Author contributions: H.K.J., J.S.B.: conception and design; H.K.J., J.K.L.: provision of study material and collection of data; H.K.J., J.K.L., S.E., J.S.B.: data analysis and interpretation; H.K.J., J.S.B.: financial support; J.K.L., E.H.S., J.E.C., J.S.B. manuscript writing; H.K.J., E.H.S and J.S.B.: final approval.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS January 28, 2010.
Alzheimer's disease (AD) is characterized by the deposition of amyloid-β peptide (Aβ) and the formation of neurofibrillary tangles. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) has been suggested as a potential therapeutic approach to prevent various neurodegenerative disorders, including AD. However, the actual therapeutic impact of BM-MSCs and their mechanism of action in AD have not yet been ascertained. The aim of this study was therefore to evaluate the therapeutic effect of BM-MSC transplantation on the neuropathology and memory deficits in amyloid precursor protein (APP) and presenilin one (PS1) double-transgenic mice. Here we show that intracerebral transplantation of BM-MSCs into APP/PS1 mice significantly reduced amyloid β-peptide (Aβ) deposition. Interestingly, these effects were associated with restoration of defective microglial function, as evidenced by increased Aβ-degrading factors, decreased inflammatory responses, and elevation of alternatively activated microglial markers. Furthermore, APP/PS1 mice treated with BM-MSCs had decreased tau hyperphosphorylation and improved cognitive function. In conclusion, BM-MSCs can modulate immune/inflammatory responses in AD mice, ameliorate their pathophysiology, and improve the cognitive decline associated with Aβ deposits. These results demonstrate that BM-MSCs are a potential new therapeutic agent for AD. STEM CELLS 2010;28:329–343
Alzheimer's disease (AD) is the most common form of dementia, affecting more than 18 million people worldwide. With increased life expectancy, this number is expected to rise in the future. AD is characterized by progressive memory deficits, cognitive impairment, and personality changes associated with the degeneration of multiple neuronal types and pathologically by the presence of neuritic plaques and neurofibrillary tangles . Amyloid β-peptide (Aβ) appears to play a key pathogenic role in AD, and studies have connected Aβ plaques with the formation of intercellular tau tangles, another neurotoxic feature of AD [2–4]. Currently, no treatment is available to cure or prevent the neuronal cell death that results in inevitable decline in AD patients.
Recent developments in stem cell technology have stimulated new prospective therapies for neurodegenerative disorders such as AD. Adult stem cells are under intense investigation as a potential therapeutic source of neurons to replace damaged or lost cells in various neurologic diseases. For example, administration of bone marrow-derived mesenchymal stem cells (BM-MSCs) has led to beneficial effects in animal models for several neurodegenerative diseases such as Parkinson's disease, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis [5, 6]. Although BM-MSC transplantation has been suggested as a potential therapeutic approach for these and other neurologic disorders [5–8], the actual therapeutic impact of BM-MSCs on AD pathology and their mechanism of action have not yet been ascertained.
The innate immune system is the vital first line of defense against a wide range of pathogens and tissue injuries, triggering inflammation through activation of microglia and macrophages. Many studies have shown that microglia are attracted to and surround senile plaques both in human AD samples and in rodent transgenic models that develop AD-related disease [9–11]. However, their exact role in the pathogenesis of AD remains to be elucidated. Some studies have also demonstrated that Aβ can activate microglia to produce cytokines and neurotoxins, hence promoting neurodegeneration [12–14]. In contrast, others have suggested that microglia are actually beneficial by secreting neurotrophic agents and eliminating toxic Aβ by phagocytosis [8, 10, 11].
Indeed, stimulation of the immune system in AD rodent models reduced Aβ burden [10, 11]. Aβ is phagocytosed by microglia and delivered to the lysosome where it is subsequently degraded [15, 16]. However, primary culture of mouse microglia indicates that these resident immune cells of the central nervous system (CNS) need to be activated prior to acquiring the ability to clear Aβ from the brain. Interestingly, the effects of BM-MSC transplantation in an ischemia mouse model were mediated through microglia and macrophage activation, and these immune cells were “alternatively activated” to play contrasting roles in response to injury . Our previous report also showed that BM-MSCs could increase microglial activation and reduce Aβ deposits in an acutely induced AD model .
Here, we examined whether intracerebral BM-MSC transplantation could have a beneficial effect in amyloid precursor protein (APP) and presenilin one (PS1) double-transgenic mice, a model of age-dependent AD. We treated APP/PS1 mice with BM-MSCs as the AD pathology progressed, and found that BM-MSC treatment promoted microglial activation, rescued cognitive impairment, and reduced Aβ and tau pathology in the brain.
MATERIALS AND METHODS
Transgenic mouse lines overexpressing the hAPP695swe (APPswe) and presenilin-1M146V (PS1) mutations, respectively, were generated at GlaxoSmithKline (Harlow, U.K., http://www.gsk.com/research/index.html) by standard techniques on a C57BL/6 background (Charles River, Margate, U.K., http://www.criver.com). APPswe mice were backcrossed onto a pure C57BL/6 background before crossing with PS1 mice to produce double heterozygous mutant mice (APP/PS1) . Given the gender-specific differences in the progression of Aβ deposition, we used only male mice in the present study. Animal protocols were approved by the Kyungpook National University Institutional Animal Care and Use Committee, and all experiments were performed according to the guidelines of the Kyungpook National University Institutional Animal Care and Use Committee. Animals were housed in a room maintained under controlled temperature and on a 12 hour/12 hour light/dark cycle.
