Effects of 6-hydroxydopamine on the Adult Neurogenesis of Dopaminergic Neurons in the Mouse Midbrain

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1 Experimental Neurobiology Vol. 18, pages 26 31, June 2009 Effects of 6-hydroxydopamine on the Adult Neurogenesis of Dopaminergic Neurons in the Mouse Midbrain Tae Woo Kim, Hyun Kim and Woong Sun* Department of Anatomy, BK21 Program, Korea University College of Medicine, Seoul , Korea ABSTRACT Recently, restricted progenitor cells have been identified in the substantia nigra (SN) of the rat and mouse, raising a hope that resident stem/progenitor cells may be useful for the therapy of Parkinson s disease. However, it is controversial whether dopamine (DA) neurons can be spontaneously or injury-dependently generated from the endogenous stem cells in the adult brain. Here, we explored the neurogenesis in C57Bl/6 adult mice under the normal and neurotoxin-injured conditions. To monitor adult neurogenesis, we injected 5-bromodeoxyuridine (BrdU) 2 weeks after striatal injection of neurotoxin 6-hydroxydopamine (6-OHDA), and sacrificed the animals 6 weeks after 6-OHDA injection. Whereas the number of BrdU-labeled cells was slightly increased in ipsilateral side than contralateral side of the midbrain, none of BrdUlabeled cells, however, exhibited neuronal markers, NeuN or DCX. Instead, BrdUlabeled cells expressed glial markers such as GFAP (astrocyte), Olig2 (oligodendrocyte) and Iba-1 (microglia). Especially, larger portion of BrdU-labeled cells in the ipsilateral side exhibited microglial marker, indicating that increased cell production in response to the 6-OHDA injection is not related to the adult neurogenesis. Key words: Parkinson s disease, 6-OHDA, mice, adult neurogenesis, dopaminergic neurons INTRODUCTION *To whom correspondence should be addressed. TEL: , FAX: woongsun@korea.ac.kr Received December 3, 2008 Accepted for publication June 16, 2009 Parkinson s disease (PD) is one of the most common neurodegenerative diseases characterized by a progressive loss of substania nigral (SN) dopaminergic neurons (DA), which leads to the typical PD symptoms including tremor, rigidity and bradykinesia. Although a pharmacological replacement of dopamine by L-DOPA alleviates these motor disorders, long-term treatment is complicated by the emergence of the dyskinesia and fluctuations in motor functions. In this respect, replacement of DA producing cells may be more therapeutically beneficial (Winkler et al., 2005). However, clinical outcome of this strategy has been varied, and the use of fetal tissue is limited in terms of availability and ethical concerns (Björklund et al., 2003; Freed et al., 2003). Recently it has been demonstrated that adult mammalian brain maintains the ability for neurogenesis (Van Praag et al., 2002; Doetsch et al., 2005). Especially, the dentate gyrus of the hippocampus and the subventricular zone (SVZ) of the

2 Effects of 6-hydroxydopamine on the Adult Neurogenesis of Dopaminergic Neurons in the Mouse Midbrain 27 lateral ventricles produce 1,000 10,000 new neurons daily in the rat (Cameron et al., 2001). More recently, restricted progenitor cells have been identified in the substantia nigra (SN) of the rat and mice, and it appears that the proliferation of these local neural progenitor cells in SN is increased in response to the SN lesion (Kay et al., 2000), enriched environments (Steiner et al., 2006), and neurotrophic factors such as GDNF (Kirik et al., 2001). These results greatly raised the possibility that endogenous adult neural progenitor cells may be utilized as an alternative source for DA neurons, bypassing the need for cell transplantation. However, these findings are controversial and some studies failed to identify adult-generated DA neurons under normal or pathological conditions (Kay et al., 2000; Lie et al., 2002; Zhao et la., 2003). Furthermore, most studies were executed with rat, and little is known whether DA neurogenesis is preserved in the other mammalian species including mice. Therefore, here we tested whether DA neurons can be produced in the adult mouse brain either