Increased Host Neuronal Survival and Motor Function in BMT Parkinsonian Mice: Involvement of...

Increased Host Neuronal Survival andMotor Function in BMT Parkinsonian

Mice: Involvement ofImmunosuppression

GILMOR I. KESHET,1 RAVI J. TOLWANI,2 ANGELICA TREJO,1 PEGGY KRAFT,1

REGIS DOYONNAS,1 CAROL CLAYBERGER,3,4 JAMES M. WEIMANN,1

AND HELEN M. BLAU1*1Baxter Laboratory for Genetic Pharmacology, Stanford University School of Medicine,

Department of Microbiology and Immunology, Stanford University,Stanford, California 94305

2Department of Comparative Medicine, Stanford University, Stanford, California 943053Department of Pediatrics – Immunology & Transplant Biology, Stanford University,

Stanford, California 943054Department of Cardiothoracic Surgery, Stanford University, Stanford, California 94305

ABSTRACTWe examined the potential of bone marrow transplantation (BMT) to rescue dopaminer-

gic neurons in a mouse model of Parkinson’s disease (PD). A BMT from mice transgenic forgreen fluorescent protein (GFP�) given either before or after administration of the neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) led to the accumulation of trans-planted adult GFP� bone-marrow-derived cells (BMDC) in the substantia nigra, wheredopaminergic neurodegeneration occurs in PD. Post-BMT, mice exposed to MPTP had sub-stantially greater numbers of endogenous tyrosine hydroxylase-positive neuronal cell bodiesin the substantia nigra and increased dopamine transporter-positive projections into thestriatum compared to controls. Moreover, motor function was restored to normal within 1month post-MPTP in BMT-treated mice assayed by a rotarod behavioral test. The effect ofBMT on PD was indirect, as no evidence of BMDC fusion with or transdifferentiation intodopaminergic neurons was observed. BMDC activated by BMT or associated factors couldplay a trophic role in rescuing damaged cells. Alternatively, the beneficial effects of BMT aredue to immunosuppression reflected by a reduction in the proportion of T-cells and areduction of T-cell proliferation in BMT mice. These findings highlight that when immuno-suppression is required for transplantation studies, the amelioration of symptoms may not bedue to the transplant itself. Further, they suggest that the immune system plays a role in thedevelopment of characteristics typical of PD. J. Comp. Neurol. 504:690–701, 2007.© 2007 Wiley-Liss, Inc.

Indexing terms: Parkinson’s disease; immunosuppression; bone marrow transplant; dopaminergic

neurons; rotarod motor function assays

This article includes Supplementary Material available via the Internetat http://www.interscience.wiley.com/jpages/0021-9967/suppmat.

Grant number: NIH K01-RR00129 (to R.J.T.); Grant sponsor: NationalInstitutes of Health (NIH); Grant numbers: AG09521, AG20961, HL65572,HD18179; Grant sponsor: Ellison Foundation; Grant number: AG-SS-0817-01; Grant sponsor: Baxter Foundation (to H.M.B.).

Current address for G.I. Keshet: Cancer Research Center, Chaim ShebaMedical Center, Tel Hashomer, Israel.

*Correspondence to: Helen M. Blau, Baxter Laboratory for Genetic Phar-macology and Departments of Microbiology and Immunology, StanfordUniversity, Stanford, CA 94305.

Received 18 April 2006; Revised 6 July 2007; Accepted 14 July 2007DOI 10.1002/cne.21483Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 504:690–701 (2007)

© 2007 WILEY-LISS, INC.

Parkinson’s disease (PD) is a debilitating neurodegen-erative disorder. Its primary clinical features includetremor, slowness of movement, stiffness, and postural in-stability (Fahn and Przedborski, 2000). These symptomsare primarily attributable to the degeneration of nigrostri-atal dopaminergic neurons in the substantia nigra parscompacta (SNpc), loss of their projecting nerve fibers intothe striatum, and the depletion of dopamine (Hornyk-iewicz and Kish, 1987). At the onset of these symptoms theSNpc dopaminergic neurons and striatal projections aredepleted by 60% and 80%, respectively (Dauer and Przed-borski, 2003). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) induces symptoms in humans that are virtuallyindistinguishable from PD (Langston and Irwin, 1986). In-deed, recognition of MPTP as a neurotoxin first occurredearly in 1982 when contamination of street preparations ofmeperidine with MPTP administered intravenously causeda profound Parkinsonian syndrome (Langston et al., 1983).In the mouse, MPTP causes selective neuropathology thatresults in neurochemical and behavioral deficits closely rep-licating those of PD (Langston, 1985; Kaakkola and Tera-vainen, 1990; Heikkila and Sonsalla, 1992; Tolwani et al.,1999). As a result, the MPTP mouse model is extensivelyused to evaluate methods for intervening in the neurodegen-erative process characteristic of PD. Data from several lab-oratories provide evidence that grafts of either hematopoieticor neural stem cells can ameliorate the symptoms of variousneurodegenerative diseases by mechanisms other than cellreplacement (Li et al., 2001; Ourednik et al., 2002; Llado etal., 2004). Therefore, we designed experiments to testwhether bone marrow transplant (BMT) could have a bene-ficial effect on the dopaminergic neurons affected by PD,either by cell fusion, transdifferentiation, or an indirectmechanism.