Isolation and Culture of BM-MSCs
Tibias and femurs were dissected from 4 to 6-week-old C57BL/6 mice. Bone marrow was harvested, and single-cell suspensions were obtained using a 40 μm cell strainer (Becton–Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Approximately 107 cells were plated in 75-cm2 flasks containing MesenCult MSC Basal Medium and Mesenchymal Stem Cell Stimulatory Supplements (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) with antibiotics according to our previous report . The cell cultures were grown for 1 week, and the plastic-adherent population (BM-MSC) was used for subsequent experiments.
Surgery for Implantation of Guide Cannula
One week before the first injection with BM-MSCs, each mouse underwent surgery to implant a guide cannula into its brain. Briefly, after anesthesia with a combination of 100 mg/kg ketamine and 10 mg/kg xylazine, a stainless steel cannula was implanted in the animal's hippocampus using a stereotaxic frame (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com). The guide cannula was fixed in the hippocampal brain region according to the following coordinates: 1.6 mm posterior to the bregma, 1.7 mm bilateral to the midline, and 1.2 mm ventral to the skull surface. The guide cannula allows the formation of a stable aperture in the brain through which treatments can be administrated. In the absence of treatment, the guide cannula contains an obturator that prevents the aperture from being filled in by surrounding tissue.
Transplantation of BM-MSCs in APP/PS1 Double Mutant Mice
BM-MSC suspensions or phosphate-buffer saline (PBS) were transplanted biweekly for 1 month (n = 15 per group). Mice were transplanted starting at 7 months 1 week of age, and finished at 8 months 1 week of age (single or triple-intracerebral injection for 1 month). To determine whether the results observed after transplantation were directly attributable to BM-MSCs, we used NIH 3T3 cells (non-neuronal cells with BM-MSC-like morphology) as a nonstem cell control. As controls, 3 μl of PBS containing 1 × 104 nonviable BM-MSCs rendered by repeated freezing and thawing was implanted. The tip of the injection cannula projected beyond the guide cannula by 1 mm. It was connected by flexible polyethylene tubing to the microinjection system, which housed a 25 μl Hamilton syringe. Three μl of the cell suspension (approximately 1 × 104 cells) were injected into the hippocampus bilaterally. The cell suspension was delivered at a rate of 0.3 μl/minutes. In the control groups, 3 μl of PBS, dead cells or NIH 3T3 cells were implanted. After surgery, each mouse was kept in an individual cage to prevent the removal of the guide cannula by other animals.
Mice were killed after behavioral testing. The mice were anesthetized with 2.5% avertin in PBS. Animals were immediately cardiac perfused with 4% paraformaldehyde in PBS. After perfusion, brains were removed, postfixed overnight at 4°C, and incubated in 30% sucrose at 4°C until equilibrated. Sequential 30 μm or 14 μm coronal sections were taken on a cryostat (CM3050S; Leica, Heerbrugg, Switzerland, http://www.leica.com) and stored at −20°C.
Thioflavin S Staining
Free-floating sections were incubated for 5 minutes at a concentration of 0.5% thioflavin S (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) dissolved in 50% ethanol, and then washed twice with 50% ethanol for 5 minutes each, and once with tap water for 5 minutes, and mounted with mounting medium.
Free-floating sections were incubated for 1 hour in PBS containing 5% normal goat serum, 2% bovine serum albumin (BSA), and 0.4% Triton X-100. In the same buffer solution, the sections were then incubated for 24 hours in primary antibodies at 4°C. The following antibodies were used: 20G10 (mouse, diluted 1:1,000) raised against the 35-42 Aβ fragment, and selected for C-terminal Aβ 42 specificity, G30 (rabbit, diluted 1:1,000) raised against CMVGGVV for Aβ 40, anti-Iba-1 (rabbit, diluted 1:500, Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english), anti AMCase (YM-1; goat, diluted 1:100, Santa Cruz), anti-Interleukin (IL)-4 (goat, diluted 1:250, Santa Cruz), and anti-AT8 (rabbit, diluted 1:500, Pierce, Rockford, IL, http://www.piercenet.com). For visualization, the primary antibody was developed by incubating with Alexa Fluor 488-, 594-, or 633-conjugated secondary antibodies for 1 hour at room temperature or by incubating with biotinylated secondary antibodies against the corresponding species. This was followed by BCIP/NBT (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) or DAB (Vector Laboratories) according to the manufacturer's instructions for alkaline phosphatase or peroxidase labeling. For double fluorescence labeling with Aβ and microglia, the brain sections were incubated with 0.5% thioflavin S (Sigma-Aldrich) in 50% ethanol for 10 minutes. Sections were then washed twice in 50% ethanol for 5 minutes each, once with water for 5 minutes, once with tris buffered saline (TBS) with 0.1% Triton X-100 for 15 minutes, and once with TBS with 0.1% Triton X-100 and 2% BSA for 30 minutes. The sections were then incubated with primary antibodies as described above. For some experiments, after thioflavin S staining tissue sections were stained overnight at 4°C with specified combinations of the following primary antibodies: goat anti-AMCase (1:100), goat anti-IL-4 (1:250), and rabbit anti-Iba-1 (1:500), followed by the corresponding Alexa 546/Alexa 633-conjugated secondary antibodies. The sections were analyzed with a laser scanning confocal microscope equipped with Fluoview FV1000 imaging software (FV1000, Olympus, Tokyo, Japan, http://www.olympus-global.com) or with an Olympus BX51 microscope.