spontaneously or following SN lesion. To selectively damage SN DA neurons, we injected 6- hydroxydopamine (6-OHDA) unilaterally into the striatum. Since striatally innervating DA neurons selectively uptake neurotoxin 6-OHDA, it progressively damages nigral DA neurons (Sauer and Oertel, 1994). This model elicits relatively slow SN DA neuronal degeneration, which is one of the most prominent features of PD. MATERIALS AND METHODS Animals and surgery Adult male C57Bl/6 mice were used. The animals received a chloral hydrate anesthesia (0.5 mg/kg) and were placed in a stereotaxic apparatus (Sterting). The skull was opened with a drill and 6- hydroxydopamine (6-OHDA, 15 μg/1.5 μl, Sigma) dissolved in 1 PBS was unilaterally injected during 10 minutes into the right striatum (A.P mm, M.L 0.2 mm, D.V mm, according to the atlas of Paxinos and Watson). Two weeks after 6-hydroxydopamine injections, the thymidine analog BrdU (Sigma) was given single i.p injections 3 times (200 mg/kg in 0.9% saline) with 2 day intervals. Histology For immunohistochemical analysis, animals were perfused with 4% paraformaldehyde, and the brains were postfixed in the same fixative for 24 hour. Brains were the cryoprotected in 30% sucrose in PBS and sectioned (40 μm), and subsequent immunostaining was performed by free-floating method. DNA was denatured by incubation in 1 N HCL at 37 o C for 30 min and sections were incubated with 3% BSA in 0.1% triton X-100 in PBS for 30 min. The following primary antibodies were applied overnight: anti-brdu (AbD serotec, 1:500), anti-th (Chemicon, 1:1,000), anti-gfap (Chemicon, 1: 500), Iba1 (Wako, 1:2,000), Olig2 (IBL, 1:1,000). After several washes in PBS, appropriate secondary antibodies were applied for 30 minutes. Subsequently sections were washed, mounted, and observed with confocal laser scanning microscope (Zeiss, Germany). RESULTS Time course changes in the SN DA neuron death after 6-OHDA treatment in mice Because little attempt has been made to quantify and characterize the striatal injection of 6-OHDAinduced mouse PD model, we first characterized the time-course changes in the DA neuronal death in mice. Following a unilateral injection of neurotoxin 6-OHDA into the striatum, the extent of DA neuronal death was determined from serial sections containing SN which are visualized by TH immunohistochemistry (Fig. 1). Quantification of TH immunoreactive (TH+) neurons in the midbrain of 6-OHDA injected side showed a progressive decrease in the total number of TH+ cells, compared with the contralateral side. Especially, the reduction of TH+ cell number was striking between 2 4 weeks after 6-OHDA treatment. These time-course changes were similar to those of previous reports in the rat (Kramer et al., 2004; Mohapel et al., 2005). Treatment of 6-OHDA increases new cell production in the midbrain Following 2 weeks of 6-OHDA treatment when is the peak period of SN neuronal loss, we injected BrdU (50 mg/kg) for 3 times with 2-day interval,

3 28 Tae Woo Kim, et al. Fig. 1. Immunolabeling of tyrosine hydroxylase (TH) in the substantia nigra in midbrain 0 (A), 2 (B), 4 (C), 6 (D) weeks after 6-OHDA injection. Quantifiation of TH-immunoreactive (TH+) dopaminergic neurons in the midbrain (E). Data are expressed as mean ±SE, n=3. *p<0.05 in t-test comparison with control (CON). VTA: ventral tagmental area, SN: substnatia nigra. Fig. 2. BrdU labeling of proliferating cells after 6-OHDA injection. (A) Schematic summary for experimental schedule. Two weeks after 6-OHDA injection, animals received BrdU (200 mg/kg) three times with 24 hr interval. Animals were sacrificed by 6 weeks after 6-OHDA injection. BrdU labeled cells in the contralateral (CON, B) and ipsilateral (IPSI, C) sides of midbrain. (D) Quantification of BrdU+ cells in contralateral and ipsilateral midbrain. and examined whether SN lesion promotes new cell production in the midbrain (Fig. 2A). Six weeks after 6-OHDA injection, animals were sacrificed and the numbers of BrdU-labeled cells were quantified in the contralateral (CON) vs. ipsilateral (IPSI) sides of the midbrain. The number of BrdU-labeled