stem digm was used to identify newborn type-1 cells (Fig. 4a). cell populations after irradiation (BrdU+ type-1 cells: The number of Ki67-labeled type-1 cells (Fig. 4b) was irradiation, p < 0.001; p21 genotype, p = not significant; Official journal of the Cell Death Differentiation Association No. of BrdU+/nestin+/GFAP+ cells No. of DCX+ apoptotic cells No. of BrdU+/nestin+/GFAP− cells Li and Wong Cell Death Discovery (2019) 5:15 Page 4 of 12 a p21 DAPI 0 Gy 20 µm 5 Gy 40 µm GFAP p21 DAPI Merged 10 µm Nestin Tbr2 DCX p21 DAPI Merged 10 µm Fig. 2 Irradiation results in upregulation of p21 in neural progenitors (NPCs) in dentate gyrus. a There is absence of p21 immunoreactivity in nonirradiated dentate gyrus whereas p21 nuclear immunostaining is apparent in both granule and subgranular cells at 9 weeks after 5 Gy (p21, red; DAPI, blue). b A type-1 cell (arrow) demonstrates p21 nuclear immunoreactivity after irradiation (nestin, green; GFAP, white; p21, red; DAPI, blue). c Some type-2a (Tbr2+/DCX−, arrowhead), type-2b (arrows, Tbr2+/DCX+), and type-3 neural progenitors (open arrow, Tbr2−/DCX+) demonstrate p21 nuclear immunoreactivity (Tbr2, green; DCX, white; p21, red; DAPI, blue) after irradiation Ki67+ type-1 cells: Ki67+ type-1 cells: irradiation, controls, p21 loss was associated with an increase in total p < 0.0001; p21 genotype, p = not significant; two-way BrdU+ cells (p21 genotype, p < 0.05; time after BrdU, p ANOVA, Fig. 4c, d). < 0.0001; interaction, p = not significant; two-way ANOVA; Fig. 6a), BrdU+ type-1 cells (p21 genotype, p p21 deficiency results in differential ablation of < 0.05; time after BrdU, p < 0.0001; interaction, p = not proliferative NPCs after irradiation significant; Fig. 6c) and BrdU+ type-3 cells (p21 genotype, To determine the influence of p21 on cell fate of NPCs, p < 0.001; time after BrdU, p < 0.0001; interaction, p < mice were given a single injection of BrdU (150 mg/kg) at 0.01; Fig. 6i), but not type-2a (p21 genotype, p = not 4 weeks after 0 or 5 Gy, and BrdU-labeled NPC sub- significant; time after BrdU, p < 0.01; Fig. 6e) and type-2b populations were quantified at 2 h, 2 days, 1, and 5 weeks cells (p21 genotype, p = not significant; time after BrdU, after BrdU. Using this BrdU paradigm, cells that p < 0.01; Fig. 6g), compared to p21+/+ mice. demonstrated BrdU incorporation at 2 h were primarily A dose of 5 Gy resulted in loss of total BrdU+ cells in proliferating cells. BrdU+ cells at 2 days represented a both p21+/+ mice and p21−/− mice (p21+/+ mice: blend of proliferating and newborn cells, and those at irradiation, p < 0.05; time after BrdU, p < 0.001; interaction, 1 week and 5 weeks after BrdU were expected to be p = not significant; p21−/− mice: irradiation, p < 0.001; mostly newborn cells. Tbr2 and DCX immunohis- time after BrdU, p < 0.0001; interaction, p = 0.01; Fig. 6a, b tochemistry was used to differentiate BrdU+ inter- and Supplementary Fig. 1a, b). Among the different NPC mediate NPCs into type-2a (Tbr2+/DCX−), type-2b subpopulations in p21+/+ mice, there was loss of BrdU+ (Tbr2+/DCX+), and type-3 (Tbr2−/DCX+) cells type-2a cells (irradiation, p < 0.05; time after BrdU, p < (Fig. 5a). 0.05; interaction, p = not significant; Fig. 6e, f and Sup- The number of BrdU-labeled cells declined over the plementary Fig. 1e) but not BrdU+ type-1 (irradiation, p 5 weeks after BrdU injection in nonirradiated mice and = not significant; time after BrdU, p < 0.01; Fig. 6c, d irradiated mice regardless of genotype (Fig. 6). In 0-Gy and Supplementary Fig. 1c), BrdU+ type-2b (irradiation, Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 5 of 12 a 0 Gy 5 Gy DCX/DAPI p21+/+ 100 µm p21−/− Ki67 DCX DAPI Merged 20 µm Merged BrdU NeuN DAPI 10 µm d e 0 Gy 2000 0 Gy 1000 5 Gy 5 Gy † 1500 750 500 250 0 0 p21+/+ p21−/− p21+/+ p21−/− f h 0 Gy 1000 1000 0 Gy 0 Gy 5 Gy 5 Gy 5 Gy 0 0 0 p21+/+ p21−/− p21+/+ p21−/− p21+/+ p21−/− Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association No. of DCX+ cells No. of Ki67+ cells No. of Ki67+/DCX+ cells No. of BrdU+ cells No. of BrdU+/NeuN+ cells Li and Wong Cell Death Discovery (2019) 5:15 Page 6 of 12 (see figure on previous page) Fig. 3 Inhibition of neurogenesis after irradiation is independent of p21. a Loss of DCX+ cells in dentate gyrus is apparent in both p21+/+mice and p21−/− mice at 9 weeks after irradiation (DCX, green; DAPI, blue). b A proliferating neuroblast (arrow) demonstrates dual Ki67/DCX staining (Ki67, red; DCX, green; DAPI, blue). c A newborn neuron (arrow) demonstrates dual BrdU/NeuN immunoreactivity (BrdU, green; NeuN, red; DAPI, blue). d, e At 9 weeks after 5 Gy, there is loss of Ki67+ and BrdU+ cells in dentate gyrus independent of p21 genotype. f−h Absence of p21 results in an increase in DCX+ and BrdU+/NeuN+ cells in nonirradiated controls, but loss of DCX+ (f), Ki67+/DCX+ (g) and BrdU+/NeuN+ (h) cells after irradiation is independent of p21 genotype. BrdU (50 mg/kg/day × 7 consecutive days) was given at 4 weeks and animals were killed at 9 weeks after 0 or 5 Gy. Data are represented as mean ± SEM and analyzed using two-way ANOVA, *p < 0.05, †p < 0.01, §p < 0.001, post hoc Bonferroni test with 3-4 mice per genotype per time point p = not significant; time after BrdU, p = not significant; decreased type-2a cell proliferation (p21 genotype, p < Fig. 6g, h and Supplementary Fig. 1g) and BrdU+ type-3 0.05, time after BrdU, p < 0.01, interaction, p = not sig- cells (irradiation, p = not significant; time after BrdU, p = nificant; Fig. 7a, b) but increased type-3 cell proliferation not significant; Fig. 6i, j and Supplementary Fig. 1i) after or enhanced differentiation into type-3 cells (p21 geno- 5 Gy. type, p < 0.05, time after BrdU, p < 0.0001, interaction, p < In p21−/− mice after irradiation, there was loss of 0.01; Fig. 7a, b). After irradiation, the effect of p21 loss different BrdU+ NPC subpopulations, namely loss of resulted in increased % BrdU+ type-2a cells (p21 geno- BrdU+ type-1 cells (irradiation, p < 0.01; time after BrdU, type, p < 0.01, time after BrdU, p < 0.0001, interaction, p < p < 0.001; interaction, p < 0.05; Fig. 6c, d and Supple- 0.01) but decreased % BrdU+ type-2b cells (p21 genotype, mentary Fig. 1d), BrdU+ type-2b cells (irradiation, p < p < 0.01, time after BrdU, p < 0.01, interaction, p = not 0.0001, time after BrdU, p < 0.001; interaction, p = 0.01; significant), and % BrdU+ type-3 cells appeared unper- Fig. 6g, h and Supplementary Fig. 1h) and BrdU+ type-3 turbed (p21 genotype, p = not significant, time after BrdU, cells (irradiation, p < 0.0001; time after BrdU, p < 0.0001; p < 0.0001; Fig. 7c, d). These results again demonstrate the interaction, p < 0.0001; Fig. 6i, j and Supplementary differential effects of p21 on neuronal development within Fig. 1j) but not type-2a cells (irradiation, p = not sig- the three stages of intermediate NPC development fol- nificant; time after BrdU, p < 0.05; Fig. 6e, f and Supple- lowing irradiation. We were unable to detect any mentary Fig. 1f). In p21+/+ mice, BrdU+ type-2b cells at BrdU+/Tbr2+ cells at 5 weeks consistent with culling of 2 h and 2 days were reduced after 5 Gy to 50.0 ± 8.3% and type-2 cells due to death and/or differentiation over a few 12,17 23.9 ± 20.3% respectively compared to controls whereas weeks after BrdU incorporation . they were markedly reduced to 1.8 ± 1.8 and 4.7 ± 2.3% To further assess the role of p21 on inhibition of NPC respectively in p21−/− mice after irradiation (Supple- proliferation after irradiation, we determined the number mentary Fig. 1a-j). of BrdU doublets, type-1 (GFAP+/nestin+) BrdU doub- After irradiation, the effect of p21 genotype demon- lets (Fig. 5b) and type-2 (Tbr2+) BrdU doublets (Fig. 5c) strated a significant independent effect on the total at 2 h after BrdU. A BrdU doublet was defined as two number of BrdU+ cells (time after BrdU, p < 0.0001; p21 abutting DAPI-stained nuclei that demonstrated BrdU genotype, p < 0.01; interaction, p < 0.001; Fig. 6b), and incorporation . The number of type-1 BrdU