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Comparative Study
. 2009 Feb 11;29(6):1874-86.
doi: 10.1523/JNEUROSCI.3095-08.2009.

Pten deletion in adult neural stem/progenitor cells enhances constitutive neurogenesis

Caroline Gregorian  1 , Jonathan Nakashima, Janel Le Belle, John Ohab, Rachel Kim, Annie Liu, Kate Barzan Smith, Matthias Groszer, A Denise Garcia, Michael V Sofroniew, S Thomas Carmichael, Harley I Kornblum, Xin Liu, Hong Wu
Affiliations
Comparative Study

Pten deletion in adult neural stem/progenitor cells enhances constitutive neurogenesis

Caroline Gregorian et al. J Neurosci. .

Abstract

Here we show that conditional deletion of Pten in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to constitutive neurogenesis in the olfactory bulb. As a result, Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, in contrast to the olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.

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Figures

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mGFAP-Cre line for conditionally _targeting adult neural stem/progenitor cells. a, b, e, f, h, Survey images of coronal sections of the SEZ (a, b, e), RMS (f), and OB (h) stained by bright-field immunohistochemistry. A small number of cells in the SEZ express Cre (a, low mag; b, high mag). Scale bars, 80 μm. c, d, Confocal micrographs of single optical slices through cells in the SEZ that are double stained by immunofluorescence for Cre and GFAP (c) or Cre and Nestin (d). Individual channels and orthogonal analysis show that all Cre-expressing cells in the SEZ also express GFAP (c) and Nestin (d). Orthogonal images (ortho) show three-dimensional analysis of individual cells at specific sites marked by intersecting x, y, and z axes. Scale bars, 20 μm. Many cells express the reporter protein β-gal (e) in the neurogenic proliferative regions of the SEZ. Migrating neuroblasts in the RMS (f) and granule neurons in the OB (h) also express β-gal. Box in g indicates the region shown in h. Arrows in a, b, and e indicate representative Cre or β-gal-positive cells in SEZ. SEZ, Subependmyal zone; LV, left ventricle; STR, striatum; RMS, rostral migratory stream; OB, olfactory bulb; GCL, granule cell layer; Gl, glomeruli.
Figure 2.
Figure 2.
Pten deletion in clonal neurosphere cultures enhances stem cell self-renewal and neurogenesis over long-term serial clonal passages compared with the gradual senescence and decreased neurogenesis in wild-type cultures. a, Neurosphere cultures generated from the adult SVZ of Pten knock-out (Mutant) and wild-type (Control) mice were cultured at clonal density (1000 cells/ml), passaged, and reseeded at clonal density every 2 weeks. Clonal neurosphere numbers, representative of the number of stem cells present in the culture, were significantly higher in mutant cultures at all passages (p < 0.001). b, The clonal neurospheres were then dissociated and the total cell number was counted at each passage followed by reculturing at clonal density. After the first passage, mutant cultures consistently produced higher total cell numbers even after long-term serial clonal passaging while control cultures produced decreased cell numbers with time, eventually senescing by passage 24. Data are means ± SEM; n = 4. c, Immunocytochemistry for TUJ1+ cells (green) and the counterstain Hoescht (blue) are shown at passage 4 and 16 indicating the clonal neurospheres from mutant mice maintained their robust neurogenesis even at late clonal passages while the ability to generate neurons was dramatically attenuated in control cultures over time. Scale bar, 60 μm. n = 4.
Figure 3.
Figure 3.
mGFAP-Cre-mediated Pten deletion leads to expansion of adult neural stem cells and their progenies in vivo. Survey images of coronal sections of control and mutant SEZ. a, Compared with littermate controls, mutant mice showed increased GFAP and P-S6-positive (labeled cells which co-localized in the SEZ. DAPI counter stain is used to visualize nuclei. Scale bars: top, 150 μm; bottom, 25 μm. b–d, Images of H&E (b), Ki-67 (c), and DCX (d) expression demonstrate increased proliferation in mutant SEZ compared with control regions. Scale bar, 60 μm. n = 5. SEZ, Subependymal zone; LV, lateral ventricle; CC, corpus callosum; STR, striatum; H&E, hematoxylin and eosin; DCX, doublecortin.
Figure 4.
Figure 4.