We and others have previously shown that followingBMT the circulatory system can be reconstituted withblood cells that have the capacity to cross the blood–brainbarrier and enter the brains of mouse or human adults(Brazelton et al., 2000; Mezey et al., 2000, 2003; Weimannet al., 2003a,b). In mice, donor cells within the bone mar-row that enter the brain can be tracked using geneticmarkers: sex-chromosome differences of donor and recip-ient or expression of green fluorescent protein (GFP).Bone-marrow-derived cells (BMDC) have previously beenshown to migrate to disparate sites of neuronal degener-ation (Kreutzberg, 1996) and have either beneficial ordeleterious effects (Hauben et al., 2000; Munch et al.,2003; Scali et al., 2003; Ziv et al., 2006). These diverseeffects could be a consequence of increasing the levels andtypes of cytokines and growth factors that are releasedlocally or into the serum, not only in response to differentneurodegenerative conditions but also by the BMT proce-dure itself (Baek et al., 2004).

Here we test the hypothesis that BMT can amelioratethe selective neurodegeneration characteristic of PD in-duced in mice by the neurotoxin MPTP. Our findingsdemonstrate that BMT prior to or after MPTP adminis-tration significantly improved certain hallmarks of PD,such as the levels of dopamine transporter (DAT) in thestriatum and numbers of endogenous tyrosinehydroxylase-positive (TH�) neurons in the SNpc. In addi-tion, motor function, which was significantly impaired byMPTP, was rendered normal when assayed by the accel-erating rotarod assay. Post-BMT, donor cells from thecirculation migrated to the lesion. We found no evidence

that these cells became dopaminergic neurons or fusedwith neurons. However, when immune function was eval-uated using the T cell mitogen concanavalin A (ConA)assay and the proportion of T-cells determined by flowcytometry, we found evidence for BMT-induced immuno-suppression. Taken together, the data presented here sug-gest that the BMT procedure induces immunosuppres-sion, possibly of a subset of T-cells that in turn promotesthe survival and recovery of the endogenous damageddopaminergic neurons, thereby alleviating biochemicaland behavioral deficits characteristic of PD. In addition,these results suggest that the state of the immune systemmay play a role in the development of the symptomscharacteristic of PD.

MATERIALS AND METHODS

Control and treatment groups

Animals were divided into six groups, animals in threeof the groups did not receive a BMT while animals in theother three groups received full body myeloablative irra-diation (9.6 Gy) followed by total BMT. Three controlgroups were included within the nontransplanted ani-mals. The vehicle control animals received a saline injec-tion instead of MPTP (saline, n � 33). To ensure that theMPTP was killing the dopaminergic neurons in the SNpc,some animals received only MPTP in saline (MPTP, n �23). To exclude the possibility that irradiation to the braincould alter the conversion of MPTP to MPP� or the bio-availability of MPP�, a group of mice had only their headirradiated followed by an injection of MPTP (head irradi-ation, n � 5). The head irradiated followed by MPTP groupserved as a control to demonstrate that the irradiationprocedure did not alter the toxicity of MPTP. The othermajor group of animals all received BMT. To control forthe possibility that the BMT procedure could alter thenumber of dopaminergic neurons in the SNpc or theirprojections to the striatum, BMT control animals receiveda saline injection instead of MPTP either 8 weeks afterBMT or 1 week prior (saline/BMT, n � 33). The experi-mental animals consisted of two groups. One group re-ceived BMT at 8 weeks of age and MPTP injection at 16weeks of age (BMT/MPTP, n � 35) and the other groupwas injected with MPTP at 14 weeks of age and receivedBMT 1 week later (MPTP/BMT, n � 53).

Bone marrow transplantation (BMT)

All animal protocols where approved by the Institu-tional Animal Care and Use Committee at Stanford Uni-versity. Total bone marrow was harvested from 8-week-old, male C57BL/6 transgenic mice that ubiquitouslyexpressed enhanced GFP, a gift from M. Okabe (OsakaUniversity; Okabe et al., 1997), and transplanted intolethally irradiated adult C57BL/6 mice (Jackson Labora-tories, Bar Harbor, ME) (see Brazelton et al., 2000; Mezeyet al., 2000). Following euthanasia, donor mice werebriefly immersed in 70% ethanol, their skin peeled backfrom a midline, circumferential incision, the femurs, tib-ias, and humeri removed, and all muscle removed with arazor blade. These bones were placed in 10 mL of calcium-and magnesium-free, Hank’s balanced salt solution(HBSS; Gibco BRL, Gaithersburg, MD) with 2% fetal calfserum (FCS; HyClone, Logan, UT) on ice for up to 90minutes. The tips of the bones were removed and a 25G

The Journal of Comparative Neurology. DOI 10.1002/cne

691ENHANCED NEURON FUNCTION IN PD

needle containing 1 mL of ice-cold HBSS with 2% FCS wasinserted into the marrow cavity and used to wash themarrow out into a sterile culture dish. Marrow fragmentswere dissociated by triturating through the 25G needleand the resulting suspension was filtered through sterile70-�m nylon mesh (BD Biosciences, San Jose, CA). Thefiltrate was cooled on ice, spun for 5 minutes at 250g, andthe pellet was resuspended in ice-cold HBSS with 2% FCSat 8 � 106 nucleated cells per mL. Simultaneously,8-week-old C57BL/6 mice (Jackson Laboratories) were le-thally irradiated with two doses of 4.8 Gy, 3 hours apart.Each irradiated recipient received 125 �L of the unfrac-tionated bone marrow cell suspension by tail vein injectionwithin 2 hours of the second irradiation dose.

MPTP administration

MPTP (Sigma, St. Louis, MO) was administered toadult C57BL/6 mice. The dosing regimen consisted of fourintraperitoneal (i.p.) injections of 20 mg/kg (free base)MPTP at 3-hour intervals.