doublets type-2b cells (time after BrdU, p < 0.001; p21 genotype, p increased in both nonirradiated and irradiated p21−/− < 0.001; interaction, p < 0.01; Fig. 6h), but not BrdU+ mice compared to wild-type controls, and irradiation did type-1 (time after BrdU, p < 0.05; p21 genotype, p = not not alter their numbers (irradiation, p = not significant; significant; Fig. 6d), BrdU+ type-2a (time after BrdU, p < p21 genotype, p = 0.001, two-way ANOVA; Supplemen- 0.0001; p21 genotype, p = not significant; Fig. 6f) or BrdU tary Fig. 2). In contrast, the number of type-2 BrdU type-3 cells (time after BrdU, p = not significant; p21 doublets remained altered with p21 loss and a 5 Gy-dose genotype, p = not significant; Fig. 6j). reduced the number of type-2 BrdU doublets in both p21 To further discern the influence of p21 genotype on +/+ mice and p21−/− mice (irradiation, p < 0.001; p21 neuronal development within the stages of transiently genotype, p = not significant). amplifying or intermediate NPCs after irradiation, we determined the distribution of BrdU+ type-2a, type-2b, Discussion and type-3 cells among all BrdU+/Tbr2+ and BrdU Inhibition of hippocampal neurogenesis is implicated in +/DCX+ cells (Fig. 7). In nonirradiated wild-type mice, neurocognitive decline following radiation treatment for the results are consistent with increasing neuronal dif- brain tumors . How DNA damage following ionizing ferentiation with progression through type-2a, type-2b, radiation results in disruption of neuronal development and type-3 cells over the 5 weeks after BrdU (Fig. 7a). remains unclear. The tumor suppressor p53 is known to With p21 loss in nonirradiated mice, there appeared to regulate cellular response after DNA damage .It Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 7 of 12 negatively regulates proliferation and self-renewal of irradiated hippocampus was not altered by p53 genotype 20–22 12 neural stem cells . We recently showed that p53 loss of recipient mice . Thus the effect of p53 on disruption resulted in increased ablation of neural stem cells and of neuronal development after irradiation is likely to be profound inhibition of neurogenesis in mouse dentate independent of damage of the neural stem cell niche. gyrus after irradiation despite lack of NPC apoptosis . How p53 regulates DNA damage response in NPCs Damage of the neurovascular niche plays a role in remains unclear. A well-described downstream effector of 23–25 impaired neurogenesis after irradiation . Impaired p53 is p21. In young adult mouse brain, p21 loss is neuronal differentiation of NPCs transplanted into the associated with increased NPC proliferation but BrdU Nestin GFAP Merged 20 µm Ki67 Nestin GFAP Merged 10 µm 100 100 800 0 Gy 0 Gy 0 Gy 5 Gy 5 Gy 5 Gy 80 80 60 60 * * 40 40 20 20 0 0 p21+/+ p21−/− p21+/+ p21−/− p21+/+ p21−/− 160 160 0 Gy 160 0 Gy 0 Gy 5 Gy 5 Gy 5 Gy 140 140 140 † † † 120 120 120 * * 100 100 100 80 80 80 60 60 60 40 40 40 20 20 0 0 p21+/+ p21−/− p21+/+ p21−/− p21+/+ p21−/− Fig. 4 Irradiation results in depletion of proliferating and newborn type-1 cells independent of p21. a A newborn type-1 cell (arrow) with characteristic morphology in subgranular zone demonstrates BrdU incorporation (green), and nestin (red) and GFAP immunoreactivity (white). b A proliferating type-1 cell (arrow) is Ki67+ (green), nestin (red) and GFAP+ (white). An arrowhead shows a Ki67+ cell which is negative for nestin and GFAP. c, d Following a dose of 5 Gy, there is loss of newborn (BrdU+) and proliferating (Ki67+) type-1 cells independent of p21 genotype, and the percent reduction in total, newborn and proliferating type-1 cells after 5 Gy shown in (d). BrdU (50 mg/kg/day × 7 consecutive days) was given at 4 weeks and animals were killed at 9 weeks after irradiation. Data are represented as mean ± SEM and analyzed using two-way ANOVA, *p < 0.05, †p < 0.01, post hoc Bonferroni test, with 3-4 mice per genotype per time point Official journal of the Cell Death Differentiation Association No. of type-1 cells No. of type-1 cells (%) No. of BrdU+ type-1 cells (%) No. of BrdU+ type-1 cells No. of Ki67+ type-1 cells (%) No. of Ki67+ type-1 cells Li and Wong Cell Death Discovery (2019) 5:15 Page 8 of 12 a BrdU Tbr2 DCX Merged Type-2a 5 µm Type-2b 5 µm Type-3 5 µm BrdU Nestin GFAP Merged 20 µm BrdU Tbr2 Merged 10 µm Fig. 5 Pulsed BrdU injections label intermediate NPCs. a Immunohistochemistry for Tbr2 and DCX is used to sort BrdU-labeled intermediate NPCs (arrows) into type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+) and type-3 cells (BrdU, green; Tbr2, red; DCX, white). b, c Some BrdU+NPCs are seen as BrdU doublets, defined as two abutting BrdU+ nuclei identified on DAPI. A type-1 BrdU doublet (b, arrow; BrdU, green) is nestin+ (red) and GFAP+ (white), and a type-2 BrdU doublet is Tbr2+ (c, arrows, BrdU, green; Tbr2, red) neurospheres from p21−/− mice demonstrated limited of apoptosis . Neural stem cells are known to be apop- 13 14 in vitro self-renewal . It has been proposed that tosis resistant after irradiation . Here, irradiation resulted p21 serves to keep adult neural stem cells in relative in an increase in p21 immunoreactivity in type-1 cells and quiescence for life-long maintenance of self-renewal. intermediate NPCs. There was no evidence of acquisition Here, p21 loss was associated with increased cell pro- of apoptosis radiosensitivity in neural stem cells with p21 liferation in the dentate gyrus, including enhanced pro- loss, and we also observed no altered apoptosis radio- liferation of type-1 cells and neuroblasts. We also sensitivity in proliferating type-2 cells and neuroblasts in observed an increase in newborn neurons, consistent with the absence of p21. In irradiated p21−/− embryonic an inhibitory effect of p21 on neurogenesis in the dentate mouse cortex, there was decreased not increased apop- gyrus. tosis . Whether these differences in p21-dependent Stem cells may have distinct mechanisms from pro- responses post-DNA damage are related to differences genitors to mitigate DNA damage to preserve self-renewal in tissue-specific stem cell radiobiology, developmental or and differentiation . Adult stem cells are known to be in vitro conditions, remain to be investigated. resistant to apoptosis after irradiation. p21 may play a role Neural stem cells and NPCs are known to exhibit dif- in the regulation of DNA damage response of stem cells . ferential responses to physiologic stresses and external In hematopoietic and mammary stem cells in vitro, irra- insults . Our results here provide evidence for differential diation was shown to result in activation of p21 inde- effects of p21 on NPC cell fate after irradiation. In wild- pendent of p53, and subsequent inhibition of basal activity type mice, the predominant effect of irradiation appears to of p53. This p21-dependent response in stem cells after be death of type-2a cells and/or inhibition of their pro- DNA damage has been postulated to result in inhibition liferation. In contrast, the major effect of irradiation with Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 9 of 12 Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 10 of 12 (see figure on previous page) Fig. 6 Loss of p21 results in differential ablation of NPCs after irradiation. a The number of BrdU+ cells in nonirradiated p21−/− mice is increased compared to nonirradiated p21+/+ mice (p21 genotype, p < 0.05). b Loss of BrdU+ cells after irradiation is p21 genotype dependent (p21 genotype, p < 0.01). c, d For BrdU+type-1 (nestin+/GFAP+) cells, their decline over time is p21 dependent in nonirradiated mice (c, p21 genotype, p < 0.05) but not after irradiation (d). e, f The number of BrdU+type-2a (Tbr2+/DCX−) cells over time after BrdU is independent of p21 in nonirradiated (e) and irradiated mice (f). g, h The decline in BrdU+type-2b (Tbr2+/DCX+) cells after BrdU in nonirradiated mice is independent of p21 (g) whereas there is a marked loss of BrdU+ type-2b cells in p21−/− mice after 5 Gy compared to wild-type