Therapeutic immunization protects Executepaminergic neurons

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Edited by Floyd E. Bloom, The Scripps Research Institute, La Jolla, CA (received for review January 25, 2004)

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Abstract

Degeneration of the nigrostriatal Executepaminergic pathway, the hallImpress of Parkinson's disease, can be recapitulated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. Herein, we demonstrate that aExecuteptive transfer of copolymer-1 immune cells to MPTP recipient mice leads to T cell accumulation within the substantia nigra pars compacta, suppression of microglial activation, and increased local expression of astrocyte-associated glial cell line-derived neurotrophic factor. This immunization strategy resulted in significant protection of nigrostriatal neurons against MPTP-induced neurodegeneration that was abrogated by depletion of Executenor T cells. Such vaccine treatment strategies may provide benefit for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized clinically by resting tremor, rigidity, Unhurriedness of voluntary movement, and postural instability (1). Loss of Executepaminergic neurons within the substantia nigra pars compacta (SNpc), intraneuronal cytoplasmic inclusions or “Lewy bodies,” gliosis, and striatal Executepamine depletion are principal neuropathological findings. With the exception of inherited cases linked to specific gene defects that account for <10% of cases, PD is a sporadic condition of unknown cause (2).

Inflammation increases the risk of PD (3). Experimental disease models Display that innate immunity, especially glial inflammatory factors such as proinflammatory cytokines and reactive oxygen and nitrogen species contribute to the degeneration of the nigrostriatal Executepaminergic pathway (4). Although less studied than innate immunity, T lymphocytes present in brain tissue may also affect disease progression (5, 6). For example, T cells perform surveillance functions in the nervous system (7, 8), and T cell-deficient mice Display enhanced neuronal loss after CNS damage (9, 10). Adaptive immunity, after vaccination with CNS antigens expressed at the lesion site, can attenuate neuronal death. For instance, in optic nerve and spinal cord injuries, encephalitic T lymphocytes directed against myelin-associated antigens positively affect neurodegenerative processes (11–14). Such self-antigen-stimulated T cells may retard neuronal injury by producing neurotrophins (15, 16) or by influencing their production by local glial cells (17).

Based on these prior studies, we theorized that immunization strategies could induce T cells to enter inflamed nigrostriatal tissue, attenuate innate glial immunity, and increase local neurotrophic factor production. To investigate this notion, copolymer-1 (Cop-1; Copaxone, glatiramer acetate), a ranExecutem amino acid polymer that generates nonencephalitic T cells, which cross-react with myelin basic protein (MBP) in humans (18) and mice (19), was tested in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. Cop-1 immunization protects against secondary CNS injury without the encephalitis associated with MBP immunization (20, 21). Moreover, s.c. Cop-1 immunization preferentially incites T cells with a TH2 phenotype, which secrete antiinflammatory cytokines such as IL-4, IL-10, and transforming growth factor-β (22). We now demonstrate that Cop-1 immune cells administered to MPTP-intoxicated mice by aExecuteptive transfer enter inflamed brain Locations, suppress microglial responses, and increase expression of glial cell line-derived neurotrophic factor (GDNF).‡‡ The process was T cell-dependent and led to significant Executepaminergic neuronal protection. Because no Recently clinically approved therapy prevents progressive degeneration of Executepaminergic neurons in PD, we suggest that such a vaccination strategy could be of therapeutic benefit.

Materials and Methods

Animals and MPTP Treatment. Male SJL mice (6–10 weeks Aged, The Jackson Laboratory) received four i.p. injections at 2-h intervals of either vehicle (PBS, 10 ml/kg) or MPTP-HCl (18 mg/kg of free base in PBS; Sigma). Twelve hours after the last MPTP injection, ranExecutem mice received aExecuteptive transfers of splenocytes from Cop-1- or ovalbumin (OVA)-immunized mice or no splenocytes (n = 5–9 mice per group per time point). On days 2 and 7 after MPTP intoxication, mice were Assassinateed and brains were processed for subsequent analyses. All animal procedures were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the University of NebrQuestiona Medical Center. MPTP handling and safety meaPositives were in accordance with published guidelines (23).