Tissue preparation

Mice were deeply anesthetized with pentobarbital (150mg/kg, i.p.) and perfused transcardially with potassiumphosphate (PB) buffer, 0.1 M, pH 7.4, for 3 minutes andthen with freshly prepared 4% paraformaldehyde in 1�PB for 30 minutes. Fixation by perfusion fixing was nec-essary as freezing alone did not retain the small GFPmolecule within the cell. Brains were removed, postfixedin 1.5% paraformaldehyde solution in 1� PB overnightand cryoprotected in increasing concentrations of sucrosesolutions (10%, 20%, and 30%), and stored in 25% glyceroland 30% ethylene glycol (Sigma) in PB at �20°C untilsectioned.

Immunofluorescence

Free-floating coronal 50–60-�m sections were washedand incubated for 2 hours at room temperature in stainingsolution (3% bovine serum albumin (BSA), 2.5% goat se-rum, 0.3% Triton X-100 in PBS). Sections were then incu-bated overnight with the following primary antibodiesdiluted in staining solution: rabbit anti-TH polyclonal an-tibody (1:1,000, AB152, Chemicon, Temecula, CA). Theimmunogen was denatured TH from rat pheochromocy-toma. Specificity assessed by Western blot, AB152 selec-tively labels a single band at �62 kDa (reduced) corre-sponding to TH. Positive control: brain (corpus striatum,sympathetic nerve terminals) and adrenal glands. Nega-tive control: liver. The rat anti-DAT monoclonal antibody(1:400, Chemicon, MAB369) was generated against theN-terminus of human dopamine transporter (amino acids1–66) fused to glutathione S-transferase. This antibody(IgG2akappa) shows no cross-reactivity to the closely re-lated serotonin and norepinephrine transporters (Miller,1997, #215). Immunolocalization of DAT on paraformal-dehyde fixed frozen sections of human brain usingMAB369 shows dense punctate staining throughout thecaudate, putamen and accumbens (Miller et al., 1997).Positive control: brain (caudate, putamen, and nucleusaccumbens). The rabbit anti-GFP polyclonal antibody (1:2,000, Molecular Probes, Eugene, OR; Invitrogen, LaJolla, CA, A6455) was raised against GFP isolated directlyfrom Aequorea victoria. This antibody does not immuno-label cells or structures in control mouse brain.

Sections were washed in staining solution and thenincubated at room temperature for 2 hours with secondaryantibodies. Sections were washed and mounted with Flu-oromount G (Southern Biotechnology, Birmingham, AL).Each section was analyzed using a Zeiss 510 Laser Scan-ning Confocal microscope with sequential laser excitation.

Flow cytometry

Peripheral blood cells from BMT or wildtype mice wereblocked for nonantigen-specific binding of immunoglobu-lins to the FcgIII/II/I receptors using a rat antimouseCD16/CD32 antibody that reacts specifically with a com-mon nonpolymorphic epitope on the extracellular domainof the mouse FcgIII/II receptors (clone 2.4G2, 1:1,000, BDBioscience, #553142). Cells were then incubated for 15minutes on ice with antibodies against CD45-FITC (ratantimouse pan-hematopoietic antibody recognizing theleukocyte common antigen, clone 30-F11), CD3e-Biotin(hamster antimouse antibody that reacts with the 25-Kda� chain of T-cell receptor-associated CD3 complex ex-pressed on thymocytes, T lymphocytes, and NK-T cells,clone 145-2C11), CD11b (rat antimouse antibody that re-acts with the 170 kDa �[M] chain of Mac1 (�[M]2 inte-grin) on granulocytes, monocyte/macrophages, myeloid-derived dendritic cells, natural killer cells, microglia, andB-1 cells, clone M1/70), CD45R/B220 (rat antimouse anti-body reacting with an epitope on the extracellular domainof CD45 glycoprotein that is dependent on specific carbo-hydrate residues expressed on B-lymphocytes at allstages, clone RA3-6B2), and Gr1 (rat antimouse antibodythat reacts on a common epitope on Ly-6G and Ly-6Cdirectly correlated with granulocyte differentiation andmaturation, clone RB6-8C5). All antibodies were directlyconjugated to PE or APC (all at 1:100, BD Biosciences,#559971). Cells were analyzed by flow cytometry (FACS-calibur, Becton Dickinson, San Jose, CA) and percentageswere determined on viable, PI-negative (propidium iodine,Sigma), and CD45� gated cells.

DAT-immunoreactive (-ir) density in thestriatum

Every fourth coronal section throughout the striatumwas immunostained for DAT using rat anti-DAT monoclo-nal antibody (1:400, Chemicon, MAB369). Images wereprepared using a Zeiss axioplan fluorescence microscopeattached to a CCD camera. The intensity of DAT-ir wasmeasured as the average pixel intensity for the entirestriatum in that section using OpenLab software (Impro-vision, Coventry, UK). The intensity in a random part ofthe cortex of each section was considered the backgroundand was subtracted from the measurement. Both thestaining and the analysis were done blindly. The bregmacoordinates for the striatum are 1.7 to –1.5 mm.

Immunostaining for dopaminergic neurons

Counts for SNpc neurons, stained for TH-ir, were donein every fourth coronal section throughout the SNpc usingrabbit anti-TH polyclonal antibody (1:1,000, Chemicon,AB152). TH-ir was visualized by Vectastain ABC-peroxidase kit with DAB substrate solution (Vector, Bur-lingame, CA) according to the manufacturer’s instructionsand counterstained with cresyl violet.


