Although many lines of evidence have demonstrated that NPCs are present in the adult CNS, the dynamic responses of NPCs to neurodegeneration at disease onset and progression across lifespan remain largely unexplored. Animal models mimicking human degenerative diseases would be particularly useful to gain the significant insights in this respect (39
). To this end, we have focused our effort to analyze the organization and distribution of NPCs in the ALS-like transgenic mouse model (Bi-Tg) compared with the age-matched littermate pNes-Tg mouse controls. Selection of the Bi-Tg mouse model for this study was primarily based on two major characteristics of the newly generated mouse line through breeding: First, the Bi-Tg mice express mutant G93A-SOD1 and develop ALS-like disease mimicking human ALS. More importantly, the phenotype and pathology of the Bi-Tg mice across their lifespan are very similar to the parental mutant G93A-SOD1 transgenic mouse line previously reported (9
); Second, the Bi-Tg mice contain nestin promoter controlled LacZ reporter gene, which specifically expresses LacZ protein product in NPCs (33
). Together, the unique features of the Bi-Tg mice allow us to identify and characterize the responses of NPCs in the adult CNS to motor neuron degeneration in the ALS-like model.
The present study using the Bi-Tg mice demonstrates three major findings of NPCs in the adult CNS in relation to ALS-like disease: 1). Mutant SOD1-mediated motor neuron degeneration enhances NPC proliferation in the EZ of the Bi-Tg mouse spinal cord; 2). Motor neuron degeneration promotes NPC migration from the EZ toward the dorsal horn, and subsequently to the ventral horn during ALS disease onset and progression; 3). Motor neuron degeneration increases the generation of neuron-like cells from NPCs in the spinal cord compared with the basal levels of neurogenesis. Thus, this study provides compelling evidence that the pathological processes of motor neuron degeneration stimulate NPC proliferation, migration and neurogenesis in ALS-like mouse model.
In light of these findings, we have also identified and characterized two closely related populations of NPCs in the adult mouse spinal cord. One population of NPCs was proliferative as determined with BrdU incorporation. These NPCs were at an undifferentiated state and were primarily localized in the EZ (, and ). Notably, the LacZ staining intensity was evidently increased in the EZ of ALS-like mice (Bi-Tg mice), particularly during the disease onset and progression, compared to that of age-matched normal littermate controls ( and ). The LacZ staining and BrdU labeling together suggested that there was an increase of NPC proliferation in response to motor neuron degeneration ( and ). Another population of NPCs was distributed sparsely, but unevenly across spinal cord, majority of which were in the dorsal horn regions. These NPCs were not proliferative, because they were not labeled with BrdU pulsing for 15 days (). Interestingly, the number of NPCs distributed in the dorsal horn region (Lamina I–III areas) was much more than in the ventral horn and other regions ( and ). Most significantly, there was a dramatic increase in the number of NPCs in both the dorsal and ventral horn regions in the ALS-like mice compared to normal control mice (). To the best of our knowledge, this is the very first report on the organization and distribution of NPCs in the normal and ALS-like adult mouse spinal cords. The significance of the predominant distribution of NPCs in dorsal horn region compared to the ventral horn region remains unknown. Based on the distribution (), migratory patterns ( and ), and long-term of BrdU pulse and chase labeling (), we concluded that the NPCs in the dorsal and ventral regions were derived from the proliferative NPCs in the EZ. Once migrated out of the EZ, the NPCs lost proliferative ability but maintained migratory function. The increased NPC proliferation in the EZ and the increased number of NPCs in the dorsal and ventral horn regions are the specific responses to motor neuron degeneration in the ALS-like mice.