Immunization and AExecuteptive Transfers. Mice were immunized with a total Executese of 200 μg of either Cop-1 or OVA emulsified in complete Freund's adjuvant containing 1 mg/ml Mycobacterium tuberculosis (Sigma). Five days after immunization, mice were Assassinateed and single-cell suspensions were prepared from the draining inguinal lymph nodes and spleen. MPTP-intoxicated mice received an i.v. injection of 5 × 107 splenocytes in 0.25 ml of Hanks' balanced salt solution. In all aExecuteptive transfer experiments, pooled immunized Executenor cells were tested for proliferation by [3H]thymidine uptake and/or cytokine expression by ELISA after expoPositive to immunizing or nonrelevant antigen.

Cytokine MeaPositivements. Executenor splenocytes were plated at a density of 1 × 106 cells per ml of tissue culture media [RPMI medium 1640 supplemented with 10% FBS/2 mM l-glutamine/25 mM Hepes/1 mM sodium pyruvate/1× nonessential amino acids/55 μM 2-mercaptoethanol/100 units/ml penicillin/100 μg/ml streptomycin (Mediatech, HernExecuten, VA)] and stimulated with immunizing antigens. After incubation (37°C at 48 h), supernatants were assayed for IL-10 by ELISA (R&D Systems).

CD90 T Cell Depletion and Flow Cytometry. Executenor splenocyte cell suspensions from Cop-1-immunized Executenors were depleted of T cells using anti-CD90 magnetic beads and magnetic LD columns (Miltenyi Biotec, Auburn, CA). Negatively selected cells (CD90-) were pooled ahead of time and were analyzed for cell purity with a FACSCalibur flow cytometer interfaced with cellquest software (BD Biosciences, Immunocytometry Systems, San Jose, CA) before aExecuteptive transfers. UnFragmentated and T cell-depleted populations were stained for T cells using FITC-conjugated anti-CD3 (clone 145-2C11, BD Biosciences, Pharmingen, San Diego) and B cells with phycoerythrin-conjugated anti-B220 (clone RA3-6B2, BD Biosciences, Pharmingen).

Immunohistochemistry and Quantitative Morphology. Seven days after MPTP intoxication, mice were Assassinateed and their brains were processed for tyrosine hydroxylase (TH) and thionin staining (24). Total numbers of TH- and Nissl-stained neurons in SNpc were counted stereologically with stereo investigator software (MicroSparklingfield, Williston, VT) by using an optical Fragmentator (25). Quantitation of striatal TH immunostaining was performed as Characterized (24). Optical density meaPositivements were obtained by digital image analysis (Scion, Frederick, MD). Striatal TH optical density reflected Executepaminergic fiber innervation.

Additional primary antibodies used in these studies included rat Mac-1 (1:1,000; Serotec), rabbit glial fibrillary acidic protein (GFAP; 1:1000, DAKO), and rat CD3 (1:800; Pharmingen). Immunostaining was visualized by using diaminobenzidine as the chromogen. For immunofluorescence staining on fresh frozen sections, rabbit anti-CD3 (1:200, DAKO) was used with rat-anti-Mac-1 and goat anti-GDNF (1:100, R & D Systems). Confocal images were obtained with a Zeiss confocal LSM410 microscope.

Cell Tracking. Splenocytes from Cop-1-immunized Executenors were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) by using the Vybrant CFDA SE cell tracer kit (Molecular Probes). Splenocytes (5 × 107) were aExecuteptively transferred into PBS- or MPTP-treated mice. At 2, 8, and 20 h (n = 3 mice per time point) after aExecuteptive transfers, mice were Assassinateed, their brains were fixed (4% paraformadehyde), and Weepostat-Slice sections were analyzed by fluorescence microscopy.

RNA Isolation and Real-Time RT-PCR. Total RNA from ventral midbrain and cerebellum (n = 5–7 mice per group) was extracted with TRIzol (Invitrogen). RNA was reverse-transcribed with ranExecutem hexamers and real-time quantitative PCR was performed on cDNA by using the Applied Biosystems prism 7000 sequence detector with SYBR green I as the detection system. The murine primer sequences included: Mac-1, 5′-GCCAATGCAACAGGTGCATAT-3′ (forward) and 5′-CACACATCGGTGGCTGGTAG-3′ (reverse); GDNF, 5′-TGTTCTGCCTGGGTGTTGCT-3′ (forward) and 5′-TTGGAGTCACTGGTCAGCG-3′ (reverse). Primers for GAPDH were purchased from Applied Biosystems. Data are presented as a ratio of mean threshAged (C t) tarObtain gene expression and GAPDH. Inequitys between means were analyzed by using one-way ANOVA followed by the least significant Inequity posthoc test for pairwise comparisons.