692 G.I. KESHET ET AL.

Unbiased stereological estimation of DAneurons in the SN

TH-stained dopaminergic neurons were counted usingthe optical fractionator method of unbiased stereology(West et al., 1991; Chan et al., 1997) and a Stereo Inves-tigator software image analysis system (MicroBright-Field, Colchester, VT). SNpc was outlined (contour area)to determine its boundaries for sampling. The SNpc wasdelineated from the ventral tegmental area (VTA) and theSN pars reticularis using landmarks, such as cerebralpeduncle and medial lemniscus as previously described(Chan et al., 1997) (bregma –2.8 to –3.8 mm). Dopaminer-gic neurons were counted using the 100� oil objective onan Axioskop microscope (Zeiss, Germany). With the totalsection thickness used as the dissector height, neuronswere only sampled if their cell body was ‘capped’ (e.g., cellbody not cut at the top of the section). Counting frames of50 � 50 �m were spaced randomly at intervals of 100 �100 �m in order to facilitate sampling of 25% of eachsection. The estimated total number of TH-ir neuronswere determined by the formula: Total number of DAneurons per each SNpc � (total number of caps) � 4 (everyfourth section analyzed) � 4 (25% area sampled). Both thestaining and the stereology were done blindly.

Evaluation of behavioral deficits followingMPTP administration and recovery

The accelerating rotarod was used to evaluate locomotorability in the mice following MPTP and BMT (Pekhletskiet al., 1996; Rozas et al., 1998; Sedelis et al., 2001). Themice underwent a 4-consecutive-day training period, con-sisting of three trials of 5 minutes each per day, starting at13 weeks of age (modified from Pekhletski et al., 1996).This training period assures that all mice receive the samelength of training time. MPTP was administered 3 daysfollowing the training phase and the mice were tested oncea week. Each mouse was tested three times in one sessionwith 5-minute resting intervals between tests. The ro-tarod was set to accelerate from 2 rpm to 30 rpm over a5-minute period. Each mouse was scored, in seconds, forthe period of time it was able to stay on the rotating rod.

Proliferative response to ConA

The proliferative response of spleen cells to the T-cellmitogen concanavalin A (ConA) was determined by themethod of Sopori et al. (1987). Briefly, spleens were re-moved from three control 20-week-old mice and three 20-week-old BMT mice that had received BMT 9 weeks beforeand single cell suspensions were prepared. After washing,cells were resuspended in RPMI 1640 supplemented with10% fetal bovine serum, 2 mM L-glutamine, 100 units/mLpenicillin, and 100 �g/mL streptomycin. Cells (2.5 �105/50 �L) were added to flat-bottom microtiter plates andwere incubated with or without ConA 5 �g/mL (Sigma) at37°C in a 5% CO2 incubator. After 24 or 48 hours ofincubation, the cells were pulsed with 1 Ci of [3H]-thymidine for 24 hours before harvesting. The cells wereharvested onto filter paper by a Tomtec Mach III har-vester and counted in a liquid scintillation counter(PerkinElmer, Boston, MA, 1450 microbeta liquid scintil-lation counter). The 3H-thymidine incorporation (cpm)given by the scintillation counter was then transferredinto Excel for analysis. The largest and smallest outliers

were removed from each group from analysis and themedian number was obtained and graphed.

Statistical analysis

All the values are reported as a mean SEM. Statisti-cal analysis was performed using one-way analysis of vari-ance (ANOVA) followed by Bonferroni t-test to determinedifferences between groups. A 2 � 3 ANOVA was used tocompare the effects and interactions of BMT and MPTP. AP-value �0.05 was considered significant.

Photomicrograph production

All photomicrographs were composed and edited in Pho-toshop 7.0 (Adobe Systems, San Jose, CA) with no en-hancement in contrast and brightness.

RESULTS

To test the hypothesis that BMT can ameliorate classi-cal symptoms of PD, we used two transplantation para-digms in mice that received MPTP, designated MPTP-mice (Fig. 1). First, to test if BMT could have a protectiveeffect, adult mice were lethally irradiated (9.6 Gy) andimmediately transplanted with age-matched bone mar-row. MPTP was administered 8 weeks post-BMT, when

Fig. 1. Experimental design. A: Outline of the paradigms used inthis study in which mice were transplanted with bone marrow eitherbefore or after MPTP treatment. B: Schematic representation describ-ing a dopaminergic neuron with its cell body in the SNpc and axonalprojections into the striatum.



reconstitution of the bone marrow is complete. In a secondexperimental paradigm, which more closely approximatesa therapeutic situation, BMT was given 1 week afterMPTP injection. This 1-week timepoint was chosen be-cause the degenerative effects of MPTP on SNpc dopami-nergic neurons have resolved after 4 days with no furtherdecrease in dopamine expression (Jackson-Lewis et al.,1995). In both paradigms BMDC could be tracked by usingbone marrow from transgenic GFP donor mice (Weimannet al., 2003b).

To validate the PD model we compared mice that re-ceived MPTP (MPTP-mice) to mice that received an injec-tion of saline instead of MPTP (saline-mice). The regimenwe used resulted in a reduction of immunoreactivity of THand DAT in the striatum 1 week after MPTP administra-tion as assessed by immunofluorescence (Suppl. Fig. 1A–D). Western blot analyses confirmed that the levels of THin the striatum were diminished a week after MPTP(Suppl. Fig. 1E). In subsequent experiments we examinedwhether BMT could ameliorate these effects.