animals (h, p21 genotype, p < 0.001). i, j BrdU+ type-3 (Tbr2−/DCX+) cells are increased in nonirradiated p21−/− mice compared to wild-type mice (i, p21 genotype, p< 0.001), but their loss after irradiation (j)is p21 genotype independent. A single dose of BrdU (150 mg/kg) was given at 4 weeks after 0 or 5 Gy. Cells were counted at 2 h, 2 days, 1 and 5 weeks after BrdU. Data are represented as mean ± SEM and analyzed using two-way ANOVA, *p < 0.05, †p < 0.01, §p < 0.001, post hoc Bonferroni test with 3−5 mice per genotype per time point ab p21+/+, 0 Gy p21−/−, 0 Gy 100 100 80 80 60 60 40 40 20 20 0 0 2 h 2 days 1 week 5 weeks 2 h 2 days 1 week 5 weeks Type-2a Type-2b Type-3 p21+/+, 5 Gy p21−/−, 5 Gy 80 80 60 60 40 40 20 20 0 0 2 h 2 days 1 week 5 weeks 2 h 2 days 1 week 5 weeks Fig. 7 Loss of p21 alters disruption of neuronal progression over the three stages of intermediate NPCs after irradiation. a−d The distribution of BrdU+ type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+) and type-3 (Tbr2−/DCX+) cells as percent total intermediate NPCs (Tbr2+or DCX+) is disrupted with p21 loss and after irradiation. A single dose of BrdU (150 mg/kg) was given at 4 weeks after 0 or 5 Gy. Data are represented as mean ± SEM with 3−5 mice per genotype per time point p21 loss was increased culling of proliferative type-1, type- loss of about 50% of newborn neurons and was con- 2b, and type-3 NPCs. There is in vitro evidence that sidered optimal to discern the effect of genotype on activation of p21 in certain stem cells results in induction inhibition of neurogenesis after irradiation. Although our of symmetric self-renewing divisions after irradiation . results appeared to internally consistent, no BrdU+ type- Although we did not address self-renewal directly, the 2 cells were observed at 5 weeks using the single BrdU absence of any increase in type-1 cells at 2 days compared injection paradigm. Further information may be obtained to at 2 h after BrdU provided no evidence to suggest using other fate mapping methods and additional sets of induced symmetrical division of type-1 cells after irra- phenotypic markers for NPCs. diation. We also did not observe any p21-dependent In conclusion, there is increased hippocampal neuro- effects on type-1 cell fate after irradiation. genesis associated with p21 loss which may be due to Several methods including thymidine analogs, retro- enhanced NPC proliferation and/or neuronal differentia- virus, and transgenic animals have been used to study tion particularly in the latter stages of intermediate NPC 1,31 adult hippocampal neurogensis . These different fate development. Irradiation results in inhibition of neuro- mapping approaches may contribute to the various genesis and loss of newborn neural stem cells. These models of NPC behavior proposed. Here we used BrdU effects appear to be independent of p21. In wild-type injection paradigms to assess neuronal development. We mice, the most pronounced effects of irradiation is death chose a dose of 5 Gy in our study as this dose resulted in and/or inhibition of proliferation of type-2a cells. In Official journal of the Cell Death Differentiation Association Percent intermediate NPC Percent intermediate NPC Percent intermediate NPC Percent intermediate NPC Li and Wong Cell Death Discovery (2019) 5:15 Page 11 of 12 contrast, there is increased ablation of proliferative type-1, NPCs, immature and mature neurons were identified by type-2b, and type-3 NPCs after irradiation with p21 loss. phenotypic markers using immunohistochemistry . Pri- Our results are thus consistent with differential effects of mary antibodies included those against DCX (1:2000, p21 on NPC cell fate after irradiation. Loss of p21 how- Abcam, Cambridge, MA, USA; Cat# ab18723), GFAP ever does not alter the ultimate inhibition of production (1:200, DakoCytomation, Copenhagen, Denmark; Cat# of new neurons after irradiation. Z0334), nestin (1:200, Millipore, Billerica, MA, USA; Cat# MAB353), NeuN (1:500, Millipore; Cat# MAB377), and Materials and methods Tbr2 (1:200, Abcam; Cat# ab23345). Secondary antibodies Animals were conjugated to Cy2, Cy3 (1:200; Jackson ImmunoR- C57 mice wild type (+/+) or knockout of the p21 gene esearch, West Grove, PA, USA; Cat# 712225150, were obtained from the Jackson Laboratory (Bar Harbor, cat#712165150) or Alexa Fluor 647 (1:200, Invitrogen, ME, USA). Mouse colonies were generated by littermate Burlington, Ontario, Canada; Cat# A31573). Colocaliza- inbreeding and genotyped by PCR . To avoid the tion of phenotypic markers with BrdU (1:200, Abcam; potential confounding influence of sex on neurogenesis , Cat# ab6326) or Ki67 (1:1000, Novocastra, Newcastle, UK; only male mice were used in the study. All animal pro- Cat# NCL-Ki67p) in selected sections were evaluated cedures (protocol number: 18-156) were approved by the using a confocal laser scanning microscope (Zeiss institutional animal care committee and performed LSM700, Carl Zeiss AG Corporate, Oberkochen, Ger- according to the Canadian Council on Animal Care many). A BrdU doublet was defined as two abutting guidelines. DAPI-stained nuclei that demonstrated BrdU incorporation . Irradiation Animals were irradiated at the age of 10 weeks. They Stereological analysis were anesthetized using an intraperitoneal injection of Apoptotic cells and cells labeled using different phe- ketamine (75 mg/kg) and xylazine (6 mg/kg) and posi- notypic markers were counted within the dentate gyrus tioned in a customized jig with the entire hippocampus including a 50-µm margin of the hilus . Cell counting irradiated using an anterior-posterior and posterior- was performed using a Zeiss Imager M1 microscope (Carl anterior pair of 160 kV X-ray beam (Model CP160, Faxi- Zeiss AG Corporate) with the Stereo Investigator software tron X-ray, Wheeling, IL, USA) defined by an 8-mm (MBF Bioscience, Williston, VT, USA). The observers diameter lead cut-out were blinded to the experimental groups. Details of ste- . Control (0 Gy) mice were sham- irradiated. A single dose of 5 Gy was used to evaluate the reology were as described previously . Apoptotic cells effects of the p21 genotype on neuronal development after and NPCs were counted using a counting frame and a irradiation. Three different BrdU incorporation schedules sampling grid of 75 × 75 µm at a magnification of ×630. were used for cell fate mapping as described in the Results Every seventh section was used as the periodicity of sec- section. BrdU was administered by intraperitoneal tions sampled. For all stereology data, the coefficient of injection. error ranged from 0.03 to 0.06. Histopathology and immunohistochemistry Statistical analysis Under anesthesia with ketamine and xylazine, mice Cell population analysis represented data from 3 to 5 underwent intracardiac perfusion with 0.9% saline fol- mice per genotype per dose per time point. All data lowed by 4% paraformaldehyde in PBS. The brain was represented the mean ± SEM. The significance of two dissected out, post-fixed for 2 days and cryoprotected in independent variables such as irradiation and p21-geno- PBS with 30% sucrose. Coronal sections containing the type, or p21-genotype and time after BrdU on the various hippocampus were cut at 40-μm thickness and stored in end-points was determined using two-way ANOVA. Pair- tissue cryoprotectant solution in 96-well plates at −20 °C wise comparisons were based on post hoc analysis with prior to immunohistochemistry. Bonferroni correction for multiple comparisons. Differ- Apoptotic cells were identified based on characteristic ences were considered significant for p < 0.05. Statistical nuclear condensation and fragmentation of apoptosis analyses were performed with the GraphPad Prism 5 11,17 using DAPI . The apoptotic response was further (GraphPad Software, La Jolla, CA, USA). characterized using TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA; Cat# 11684795910) and caspase-3 (1:1000; Cell Signaling Technology, Dan- Acknowledgements vers, MA, USA; Cat# 9661) immunohistochemistry as This work was supported by the Cancer Research Society and the Ontario previously described . Association of Radiation Oncologists (C.S.W.). Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 12 of 12 Authors’ contributions 13. Kippin, T. E.,Martens,D. J.&vander Kooy,D.p21 loss compromisesthe Conception and design: C.S.W.; Development of methodology: Y.-Q.L., C.S.W.; relative quiescence of forebrain stem cell proliferation leading to exhaustion of Acquisition of data: Y.-Q.L., C.S.W.; Analysis and interpretation of data: Y.-Q.L., their proliferation capacity. Genes Dev. 19,756–767 (2005). C.S.W.; Writing, review, and/or revision of the manuscript: Y.-Q.L., C.S.W.; 14. Li, Y. Q., Cheng, Z. & Wong, S. Differential apoptosis radiosensitivity of neural Administrative, technical, or material support: Y.-Q.L., C.S.W.; Study supervision: progenitors in adult mouse hippocampus. Int. J. Mol. Sci. 17, 970 (2016). C.S.W.; Other (oversight of every aspect of the research): C.S.W. 15. Cheng, Z., Zheng, Y. Z., Li, Y. Q. & Wong, C. S. Cellular senescence in mouse hippocampus after irradiation and the role of p53 and p21. J. Neuropath. Exp. Neurol. 76,260–269 (2017). Conflict of interest 16. Hodge, R. D. et al. Tbr2 is essential for hippocampal lineage progression from The authors declare that they have no conflict of interest. neural stem cells to intermediate progenitors and neurons. J. Neurosci. 32, 6275–6287 (2012). 17. Sierra, A. et al. Microglia shape adult hippocampal neurogenesis through Publisher's note apoptosis-coupled phagocytosis. Cell Stem Cell 7,483–495 (2010). 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Roles of p53 and p27(Kip1) in the regulation of neuro- genesis in the murine adult subventricular zone. Eur. J. Neurosci. 34,1040–1052 References (2011). 1. Bonaguidi, M. A., Song, J., Ming, G. L. & Song, H. A unifying hypothesis on 23. Monje,M.L., Mizumatsu, S.,Fike, J. R. & Palmer, T. D. Irradiation induces neural mammalian neural stem cell properties in the adult hippocampus. Curr. Opin. precursor-cell dysfunction. Nat. Med. 8,955–962 (2002). Neurobiol. 22,754–761 (2012). 24. Monje,M.L., Toda,H.&Palmer,T.D.Inflammatory blockade restores adult 2. Goncalves, J. T.,Schafer, S.T.&Gage,F.H.Adult neurogenesis in the hippo- hippocampal neurogenesis. Science 302, 1760–1765 (2003). campus:fromstemcells to behavior. Cell 167,897–914 (2016). 25. Belarbi,K., Jopson,T., Arellano,C., Fike, J.R. & Rosi, S.CCR2deficiency prevents 3. van Praag, H., Kempermann, G. & Gage, F. H. 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Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Effects of p21 on adult hippocampal neuronal development after irradiation

Cell Death Discovery , Volume 5 (1) – Jul 18, 2018

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Springer Journals
Copyright
Copyright © 2018 by The Author(s)
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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2058-7716
DOI
10.1038/s41420-018-0081-2
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Abstract

Inhibition of hippocampal neurogenesis is implicated in neurocognitive impairment after cranial irradiation. We recently demonstrated that disruption of neuronal development after DNA damage was regulated by p53. The cyclin- dependent kinase inhibitor 1 or p21, a downstream effector p53, mediates cell cycle arrest in response to DNA damage. There is evidence that p21 negatively regulates proliferation of neural progenitors (NPCs). Here we characterized the effects of p21 on disruption of neuronal development in the hippocampal dentate gyrus after irradiation. We irradiated young adult mice wild type (+/+) or knockout (−/−) of the Cdkn1a (p21) gene, and used different bromodeoxyuridine (BrdU) paradigms for cell fate mapping. The acute apoptotic response of NPCs in the subgranular zone of the dentate gyrus was independent of p21 after irradiation. In nonirradiated mice, p21 knockout resulted in an increase in neuroblast proliferation and neurogenesis. At 9 weeks after 5Gy, NPCs in the subgranular zone demonstrated increased p21 expression. Loss of newborn type-1 cells and disruption of hippocampal neurogenesis was evident at 9 weeks after irradiation, and these effects were independent of p21 genotype status. Within the developmental milestones of NPCs, irradiation resulted in loss of early intermediate NPCs (type-2a cells) in wild-type mice, whereas the principal effect of irradiation with p21 loss was culling of proliferating late intermediate (type-2b cells) and neuroblasts. These results suggest that p21 exerts differential effects on cell fate of NPCs after irradiation. p21 may serve to protect proliferating late NPCs but does not alter the ultimate inhibition of new neuron production after DNA damage. Introduction radiation . Neurogenesis is associated with hippocampal 5,6 Multipotent neural stem cells and/or neural progenitor function of learning and memory . Inhibition of neuro- cells (NPCs) are present in the adult mammalian central genesis is implicated in neurocognitive decline following nervous system. In the adult mammalian brain, the den- radiation treatment for brain tumors . How DNA damage tate gyrus of the hippocampus represents an area where following ionizing radiation leads to impaired neuronal NPCs continue to generate new neurons which become development in the adult hippocampus remains unclear . 1,2 integrated into the neuronal circuitry . In the commonly accepted model of hippocampal neu- Many physiologic conditions such as an enriched ronal development, radial glial-like cells or type-1 cells are environment and exercise have been reported to result in thought to be the neural stem cells .Theygiveriseto enhanced adult neurogenesis . Neuronal development in transient amplifying or intermediate NPCs (type-2a, type- the adult hippocampus is disrupted in various pathologic 2b, and type-3 cells) which differ by their potential for 1,2 8 conditions and brain injuries including after ionizing proliferation and increasing neuronal differentiation . NPCs in the adult mouse hippocampus are known to undergo apoptosis after irradiation , a response mediated by the 10,11 tumor suppressor p53 . Despite the absence of NPC Correspondence: C. S. Wong (shun.wong@sunnybrook.ca) Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, apoptosis, p53 loss resulted in increased ablation of new- Canada born type-1 cells and profound inhibition of adult neuro- Departments of Radiation Oncology and Medical Biophysics, University of genesis after irradiation . Activation of p53 after irradiation Toronto, Toronto, ON, Canada Edited by A. Rufini © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Li and Wong Cell Death Discovery (2019) 5:15 Page 2 of 12 results in upregulation of its downstream effector, the Expression of p21 is increased in neural progenitors cyclin-dependent kinase inhibitor 1 or p21. There is evi- after irradiation dence that p21 negatively regulates NPC proliferation . Irradiation is known to result in upregulation of p21 in Here we asked whether p21 might play a role in disruption mouse huppocampus . In nonirradiated dentate gyrus, of hippocampal neuronal development after irradiation. there was no evidence of p21 immunoreactivity. At Using mice wild type (+/+)or knockout(−/−)of the p21 9 weeks after 5 Gy, we observed a generalized increase in gene, p21 was found to have differential effects on cell fate p21 nuclear immunoreactivity in the granular cell layer of of NPCs, and specifically on disruption of the intermediate dentate gyrus (Fig. 2a). Since cells in the subgranular zone NPC stages of neuronal development after irradiation. Loss also expressed p21 immunoreactivity, sections were of p21 however did not alter the extent of inhibition of stained for phenotypic markers of NPCs to determine if production of new neurons after irradiation. there was differential expression of p21 in NPCs after irradiation. Among the nestin+/GFAP+ type-1 cells