- Email: [email protected]
2 mouse model of Huntington's disease
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Physical activity fails to rescue hippocampal neurogenesis deficits in the R6/2 mouse model of Huntington's disease Zacharias Kohl a,1 , Mahesh Kandasamy a,1 , Beate Winner a , Robert Aigner a , Claudia Gross a , Sebastien Couillard-Despres a,b , Ulrich Bogdahn a , Ludwig Aigner a,b,⁎, Jürgen Winkler a,⁎ a
Department of Neurology, University of Regensburg, Universitätsstr. 84, 93053 Regensburg, Germany Volkswagen-Foundation Junior Group, University of Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to a
Accepted 9 April 2007
mutation in the huntingtin gene leading to protein aggregation in neurons. The generation
Available online 21 April 2007
of new neurons in neurogenic regions, such as the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus, is affected by these aggregation
Keywords:
processes. In particular, hippocampal neurogenesis is reduced in the R6/2 transgenic mouse
Neurogenesis
model of HD. Since physical activity stimulates adult hippocampal neurogenesis, we
Running
examined whether running is capable to rescue the impaired hippocampal neurogenesis in
Dentate gyrus
R6/2 mice. Proliferation of hippocampal cells measured by proliferating cell nuclear antigen
Huntingtin
(PCNA) marker was reduced in R6/2 animals by 64% compared to wild type mice.
Neuronal precursor
Accordingly, newly generated neurons labeled with doublecortin (DCX) were diminished
Doublecortin
by 60% in the hippocampus of R6/2 mice. Furthermore, the number of newly generated mature neurons was decreased by 76%. Within the hippocampus of wild type animals, a four-week running period resulted in a doubling of PCNA-, DCX-, and bromo-deoxyuridine (BrdU)-labeled cells. However, physical exercise failed to stimulate proliferation and survival of newly generated neurons in R6/2 transgenic mouse model of HD. These findings suggest that mutant huntingtin alters the hippocampal microenvironment thus resulting in an impaired neurogenesis. Importantly, this adverse microenvironment impeded neurogenesis upregulation such as induced by physical exercise. Future studies need to decipher the molecular pathways involved in repressing the generation of new neurons after physical activity in huntingtin transgenic rodents. © 2007 Elsevier B.V. All rights reserved.
⁎ Corresponding authors. Department of Neurology, University of Regensburg, Universitaetsstr. 84, 93053 Regensburg, Germany. Fax: +49 941 941 3005. E-mail addresses: [email protected] (L. Aigner), [email protected] (J. Winkler). 1 Authors contributed equally. 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.04.039
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1.
Introduction
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide repeat expansion within the HD gene encoding an extended polyglutamine tract in the huntingtin protein (The Huntington's Disease Collaborative Research Group, 1993). Clinically, HD is characterized by involuntary choreatic movements, cognitive decline and psychiatric symptoms (Myers et al., 1998). In humans, the disease inducing mutant huntingtin protein contains more than 39 glutamine repeats and forms protein aggregates in neurons (Trottier et al., 1995). Emerging evidence suggests that the mutant protein leads to neuronal damage by a gain of toxic function inducing neuronal loss and gliosis in the neostriatum (de la Monte et al., 1988; Trushina et al., 2004). The presence of neural stem cells in the adult central nervous system (CNS) has comprehensively been documented in rodents and in humans (Altman and Das, 1965; Eriksson et al., 1998). Throughout the mammalian phylogeny, this process is mainly restricted to two neurogenic regions of the CNS, namely the subventricular zone (SVZ) of the lateral ventricle wall and the dentate gyrus (DG) of the hippocampus (Lois and Alvarez-Buylla, 1993). In the subgranular lining of the hippocampal DG, cells divide and continuously generate a pool of neuronal precursor cells that functionally integrate into the overlaying hippocampal granule cell layer (Altman and Das, 1965; Kuhn et al., 1996; van Praag et al., 2002). Interestingly, hippocampal neurogenesis in the adult CNS is modulated by numerous molecular and environmental factors (reviewed in Ming and Song, 2005). Increased physical activity is one of the most robust and specific stimuli for adult neurogenesis in the hippocampus (van Praag et al., 1999; Brown et al., 2003a; Couillard-Despres et al., 2005). After a four-week period of voluntary running, adult healthy mice showed a 2-fold increase in surviving BrdU-labeled neurons in the DG, as compared to non-running mice (van Praag et al., 1999). Moreover, physical exercise was able to reverse ethanol associated reduction of hippocampal cell proliferation (Crews et al., 2004; Redila et al., 2006). Several molecules involved in mediating the exercise-induced neurogenesis have been deciphered. Brain derived neurotrophic factor (BDNF) has been shown to accumulate in the hippocampus after running and furthermore is known to play an important role for the survival of newly generated neurons (Neeper et al., 1996). Stimulation of endogenous neural precursor cell proliferation may be an ideal mean for cell-based therapeutic approaches in neurodegenerative disorders (Lie et al., 2004). Neurogenesis has been shown to be impaired not only in models of HD, but in models of Parkinson's and Alzheimer's disease as well (Hoglinger et al., 2004; Wen et al., 2004; Winner et al., 2004; Gil et al., 2005). To study the potential of an endogenous cell-based strategy in HD, we analyzed the modulation of neurogenesis after increased physical exercise in the widely used R6/2 transgenic mouse model. R6/2 transgenic mice express exon 1 of the human HD gene with 130–150 CAG repeat length (Mangiarini et al., 1996). R6/2 transgenic mice show distinct motor symptoms and cognitive
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deficits at the age of 8 weeks, progressive weight loss and premature death at the age of 3–4 months (Mangiarini et al., 1996; Carter et al., 1999; Murphy et al., 2000). Neuropathological features of R6/2 mice consist of decreased brain size with a reduced number of striatal neurons (Mangiarini et al., 1996; Stack et al., 2005). Inclusions of mutant huntingtin have been observed in neuronal nuclei and cytoplasm of R6/2 mice (Davies et al., 1997; Stack et al., 2005). Moreover, in this model of HD, impaired proliferation and survival of newly generated hippocampal neurons have also been observed (Gil et al., 2005). In the present study, we determined how mutant huntingtin affects proliferation, survival and differentiation of hippocampal neuronal precursor cells using the R6/2 mice as compared to control wild type animals under standard conditions. Since physical exercise is capable to rescue a hippocampal proliferation deficit in toxic models the present study aimed to test this paradigm in an animal model of HD.
2.
Results
2.1.
Mutant huntingtin expression in the DG of R6/2 mice
Stability of the transgene was confirmed and the mean repeat length in animals of both transgenic groups (R6/2non-run, R6/ 2run) was found to be 131.4 ± 3.6 CAG triplets (range: 125–139; p = 0.68). To determine whether mutant huntingtin was expressed in the hippocampus, we used an α-huntingtin antibody detecting the N-terminus of human mutant huntingtin with a repeat expansion N82 repeats (MAB5374; Gutekunst et al., 1999). We detected an intranuclear immunoreactivity in the vast majority of granule cells of the DG in R6/2 animals. The intensity of intracellular mutant huntingtin immunoreactivity within the granule cells was more pronounced towards the molecular layer of the DG. Interestingly, mutant huntingtin was not detected in DCX-expressing neuronal progenitor cells of R6/2 animals (Fig. 1B). No immunoreactivity of mutant human huntingtin was observed in the hippocampus of WT mice (Fig. 1A).
2.2. Hippocampal cell proliferation remained reduced in running R6/2 To study the effect of physical activity on proliferating cells in the DG of the hippocampus, we compared PCNA labeled cells in running and non-running mice of the transgenic (R6/2) and wild type (WT) groups. In WT mice, running doubled the number of PCNA labeled cells detected in the DG of the hippocampus (WTnon-run: 874 ± 441 vs. WTrun: 1549 ± 588; p < 0.05, Table 1, Fig. 2A). Cell proliferation in the DG of R6/ 2non-run mice was decreased to less than 40% of the level found in WTnon-run animals (874 ± 441 in WTnon-run vs. 319 ± 178 in R6/ 2non-run, p < 0.05). In contrast to the strong increase detected in the WT group, the cell proliferation in the DG in R6/2run animals after physical exercise did not significantly change in R6/2non-run mice (319 ± 178 in R6/2non-run vs. 503 ± 192 in R6/2run, p = 0.38). Thus, hippocampal precursor cells of huntingtin
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In accordance with our previous data (Couillard-Despres et al., 2005), WT mice, following physical activity, showed a greater than two-fold increase in the number of DCX-labeled cells (2302 ± 526 in WTnon-run vs. 5499 ± 1272 in WTrun, p < 0.001, Table 1, Fig. 2F). A 50% decrease of neural precursor cells was detected in the DG of R6/2 animals housed under standard conditions (2302 ± 526 cells in WTnon-run vs. 983 ± 254 in R6/2non-run, p < 0.01). In contrast to WT mice, running did not significantly alter the number of DCX positive neural precursor cells in the DG of R6/2 animals (983 ± 254 cells in R6/2non-run vs. 1580 ± 441 cells in R6/2run, p = 0.13, Figs. 2G–J). This finding suggested that physical exercise failed to increase the number of neural precursor cells in the DG of R6/2 mice.