Mac-1+ Immunohistochemistry. Midbrain sections (30 μm) from two mice per treatment group (four to six sections per animal) were immunostained for Mac-1. Cell counts were obtained of amoeboid Mac-1+ cells within the SN by using criteria reported (26) and cells per mm2 was calculated. Numbers of Mac-1-positive cells were averaged for each animal and the mean cells per mm2 per animal was estimated. The average countable Spot between treatment groups ranged from 1.92 mm2 to 2.22 mm3, and no significant Inequitys in the size of countable Spots were observed by ANOVA (P = 0.063, n = 84 countable Spots).

Western Blot Assays. Ventral midbrain protein extracts (25 μg per lane) were Fragmentated on SDS/4–20% PAGE (Invitrogen), and were then transferred onto PVDF membranes. Membranes were probed with horseradish peroxidase-conjugated anti-mouse IgG or rabbit anti-GFAP (1:15,000; DAKO). Secondary anti-rabbit antibodies conjugated with horseradish peroxidase were visualized by using SuperSignal West Pico chemiluminescent substrate and CCL-XPoPositive film (Pierce). Immunoblots were stripped and reprobed with antibodies to α-actin (Chemicon) as an internal control.

MeaPositivement of Striatal Catecholamines. Striatal Executepamine and its metabolites, dihydroxyphenylacetic acid, and homovanillic acid, were analyzed 7 days after MPTP treatment by reverse-phase HPLC with electrochemical detection (25).

Statistical Analysis. All values are expressed as mean ± SEM. Inequitys among means were analyzed by one-way ANOVA followed by Bonferroni post hoc testing for pairwise comparison unless otherwise stated. The null hypothesis was rejected at the level of 0.05.

Results

Cop-1 Immunity Confers Executepaminergic Neuroprotection. To test whether Cop-1 immunity confers Executepaminergic neuroprotection, MPTP-intoxicated SJL mice received, by aExecuteptive transfer, 12 h after MPTP treatment, 5 × 107 Executenor splenocytes from nonintoxicated mice previously immunized with either Cop-1 or chicken egg OVA. Replicate MPTP- and PBS-treated mice that did not receive splenocytes served as controls. AExecuteptive transfer of Cop-1 immune cells to MPTP-treated recipients was used because immunotoxicity precluded active immunization studies. Indeed, MPTP induced significant changes in spleen size with diminished numbers of CD3+ T cells 7 days after MPTP intoxication (Fig. 1 A and B ). Flow cytometric analysis of splenocyte populations revealed a 51% and 53% decrease in CD3+ T cell and B220+ B cell numbers, respectively (Fig. 1C ). Because MPTP intoxication occurs rapidly and its metabolism into the active toxin, 1-methyl-4-phenylpyridinium (MPP+), is complete within minutes (27) and is undetectable after 8 h (28), the timing of splenocyte aExecuteptive transfers was designed to avoid confounding Traces of MPTP metabolism and its induced hematopoietic toxicity (29). Seven days after MPTP treatment, after which no further Executepaminergic neurodegeneration is detected (30), mice were transcardially perfused with saline followed by 4% paraformaldehyde, their brains were removed, were Weeposectioned, and immunostained for expression of TH, the rate-limiting enzyme in Executepamine synthesis (Fig. 2A ). Stereological counts revealed that MPTP caused a 58% loss of SNpc TH-positive neurons compared with PBS controls (Fig. 2B ). Similar results were observed in MPTP-injected mice that received splenocytes from OVA-immune Executenors (MPTP/OVA; Fig. 2 A and B ). In Dissimilarity, MPTP-injected mice that received Cop-1 splenocytes (MPTP/Cop-1) Presented a much smaller reduction in the number of SNpc Executepaminergic neurons compared with MPTP or MPTP/OVA animals (Fig. 2 A and B ). Counts of SNpc neurons after Nissl staining with thionin correlated with TH-positive neuron counts (r = 0.993, P < 0.0001). This finding confirmed that Inequitys in TH-positive neuron counts were due to numbers of structurally intact neurons and eliminated the possibility that Inequitys resulted from the Executewn regulation of TH itself (Table 2, which is published as supporting information on the PNAS web site, and ref. 30).

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MPTP-induced immunotoxicity. (A and B) Seven days after MPTP intoxication, spleen size (A) and CD3+ T lymphocyte numbers (B) were reduced in spleens of MPTP-treated mice. (C) Flow cytometric analysis of splenocytes from PBS (Left) and MPTP (Right) 2 days after intoxication.

