View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Stimulated increase of BrdU+ cells by G-CSF. (A) WBC counts of AD mice administrated with PBS or G-CSF. The WBC counts were analyzed for mice after their treatment for 5 d with PBS or G-CSF (n = 8 for each group). (B and C) Immunostaining for BrdU+ cells in a coronal brain section after BrdU incorporation of AD mice treated with G-CSF. The boxed area in B is magnified in C. The arrows indicate the injection site. Bar, 100 µm. (D) Comparison of the average numbers of BrdU+ cells in the hemispheres of AD mice receiving BrdU and treated with PBS or G-CSF for 5 d (n = 5 for each group). The cell numbers were determined by immunostaining with anti-BrdU, as represented in B and C. (E) Distance dependence of the distribution of BrdU+ cells. The numbers of BrdU+ cells at different distances radially from the injection sites of the individual AD mice treated with G-CSF or PBS for 5 d and incorporated with BrdU were counted in the confocal microscope and averaged (n = 5 for each group). (F) Double immunostaining of BrdU and CD34. The brain sections from the AD mice with G-CSF treatment and BrdU incorporation were double stained with anti-BrdU (green) and anti-CD34 (red). Bar, 100 µm. Data in A, D, and E are means ± SEM. *, P < 0.05. DG, dentate gyrus.

G-CSF also improved the neurological behavior of a chronic AD mouse model, Tg2576
The second AD mouse model we used to test the therapeutic effect of G-CSF was Tg2576. Tg2576 mice overexpressed the human amyloid precursor protein with the Swedish mutation, and they developed AD-like features such as memory deficits and Aß plaques in the brain (17, 18). The amyloid plaques began to accumulate in the brains of Tg2576 mice around the age of 9 mo, when the AD-like features started to develop (17). We initially checked the brains of 12-mo-old Tg2576 mice for the existence of Aß aggregates and their learning/memory capabilities. Indeed, the Tg2576 mouse brains contained Aß depositions throughout the cortex (Fig. 3 A) and hippocampus (Fig. 3 B). These Tg2576 mice also exhibited significantly impaired learning/memory in the water maze test (compare the latencies of WTPBS with Tg2576PBS from session 1; Fig. 3 C). The Tg2576 mice were then treated with PBS or G-CSF, respectively, for 5 d. As shown in Fig. 3 C, although the latencies were similar between the wild-type mice treated with G-CSF and PBS, respectively, throughout the test (sessions 1–6; Fig. 3 C), G-CSF treatment significantly reduced the latency of the Tg2576 mice when compared with the PBS-treated controls (Fig. 3 C). Similar to the experiments with the Aß injection AD model, the G-CSF treatment also significantly increased the number of BrdU⁺-proliferating cells in the damaged brains of Tg2576 mice (Fig. 3 D), which were spread throughout the cortex and hippocampus (not depicted). Thus, G-CSF also appeared to be an effective agent in rescuing the cognitive function of mice with a chronic pattern of AD development.

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Rescue of the cognitive function of Tg2576 by G-CSF treatment. (A and B) Immunostaining of Aß aggregates (brown) in the cortex (A) and hippocampus (B) of a 12-mo-old Tg2576 mouse. Bars, 200 µm. (C) The cognitive function of Tg2576 mice after subcutaneous administration of PBS or G-CSF was tested by the water maze learning task. The latencies from the tests of the wild-type control mice treated with PBS or G-CSF are included for comparison (n = 9 for the Tg2576G-CSF group; n = 7 for the other three groups). The test data of one mouse unresponsive to the G-CSF treatment was not included in this figure. (D) Average numbers of BrdU+ cells, as determined by anti-BrdU immunostaining, in the hemispheres of Tg2576 mice receiving BrdU and treated with PBS or G-CSF for 5 d (n = 5 for each group). Data are means ± SEM. *, P < 0.05. CA1, hippocampal CA1 layer; CA3, hippocampal CA3 layer; CX, cortex; DG, dentate gyrus; LV, lateral ventricle.

