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发表于 2009-3-5 00:14 |只看该作者 |倒序浏览 |打印

作者：Liying Chia, Yan Kea, Chun Luoa, Baolin Lib, David Gozalc, Balaraman Kalyanaramand, Rugao Liua作者单位：a Department of Anatomy and Cell Biology, University of North Dakota School of Medicine, Grand Forks, North Dakota;b Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana;c Kosair Childrens Hospital Research Institute, Department of Pediatrics, University of Louisville School of  - z' p6 h: C! A
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      【摘要】
   The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter¨Ccontrolled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by bromodeoxyurindine incorporation and LacZ staining was restricted to the ependymal zone surrounding the central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALS-like mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS. , J# c4 _4 d% q# w+ [
      【关键词】 Neural progenitor cells Radial glia Glial progenitor cells Motor neurons Nestin Mutant SOD Amyotrophic lateral sclerosis" h$ v) _# V2 X/ C5 \  U  m
   INTRODUCTION
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The mechanisms of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown, and effective therapy for ALS is not yet available  to analyze the responses of NPCs to motor neuron degeneration in the ALS-like mice.9 [9 B, P6 G0 D, x+ |  i  R" U

MATERIALS AND METHODS

Transgenic Mouse Lines

Nestin promoter¨Cdriven LacZ reporter transgenic mice (pNes-Tg) and mutant SOD1-mimicking human ALS transgenic mice (G93A-SOD1-Tg) (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were used to generate bitransgenic mice containing both LacZ and G93A-SOD1 (BiTg) through heterozygous breeding. Transgenic progeny were identified by regular polymerase chain reaction amplification of tail DNA using specific primers. Like mutant G93A-SOD1 transgenic mice, the BiTg mice had ALS-like disease onset and disease progression at 70 to 90 and 100 to 130 days of age, respectively. Because mutant SOD1-mediated motor neuron degeneration in ALS is inherited in an autosomal-dominant fashion, the transgenic mice used in this study were heterozygous. Age-matched littermates of pNes-Tg mice were used as controls. The experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the National Institutes of Health guideline for the care and use of laboratory animals.3 w  C& n# B; d4 {6 U" U8 V+ |
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In Vivo 5-Bromodeoxyuridine Labeling

Bromodeoxyurindine (BrdU) at 50 mg/kg per day was intraperitoneally (IP) administered for 4, 9, and 14 days, respectively, to adult BiTg and age-matched pNes-Tg mice. Spinal cords were dissected out 1 day after the last injection of BrdU and processed for BrdU immunohistochemical staining as described in the following section. To identify and define the increased number of NPCs in the dorsal and ventral regions derived from the ependymal zone surrounding the central canal (EZ) of the ALS-like spinal cords, mice were pulsed with 50 mg/kg per day of BrdU IP for 25 days followed by 5 days of chasing. The percentage of BrdU-labeled cells in LacZ-positive cells was determined after LacZ staining and BrdU immunohistochemical staining.& S. [+ ~( E: K; z4 f* w' ^

LacZ Staining, Immunohistochemical Staining, Image Analysis, and Quantification: y- f- O" G, x2 t

The lumbar region of the spinal cord was used to analyze the organization and distribution of NPCs in response to motor neuron degeneration. For LacZ staining, sections (12 µm) were incubated in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) solution for 16 hours at room temperature as previously described. For immunohistochemical staining, sections were incubated in blocking buffer (10% goat serum/0.2% triton X-100 in 1x phosphate-buffered saline , pH 7.5) for 1 hour at room temperature. Primary antibody was then added to the blocking buffer (1:250), and the section was incubated at 4¡ãC overnight. The next day, sections were washed five times (5 minutes each) in 1x PBS (pH 7.5) containing 0.5% triton X-100, followed by incubation with specific fluorescein-conjugated secondary antibody for 2 hours at room temperature. After extensive washes, sections were covered with antifade medium and sealed for fluorescent microscopic analysis. For negative control staining, sections were incubated without primary antibody.1 {4 l+ R5 d& r* E
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All images were collected and analyzed with a Nikon fluorescent microscope E800 equipped with the Spot digital camera and Photoshop software.
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Quantifications of LacZ staining intensity at the EZ of BiTg mouse spinal cord during disease-free, disease-onset, and disease-progression stages compared with age-matched littermate control pNes-Tg mice were performed with the NIH software Image J. At least three sections per mouse and three mice at specific ages were analyzed. The arbitrary units were used to express the LacZ staining intensity of EZ. To quantify the distribution of NPCs in the adult ALS-like mice compared with age-matched controls, all LacZ-positive cells (nuclei) in the dorsal and ventral horn regions (five sections per region per mouse and three mice per group) were manually counted. Statistical analysis was performed using the paired Student¡¯s t-test. p 5 ^3 ]5 q# ~& v