Western Blot Analysis
Different brain regions (cerebral cortex, hippocampus) were isolated from wild type (WT) and BM-MSC or control-infused APP/PS1 mice after behavioral testing. The brain tissues were weighed and sonicated in 10X volume of Laemmli's lysis buffer plus protease inhibitors. Equal amounts of protein samples (∼80 μg) in SDS sample buffer were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred electrophoretically to immunoblotting polyvinylidene difluoride (PVDF) membranes. The membranes were pretreated with blocking solution (5% skim milk, 0.1% Tween 20 in PBS) for 1 hour at room temperature and reacted with primary antibodies against 6E10 (1:500 dilution; Signet, http://www.signetlabs.com) and β-actin (1:500 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) in blocking solution overnight at 4°C. They were then washed with a washing solution (0.1% Tween 20 in PBS) five times for 10 minutes each and reacted with horseradish peroxidase-conjugated secondary antibodies against mouse IgG in blocking solution for 1 hour. The membranes were washed again with washing solution five times for 10 minutes each, and the protein signals were detected by chemiluminescence exposed to x-ray film. Densitometric measurements were made from the film using an imaging densitometer (Bio-Rad, Hercules, CA, http://www.bio-rad.com), and then quantified using Bio-Rad analysis software. For quantification of relative protein expression, the optical density of the protein band of interest was normalized to the optical density of β-actin on the same gel.
Aβ 40 and Aβ 42 enzyme-linked immunosorbent assays (ELISAs) were performed using fluorescent-based ELISA kits (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and appropriate Aβ standards according to the manufacturer's protocol. The hippocampus and frontal cortex from one hemisphere were homogenized in guanidine buffer with a final concentration of 50 mM Tris and 5 M guanidine HCl, pH 8.0. Homogenates were mixed at room temperature for 4 hours. After mixture, homogenates were diluted in PBS containing 5% BSA, 0.03% Tween 20, and protease inhibitor cocktail (Calbiochem, San Diego, CA, http://www.emdbiosciences.com). Each Aβ standard and experimental sample was run in duplicate and the results were averaged.
Quantitative Real-Time PCR
RNA extraction was performed with the RNeasy Lipid Tissue Mini kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer's instructions. RNA samples from four individual animals per experimental group were used to prepare cDNA for reverse transcription polymerase chain reaction (RT-PCR) using the oligo (dT)12–18 primers and SuperScript III RT (Invitrogen). The cDNA was quantified using the QuantiTect SYBR Green PCR Kit (Qiagen). The PCR primers used are described in the Supporting Table. For each investigated transcript, a mixture of the following reaction components was prepared to the indicated end-concentration: forward primer (five pM), reverse primer (five pM), and QuantiTect SYBR Green PCR Master mix. The 10 μl master mix was added to a 0.1 ml tube, and 5 μl volume containing 100 ng reverse transcribed total RNA was added as PCR template. The tubes were closed, centrifuged, and placed into the Corbett Research RG-6000 real-time PCR machine (Corbett Life Science, Sydney, Australia, http://www.corbettlifescience.com).
We used the Morris water maze task to assess spatial memory performance . The water maze was a white tank (1.0 m diameter, 30 cm height) filled to a depth of 20 cm (22 to 24°C). White, opaque, nontoxic paint was added to the water to hinder visibility. A submerged Plexiglas platform (10 cm diameter; 6 to 8 mm below the surface of the water) was located at a fixed potion throughout the training session. The position of the platform was varied from mouse to mouse while being counterbalanced across experiment groups. One day before training, all mice were habituated to the maze. The animals were subjected to four trials per day. A training session consisted of a series of four trials per day for 10 consecutive days (total 40 trials). In each of the four trials, the animals were placed at different starting positions equally spaced around the perimeter of the pool in random order. The mouse was given 60 seconds to find the submerged platform. If the mouse did not mount the platform within 60 seconds, it was guided to the platform. The time to mount the platform was recorded as the latency for each trial. Mice were allowed to remain on the platform for 10 seconds before being returned to a home cage. A single probe trial, in which the platform was removed, was performed after the hidden platform task had been completed (day 11). Each mouse was placed into one quadrant of the pool and allowed to swim for 60 seconds. All trials were recorded using a charge-coupled device (CCD) camera connected to a video monitor and a computer. The test was run using Image J 1.34s software. All apparatus used in this study were made by O'Hara & Company (Tokyo, Japan, http://www. ohara-time.co.jp/company.html).
The Student's t test was used to compare the two groups, whereas the Tukey's honestly significant differences test and repeated measures analysis of variance test was used for multigroup comparisons according to the SAS statistical package (release 9.1; SAS Institute Inc., Cary, NC). p < .05 was considered to be significant.