Increase in DAT-ir in the striatum of MPTP-mice transplanted with bone marrow

relative to nontransplanted mice

The loss of dopaminergic neurons that project fromthe SNpc to the striatum is the main cause of PD, asthey are essential to motor function. We examined theexpression pattern of DAT, a functional marker of do-paminergic axons and terminals. In order to evaluatemany animals and sections the measurement of DATimmunofluorescence rather than dopamine levels in theentire striata was performed. DAT was measured byimmunofluorescence (DAT-ir) in the striata in everyfourth section using an epifluorescent microscopeequipped with a CCD camera (bregma 1.7 to –1.5 mm)(Fig. 1B). The data represent the percentage of themean density of DAT-ir in the striata, which was inter-nally controlled in each section examined by subtractingthe signal in the neighboring cortex that lacks DAT(Fig. 2). First, as one control, nontransplanted micereceived an injection of saline (saline) instead of MPTPand the mean fluorescence in sections of the striatumwas designated as 100% (Fig. 2A,E). Mice that receivedMPTP (MPTP) but not BMT had only 10% of the DATstaining detected in the saline control mice 4 weeksafter MPTP administration (Fig. 2B,E). Mice that re-ceived BMT either 8 weeks before MPTP (BMT/MPTP;Fig. 2C,E) or 1 week after MPTP (MPTP/BMT; Fig.2D,E) led to an increase of DAT-ir of 3-fold (33.5%) and4-fold (41.4%) compared to MPTP-only controls, respec-tively. Thus, mice that received BMT either before orafter MPTP administration had significantly higherDAT-ir when compared to nontransplanted MPTPmice, (P � 0.01 and P � 0.001, respectively). These micewere also different from saline controls (P � 0.001).Mice that received only BMT, but no lesion (saline/BMT), showed a nonsignificant reduction in DAT-ircompared to saline controls (85%) (Fig. 2E and see datafor Fig. 2 in Suppl. Table 1). A 2 � 3 ANOVA revealedthat BMT alone does not have an effect on DAT inten-sity in the striatum, and there is a significant interac-tion between BMT and MPTP (P � 0.0002). StriatalDAT-ir was higher, but statistically indistinguishable,in mice transplanted after MPTP, rather than before

MPTP administration. Taken together, these data sug-gest that BMT prior to and especially after MPTP isassociated with preservation of endogenous dopaminer-gic input into the striatum.

Increase in TH-expressing cells in thesubstantia nigra of MPTP-mice transplanted

with bone marrow relative tonontransplanted mice

The projections to the striatum originate in the SNpc.Thus, we compared the numbers of dopaminergic neuronsin the SNpc by immunostaining for tyrosine hydroxylase(TH-ir) cells. TH-ir cells were quantified using an opticalfractionator, an unbiased stereological technique (West etal., 1991; Chan et al., 1997). Two sets of control mice wereanalyzed: one received saline instead of MPTP (saline;10,635 2,288) (Fig. 3A,E), and the other received salineinstead of MPTP as well as a bone marrow transplant(saline/BMT; 9,904 2,602) (Fig. 3E). In Paradigm 1 (Fig.1A) mice that received MPTP only without BMT hadgreatly diminished endogenous neuronal numbers (Fig.3B,E; 2,816 1,813). In mice that were transplanted withbone marrow before MPTP administration, double thenumber of TH-ir cells were present in the SNpc relative tomice that received MPTP (Fig. 3C,E; 5,963 845). Theseresults demonstrate a 2-fold increase from 30% (MPTPonly) to 60% (BMT followed by MPTP) of the number ofneurons observed in untreated controls and is statisticallythe same as saline controls. In Paradigm 2 (Fig. 1A), inmice given BMT 1 week after MPTP, 90% of dopaminergicneurons (9,395 2,292) were present by comparison withMPTP only mice in which only 30% of the dopaminergicneurons remained (P � 0.03) and, importantly, are statis-tically the same as the saline controls (Fig. 3D,E). A 2 � 3ANOVA showed no effect of BMT on TH-ir and a stronginteraction between BMT and MPTP (see data for Fig. 3 inSuppl. Table 1). These results with TH-ir, like those ob-tained for DAT-ir above, strongly suggest that BMT eitherbefore and especially after MPTP damage promotes thesurvival both of dopaminergic projections into the stria-tum, as well as the endogenous dopaminergic neurons inthe SNpc.

BMT before MPTP administration results infull recovery of motor function

We tested whether the histological evidence of dopami-nergic neuron recovery observed in the SNpc and theconsequent high levels of DAT-ir detected in the striatumin BMT-treated MPTP-mice also resulted in behavioralrecovery. For this purpose the motor performance of themice was assessed using a learned accelerating rotarodtest (Sedelis et al., 2001). All mice, with and without BMT,were trained on the rotarod on each of 4 consecutive daysprior to MPTP administration according to standard pro-tocols (Pekhletski et al., 1996). This regimen results in atotal of 1 hour of exercise over the 4-day training periodand is well below the levels of exercise previously shown toalter MPP� uptake (Fisher et al., 2004). MPTP was thenadministered and the mice assessed on the acceleratingrotarod beginning 2 days after MPTP administration andweekly thereafter. Mice were tested three times per ses-sion and the length of time on the accelerating rotarod wasrecorded.



Complete recovery of behavior was observed in Para-digm 1, in which BMT was performed 8 weeks beforeMPTP administration (Fig. 4). Mice were tested 2, 8, 15,and 20 days after MPTP treatment. At 2 days after MPTP

administration there was no detectable motor differencebetween BMT and non-BMT-treated mice and both scoredpoorly on the rotarod test. BMT-treated mice began toexhibit readily apparent, significant behavioral recovery 8

Fig. 2. Increase in DAT-ir in the striatum of MPTP-mice trans-planted with bone marrow relative to nontransplanted-mice. Repre-sentative striatal sections of (A) saline control, (B) MPTP, and (C)mice given MPTP 8 weeks after BMT or (D) 1 week before BMT. Themice were sacrificed 4 weeks after MPTP or saline injections. Everyfourth 50-�m coronal section was stained with anti-DAT antibody.Str, striatum; OT, olfactory tubercle. E: DAT-ir densitometry wasdone using OpenLab software. Data represent the mean density as apercentage of control SEM. The mean intensity in nonimmunoreac-

tive areas (cortex), the background, was subtracted from the meanintensity of the striatum. ANOVA F � 105.9, P � 0.001. Bonferronit-test revealed no difference between saline and saline after BMT; nodifference between MPTP/BMT and BMT/MPTP. *Saline and Sal/BMT and were different from all three MPTP groups P � 0.001.**MPTP/BMT was different from MPTP P � 0.001 and **BMT/MPTPwas different from MPTP P � 0.01; n � 3 mice each from differentexperiments. Scale bar � 1 mm.



