NPC1 Gene Deficiency Leads to Lack of Neural Stem Cell Self-Renewal and Abnormal - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Se-Ran Yanga, Sun-Jung Kima, Kyoung-Hee Byunb, Brian Hutchinsonb, Bong-Hee Leeb, Makoto Michikawac, Yong-Soon Leea, Kyung-Sun Kanga作者单位：a Laboratory of Stem Cell and Tumor Biology, Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University, Seoul, Korea;b Department of Anatomy and Neurobiology, College of Medicine, Cheju National University, Cheju, Korea;c Department of Alzheimers Disease Resea

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      【摘要】/ }7 u* g" v: f7 @( m8 u1 D
   Neural stem cells (NSCs) are capable of giving rise to neurons, glia, and astrocytes. Although self-renewal and differentiation in NSCs are regulated by many genes, such as Notch and Numb, little is known about the role of defective genes on the self-renewal and differentiation of NSCs from developing brain. The Niemann-Pick type C1 (NPC1) disease is a neurodegenerative disease caused by a mutation of the NPC1 gene that affects the function of the NPC1 protein. The ability of NSC self-renewal and differentiation was investigated using a model of NPC1 disease. The NPC1 disorder significantly affected the self-renewal ability of NSCs, as well as the differentiation. NSCs from NPC1¨C/¨C mice showed impaired self-renewal ability compared with the NPC1 /  mice. These alterations were accompanied by the enhanced activity of p38 mitogen-activated protein kinases (MAPKs). Further, the specific p38 MAPK inhibitor SB202190 improved the self-renewal ability of NSCs from NPC¨C/¨C mice. This indicated that the NPC1 deficiency can lead to lack of self-renewal and altered differentiation of NSCs mediated by the activation of p38 MAPK, impairing the generation of neurospheres from NPC1¨C/¨C Thus, the NPC1 gene may play a crucial role in NSC self-renewal associated with p38 MAPK.
      【关键词】 Neural stem cells Astrocytes Niemann-Pick type C p mitogen-activated protein kinase
   INTRODUCTION$ I3 w( N* E  `% z" J3 d# ?
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Neural stem cells (NSCs) are multipotent cells that are able to renew themselves and differentiate into neural and glial lineages in vitro. The NSC clonal aggregates, often called neurospheres, may provide an unlimited source of cells for grafting into patients with neurodegenerative diseases .
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Many factors, such as the well-known signaling of Notch and Numb, are crucial in determining the fate of NSCs .
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Niemann-Pick type C1 (NPC1) is an autosomal-recessive, lysosomal lipid storage disease characterized by defective trafficking of intracellular cholesterol and lysosomal accumulation of unesterified cholesterol gangliosides and other lipids .1 |. t2 a+ _! `; k3 b5 m5 ]8 L

There is an increasing interest in the importance of glial cells for neuronal survival. Astrocytes that ensheath the synapses within the CNS are known to play an important role in synapse formation . However, whether deficiency of certain genes may affect self-renewal and differentiation in NSC fate remains unclear. This study investigated whether gene mutation can affect self-renewal of NSCs in NPC knockout mice that are mutated in NPC1 protein-mutated mice and conclusively showed that NSCs isolated from NPC¨C/¨C mice were unable to self-renew and altered differentiation by the activation of p38 MAPK signaling.

MATERIALS AND METHODS; q; b4 Q( |9 M
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Mice
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A breeding pair of BALB/c NPC1NIH mice, heterozygous for NPC1 (NPC1¨C/ ), were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). For genotyping of cultured NSCs prepared from fetal mice, DNA was isolated from tail tips of each mouse and polymerase chain reaction was performed as described in Loftus et al. .  j# ^; Y( e" a3 Z
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Neurosphere Cell Culture3 x/ |  b6 o+ Q/ B! m4 s7 b" ~  q/ h$ I8 |2 `
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For neurospheres, fetal cerebral cells from NPC transgenic mice were collected at day E16. After trypsin dissociation, primary cells were seeded at the concentration of 1 cell per microliter and expanded in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM)/F12 medium (Gibco, Carlsbad, CA, http://www.invitrogen.com) supplemented with epidermal growth factor (EGF) (10 ng/ml) (Roche Applied Science, Mannheim, Germany, https://www.roche-applied-science.com/) and basic fibroblast growth factor (bFGF) (20 ng/ml) (Roche Applied Science), 2% B27 supplement (Gibco), and penicillin-streptomycin-neomycin antibiotic mixture (100 mg/ml, Gibco). The ratio of the number of spheres formed after 7 days (formation of secondary spheres) and 21 days (formation of tertiary spheres) in vitro to the number of cells plated is the NSC frequency. Subsequent passaging of primary spheres was performed by mechanically dissociating collected spheres (centrifuged for 5 minutes at 100g) into a single-cell suspension and replating in basal media containing EGF and bFGF. After NSCs were treated with MAPK inhibitors PD 980590 and SB202102 (Tocris, Avonmouth, U.K., http://www.tocris.com/), they were differentiated by transfer to chamber slides coated with poly-D-lysine (Nunc, Roskide, Denmark, http://www.nuncbrand.com) in basal medium with 1% fetal bovine serum (Gibco) for 5 to 6 days and then assessed by immunocytochemistry for astrocytes.