2.3. The decreased survival of newborn hippocampal neurons remained unchanged in running R6/2 mice
Fig. 1 – Expression of mutant huntingtin in the DG of WT (A) and R6/2 mice (B). (A) In the DG of WT mice no specific staining for mutant huntingtin is present. (B) Immunoreactivity of mutant human huntingtin (bright green) can be detected in almost every mature granule cell (blue, nuclear staining) in the DG of an R6/2 animal representing huntingtin aggregates. Mutant huntingtin is not expressed in DCX-positive cells (red) indicating that the transgene is not expressed in developing neurons. It is important to note that there are fewer DCX-immunopositive cells present in the DG of R6/2 mice compared to WT mice. Scale bar represents 20 μm.
mutant animals did not show the expected physiological response of an increased proliferation after physical exercise (Figs. 2B–E).
2.2. Running did not increase the number of neural progenitor cells in R6/2 mice Previously, the neural precursor marker DCX has been established as a valid marker reflecting the amount of neurogenesis (Couillard-Despres et al., 2005). To determine the effect of the running paradigm on the generation of neural precursor cells, we further analyzed the number of DCXlabeled cells in the DG at the end of the running period.
We further analyzed in the four experimental groups the survival of newly generated cells in the DG of the hippocampus by determining the number of labeled cells remaining 4 weeks after BrdU injection. In WT mice, we observed a more than two-fold increase of BrdU-labeled cells in WTrun mice compared to mice from the WTnon-run group (861 ± 247 in WTnon-run vs. 2130 ± 789 in WTrun, p < 0.001, Table 1, Figs. 2K, L, M). To quantify the number of newborn neurons surviving after 4 weeks, colocalization analysis of BrdU with the mature neuron marker NeuN was performed. BrdU-labeled cells detected in WTrun and WTnon-run mice showed a high percentage of neuronal differentiation (neuronal phenotype: 84%–95%). The total number of newly generated neurons, i.e. BrdU+/NeuN+, coexpression was 2.5-fold increased in WTrun animals (759 ± 215 in WTnon-run vs. 1965 ± 676 in WTrun, p < 0.001, Table 1, Fig. 3). This implies that net neurogenesis was strongly stimulated after physical exercise in our WT mice, in agreement with previous studies (van Praag et al., 1999).
Table 1
PCNA+ cells DCX+ cells BrdU+ cells % BrdU+/NeuN+ cells BrdU+/NeuN+ total cell number
WTnon-run
WTrun
R6/2non-run
R6/2run
874 ± 441 2302 ± 526 861 ± 247 88.6 ± 5.1
1549 ± 588a 5499 ± 1272a 2130 ± 789a 93.4 ± 6.5
319 ± 178b 983 ± 254b 204 ± 52b 83.8 ± 2.9
503 ± 192 1580 ± 441 258 ± 47 95.3 ± 3.7c
759 ± 215
1965 ± 676a
172 ± 46b
246 ± 44
Morphological quantification of neurogenesis in the DG of nonrunning and running WT and R6/2 mice comprises the following measures: total number of proliferating cells (PCNA+), number of neural precursor cells (DCX+), number of surviving newly generated cells (BrdU+), percentage of newly generated neurons (% BrdU+/ NeuN+ cells) and total number of new neurons (BrdU+/NeuN+ total cell number). Analyzing the DG volumes of non-running and running WT and R6/2 mice revealed no significant difference between the groups. All measures given as mean ± SD. a Indicates significance comparing WTnon-run vs. WTrun; bindicates significance comparing WTnon-run vs. R6/2non-run; cindicates significance comparing R6/2non-run vs. R6/2run.
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Fig. 2 – Numbers of proliferating and developing neuronal precursor cells are increased in WT, but not in R6/2 mice after 4 weeks of running. (A) Reduced numbers of proliferating cells in the DG of the hippocampus determined by PCNA staining in R6/2run and R6/2non-run animals. Physiological increase of proliferating cells in the DG of WTrun mice. (B–E) Cell proliferation in the DG of WT mice is significantly increased after physical activity. In contrast, R6/2non-run exhibit a reduced proliferation rate compared to WTnon-run that remains unchanged in R6/2run mice. (F) Reduced numbers of DCX-expressing neural precursor cells in R6/2run and R6/2non-run mice. Running increases number of neural precursor cells in WTrun animals. (G–J) DCX-labeled neural precursor cells in R6/2 animals show less dendritic arborization compared to WT. The number of DCX-labeled neural precursors is increased after physical activity in WTrun only but not in R6/2run mice. (K) Reduced surviving newborn BrdU-positive cells in R6/2run and R6/2non-run animals. In contrast, WTrun mice only show an increase of surviving newborn cells. (L–O) BrdU-labeled surviving newborn cells in the DG of the hippocampus are significantly increased in WTrun compared to WTnon-run. R6/2 mice show a strong reduction of BrdU-labeled cell numbers in R6/2non-run compared to WTnon-run and a lack of increase in R6/2run mice. Error bars represent SD, * indicating p < 0.05, n.s. indicating no significant difference. Scale bars represent 50 μm.
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2.4.
Increased TUNEL-positive cells in the DG of R6/2 mice
The detection of terminal deoxynucleotidyltransferasemediated dUTP nick-end labeling (TUNEL)-positive cells in regions of adult neurogenesis has been widely used to determine modulations of cell death (Biebl et al., 2000, 2005). There were more TUNEL stained profiles in the DG of R6/2 mice compared to WT mice indicating an increased cell death in the presence of mutant huntingtin (Fig. 4).
3.
Discussion
3.1. Physical activity did not upregulate hippocampal cell proliferation in R6/2 mice
Fig. 3 – The decreased numbers of newly generated DG neurons in R6/2 mice remain unchanged in R6/2run animals. (A) The total number of newly generated neurons is increased in WTrun animals compared to WTnon-run mice. Error bars represent SD, * indicating p < 0.05; n.s., no significant level. (B) Confocal 3-D analysis shows a double labeling of BrdU (red) and NeuN (green) in a subgranular cell of the DG representing a newly generated mature hippocampal neuron. Scale bar indicates 50 μm.
By analyzing the number of surviving newborn cells in the DG of R6/2non-run animals, we detected a 4-fold decrease of BrdU-labeled cells compared to WTnon-run animals (205 ± 52 in R6/2non-run vs. 861 ± 247 in WTnon-run, p < 0.05, Table 1, Figs. 2L, N). Hence, the net number of newly generated neurons (BrdU+/ NeuN+) showed a reduction of 75% in the R6/2non-run mice compared to WTnon-run animals (172 ± 46 in R6/2non-run vs. 759 ± 215 in WTnon-run, p < 0.01, Fig. 3). Thus, mutant huntingtin expression severely impaired hippocampal neurogenesis. To determine whether the four-week running period resulted in an increase of hippocampal neurogenesis in R6/2 mice, we compared the total number of BrdU-labeled cells in both R6/2 groups. The number of BrdU-labeled cells in R6/2non-run and R6/2run mice was not significantly altered after physical exercise (205 ± 52 in R6/2non-run vs. 258 ± 47 in R6/2run, p = 0.82, Table 1, Figs. 2K, N, O). The percentage of BrdU/NeuN co-labeled cells increased however, revealing a significant shift towards a neuronal phenotype in R6/2run mice (84 ± 2.9% in R6/2non-run vs. 95 ± 3.7% in R6/2run, p < 0.05, Table 1). Nevertheless, the total number of newly generated neurons did not significantly differ in the R6/2run compared to R6/2non-run (172 ± 46 BrdU+/NeuN+ cells in R6/2non-run vs. 246 ± 44 in R6/2run, p = 0.70, Table 1, Fig. 3).