BM-MSC Transplantation Can Reduce Aβ Deposition in the Plaques of APP/PS1 Mice
To determine whether BM-MSC transplantation could delay or diminish Aβ plaque formation, we used APP/PS1 male mice. These animals were transplanted in the hippocampus with BM-MSCs beginning at 7 months 1 week of age (see Methods), as the AD pathology progressed . At 9 months of age, mice were killed and evaluated for changes in AD-like pathology. To evaluate Aβ pathology, we first used thioflavin S staining to see whether reduced Aβ aggregation could be observed in the brains of the BM-MSC transplanted mice. We observed a dramatic reduction in Aβ deposition in the cortex and hippocampus of the treated animals as compared with controls-infused counterparts (Fig. 1A, 1B). Quantitative image analysis (Fig. 1C, 1D) revealed statistically significant differences for each brain region examined (p < .01) between APP/PS1 mice treated with BM-MSCs and PBS, respectively. The average volume and area occupied by the Aβ plaques also were reduced significantly in both the cortex and hippocampus of BM-MSC transplanted mice compared with the control group. To further confirm that the aggregated protein we observed by thioflavin S staining was indeed aggregated Aβ peptide, the Aβ level was analyzed by immunoblotting (n = 4 for each group). Western blotting with a 6E10 antibody, recognizing the APP and C-terminal fragments (CTFs), showed a decrease in the cortex and hippocampus of BM-MSC-transplanted APP/PS1 mice compared with the PBS-injected animals (Fig. 1E, 1F). The reductions in APP were quantified and standardized according to the amount of β-actin protein, and were statistically significant (p < .05).
The levels of Aβ 40 and 42 are elevated early in dementia, and this change has been strongly correlated with cognitive decline . Aβ 42 is the most toxic Aβ isoform, and can elicit immunologic responses  and cognitive deficits [19, 20]. We thus examined the brain sections of our treated and control mice with anti-Aβ 40 (G30) and 42 antibodies (20G10). In the BM-MSC-treated group, the fraction of G30 and 20G10 positive plaques was dramatically lower than those in the PBS-transplanted group (Fig. 2A, 2C). Quantitative image analysis showed that the area occupied by the plaques was significantly smaller in the BM-MSC-treated APP/PS1 mice than in their age-matched PBS-infused counterparts (p < .05; Fig. 2B; p < .01; Fig. 2D). In addition, we also analyzed cerebral Aβ 40 and 42 levels by Aβ sandwich ELISA. (n = 3 for each group). The results showed that the hippocampus of BM-MSC-treated mice had less Aβ 40 and 42 (Fig. 2E, 2F). Quantitatively, the levels were decreased by 31% and 44%, respectively, in the hippocampus of BM-MSC-treated mice (p < .01). BM-MSC transplanted APP/PS1 mice also showed attenuation of Aβ 40 and 42 in the cortex compared with PBS-transplanted mice, although this did not reach statistical significance (Fig. 2E, 2F). These differences might be related to the fact that the BM-MSCs were transplanted into the hippocampus, not the cortex, or a preferential therapeutic effect of BM-MSC in this brain region. Our data provide very clear evidence that transplantation of BM-MSCs prevents the formation of deposition and clears the Aβ deposits in the brain of this AD model mice.
BM-MSC Transplantation Stimulates Microglial Activation in the Brains of APP/PS1 Mice
Recently, we demonstrated that microglia are activated by BM-MSC transplantation and that these activated microglia led to decreased Aβ deposits in an acutely induced AD mouse model . Based on this report, we examined whether BM-MSC treatment modulated microglia activity in the APP/PS1 AD model. To determine the number of microglia, 30 μm thick brain sections were labeled using an anti-Iba-1 antibody. Iba-1 positive cells were more numerous in the brains of BM-MSC-treated APP/PS1 mice than in PBS-treated animals (Fig. 3A, 3C). The area of Iba-1 immunoreactive cells was quantified in the cortex and hippocampus using Metamorph 7.1.2 software. Compared with PBS-treated mice, both the cortex and hippocampus exhibited a significant increase of 1.74- and 4.27-fold, respectively, in the number of Iba-1 positive cells in the BM-MSC-treated animals (p < .01; Fig. 3B, 3D). Although the number of microglia increased in the cortex and hippocampus of BM-MSC-treated APP/PS1 mice compared with PBS-injected animals, as with the Aβ results, the changes were more significant in the hippocampus than the cortex. Overall, these results demonstrated that BM-MSC treatment increases the microglia population in the central nervous system of APP/PS1 mice.
To validate whether the decreased Aβ depositions and increased microglia are stem cell-specific, we transplanted NIH 3T3 cells into APP/PS1 mice (this study), as well as “dead” BM-MSC into the acutely induced AD model in our previous study . Aβ deposits and microglia activation in NIH 3T3 cell transplanted APP/PS1 mice were similar to that of PBS-transplanted APP/PS1 mice (data not shown). Although dead BM-MSC transplantation activated microglia slightly, this injection did not promote the reduction of Aβ in an acutely induced AD mouse model . These results suggest that the effect of reducing Aβ deposits and increasing microglia activation are BM-MSC specific. They also showed that Aβ deposits and microglia activation in mice injected with a single injection of BM-MSC APP/PS1 were similar to PBS-injected mice (data not shown).
Next, we examined whether BM-MSC transplantation had a sustained effect on microglia activation in APP/PS1 mice. For these studies we assessed microglia activation at 6 weeks after the final treatment, and found that these cells were still increased in the BM-MSC transplanted mice compared with the PBS-treated APP/PS1 mice (Supporting Fig. S1). These data indicated that after BM-MSC transplantation, microglia activation was maintained even for 6 weeks, although the effect was slightly reduced over time.