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Enhancement of neurogenesis in AD mice by G-CSF. (A–H) Brain sections of the acute AD mice treated with G-CSF were double stained with anti-BrdU/anti-MAP2 (A–D) or with anti-BrdU/anti-NeuN (E–H). A and E provide low-magnification pictures of the staining patterns. The boxed areas in A and E are magnified in B–D and F–H, respectively. The arrows point to one example each of BrdU+/MAP2+ and BrdU+/NeuN+ cells. Bars, 50 µm. (I and J) Comparisons of the average percentages of NeuN+/BrdU+ cells in the hemispheres of Aß-induced AD mice (I) or Tg2576 mice (J) treated with PBS or G-CSF for 5 d (n = 5 for each group). The cell numbers were determined by immunostaining, as represented in A–H. Data are means ± SEM. DG, dentate gyrus.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Effects of G-CSF treatment on the brain levels of ACh and Aß. (A) Extents of ACh release in the brains of Tg2576 mice after subcutaneous administration with PBS or G-CSF. The extents are expressed as the percentages of the brain ACh level of the wild-type mice (n = 5 for each group). Note the relatively lower level of ACh in the brains of Tg2576 mice compared with the wild type, as well as the significant increase of the brain ACh amount of Tg2576 upon treatment with G-CSF but not with PBS. *, P < 0.05. (B) The levels of SDS-soluble Aß (SDS-Aß, left) and SDS-insoluble/formic acid–soluble Aß (FA-Aß, right) in the brains of untreated Tg2576 mice in comparison with Tg2576 mice after subcutaneous administration with PBS or G-CSF (n = 5 for each group). Note the insignificant differences among the three groups (P > 0.1 according to the Student's t test). (C and D) The Aß plaque burden in the brains of untreated Tg2576 mice in comparison with Tg2576 mice after subcutaneous administration with PBS or G-CSF. The distribution patterns of the Aß plaques in the hippocampus of PBS- and G-CSF–treated Tg2576 mice, respectively, are represented in C. Bars, 200 µm. The quantitative image analysis of the total areas of the Aß plaque burden in the hippocampus or cortex of untreated, PBS-treated, and G-CSF–treated Tg2576 mice, respectively, is shown in D (n = 3 for each group). Note the insignificant differences among the three groups (P > 0.1 according to the Student's t test). (E) Long-lasting effect of G-CSF rescue of Tg2576 mice. The water maze test was applied to measure the cognitive functions of Tg2576 mice 3 mo after the subcutaneous administration of PBS or G-CSF. The latencies of the Tg2576 mice before the treatment with PBS or G-CSF (the controls) are included for comparison (n = 5 for each group). Data in A, B, D, and E are means ± SEM. *, P < 0.05.

One or a combination of the following could explain the cellular basis for rescue of the learning/memory deficiency in AD mice by G-CSF. First, similar to that proposed for the G-CSF effect on mice with ischemic heart disease or cerebral ischemia/stroke (8, 11), G-CSF could induce mobilization of bone marrow–derived HSCs that invaded the mouse brain areas damaged by the Aß aggregates. As suggested by the significant increases of BrdU⁺/NeuN⁺ and BrdU⁺/MAP2⁺ cells near the Aß aggregates in the brains of the acute AD mice or in the cortex/hippocampus of Tg2576 mice (Figs. 2 and 4), the HSCs differentiated into neuronal cells, thus leading to the functional and structural recovery of the brains of AD mice. Further, the fact that at least a portion (20%) of the BrdU⁺ cells in the brains from an acute AD mouse model treated with G-CSF were also CD34⁺, as exemplified in Fig. 2 F, provided the first direct evidence for G-CSF–induced mobilization of HSCs into the damaged brain regions. The different cytokines and trophic factors produced by HSCs within the cerebral tissue or within the microvasculature of an injured brain could also stimulate the proliferation, migration, and differentiation of neuronal progenitor cells (20–22), thus contributing to the functional and structural recoveries of the damaged brain.

Second, neuronal stem cells (NSCs) have been shown to reside in several regions in the adult brain, and they give rise to mature neuronal cells (23). Interestingly, G-CSF could induce the differentiation of NSCs in vitro, and this effect paralleled the in vivo finding that G-CSF also induced neurogenesis and subsequently enhanced functional recovery after cortical cerebral ischemia (12). Thus, G-CSF might also have enhanced the neurogenesis of NSCs near the Aß aggregates and/or damaged brain regions of the AD mice. Finally, part of the rescuing effect of G-CSF on the AD mice might be caused by a direct protective role of G-CSF against programmed death of the neurons (24).