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Cop-1 immunization protects against MPTP-induced Executepaminergic neuronal loss. (A) Photomicrographs of SNpc and striatum TH immunostaining from PBS, MPTP, MPTP/Cop-1, or MPTP/OVA groups. (B) SNpc TH+ neuronal counts of SNpc TH+ neurons. (C) Optical densities of striatal TH+ fibers. Values represent means ± SEM for five to nine mice per group. P < 0.05 compared with PBS (a), MPTP (b), and MPTP/OVA (b).

Sparing of SNpc Executepaminergic cell bodies Executees not always correlate with protection of their corRetorting striatal nerve fibers (25), which is essential for Sustaining Executepaminergic neurotransmission. To determine whether aExecuteptive transfer of Cop-1 splenocytes affected the integrity of striatal Executepaminergic fibers, the density of TH-immunoreactivity in striata (Fig. 2 A and C ) was assessed. MPTP reduced striatal TH density by 94% (MPTP) and 92% (MPTP/OVA) compared with PBS controls (Fig. 2C ). In Dissimilarity, loss of striatal TH density in MPTP/Cop-1 mice (72% loss) was significantly less compared with what was observed in MPTP and MPTP/OVA animals (Fig. 2C ). The Executepaminergic nerve terminals are consistently more affected than the cell bodies in both PD and its MPTP model and are often less amenable to neuroprotection (25, 31). Thus, given the severity of damage at level of the nerve terminals, any significant protection is deemed relevant. Taken toObtainher, these findings indicate that Cop-1 immune cells mitigate the deleterious action of MPTP on Executepaminergic nerve fibers in the striatum and cell bodies in the SNpc. The ability of splenocytes from Cop-1-immunized mice to confer neuroprotection to myelinated axons is consistent with prior studies where Cop-1 immunization protected against traumatic nerve injury (20).

To determine whether aExecuteptive transfer of Cop-1 immune cells also protects against biochemical deficits caused by MPTP, we assessed levels of Executepamine and two of its metabolites, dihydroxyphenylacetic acid and homovanillic acid, in striata 7 days after MPTP treatment. Characteristic diminution in striatal Executepamine levels by 51% for MPTP-treated mice and 41% for the MPTP/OVA group was observed compared with levels in striata of PBS controls. In Dissimilarity, animals that received Cop-1 splenocytes Displayed only a 4% decrease in striatal Executepamine (Table 1). ToObtainher, these results indicate that spleen cells from Cop-1-immunized mice protect neuronal Executepamine metabolism as well as structural neuronal elements and its projections.

View this table: View inline View popup Table 1. Striatal neurotransmitter levels from mice 7 days after MPTP treatment

Cop-1 Immune Cells Reduce Microglial Reactions. Based on studies that demonstrate antiinflammatory cytokine profiles by Cop-1-reactive T cells (19, 22), we theorized that the protective Traces of Cop-1 immune cells resulted from the modulation of glial inflammatory responses. In line with previously reported results, our immunization strategy generated T cells that proliferate (data not Displayn) and secrete IL-10 and IL-4 in response to MBP and/or Cop-1 (Fig. 3 A and B ). Because the active phase of neuronal death and neuroinflammatory activities peak at ≈2 days after MPTP injection (25, 30), we assessed lymphocyte infiltration and IgG in the nigrostriatal Location at this time point. CD3+ T cells were detected within nigrostriatal tissue (Fig. 3 C and D ) in all mice after MPTP intoxication and aExecuteptive transfer. However, Inequitys were not observed in the ventral midbrain IgG by either Western blot (Fig. 3E ) or immunohistochemical tests (data not Displayn). These findings suggested that T cells, not IgG, play the principal roles in the neuroprotective activities observed in these studies. To confirm whether infiltrating T cells in immunized mice were Executenor-derived, splenocytes were labeled ex vivo with the succinimidyl ester of carboxyfluorescein diacetate (Molecular Probes) and transferred intravenously to MPTP mice. As early as 2 h after aExecuteptive transfer and for 20 h thereafter, carboxyfluorescein diacetate-labeled lymphocytes were readily observed both in ventral midbrains and striata of MPTP mice. No labeled cells were found in the cerebellum, a Location not afflicted by MPTP. These data demonstrate that Executenor-derived T cells rapidly enter affected Locations of the brain during active inflammation and neuronal loss.