Immunocytochemistry

Neurospheres were fixed immediately in 4% paraformaldehyde and preincubated for 1 hour with normal goat serum (10%) (Zymed Laboratories Inc, San Francisco, http://www.zymed.com). The neurospheres were performed as described previously, using known marker for astrocytes (anti¨Cglial fibrillary acidic protein , 1:200, Chemicon, Temecula, CA, http://www.chemicon.com), and were incubated with peroxidase-conjugated secondary antibody (anti-mouse TRITC, 1:200, Zymed Laboratories Inc). Images were captured on a Nikon Diaphot microscope using a Progress camera.) _  L2 j- l' j' P) Z1 q" T

Western Blot Analysis
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Neurosphere-derived cells were lysed with a buffer (150 Mm NaCl, 20 Mm Tris-HCl, 1 mM EDTA) containing protein inhibitors (1 µg/ml aprotonin, 1 µM leupeptin, 1 mM PMSF) and protease inhibitors (1 mM NaOV3, 1 mM NaF). Collected proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, hybridized with antibody to GFAP (1:1,000, Chemicon), MKK3 (1:1,000, Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), pMKK3/6 (1:1,000, Cell Signaling Technology), and p38 (1:1,000, Cell Signaling Technology), phospho-p38 (1:1,000, Promega, Madison, WI, http://www.promega.com), phospho-ERK1/2 (1:1,000, Promega), ERK1/2 (1:1,000, Zymed Laboratories Inc), and B-actin (1: 5,000, Sigma), and detected by chemiluminescence.

Statistical Analysis

Statistical analysis was performed using analysis of variance followed by Duncan¡¯s multiple-range test.* B- s$ h" o: r$ @
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RESULTS
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Multipotent Neurospheres from NPC¨C/¨C Mice Significantly Showed Lack of Self-Renewal Ability
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NSC self-renewal in the presence of a deficiency of NPC1 gene was assessed in isolated cells from brain cerebral cortices of NPC1 / , NPC1 /¨C, and NPC1¨C/¨C mice at embryonic day 16 (E16). The genotype was determined by tail DNA analysis (Fig. 1). The cells were plated as single cells at a density of 5 x 104 cells/ml in 24 wells with DMEM/F12 medium containing bFGF and EGF. After incubation for 7 days, cerebral cells from NPC1 / , NPC1 /¨C, and NPC1¨C/¨C mice generated multipotent neurospheres, but neurospheres from NPC1 /¨Cand NPC1¨C/¨C mice were formed at significantly lower numbers and smaller diameters (p

Figure 1. Genotyping analysis with tail-DNA by polymerase chain reaction as described in Materials and Methods. NPC¨C/¨C is presented in 475 bp, NPC /  in 173 bp, and NPC /¨C in both 475 bp and 173 bp. Abbreviation: NPC1, Niemann-Pick type C.
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Figure 2. Neural stem cells (NSCs) require Niemann-Pick type C (NPC) to self-renew normally. Images show typical neurospheres that formed after 7 days from E16 fetal mice brain. (A): Generation of NPC¨C/¨C E16 cerebral cells; the cells that formed multipotent neurospheres were significantly reduced relative to wild-type cells. (B, C): Self-renewal capacity is expressed as the number and diameter of secondary and tertiary neurospheres generated per primary neurospheres on subcloning.8 j" L) L: u6 o  r* |3 ?
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NPC1 Deficiency¨CMediated Activation of the MAPK Impaired the Generation of Neurospheres from NPC1¨C/¨C