Pten deletion leads to enhanced migration of SEZ-born progenitors in RMS. a, Representative images of RMS stained for BrdU, P-S6, and their colocalization revealed an increase in Pten null SEZ-born progenitors migrating in RMS when compared with control and shows the BrdU+ population in mutant RMS is Pten null. Scale bar, 100 μm. b, Survey images of sagittal sections of control and mutant RMS stained with DCX demonstrate an increase in the number of migrating neuroblasts in mutant RMS compared with control. DAPI counter stain is used to visualize nuclei. Scale bar, 400 μm. n = 6. DCX, Doublecortin; RMS, rostral migratory stream; OB, olfactory bulb.
Figure 5.
Figure 5.
Increased OB mass and enhanced proliferation and migration of SEZ-born progenitors to the GCL of OB in mutant mice. a, Enlarged OB of mutant mice. Photo shows a brain hemisphere from age-matched control and mutant mice. Scale bar, 1 mm. Panel below depicts the progressive increase in the ratio of mutant/control OB weight. n = 40. b, Histological analysis of OB section from control and mutant mice reveals normal histoarchitecture. Scale bar, 500 μm. c, GCL volume is significantly increased in mutant mice (*p < 0.05) whereas no significant change is observed in EPL (p > 0.05). Data are means ± SEM; n = 10. d, SEZ-born neuroblast migration was analyzed by BrdU pulse-labeling. Mice were injected with 200 mg/kg BrdU peritoneally, and distribution of BrdU-labeled cells was determined 2 weeks after injection. An increased number of BrdU-labeled cells within mutant GCL indicated Pten mutant mice had a significant increase in cell number at 2 weeks post injection. Scale bar, 100 μm. n = 5. e, Representative GCL images show enhanced immunoreactivity for NeuN and increased NeuN/P-S6 double positive cells in mutant when compared with a matched control region. DAPI is used as counterstain to visualize nuclei. Scale bar, 10 μm. OB, Olfactory bulb; GCL, granule cell layer; EPL, external plexiform layer.
Figure 6.
Figure 6.
Enhanced olfactory habituation and recovery after epithelium injury in Ptenloxp/loxp;mGFAP-Cre+ mice. a, b, Control and mutant mice were subjected to the olfactory habituation test. Pretraining of wild-type control mice to a cotton swab soaked in water habituated mice to the presence of the swab in their cage. This was manifested as a decline in the number of sniffs with subsequent exposures to the swab. During odorant testing, the cotton swab was laced with 50 μm isoamyl acetate or 50 μm hexyl alcohol and introduced on three successive trials. The fact that the control mice sniffed the isoamyl acetate- and hexyl alcohol-laced cotton swabs more times than during the third exposure to the water swab indicated that the animal was able to smell these odorants. a, Compared with control, mutant mice sniffed the swabs less frequently in the second and third exposures (all three conditions) signifying they habituated to the novel odorant faster than the control. n = 14 (control) and 12 (mutant). b, Both control and mutant mice lost their ability to detect odorants at 2 weeks post treatment, whereas the ability of mutant mice to detect odorants began recovering at 4 weeks post treatment, and their ability to detect odorants was restored to pre-treatment levels by week 6. Data are means ± SEM; n = 8.
Figure 7.
Figure 7.
Pten deletion enhances post-stroke neuroblast migration. a, DCX labeling in peri-infarct cortex and SEZ. DCX+ cells in control and mutant 7 d after stroke. Right panel shows stereological quantification of DCX+ cells in peri-infarct cortex. Scale bars: top, 100 μm; bottom, 25 μm. n = 6. b, Representative images of P-S6 staining from both hemispheres of mutant mouse. Left panel shows hemisphere containing peri-infarct cortex which has increased staining for p-S6 in peri-infarct cortex when compared with uninjured cortex in the same animal (right panel). DAPI is used as counterstain to visualize nuclei. Scale bar, 50 μm. CTX, Cortex; CC, corpus callosum.
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References

    1. Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002;8:963–970. - PubMed
    1. Backman SA, Stambolic V, Suzuki A, Haight J, Elia A, Pretorius J, Tsao MS, Shannon P, Bolon B, Ivy GO, Mak TW. Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease. Nat Genet. 2001;29:396–403. - PubMed
    1. Baker H, Cummings DM, Munger SD, Margolis JW, Franzen L, Reed RR, Margolis FL. _targeted deletion of a cyclic nucleotide-gated channel subunit (OCNC1): biochemical and morphological consequences in adult mice. J Neurosci. 1999;19:9313–9321. - PMC - PubMed
    1. Bhattacharyya A, Svendsen CN. Human neural stem cells: a new tool for studying cortical development in Down's syndrome. Genes Brain Behav. 2003;2:179–186. - PubMed
    1. Brunjes PC. Unilateral naris closure and olfactory system development. Brain Res Brain Res Rev. 1994;19:146–160. - PubMed

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