Fig. 3. Increase in TH-expressing cells in the substantia nigra ofMPTP-mice transplanted with bone marrow relative tonontransplanted-mice. Representative section of SNpc of (A) saline,(B) MPTP, and (C) mice given MPTP 8 weeks after BMT or (D) 1 weekbefore BMT. Mice were sacrificed 4 weeks after MPTP or salineadministration. Every fourth 50-�m coronal section was stained withanti-TH antibody. Numbers of neurons in SNpc (E) as counted by

optical fractionator. Data represent the mean SEM. ANOVA F �7.62, P � 0.004. *Bonferroni t-test revealed that the only group thatdiffered from saline was the MPTP group (P � 0.01). All three BMTgroups did not differ from saline. Three mice were used in eachexperiment. Scale bar � 300 �m. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]



days following MPTP and were behaviorally and statisti-cally similar to the saline control animals (Fig. 4 and datafor Fig. 4 in Suppl. Table 1). The recovery of BMT/MPTPmice compared to MPTP mice 20 days following MPTPadministration is demonstrated online in SupplementaryVideo 1. Interestingly, both BMT groups showed a smalldecrease in motor performance on day 15 that promptlyreturned to normal levels by day 20.

To rule out the possibility that the irradiation to thehead that occurs during BMT before administration of theneurotoxin, MPTP, could be the basis for the ameliorationof motor performance in Paradigm 1, a group of miceunderwent head-irradiation while the rest of the body wasshielded with lead. No bone marrow was transplanted.Head-irradiation at levels comparable to the dose given toBMT-injected mice did not improve motor function follow-ing MPTP administration (P � 0.05) (Fig. 4). These resultsshow that control MPTP-mice that received head irradia-tion only without BMT performed as poorly as the MPTP-mice that did not receive any additional treatment. Thesefindings are particularly relevant to Paradigm 1, becausethe irradiation that is a necessary component of the BMTprocedure occurred prior to MPTP administration. Thepersistent loss of function with head irradiation showsthat irradiation could not have rendered the brain resis-tant to the MPTP toxin. Moreover, irradiation cannot ac-count for the full recovery of motor function observed 2months after BMT.

BMT after MPTP administration results infull recovery of motor function

In Paradigm 2, as in Paradigm 1, mice were trained onthe rotarod before MPTP administration and their motor

function assessed beginning 2 days after MPTP adminis-tration and weekly thereafter. In this case, bone marrowwas transplanted 6 days after MPTP administration(Fig. 5).

The motor recovery of the MPTP mice correlated withthe time course of peripheral blood reconstitution follow-ing BMT. MPTP-mice began to show motor improvement9 days after BMT, compared to nontransplanted MPTPmice (Fig. 5, and data for Fig. 5 in Suppl. Table 1). Recon-stitution of the blood with donor-derived cells was wellunderway at this time as determined by FACS analyses,which showed 75.9 14%, donor-derived GFP� blood cells(n � 4) at 2 weeks post-BMT (data not shown). The timebefore mice fell off the revolving rod differed significantlyfor mice that had received BMT from those which had not.BMT-mice tested 15 and 20 days after MPTP administra-tion exhibited progressively greater motor performance(P � 0.05). Thus, in Paradigm 2, by day 15 mice that hadreceived BMT after MPTP exhibited behavior similar tothe saline controls. By day 20 they were indistinguishablefrom saline controls and very different from MPTP mice(P � 0.01). These results demonstrate that in this para-digm as in Paradigm 1 full recovery of motor functionoccurs following BMT.

BMDC accumulate in the MPTP-lesionedsubstantia nigra

The recovery of biochemical deficits and motor functionin BMT/MPTP mice suggested that transplanted GFP�-BMDC had migrated to the injured areas. Consequently,the accumulation of donor BMDC in these sites was as-sessed. We hypothesized that the presence of such cellscould be responsible for the increased survival of endoge-

Fig. 4. BMT before MPTP administration results in full recovery ofmotor function (Paradigm 1). Mice were tested using the rotarod test2, 8, 15, and 20 days after MPTP administration. Data represent themean time SEM spent on the rotarod before falling. ANOVA: P �0.0002 for each day. Bonferroni t-test: Saline vs. BMT/Saline and headirr/MPTP vs. MPTP show no differences. BMT/MPTP is different fromSaline only on day 2 but not different thereafter (*). Data from threeexperiments, n � 5 in each group (see supplemental data for statistics).

Fig. 5. BMT after MPTP administration results in full recovery ofmotor function (Paradigm 2). Mice were tested using the rotarod test2, 8, 15, and 20 days after MPTP administration. Data represent themean time SEM spent on the rotarod before falling. ANOVA: P �0.0001 for day 20. Bonferroni t-test: Saline vs. MPTP/BMT are differ-ent on days 2 and 8 P � 0.05, but not different on days 15 and 20 (*).Data from three experiments, n � 5 in each group (see supplementaldata for statistics).



nous SN neurons or the sprouting and maintenance of theaxons of these neurons induced in the striatum, therebyenhancing the dopaminergic system. Immunofluorescenceanalysis of brain sections revealed an accumulation ofGFP�-BMDC in SN damaged by MPTP (Fig. 6). Therewere 352 75 GFP� cells in the SNpc of BMT/MPTPmice, as compared with 85 22 GFP� cells in the SNpc ofBMT-treated mice that did not receive MPTP (n � 3; P �0.03). Moreover, these GFP� BMDC had the characteris-tic morphology of microglia with many fine ramified pro-cesses (see Streit et al., 1988; Weimann et al., 2003b) andwere often observed in close proximity to TH� dopaminer-gic cells in the SNpc of MPTP-mice (Fig. 7). An accumu-lation of the GFP�-BMDC was observed in the injured SN,not the striatum (data not shown). The basis for the lackof detection of GFP� cells in the striatum may be due tothe fact that the cell bodies of these neurons are in the SN,whereas their extensions project into the striatum.