Because several different MAPK-dependent pathways mediate the cell differentiation of astroglia, we extracted and examined several MAPK pathway¨Crelated proteins from NSCs from NPC1 / , NPC1 /¨C, and NPC1¨C/¨C mice. The levels of MKK3, p38, pMKK3/6, and pp38 in the NSCs from NPC1 /¨Cand NPC¨C/¨C were significantly higher than those in the NPC1 /  (Fig. 3A). Moreover, the levels of ERK1/2 and phosphorylated ERK1/2 were also increased in NSCs from NPC1¨C/¨Ccompared with those in the cells from NPC1 /  (Fig. 3C).# H% W/ p5 \. W4 _) N7 S5 a* d

Figure 3. Western blotting results from Niemann-Pick type C (NPC) neurosphere-derived cell. (A): Expression of protein level with various antibodies, GFAP, pMKK3/6, MKK3, pp38, p38, pERK1/2, and ERK1/2 in wild-type, NPC /¨C, and NPC ¨C/¨C mice (protein, 25 µg/well). Values are the mean ¡À standard deviation for three to five independent experiments. (B, C): Protein level was determined by densitometry programs, Image J analysis software. Values are the mean ¡À standard deviation for three to five independent experiments. (B): Protein expression of GFAP/ß-actin. (C): Open bar represented expression of pERK1/ERK1, and closed bar represented expression of pERK2/ERK2.- a8 y3 [  ~* H6 Z3 z5 L
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Self-Renewal Ability of NSCs Was Improved by p38 MAPK Inhibitor in NPC¨C/¨C Mice¨CDerived Neurospheres5 W0 W% k! }/ m  Z
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To determine if the higher levels of MAPK were impairing the self-renewal, proliferation, and differentiation of the neurospheres from NPC1¨C/¨Cmice, NSCs were treated with a MAPK kinase (MEK) inhibitor (2 µM PD98059) or a p38 MAPK inhibitor (2 µM SB202190), respectively. When cells were treated with SB202190, they gave rise to larger secondary and tertiary neurospheres and significantly greater numbers of neurospheres compared with the untreated neurospheres from NPC¨C/¨C mice. Treatment with the MEK inhibitor PD98059 also could increase sizes of neurospheres from NPC¨C/¨C mice. However, there was no significant difference in the number of neurospheres between the PD98059-treated group and control group (Fig. 4A). Quantitative results of these treatments are shown in Figures 4B and 4C.
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Figure 4. MAPK inhibitors increase neurosphere formation from derived cells in NPC¨C/¨C. (A): Cerebral cells from E16 NPC¨C/¨C mice were dissociated and cultured to generate neurospheres in the presence of MAPK kinase inhibitor (2 µM PD98059) and p38 MAPK inhibitor (2 µM SB202190). (B, C): Self-renewal capacity is expressed as the number and diameter of secondary and tertiary neurospheres generated per primary neurosphere on subcloning. Abbreviations: MAPK, mitogen-activated protein kinase; NPC1, Niemann-Pick type C., f3 C' M+ u5 s7 `7 V- H* W4 }
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The effect of PD98059 and SB202190 treatment on the expression levels of the proteins in the NSCs from NPC1¨C/¨C mice was assessed. As shown in Figure 5, protein expression of GFAP did not change after either treatment; however, the activation of the phosphorylated MAPK-related proteins, including pMKK3/6, pp38 in NPC1¨C/¨C cells treated with SB202190, was significantly inhibited. Even though the phosphorylated pERK was highly activated in NPC /¨Cand NSCs from NPC¨C/¨C compared with NPC / , this was not the case after treatment with PD98059 in NSCs from NPC¨C/¨C9 {! K# x/ y+ `! Z, _

Figure 5. (A): Expression of GFAP, pMKK3/6, pp38, and pERK1/2 was reduced in the neurosphere-derived cells treated with MAPK inhibitors PD98059 (MAPK kinase inhibitor) and SB202190 (P38 inhibitor). Western blotting was done on neurosphere-derived cells (25 µg of protein) from each mouse. (B): Protein level of pp38/p38 was determined by densitometry programs, Image J analysis software. Values are the mean ¡À standard deviation for three to five independent experiments. Abbreviations: MAPK, mitogen-activated protein kinase; NPC1, Niemann-Pick type C.9 J! B6 b5 u. C8 L* z
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NSCs from NPC1 / and NPC1¨C/¨C Mice Give Rise to Different Astrocytes
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To examine differentiation ability into astrocytes from neurospheres derived from NPC /  or NPC¨C/¨Cmice, differentiation of neurospheres was induced by adding 1% fetal bovine serum to culture medium in the absence of bFGF and EGF. As shown in Figure 6, the NSCs from NPC1 / and NPC1¨C/¨C mice showed a different pattern of immunoreactivity against GFAP, a marker of astrocytes. Astrocytes differentiated from NSCs of NPC1 /  showed typical polygonal morphology. The astrocytes derived from NSCs of NPC1¨C/¨C had larger diameters,
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Figure 6. Generated neurospheres were differentiated by 1% fetal bovine serum; after 7 days, the spheres were differentiated into astrocytes (GFAP) between wild-type ( / ) and knockout (¨C/¨C). Scale bars = 100 µm. appeared clumpy, and had rounded appearance, results that are consistent with those of literature reports .