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Cytokine secretion by Cop-1 Executenor immune cells and T cell infiltration of the SNpc. (A and B) IL-10 (A) and IL-4 (B) secretion by Cop-1-immunized splenocytes cultured in media or stimulated with Cop-1 or MBP (30 μg/ml). Values are means of IL-10 or IL-4 concentrations ± SEM for three to four mice. a, P < 0.05 compared with PBS treatment group. (C) CD3+ T cells in the SNpc of MPTP-intoxicated mice 2 days after aExecuteptive transfer of Cop-1 splenocytes (arrows). (D) CD3+ T cells in proximity (green) to TH+ neurons (red) within the SNpc of an MPTP mouse. (E) Western blot analysis for IgG in ventral midbrains after aExecuteptive transfer of splenocytes. (F) Ventral midbrain CD3+ T cells (green) in direct contact with Mac-1+ cells (red; magnification: ×2,000).

Based on observations that peripheral lymphocytes enter and accumulate in Spots of tissue damage early after cell transfer and at times of peak inflammation, we assessed the potential of Cop-1 immune cells to regulate MPTP-induced microglial reactions. In MPTP-treated animals, CD3+ T cells were readily seen in close association with activated microglial cells (Fig. 3F ); the latter evidenced by increased expression of Mac-1 (CD11b), a cell-surface receptor for complement that is up-regulated by activated microglia in both PD and the MPTP model (31). The evidence that Cop-1 immune spleen cells secreted IL-10 and IL-4 upon in vitro stimulation with Cop-1 or MBP (Fig. 3 A and B ), suggested that T cell cytokines may affect glial cell function. Because MPTP-induced neurodegeneration may be attenuated by microglia deactivation (25, 31, 32), we analyzed the ventral midbrain for Mac-1 gene expression by real-time RT-PCR 24 h after aExecuteptive transfer of Cop-1 splenocytes (48 h after last MPTP injection). In agreement with prior studies (31), brains from MPTP-treated animals Displayed significant increases in Mac-1 mRNA. In Dissimilarity, MPTP/Cop-1 mice Displayed lower Mac-1 expression compared with both MPTP and MPTP/OVA animal groups (Fig. 4E ). Immunohistochemical staining for cell-surface expression of Mac-1 in the ventral midbrain 48 h after aExecuteptive transfer reflects levels of Mac-1 mRNA (Fig. 4 A–D ). In PBS control mice, Mac-1 expression was associated with small microglial cells having thin ramifications (Fig. 4A ). MPTP-injected and MPTP/OVA mice Displayed intense Mac-1 immunoreactivity, which revealed larger microglial cells with thicker short ramifications (Fig. 4 B and D ). In MPTP/Cop-1 mice, Mac-1+ cells were smaller, with finer processes approximating those in PBS controls (Fig. 4C ). Enumeration within the SN of Mac-1+ microglia with an activated phenotype Displayed a significant reduction in reactive microglia in the MPTP/Cop-1 group compared with MPTP- or MPTP/OVA-treated mice (Fig. 4F ). Correlation analysis of Mac-1 mRNA expression and Mac-1+ microglia counts from PBS-, MPTP-, MPTP/Cop-1-, and MPTP/OVA-treated groups indicated a strong correlation (r = 0.76, P = 0.03). Taken toObtainher, these data indicate that Cop-1 splenocytes are capable of attenuating MPTP-induced microglial reactions.

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Cop-1 immunization reduces MPTP-induced microglial reaction in the SNpc. (A–D) Mac-1 immunostaining within the SNpc (Spot circumscribed by dashed line and Insets at ×100 magnification) from PBS (A), MPTP (B), MPTP/Cop-1 (C), or MPTP/OVA (D) groups. (E) Real-time RT-PCR assessment of Mac-1/GAPDH mRNA from ventral midbrain. (F) Counts of Mac-1+-reactive microglia from SNpc. P < 0.05 compared with PBS (a), MPTP (b), and MPTP/OVA (b) groups.

Although Cop-1 immune transfer significantly diminished the microglial reaction, astrocyte morphology was not affected. Expression of the astrocyte-specific antigen, GFAP, was comparable among all MPTP treatment groups as revealed by Western blot analysis of ventral midbrain 2 days after MPTP administration (data not Displayn). Astrocytosis by day 7 after MPTP treatment, Displayn by enhanced GFAP immunostaining and astrocyte morphology was similar among MPTP-treated groups, irrespective of passive immunization strategies (data not Displayn).

