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Keywords:

  • fluorescence-activated cell sorting;
  • neural stem cell;
  • Parkinson’s disease;
  • subventricular zone;
  • α-synuclein

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2010) 115, 854–863.

Abstract

α-Synuclein (α-syn) is a key protein in Parkinson’s disease (PD), and its abnormal accumulation is implicated only not in the loss of dopaminergic neurons in the substantia nigra but also in impairment of olfactory bulb (OB) in PD. Olfactory dysfunction could arise from these OB changes as an early symptom in PD. We reported previously the impairment of neuronal stem cell (NSC) proliferation in the subventricular zone, which is upstream of OB in PD models. Reduction of NSC generation could potentially lead to olfactory dysfunction, which is commonly associated with and precedes the motor symptoms by several years in PD. Here, we investigated neurosphere formation in vitro and migration of NSCs in vivo after transduction of α-syn-encoding retroviral vector to characterize the function of α-syn in NSC. Over-expression of α-syn caused less effective formation of neurospheres and induced morphological changes. Fluorescence-activated cell sorting showed diminished NSC cell cycle progression induced by over-expression of α-syn. Intriguingly, suppression of NSC migration along the rostral migratory stream was observed when the α-syn-encoding vector was directly injected into the subventricular zone of mice in vivo. These results indicate that α-syn affects the generation of NSC and suggest that this protein could serve as a tool for the design of potentially useful therapy for PD patients.

Abbreviations used:
BrdU

5′-bromo-2′-deoxyuridine

α-syn

α-synuclein

DA

dopaminergic

eGFP

enhanced green fluorescent protein

FACS

fluorescence-activated cell sorting

GFAP

glial fibrillary acidic protein

GL

glomerular layer

MOI

multiplicity of infection

NS

neurosphere

NSCs

neural stem cells

OB

olfactory bulb

PBS

phosphate-buffered saline

PD

Parkinson’s disease

PSA-NCAM

polysialic acid-neural cell adhesion molecule

RMS

rostral migratory stream

rRV

recombinant retroviral

SGZ

subgranular zone

SVZ

subventricular zone

TH

tyrosine hydroxylase

Missense mutations (A53T, A30P, and E46K) (Polymeropoulos et al. 1997; Kruger et al. 1998; Zarranz et al. 2004) and multiplication of the α-synuclein (α-syn) gene (Singleton et al. 2003; Nishioka et al. 2006) cause rare inherited forms of Parkinson’s disease (PD). Lewy bodies, the pathological hallmark of sporadic PD found in surviving dopaminergic (DA) neurons, is mainly composed of α-syn protein (Shults 2006). Abnormal accumulation of α-syn in various brain regions is involved in the pathogenesis of various neurodegenerative diseases such as PD, dementia with Lewy bodies, and multiple system atrophy, which are termed collectively as α-synucleinopathies. However, the pathophysiological mechanisms through which the accumulated α-syn causes neuronal dysfunctions are not fully understood.

Recently, abnormal deposition of α-syn was demonstrated in fetal nigral transplants in advanced PD brains (Kordower et al. 2008a,b; Li et al. 2008). Neuron-to-neuron (and neuron-to-neural stem cell, NSC) transmission of α-syn was shown experimentally to induce inclusion formation and neuronal cell death (Desplats et al. 2009). Moreover, there is increasing evidence that α-syn might contribute to the neurodegenerative phenotype by interfering with adult neurogenesis (Winner et al. 2004; Crews et al. 2008; Marxreiter et al. 2009). Impaired neurogenesis mainly reflected reduced survival of NSCs in the olfactory bulb (OB) and the hippocampal subgranular zone (SGZ) of human α-syn-transgenic mice (Winner et al. 2004; Crews et al. 2008). These studies suggest that α-syn protein can be transmitted to virtually all brain cells, which in turn affects the fate and function of NSCs and mature neurons. Studies from our laboratories also showed impairment of NSC proliferation in the subventricular zone (SVZ), which is upstream of OB in PD models (Oizumi et al. 2008).