RESULTS

Organization and Distribution of NPCs in the Normal and ALS-Like Adult Mouse Spinal Cord

A combination of LacZ reporter (LacZ), nestin expression (Nestin), and BrdU labeling (BrdU) was used to identify NPCs in the adult ALS-like mouse spinal cord . More significantly, there was an increase of LacZ staining intensity (Figs. 2A¨C2C) in the EZ of the ALS-like mice (BiTg) compared with the age-matched control mice (pNes-Tg), suggesting that there is an increase of NPC proliferation.3 @7 q! C5 W8 {1 F6 l9 m* \8 j
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Figure 1. Identification and characterization of NPCs in the adult ALS-like (BiTg) and normal (pNes-Tg) mouse spinal cord. Identification of NPCs in the ependymal zone surrounding central canal region of adult mouse spinal cord was performed by LacZ staining and nestin immunostaining (A). Characterization of NPCs in the ependymal zone surrounding central canal (B, C), dorsal horn region (D, E), and ventral horn region (F) of pNes-Tg (B, D) and BiTg (C, E, F) mouse spinal cord was conducted by LacZ staining and BrdU labeling. Notably, some of the LacZ positively-stained NPCs in the ependymal zone were labeled with BrdU. In contrast, the NPCs from dorsal and ventral horn regions of the pNes-Tg and BiTg mouse spinal cord were not labeled with BrdU. Abbreviations: ALS, amyotrophic lateral sclerosis; BrdU, bromodeoxyurindine; C.C., central canal; D.H., dorsal horn; NPC, neural progenitor cell; V.H., ventral horn.

Figure 2. LacZ staining intensity in the ependymal zone surrounding the central canal (C.C.) of the pNes-Tg and BiTg mouse spinal cord. The LacZ staining intensity in the ependymal zone surrounding central canal regions of amyotrophic lateral sclerosis-like (B) (BiTg) mouse spinal cord during disease-free (40 days), disease-onset (70 days), and disease-progression (120 days) stages was compared with age-matched littermate control mice (A) (pNes-Tg). (C): The relative LacZ staining intensity in the ependymal zone surrounding central canal regions is shown (five sections per mouse, n = 3 mice; *p
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In addition, we have also identified the LacZ-stained NPCs distributed in other areas of the adult mouse spinal cord (Figs. 1D-1F, 3A-3E). Most NPCs were found in Lamina I¨CIII of the dorsal horns, although there were a few NPCs sparsely distributed in other regions of the normal control spinal cord. Notably, NPCs outside of the EZ were not labeled with BrdU (Figs. 1D¨C1F) in contrast to the NPCs in the EZ (Figs. 1B, 1C), suggesting that the NPCs outside of the EZ were not proliferative. Systematic analyses with different ages of animals showed that there were more NPCs in the dorsal horn region than in the ventral horn region of the spinal cord (Fig. 3). In particular, there were more NPCs in the ALS-like (BiTg) mouse spinal cord than that of the age-matched littermate control mice (pNes-Tg), suggesting that the increased number of NPCs was associated with the motor neuron degeneration in the ALS-like mouse model (Figs. 1D¨C1F, 3).2 J" J0 Z% h# }# A* `

Figure 3. Organization and distribution of NPCs in the lumbar regions of the pNes-Tg and BiTg mouse spinal cord. The representative distribution of NPCs in the dorsal horn region (A, B) and ventral horn region (C) of the pNes-Tg (A) and BiTg (B, C) mouse spinal cord corresponding to clinical disease-free (40 days), disease-onset (70 days), and disease-progression (120 days) stages was characterized by LacZ staining. The number of NPCs in the dorsal horn region (D) and ventral horn region (E) of the adult mouse spinal cord was counted and statistically analyzed from five sections per lumbar region per mouse (n = 3 mice). Abbreviations: BiTg, amyotrophic lateral sclerosis-like; C.C., central canal; D.H., dorsal horn; NPC, neural progenitor cell; pNes-Tg, age-matched littermate control; V.H., ventral horn.' }* ]6 O$ w/ I9 X5 p6 ^! Q1 x! v