Decreased Aβ Deposition Following Transplantation of BM-MSCs Is Related to Restoring Aβ Clearance by Microglia
In the previous experiment (Fig. 3), we observed that BM-MSC treatment increased the number of microglia in the brains of APP/PS1 mice. We next wanted to know the relationship between increased microglia and decreased Aβ deposits. Therefore, the Aβ deposits and microglia were coimmunostained using thioflavin S and Iba-1 antibodies, respectively. Confocal microscopy for Iba-1 revealed that many of the microglia in the BM-MSC-treated APP/PS1 mice were close to Aβ plaques, and they often colocalized with Aβ deposits (Fig. 4B) compared with PBS-injected animals (Fig. 4A). In fact, sometimes the activated microglia in BM-MSC-treated mice contained Aβ (data not shown). Microglia secrete proteolytic enzymes that degrade Aβ, such as insulin-degrading enzyme (IDE), neprilysin (NEP), matrix metalloproteinase 9 (MMP-9), and plasminogen [21, 22].
In addition to secretion of Aβ-degrading enzymes, microglia also express receptors that promote the clearance and phagocytosis of Aβ, such as class A scavenger receptor, CD36, and receptor for advanced-glycosylation endproducts (RAGE) [23, 24]. Microglia can also restrict senile plaque formation by phagocytosing Aβ. Previous reports have shown that as AD mice age, their microglia become dysfunctional and exhibit a significant reduction in expression of their Aβ-degrading enzymes and Aβ-binding receptors . Our APP/PS1 mouse models also exhibited microglia dysfunction with age (Supporting Fig. S2). For this reason, we decided to investigate whether BM-MSC grafting could modulate the expression of Aβ-degrading enzymes and Aβ-phagocytosis related receptors. To test this hypothesis, we measured expression of these proteolytic enzymes and receptors using quantitative real-time PCR analysis. Messenger RNA (mRNA) expression levels of the Aβ-degrading enzymes NEP, IDE, and MMP9, were increased in the cortex of BM-MSC-treated mice as compared with the PBS-infused counterparts (Fig. 4C, 1.57-, 1.78-, and 2.30-fold increase compared with PBS-treated mice, respectively; p < .05). Similarly, mRNA expressions were elevated in the hippocampus after BM-MSC treatment (Fig. 4C, 2.69-, 2.64-, and 1.87-fold increase compared with PBS-treated mice; p < .01). We also examined whether BM-MSC treatment affected expression of Aβ-binding receptors. Compared with PBS-treated mice, both the cortex and hippocampal areas of BM-MSC-treated animals exhibited a significant increase (1.88- and 1.71-fold, respectively) of SRB1 (p < .01; Fig. 4D). There were no significant differences in expression of other Aβ-binding receptors observed between the groups, although we found slight increases of some in the BM-MSC-treated animals. These data indicate that BM-MSC treatment restores the reduced expression of Aβ-degrading enzymes in the APP/PS1 mice and increases expression of some Aβ-binding receptors.
BM-MSC Treatment Switches the Microglial Phenotype From Classic to the Alternative Form in the APP/PS1 Mice
Microglia can adopt several different phenotypes. For example, microglia activation by Aβ peptides has been associated with the production of proinflammatory and potentially toxic cytokines , and proinflammatory cytokines are upregulated in the brains of triple transgenic AD mice . To examine this in our models, we quantified the mRNA expression levels of several proinflammatory factors, tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, and IL-6. Compared with WT mice, both the cortex and hippocampus of PBS-treated APP/PS1 mice had significantly increased TNF-α (3.47- and 4.28-fold, respectively; p < .01). In contrast, the expression of this cytokine was dramatically decreased in BM-MSC-transplanted APP/PS1 mice (p < .05; Fig. 5A). Similarly, the levels of IL-1β mRNA in the cortex and hippocampus of PBS-injected APP/PS1 mice were also increased (2.01- and 3.86-fold; p < .01) compared with WT mice, and reduced following BM-MSC transplantation (p < .01; Fig. 5B). The expression of IL-6 was not different between the groups (data not shown).
Surprisingly, reduced expression of these proinflammatory cytokines occurred despite clear evidence of microglia activation following BM-MSC transplantation (Figs. 3, 5A, 5B). Thus, we wondered whether these activated microglia displayed an alternative phenotype. Alternative activation of microglia is commonly considered part of the repair process and extracellular matrix reorganization that begins during or after the first stages of an acute innate immune response . It is well established that IL-4 is associated with the alternative activation of the macrophage/microglia cell population . YM-1 and Arg-1 are also strongly expressed in alternatively activated macrophages/microglia . To examine whether the activated microglia observed following BM-MSC transplantation expressed these alternative markers, we examined the expression of the IL-4, YM-1, and Arg-1 mRNAs. The results revealed clear induction in the expression of IL-4, YM-1, and Arg-1 mRNA in the BM-MSC-treated mice compared with the PBS-infused group (p < .05; Fig. 5C–5E). Next, we examined whether the activated microglia induced by BM-MSC treatment maintained high level expression of these neuroprotective markers over time (6 weeks after transplant). In BM-MSC-treated mice, the mRNA levels of IL-4, YM-1, and Arg-1 were slightly decreased as compared with 2 weeks following transplantation, but were still higher than in the PBS-infused animals (Supporting Fig. S3). These data indicate activation of an alternative microglia population following transplantation of BM-MSC. These cells can be maintained for up to 6 weeks and elicit a neuroprotective effect.