Hyposmia (reduced sensitivity to odors) is a common symptom in PD patients and precedes the emergence of cardinal motor symptoms in most cases (Ross et al. 2008; Ponsen et al. 2009). The presence of twice as many tyrosine hydroxylase (TH)-immunoreactive neurons in the OB of PD patients, compared with the age-matched controls (Huisman et al. 2004), may explain the olfactory dysfunction in PD, because dopamine inhibits olfactory transmission in OB. We reported recently that the Ser129-phosphorylated α-syn deposits were prominently found in the OB of patients with Lewy body-related α-synucleinopathy, while the α-syn rarely co-localized with TH-positive DA cells (Sengoku et al. 2008). Another group reported that α-syn pathology in OB correlated with that in other brain regions in patients with Lewy body-related α-synucleinopathy (Beach et al. 2009). Furthermore, olfactory deficits in the presence of proteinase K-resistant α-syn inclusions were demonstrated in human α-syn-transgenic mice (Fleming et al. 2008).

The present study was designed to further analyze the effect of α-syn on NSCs. Specifically, we investigated neurosphere formation in vitro and migration of NSCs in vivo following the transduction with α-syn-encoding retroviral vector. The proliferative state of NSCs was examined in vitro by using fluorescence-activated cell sorting (FACS) following recombinant retroviral (rRV) vector-mediated over-expression of human α-syn. In other experiments, the human α-syn-encoding vector was injected into the SVZ of mice and its in vivo effect on NSCs migration from the SVZ to OB was analyzed. The differentiation of the genetically modified NSCs was also investigated in OB.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Preparation of rRV vectors

The vesicular somatitis virus G protein-enveloped rRV vectors (Suzuki et al. 2002) were used in this study. We reported previously that this pseudotyped rRV was useful for introducing foreign genes into NSCs in the SVZ of mice in vivo (Yamada et al. 2004). A high-titre rRV vector carrying eGFP cDNA [named as rRV–enhanced green fluorescent protein (eGFP)] or α-syn-ires-eGFP expression cassette (rRV-α-syn-ires-eGFP) was produced as described previously (Suzuki et al. 2002). The titres of both rRV vectors were 1.7 × 108 cells transducing units/mL.

Preparation of neurosphere and transduction with the rRV vector

The fetal striatal tissues (E13.5) were collected and dissociated mechanically with a Pasteur pipette in Dulbecco’s modified Eagle medium/F12 medium (Invitrogen Corp., Carlsbad, CA, USA) containing N-2 supplement (Invitrogen Corp.), 20 ng/mL epidermal growth factor (PeproTech Inc., Rocky Hill, NJ, USA), 20 ng/mL basic fibroblast growth factor (PeproTech Inc.), and antibiotics (penicillin and streptomycin) (Invitrogen Corp.). The isolated NSCs (1.5 × 106 cells/mL) were cultured at 37°C for 7 days. The primary neurosphere (NS), formed at 7 days in vitro, were collected and dissociated mechanically. These NSCs (5 × 104 cells) were then transduced with 1 (calculated multiplicity of infection, MOI = 3.4), 5 (MOI = 17), or 10 μL (MOI = 34) of either rRV-eGFP or rRV-α-syn-ires-eGFP and cultured for another 36 h (for western blotting and FACS) or 7 days (for secondary NS formation).

FACS analysis for cell cycle state of NSC culture

For FACS analysis, 5′-bromo-2′-deoxyuridine (BrdU; 10 μM) was added to the secondary NS culture at 24 h post-transduction with rRV vector. After 12 h of the following culture, the NSCs were stained by anti-BrdU antibody and 7-amino-actinomycin D (BrdU Flow Kit; BD Biosciences, San Jose, CA, USA) and analyzed by using BD FACSAria (BD Biosciences). The signal of a doublet, which was detected when two adjacent cells at G0/G1-phase (2N) passed through the detection module, was carefully distinguished from that of single cell at G2/M-phase (4N).

