Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease

Abstract

Degeneration of the nigrostriatal dopaminergic pathway, the hallmark of Parkinson's disease, can be recapitulated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. Herein, we demonstrate that adoptive 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 donor T cells. Such vaccine treatment strategies may provide benefit for Parkinson's disease.

Fig. 1.

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.

Fig. 2.

Cop-1 immunization protects against MPTP-induced dopaminergic 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).

Fig. 3.

Cytokine secretion by Cop-1 donor 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 adoptive 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 adoptive transfer of splenocytes. (F) Ventral midbrain CD3⁺ T cells (green) in direct contact with Mac-1⁺ cells (red; magnification: ×2,000).

Fig. 4.

Cop-1 immunization reduces MPTP-induced microglial reaction in the SNpc. (A–D) Mac-1 immunostaining within the SNpc (area 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.

Fig. 5.

T cell depletion ablates Cop-1-mediated dopaminergic neuroprotection. (A) Flow cytometric analysis of Cop-1 immune splenocytes before (unfractionated) 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.

Fig. 6.

GDNF expression in MPTP-intoxicated mice after adoptive 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 showing 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.