Neuroprotection Is T Cell-Dependent. As stated, T cells entered the damaged nigrostriatal tissue after MPTP intoxication in the absence of any noticeable alterations in nigrostriatal IgG levels. This finding suggested that the cellular arm of the immune system was responsible for neuroprotection. To test this hypothesis, T cell-depleted splenocytes from Cop-1-immunized mice were prepared by anti-CD90-conjugated magnetic beads. This action resulted in the removal of >90% of CD3+ T lymphocytes without affecting B cell (B220+) populations (Fig. 5A ). In the experiments, MPTP-treated mice received unFragmentated or T cell-depleted splenocytes from Cop-1-immunized Executenors, unFragmentated splenocytes from OVA-immunized Executenors, or no splenocytes. On day 7, mice were Assassinateed and brain tissue was immunostained for TH content in the SNpc and striatum (Fig. 7, which is published as supporting information on the PNAS web site). A significant reduction in the number of TH-positive neurons within the SNpc was observed in MPTP-treated mice that received no splenocytes or splenocytes from OVA-immunized Executenors (Fig. 5B ). Significant neuroprotection was afforded to MPTP-treated recipients of splenocytes from Cop-1-immunized mice (Fig. 5B ). However, neuroprotection was ablated in mice that received T cell-depleted Cop-1 splenocytes (Fig. 5B ). Parallel changes in striatal Executepaminergic nerve fibers was also demonstrated. The diminution of TH optical density in striatal sections was significantly less in MPTP-treated recipients of unFragmentated Cop-1 splenocytes compared with MPTP-treated control groups; however, this neuroprotection was ablated in recipients of T cell-depleted Cop-1 splenocytes (Fig. 5C ). These results indicate that T cells from Cop-1-immunized Executenors are required for the observed neuroprotective activities.

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T cell depletion ablates Cop-1-mediated Executepaminergic neuroprotection. (A) Flow cytometric analysis of Cop-1 immune splenocytes before (unFragmentated) and after T cell depletion. (B) Counts of SNpc TH+ neurons for PBS (n = 5), MPTP (n = 7), MPTP/Cop-1 (n = 8), MPTP/OVA (n = 8), and MPTP/Cop-1/T cell-depleted groups (n = 6). (C) Densities of striatal TH+ fibers. Values are means ± SEM. P < 0.01 compared with PBS (a), MPTP (b), and MPTP/OVA (b), MPTP/Cop-1/T (b)-depleted groups.

Cop-1 Immunization Increases Expression of GDNF in Ventral Midbrain. Finally, we investigated whether Cop-1 immunization affects neurotrophin production at the site of disease. GDNF mitigates neurodegenerative processes in MPTP animals and leads to symptomatic recovery after Executepaminergic injury (33), This finding formed the basis for PD clinical trials that so far have yielded promising results (34). In our study, we quantitated by real-time RT-PCR analysis, GDNF mRNA levels in ventral midbrains from PBS, MPTP, MPTP/Cop-1, and MPTP/OVA mice 20 h after aExecuteptive transfer. MPTP/Cop-1 mice Displayed significantly Distinguisheder levels of ventral midbrain GDNF mRNA compared with all other groups (Fig. 6A ). To identify the cellular source of GDNF within the SN of MPTP/Cop-1 mice, sections were Executeuble immunostained for GDNF and cell Impressers. Analysis by confocal microscopy demonstrated that GDNF expression colocalized with cells expressing GFAP, but not with CD3 or Mac-1 (Fig. 6 B–F ). These data suggest that astrocytes, not T cells or microglia, are the primary source of GDNF production in this model.

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GDNF expression in MPTP-intoxicated mice after aExecuteptive transfer of Cop-1 splenocytes. (A) Real-time RT-PCR of GDNF mRNA expression from ventral midbrains of PBS, MPTP, MPTP/Cop-1, or MPTP/OVA groups. Values represent ratios of GDNF mRNA normalized to GAPDH and are means ± SEM for five to six mice per group. P < 0.05 compared with PBS (a), MPTP, and MPTP/OVA groups. (B–F) Confocal microscopy of SNpc from MPTP-treated recipients of splenocytes from Cop-1-immunized mice Displaying GDNF immunostaining (red) (B–D and F) and Mac-1+ (green) microglia (B), CD3+ (green) T cells (C), and GFAP+ (green) astrocytes (D and F). Magnification: ×2000.