cells in the IPSI side was slightly but significantly increased comparing to the CON side (Fig. 2B D). These results may imply that proliferation of adult neural progenitor cells was up-regulated in response to the SN lesion. Most newly produced cells are differentiated into the non-neuronal glial cells Next, we assessed the fate of these newly produced cells in the CON and IPSI of SN by double immunofluorescence labeling of BrdU with markers for astrocytes (GFAP), oligodendrocytes (Olig2), microglia (Iba1) and neurons (NeuN) (Fig. 3). In CON side, large proportion of BrdU-labeled cells (35%) did not express any markers, suggesting that they maintain at undifferentiated stages. Although 18 26% of BrdU-labeled cells exhibited one of any

4 Effects of 6-hydroxydopamine on the Adult Neurogenesis of Dopaminergic Neurons in the Mouse Midbrain 29 Fig. 3. Double immunofluorescence labeling of BrdU (red) with markers for specific neural cell types (green) such as GFAP (A, astrocytes), Olig2 (B, oligodendrocytes), Iba1 (C, microglia), and NeuN (D, neurons). Nuclei were counter-stained with Hoechst33342 (blue). Proportions of double-labeled cells in the CON (E) and IPSI (F) sides. Numbers indicate the percentage of co-labeled cells. glial markers, we failed to observe any NeuNlabeled, BrdU+ cells. Similarly, we also failed to observe any NeuN and BrdU double labeled cells in the ipsilateral side, indicating that neurons were not produced in the adult midbrain area under normal or lesioned conditions. We also failed to observe any DCX- or TH-labeled BrdU+ cells, while newly produced cells in the dentate gyrus (DG) of hippocampal formation and subventricular zone of lateral ventricle (SVZ)-rostral migratory stream (RMS)-olfactory bulb (OB) systems were readily labeled with these markers (data not shown), suggesting the absence of NeuN+ cells in SN was not due to that they are at different differentiation stages of the neuronal cells, or there was technical problems. While neurons were not generated, substantially larger portion of BrdU+ cells in the IPSI side exhibited microglial marker, suggesting that increased cell proliferation on 2 3 week after 6-OHDA injection was related to the microlglial proliferation. Another noticeable difference we found was that the proportion of undifferentiated BrdU+ cells was markedly reduced, which might indicate that injury condition influenced the glial differentiation of neural progenitor cells. DISCUSSION There are reports demonstrating that DA neurons in the SN is produced spontaneously or in response to the nigral injury induced by neurotoxin MPTP or 6-OHDA in rats and mice (Lie et al., 2002; Zhao et al., 2003; Shan et al., 2006). Here we tested whether striatal injection of 6-OHDA which evokes relatively slow progression of DA neuronal degeneration taking >4-week to complete. There have been great debates on the neurogenic potentials in mammalian midbrain. Comparing to animal PD model generated by striatal injection of 6-OHDA, systemic injection of MPTP or nigral injection of 6-OHDA induce rapid DA neuronal death (Dauer and Przedborski, 2003). As well as time course of neurodegeneration, it appears that these models induce different cell death cascades and different glial reactions (Depino et al., 2003; Hunter et al., 2007). Considering that DA neuronal death in human PD patients is a very slow and progressive process, it is believed that striatal 6-OHDA injection model more faithfully mimic human PD at least in some aspects. To this end, here we examined the possible DA neurogenesis in response to the striatal 6-OHDA injection. However, different from MPTP injection in mice, this model did not promote