By immunohistochemistry, the expression of IL-4 was further studied in BM-MSC and PBS-treated APP/PS1 mice. In the BM-MSC-treated mice, IL-4-positive cells were localized predominantly around amyloid deposits. In contrast, in PBS-treated mice, there were nearly no IL-4 positive cells near amyloid plaques (Fig. 5F). Triple labeling of anti-YM-1, thioflavin S, and anti-Iba-1 in BM-MSC-treated mice demonstrated the existence of YM-1 positive microglia near the Aβ plaques (Fig. 5G). These results indicate that the alternatively activated microglia surround Aβ plaques following BM-MSC treatment.
BM-MSC Transplantation Is Able to Reduce Tau Hyperphosphorylation in APP/PS1 Mice
Recent studies have shown that tau is a necessary component of Aβ-induced cognitive dysfunction , suggesting that the interaction between Aβ and tau may be central to the development of AD dementia. Previous studies have also shown that APP/PS1 transgenic mice develop age-related accumulation of plaques and tangles in disease relevant regions , and a growing body of evidence suggests that Aβ and tau may be mechanistically linked . Our previous results (Figs. 1, 2) showed that BM-MSC treatment ameliorated Aβ depositions, including aggregated Aβ 40 and 42. Thus, we examined whether BM-MSC treatment also affected the hyperphosphorylated tau pathology. To assess this issue, we stained brain sections from PBS- or BM-MSC-treated APP/PS1 mice and age-matched WT littermates using anti-AT8 antibody, and found that BM-MSC treatment led to a significant reduction of hyperphosphorylated tau in the hippocampus and cortex compared with PBS controls (Fig. 6). Compared with PBS-treated mice, both the cortex and hippocampus exhibited a significant decrease of 35% and 39%, respectively, in the area occupied by AT8 positive cells in the BM-MSC-treated animals (p < .05; Fig. 6C). These results show that BM-MSC treatment is able to reduce hyperphosphorylated tau levels in a pattern similar to aggregated Aβ.
BM-MSC Treatment Ameliorates Spatial Learning and Memory Impairments
To assess whether BM-MSC treatment could improve spatial memory of transgenic AD mice, PBS-treated, and BM-MSC-treated APP/PS1 mice and their control (nontransplanted APP/PS1 and WT) littermates were tested at 3 days after the last treatment for spatial learning using the hidden platform version of the Morris water maze test . This task is a hippocampus-dependent cognitive task that requires spatial memory. Spatial memory was assessed by determining the escape latency in the hidden platform test of four trials per day. As shown in Figure 7A, the APP/PS1 mice (injected with PBS) showed significant memory deficits compared with WT (PBS-injected) mice. Notably, we found that APP/PS1 mice treated with BM-MSCs performed significantly better on the water maze test than PBS-treated counterparts (p < .01; Fig. 7A). Representative navigation paths at day 10 of training demonstrated that spatial learning acquisition was impaired in the APP/PS1 mice that received PBS relative to the animals injected with BM-MSCs, which displayed a navigation pattern similar to control animals (Fig. 7B). On the final day, a probe test was performed in which the platform was removed to assess whether the animals used a nonspatial strategy to find the platform. During the probe trial, we calculated the number of times each animal entered the small target zone during the 60-second test. Animals treated with BM-MSCs averaged 2 ± 0.37 entries into the small target zone; animals infused with PBS averaged 0.67 ± 0.24; and PBS-treated WT mice averaged 2.56 ± 0.6 entries into the target zone (p < .01; Fig. 7C). These results indicated that BM-MSC transplantation is able to reduce the cognitive impairment of spatial memory associated with the accumulation of Aβ peptide.
In this study, we employed a transgenic mouse model of AD that overexpresses the FAD-linked APP and PS1 transgenes to test the hypothesis that BM-MSCs could modulate Aβ deposition and cognitive decline in vivo. The hippocampus plays a major role in memory and spatial navigation, and in AD patients the hippocampus is one of the first regions of the brain to suffer damage. For this reason, we transplanted BM-MSCs into the hippocampus. We found that BM-MSC treatment was very effective in reducing Aβ deposits and tau hyperphosphorylation in APP/PS1 double-transgenic mice. The BM-MSC-treated APP/PS1 mice also showed significantly increased levels of microglial cells, correlating with enhanced Aβ-clearance in cortical and hippocampal regions, and improved spatial learning and memory when compared with the PBS-treated APP/PS1 mice. Furthermore, the BM-MSC-treated APP/PS1 mice exhibited decreased levels of neurotoxic cytokines normally associated with classically activated microglia, and instead had increased levels of neuroprotective cytokines that are associated with alternatively activated microglia. Taken together, these observations suggest new possibilities for a potentially effective therapy for AD.
The effect of BM-MSCs on reducing Aβ accumulation is likely attributable to restoration or enhancement of the Aβ-clearance pathway via microglial cells. Increasing evidence indicates that microglia may play a protective role in AD by mediating clearance of Aβ. Moreover, many studies support the idea that bone marrow-derived cells are able to cross the blood-brain barrier and differentiate into microglia [10, 11]. Notably, our previous report showed that BM-MSC transplantation induced endogenous microglia/macrophage activation rather than differentiation of microglia/macrophage from BM-MSC . Moreover, the activated microglia were able to phagocytose Aβ [8, 11], and their selective ablation resulted in an increased amyloid burden . Similarly, CC chemokine receptor-2 (CCR2) gene deficiency was found to impair microglia recruitment and increase amyloid deposits in APP mice . These newly recruited microglia cells are specifically attracted to the Aβ 40/42 isoforms in vivo, and they participate in the elimination of these proteins by phagocytosis . Although, in our experiment the number of microglia greatly increased in the cortex and hippocampus of BM-MSC-treated animals compared with PBS-injected APP/PS1 mice, the fold changes were higher in the hippocampus than the cortex (Fig. 3). These differences are likely caused by the fact that the BM-MSCs were transplanted into the hippocampus.