Stereotaxic injection of rRV vector into the SVZ of mice

Mice were anesthetized with sodium pentobarbital (50 mg/kg body weight, intraperitoneally, i.p.) and positioned in a stereotaxic frame. The skull was exposed, and a small portion of the skull over the SVZ was removed unilaterally using a dental drill. Subsequently, rRV-eGFP or rRV-α-syn-ires-eGFP was injected unilaterally into the SVZ (3 μL, 1.7 mm lateral from the bregma, 3.0 mm below the dural surface, tooth bar = 0.0 mm) through a 5-μL Hamilton microsyringe.

The rRV vector-injected mice were killed at 2, 7, or 21 days post-injection. BrdU was injected (50 mg/kg body weight; i.p.) at 2 h before killing. Mice were deeply anesthetized with sodium pentobarbital (250 mg/kg body weight, i.p.) and perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed from the skull, post-fixed overnight in 4% paraformaldehyde in PBS, and immersed in PBS containing 30% sucrose until sinking. Sagittal sections that included the SVZ, rostral migratory stream (RMS), and OB were cut serially at 20 μm thickness using a cryostat (CM1900, Leica Microsystems, Wetzlar, Germany).

Primary antibodies for immunohistochemistry

The primary antibodies used for immunohistochemistry were as follow; mouse monoclonal anti-human α-syn (clone LB509; diluted at 1 : 100; Invitrogen Corp.), rabbit anti-GFP (1 : 100; Millipore Corp., Temecula, CA, USA), rat anti-BrdU (clone BU1/75; 1 : 400; OBT, Oxford, UK), rabbit monoclonal anti-activated caspase 3 (1 : 500; BD Biosciences), sheep anti-TH (1 : 5,000; Calbiochem, San Diego, CA, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (1 : 10 000; kindly gifted from Dr. Akiyama at Psychiatric Research Institute, Tokyo, Japan), mouse monoclonal anti-polysialic acid-neural cell adhesion molecule (PSA-NCAM) (1 : 2,000; kindly gifted from Dr. Seki at Juntendo University, Tokyo, Japan), rabbit anti-Pax6 (1 : 500; Covance Inc., Emeryville, CA, USA), and mouse monoclonal anti-Tuj1 (neuronal class III β-tubulin) (1 : 500; Covance Inc.) antibodies.

Cell counting

Following transduction with the rRV vectors, the numbers of the secondary NS (> 100 μm in diameter) and GFAP-positive cells in 9 cultures of secondary NS and in 4–6 cultures of GFAP were counted manually at 7 days. The number of GFAP-positive cells was expressed as percentage over that of Hoechst 33258-positive cells. The number of Hoechst 33258-positive cells per culture was 98.2 ± 7.4 (± SEM).

The SVZ, RMS, and OB areas were defined for cell counting as outlined in Fig. 4u (a representative sagittal section stained with hematoxylin). The SVZ was defined as the peristriatal region along the lateral ventricle (1417-μm long; Fig. 4u). The RMS was defined as the region not adjacent to the striatum between the SVZ and OB (1582-μm long; 2345 μm away from the lateral ventricle; Fig. 4u). The OB was defined as the region that can be distinguished anatomically from the neighboring structures. All cells in these areas were counted as described below. The numbers of eGFP-, BrdU-, and activated caspase 3-positive cells in the SVZ were counted manually in five sagittal sections at 80 μm intervals per mouse, and the number of eGFP-PSA-NCAM-double-positive cells in the SVZ was counted manually in three sagittal sections at 160 μm intervals per mouse (in the area 0.88–1.20 mm lateral from the bregma; four mice per group). The numbers of eGFP-, BrdU-, and activated caspase 3-positive cells in the RMS and OB were counted manually in five sagittal sections at 80 μm intervals per mouse, and the number of eGFP-PSA-NCAM-double-positive cells in the RMS and OB was counted manually in three sagittal sections at 160 μm intervals per mouse (in the area 0.64–0.96 mm lateral from the bregma; four mice per group). The numbers of eGFP-, eGFP-TH-double-positive cells in the granular cell layer, mitral cell layer, and glomerular (GL) of the OB were counted manually in 15–18 sagittal sections at 80 μm intervals per mouse, and the number of eGFP-Tuj1-double-positive cells in the GL of the OB was counted manually in three sagittal sections at 160 μm intervals per mouse (in the area 0.12–1.48 mm lateral from the bregma; four mice per group).

