Neurological disorders are the leading source of disability. Currently there are no treatments that promote repair and recovery in the brain from any major neurological disorder, including stroke, degenerative disease, and trauma. NSCs are cells that can give rise to the major components of the brain and spinal cord, thus attractive resources for repairing neurological disorders (Lindvall and Kokaia, 2006
). The major challenges of NSC-mediated neuronal repair are 1) to understand the key pathways involved in controlling NSC self-renewal, proliferation, survival and differentiation, 2) to develop methodologies for manipulating these pathways towards therapeutic use, and 3) to test the potential benefit of NSCs and their progenies in human disease-relevant model systems before being used on human patients.
In this study we demonstrated that deletion of Pten in adult NSCs is not tumorigenic but promotes NSC expansion. Progeny from Pten null NSCs follow the endogenous migratory cues, differentiate, and integrate into the existing circuitry. Under conditions of acute damage, such as olfactory epithelium ablation, animals with Pten NSC deletion exhibited faster recovery. These data suggest that conditional manipulation of the PTEN/PI3K pathway in NSCs may be beneficial for NSC expansion, proliferation, and survival, in turn, promoting recovery and the integration of newly born neurons to existing or damaged neuronal networks associated with constitutive neurogenesis.
Stem cell self-renewal is known to be regulated by different signaling pathways, but the effect of a specific pathway may depend on the developmental stage or lineage of a stem cell population. For example, deletion of p21 in adult neural stem cells results in a transient increase in neurospheres formation with a subsequent decline in stem cell numbers; a phenomenon known as stem cell “exhaustion” (Kippin et al., 2005
). Although, in a similar fashion, PTEN loss leads to the “exhaustion” of adult hematopoietic stem cells (Yilmaz et al., 2006; Zhang et al., 2006; and our unpublished observation), conditional deletion of Pten
in both embryonic and adult NSC promotes a sustained self-renewal and stem/progenitor expansion (Groszer et al., 2006
; Groszer et al., 2001
; and this study). Significantly, we observed that the mutant cultures maintain an enhanced self-renewal, proliferation/survival rate, and neurogenic potential over extremely long-term expansion times under clonal conditions, suggesting that the role of PTEN in regulating stem cell activity may be modulated by factors specific to the neural stem cell lineage. Therefore, further study of these lineage- or developmental stage-specific mediators of self-renewal pathways will be increasingly critical for our understanding of the underlying mechanisms of stem cell self-renewal and therapeutic manipulations.
Our previous studies and those by others demonstrated that Pten
deletion in the embryonic brain leads to abnormal histoarchitecture with severe layering defects (Backman et al., 2001
; Groszer et al., 2001
; Marino et al., 2002
; Yue et al., 2005
). However, as Pten
deletion happens in nearly 100% of the neural stem/progenitor cells, it is unclear whether the abnormal phenotypes observed are caused by intrinsic migratory defects of Pten
-null neurons, extrinsic microenvironmental cues provided by Pten
null radial glia, or perhaps both (Groszer et al., 2001
). In this study, we demonstrated that in the presence of wild-type environmental cues, Pten
null adult NSCs and their progenies can follow the endogenous migration pathway, undergo differentiation, and reach their normal target. This result is consistent with a previous study on Pten
heterozygous mice showing that Pten+/−
cells migrated to the outer layers of the OB more rapidly and incorporated at the same sites as Pten+/+
cells (Li et al., 2002
). Therefore, the extrinsic microenvironment cues plays a more predominant role in controlling the migratory behavior of SEZ-derived Pten
null NSCs and their progenies.
Nearly 90% of the GCs in the OB are SEZ-derived and incorporated postnatally, and their survival depends on incoming activities (Brunjes, 1994
; Rosselli-Austin and Williams, 1990
). GCs are also known to extensively shape mitral cell response to odors (Yokoi et al., 1995
), and there is evidence that the OB circuitry maximizes differences in odor representations (Dulac, 2005
; Friedrich and Laurent, 2001
). Animals carrying a mutation in the cyclic nucleotide gated channel are not able to transduce the signal from the olfactory receptors in the olfactory neurons (Baker et al., 1999
) and have smaller GCL volume and OB mass (Petreanu and Alvarez-Buylla, 2002
). The lack of electrical activity in the OB of these mice dramatically reduced the survival of newly generated neurons of the GCL, suggesting that peripheral olfactory epithelium plays a critical role in the survival of GCL neurons in the OB (Petreanu and Alvarez-Buylla, 2002
). Our results showed that PTEN loss in SEZ NSCs leads to an increase in GCL volume and OB size as well as an improvement in olfactory function, suggesting that the Pten
null SEZ-derived GCL neurons are able to integrate into the existing circuitry, receive electrical input from olfactory epithelium and are thus functional. Because Pten
conditional deletion in adult NSCs also enhances the recovery of olfactory function after ablation of olfactory epithelium, our data also suggest an important role for SEZ-derived neurons in reestablishment of the connection between OB and olfactory epithelium during acute damage via a currently unknown mechanism.
SEZ-born progenitors are known to play a role in post-ischemic injury recovery (Ohab et al., 2006
). In a stroke-injury model, Pten
conditional knockout animals showed a robust increase in cell proliferation and DCX+
neuroblast migration from the SEZ over long distances to the peri-infarct cortex, suggesting Pten
null neuroblasts are responsive to chemotactic stimulation and can migrate to the injury site. Although the SEZ DCX volume did not increase in PTEN conditional knockout mice post-stroke when compared to non-stroke animals (Supplementary Figure 10B
), the significantly enhanced proliferation and migration of Pten
null neuroblasts to the peri-infarct cortex suggested that Pten
null neuroblasts are primed for the signals that induce post-stroke neurogenesis. Pten
deletion might have been expected to promote neuroblast survival as it potentiates the action of the PI3K/Akt pathway, a common downstream signaling pathway for many of the neural stem cell survival and differentiation growth factors (Greenberg and Jin, 2006
). Interestingly, Pten
conditional deletion in these neuroblasts is not sufficient to promote their long-term differentiation/survival and functional integration once they have migrated into peri-infarct cortex. Thus, in addition to soluble growth factor signaling, the cellular environment of the peri-infarct cortex may exert an important degree of control over cell survival in newly born neurons after stroke.
Stem cell-based therapies, including the promotion of endogenous neurogenesis or transplantation of stem/progenitor cells, are limited in large part by the death of the progenitor pool (Goldman, 2005
). In post-stroke neurogenesis, despite a robust generation of newly born neurons, ninety percent of them eventually die (Ohab et al., 2006
). After stem/progenitor transplantation, a similar result is seen in which most transplanted cells die or fail to differentiate. The differential roles of PTEN defined in the present study highlight the key differences between constitutive/normal and injury-induced neurogenesis. PTEN inactivation alone is sufficient to promote a significant increase in neurogenesis, neuronal survival and improved function in the olfactory bulb but insufficient to promote long-term survival after stroke. Though both post-stroke and normal neurogenesis involve growth factor signaling within glial and vascular niches (Hagg, 2007
; Nilsson et al., 1999
; Ohab et al., 2006
; Puche and Baker, 2007
; Teramoto et al., 2003
) post-stroke neurogenesis requires additional, likely, cellular signals that are independent of the common PI3K-AKT-mediated growth factor signaling pathway. In designing successful stem cell based therapies it will be important to determine both the molecular and cellular constituents that support survival and differentiation of stem cell progenies.