5 30 Tae Woo Kim, et al. substantial DA neurogenesis. In fact, although generation of new DA neurons has been demonstrated using systemic injection of MPTP or nigral injection of 6-OHDA in the rat models of PD, it was reported that striatal injection of 6-OHDA did not result in the generation of new DA neurons, similarly to our result from mice (Frielingsdorf et al., 2004). Therefore, it appears that the MPTP and 6-OHDA differentially impact on the proliferation/differentiation of neural progenitor cells in the SN. Although the origin of new DA neurons following SN injury is yet controversial, it is possible that marked and rapid injury of SN by MPTP may promote neural progenitor cell differentiation in the other regions but recruit them to the injured SN, while relatively mild SN lesion by 6-OHDA fails to do so. Supporting this notion, rapid and marked injury of non-neurogenic regions by focal ischemia or traumatic brain injury can promote the proliferation of neural stem/progenitor cell in SVZ, and re-route these newly produced neuronal cells to the injury sites (Komitova et al., 2005; Xu et al., 2005; Zhang et al., 2008). To clarify this issue, it is required to trace the lineage of newly produced DA neurons in the adult brain with local virus infection or genetic manipulations. While DA neurogenesis is unclear in rodents, newts appear to maintain complete potency to regenerate DA neuron in association with stem cell proliferation and genesis of new DA neurons in the midbrain (Parish et al., 2007). Thus, it appears that the ability to produce new DA neurons distinguished or markedly reduced during evolution, and thus it is important to evaluate the extent of new cell production in the human SN for therapeutic application. Because damaged neurons can be labeled with BrdU (Coronas et al., 2004; Hoglinger et al., 2004), we do not rule out the possibility that severe injury induced by the MPTP might promote the nonspecific BrdU labeling in injured neurons. Furthermore, it is known that the fusion of neurons with cells expressing BrdU can produce double labeling of BrdU and neuronal markers in a cell without actual adult neurogenesis (El-Khodor et al., 2003). Such cell fusion rate is different depending on the brain region, and cell fusion in the cerebellum appears to be considerably higher than other brain regions (Johansson et al., 2008). Currently cell fusion rate in the SN is not known. Although the mechanism is yet unclear, microglial cells appear to be major cell type involved in the cell fusion (James et al., 2006). Since we found that 6-OHDA injection enhanced the proliferation of microglial cells in SN, possible fusion of newly produced, BrdU+ microglial cells and damaged neurons can generate double labeled cells without neurogenesis. Considering that DA neurogenesis in the SN is very low even in the MPTP-treated model, it is important to completely exclude above possibilities. Conversely, we cannot rule out the possibility that we missed the low level of DA neurogenesis. Since we tested the neurogenesis on 2 3 week after 6-OHDA injury, DA neurons may be produced with different time windows. Steiner et al. (2006) demonstrated that MPTP-injured rats exhibited functional recovery with increased number of newly produced DA neurons by 7-weeks. Therefore, exploration of longer period after 6-OHDA injection in mice may be required in the future. Considering that the SN DA neurons are critical for the fine tuning of motor performance and the loss of these neurons elicits Parkinson s disease in human, spontaneous renewal of this population received great attention. However, our results with other observations suggest that the ability to generate new DA neurons is greatly limited. Although we failed to observe DA neurogenesis, we did observe that midbrain injury enhanced differentiation of undifferentiated cells into glial cells. Furthermore, treatment of PDGF, BDNF or dopamine D3 receptor agonist is reported to promote neurogenesis in a rat model of PD, suggesting that some inducible factors/neurotransmitter signaling may alter the fate of neural stem cells (Mohapel et al., 2005; Van Kampen and Eckman, 2006). Therefore, identification of new factors to enhance the endogenous DA neurogenic potential may greatly strengthen the possibility to cure PD with endogenous DA neuron production. ACKNOWLEDGMENTS This study was supported by a grant of the Korean Health 21 R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea (A A N A).

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