image

Figure 4.  Transduction of enhanced green fluorescent protein (eGFP) and α-syn by the recombinant retroviral (rRV) vector in vivo. An overview of the virally labeled eGFP-positive neural stem cells in the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) (a and j). Sections were immunostained for human α-syn (d and m), and merged with eGFP (g and p). The indicated areas in (a), (d), (g), (j), (m), and (p) are enlarged in (b), (c), (e), (f), (h), (i), (k), (l), (n), (o), (q), and (r). All eGFP-positive cells were immunopositive for human α-syn (p–r) in the rRV-α-syn-ires-eGFP mice. Sections were prepared at 7 (s) and 21 (t) days post-injection of rRV-α-syn-ires-eGFP and immunostained for human α-syn. The areas of SVZ, RMS, and OB for cell counting were defined in sagittal sections stained with hematoxylin, as shown in (u). The SVZ was defined as the peristriatal region along the lateral ventricle (1417-μm long). The RMS was defined as the region that is not adjacent to the striatum between the SVZ and OB (1582-μm long; 2345-μm away from the lateral ventricle). The OB was defined as the region that can be distinguished anatomically from the neighboring structures. Scale bar in (g), 200-μm (applicable to a, d, and g); in (i), 20-μm (b, c, e, f, h, and i); in (p), 200-μm (j, m, and p); in (r), 20-μm (k, l, n, o, q, and r); in (t), 20-μm (s, t); and in (u), 500-μm (s).

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Statistical analysis

All data are expressed as mean ± SEM. The two-tailed Student’s t-test was used to evaluate differences between groups. In cell counting in vivo studies, the p value was calculated using the mean values of individual animals. A p value < 0.05 denoted the presence of a statistically significant difference.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Over-expression of α-syn in NSC culture induced by a pseudotyped retroviral vector

The high-titre vesicular somatitis virus G protein-pseudotyped rRV-eGFP and rRV-α-syn-ires-eGFP were produced (the titres of both rRV vectors were 1.7 × 108 cells transducing units/mL). Cultures of NSCs (NS) were prepared from the fetal striatal tissue of normal C57BL/6 mice. The primary NS, formed at 7 days in vitro, were collected and dissociated mechanically. These NSCs (5 × 104 cells) were then transduced with 1 (MOI = 3.4), 5 (MOI = 17), or 10 μL (MOI = 34) of either rRV-eGFP or rRV-α-syn-ires-eGFP and cultured for another 36 h. Western blotting revealed a dose-dependent increase in the protein amount of both eGFP (when transduced with rRV-eGFP and rRV-α-syn-ires-eGFP; Fig. 1a) and α-syn (RV-α-syn-ires-eGFP; Fig. 1b). The protein level of eGFP was comparable between the rRV-eGFP- and the rRV-α-syn-ires-eGFP-transduced cells (Fig. 1a). When the dissociated primary NSCs were transduced with 1 μL of rRV-eGFP, they formed secondary NS in 7 days of the following culture with no critical differences compared with non-transduced control NSCs (Fig. 2a, b, d, and e). However, NSCs transduced with 1 μL of rRV-α-syn-ires-eGFP did not form secondary NS efficiently in 7 days; they were attached to the culture dish and showed apparent morphological changes (Fig. 2c and f). The immunoreactivity to human α-syn was not different between the 36-h and 7-days post-transduction groups (Fig. 2g–j). We counted the number of the secondary NS formed at 7 days following transduction with the rRV vectors (Fig. 2k). NS formation was reduced to 77.3% in the rRV-α-syn-ires-eGFP group compared with the rRV-eGFP group. Transduction with 5 μL of rRV-α-syn-ires-eGFP resulted in a marked reduction in NS formation (3.05% compared with the rRV-eGFP group; Fig. 2k). Next, NSCs attached spontaneously to the culture dish were immunostained for GFAP. As shown in Fig. 2l and m–r, transduction with rRV-α-syn-ires-eGFP resulted in a significant and dose-dependent increase in the number of NSCs, which were strongly immunoreactive to GFAP with fibrillary morphology; each fibrillary structure represented a single cell (Fig. 2p–r). These cells were fewer in number in the rRV-eGFP group (Fig. 2l). The majority of the rRV-eGFP-transduced NSCs were spherical in shape, faintly immunoreactive to GFAP; and each was composed of several cells (Fig. 2m–o).