Inhibition of T-lymphocyte proliferation inBMT mice

The response to the T-cell mitogen ConA is an estab-lished criterion for cell-mediated immunity (Sopori et al.,

1987). To determine whether the BMT procedure affectsthe cell-mediated immune response, spleen cells weretaken 9 weeks following BMT and cultured with ConA for3 days. Then T-cell proliferation was determined by incor-poration of [3H]-thymidine into the cells. The counts in themedium-only sample (background) were subtracted fromthe samples containing cells to yield the final result. A5-fold decrease in the ConA responsiveness was observedin the cells taken from mice following BMT (Fig. 8A) (P �0.001). This result showing immunosuppression in BMTmice was consistent between culture days 2 and 3. FACSanalysis of peripheral blood (Fig. 8B) as well as spleeno-cytes (data not shown) taken from BMT mice revealed alower number of T-cells (CD3�) when compared to controlmice. The number of B-cells (B220�), monocytes (Mac1),and granulocytes (Gr1), however, was not significantlychanged. These data suggest that the BMT mice remainedimmunocompromised even 8 weeks following the BMTprocedure.

DISCUSSION

Our data demonstrate an unexpected, marked func-tional recovery resulting from BMT in the MPTP mousemodel of PD, a drug-induced PD first described in humans(Langston et al., 1983), Increased numbers of TH� neu-rons were detected in the SNpc and increased DAT levelswere measured in the striatum compared to MPTP-micethat did not receive BMT.

Recovery of motor function assayed by the classicallearned accelerating-rotarod test was evident in MPTP-mice that received BMT either before or after MPTP. Thefunctional recovery we observed paralleled that of bio-chemical recovery in assays of DAT in the striatum andTH in the SNpc, two anatomically distinct regions affected

Fig. 6. BMDC accumulate in the MPTP-lesioned substantia nigra.Laser scanning confocal microscopic analysis of the SNpc in 60-�mbrain sections from BMT mice that were either saline-injected but notMPTP (A) or MPTP-injected 8 weeks after BMT (B), showingGFP�BMDC accumulation in the SNpc of the MPTP mouse as com-pared to the saline-control. The sections were stained with an anti-body against TH (Texas red, red). No antibody against GFP was used.Scale bar � 30 �m.

Fig. 7. (A–D) GFP� cells in proximity to TH� cells in the injuredSNpc. Laser scanning confocal microscopic analysis of the SN in a1-�m-thick optical section acquired from 60-�m brain sections show-ing BMDC that reside in proximity to DA neurons in the injured SN.The sections were obtained from MPTP-mice and were immuno-stained with an antibody against TH (Texas red, red) and with arabbit anti-GFP antibody (Alexa 488, green). Nuclei stained in B withDAPI (blue). Scale bars � 20 �m.



by PD. The depletion of striatal fiber density by 90% andof SNpc dopaminergic neurons by 70% is in accordancewith findings by others in MPTP mice (Liker et al., 2003;Jakowec et al., 2004) and in PD patients (Koller, 1992;Hornykiewicz, 1998). The marked increase in endogenousstriatal DAT� fibers and endogenous TH� neurons in theSNpc in MPTP mice relative to non-MPTP controls inresponse to BMT was not anticipated. It should be notedthat in mice and humans only 20% of dopaminergic pro-jections to the striatum (assayed by DAT) and 40% ofnormal levels of dopaminergic cell numbers (assayed byTH) are required for normal behavior (Koller, 1992; Forno,1996; Ho and Blum, 1998; Tillerson et al., 2003). Thesereports suggest that the extent of motor function recoveryobserved in our studies can be accounted for by the ana-tomical recovery assessed by DAT (�30%) and TH(�60%).

Importantly, the increased DAT-ir detected in the stri-atum 1 month after MPTP administration is not due tospontaneous recovery. Spontaneous striatal DAT recoveryhas not been reported in the mouse until 3 months afterMPTP using a similar regimen to that used in this study(Bezard et al., 2000; Jakowec et al., 2004). Notably, SNpcdopaminergic neuron number remained low and stableeven 7 months after MPTP (Bezard et al., 2000) and thecell bodies that provided projections into the striatum

derived from a different region of the brain, the VTA (seeFig. 3) and not the SN. Our results demonstrating reducedTH cells in SN, as well as reduced DAT in the striatum inMPTP-treated mice, are consistent with persistent dam-age that spanned the entire duration of the experiments.Thus, the anatomical recovery reported in this study can-not be explained by spontaneous recovery (Ho and Blum,1998). Exercise-induced behavioral and neurochemical re-covery of dopamine function after MPTP treatment hasbeen reported in rodents (Tillerson et al., 2003; Fisher etal., 2004). We were concerned that the amount of exercisethat the mice received during the behavioral testing in thepresent study may confound our observations. However,the nature of the testing did not require forced runningand was for a much shorter duration than reported inother studies. Statistical analysis of the control mice(BMT no MPTP; head irradiation and MPTP; and MPTPalone) strongly argues against an effect of the testingprotocol on the recovery of motor function due to BMT.