Results (Fig. 2b), 10.1 ± 2.0% demonstrated p21 nuclear immu- Apoptosis of neural progenitors after irradiation is nostaining after 5 Gy, and 12.0 ± 3.8% of p21+ subganular independent of p21 cells were nestin+/GFAP+. Within hours after irradiation, there is a robust p53- We used immunohistochemistry for T-box transcription mediated apoptotic response of NPCs in the subgranular factor (Tbr2) and DCX to further sort intermediate NPCs zone of the dentate gyrus . Two apoptosis radiosensitive into type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+), and NPC subpopulations, proliferating type-2 cells and non- type-3 (Tbr2−/DCX+) cells. Of the p21+ subgranular cells, proliferating neuroblasts (type-3 cells) have been descri- 9.2 ± 1.6% were Tbr2+, and 10.4 ± 3.7% were DCX+.Of bed .We first determined whether p21, a downstream the type-2a, type-2b, and type-3 cells, 32.5 ± 10.3%, 56.7 ± effector of p53, plays a role in radiation-induced apopto- 3.3%, and 14.3 ± 6% respectively demonstrated p21 sis. Using nonbiased stereology, we compared the number immunoreactivity after irradiation (Fig. 2c). of apoptotic cells at 8 h, the peak apoptotic response after 5Gy in the dentate gyrus of p21−/− mice with p21+/+ Loss of p21 does not alter inhibition of newborn neurons mice. Apoptotic cells with characteristic morphology on after irradiation 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining To determine whether p21 plays a role in inhibition of and positive (+) for terminal deoxynucleotidyl transferase hippocampal neurogenesis after irradiation, we compared dUTP nick-end labeling (TUNEL) or caspase-3 (Fig. 1a, b) the number of neuroblasts and newborn neurons in p21 were readily observed in the subgranular zone of both +/+ mice and p21−/− mice after 0 or 5 Gy (Fig. 3) using irradiated p21+/+ mice and p21−/− mice. Using dou- stereologic estimates. BrdU (50 mg/kg) was given daily for blecortin (DCX) to label neuroblasts, we observed no 7 consecutive days at 4 weeks after cranial irradiation. The difference in the number of TUNEL+/DCX+ and caspase- number of DCX+ cells and BrdU-labeled NeuN+ cells 3+/DCX+ cells in p21−/− mice compared to p21+/+ were determined at 9 weeks after irradiation or at 4 weeks mice at 8 h after irradiation (Fig. 1c). after the last BrdU injection. Ki67 was used as a marker Since only a fraction of apoptotic cells expressed nestin, for proliferating cells. Compared to controls, there was an a marker of early NPCs , we determined loss of pro- apparent loss of DCX+ cells in both p21+/+ mice and liferating type-2 cells at 24 h to provide evidence for p21−/− mice after irradiation consistent with inhibition apoptosis in this apoptotic-sensitive NPC subpopula- of neurogenesis (Fig. 3a). Regardless of p21 genotype, tion . Bromodeoxyuridine (BrdU, 50 mg/kg) was given irradiation resulted in loss of total Ki67+ cells (irradia- every 2 h for four doses, and animals were given a single tion, p < 0.001; p21 genotype, p = not significant; two-way dose of 5 Gy immediately after the final BrdU injection. ANOVA; Fig. 3b, d) and BrdU+ cells (irradiation, p < 0.01; No change in the number of BrdU+/GFAP+/nestin+ p21 genotype, p = not significant; Fig. 3c, e). Consistent cells with characteristic radial glia morphology (type- with the negative effects of p21 on neurogenesis ,an 1 cells) in irradiated p21+/+ mice and p21−/− mice at increase in DCX+ cells and BrdU+/NeuN+ cells was 24 h was observed compared to their respective 0-Gy observed in control nonirradiated p21−/− mice com- controls (Fig. 1d). A marked loss of BrdU+/nestin+ but pared to wild-type mice. Irradiation resulted in loss of GFAP-negative (−) cells or proliferating type-2 cells was DCX cells (irradiation, p < 0.001; p21 genotype, p = not observed in irradiated p21+/+ mice and p21−/− mice significant; Fig. 3f), Ki67+/DCX+ cells (irradiation, p < after 5 Gy compared to 0-Gy genotype controls (irradia- 0.01; p21 genotype, p = not significant; Fig. 3g), and tion, p = 0.001; p21 genotype, p = 0.8; two-way ANOVA; BrdU+/NeuN+ cells (irradiation, p < 0.01; p21 genotype, Fig. 1e). These results are consistent with the lack of a p = not significant; two-way ANOVA; Fig. 3h) indepen- role for p21 in the apoptotic response of NPCs after dent of the p21 genotype. Taken together, although p21 irradiation. loss increases neurogenesis in the absence of irradiation, it Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 3 of 12 TUNEL DCX DAPI Merged 5 µm b Caspase-3 DCX DAPI Merged 10 µm p21+/+ p21−/− TUNEL Caspase-3 de 120 4000 0 Gy 0 Gy 5 Gy 5 Gy 0 0 p21+/+ p21−/− p21+/+ p21−/− Fig. 1 Irradiation induces apoptosis of neuronal progenitors in the subgranular zone of dentate gyrus independent of p21. a A TUNEL+ cell (arrow, green) is DCX+ (red, arrow) and demonstrates characteristic nuclear condensation (DAPI, blue). b A caspase-3+ cell (arrow, red) shows perinuclear DCX immunoreactivity (green) and nuclear condensation on DAPI (blue). c There is no difference in the number of DCX+ apoptotic cells labeled by TUNEL or caspase-3 at 8 h after 5 Gy between wild-type (+/+) mice and p21−/− mice. d At 24 h after 5 Gy, there is no loss of BrdU +/nestin+/GFAP+ cells in dentate gyrus of p21+/+ mice and p21−/− mice. e A marked reduction in BrdU+/nestin+/GFAP− cells is noted at 24 h after 5 Gy regardless of p21 genotype. BrdU (50 mg/kg) was given every 2 h for four doses, and animals were irradiated with a single dose of 0 or 5 Gy immediately after the final BrdU injection. Data are represented as mean ± SEM and analyzed using two-way ANOVA, †p < 0.01, §p < 0.001, post hoc Bonferroni comparison with 3−4 mice per experimental group does not have an effect on inhibition of neurogensis after estimated by stereology to determine the effects of p21 on irradiation. proliferating type-1 cells after irradiation. In nonirradiated mice, p21 loss did not appear to alter the total number of Loss of p21 does not alter loss of neural stem cells type-1 cells, number of BrdU+ type-1 cells, and number of after irradiation Ki67+ type-1 cells. A 5 Gy dose resulted in loss of BrdU+ We next asked if p21 loss might alter the ablation of type-1 and Ki67+ type-1 cells but the p21 genotype did type-1 cells after irradiation. The same 7-day BrdU para- not have an effect on loss of these neural stem digm was used to identify newborn type-1 cells (Fig. 4a). cell populations after irradiation (BrdU+ type-1 cells: The number of Ki67-labeled type-1 cells (Fig. 4b) was irradiation, p < 0.001; p21 genotype, p = not significant; Official journal of the Cell Death Differentiation Association No. of BrdU+/nestin+/GFAP+ cells No. of DCX+ apoptotic cells No. of BrdU+/nestin+/GFAP− cells Li and Wong Cell Death Discovery (2019) 5:15 Page 4 of 12 a p21 DAPI 0 Gy 20 µm 5 Gy 40 µm GFAP p21 DAPI Merged 10 µm Nestin Tbr2 DCX p21 DAPI Merged 10 µm Fig. 2 Irradiation results in upregulation of p21 in neural progenitors (NPCs) in dentate gyrus. a There is absence of p21 immunoreactivity in nonirradiated dentate gyrus whereas p21 nuclear immunostaining is apparent in both granule and subgranular cells at 9 weeks after 5 Gy (p21, red; DAPI, blue). b A type-1 cell (arrow) demonstrates p21 nuclear immunoreactivity after irradiation (nestin, green; GFAP, white; p21, red; DAPI, blue). c Some type-2a (Tbr2+/DCX−, arrowhead), type-2b (arrows, Tbr2+/DCX+), and type-3 neural progenitors (open arrow, Tbr2−/DCX+) demonstrate p21 nuclear immunoreactivity (Tbr2, green; DCX, white; p21, red; DAPI, blue) after irradiation Ki67+ type-1 cells: Ki67+ type-1 cells: irradiation, controls, p21 loss was associated with an increase in total p < 0.0001; p21 genotype, p = not significant; two-way BrdU+ cells (p21 genotype, p < 0.05; time after BrdU, p ANOVA, Fig. 4c, d). < 0.0001; interaction, p = not significant; two-way ANOVA; Fig. 6a), BrdU+ type-1 cells (p21 genotype, p p21 deficiency results in differential ablation of < 0.05; time after BrdU, p < 0.0001; interaction, p = not proliferative NPCs after irradiation significant; Fig. 6c) and