Based on the well-established clinical and pathological manifestations, we have analyzed the distribution of NPCs in the spinal cord of the BiTg mice at 40, 70, and 120 days of age, which correspond to disease-free, disease-onset, and disease-progression stages . Notably, there were dynamic changes of NPCs in the spinal cord of the BiTg mice during the ALS-like disease onset and progression compared with the control pNes-Tg mice and BiTg mice at the clinical disease-free stage (Fig. 3). The distribution of NPCs in the dorsal horn and ventral horn regions at different time points is shown in Figures 3D and 3E, respectively. The increased number of NPCs in the dorsal and ventral horn areas during the ALS-like disease onset and progression was not from de novo proliferation, because these NPCs were not labeled with BrdU even up to 15 days of pulsing (Fig. 1). However, these cells were highly migratory and could mobilize an immediate response to spinal cord injury (unpublished data) and motor neuron degeneration in the ALS-like mice. This is the first report on the identification of the dormant NPCs in the dorsal and ventral horn regions of the adult spinal cord. The increased number of NPCs in these areas of the BiTg mouse spinal cord was largely attributed to the migration of pre-existing NPCs from the EZ (Figs. 4¨C6)., E; A( t" H. W' {& d8 w

Figure 4. LacZ staining images demonstrating NPC migration patterns. A few NPCs in control pNes-Tg mice at 70 days of age (A) and increased number of NPCs in BiTg mice during disease-free (B) (40 days of age), disease-onset (C) (70 days of age), and disease-progression (D) (120 days of age) stages migrated out from the ependymal zone surrounding the central canal toward dorsal direction and subsequently to the ventral regions. Abbreviations: BiTg, amyotrophic lateral sclerosis-like; NPC, neural progenitor cell; pNes-Tg, age-matched littermate control.( s2 \6 ^8 V0 ~, K
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Figure 5. Migration and migratory paths of NPCs in the adult mouse spinal cord in response to motor neuron degeneration in ALS-like mice. The migration of NPCs characterized by LacZ staining from the ependymal zone surrounding central canal region to the dorsal direction and subsequently to ventral direction in control pNes-Tg mice (A) (70 days of age) and in ALS-like BiTg mice during disease-free (B) (40 days of age), disease-onset (C) (70 days of age), and disease-progression (D) (120 days of age) stages. The migratory paths of NPCs at normal and clinical disease-free (E), disease-onset (F), and disease-progression (G) stages are shown. Abbreviations: BiTg, amyotrophic lateral sclerosis-like; NPC, neural progenitor cell; pNes-Tg, age-matched littermate control.' A' V: O  F! \8 h# \& g) U
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Figure 6. Identification and determination of the source of increased NPCs in the dorsal and ventral horn regions of the adult BiTg mouse spinal cords in response to motor neuron degeneration in ALS-like mice. Analysis of the source of increased NPCs in the dorsal and ventral horn regions of the ALS-like mouse spinal cords was carried out by 25 days of BrdU pulse labeling and 5 days of chasing experiments. (A¨CD): The BrdU-labeled and LacZ-stained NPCs in the upper region of central canal, dorsal horn, and ventral horn regions of the 115-day-old BiTg mice. (E): The ratio of BrdU-labeled LacZ-positive cells in the 115-day-old BiTg mice is shown (three sections per mouse, n = three mice). Abbreviations: BiTg, amyotrophic lateral sclerosis-like; BrdU, bromode-oxyurindine; NPC, neural progenitor cell; pNes-Tg, age-matched littermate control.8 r3 B2 V- U- Q- [; J9 K! n! ~4 y5 v
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Migration and Migratory Paths of NPCs in the Adult ALS-Like Mouse Spinal Cord During Clinical Disease-Free, Disease-Onset, and Disease-Progression Stages' i6 Z% p+ o! v* U
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In a separate study, we showed that spinal cord injury promoted migration of pre-existing NPCs from the central canal toward the dorsal region, where the lesion occurs (unpublished data). We hypothesized that NPCs generated in the EZ of ALS-like mouse spinal cord might migrate to the ventral direction directly in response to motor neuron degeneration. To this end, BiTg mice were assessed to characterize NPC migration and migratory paths during ALS-like disease onset and progression compared with age-matched littermate control (pNes-Tg) mice. In contrast with our original assumption, NPCs from the EZ did not migrate directly toward the degenerated ventral motor neuron domain in the ALS-like mice. Instead, NPCs initially migrated from the EZ toward the dorsal horn direction, and then some of them migrated to the ventral horn regions (Figs. 4A¨C4D, 5A¨C5D). More significantly, the number of NPCs migrating out from the EZ to the dorsal and from dorsal to ventral regions was dramatically increased during ALS-like disease onset and progression (Figs. 4C, 4D, 5C, 5D). In fact, even in the disease-free stage (40 days of age), the number of NPCs migrating in the dorsal region in ALS-like mice was also increased compared with age-matched control mice (Figs. 4B, 5B, and data not shown). In contrast, there are only a few NPCs migrating out from the EZ to the dorsal horn, and no NPCs were detected migrating further toward the ventral horn in normal adult (pNes-Tg) mouse spinal cord (Figs. 4A, 5A).
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The migratory paths of NPCs in normal clinical disease-free, disease-onset, and disease-progression stages are presented in Figures 5E through 5G based on the analyses of NPC organization and distribution in different stages of ALS-like mice (Figs. 4A¨C4D, 5A¨C5D). To identify and confirm vigorously that the increased number of NPCs in the dorsal and ventral regions was derived from the EZ of the central canal, we further carried out experiments with 25 days of BrdU pulse labeling and 5 days of chasing in the ALS-like (BiTg) mice. We demonstrated that there was an increase of BrdU labeling in the LacZ-positive cells located in the upper central canal (Figs. 6A, 6B, 6E), dorsal horn (Figs. 6C, 6E), and ventral horn (Figs. 6D, 6E) regions. Thus, the increased migration of NPCs from the EZ led to the increased numbers of NPCs in the dorsal and ventral areas of the spinal cord during the ALS-like disease onset and progression (Figs. 3, 4). More interestingly, several NPCs that migrated to the ventral horn regions were in the vicinity of degenerated motor neurons in the adult BiTg mouse spinal cord (Fig. 8F). Although the specific mechanisms of NPC migration and NPC migratory paths in response to motor neuron degeneration remain to be determined, it is likely that these NPCs were mobilized to attempt to functionally replace (repair) the degenerated motor neurons.