Notably, the treatment of patients with AD has been based largely on a strategy of enhancing the ACh-mediated transmission. Symptomatic benefits (cognitive, functional, and behavioral) have been observed in multiple clinical trials with agents known to inhibit acetylcholinesterase, which catalyses the breakdown of ACh. Our data in Fig. 5 A suggested that the rescue of the cognitive function of Tg2576 by G-CSF mice was partly caused by the increased level of ACh, likely as the result of a release of ACh from cholinergic neurons in the population of newly generated neuronal cells, as stimulated by G-CSF.

Our data have suggested the feasibility of using G-CSF as a novel therapeutic strategy for the treatment of AD. When compared with the drugs previously applied to Aß-induced AD animal models (e.g., ferulic acid [15] and cannabinoids [25]), the rescuing effect of G-CSF in the behavior test is at least equal. G-CSF is also as effective as other therapies such as the use of Aß antibody (26). Notably, our data in Fig. 5 E showed that the rescuing effect of G-CSF may be long-lasting. We propose that G-CSF treatment of human AD patients would also mobilize autologous HSCs into circulation, enhance their translocation into the damaged brains, substantially repair the lesion areas, and, thus, rescue the memory dysfunction of the patients. The noninvasive nature of G-CSF treatment, when compared with the common practice of stem cell transplantation, makes it a potentially ideal AD drug for clinical trials. The combination of G-CSF with one or more of the other therapeutic ways would be another perspective direction for AD treatment.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
AD mouse models.
Two different mouse models of AD were generated and/or bred for the test of G-CSF therapy. The acute Aß-induced model was generated according to a previously described protocol (14, 15). 8-wk-old C57BL/6 male mice were purchased from the National Laboratorial Animal Center and bred at the Animal Facility of the Institute of Molecular Biology (IMB) at Academia Sinica. Experimental procedures for handling the mice followed the guidelines of the IMB. Animals were housed in a room maintained on a 12-h/12-h light/dark cycle (light on at 7:00 a.m.). The Aß aggregate was prepared from a solution of 10 mM of soluble Aß_(1–42) (Sigma-Aldrich) in 0.01 M PBS, pH 7.4. The solution was incubated at 37°C for 3 d to form the aggregated Aß and stored at –70°C. Animals were intraperitoneally anesthetized with 40 mg/kg sodium pentobarbital, and the injection of aggregated Aß was made bilaterally into the dorsal hippocampus using a 26-gauge needle connected to a microsyringe (Hamilton). The animals were subjected to stereotaxic surgery with the incisor bar set at the following coordinates: 2 mm posterior to the bregma, 2.1 mm bilateral to the midline, and 1.8 mm ventral to the skull surface. The volume of injection was 1 µl of aggregated Aß or 1 µl PBS, and 7 d were allowed for AD symptoms to develop in the subjects. For the chronic AD model, Tg2576 mice were purchased from Taconic and were bred as described in this section.

G-CSF treatment.
For the acute AD model, 7 d after injection of the aggregated Aß, mice were subcutaneously injected with 50 µg/kg of recombinant human G-CSF (Amgen Biologicals) once daily for 5 consecutive days (7, 11). The control animals were injected with PBS in parallel. More details are described in Fig. 1 A. Tg2576 mice were treated in a similar way.

Behavioral measurements.
For spatial learning in the Morris water maze learning task, which was used as previously described (27), the animals were subjected to four trials per session and two sessions per day, with one session given in the morning and the other given in the afternoon. For a complete test, a total of six sessions over 3 d were given. In each of the four trials, the animals were placed at four different starting positions equally spaced around the perimeter of the pool in a random order. They were allowed 120 s to find the platform. If an animal could not find the platform in 120 s, it was guided to the platform. After mounting the platform, the animals were allowed to stay there for 20 s. The time that an individual mice took to reach the platform was recorded as the escape latency. For all the behavior tests in this study, the native mice received the spatial training for six sessions over 3 d to learn the location of a submerged platform in the pool. 7 d after the Aß injection, the water maze test (six sessions over 3 d) was used to confirm the AD symptoms. The same water maze test was also used to check the behavioral performances of mice after G-CSF or PBS treatment. For Aß-injected mice, the waiting period before the test was 14 d after the first injection (Fig. 1 E). For Tg2576 mice, the waiting period was either 14 d (Fig. 3 C) or 3 mo (Fig. 5 E).

