A critical role for alpha-synuclein in development and function of T lymphocytes

Abstract

Alpha-synuclein is highly expressed in the central nervous system and plays an important role in pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia. Previous studies have demonstrated the expression of α-synuclein in hematopoietic elements and peripheral blood mononuclear cells, although its roles in hematopoiesis and adaptive immunity are not studied. Using an α-synuclein knock out (KO) mouse model, we have recently shown that α-synuclein deficiency is associated with a mild defect in late stages of hematopoiesis. More importantly, we demonstrated a marked defect in B lymphocyte development and IgG, but not IgM production in these mice. Here we show a marked defect in development of T lymphocytes in α-synuclein KO mice demonstrated by a significant increase in the number of CD4 and CD8 double negative thymocytes and significant decreases in the number of CD4 single positive and CD8 single positive T cells. This resulted in markedly reduced peripheral T lymphocytes. Interestingly, splenic CD4(+) and CD8(+) T cells that developed in α-synuclein KO mice had a hyperactivated state with higher expression of early activation markers and increased IL-2 production. Moreover, splenic CD4(+) T cells from α-synuclein KO mice produced lower levels of IL-4 upon antigenic stimulation suggesting a defective Th2 differentiation. Our data demonstrate an important role for α-synuclein in development of T lymphocytes and regulation of their phenotype and function.

Keywords: CD4; CD8; Lymphopoiesis; T cells; Treg; α-Synuclein.

Figure 1

Alpha-synuclein deficiency does not affect the morphology hematopoietic elements. Representative sections of bone marrow from WT (A) and α-synuclein KO (B) mice were examined by H&E staining at 400X magnification (bone marrow sections from 10 mice of each strain were examined).

Figure 2

Alpha-synuclein KO mice have defective thymic maturation. Representative flow cytometry panels of thymocytes stained with anti-CD4 and anti-CD8 antibodies (A). Absolute numbers (B) and percentages (C) of thymocytes from α-synuclein KO and WT mice at different stages of development (DN: double negative, DP: double positive, SP: single positive, n=5 for each strain).

Figure 3

Alpha-synuclein KO mice have significantly reduced number of mature splenic T lymphocytes. . Absolute numbers (A) and percentages (B) of splenic CD4⁺ and CD8⁺ T cells from α-synuclein KO and WT mice (n=5 for each strain).

Figure 4

Alpha-synuclein KO mice have reduced number of T lymphocytes associated with disorganized thymus and spleen architecture. (A) Representative section of thymus, examined by H&E staining at 40X magnification showing increased size of cortex in relation to medulla in α-synuclein KO as compared to WT animals. (B) Representative sections of spleen from α-synuclein KO and WT animals examined by H&E staining and after immunohistochemical staining with anti-CD3 antibody at 40X magnification (Data are representative of 5 independent experiments).

Figure 5

Peripheral T cells in α-synuclein KO mice have a hyperactivated state. (A) Naïve splenic T cells from α-synuclein KO and WT mice were analyzed by flow cytometry using antibodies against CD4, CD8 and one of early activation markers CD69 (A), CD49d (B), or late activation marker CD44 (C), or lymph node homing marker CD62L (D).

Figure 6

Defective Th2 differentiation in α–synuclein KO mice. Purified naïve splenic CD4⁺ T cells from α-synuclein KO and WT mice were activated in vitro with anti-CD3/anti-CD28 mAb-coated beads and IL-4 (A), IFN-γ (B) and IL-2 (C) production levels were analyzed after 48 hours.

Figure 7

Development of Foxp3⁺ CD4⁺ regulatory T cells (Tregs) in α-synuclein KO mice. Single cell suspensions were obtained from thymi and spleens of α-synuclein KO and WT mice and stained with antibodies against CD4, CD8 and Foxp3 (intracellular). Percentage of Foxp3⁺ population was analyzed in CD4⁺ CD8⁻ population.