image

Figure 1.  Western blotting for neural stem cells transduced with recombinant retroviral (rRV)–enhanced green fluorescent protein (eGFP) or rRV-α-syn-ires-eGFP. Cell extracts were prepared form neural stem cells transduced with rRV-α-syn-ires-eGFP (lanes 1–3), rRV-eGFP (lanes 4–6), or no rRV vector (lane 7). Signals were obtained by using anti-eGFP (a), anti-α-syn (b), and anti-actin antibodies (c). The multiplicity of infection was 3.4 (lanes 1 and 4), 17 (lanes 2 and 5), and 34 (lanes 3 and 6). Representative data of four experiments with similar results.

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image

Figure 2.  Effects of α-syn over-expression on secondary neurosphere formation and morphology of neural stem cells (NSCs). Representative photomicrographs of the secondary neurospheres formed at 7 days after transduction of no recombinant retroviral (rRV) vector (a and d; indicated by Non), rRV-enhanced green fluorescent protein (eGFP) (b and e; eGFP), or rRV-α-syn-ires-eGFP (c and f; α-syn). Cultured cells were immunostained for α-syn at 36 h (h) and 7 days (j) following transduction with rRV-α-syn-ires-eGFP, and merged with eGFP fluorescence (g and i). The number of secondary neurospheres was counted (k). Few secondary neurospheres were observed following over-expression of α-syn. The NSCs attached to the culture dish were immunostained for GFAP (l–r). The number of GFAP-positive morphologically–altered NSCs (shown in p–r) was counted (l). The majority of the rRV-eGFP-transduced cells exhibited a sphere-like structure (m–o). Scale bar in (a), 100 μm (applicable to a–c); in (d), 100 μm (d–f); in (g and i), 100 μm (g–j); and in (p), 200 μm (m–r). ***p < 0.001, by two-tailed Student’s t-test.

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FACS analysis for cell cycle state of retrovirally α-syn-transduced NSCs

Next, at 36 h post-transduction, the eGFP-positive NSCs were collected and analyzed by FACS for their cell cycle state. About 40% of NSCs were positive for eGFP at that time point in both rRV-eGFP- and rRV-α-syn-ires-eGFP-transduced groups (5 μL each of the rRV vector was transduced). BrdU (added 12 h prior to the FACS) was used as a marker for S-phase and 7-amino-actinomycin D was used to distinguish G0/G1-phase from G2/M-phase (Figure S1a and b). Control experiments using non-transduced primary NS showed active DNA synthesis at 7 days in vitro (Figure S1c and d). As shown in Fig. 3, over-expression of α-syn significantly increased G0/G1-phase cells (Fig. 3a) and decreased S-phase cells (Fig. 3b), but had no effect on the number of G2/M-phase cells (Fig. 3c). Furthermore, over-expression of α-syn did not alter the number of apoptotic cells (data not shown). These results suggest that ectopic expression of α-syn diminished the cell cycle entry and induced differentiation of NSCs.

image

Figure 3.  Effects of α-syn over-expression on the cell cycle of neural stem cells in vitro. The recombinant retroviral-mediated over-expression of α-syn was manifested by a significant increase in the proportion of G0/G1-phase cells (a) and decrease in the proportion of S-phase cells (b). Data are mean ± SEM of four experiments. *p < 0.05, ***p < 0.001, by two-tailed Student’s t-test.