Our study demonstrates that a four-week period of physical activity efficiently stimulates proliferation of neural stem cells in the DG of the hippocampus in WT mice, whereas it failed in the HD mouse model R6/2. The stimulating effect of running on hippocampal cell proliferation has been described initially in C57BL/6 mice using BrdU labeling in a proliferation paradigm (van Praag et al., 1999). The increase of PCNA expressing profiles described in our study (77% in WTrun) is higher than in the initial study (54%; van Praag et al., 1999). But more importantly, running could not rescue the decreased cell proliferation observed in the DG of R6/2 animals at 9 weeks of age. This failure suggests that mechanisms inducing hippocampal neural precursor proliferation following physical exercise were inhibited or neutralized by the presence of mutant huntingtin protein.
Fig. 4 – Increased cell death in the DG of R6/2 mice. TUNEL staining of the DG in WT and R6/2 animals shows an increase of TUNEL-positive profiles in animals carrying the mutant huntingtin (B) compared to WT mice (A). Inserts display TUNEL-positive cells of WT (A) and R6/2 (B) at higher magnification. Scale bar represents 50 μm.
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The proliferation of hippocampal precursor cells in transgenic R6/2non-run mice was assessed at a symptomatic stage (9 weeks) and we detected a 64% reduction of PCNA positive cells compared to WTnon-run. This effect was more pronounced than in a previous study observing only a 36% decrease of PCNA labeled cells in 11.5 week old symptomatic R6/2 animals (Gil et al., 2005) and might be explained due to different staining protocols. Furthermore, in mice of the R6/1 strain that carry the human HD gene with a shorter CAG repeat expansion of about 115, a reduction of hippocampal cell proliferation was observed by approximately 50% at a symptomatic age of 22 weeks (Lazic et al., 2004). Taken together, current data converge in describing a severely reduced hippocampal precursor proliferation at the various disease stages in the presence of mutant huntingtin. What are potential underlying mechanisms for the decreased rate of hippocampal cell proliferation in HD animal models? Mutant huntingtin is involved in a broad variety of intrinsic cell functions including transcriptional regulation, protein–protein interactions, protein trafficking and proteasomal actions (reviewed in Landles and Bates, 2004). While immunoreactivity for mutant human huntingtin was present in the vast majority of granule cells of the DG in R6/2 mice (Fig. 2B), co-labeling with DCX could not be detected (Fig. 1B). This expression pattern for mutant huntingtin within the DG could be due to a different expression level in neural precursor cells compared to mature DG neurons. Alternatively, accumulation of mutant huntingtin in newly generated cells may not be advanced as far as compared to mature neurons. Neurospheres derived from striatal and cortical tissues from neonatal R6/2 mice expressed wild type and mutant expanded huntingtin in the nucleus and cytoplasm of neural precursor and stem cells (Chu-LaGraff et al., 2001). Since proliferation and differentiation of SVZ derived adult neural precursor cells of R6/2 mice were not affected in vitro (Phillips et al., 2005), the impact of mutant huntingtin on the local microenvironment may be more detrimental in the hippocampus than the sole intrinsic presence of huntingtin in adult neural stem cells. Furthermore, disturbed cell–cell interactions and impaired synaptic connectivity were observed within the hippocampus of R6/2 mice (Murphy et al., 2000). Reduced neural stem cell proliferation may also derive from proliferation-inhibiting factors. We have recently demonstrated that transforming growth factor (TGF)-beta1 inhibits neural stem cell proliferation and neurogenesis (Wachs et al., 2006). TGF-beta1 levels might be upregulated in R6/2 mice as a result of pathologyinduced microglia activation. Furthermore, stress is known to downregulate hippocampal cell proliferation (reviewed in Mirescu and Gould, 2006). Increased glucocorticoid levels, indicating elevated stress levels, have been described in R6/2 mice (Bjorkqvist et al., 2006). Hence, our results together with recent reports underscore the crucial role of the microenvironment for the generation of new hippocampal cells in transgenic HD mice.
3.2. The number of hippocampal neuronal precursor cells was not increased in R6/2 mice after physical activity DCX is a valid marker to measure modulation of neurogenesis (Couillard-Despres et al., 2005, 2006). In contrast to WT
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animals, the number of neuronal precursors in R6/2 mice failed to increase after wheel running. We detected a 67% reduction in the number of DCX-labeled cells in the DG of R6/ 2non-run animals in agreement with a previous study (Gil et al., 2005). The hippocampal DCX-expressing cells in R6/2 mice are smaller in size with less dendritic branching (Figs. 2I, J). DCXlabeled cells can be divided in two categories according to their morphology (Brown et al., 2003b; Winner et al., 2004). Based on this distinction, the DCX positive cells in R6/2 mice resemble “early” progenitor cells characterized by short processes oriented parallel to the granule cell layer. This observation is consistent with a previous study who quantified the extent of dendritic branching in DCX-labeled hippocampal neuronal progenitor cells in R6/2 mice and observed a reduction of almost 50% (Phillips et al., 2005). Our findings therefore indicate that, besides the proliferation of hippocampal precursor cells, the maturation towards a neuronal fate is also disturbed by the presence of mutant huntingtin. The vascular endothelial growth factor (VEGF), known to increase the number of DCX-labeled precursor cells in the DG of running mice (Fabel et al., 2003), might be one of the involved factors. However, its impact on hippocampal precursors in R6/2 mice is not yet known.
3.3. Reduced survival of newly generated hippocampal neurons after running in R6/2 The survival of newly generated neurons in the DG was strongly reduced in R6/2 mice by 77% and no rescue effect was detected after running. The reduction of newly generated neurons is even more pronounced than the decrease of hippocampal cell proliferation. This suggests that the survival of newly generated neurons is also diminished besides a reduced cell turnover in the DG. The reduction of survival in standard housing conditions is similar for both R6/2 and R6/1 mice (Gil et al., 2005; Lazic et al., 2006). One factor potentially capable of increasing the survival of newborn hippocampal cells is the neurotrophin brain derived neurotrophic factor (BDNF; Lee et al., 2002). BDNF levels are increased after voluntary exercise in rodents (Neeper et al., 1996), and BDNF seems to have mainly a survival promoting effect on newborn hippocampal neurons (Sairanen et al., 2005). A reduction of cortical and hippocampal BDNF levels was observed in R6/1 mice (Spires et al., 2004). However, a recent study investigating BDNF in the hippocampus of R6/1 mice revealed unchanged BDNF protein levels but decreased BDNF mRNA expression. Moreover, a running paradigm did not result in an increase of BDNF in R6/1 mice in contrast to wild type animals (Pang et al., 2006). Interestingly, we found a more enhanced neuronal fate in surviving newly generated DG cells in R6/2run mice as compared to new ones of the WT mice. This exerciseinduced shift towards mature neurons could be modulated by BDNF independent mechanisms. To further evaluate the underlying mechanism of a reduced number of newly generated neurons in R6/2 mice, we detected an increase in TUNEL-positive profiles in the hippocampus of R6/2 mice. It is known that cell death is physiologically present within neurogenic regions of adult mammalians and that the apoptotic elimination of neural
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progenitors plays an important role in the dynamics of adult neurogenesis (Winner et al., 2002; Kuhn et al., 2005). Furthermore, pathological conditions such as aggregation of alphasynuclein are associated with an increased cell death in neurogenic regions of the adult forebrain (Winner et al., 2004, in press).
7 litters were kept in normal light–dark cycle of 12 h and had free access to food and water. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the local governmental commission for animal health.
3.4.
4.2.
Modulation of hippocampal neurogenesis in R6/2 mice
Running experiment
Several studies failed to induce hippocampal neurogenesis in transgenic HD mice. Asialoerythropoetin, known to be neuroprotective and to promote cell proliferation, failed to increase the number newborn cells in the DG (Gil et al., 2004). Moreover, administration of fibroblast growth factor (FGF)-2 did not affect hippocampal neurogenesis in R6/2 mice, though it increased proliferation of neural precursor cells in the SVZ (Jin et al., 2005). Furthermore, kainic acid induced seizures, also a strong upregulator of hippocampal neurogenesis in mice, were not able to increase hippocampal neurogenesis in R6/2 mice (Phillips et al., 2005). In contrast, complex enriched housing conditions have been reported to increase hippocampal neurogenesis (Kempermann et al., 1997) and was successful in delaying the onset of symptoms in R6/1 and R6/2 mice (van Dellen et al., 2000; Hockly et al., 2002). A recent study reported a higher rate of surviving newborn cells in the DG of R6/1 mice after 20 weeks of environmental enrichment, while the number of surviving new neurons remained unchanged (Lazic et al., 2006). These results suggest that environmental enrichment could partly rescue impairments of hippocampal neurogenesis. Discrepancies observed following the various paradigms to stimulated neurogenesis in the HD mouse models may originate from their influence on different regulators of hippocampal neurogenesis (Olson et al., 2006). In summary, we demonstrate that R6/2 mice show a reduced neurogenesis in the DG of the hippocampus due to decreased proliferation and survival of neural precursor cells. Furthermore, physical activity, which is known to strongly enhance hippocampal neurogenesis in wild type mice, was not able to increase neither the proliferation nor the survival of newly generated neurons in transgenic HD mice. Our findings suggest that the local microenvironment present in the CNS of R6/2 mice affects the generation of new hippocampal neuronal precursors and strongly interferes with their proliferation, their survival and the hippocampal neurogenesis regulatory mechanisms.