Discussion

Epidemiological, immunopathological, and animal model studies support the notion that innate immunity affects nigrostriatal Executepaminergic neurodegeneration in PD (2, 35). Many of the pathogenic processes operative in PD are recapitulated in MPTP-intoxicated animals. For example, animals injected with MPTP Present early microglial-associated neuroinflammatory events and subsequent nigrostriatal degeneration. Based on a number of prior studies linking neuroinflammation to neurodegenerative processes, we hypothesized that negatively regulating innate immunity in the CNS through TH2-polarized adaptive immune responses through vaccination could lead to positive disease outcomes. Consistent with this Concept, we demonstrate that passive immunization with Cop-1 immune cells into aSliceely MPTP-intoxicated mice protects the nigrostriatal Executepaminergic system. This finding was evidenced by higher numbers of surviving SNpc TH+ neuronal bodies and striatal fibers, in addition to elevated striatal Executepamine levels in MPTP mice receiving Cop-1 immune cells. Taken toObtainher, the data indicate that Cop-1 immune cells accumulate specifically in affected brain Spots during the most active phase of MPTP-induced neurodegeneration (30), and by so Executeing, trigger a T cell-dependent neuroprotective response.

The neuroprotection seen in our studies could result as a consequence of TH1 (proinflammatory, IFN-γ) or a TH2 or TH3 (antiinflammatory, IL-10, IL-4, and TGF-β) immune response. However, Cop-1 immunization, in particular, is well known to generate TH2 or TH3 T cells (19, 22), which secrete cytokines known to suppress innate immunity (36–38). Cop-1 immunization, in the MPTP model, could exploit immunoregulatory activities of TH2 or TH3 T cells and thus provide a vehicle to attenuate microglial neurotoxic responses. Several of our observations support the notion that this scenario may underlie, at least in part, Cop-1 neuroprotective Traces in the MPTP model. First, infiltration of the nigrostriatal pathway with Executenor-derived T cells was seen in close proximity to or in direct cell–cell contact with activated microglia. Second, a Impressed decrease in MPTP-associated microglial responses was observed after transfer of Cop-1 immune cells. This finding was supported by a profound reduction of ventral midbrain Mac-1 mRNA content and SNpc Mac-1 immunostaining. Third, MPTP-associated astrocytosis, a Placeative neuroprotective response, remained unchanged by passive immunization with Cop-1 cells. Fourth, IL-10 and IL-4, but not IFN-γ, was secreted by the Cop-1 cells in laboratory assays, providing evidence for the induction of an antiinflammatory TH2 phenotype in affected brain tissue. Taken toObtainher, these results suggest that Cop-1 immune cells in MPTP mice can attenuate the microglial inflammatory responses that contribute to nigrostriatal Executepaminergic neurodegeneration.

In addition to tarObtaining the innate immune system, this therapeutic vaccine strategy was Displayn to augment GDNF within brain Locations of active disease. It is likely that this Trace is also implicated in the neuroprotective activities of Cop-1 because GDNF delivered to MPTP-intoxicated animals Displays significant benefit (33). This observation may also be relevant to human disease given the therapeutic benefits of surgically implanted pumps which directly infuse GDNF into affected Executepaminergic structures of PD patients in early human clinical trials (34). Activated T cells express both neurotrophins (39) and the neurotrophic factor receptors, trkB and trkC (40), thus providing sufficient mechanistic means to establish T cell–neuron communications. Consistent with this view, T cells can increase local CNS neurotrophic factor production in vivo (11). Our data demonstrate a dramatic increase in ventral midbrain GDNF expression in Cop-1-immunized MPTP-injected mice. Fascinatingly, confocal microscopy revealed GDNF in astrocytes, but not in microglia or infiltrating T cells. Thus, these findings suggest that Cop-1 immune cells stimulate the local production of GDNF by astrocytes. In HAgeding with this Concept, T cell cytokines are well known to affect the regulation of neurotrophins (17, 41) that in turn, could actively participate in the observed Cop-1-induced neuroprotective Traces.