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Effect of over-expression of α-syn on NSC migration in vivo

Normal mice were injected intra-SVZ with 3 μL of either rRV-eGFP or rRV-α-syn-ires-eGFP (unilaterally), as reported previously (Yamada et al. 2004), and then killed 7 days later. Virally-labeled NSCs were detected immunohistochemically (Fig. 4). The eGFP-positive NSCs were observed in the SVZ, RMS, and OB (Fig. 4a–c, j–l) in both rRV-eGFP and rRV-α-syn-ires-eGFP groups. All eGFP-positive NSCs in rRV-α-syn-ires-eGFP-injected mice co-expressed human α-syn (Fig. 4j–r). We also counted the number of the eGFP-positive cells in the SVZ, RMS, and OB at 2, 7, and 21 days post-injection of either rRV-eGFP or rRV-α-syn-ires-eGFP. As shown in Fig. 5a, there were no significant differences in the number of eGFP-positive cells in any region between the rRV-eGFP and rRV-α-syn-ires-eGFP groups at 2 days post-injection. However, at 7 days post-injection, there was a significantly larger number of eGFP-positive cells in the SVZ (rRV-eGFP group, 70.3 ± 12.8; rRV-α-syn-ires-eGFP group, 154.3 ± 15.8; p = 0.0144) and smaller number in the OB (rRV-eGFP group, 400.0 ± 27.6; rRV-α-syn-ires-eGFP group, 317.0 ± 34.4; p = 0.0374) in the rRV-α-syn-ires-eGFP-injected mice compared with the rRV-eGFP-injected control mice (Fig. 5b). There were no significant differences between the groups at 21 days post-injection; more than 95% of labeled cells have migrated to the OB (Fig. 5c). The immunoreactivity to human α-syn was not different between 7- and 21-days post-injection rRV-α-syn-ires-eGFP groups (Fig. 4s and t). These results indicate that over-expression of α-syn seems to retard the migration of NSCs to the OB.

image

Figure 5.  Numbers of enhanced green fluorescent protein (eGFP)-positive neural stem cells in the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) of recombinant retroviral (rRV)-injected mice at 2 (a), 7 (b), and 21 days post-injection (c). Open bars: rRV-eGFP group; solid bars: rRV-α-syn-ires-eGFP group. There were significant differences in the SVZ and OB at 7 days post-injection (b). Data are mean ± SEM of four experiments. *p < 0.05, by two-tailed Student’s t-test.

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α-syn has no effect on BrdU-positive and apoptotic cells

Next, we investigated cell proliferation and apoptotic cell loss in the SVZ, RMS, and OB of rRV-injected mice. BrdU was injected i.p. at 2 h before killing. α-Syn had no effect on the number of BrdU-positive cells irrespective of the brain region and post-injection time of examination (Figure S2a–c). For example, there were 307.3 ± 22.6 cells (rRV-eGFP group) and 281.3 ± 9.1 cells (rRV-α-syn-ires-eGFP group) in the SVZ and 246.0 ± 50.0 cells (rRV-eGFP group) and 240.7 ± 26.0 cells (rRV-α-syn-ires-eGFP group) in the OB at 7 days post-injection. Similarly, α-syn had no effect on the number of activated caspase 3-immunoreactive cells irrespective of the region and post-injection time of examination (Figure S3). Terminal deoxynucleotidyl transferase-dUTP nick end labeling staining of the sections also found no significant differences (data not shown). These results suggest that the expression of α-syn in NSCs does not alter the proliferation and survival of rRV-α-syn-ires-eGFP-transduced and non-transduced intact NSCs.

Phenotype of virally-labeled NSCs in the SVZ, RMS, and OB

We performed immunostaining for GFAP and PSA-NCAM using the sections prepared at 7 days post-injection of rRV vectors. The eGFP-GFAP-double-positive cells were scarcely observed in any region in both rRV-eGFP and rRV-α-syn-ires-eGFP groups (Fig. 6a and b). We found that a substantial proportion of eGFP-positive cells was immunolabeled with anti-PSA-NCAM in the SVZ (rRV-eGFP group, 68.8 ± 6.58%; rRV-α-syn-ires-eGFP group, 72.4 ± 1.80%; p = 0.6283), RMS (rRV-eGFP group, 71.1 ± 2.74%; rRV-α-syn-ires-eGFP group, 80.2 ± 0.45%; p = 0.0314), and OB in both groups (rRV-eGFP group, 76.2 ± 4.70%; rRV-α-syn-ires-eGFP group, 84.2 ± 2.49%; p = 0.2070) (Fig. 6c and d). Importantly, the number of eGFP-PSA-NCAM-double-positive cells was significantly larger in the SVZ of the rRV-α-syn-ires-eGFP-injected mice compared with the rRV-eGFP-injected mice (Fig. 6e).