Four hypotheses could account for the observed recoveryby BMT of the MPTP-lesioned nigrastriatal pathway. 1)Transdifferentiation of BMDC into dopaminergic neuronsas reported for satellite cells and lung epithelium (La-Barge and Blau, 2002; Harris et al., 2004). 2) Fusion ofBMDC with dopaminergic neurons, as reported for hepa-tocytes, skeletal muscle, or Purkinje neurons in the brain(Gussoni et al., 1999; Lagasse et al., 2000; Alvarez-Doladoet al., 2003; Weimann et al., 2003a,b). 3) After BMT,BMDC could produce trophic factors that promote thesurvival of endogenous dopaminergic neuronal cell bodiesin the SN, the source for the fibers that innervate thestriatum, or the resprouting and outgrowth of axons fromthe SN. 4) Immunosuppression could protect from or aidin the recovery of dopaminergic neurons exposed toMPTP.

The etiology of the recovery of dopaminergic proteins inthe damaged host cells and concordant recovery of motorfunction differs from reported effects of BMT derivativeson tissue restoration. GFP�-BMDC accumulate in theSNpc and are in proximity to host nigral neurons, makingit feasible for them to exert an influence on the cell bodiesof nigral DA neurons. BMDC such as microglia have beenreported by others to migrate to sites of brain injury suchas in cerebral ischemia (Priller et al., 2001). The trans-planted cells described in this report do not transdifferen-tiate, i.e., activate gene characteristics of the tissues inwhich they reside. In addition, the donor BMDC do notfuse with dopaminergic neurons. Possibly the BMDC pro-vide trophic support leading to the recovery of damageddopaminergic cells. This would imply, however, that theBMDC in SN and striatum posttransplant differ fromendogenous BMDC. In theory, the BMT procedure itselfcould alter the blood cells, mobilizing them to acquire an‘activated’ state in a manner not seen with endogenousBMDC. If this were the case, our findings for BMDC wouldconcur with those observed for neural stem cells im-planted in the midbrain that were reported to rescue de-generating TH� dopaminergic neurons as opposed to re-placing damaged neurons (Ourednik et al., 2002). Possiblythis mode of cell rescue is more prevalent than originallyanticipated.

In recent years the long-held tenet that immune systemsurveillance of the CNS does not occur has been eclipsedby the realization that cells of the immune system areconstantly monitoring the CNS. After insult or injury

Fig. 8. Reduced number of T cells in BMT mice 8 weeks post BMT.A: Proliferation of T-cells from spleen in response to ConA as deter-mined by incorporation of 3H-thymidine into cells following 72 hoursincubation in ConA. Background counts subtracted (n � 10, SEM,*P � 0.001). B: Percentage of T-cells (CD3�) within the gated CD45�

population in peripheral blood and spleen (n � 3, SEM, **P �0.005).



microglia (macrophage lineage) can migrate to the CNS(Imai et al., 1999). In addition, T-cells are now known toroutinely enter the CNS (Engelhardt, 2006). Interestingly,recent studies clearly show that neurons express bothmajor histocompatibility antigens (MHC) class I and II(Boulanger and Shatz, 2004; Liu et al., 2006). MHC classII is expressed by neurons and may mediate a form ofadaptive response in the EAE model of multiple sclerosis.Given the amount of interplay between neurons and theimmune system, a more likely mechanism that could ex-plain the results reported here is that the BMT procedureinduces a form of immunosuppression within the T-cellpopulation. Even 9 weeks following BMT, T-cell activitywas decreased to 20% of normal. This was determined byassaying proliferative activity in response to the mitogenConA. In addition, flow cytometry revealed a decrease inthe number of T-cells. By contrast, the number of B-cells,monocytes, and granulocytes was not reduced. The find-ings presented here raise questions regarding the rela-tionship between immune system function and progres-sion of neurodegenerative disease. There are a growingnumber of reports suggesting the involvement of the im-mune system in neurodegeneration. Immunosuppressionhas been indicated in a mouse model of lupus and in a ratmodel of global ischemia attenuated neurodegeneration(Sakic et al., 2000; Sinigaglia-Coimbra et al., 2002; Balloket al., 2004). Interestingly, direct transplantation of cells,especially stem cells, can result in immunosuppression(Park et al., 2002; Pluchino et al., 2005). In addition, inrecent years immunosuppressants including cyclosporineA, FK-506, and their analogs were found to protect thestriatum from MPTP-induced dopamine depletion (Kita-mura et al., 1994; Borlongan et al., 1996; Matsuura et al.,1997). Directly relevant to our study has been the demon-stration that the immune system was found to be involvedin PD as well as in MPTP intoxication (Orr et al., 2002,2005; Benner et al., 2004; Miklossy et al., 2006). Immu-nophilins, which are chaperones for compounds such asFK506 and cyclosporine, can reduce the degenerative ef-fects of MPTP (Costantini et al., 2001; Barik and Bitko,2006). Together, these results suggest that T-cells have adeleterious effect in MPTP-induced parkinsonism.

These findings have three major implications. First,when studying the contribution of allogeneic cells trans-planted into animals to the recovery of these animals fromdiseases, researchers should be aware of the role of immu-nosuppression in this recovery. Second, the immune re-sponse may play a deleterious role in CNS disease pro-gression, as shown here for MPTP-induced PD. Third, arole for immunosuppression, specifically T-cell depletion,is suggested by these data, which, if true for humans, maybe useful in designing therapies for PD.

ACKNOWLEDGMENTS

We thank Drs. Theo M. Palmer and Lawrence Steinmanfor critical review of the data and valuable insights andDr. Robert C. Malenka for generous use of behavioralequipment and advice. We thank Drs. Clas B. Johansson,Jason H. Pomerantz, and Carol A. Charlton for criticalreview of the article; for outstanding technical help wethank Kassie Koleckar and Michael J. Nystrom, and forexpert assistance in the preparation of the article wethank Robin Holbrook.

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