WT and R6/2 mice at the age of 5 weeks (weight: 18–21 g) were randomly divided into two groups. The non-running group consisted of one half of the mice (WTnon-run, n = 7; R6/2non-run, n = 8) and was placed in standard housing conditions (up to 5 animals per cage). The running group (WTrun, n = 7; R6/2run, n = 6) was composed of the other half of the mice housed (up to 5 animals per cage) with free access to two running wheels in each cage. Animals were placed in the different housing environments 12 h before the start of the experiment. The running animals had free access to the wheels during 4 weeks. WT and R6/2 mice displayed normal running behavior during the duration of the running period. In particular, R6/2 mice showed no obvious motor deficits compared to WT animals. All animals were sacrificed at the end of the 4-week running period.
4.
Experimental procedures
4.5.
4.1.
Animals
Free-floating sections were treated with 0.6% H2O2 in Trisbuffered saline (TBS: 0.15 M NaCl, 0.1 M Tris–HCl, pH 7.5) for 30 min. For immunohistological detection of the incorporated BrdU, pre-treatment of tissues was performed as described previously (Brown et al., 2003b). Following extensive washes in TBS, sections were blocked with a solution composed of TBS, Triton X100 0.1%, bovine serum albumin 1%, and teleostean gelatine (Sigma, Taufkirchen, Germany) 0.2% for 2 h. This buffer was also used during the incubation with antibodies. Primary antibodies were applied overnight at 4 °C. For
Female B6CBAF1/J mice transplanted with ovaries from female B6CBATg(Hdexon1)62Gpb/1J mice (“R6/2”, Mangiarini et al., 1996) were bred to male B6CBAF1/J mice. All animals were obtained from Jackson Laboratories (Bar Harbor, USA). DNA derived from the tail was used for determining the CAG repeat length in transgenic animals using a polymerase chain reaction (PCR) assay (Mangiarini et al., 1996). Female mice (wild type (WT), n = 14; transgenic (R6/2), n = 14) derived from
4.3.
BrdU labeling
Labeling of dividing cells was performed by intraperitoneal injection of the thymidine analogue BrdU (Sigma, Steinheim, Germany) at 50 mg/kg of body weight using a sterile solution of 10 mg/ml of BrdU dissolved in a 0.9% (w/v) NaCl solution. The BrdU injections were performed daily from day 1 to 5 at the beginning of the running period.
4.4.
Tissue processing
Animals were deeply anesthetized using a ketamine (20.38 mg/ ml), xylazine (5.38 mg/ml) and acepromazine (0.29 mg/ml) mixture. Transcardial perfusion was performed with 0.9% (w/ v) NaCl solution followed by a 4% paraformaldehyde, 0.1 M sodium phosphate solution (pH 7.4). The brains were removed, post-fixed in the paraformaldehyde solution overnight at 4 °C. Tissues were then cryoprotected in a 30% (w/v) sucrose, 0.1 M sodium phosphate solution (pH 7.4). Brains were cut into 40 μm sagittal sections using a sliding microtome on dry ice. Sections were stored at −20 °C in cryoprotectant solution (ethylene glycol, glycerol, 0.1 M phosphate buffer pH 7.4, 1:1:2 by volume).
Immunostaining
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chromogenic immunodetection, sections were washed extensively and further incubated with biotin-conjugated speciesspecific secondary antibodies followed by a peroxidase–avidin complex solution from the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, USA). The peroxidase activity of immune complexes was revealed with a solution of TBS containing 0.25 mg/ml 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, USA), 0.01% (v/v) H2O2, and 0.04% (w/v) NiCl2. Sections were put on Superfrost Plus slides (Menzel, Braunschweig, Germany) and mounted in Neo Mount (Merck, Darmstadt, Germany). For epifluorescence immunodetection, sections were washed extensively and incubated with fluorochrome-conjugated species-specific secondary antibodies for 2 h. Sections were put on slides and mounted in Prolong Antifade kit (Molecular Probes, Eugene, USA). Photodocumentation was realized using a Leica microscope (Leica, Wetzlar, Germany) equipped with a Spot™ digital camera (Diagnostic Instrument Inc, Sterling Heights, USA) and epifluorescence observation was performed on a confocal scanning laser microscope (Leica TCS-NT, Wetzlar, Germany). The following antibodies and final dilutions were used. Primary antibodies: rat α-BrdU 1:500 (Oxford Biotechnology, Oxford, UK), mouse α-PCNA 1:500, goat α-DCX 1:500 (C-18; both Santa Cruz Labs, Santa Cruz, USA), mouse α-NeuN 1:500, mouse-α-huntingtin 1:500 (both Chemicon, Temecula, USA). The mouse-α-huntingtin antibody MAB5374 detects N-terminal mutant human huntingtin with a repeat length N82. Secondary antibodies: donkey α-goat, -mouse or -rat conjugated with Alexa 488 (1:1000, Molecular Probes, Eugene, USA), rhodamine X, or biotin 1:500 (Jackson Immuno Research, West Grove, USA). For labeling cell nuclei, ToPro-3 (1:2000; Molecular Probes, Eugene, OR, USA) was diluted in TBS and directly administered to the sections for 10 min followed by two washing steps. To determine cell death in the DG, the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using the Apoptag In Situ Cell Death Detection Kit (In-tergene, Purchase, NY, USA). The modified protocol for free floating sections was administered which was described previously in detail (Cooper-Kuhn and Kuhn, 2002).
4.6.
Counting procedures
All morphological analyses were performed on blind-coded slides. Every sixth section (240-μm interval) of one hemisphere was selected from each animal and processed for immunohistochemistry. To analyze cell proliferation in the DG, PCNA immunopositive cells were counted. To investigate neurogenesis, the number of neural precursor cells stained for DCX was determined, while the amount of surviving newborn neurons was measured by counting the number of BrdU-labeled cells. All cells that were stained by PCNA, DCX or BrdU antibodies were counted using a 40× objective lens of a light microscope (Leica, Wetzlar, Germany) and multiplied by 6 to obtain an estimate of the total immunopositive cell numbers. The reference volume was determined by tracing the granule cell layer of the hippocampal DG using a semi-automatic stereology system (Stereoinvestigator, MicroBrightField, Colchester, USA). All extrapolations were calculated for one hippocampus
31
and should be doubled to represent the total hippocampal values. To determine the frequency of neuronal differentiation of newborn cells, a series of every sixth brain section (240-μm interval) was stained for BrdU and NeuN by immunofluorescence and examined using the confocal laser microscope equipped with a 40× PL APO oil objective (1.25 numeric aperture) and a pinhole setting that corresponded to a focal plane of 2 μm or less. On average 50 BrdU-labeled cells per animal were analyzed for neuronal differentiation. BrdU-positive cells were characterized as solely BrdU-positive (newborn cells) and BrdU/NeuN double-positive cells (newborn neurons).
4.7.
Statistical analysis
The data are presented as mean values ± standard deviations (SD). Two-way ANOVA (genotype × physical activity) was used to test for differences in PCNA, DCX and BrdU numbers, BrdU/ NeuN percentage, total number of BrdU-labeled neurons and DG volume. For post hoc analysis a Bonferroni's correction was performed. A Student's t-test was applied for analyzing differences in repeat lengths of R6/2non-run and R6/2run animals. Statistical analysis was performed using Prism (Prism Graph Pad Software, San Diego, USA). The significance level was assumed at p < 0.05, unless otherwise indicated.