Compared to the age-matched littermate control pNes-Tg mice, the migration of NPCs from the EZ was greatly enhanced in Bi-Tg mice, even at the clinical disease free stage ( and ). The maximum migration of NPCs in the Bi-Tg mice across their lifespan occurred at disease onset stage, during which there was a maximum of NPC distribution in the dorsal horn region ( and ). At disease progression stage, more NPCs migrated to the ventral horn region. The migratory pattern of NPCs in response to motor neuron degeneration during disease onset and progression is intriguing. Because motor neuron degeneration is in the ventral motor domain, we initially hypothesized that NPCs from the EZ may migrate directly to the ventral horn direction. Different from our hypothesis, NPCs from the EZ migrated to dorsal region first, and then, some of which migrated to the ventral region subsequently. Such a temporospatially regulated migration pattern of NPCs in the adult spinal cord in response to disease and/or traumatic injury has not been reported. However, during embryonic spinal cord developmental or early postnatal stages, the oligodendrocyte precursor cells (OPCs) defined by NKX6.1, NKX2.2, or Olig1/2 were shown to have specific migratory pattern (42
). Nevertheless, the OPCs initiated from ventral domain, moved down in ventral direction briefly, and then migrated toward the dorsal direction along the peripheral area of spinal cord. The migratory patterns of OPCs at embryonic and early postnatal stages, and NPCs at adult stages, particularly during ALS-like disease onset and progression were quite different. The intrinsic mechanism(s) governing NPC migration and migratory patterns in the spinal cord of the ALS-like mice remain elusive. Further analysis with immunohistochemical staining suggested that the expression of CXCR4 in NPCs may contribute, at least partially to the NPC migration (). Several reports have demonstrated that chemokine receptors participate in NPC migration. For example, expression of CXCR4 receptor directed the migration of neural stem cells to the lesioned sites in the CNS injury model (47
). However, the increased CXCR4 receptor expression in the spinal cords of ALS-like mice appears to be the global response of neurons and glia to motor neuron degeneration by mutant SOD1 effects. With respect to the current finding in NPC migration and migratory pathways, what and how chemokine(s)/chemokine receptor(s) or other molecule(s) participate in the directionality of NPC migration remain to be defined. Functionally, the temporospatially organized migratory pathways may have advantages in repairing the dysfunctional circuitry involved in not only motor neurons, but other cell types as well.
One important, but has not been unambiguously resolved issue of the current study is the lineage of the adult NPCs we have identified with the LacZ positive staining. Apparently, the adult NPCs defined by nestin promoter controlled LacZ reporter staining are different from glial precursor cells (GPCs) identified from embryonic stages in that the GPCs do, while NPCs do not express immature and mature markers of astrocyte and oligodendrocytes (48
). Thus, the NPCs and GPCs represent two distinguished cell type populations. However, the NPCs and GPCs may be developmentally derived from the same or similar origin (progenitor cells), because both are positive in nestin staining. We showed that some of the NPCs differentiated into neurons, but not into astrocytes and oligodendrocytes in adult brain (data not shown) and spinal cord ( and , and additional discussion in next section). More importantly, the percentage of NPCs that differentiated into neurons was increased in ALS-like mice as disease onset and progression were advanced. To a large extent, the NPCs identified in this study may be derived from radial glia, that have been demonstrated to give rise to adult neural stem cells and adult neuronal cells in vivo
). Because the adult NPCs in this study differentiate primarily into neurons, not astrocytes or oligodendrocytes, we think that these adult NPCs represent a population of radial glia derivatives with a default characteristic of differentiation potential toward neuronal direction.
During the ALS-like disease onset and progression, there was an increase of neurogenesis, but not astrogenesis, oligogenesis or microgliogeneis from NPCs as detected with specific cell type markers respectively ( and ). Enhancement of neurogenesis has been observed in animal models of Alzheimer’s disease (53
) and ischemic stroke (54
). In addition, increased neurogenesis has been reported in patients with Alzheimer’s disease (18
) and Huntington’s disease (19
). In the ALS-like mouse model, we demonstrated that motor neuron degeneration promoted neurogenesis from NPCs in the mouse spinal cord. Interestingly, there were NPCs in the vicinity of some dying motor neurons (), suggesting that some factor(s) from degenerated motor neurons may induce NPC migration and differentiation. Thus, identifying the factors that promote NPC migration and differentiation may contribute to delay or prevent ALS disease onset and progression and enhance survival. Though the molecular mechanisms governing the proliferation, migration and neurogenesis of adult NPCs in the ALS-like mice remain to be defined, the present study will allow us to explore the therapeutic potential of stimulating de novo
neurogenesis for functional replacement of degenerated neurons in ALS and other neuron degenerative diseases.