Concomitant with an elevated number of microglia, the number of Aβ plaques and the area occupied by Aβ deposits were dramatically lower in the brains of BM-MSC-treated mice (Figs. 1, 2). In most cases, the plaques were smaller and obviously less dense than those of PBS-treated APP/PS1 mice. A higher number of microglia was associated with Aβ plaques, and the amount of Aβ-containing microglia also greatly increased in response to the BM-MSC transplantation (Fig. 4). These results suggest that BM-MSCs treatment either prevented the synthesis of Aβ peptide or enhanced the clearance of existing Aβ deposits through microglia activation. Supporting this concept, the decrease in the early recruitment of the microglial cells, by genetic ablation of either TLR2 receptor in a PS1/APP model  or Ccr2 in a Tg2576 mice , increased Aβ 42 levels and accelerated memory impairment. Thus, we propose that activation of microglia by BM-MSC treatment is able to act as a natural defense mechanism to prevent Aβ accumulation or reduce Aβ deposits in the central nervous system.
Some reports have previously shown that microglia are unable to eliminate Aβ plaques, release cytotoxic cytokines, and participate in plaque formation as AD progresses [36, 37]. However, Boissonneault et al.  recently reported that M-CSF increases microglial cell number and stimulates Aβ degradation. In addition, Ohtaki et al.  suggested that BM-MSC treatment increases immune responses, including microglia activation and secretion of neuroprotective cytokines in ischemia mouse models. Based on this somewhat contradictory literature, we decided to examine the relationship of elevated microglia BM-MSC-transplanted AD mice and decreased Aβ deposits in more detail. We first investigated the secretion of Aβ-degrading enzymes by microglia. Within the brain, the metabolism of Aβ is mainly regulated by the activity of two major peptides, NEP and IDE, as demonstrated by studies in knock-out animals [34, 36]. In the AD brain, NEP is reduced ∼50% relative to controls in regions of high plaque content, but is unchanged in brain areas with moderate or low plaque burden . Pérez et al.  also reported that the levels of cystolic IDE were lower in AD than normal brain. More recently, Zhao et al.  demonstrated that hippocampal IDE protein and activity were reduced in AD and that decreased levels of this enzyme increase the risk of AD. In our studies, the most striking finding was that expression of Aβ-degrading enzymes was significantly increased in BM-MSC-treated APP/PS1 mice compared with PBS-treated controls. Therefore, our results suggest that BM-MSCs enhance Aβ clearance by increasing the levels of Aβ-degrading enzymes secreted by microglia. Microglia also express receptors that promote the clearance and phagocytosis of Aβ. To examine this issue, we measured the expression of Aβ-binding receptors. Among the Aβ-binding receptors, SRB1 was significantly upregulated by BM-MSC treatment as well.
It is widely acknowledged that Aβ triggers the proinflammatory reaction of microglia. The aggregated Aβ protein, which is present in senile plaques of AD patients, activates microglia to produce neurotoxic substances that contribute to the neurodegenerative changes [41–44]. In accordance with previous reports in AD and AD animal models, we wondered whether activated microglia by BM-MSCs produce proinflammatory cytokines. To examine this hypothesis, we examined the expression of TNF-α and IL-1β, major cytokines produced by microglia in response to Aβ stimulation. The expression of TNF-α and IL-1β was significantly increased in 9-month old APP/PS1 mice compared with wild type mice. However, a remarkable decline in the expression of both TNF-α and IL-1β was detected in BM-MSC-treated mice. This was despite clear evidence of microglial activation in BM-MSC-treated APP/PS1 mice compared with PBS-treated animals (Fig. 3).
Thus, we examined whether these activated microglia displayed a unique phenotype. Alternatively activated macrophages are primarily associated with wound healing and tissue repair . IL-4 is a well described immune regulatory cytokine able to suppress inflammation  and enhance uptake of fibrillar Aβ peptides . Of direct relevance to this study, the activation of macrophages in the presence of IL-4 results in an alternatively activated phenotype . In peripheral macrophages, alternative phenotypes are also characterized by the absence of expression of cytotoxic factors and the expression of alternative markers (YM-1 and Arg-1) . Our results clearly showed that the microglia in BM-MSC-treated APP/PS1 mice expressed an alternative phenotype, as evidence by the expression of the IL-4, YM-1, and Arg-1 genes. Increasing levels of these genes were maintained for up to 6 weeks after BM-MSC treatment, consistent with improved pathologic findings and learning.
In the BM-MSC-treated mice, the IL-4 positive cells were localized predominantly around Aβ deposits. In contrast, in PBS-treated mice, there were nearly no IL-4 positive cells adjacent to the Aβ plaques. Furthermore, in BM-MSC-treated mice the microglial cells located near the Aβ plaques colabeled with YM-1. Concerning their physiologic role, the alternative activated microglia could exert a neuroprotective function. In the presence of IL-4, microglia produces growth factors such as IGF-1 . Furthermore, IL-4 has been shown to reduce Aβ toxicity in vitro and in vivo [48–50], and to enhance Aβ phagocytosis . Thus, in agreement with other reports, our results demonstrate that plaque-associated alternative activated microglia produced following BM-MSC transplantation could reduce Aβ toxicity by increasing expression of IL-4.