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Figure 6.  Immunolabeling of enhanced green fluorescent protein (eGFP)-positive cells with GFAP and polysialic acid-neural cell adhesion molecule (PSA-NCAM) in the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) at 7 days post-injection of recombinant retroviral (rRV) vectors. Sections were immunostained for GFAP (a and b) and PSA-NCAM (c and d), and merged with eGFP in rRV-eGFP group (a and c) and rRV-α-syn-ires-eGFP group (b and d). The indicated areas in upper panels are enlarged in lower panels (a–d). The number of eGFP- and PSA-NCAM-double-positive cells was significantly increased in the SVZ of the rRV-α-syn-ires-eGFP mice (e). Open bars: rRV-eGFP group; solid bars: rRV-α-syn-ires-eGFP group. There was significant difference in the SVZ (i). Data are mean ± SEM of four experiments. **p < 0.01, by two-tailed Student’s t-test. Scale bar in (a) 50 μm (applicable to a–d).

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Differentiation of α-syn-transduced cells in the OB

Finally, we examined the effect of α-syn expression on the localization of NSCs in the granular cell layer, mitral cell layer, and GL in the OB (Figure S4a). The eGFP-positive cells distributed equally in the granular cell layer and mitral cell layer in rRV-eGFP and rRV-α-syn-ires-eGFP mice (data not shown). A significantly larger number of eGFP-positive cells were noted in the GL of rRV-α-syn-ires-eGFP mice than rRV-eGFP mice (Figure S4b). Several of these eGFP-positive cells were immunoreactive with anti-Tuj1 (rRV-eGFP, 51.1 ± 0.79%; rRV-α-syn-ires-eGFP group, 53.0 ± 2.64%; p = 0.5288; Figure S4c and d), but none of them with anti-Pax6 antibody (Figure S4c). Over-expression of α-syn did not influence the differentiation of NSCs into TH-immunoreactive DA neurons (Figure S4e and f).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The present study showed that α-syn regulates cell cycle entry and differentiation of NSCs. The rRV-mediated over-expression of α-syn affected NS formation and morphological phenotype. A significantly higher proportion of α-syn-expressing NSCs exhibited quiescence at G0/G1 state compared with eGFP-alone-expressed control. Hoglinger et al. (2004) reported that the proliferation of NSCs is stimulated by DA afferents in SVZ and hippocampal SGZ of mice, and is impaired in mouse model and patients with PD. Using an experimental model, one recent study has shown augmentation of SVZ proliferation following oral administration of pramipexole, a dopamine receptor agonist (Winner et al. 2009). Our present results raise the possibility that dopamine depletion and α-syn accumulation might have similar consequences, i.e. impairment of NSC generation. The molecular target(s) of α-syn in relation to cell cycle regulation has not been determined so far; however, the present in vitro model is useful to identify the target molecule(s) and signaling pathway by comprehensive microarray study.

Intracerebral injection of rRV vectors revealed that α-syn hindered NSC migration, which was significant at 7 days post-injection but the effect lessened by day 21. We found neither reduced proliferation nor increased apoptotic loss of NSCs following injection of the rRV-α-syn-ires-eGFP, based on anti-BrdU and anti-activated caspase 3 immunoreactivities, respectively. The results also showed that α-syn-over-expressed NSCs were scarcely labeled with GFAP and BrdU at 7 days post-transduction. Accordingly, we speculate that GFAP-positive NSCs (B cells), transduced with rRV-α-syn-ires-eGFP, were perturbed to generate transit-amplifying C cells, and/or that C cells, transduced with rRV-α-syn-ires-eGFP, were compromised to give rise to PSA-NCAM-positive neuroblasts (A cells) that will be incorporated into the ‘chain’ and migrate to the OB. Ectopic α-syn might have affected differentiated neuroblasts. Rostral migration of NSCs is regulated by (i) extracellular and transmembrane proteins, including Slit (Sawamoto et al. 2006), Prokineticin (Ng et al. 2005), ErbB4 (Anton et al. 2004), and Eph (Conover et al. 2000), and (ii) subsequent reorganization of the cytoskeleton involving Doublecortin (Koizumi et al. 2006), cell division cycle 42 (Wong et al. 2001), and cyclin-dependent kinase 5 (Hirota et al. 2007). The effect of over-expression of α-syn on the intrinsic signaling pathways should be determined in future studies. Artificial recruitment of NSCs induced by intrastriatal infusion of neurotrophic factors including transforming growth factor α (Cooper and Isacson 2004; de Chevigny et al. 2008) is believed to be an alternative strategy to transplantation therapies.