Acknowledgments The study was supported by the School of Medicine at the University of Regensburg, Germany (ReForM Program) and the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell grant). Beate Winner was supported by a fellowship sponsored of the “Hochschul- und Wissenschaftsprogramm” (University of Regensburg). Mahesh Kandasamy was supported by a fellowship sponsored by the “Bayerische Forschungsstiftung”, Munich, Germany. Ludwig Aigner was supported by the Volkswagen Foundation, Hannover, Germany.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Physical activity fails to rescue hippocampal neurogenesis deficits in the R6/2 mouse model of Huntington's disease Zacharias Kohl a,1 , Mahesh Kandasamy a,1 , Beate Winner a , Robert Aigner a , Claudia Gross a , Sebastien Couillard-Despres a,b , Ulrich Bogdahn a , Ludwig Aigner a,b,⁎, Jürgen Winkler a,⁎ a
Department of Neurology, University of Regensburg, Universitätsstr. 84, 93053 Regensburg, Germany Volkswagen-Foundation Junior Group, University of Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to a
Accepted 9 April 2007
mutation in the huntingtin gene leading to protein aggregation in neurons. The generation
Available online 21 April 2007
of new neurons in neurogenic regions, such as the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus, is affected by these aggregation
Keywords:
processes. In particular, hippocampal neurogenesis is reduced in the R6/2 transgenic mouse
Neurogenesis
model of HD. Since physical activity stimulates adult hippocampal neurogenesis, we
Running
examined whether running is capable to rescue the impaired hippocampal neurogenesis in
Dentate gyrus
R6/2 mice. Proliferation of hippocampal cells measured by proliferating cell nuclear antigen
Huntingtin
(PCNA) marker was reduced in R6/2 animals by 64% compared to wild type mice.
Neuronal precursor
Accordingly, newly generated neurons labeled with doublecortin (DCX) were diminished
Doublecortin
by 60% in the hippocampus of R6/2 mice. Furthermore, the number of newly generated mature neurons was decreased by 76%. Within the hippocampus of wild type animals, a four-week running period resulted in a doubling of PCNA-, DCX-, and bromo-deoxyuridine (BrdU)-labeled cells. However, physical exercise failed to stimulate proliferation and survival of newly generated neurons in R6/2 transgenic mouse model of HD. These findings suggest that mutant huntingtin alters the hippocampal microenvironment thus resulting in an impaired neurogenesis. Importantly, this adverse microenvironment impeded neurogenesis upregulation such as induced by physical exercise. Future studies need to decipher the molecular pathways involved in repressing the generation of new neurons after physical activity in huntingtin transgenic rodents. © 2007 Elsevier B.V. All rights reserved.
⁎ Corresponding authors. Department of Neurology, University of Regensburg, Universitaetsstr. 84, 93053 Regensburg, Germany. Fax: +49 941 941 3005. E-mail addresses: [email protected] (L. Aigner), [email protected] (J. Winkler). 1 Authors contributed equally. 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.04.039
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1.
Introduction
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide repeat expansion within the HD gene encoding an extended polyglutamine tract in the huntingtin protein (The Huntington's Disease Collaborative Research Group, 1993). Clinically, HD is characterized by involuntary choreatic movements, cognitive decline and psychiatric symptoms (Myers et al., 1998). In humans, the disease inducing mutant huntingtin protein contains more than 39 glutamine repeats and forms protein aggregates in neurons (Trottier et al., 1995). Emerging evidence suggests that the mutant protein leads to neuronal damage by a gain of toxic function inducing neuronal loss and gliosis in the neostriatum (de la Monte et al., 1988; Trushina et al., 2004). The presence of neural stem cells in the adult central nervous system (CNS) has comprehensively been documented in rodents and in humans (Altman and Das, 1965; Eriksson et al., 1998). Throughout the mammalian phylogeny, this process is mainly restricted to two neurogenic regions of the CNS, namely the subventricular zone (SVZ) of the lateral ventricle wall and the dentate gyrus (DG) of the hippocampus (Lois and Alvarez-Buylla, 1993). In the subgranular lining of the hippocampal DG, cells divide and continuously generate a pool of neuronal precursor cells that functionally integrate into the overlaying hippocampal granule cell layer (Altman and Das, 1965; Kuhn et al., 1996; van Praag et al., 2002). Interestingly, hippocampal neurogenesis in the adult CNS is modulated by numerous molecular and environmental factors (reviewed in Ming and Song, 2005). Increased physical activity is one of the most robust and specific stimuli for adult neurogenesis in the hippocampus (van Praag et al., 1999; Brown et al., 2003a; Couillard-Despres et al., 2005). After a four-week period of voluntary running, adult healthy mice showed a 2-fold increase in surviving BrdU-labeled neurons in the DG, as compared to non-running mice (van Praag et al., 1999). Moreover, physical exercise was able to reverse ethanol associated reduction of hippocampal cell proliferation (Crews et al., 2004; Redila et al., 2006). Several molecules involved in mediating the exercise-induced neurogenesis have been deciphered. Brain derived neurotrophic factor (BDNF) has been shown to accumulate in the hippocampus after running and furthermore is known to play an important role for the survival of newly generated neurons (Neeper et al., 1996). Stimulation of endogenous neural precursor cell proliferation may be an ideal mean for cell-based therapeutic approaches in neurodegenerative disorders (Lie et al., 2004). Neurogenesis has been shown to be impaired not only in models of HD, but in models of Parkinson's and Alzheimer's disease as well (Hoglinger et al., 2004; Wen et al., 2004; Winner et al., 2004; Gil et al., 2005). To study the potential of an endogenous cell-based strategy in HD, we analyzed the modulation of neurogenesis after increased physical exercise in the widely used R6/2 transgenic mouse model. R6/2 transgenic mice express exon 1 of the human HD gene with 130–150 CAG repeat length (Mangiarini et al., 1996). R6/2 transgenic mice show distinct motor symptoms and cognitive
25
deficits at the age of 8 weeks, progressive weight loss and premature death at the age of 3–4 months (Mangiarini et al., 1996; Carter et al., 1999; Murphy et al., 2000). Neuropathological features of R6/2 mice consist of decreased brain size with a reduced number of striatal neurons (Mangiarini et al., 1996; Stack et al., 2005). Inclusions of mutant huntingtin have been observed in neuronal nuclei and cytoplasm of R6/2 mice (Davies et al., 1997; Stack et al., 2005). Moreover, in this model of HD, impaired proliferation and survival of newly generated hippocampal neurons have also been observed (Gil et al., 2005). In the present study, we determined how mutant huntingtin affects proliferation, survival and differentiation of hippocampal neuronal precursor cells using the R6/2 mice as compared to control wild type animals under standard conditions. Since physical exercise is capable to rescue a hippocampal proliferation deficit in toxic models the present study aimed to test this paradigm in an animal model of HD.
2.
Results
2.1.
Mutant huntingtin expression in the DG of R6/2 mice
Stability of the transgene was confirmed and the mean repeat length in animals of both transgenic groups (R6/2non-run, R6/ 2run) was found to be 131.4 ± 3.6 CAG triplets (range: 125–139; p = 0.68). To determine whether mutant huntingtin was expressed in the hippocampus, we used an α-huntingtin antibody detecting the N-terminus of human mutant huntingtin with a repeat expansion N82 repeats (MAB5374; Gutekunst et al., 1999). We detected an intranuclear immunoreactivity in the vast majority of granule cells of the DG in R6/2 animals. The intensity of intracellular mutant huntingtin immunoreactivity within the granule cells was more pronounced towards the molecular layer of the DG. Interestingly, mutant huntingtin was not detected in DCX-expressing neuronal progenitor cells of R6/2 animals (Fig. 1B). No immunoreactivity of mutant human huntingtin was observed in the hippocampus of WT mice (Fig. 1A).
2.2. Hippocampal cell proliferation remained reduced in running R6/2 To study the effect of physical activity on proliferating cells in the DG of the hippocampus, we compared PCNA labeled cells in running and non-running mice of the transgenic (R6/2) and wild type (WT) groups. In WT mice, running doubled the number of PCNA labeled cells detected in the DG of the hippocampus (WTnon-run: 874 ± 441 vs. WTrun: 1549 ± 588; p < 0.05, Table 1, Fig. 2A). Cell proliferation in the DG of R6/ 2non-run mice was decreased to less than 40% of the level found in WTnon-run animals (874 ± 441 in WTnon-run vs. 319 ± 178 in R6/ 2non-run, p < 0.05). In contrast to the strong increase detected in the WT group, the cell proliferation in the DG in R6/2run animals after physical exercise did not significantly change in R6/2non-run mice (319 ± 178 in R6/2non-run vs. 503 ± 192 in R6/2run, p = 0.38). Thus, hippocampal precursor cells of huntingtin
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In accordance with our previous data (Couillard-Despres et al., 2005), WT mice, following physical activity, showed a greater than two-fold increase in the number of DCX-labeled cells (2302 ± 526 in WTnon-run vs. 5499 ± 1272 in WTrun, p < 0.001, Table 1, Fig. 2F). A 50% decrease of neural precursor cells was detected in the DG of R6/2 animals housed under standard conditions (2302 ± 526 cells in WTnon-run vs. 983 ± 254 in R6/2non-run, p < 0.01). In contrast to WT mice, running did not significantly alter the number of DCX positive neural precursor cells in the DG of R6/2 animals (983 ± 254 cells in R6/2non-run vs. 1580 ± 441 cells in R6/2run, p = 0.13, Figs. 2G–J). This finding suggested that physical exercise failed to increase the number of neural precursor cells in the DG of R6/2 mice.