Another interesting outcome of the present study was the inhibition of tau hyperphosphorylation following BM-MSC treatment. Recent evidence points to tau as a necessary component of the Aβ-induced cognitive dysfunction , suggesting that either a direct or indirect interaction between Aβ and tau may be central to the development of AD dementia. Previous studies have shown that APP/PS1 transgenic mice develop an age-related accumulation of plaques and tangles in disease-relevant regions . Thus, we examined whether BM-MSC treatment reduced the levels of hyperphosphorylated tau. BM-MSC treatment of APP/PS1 mice showed a significant reduction of hyperphosphorylated tau in the hippocampus and cortex. Although it remains to be determined whether BM-MSCs reduce tau pathology directly or indirectly, previous studies in 3xTg-AD mice support a causal relationship between Aβ and tau. The development of tau pathology is strongly dependent on the Aβ 42 levels . Aβ immunotherapy in 3xTg-AD mice reduces not only Aβ pathology, but also clears somatodendritic tau. Furthermore, single immunization with antioligomeric-specific antibodies reduces tau as well as Aβ pathology . The mechanisms behind the inhibitory role of BM-MSC in tau phosphorylation are largely unknown. However, in our experiments, reduced phosphorylated tau levels after BM-MSC treatment may be related, at least in part, to decreased Aβ 42 levels by BM-MSCs treatment.
As reported, excessive Aβ accumulation is associated with disturbed cognitive function in an AD mouse model , and hyperphosphorylated tau leads to memory deficits and loss of functional synapses in a transgenic mouse model . To study the behavioral consequence of BM-MSC treatment, the Morris water maze task was used to detect capacity hippocampal-dependent spatial learning and memory . In our mouse model, Aβ plaques and hyperphosphorylated tau were observed in the hippocampus from 6 months of age . In the Morris water maze test, the PBS-treated APP/PS1 mice took a significantly longer time to find the hidden platform than WT mice, indicating an impairment of spatial learning memory (Fig. 7). When APP/PS1 mice were treated with BM-MSCs, they took significantly shorter times and distances to reach the platform than PBS-treated APP/PS1 mice (Fig. 7). These results suggest that BM-MSC treatment of the APP/PS1 mice significantly improves cognitive and behavioral functions. Although it remains to be determined how BM-MSCs can improve cognitive function, the beneficial effect of BM-MSCs on cognitive improvement may be related to the combined effects of decreased levels of toxic Aβ peptide and tau hyperphosphorylation. These results are very promising for the development of a new therapeutic strategy for patient exhibiting mild cognitive impairment.
Recent observations indicate that only small numbers of transplanted bone marrow cells engraft into most injured tissues and they disappear quickly [56–58]. For example, when BM-MSCs were injected into the hippocampus in immunodeficient mice, most transplanted cells disappeared within 1 week, but they activated endogenous neural stem cells . While some reports suggest the transdifferentiation of BM-MSCs into cells of neural lineages in cell replacement therapies, this is seen at low frequency in vivo, and is, therefore, unlikely to be the predominant beneficial effect of BM-MSCs transplantation . Indeed, in our previous work, we could not observe the transdifferentiation of injected BM-MSCs despite their beneficial effects in neurodegenerative disease mice models [7, 8, 59]. Recently, evidence has emerged that transplantation of BM-MSCs into a damaged brain actually promotes brain repair via trophic mechanisms resulting in the release of bioactive factors (for example, cytokines, chemokines, and neurotrophins) and modulating the immune responses . These and related observations have focused attention on the paracrine effects of BM-MSCs [56, 57]. Our unpublished data, likewise, have also shown that BM-MSCs can promote neuronal survival through the release of soluble factors. These results were obtained using three-dimensional coculture technique of BM-MSCs and primary cultured neurons.
Our current findings clearly show that microglial activation remains elevated at 6 weeks in the BM-MSC-treated animals compared with PBS-infused counterparts. Previous work would predict that, at this point after transplantation, most BM-MSC are actually dead. This raises two interesting hypotheses. First, that the effect on microglial activation is not a direct effect of BM-MSC, but rather a trophic one more likely induced by release of soluble factors from BM-MSC in response to brain damage and sustained by these factors after their death. Second, that activation of microglia with conversion to an alternative phenotype results in a feed-forward loop whereby elevation in IL-4, Arg1, and Ym-1 decreases Aβ neurotoxity and increases Aβ phagocytosis, ultimately decreasing Aβ load. Finally, although not directly evaluated, the decrease in amyloid load identified here most likely produces an improvement in mouse performance on the water maze test.
Our results strongly suggest that intracerebral BM-MSC transplantation not only reduces amyloid load and tau phosphorylation in the brain, but also prevents cognitive decline and memory impairment associated with the AD-like pathology in APP/PS1 mice. We hypothesize that this occurs by activation of an endogenous microglial population with an alternative phenotype that has neuroprotective effects. When taken together, our results provide the basis for a novel immunomodulatory strategy for AD using BM-MSCs. While the feasibility of intracerebral MSC transplantation in AD patients is unclear at the present time, further studies will be aimed at exploring which factors secreted by BM-MSCs are able to modulate microglia and rescue AD-like pathology.
This work was supported by the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084065 to H.K.J.) and World Class University program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (R32-10064; to J.S.B.).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.