Figure 7. CXCR4 expression and NPC migration in the BiTg mouse spinal cords compared with control pNes-Tg mice. Analysis of the potential role of CXCR4 expression in NPC migration was carried out by immunostaining of spinal cord sections with anti-CXCR4 antibody. There was an increase of CXCR4 staining at ALS-like disease onset (B, D) (70 days of age) compared with age-matched control pNes-Tg mice (A, C) (70 days of age). The representative staining in the central canal and adjacent areas (A, B) and dorsal horn regions (C, D) are shown. (E): The percentage of CXCR4-positive cells out of NPCs in the spinal cord sections during disease onset (70 days of age) and progression (120 days of age) is shown (*p
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Figure 8. De novo neurogenesis from NPCs in the BiTg mouse spinal cord compared with pNes-Tg mice. Analysis of neurogenesis from NPCs was carried out by immunostaining of spinal cord sections with the preneuronal marker HuC (A¨CC) and the neuronal marker NeuN (D¨CF) in the ependymal zone surrounding the central canal region (A, D), dorsal horn region (B, E), and ventral horn region (C, F). There was an increased neuronal differentiation, as ALS-like disease onset (data not shown) and progression (G and H) were advanced in the BiTg mice compared with age-matched control pNes-Tg mice (*p

Expression of CXCR4, a SDF-1 receptor, was demonstrated to associate with neural stem cell migration . For this reason, we analyzed the expression of CXCR4 to test its potential role in adult NPC migration in response to motor neuron degeneration in the ALS-like mouse model (BiTg) compared with age-matched littermate control mice (pNes-Tg). We demonstrated that the expression of CXCR4 was dramatically increased in the adult spinal cords of ALS-like mice during disease onset at the age of 70 days (Figs. 7B, 7D) and progression (data not shown) compared with the age-matched control normal mice (Figs. 7A, 7C). There was almost no detection of CXCR4 staining in age-matched littermate control and the disease-free stage of ALS mice (40 days of age) (data not shown). More importantly, some NPCs were shown to express CXCR4 as ALS-like disease onset (Figs. 7B, 7D) and progression (Fig. 7E and data not shown). The percentage of CXCR4-positive staining in the NPCs outside of the EZ in the spinal cords is shown in Figure 7E.* S* f# q. C$ @5 f/ U! X- C5 ?8 M/ i% H