BrdU labeling.
BrdU (Sigma-Aldrich), a thymidine analogue that is incorporated into the DNA of dividing cells during S phase, was used for mitotic labeling. The labeling protocol followed those previously described (28). A cumulative labeling method was used to examine the population of proliferative cells, with the mice receiving daily intraperitoneal injections of 50 mg/kg BrdU for 16 consecutive days, starting on the first day of G-CSF injection. The BrdU⁺ cells in both hemispheres of the hippocampus and cortex were digitally counted with the use of a 20x objective lens with a laser scanning confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.) via a computer imaging analysis system (Imaging Research). For each animal, 40 coronal sections (each 15-µm thick) throughout the hippocampus and cortex were analyzed. The image analysis was also used to examine the distributions of BrdU⁺ cells radially surrounding the Aß aggregates that formed near the microinjection sites.

Immunohistochemistry.
Immunohistostaining was used to identify the locations of BrdU and different proteins in the mouse brain. Double immunofluorescence staining was performed to identify MAP2, NeuN, and CD34, respectively, in BrdU⁺ cells. For this, the mouse monoclonal anti-BrdU (Chemicon) and Alex Fluor 488–conjugated goat anti–mouse antibodies (Invitrogen) were initially used, followed by treatment with the rabbit polyclonal anti-MAP2 (Chemicon) and Alex Fluor 555–conjugated donkey anti–rabbit antibodies (Invitrogen) for the identification of the neuronal dendrites. Alternatively, the anti-BrdU and Alex Fluor 555–conjugated donkey anti–mouse antibodies (Invitrogen) were used, followed by treatment with rabbit polyclonal Alex Fluor 488–conjugated anti-NeuN (Chemicon) antibodies to identify the neuronal nuclei. The rabbit polyclonal anti-CD34 (Serotec) and Alex Fluor 555–conjugated donkey anti–rabbit antibodies were used to identify HSCs. For thioflavin S staining (29), the brain sections were fixed to the slides with 4% formaldehyde in 0.1 M phosphate buffer and stained with 1% thioflavin S (Sigma-Aldrich) solution for 8 min. Sections were rapidly washed once in 100% ethanol and twice in 80% ethanol/water, rinsed for 10 min with water, and mounted with the mounting medium. All immunostaining sections were analyzed with a laser scanning confocal microscope. The green (Alex Fluor 488) and red (Alex Fluor 555) fluorochromes on the slides were excited by a laser beam at 488 and 543 nm, respectively.

ACh assay.
For measuring ACh levels in the mouse brains, the mice were killed, and their brains were quickly removed and frozen on dry ice. The brains were homogenized on ice and immediately subjected to the ACh assay. The ACh levels in the brain extracts were then determined with the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (Invitrogen), according to the manufacturer's instructions. The measurement was performed in a fluorescence microplate reader (PowerWave 340; BioTek) using excitation in the range of 530–560 nm and emission detection at 590 nm.

Quantifications of the Aß levels and Aß plaque burden.
The levels of soluble and insoluble Aß were quantified according to the procedures of Kawarabayashi et al. (30). The tissue samples were homogenized in 10 wet-weight volumes of PBS containing a cocktail of protease inhibitors (20 µg/ml each of pepstatin A, aprotinin, phosphoramidon, and leupeptin; 0.5 mM PMSF; and 1 mM EGTA). The samples were briefly sonicated (10 W, 2 x 5 s) and centrifuged at 100,000 g for 20 min at 4°C. The soluble fraction (supernatant) was used for the Aß ELISA assay. To quantitate the insoluble Aß/Aß aggregates, the SDS-insoluble pellet was sonicated and dissolved in 70% formic acid. The extract was neutralized with 0.25 M Tris, pH 8, containing 30% acetonitrile and 5 M NaOH before loading onto an ELISA plate. The protein-containing samples were subjected to assay by the Aß ELISA kit (Sigma-Aldrich), and the signals at 450 nm were detected using a multiwell plate reader.

To quantify the Aß plaque burden in the mouse brains, the method of Janus et al. (26) was followed. Eight sections from each cerebral hemisphere were immunostained with anti-Aß antibody (Chemicon) and visualized with diaminobenzidine. The plaque burden was analyzed using ImageJ software (National Institutes of Health), the brain area (hippocampus or cortex) was outlined, and the area and number of plaques in the outlined structure were recorded.

Statistical analysis.
Data are reported as means ± SEM. Independent experiments were compared using the Student's t test. Differences, indicated by asterisks, were considered statistically significant at P < 0.05.

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