2.3. The decreased survival of newborn hippocampal neurons remained unchanged in running R6/2 mice
Fig. 1 – Expression of mutant huntingtin in the DG of WT (A) and R6/2 mice (B). (A) In the DG of WT mice no specific staining for mutant huntingtin is present. (B) Immunoreactivity of mutant human huntingtin (bright green) can be detected in almost every mature granule cell (blue, nuclear staining) in the DG of an R6/2 animal representing huntingtin aggregates. Mutant huntingtin is not expressed in DCX-positive cells (red) indicating that the transgene is not expressed in developing neurons. It is important to note that there are fewer DCX-immunopositive cells present in the DG of R6/2 mice compared to WT mice. Scale bar represents 20 μm.
mutant animals did not show the expected physiological response of an increased proliferation after physical exercise (Figs. 2B–E).
2.2. Running did not increase the number of neural progenitor cells in R6/2 mice Previously, the neural precursor marker DCX has been established as a valid marker reflecting the amount of neurogenesis (Couillard-Despres et al., 2005). To determine the effect of the running paradigm on the generation of neural precursor cells, we further analyzed the number of DCXlabeled cells in the DG at the end of the running period.
We further analyzed in the four experimental groups the survival of newly generated cells in the DG of the hippocampus by determining the number of labeled cells remaining 4 weeks after BrdU injection. In WT mice, we observed a more than two-fold increase of BrdU-labeled cells in WTrun mice compared to mice from the WTnon-run group (861 ± 247 in WTnon-run vs. 2130 ± 789 in WTrun, p < 0.001, Table 1, Figs. 2K, L, M). To quantify the number of newborn neurons surviving after 4 weeks, colocalization analysis of BrdU with the mature neuron marker NeuN was performed. BrdU-labeled cells detected in WTrun and WTnon-run mice showed a high percentage of neuronal differentiation (neuronal phenotype: 84%–95%). The total number of newly generated neurons, i.e. BrdU+/NeuN+, coexpression was 2.5-fold increased in WTrun animals (759 ± 215 in WTnon-run vs. 1965 ± 676 in WTrun, p < 0.001, Table 1, Fig. 3). This implies that net neurogenesis was strongly stimulated after physical exercise in our WT mice, in agreement with previous studies (van Praag et al., 1999).
Table 1
PCNA+ cells DCX+ cells BrdU+ cells % BrdU+/NeuN+ cells BrdU+/NeuN+ total cell number
WTnon-run
WTrun
R6/2non-run
R6/2run
874 ± 441 2302 ± 526 861 ± 247 88.6 ± 5.1
1549 ± 588a 5499 ± 1272a 2130 ± 789a 93.4 ± 6.5
319 ± 178b 983 ± 254b 204 ± 52b 83.8 ± 2.9
503 ± 192 1580 ± 441 258 ± 47 95.3 ± 3.7c
759 ± 215
1965 ± 676a
172 ± 46b
246 ± 44
Morphological quantification of neurogenesis in the DG of nonrunning and running WT and R6/2 mice comprises the following measures: total number of proliferating cells (PCNA+), number of neural precursor cells (DCX+), number of surviving newly generated cells (BrdU+), percentage of newly generated neurons (% BrdU+/ NeuN+ cells) and total number of new neurons (BrdU+/NeuN+ total cell number). Analyzing the DG volumes of non-running and running WT and R6/2 mice revealed no significant difference between the groups. All measures given as mean ± SD. a Indicates significance comparing WTnon-run vs. WTrun; bindicates significance comparing WTnon-run vs. R6/2non-run; cindicates significance comparing R6/2non-run vs. R6/2run.
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27
Fig. 2 – Numbers of proliferating and developing neuronal precursor cells are increased in WT, but not in R6/2 mice after 4 weeks of running. (A) Reduced numbers of proliferating cells in the DG of the hippocampus determined by PCNA staining in R6/2run and R6/2non-run animals. Physiological increase of proliferating cells in the DG of WTrun mice. (B–E) Cell proliferation in the DG of WT mice is significantly increased after physical activity. In contrast, R6/2non-run exhibit a reduced proliferation rate compared to WTnon-run that remains unchanged in R6/2run mice. (F) Reduced numbers of DCX-expressing neural precursor cells in R6/2run and R6/2non-run mice. Running increases number of neural precursor cells in WTrun animals. (G–J) DCX-labeled neural precursor cells in R6/2 animals show less dendritic arborization compared to WT. The number of DCX-labeled neural precursors is increased after physical activity in WTrun only but not in R6/2run mice. (K) Reduced surviving newborn BrdU-positive cells in R6/2run and R6/2non-run animals. In contrast, WTrun mice only show an increase of surviving newborn cells. (L–O) BrdU-labeled surviving newborn cells in the DG of the hippocampus are significantly increased in WTrun compared to WTnon-run. R6/2 mice show a strong reduction of BrdU-labeled cell numbers in R6/2non-run compared to WTnon-run and a lack of increase in R6/2run mice. Error bars represent SD, * indicating p < 0.05, n.s. indicating no significant difference. Scale bars represent 50 μm.
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2.4.
Increased TUNEL-positive cells in the DG of R6/2 mice
The detection of terminal deoxynucleotidyltransferasemediated dUTP nick-end labeling (TUNEL)-positive cells in regions of adult neurogenesis has been widely used to determine modulations of cell death (Biebl et al., 2000, 2005). There were more TUNEL stained profiles in the DG of R6/2 mice compared to WT mice indicating an increased cell death in the presence of mutant huntingtin (Fig. 4).
3.
Discussion
3.1. Physical activity did not upregulate hippocampal cell proliferation in R6/2 mice
Fig. 3 – The decreased numbers of newly generated DG neurons in R6/2 mice remain unchanged in R6/2run animals. (A) The total number of newly generated neurons is increased in WTrun animals compared to WTnon-run mice. Error bars represent SD, * indicating p < 0.05; n.s., no significant level. (B) Confocal 3-D analysis shows a double labeling of BrdU (red) and NeuN (green) in a subgranular cell of the DG representing a newly generated mature hippocampal neuron. Scale bar indicates 50 μm.
By analyzing the number of surviving newborn cells in the DG of R6/2non-run animals, we detected a 4-fold decrease of BrdU-labeled cells compared to WTnon-run animals (205 ± 52 in R6/2non-run vs. 861 ± 247 in WTnon-run, p < 0.05, Table 1, Figs. 2L, N). Hence, the net number of newly generated neurons (BrdU+/ NeuN+) showed a reduction of 75% in the R6/2non-run mice compared to WTnon-run animals (172 ± 46 in R6/2non-run vs. 759 ± 215 in WTnon-run, p < 0.01, Fig. 3). Thus, mutant huntingtin expression severely impaired hippocampal neurogenesis. To determine whether the four-week running period resulted in an increase of hippocampal neurogenesis in R6/2 mice, we compared the total number of BrdU-labeled cells in both R6/2 groups. The number of BrdU-labeled cells in R6/2non-run and R6/2run mice was not significantly altered after physical exercise (205 ± 52 in R6/2non-run vs. 258 ± 47 in R6/2run, p = 0.82, Table 1, Figs. 2K, N, O). The percentage of BrdU/NeuN co-labeled cells increased however, revealing a significant shift towards a neuronal phenotype in R6/2run mice (84 ± 2.9% in R6/2non-run vs. 95 ± 3.7% in R6/2run, p < 0.05, Table 1). Nevertheless, the total number of newly generated neurons did not significantly differ in the R6/2run compared to R6/2non-run (172 ± 46 BrdU+/NeuN+ cells in R6/2non-run vs. 246 ± 44 in R6/2run, p = 0.70, Table 1, Fig. 3).