Mice injected with rRV-α-syn-ires-eGFP had a significantly large number of eGFP-Tuj1-double-positive cells in the GL. This is inconsistent with the observation by Marxreiter et al. (2009) that human α-syn (A30P)-transgenic mice harbored a significantly small number of newly generated neurons in the OB. We speculate that modest expression of wild-type α-syn might promote differentiation and/or survival of NSCs/mature neurons in the peripheral structure. Ectopic α-syn had no critical effect on differentiation into TH-immunoreactive DA cells, in agreement with our previous study that α-syn deposits rarely co-localized with TH-positive DA cells in OB of patients with advanced α-synucleinopathy (Sengoku et al. 2008), while it cannot be ruled out that α-syn accumulation causes reduced expression of the DA marker. It is also possible that accumulation of α-syn-expressing cells could result in perturbation of the olfactory neural circuit arrangement, potentially representing an early sign of hyposmia in patients with α-synucleinopathy.

In the present study, we also took advantage of FACS strategy to investigate cell cycle state of NSCs in vitro following transduction with the α-syn-encoding rRV vector. In the field of stem cell research, FACS has been used for efficient isolation and characterization of specific subpopulations of the NSCs derived from mouse fetal brain and embryonic stem cells (Nagato et al. 2005; Pruszak et al. 2007; Pastrana et al. 2009) and elimination of teratoma-forming cells from neural progenitors derived from induced pluripotent stem cells (Wernig et al. 2008). We performed rRV-mediated eGFP labeling of NSCs to meet the FACS analysis simultaneously with over-expression of α-syn, which had a crucial effect on NSCs in vivo (Winner et al. 2004; Crews et al. 2008; Marxreiter et al. 2009). Previous studies showed that impaired neurogenesis in human α-syn-transgenic mice was because of α-syn-induced reduced survival of NSCs in OB and hippocampal SGZ (Winner et al. 2004; Crews et al. 2008) and of terminally differentiated neurons in OB (Marxreiter et al. 2009). The present study demonstrated that over-expression of α-syn did not increase cell death in both in vitro and in vivo studies. This discrepancy might be as a result of the relatively weak expression of α-syn driven by the promoter sequence in 5′-long terminal repeat of the vector; or rather, such modest α-syn expression might have enabled the observation of early-stage NSC changes induced by accumulation of α-syn.

In conclusion, the present study demonstrated that accumulation of α-syn in NSCs exhibited a cell-autonomous influence on the generation of neural progenitors, suggesting that effective elimination of toxic species of α-syn could pave the way to halt and possibly reverse the clinical symptoms of patients with α-synucleinopathy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) to H.M.; Grants-in-Aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health, Labour and Welfare of Japan to H.M.; the Research Grant for Longevity Sciences from the Ministry of Health, Labour and Welfare of Japan to H.M.; and grants (#S0801035) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to H.M.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. FACS analysis for cell cycle state of NSCs.

Figure S2. Numbers of BrdU-immunopositive cells in the SVZ, RMS, and OB of rRV-injected mice at 2 (a), 7 (b), and 21 days post-injection (c).

Figure S3. Number of activated caspase 3-immunopositive cells in the SVZ, RMS, and OB of rRV-injected mice at 2, 7, and 21 days post-injection.

Figure S4. Differentiation of virally transduced NSCs in the OB.

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