DISCUSSION# r/ h; `! V3 \* }0 |

Self-renewal of NSCs is important to progression into multipotent lineages and maintenance of adult tissue. The polycomb family transcriptional repressor Bmi-1 has been suggested to be a requirement for self-renewal and multiple tissues, including CNS and peripheral nervous system in NSCs . However, studies have not been directed at the identification of how the implications of deficiency of certain genes can affect NSCs of self-renewal and differentiation in neurodegenerative diseases. Here, we investigated self-renewal and differentiations of NSCs using one of the neurodegenerative diseases, NPC1 animal model.

To determine whether NPC1 gene deficiency can cause self-renewal of NSCs, NSCs from mice brain at E16 were isolated and characterized. It became clear that the NSCs from NPC¨C/¨C mice lacked the ability to self-renew compared with those from the NPC / mice. Based on previous results . Further insight into the mechanism of MAPK-mediated self-renewal of NSCs was sought by studies involving the MAPK inhibitors SB 202190 for p38 MAPK and PD 98059 for pERK kinase in the NSCs from NPC¨C/¨C mice. The self-renewal ability of NSCs from NPC¨C/¨C was recovered when cells were treated with SB202190, p38 MAPK inhibitor but not PD 98059, pERK kinase inhibitor. These data suggest that hyperactivation of MKK3/6, p38 MAPK but not ERK1/2 might be related to generation and differentiation of NSCs from NPC1¨C/¨C mice. This is evidence for the possibility that there is a link between the lack of self-renewal of NSCs and some of the neuropathological symptoms seen in NPC1 patients. Thus, anti-MAPK agents could be of therapeutic importance in patients with the NPC1 disorder.

Astrocytes are considered a most important part of neurogenesis, which regulates synapse formation and synaptic transmission, reinforcing the emerging view that astrocytes have an active regulatory role rather than merely supportive roles traditionally assigned to them in the mature CNS  reported that neuropathologic alterations of astrocytes are presented in NPC1 disease. Consistent with this, the absence of NPC1 gene leads to a significant number of different phenotypes of astrocytes when NSCs from NPC¨C/¨C mice are differentiated. We suggest that these morphologically altered astrocytes are due to the deficiency of NPC1 gene through the activation of the p38 MAPK. Thus, NPC1 deficiency leads to lack of self-renewal ability and altered morphology of astrocytes through the activation of p38 MAPK, suggesting that p38 MAPK inhibitors may be an effective method for increasing self-renewal of NSCs for clinical application in NPC1 disease. It is clear that a greater understanding of the exact mechanism of self-renewal and the function of the NPC1 protein should provide better insights into the application of stem cell therapy in neurodegenerative diseases. We conclude that the NPC1 gene may be a candidate gene for controlling self-renewal of NSCs throughout life.

Figure 7. Schematic diagram for expression and function of NPC1 gene is mediated by MAPK in regulation of neuronal stem cell self-renewal and differentiation. Neuronal stem cells were isolated from brain of wild-type and NPC¨C/¨C mice; NPC1 gene affects self-renewal ability and differentiation of neuronal stem cell not through the MAPK (pERK, pp38) depending on NPC1 gene presence. MAPK inhibitors PD 98059 (MAPK kinase inhibitor) and SB202190 (p38 inhibitor) could induce improvement of self-renewal ability in neuronal stem cells from NPC¨C/¨C Abbreviations: MAPK, mitogen-activated protein kinase; NPC1, Niemann-Pick type C.
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ACKNOWLEDGMENTS

This work was supported by a grant from the Korean Science & Engineering Foundation (R01-2005-000-10190-0) and a grant from the Korean Research Foundation (KRF-005-E00076).

DISCLOSURES: L: b6 A$ `1 T. v/ j

The authors indicate no potential conflicts of interest.6 D- A& \0 Z# J% o: P0 d& g/ [
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