De Novo Neurogenesis From NPCs in Response to Motor Neuron Degeneration During the ALS-Like Disease Onset and Progression
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To further study the potential functionality of the NPCs in response to motor neuron degeneration, we analyzed the possibility of neurogenesis, astrogenesis, and oligogenesis from the NPCs in the ALS-like mouse model. An increase of de novo neurogenesis from NPCs outside of the EZ emerged during the ALS-like disease onset and progression as determined with preneuronal and neuronal markers HuC (Figs. 8A¨C8C, 7G), TuJ1 (data not shown), and NeuN (Figs. 8D¨C8F, 8H), respectively. No neurogenesis was detected in the EZ with these neuronal markers. Similarly, we also analyzed astrogenesis and oligogenesis using the specific astrocyte and oligodendrocyte markers, respectively (Fig. 9). To a large extent, there was no astrogenesis and oligogenesis detected from the NPCs using GFAP (Fig. 9A), NG-2 (Fig. 9B), and Olig-2 (Fig. 9C) immunostaining, respectively. In addition, there was no microgliogenesis from the NPCs as detected with OX-42 immunostaining (Fig. 9D). These results collectively support an increase of de novo neurogenesis from the NPCs in the adult mouse spinal cord, as motor neuron degeneration advanced in the ALS-like mice.1 [8 Q2 B, G% K6 M
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Figure 9. Gliogenesis and NPCs in the BiTg mouse spinal cords compared with pNes-Tg mice. Analyses of astrogenesis (A, B), oligogenesis (C), and microgaliogenesis (D) in the spinal cord of ALS-like (BiTg) mice with specific cell-type markers. No astrogenesis, oligogenesis, or microgaliogenesis was detected from NPCs at the disease-onset (A, B) (70 days of age) and disease-progression (C, D) (115 to 120 days of age) stages. Abbreviations: BiTg, amyotrophic lateral sclerosis-like; NPC, neural progenitor cell; pNes-Tg, age-matched littermate control." R( Z/ l7 e/ ?" c) l5 P7 s
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DISCUSSION: }0 K. `( M6 _6 W$ u- w8 Z

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 . Together, the unique features of the BiTg mice allow us to identify and characterize the responses of NPCs in the adult CNS to motor neuron degeneration in the ALS-like model.6 N9 D  ^1 N8 J  D5 c

The present study using the BiTg mice demonstrates three major findings of NPCs in the adult CNS in relation to ALS-like disease. First, mutant SOD1-mediated motor neuron degeneration enhances NPC proliferation in the EZ of the BiTg mouse spinal cord. Second, 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. Third, 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 the ALS-like mouse model.3 ?: j' g' t5 i
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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 (Figs. 1, 2, 4). Notably, the LacZ staining intensity was evidently increased in the EZ of ALS-like mice (BiTg mice), particularly during the disease onset and progression, compared with age-matched normal littermate controls (Figs. 2, 4). The LacZ staining and BrdU labeling together suggested that there was an increase of NPC proliferation in response to motor neuron degeneration (Figs. 1, 2). Another population of NPCs was distributed sparsely but unevenly across the spinal cord, most of which were in the dorsal horn regions. These NPCs were not proliferative because they were not labeled with BrdU pulsing for 15 days (Fig. 1). Interestingly, the number of NPCs distributed in the dorsal horn region (Lamina I¨CIII areas) was much more than in the ventral horn and other regions (Figs. 1, 3). 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 with normal control mice (Fig. 3). 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 with the ventral horn region remains unknown. Based on the distribution (Fig. 3), migratory patterns (Figs. 4, 5), and long term of BrdU pulse and chase labeling (Fig. 6), 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.
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Compared with the age-matched littermate control pNes-Tg mice, the migration of NPCs from the EZ was greatly enhanced in BiTg mice, even at the clinical disease-free stage (Figs. 4, 5). The maximum migration of NPCs in the BiTg mice across their lifespan occurred at disease-onset stage, during which there was a maximum of NPC distribution in the dorsal horn region (Figs. 4, 5). 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 the dorsal region first and then some migrated to the ventral region subsequently. Such a temporospatially regulated migration pattern of NPCs in the adult spinal cord in response to disease 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 . However, the increased CXCR4 receptor expression in the spinal cords of ALS-like mice seems 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 chemokines/chemokine receptors or other molecules participate in the directionality of NPC migration remains 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.# I& F; d+ _* ]5 X, }! n
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One important issue of the current study that has not been unambiguously resolved is the lineage of the adult NPCs we have identified with the LacZ-positive staining. Apparently, the adult NPCs defined by nestin promoter¨C controlled LacZ reporter staining are different from glial precursor cells (GPCs) identified from embryonic stages in that the GPCs do whereas the NPCs do not express immature and mature markers of astrocyte and oligodendrocytes . 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.& `8 m  R* j. x/ V* N$ M8 f  C, p
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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 (Figs. 8, 9). Enhancement of neurogenesis has been observed in animal models of Alzheimer¡¯s disease . 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 (Fig. 8F), suggesting that some factors 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. Although 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.

ACKNOWLEDGMENTS

This study was supported in part by U.S. Public Health Service Grants AG23923, NS45829, and HL75034 and Muscular Dystrophy Association grant 3334.
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DISCLOSURES

The authors indicate no potential conflicts of interest.
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