Our study demonstrates that a four-week period of physical activity efficiently stimulates proliferation of neural stem cells in the DG of the hippocampus in WT mice, whereas it failed in the HD mouse model R6/2. The stimulating effect of running on hippocampal cell proliferation has been described initially in C57BL/6 mice using BrdU labeling in a proliferation paradigm (van Praag et al., 1999). The increase of PCNA expressing profiles described in our study (77% in WTrun) is higher than in the initial study (54%; van Praag et al., 1999). But more importantly, running could not rescue the decreased cell proliferation observed in the DG of R6/2 animals at 9 weeks of age. This failure suggests that mechanisms inducing hippocampal neural precursor proliferation following physical exercise were inhibited or neutralized by the presence of mutant huntingtin protein.
Fig. 4 – Increased cell death in the DG of R6/2 mice. TUNEL staining of the DG in WT and R6/2 animals shows an increase of TUNEL-positive profiles in animals carrying the mutant huntingtin (B) compared to WT mice (A). Inserts display TUNEL-positive cells of WT (A) and R6/2 (B) at higher magnification. Scale bar represents 50 μm.
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The proliferation of hippocampal precursor cells in transgenic R6/2non-run mice was assessed at a symptomatic stage (9 weeks) and we detected a 64% reduction of PCNA positive cells compared to WTnon-run. This effect was more pronounced than in a previous study observing only a 36% decrease of PCNA labeled cells in 11.5 week old symptomatic R6/2 animals (Gil et al., 2005) and might be explained due to different staining protocols. Furthermore, in mice of the R6/1 strain that carry the human HD gene with a shorter CAG repeat expansion of about 115, a reduction of hippocampal cell proliferation was observed by approximately 50% at a symptomatic age of 22 weeks (Lazic et al., 2004). Taken together, current data converge in describing a severely reduced hippocampal precursor proliferation at the various disease stages in the presence of mutant huntingtin. What are potential underlying mechanisms for the decreased rate of hippocampal cell proliferation in HD animal models? Mutant huntingtin is involved in a broad variety of intrinsic cell functions including transcriptional regulation, protein–protein interactions, protein trafficking and proteasomal actions (reviewed in Landles and Bates, 2004). While immunoreactivity for mutant human huntingtin was present in the vast majority of granule cells of the DG in R6/2 mice (Fig. 2B), co-labeling with DCX could not be detected (Fig. 1B). This expression pattern for mutant huntingtin within the DG could be due to a different expression level in neural precursor cells compared to mature DG neurons. Alternatively, accumulation of mutant huntingtin in newly generated cells may not be advanced as far as compared to mature neurons. Neurospheres derived from striatal and cortical tissues from neonatal R6/2 mice expressed wild type and mutant expanded huntingtin in the nucleus and cytoplasm of neural precursor and stem cells (Chu-LaGraff et al., 2001). Since proliferation and differentiation of SVZ derived adult neural precursor cells of R6/2 mice were not affected in vitro (Phillips et al., 2005), the impact of mutant huntingtin on the local microenvironment may be more detrimental in the hippocampus than the sole intrinsic presence of huntingtin in adult neural stem cells. Furthermore, disturbed cell–cell interactions and impaired synaptic connectivity were observed within the hippocampus of R6/2 mice (Murphy et al., 2000). Reduced neural stem cell proliferation may also derive from proliferation-inhibiting factors. We have recently demonstrated that transforming growth factor (TGF)-beta1 inhibits neural stem cell proliferation and neurogenesis (Wachs et al., 2006). TGF-beta1 levels might be upregulated in R6/2 mice as a result of pathologyinduced microglia activation. Furthermore, stress is known to downregulate hippocampal cell proliferation (reviewed in Mirescu and Gould, 2006). Increased glucocorticoid levels, indicating elevated stress levels, have been described in R6/2 mice (Bjorkqvist et al., 2006). Hence, our results together with recent reports underscore the crucial role of the microenvironment for the generation of new hippocampal cells in transgenic HD mice.
3.2. The number of hippocampal neuronal precursor cells was not increased in R6/2 mice after physical activity DCX is a valid marker to measure modulation of neurogenesis (Couillard-Despres et al., 2005, 2006). In contrast to WT
29
animals, the number of neuronal precursors in R6/2 mice failed to increase after wheel running. We detected a 67% reduction in the number of DCX-labeled cells in the DG of R6/ 2non-run animals in agreement with a previous study (Gil et al., 2005). The hippocampal DCX-expressing cells in R6/2 mice are smaller in size with less dendritic branching (Figs. 2I, J). DCXlabeled cells can be divided in two categories according to their morphology (Brown et al., 2003b; Winner et al., 2004). Based on this distinction, the DCX positive cells in R6/2 mice resemble “early” progenitor cells characterized by short processes oriented parallel to the granule cell layer. This observation is consistent with a previous study who quantified the extent of dendritic branching in DCX-labeled hippocampal neuronal progenitor cells in R6/2 mice and observed a reduction of almost 50% (Phillips et al., 2005). Our findings therefore indicate that, besides the proliferation of hippocampal precursor cells, the maturation towards a neuronal fate is also disturbed by the presence of mutant huntingtin. The vascular endothelial growth factor (VEGF), known to increase the number of DCX-labeled precursor cells in the DG of running mice (Fabel et al., 2003), might be one of the involved factors. However, its impact on hippocampal precursors in R6/2 mice is not yet known.
3.3. Reduced survival of newly generated hippocampal neurons after running in R6/2 The survival of newly generated neurons in the DG was strongly reduced in R6/2 mice by 77% and no rescue effect was detected after running. The reduction of newly generated neurons is even more pronounced than the decrease of hippocampal cell proliferation. This suggests that the survival of newly generated neurons is also diminished besides a reduced cell turnover in the DG. The reduction of survival in standard housing conditions is similar for both R6/2 and R6/1 mice (Gil et al., 2005; Lazic et al., 2006). One factor potentially capable of increasing the survival of newborn hippocampal cells is the neurotrophin brain derived neurotrophic factor (BDNF; Lee et al., 2002). BDNF levels are increased after voluntary exercise in rodents (Neeper et al., 1996), and BDNF seems to have mainly a survival promoting effect on newborn hippocampal neurons (Sairanen et al., 2005). A reduction of cortical and hippocampal BDNF levels was observed in R6/1 mice (Spires et al., 2004). However, a recent study investigating BDNF in the hippocampus of R6/1 mice revealed unchanged BDNF protein levels but decreased BDNF mRNA expression. Moreover, a running paradigm did not result in an increase of BDNF in R6/1 mice in contrast to wild type animals (Pang et al., 2006). Interestingly, we found a more enhanced neuronal fate in surviving newly generated DG cells in R6/2run mice as compared to new ones of the WT mice. This exerciseinduced shift towards mature neurons could be modulated by BDNF independent mechanisms. To further evaluate the underlying mechanism of a reduced number of newly generated neurons in R6/2 mice, we detected an increase in TUNEL-positive profiles in the hippocampus of R6/2 mice. It is known that cell death is physiologically present within neurogenic regions of adult mammalians and that the apoptotic elimination of neural
30
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progenitors plays an important role in the dynamics of adult neurogenesis (Winner et al., 2002; Kuhn et al., 2005). Furthermore, pathological conditions such as aggregation of alphasynuclein are associated with an increased cell death in neurogenic regions of the adult forebrain (Winner et al., 2004, in press).
7 litters were kept in normal light–dark cycle of 12 h and had free access to food and water. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the local governmental commission for animal health.
3.4.
4.2.
Modulation of hippocampal neurogenesis in R6/2 mice
Running experiment
Several studies failed to induce hippocampal neurogenesis in transgenic HD mice. Asialoerythropoetin, known to be neuroprotective and to promote cell proliferation, failed to increase the number newborn cells in the DG (Gil et al., 2004). Moreover, administration of fibroblast growth factor (FGF)-2 did not affect hippocampal neurogenesis in R6/2 mice, though it increased proliferation of neural precursor cells in the SVZ (Jin et al., 2005). Furthermore, kainic acid induced seizures, also a strong upregulator of hippocampal neurogenesis in mice, were not able to increase hippocampal neurogenesis in R6/2 mice (Phillips et al., 2005). In contrast, complex enriched housing conditions have been reported to increase hippocampal neurogenesis (Kempermann et al., 1997) and was successful in delaying the onset of symptoms in R6/1 and R6/2 mice (van Dellen et al., 2000; Hockly et al., 2002). A recent study reported a higher rate of surviving newborn cells in the DG of R6/1 mice after 20 weeks of environmental enrichment, while the number of surviving new neurons remained unchanged (Lazic et al., 2006). These results suggest that environmental enrichment could partly rescue impairments of hippocampal neurogenesis. Discrepancies observed following the various paradigms to stimulated neurogenesis in the HD mouse models may originate from their influence on different regulators of hippocampal neurogenesis (Olson et al., 2006). In summary, we demonstrate that R6/2 mice show a reduced neurogenesis in the DG of the hippocampus due to decreased proliferation and survival of neural precursor cells. Furthermore, physical activity, which is known to strongly enhance hippocampal neurogenesis in wild type mice, was not able to increase neither the proliferation nor the survival of newly generated neurons in transgenic HD mice. Our findings suggest that the local microenvironment present in the CNS of R6/2 mice affects the generation of new hippocampal neuronal precursors and strongly interferes with their proliferation, their survival and the hippocampal neurogenesis regulatory mechanisms.