BrdU+ type-3 cells (p21 genotype, To determine the influence of p21 on cell fate of NPCs, p < 0.001; time after BrdU, p < 0.0001; interaction, p < mice were given a single injection of BrdU (150 mg/kg) at 0.01; Fig. 6i), but not type-2a (p21 genotype, p = not 4 weeks after 0 or 5 Gy, and BrdU-labeled NPC sub- significant; time after BrdU, p < 0.01; Fig. 6e) and type-2b populations were quantified at 2 h, 2 days, 1, and 5 weeks cells (p21 genotype, p = not significant; time after BrdU, after BrdU. Using this BrdU paradigm, cells that p < 0.01; Fig. 6g), compared to p21+/+ mice. demonstrated BrdU incorporation at 2 h were primarily A dose of 5 Gy resulted in loss of total BrdU+ cells in proliferating cells. BrdU+ cells at 2 days represented a both p21+/+ mice and p21−/− mice (p21+/+ mice: blend of proliferating and newborn cells, and those at irradiation, p < 0.05; time after BrdU, p < 0.001; interaction, 1 week and 5 weeks after BrdU were expected to be p = not significant; p21−/− mice: irradiation, p < 0.001; mostly newborn cells. Tbr2 and DCX immunohis- time after BrdU, p < 0.0001; interaction, p = 0.01; Fig. 6a, b tochemistry was used to differentiate BrdU+ inter- and Supplementary Fig. 1a, b). Among the different NPC mediate NPCs into type-2a (Tbr2+/DCX−), type-2b subpopulations in p21+/+ mice, there was loss of BrdU+ (Tbr2+/DCX+), and type-3 (Tbr2−/DCX+) cells type-2a cells (irradiation, p < 0.05; time after BrdU, p < (Fig. 5a). 0.05; interaction, p = not significant; Fig. 6e, f and Sup- The number of BrdU-labeled cells declined over the plementary Fig. 1e) but not BrdU+ type-1 (irradiation, p 5 weeks after BrdU injection in nonirradiated mice and = not significant; time after BrdU, p < 0.01; Fig. 6c, d irradiated mice regardless of genotype (Fig. 6). In 0-Gy and Supplementary Fig. 1c), BrdU+ type-2b (irradiation, Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 5 of 12 a 0 Gy 5 Gy DCX/DAPI p21+/+ 100 µm p21−/− Ki67 DCX DAPI Merged 20 µm Merged BrdU NeuN DAPI 10 µm d e 0 Gy 2000 0 Gy 1000 5 Gy 5 Gy † 1500 750 500 250 0 0 p21+/+ p21−/− p21+/+ p21−/− f h 0 Gy 1000 1000 0 Gy 0 Gy 5 Gy 5 Gy 5 Gy 0 0 0 p21+/+ p21−/− p21+/+ p21−/− p21+/+ p21−/− Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association No. of DCX+ cells No. of Ki67+ cells No. of Ki67+/DCX+ cells No. of BrdU+ cells No. of BrdU+/NeuN+ cells Li and Wong Cell Death Discovery (2019) 5:15 Page 6 of 12 (see figure on previous page) Fig. 3 Inhibition of neurogenesis after irradiation is independent of p21. a Loss of DCX+ cells in dentate gyrus is apparent in both p21+/+mice and p21−/− mice at 9 weeks after irradiation (DCX, green; DAPI, blue). b A proliferating neuroblast (arrow) demonstrates dual Ki67/DCX staining (Ki67, red; DCX, green; DAPI, blue). c A newborn neuron (arrow) demonstrates dual BrdU/NeuN immunoreactivity (BrdU, green; NeuN, red; DAPI, blue). d, e At 9 weeks after 5 Gy, there is loss of Ki67+ and BrdU+ cells in dentate gyrus independent of p21 genotype. f−h Absence of p21 results in an increase in DCX+ and BrdU+/NeuN+ cells in nonirradiated controls, but loss of DCX+ (f), Ki67+/DCX+ (g) and BrdU+/NeuN+ (h) cells after irradiation is independent of p21 genotype. BrdU (50 mg/kg/day × 7 consecutive days) was given at 4 weeks and animals were killed at 9 weeks after 0 or 5 Gy. Data are represented as mean ± SEM and analyzed using two-way ANOVA, *p < 0.05, †p < 0.01, §p < 0.001, post hoc Bonferroni test with 3-4 mice per genotype per time point p = not significant; time after BrdU, p = not significant; decreased type-2a cell proliferation (p21 genotype, p < Fig. 6g, h and Supplementary Fig. 1g) and BrdU+ type-3 0.05, time after BrdU, p < 0.01, interaction, p = not sig- cells (irradiation, p = not significant; time after BrdU, p = nificant; Fig. 7a, b) but increased type-3 cell proliferation not significant; Fig. 6i, j and Supplementary Fig. 1i) after or enhanced differentiation into type-3 cells (p21 geno- 5 Gy. type, p < 0.05, time after BrdU, p < 0.0001, interaction, p < In p21−/− mice after irradiation, there was loss of 0.01; Fig. 7a, b). After irradiation, the effect of p21 loss different BrdU+ NPC subpopulations, namely loss of resulted in increased % BrdU+ type-2a cells (p21 geno- BrdU+ type-1 cells (irradiation, p < 0.01; time after BrdU, type, p < 0.01, time after BrdU, p < 0.0001, interaction, p < p < 0.001; interaction, p < 0.05; Fig. 6c, d and Supple- 0.01) but decreased % BrdU+ type-2b cells (p21 genotype, mentary Fig. 1d), BrdU+ type-2b cells (irradiation, p < p < 0.01, time after BrdU, p < 0.01, interaction, p = not 0.0001, time after BrdU, p < 0.001; interaction, p = 0.01; significant), and % BrdU+ type-3 cells appeared unper- Fig. 6g, h and Supplementary Fig. 1h) and BrdU+ type-3 turbed (p21 genotype, p = not significant, time after BrdU, cells (irradiation, p < 0.0001; time after BrdU, p < 0.0001; p < 0.0001; Fig. 7c, d). These results again demonstrate the interaction, p < 0.0001; Fig. 6i, j and Supplementary differential effects of p21 on neuronal development within Fig. 1j) but not type-2a cells (irradiation, p = not sig- the three stages of intermediate NPC development fol- nificant; time after BrdU, p < 0.05; Fig. 6e, f and Supple- lowing irradiation. We were unable to detect any mentary Fig. 1f). In p21+/+ mice, BrdU+ type-2b cells at BrdU+/Tbr2+ cells at 5 weeks consistent with culling of 2 h and 2 days were reduced after 5 Gy to 50.0 ± 8.3% and type-2 cells due to death and/or differentiation over a few 12,17 23.9 ± 20.3% respectively compared to controls whereas weeks after BrdU incorporation . they were markedly reduced to 1.8 ± 1.8 and 4.7 ± 2.3% To further assess the role of p21 on inhibition of NPC respectively in p21−/− mice after irradiation (Supple- proliferation after irradiation, we determined the number mentary Fig. 1a-j). of BrdU doublets, type-1 (GFAP+/nestin+) BrdU doub- After irradiation, the effect of p21 genotype demon- lets (Fig. 5b) and type-2 (Tbr2+) BrdU doublets (Fig. 5c) strated a significant independent effect on the total at 2 h after BrdU. A BrdU doublet was defined as two number of BrdU+ cells (time after BrdU, p < 0.0001; p21 abutting DAPI-stained nuclei that demonstrated BrdU genotype, p < 0.01; interaction, p < 0.001; Fig. 6b), and incorporation . The number of type-1 BrdU doublets type-2b cells (time after BrdU, p < 0.001; p21 genotype, p increased in both nonirradiated and irradiated p21−/− < 0.001; interaction, p < 0.01; Fig. 6h), but not BrdU+ mice compared to wild-type controls, and irradiation did type-1 (time after BrdU, p < 0.05; p21 genotype, p = not not alter their numbers (irradiation, p = not significant; significant; Fig. 6d), BrdU+ type-2a (time after BrdU, p < p21 genotype, p = 0.001, two-way ANOVA; Supplemen- 0.0001; p21 genotype, p = not significant; Fig. 6f) or BrdU tary Fig. 2). In contrast, the number of type-2 BrdU type-3 cells (time after BrdU, p = not significant; p21 doublets remained altered with p21 loss and a 5 Gy-dose genotype, p = not significant; Fig. 6j). reduced the number of type-2 BrdU doublets in both p21 To further discern the influence of p21 genotype on +/+ mice and p21−/− mice (irradiation, p < 0.001; p21 neuronal development within the stages of transiently genotype, p = not significant). amplifying or intermediate NPCs after irradiation, we determined the distribution of BrdU+ type-2a, type-2b, Discussion and type-3 cells among all BrdU+/Tbr2+ and BrdU Inhibition of hippocampal neurogenesis is implicated in +/DCX+ cells (Fig. 7). In nonirradiated wild-type mice, neurocognitive decline following radiation treatment for the results are consistent with increasing neuronal dif- brain tumors . How DNA damage following ionizing ferentiation with progression through type-2a, type-2b, radiation results in disruption of neuronal development and type-3 cells over the 5 weeks after BrdU (Fig. 7a). remains unclear. The tumor suppressor p53 is known to With p21 loss in nonirradiated mice, there appeared to regulate cellular response after DNA