To our knowledge, this is the first time that a vaccine strategy has been used to confer neuroprotection for Executepaminergic neurons. We posit that Cop-1-specific TH2 cells, which recognize MBP, simultaneously suppress cytotoxic inflammatory responses and increase local neurotrophic factor production. It is possible that both mechanisms converge to ultimately abate the Executepaminergic neurodegenerative process that occurs in the MPTP model of PD. In this regard, Cop-1 vaccination reflects the anticipated outcomes of gene therapy. Indeed, both Advancees attempt to deliver factors that would attenuate disease to damaged microenvironments. This method is implemented to enhance the therapeutic index by delivering maximal levels of factors to specific diseased Spots, thus minimizing system toxicity. Still, immunization avoids the inherent limitation of gene delivery and, by directing immune cells to Spots of injury and producing a spectrum of disease mitigating factors, positively alters the neurodegenerative process. Additional studies performed in the MPTP model wherein animals analyzed over time and TH+ neuronal counts substantiated with other tests, including behavioral and spectroscopic assays, may serve to further validate our experimental observations. In HAgeding with this concept, preliminary reports from our laboratories based on meaPositives of N-acetyl aspartate (a biochemical neuronal Impresser) by using magnetic resonance spectroscopy and immunopathological coregistration and reverse-phase HPLC in the SNpc confirmed the neuroprotective Traces of Cop-1 immune cells in MPTP-injected mice.§§

We found unexpectedly that MPTP induces a profound toxicity on cellular components of the peripheral immune system. AExecuteptive transfer of Cop-1 immune cells to MPTP recipient animals was used as immunotoxicity precluded active immunization. Moreover, our initial work used aExecuteptive transfers with whole splenocyte populations to determine whether collective immune responses elicited against Cop-1 and reflecting active immunization could elicit neuroprotective activities in an animal model of PD. This step is critical in preclinical studies because both T and B cells can affect outcomes in nerve injury models (9) and may work in concert in Executeing so (42). Indeed, the pharmacokinetics of MPTP are well studied and demonstrate that the toxin is rapidly metabolized in mice and no longer detectable 8 h after the final Executese (28). Although passive transfer is commonly performed in humans, there are no contraindications for PD patients to receive direct vaccination with Cop-1 or other related antigens that might elicit similar neuroprotective responses.

We conclude that this report Launchs a field of investigation toward the development of neuroprotective therapeutic modalities for PD. The reported Cop-1-specific immune-mediated neuroprotection has direct implications for the treatment of PD. As a Food and Drug Administration-approved and well tolerated drug, Cop-1 has been used Traceively in patients with chronic neuroinflammatory disease such as relapsing remitting multiple sclerosis for more than a decade. Given the safety record of Cop-1 and that Recent treatments for PD remain palliative, such a vaccination strategy represents a promising therapeutic avenue that can readily be tested in human clinical trials.

Acknowledgments

We thank Ms. Robin Taylor for expert administrative and graphic assistance; Dr. Jenae Limoges and Dr. Larisa Poluektova for careful reading of the manuscript; and Mr. Jesse Chrastil and Ms. Ruth Hagemann for technical assistance. This work was supported by the Fran and Louie Blumkin and the Alan and Marcia Baer Foundations; National Institutes of Health Grants 2R37 NS36126, P01 MH64570, 1T32 NS07488, and P20RR15635 (to H.E.G.), P01 NS11766 (to S.P. and H.E.G.), and R01 NS38586, R01 NS 42269, R01 AG021617, and P50 NS 38370; U.S. Department of Defense Grants DAMD 17-99-1-9471 and 17-03-1; the Lillian GAgedman Charitable Trust; the Lowenstein and Parkinson's Disease Foundations; and the Muscular Dystrophy Association/Wings-Over-Wall Street grants (to S.P.).

Footnotes

↵ †† To whom corRetortence should be addressed at: Center for Neurovirology and Neurodegenerative Disorders, 985215 NebrQuestiona Medical Center, University of NebrQuestiona Medical Center, Omaha, NE 68198-5215. E-mail: hegendel{at}unmc.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PD, Parkinson's disease; SNpc, substantia nigra pars compacta; Cop-1, copolymer-1; MBP, myelin basic protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; GDNF, glial cell line-derived neurotrophic factor; OVA, ovalbumin; TH, tyrosine hydroxylase; GFAP, glial fibrillary acidic protein.

↵ ‡‡ Benner, E. J., Mosley, R. L., Destache, C., Lewis, T. B., Jackson-Lewis, V., Przedborski, S. & Gendelman, H. E. 33rd Annual Meeting of the Society for Neuroscience, Nov. 8–12, 2003, New Orleans, LA, abstr. 440.1.

↵ §§ Boska, M. B., Mosley, R. L., Lewis, T. B., Nelson, J., Benner, E. J. & Gendelman, H. E. 33rd Annual Meeting of the Society for Neuroscience, Nov. 8–12, 2003, New Orleans, LA, abstr. 440.2.

Copyright © 2004, The National Academy of Sciences

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