WT and R6/2 mice at the age of 5 weeks (weight: 18–21 g) were randomly divided into two groups. The non-running group consisted of one half of the mice (WTnon-run, n = 7; R6/2non-run, n = 8) and was placed in standard housing conditions (up to 5 animals per cage). The running group (WTrun, n = 7; R6/2run, n = 6) was composed of the other half of the mice housed (up to 5 animals per cage) with free access to two running wheels in each cage. Animals were placed in the different housing environments 12 h before the start of the experiment. The running animals had free access to the wheels during 4 weeks. WT and R6/2 mice displayed normal running behavior during the duration of the running period. In particular, R6/2 mice showed no obvious motor deficits compared to WT animals. All animals were sacrificed at the end of the 4-week running period.
4.
Experimental procedures
4.5.
4.1.
Animals
Free-floating sections were treated with 0.6% H2O2 in Trisbuffered saline (TBS: 0.15 M NaCl, 0.1 M Tris–HCl, pH 7.5) for 30 min. For immunohistological detection of the incorporated BrdU, pre-treatment of tissues was performed as described previously (Brown et al., 2003b). Following extensive washes in TBS, sections were blocked with a solution composed of TBS, Triton X100 0.1%, bovine serum albumin 1%, and teleostean gelatine (Sigma, Taufkirchen, Germany) 0.2% for 2 h. This buffer was also used during the incubation with antibodies. Primary antibodies were applied overnight at 4 °C. For
Female B6CBAF1/J mice transplanted with ovaries from female B6CBATg(Hdexon1)62Gpb/1J mice (“R6/2”, Mangiarini et al., 1996) were bred to male B6CBAF1/J mice. All animals were obtained from Jackson Laboratories (Bar Harbor, USA). DNA derived from the tail was used for determining the CAG repeat length in transgenic animals using a polymerase chain reaction (PCR) assay (Mangiarini et al., 1996). Female mice (wild type (WT), n = 14; transgenic (R6/2), n = 14) derived from
4.3.
BrdU labeling
Labeling of dividing cells was performed by intraperitoneal injection of the thymidine analogue BrdU (Sigma, Steinheim, Germany) at 50 mg/kg of body weight using a sterile solution of 10 mg/ml of BrdU dissolved in a 0.9% (w/v) NaCl solution. The BrdU injections were performed daily from day 1 to 5 at the beginning of the running period.
4.4.
Tissue processing
Animals were deeply anesthetized using a ketamine (20.38 mg/ ml), xylazine (5.38 mg/ml) and acepromazine (0.29 mg/ml) mixture. Transcardial perfusion was performed with 0.9% (w/ v) NaCl solution followed by a 4% paraformaldehyde, 0.1 M sodium phosphate solution (pH 7.4). The brains were removed, post-fixed in the paraformaldehyde solution overnight at 4 °C. Tissues were then cryoprotected in a 30% (w/v) sucrose, 0.1 M sodium phosphate solution (pH 7.4). Brains were cut into 40 μm sagittal sections using a sliding microtome on dry ice. Sections were stored at −20 °C in cryoprotectant solution (ethylene glycol, glycerol, 0.1 M phosphate buffer pH 7.4, 1:1:2 by volume).
Immunostaining
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chromogenic immunodetection, sections were washed extensively and further incubated with biotin-conjugated speciesspecific secondary antibodies followed by a peroxidase–avidin complex solution from the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, USA). The peroxidase activity of immune complexes was revealed with a solution of TBS containing 0.25 mg/ml 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, USA), 0.01% (v/v) H2O2, and 0.04% (w/v) NiCl2. Sections were put on Superfrost Plus slides (Menzel, Braunschweig, Germany) and mounted in Neo Mount (Merck, Darmstadt, Germany). For epifluorescence immunodetection, sections were washed extensively and incubated with fluorochrome-conjugated species-specific secondary antibodies for 2 h. Sections were put on slides and mounted in Prolong Antifade kit (Molecular Probes, Eugene, USA). Photodocumentation was realized using a Leica microscope (Leica, Wetzlar, Germany) equipped with a Spot™ digital camera (Diagnostic Instrument Inc, Sterling Heights, USA) and epifluorescence observation was performed on a confocal scanning laser microscope (Leica TCS-NT, Wetzlar, Germany). The following antibodies and final dilutions were used. Primary antibodies: rat α-BrdU 1:500 (Oxford Biotechnology, Oxford, UK), mouse α-PCNA 1:500, goat α-DCX 1:500 (C-18; both Santa Cruz Labs, Santa Cruz, USA), mouse α-NeuN 1:500, mouse-α-huntingtin 1:500 (both Chemicon, Temecula, USA). The mouse-α-huntingtin antibody MAB5374 detects N-terminal mutant human huntingtin with a repeat length N82. Secondary antibodies: donkey α-goat, -mouse or -rat conjugated with Alexa 488 (1:1000, Molecular Probes, Eugene, USA), rhodamine X, or biotin 1:500 (Jackson Immuno Research, West Grove, USA). For labeling cell nuclei, ToPro-3 (1:2000; Molecular Probes, Eugene, OR, USA) was diluted in TBS and directly administered to the sections for 10 min followed by two washing steps. To determine cell death in the DG, the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using the Apoptag In Situ Cell Death Detection Kit (In-tergene, Purchase, NY, USA). The modified protocol for free floating sections was administered which was described previously in detail (Cooper-Kuhn and Kuhn, 2002).
4.6.
Counting procedures
All morphological analyses were performed on blind-coded slides. Every sixth section (240-μm interval) of one hemisphere was selected from each animal and processed for immunohistochemistry. To analyze cell proliferation in the DG, PCNA immunopositive cells were counted. To investigate neurogenesis, the number of neural precursor cells stained for DCX was determined, while the amount of surviving newborn neurons was measured by counting the number of BrdU-labeled cells. All cells that were stained by PCNA, DCX or BrdU antibodies were counted using a 40× objective lens of a light microscope (Leica, Wetzlar, Germany) and multiplied by 6 to obtain an estimate of the total immunopositive cell numbers. The reference volume was determined by tracing the granule cell layer of the hippocampal DG using a semi-automatic stereology system (Stereoinvestigator, MicroBrightField, Colchester, USA). All extrapolations were calculated for one hippocampus
31
and should be doubled to represent the total hippocampal values. To determine the frequency of neuronal differentiation of newborn cells, a series of every sixth brain section (240-μm interval) was stained for BrdU and NeuN by immunofluorescence and examined using the confocal laser microscope equipped with a 40× PL APO oil objective (1.25 numeric aperture) and a pinhole setting that corresponded to a focal plane of 2 μm or less. On average 50 BrdU-labeled cells per animal were analyzed for neuronal differentiation. BrdU-positive cells were characterized as solely BrdU-positive (newborn cells) and BrdU/NeuN double-positive cells (newborn neurons).
4.7.
Statistical analysis
The data are presented as mean values ± standard deviations (SD). Two-way ANOVA (genotype × physical activity) was used to test for differences in PCNA, DCX and BrdU numbers, BrdU/ NeuN percentage, total number of BrdU-labeled neurons and DG volume. For post hoc analysis a Bonferroni's correction was performed. A Student's t-test was applied for analyzing differences in repeat lengths of R6/2non-run and R6/2run animals. Statistical analysis was performed using Prism (Prism Graph Pad Software, San Diego, USA). The significance level was assumed at p < 0.05, unless otherwise indicated.
Acknowledgments The study was supported by the School of Medicine at the University of Regensburg, Germany (ReForM Program) and the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell grant). Beate Winner was supported by a fellowship sponsored of the “Hochschul- und Wissenschaftsprogramm” (University of Regensburg). Mahesh Kandasamy was supported by a fellowship sponsored by the “Bayerische Forschungsstiftung”, Munich, Germany. Ludwig Aigner was supported by the Volkswagen Foundation, Hannover, Germany.
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