damage .It Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 7 of 12 negatively regulates proliferation and self-renewal of irradiated hippocampus was not altered by p53 genotype 20–22 12 neural stem cells . We recently showed that p53 loss of recipient mice . Thus the effect of p53 on disruption resulted in increased ablation of neural stem cells and of neuronal development after irradiation is likely to be profound inhibition of neurogenesis in mouse dentate independent of damage of the neural stem cell niche. gyrus after irradiation despite lack of NPC apoptosis . How p53 regulates DNA damage response in NPCs Damage of the neurovascular niche plays a role in remains unclear. A well-described downstream effector of 23–25 impaired neurogenesis after irradiation . Impaired p53 is p21. In young adult mouse brain, p21 loss is neuronal differentiation of NPCs transplanted into the associated with increased NPC proliferation but BrdU Nestin GFAP Merged 20 µm Ki67 Nestin GFAP Merged 10 µm 100 100 800 0 Gy 0 Gy 0 Gy 5 Gy 5 Gy 5 Gy 80 80 60 60 * * 40 40 20 20 0 0 p21+/+ p21−/− p21+/+ p21−/− p21+/+ p21−/− 160 160 0 Gy 160 0 Gy 0 Gy 5 Gy 5 Gy 5 Gy 140 140 140 † † † 120 120 120 * * 100 100 100 80 80 80 60 60 60 40 40 40 20 20 0 0 p21+/+ p21−/− p21+/+ p21−/− p21+/+ p21−/− Fig. 4 Irradiation results in depletion of proliferating and newborn type-1 cells independent of p21. a A newborn type-1 cell (arrow) with characteristic morphology in subgranular zone demonstrates BrdU incorporation (green), and nestin (red) and GFAP immunoreactivity (white). b A proliferating type-1 cell (arrow) is Ki67+ (green), nestin (red) and GFAP+ (white). An arrowhead shows a Ki67+ cell which is negative for nestin and GFAP. c, d Following a dose of 5 Gy, there is loss of newborn (BrdU+) and proliferating (Ki67+) type-1 cells independent of p21 genotype, and the percent reduction in total, newborn and proliferating type-1 cells after 5 Gy shown in (d). BrdU (50 mg/kg/day × 7 consecutive days) was given at 4 weeks and animals were killed at 9 weeks after irradiation. Data are represented as mean ± SEM and analyzed using two-way ANOVA, *p < 0.05, †p < 0.01, post hoc Bonferroni test, with 3-4 mice per genotype per time point Official journal of the Cell Death Differentiation Association No. of type-1 cells No. of type-1 cells (%) No. of BrdU+ type-1 cells (%) No. of BrdU+ type-1 cells No. of Ki67+ type-1 cells (%) No. of Ki67+ type-1 cells Li and Wong Cell Death Discovery (2019) 5:15 Page 8 of 12 a BrdU Tbr2 DCX Merged Type-2a 5 µm Type-2b 5 µm Type-3 5 µm BrdU Nestin GFAP Merged 20 µm BrdU Tbr2 Merged 10 µm Fig. 5 Pulsed BrdU injections label intermediate NPCs. a Immunohistochemistry for Tbr2 and DCX is used to sort BrdU-labeled intermediate NPCs (arrows) into type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+) and type-3 cells (BrdU, green; Tbr2, red; DCX, white). b, c Some BrdU+NPCs are seen as BrdU doublets, defined as two abutting BrdU+ nuclei identified on DAPI. A type-1 BrdU doublet (b, arrow; BrdU, green) is nestin+ (red) and GFAP+ (white), and a type-2 BrdU doublet is Tbr2+ (c, arrows, BrdU, green; Tbr2, red) neurospheres from p21−/− mice demonstrated limited of apoptosis . Neural stem cells are known to be apop- 13 14 in vitro self-renewal . It has been proposed that tosis resistant after irradiation . Here, irradiation resulted p21 serves to keep adult neural stem cells in relative in an increase in p21 immunoreactivity in type-1 cells and quiescence for life-long maintenance of self-renewal. intermediate NPCs. There was no evidence of acquisition Here, p21 loss was associated with increased cell pro- of apoptosis radiosensitivity in neural stem cells with p21 liferation in the dentate gyrus, including enhanced pro- loss, and we also observed no altered apoptosis radio- liferation of type-1 cells and neuroblasts. We also sensitivity in proliferating type-2 cells and neuroblasts in observed an increase in newborn neurons, consistent with the absence of p21. In irradiated p21−/− embryonic an inhibitory effect of p21 on neurogenesis in the dentate mouse cortex, there was decreased not increased apop- gyrus. tosis . Whether these differences in p21-dependent Stem cells may have distinct mechanisms from pro- responses post-DNA damage are related to differences genitors to mitigate DNA damage to preserve self-renewal in tissue-specific stem cell radiobiology, developmental or and differentiation . Adult stem cells are known to be in vitro conditions, remain to be investigated. resistant to apoptosis after irradiation. p21 may play a role Neural stem cells and NPCs are known to exhibit dif- in the regulation of DNA damage response of stem cells . ferential responses to physiologic stresses and external In hematopoietic and mammary stem cells in vitro, irra- insults . Our results here provide evidence for differential diation was shown to result in activation of p21 inde- effects of p21 on NPC cell fate after irradiation. In wild- pendent of p53, and subsequent inhibition of basal activity type mice, the predominant effect of irradiation appears to of p53. This p21-dependent response in stem cells after be death of type-2a cells and/or inhibition of their pro- DNA damage has been postulated to result in inhibition liferation. In contrast, the major effect of irradiation with Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 9 of 12 Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 10 of 12 (see figure on previous page) Fig. 6 Loss of p21 results in differential ablation of NPCs after irradiation. a The number of BrdU+ cells in nonirradiated p21−/− mice is increased compared to nonirradiated p21+/+ mice (p21 genotype, p < 0.05). b Loss of BrdU+ cells after irradiation is p21 genotype dependent (p21 genotype, p < 0.01). c, d For BrdU+type-1 (nestin+/GFAP+) cells, their decline over time is p21 dependent in nonirradiated mice (c, p21 genotype, p < 0.05) but not after irradiation (d). e, f The number of BrdU+type-2a (Tbr2+/DCX−) cells over time after BrdU is independent of p21 in nonirradiated (e) and irradiated mice (f). g, h The decline in BrdU+type-2b (Tbr2+/DCX+) cells after BrdU in nonirradiated mice is independent of p21 (g) whereas there is a marked loss of BrdU+ type-2b cells in p21−/− mice after 5 Gy compared to wild-type animals (h, p21 genotype, p < 0.001). i, j BrdU+ type-3 (Tbr2−/DCX+) cells are increased in nonirradiated p21−/− mice compared to wild-type mice (i, p21 genotype, p< 0.001), but their loss after irradiation (j)is p21 genotype independent. A single dose of BrdU (150 mg/kg) was given at 4 weeks after 0 or 5 Gy. Cells were counted at 2 h, 2 days, 1 and 5 weeks after BrdU. Data are represented as mean ± SEM and analyzed using two-way ANOVA, *p < 0.05, †p < 0.01, §p < 0.001, post hoc Bonferroni test with 3−5 mice per genotype per time point ab p21+/+, 0 Gy p21−/−, 0 Gy 100 100 80 80 60 60 40 40 20 20 0 0 2 h 2 days 1 week 5 weeks 2 h 2 days 1 week 5 weeks Type-2a Type-2b Type-3 p21+/+, 5 Gy p21−/−, 5 Gy 80 80 60 60 40 40 20 20 0 0 2 h 2 days 1 week 5 weeks 2 h 2 days 1 week 5 weeks Fig. 7 Loss of p21 alters disruption of neuronal progression over the three stages of intermediate NPCs after irradiation. a−d The distribution of BrdU+ type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+) and type-3 (Tbr2−/DCX+) cells as percent total intermediate NPCs (Tbr2+or DCX+) is disrupted with p21 loss and after irradiation. A single dose of BrdU (150 mg/kg) was given at 4 weeks after 0 or 5 Gy. Data are represented as mean ± SEM with 3−5 mice per genotype per time point p21 loss was increased culling of proliferative type-1, type- loss of about 50% of newborn neurons and was con- 2b, and type-3 NPCs. There is in vitro evidence that sidered optimal to discern the effect of genotype on activation of p21 in certain stem cells results in induction inhibition of neurogenesis after irradiation. Although our of symmetric self-renewing divisions after irradiation . results appeared to internally consistent, no BrdU+ type- Although we did not address self-renewal directly, the 2 cells were observed at 5 weeks using the single BrdU absence of any increase in type-1 cells at 2 days compared injection paradigm. Further information may be obtained to at 2 h after BrdU provided no evidence to suggest using other fate mapping methods and additional sets of induced symmetrical division of type-1 cells after irra- phenotypic markers for NPCs. diation. We also did not observe any p21-dependent In conclusion, there is increased hippocampal neuro- effects on type-1 cell fate after irradiation. genesis associated with p21 loss which may be due to Several methods including thymidine analogs, retro- enhanced NPC proliferation and/or neuronal differentia- virus, and transgenic animals have been used to study tion particularly in the latter stages of intermediate NPC 1,31 adult hippocampal neurogensis . These different fate development. Irradiation results in inhibition of neuro- mapping approaches may contribute to the various genesis and loss of newborn neural stem cells. These models of NPC behavior proposed. Here we used BrdU effects appear to be independent of p21. In wild-type injection paradigms to assess neuronal development. We mice, the most pronounced effects of irradiation is death chose a dose of 5 Gy in our study as this dose resulted in and/or inhibition of proliferation of type-2a cells. In Official journal of the Cell Death Differentiation Association Percent intermediate NPC Percent intermediate NPC Percent intermediate NPC Percent intermediate NPC Li and Wong Cell Death Discovery (2019) 5:15 Page 11 of 12 contrast, there is increased ablation of proliferative type-1, NPCs, immature and mature neurons were identified by type-2b, and type-3 NPCs after irradiation with p21 loss. phenotypic markers using immunohistochemistry . Pri- Our results are thus consistent with differential effects of mary antibodies included those against DCX (1:2000, p21 on NPC cell fate after irradiation. Loss of p21 how- Abcam, Cambridge, MA, USA; Cat# ab18723), GFAP ever does not alter the ultimate inhibition of production (1:200, DakoCytomation, Copenhagen, Denmark; Cat# of new neurons after irradiation. Z0334), nestin (1:200, Millipore, Billerica, MA, USA; Cat# MAB353), NeuN (1:500, Millipore; Cat# MAB377), and Materials and methods Tbr2 (1:200, Abcam; Cat# ab23345). Secondary antibodies Animals were conjugated to Cy2, Cy3 (1:200; Jackson ImmunoR- C57 mice wild type (+/+) or knockout of the p21 gene esearch, West Grove, PA, USA; Cat# 712225150, were obtained from the Jackson Laboratory (Bar Harbor, cat#712165150) or Alexa Fluor 647 (1:200, Invitrogen, ME, USA). Mouse colonies were generated by littermate Burlington, Ontario, Canada; Cat# A31573). Colocaliza- inbreeding and genotyped by PCR . To avoid the tion of phenotypic markers with BrdU (1:200, Abcam; potential confounding influence of sex on neurogenesis , Cat# ab6326) or Ki67 (1:1000, Novocastra, Newcastle, UK; only male mice were used in the study. All animal pro- Cat# NCL-Ki67p) in selected sections were evaluated cedures (protocol number: 18-156) were approved by the using a confocal laser scanning microscope (Zeiss institutional animal care committee and performed LSM700, Carl Zeiss AG Corporate, Oberkochen, Ger- according to the Canadian Council on Animal Care many). A BrdU doublet was defined as two abutting guidelines. DAPI-stained nuclei that demonstrated BrdU incorporation . Irradiation Animals were irradiated at the age of 10 weeks. They Stereological analysis were anesthetized using an intraperitoneal injection of Apoptotic cells and cells labeled using different phe- ketamine (75 mg/kg) and xylazine (6 mg/kg) and posi- notypic markers were counted within the dentate gyrus tioned in a customized jig with the entire hippocampus including a 50-µm margin of the hilus . Cell counting irradiated using an anterior-posterior and posterior- was performed using a Zeiss Imager M1 microscope (Carl anterior pair of 160 kV X-ray beam (Model CP160, Faxi- Zeiss AG Corporate) with the Stereo Investigator software tron X-ray, Wheeling, IL, USA) defined by an 8-mm (MBF Bioscience, Williston, VT, USA). The observers diameter lead cut-out were blinded to the experimental groups. Details of ste- . Control (0 Gy) mice were sham- irradiated. A single dose of 5 Gy was used to evaluate the reology were as described previously . Apoptotic cells effects of the p21 genotype on neuronal development after and NPCs were counted using a counting frame and a irradiation. Three different BrdU incorporation schedules sampling grid of 75 × 75 µm at a magnification of ×630. were used for cell fate mapping as described in the Results Every seventh section was used as the periodicity of sec- section. BrdU was administered by intraperitoneal tions sampled. For all stereology data, the coefficient of injection. error ranged from 0.03 to 0.06. Histopathology and immunohistochemistry Statistical analysis Under anesthesia with ketamine and xylazine, mice Cell population analysis represented data from 3 to 5 underwent intracardiac perfusion with 0.9% saline fol- mice per genotype per dose per time point. All data lowed by 4% paraformaldehyde in PBS. The brain was represented the mean ± SEM. The significance of two dissected out, post-fixed for 2 days and cryoprotected in independent variables such as irradiation and p21-geno- PBS with 30% sucrose. Coronal sections containing the type, or p21-genotype and time after BrdU on the various hippocampus were cut at 40-μm thickness and stored in end-points was determined using two-way ANOVA. Pair- tissue cryoprotectant solution in 96-well plates at −20 °C wise comparisons were based on post hoc analysis with prior to immunohistochemistry. Bonferroni correction for multiple comparisons. Differ- Apoptotic cells were identified based on characteristic ences were considered significant for p < 0.05. Statistical nuclear condensation and fragmentation of apoptosis analyses were performed with the GraphPad Prism 5 11,17 using DAPI . The apoptotic response was further (GraphPad Software, La Jolla, CA, USA). characterized using TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA; Cat# 11684795910) and caspase-3 (1:1000; Cell Signaling Technology, Dan- Acknowledgements vers, MA, USA; Cat# 9661) immunohistochemistry as This work was supported by the Cancer Research Society and the Ontario previously described . Association of Radiation Oncologists (C.S.W.). Official journal of the Cell Death Differentiation Association Li and Wong Cell Death Discovery (2019) 5:15 Page 12 of 12 Authors’ contributions 13. Kippin, T. E.,Martens,D. J.&vander Kooy,D.p21 loss compromisesthe Conception and design: C.S.W.; Development of methodology: Y.-Q.L., C.S.W.; relative quiescence of forebrain stem cell proliferation leading to exhaustion of Acquisition of data: Y.-Q.L., C.S.W.; Analysis and interpretation of data: Y.-Q.L., their proliferation capacity. Genes Dev. 19,756–767 (2005). C.S.W.; Writing, review, and/or revision of the manuscript: Y.-Q.L., C.S.W.; 14. Li, Y. Q., Cheng, Z. & Wong, S. Differential apoptosis radiosensitivity of neural Administrative, technical, or material support: Y.-Q.L., C.S.W.; Study supervision: progenitors in adult mouse hippocampus. Int. J. Mol. Sci. 17, 970 (2016). C.S.W.; Other (oversight of every aspect of the research): C.S.W. 15. Cheng, Z., Zheng, Y. Z., Li, Y. Q. & Wong, C. S. Cellular senescence in mouse hippocampus after irradiation and the role of p53 and p21. J. Neuropath. Exp. Neurol. 76,260–269 (2017). Conflict of interest 16. Hodge, R. D. et al. Tbr2 is essential for hippocampal lineage progression from The authors declare that they have no conflict of interest. neural stem cells to intermediate progenitors and neurons. J. Neurosci. 32, 6275–6287 (2012). 17. Sierra, A. et al. Microglia shape adult hippocampal neurogenesis through Publisher's note apoptosis-coupled phagocytosis. Cell Stem Cell 7,483–495 (2010). 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