Calpain mediated expansion of CD4+ cytotoxic T cells in rodent models of Parkinson's disease

Abstract

Parkinson's disease (PD), a debilitating progressive degenerative movement disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra (SN), afflicts approximately one million people in the U.S., including a significant number of Veterans. Disease characteristics include tremor, rigidity, postural instability, bradykinesia, and at a cellular level, glial cell activation and Lewy body inclusions in DA neurons. The most potent medical/surgical treatments do not ultimately prevent disease progression. Therefore, new therapies must be developed to halt progression of the disease. While the mechanisms of the degenerative process in PD remain elusive, chronic inflammation, a common factor in many neurodegenerative diseases, has been implicated with associated accumulation of toxic aggregated α-synuclein in neurons. Calpain, a calcium-activated cysteine neutral protease, plays a pivotal role in SN and spinal cord degeneration in PD via its role in α-synuclein aggregation, activation/migration of microglia and T cells, and upregulation of inflammatory processes. Here we report an increased expression of a subset of CD4+ T cells in rodent models of PD, including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mice and DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride]/6-hydroxydopamine rats, which produced higher levels of perforin and granzyme B - typically found in cytotoxic T cells. Importantly, the CD4+ cytotoxic subtype was attenuated following calpain inhibition in MPTP mice, suggesting that calpain and this distinct CD4+ T cell subset may have critical roles in the inflammatory process, disease progression, and neurodegeneration in PD.

Keywords: CD4+ cytotoxic T cells; Calpain; Granzyme B; MPTP; Parkinson's disease; Perforin.

Figure 1:. Calpain activation and dysregulation of the pro- and anti-apoptotic mitochondrial membrane proteins Bax and Bcl-2 in subacute MPTP mice; calpeptin (Cp) pretreatment is protective.

Calpain over-activation and expression of Bax/Bcl-2 was compared in SC in subacute MPTP mice (MPTP without/with Cp (25 μg/kg, s.c., 30 min before MPTP) on day 7 after last injection. (A) Western blot analysis of SC (data presented in cervical) showed parallel increases in calpain expression and activity in MPTP, which were attenuated by Cp treatment. (B) Quantitative data regarding Cp treatment and reduction of active calpain (76 kDa) and calpain-specific SBDP (145 kDa) in MPTP compared to controls (*p<0.05). (C) Western blot analysis of SC (cervical SC) for Bax and Bcl-2 proteins after Cp treatment, and (D) Quantification of the Bax/Bcl-2 ratio showing elevated Bax (cell death) and Cp-mediated protection (low Bax), suggesting that Cp treatment attenuated calpain expression/activity and protected neurons in MPTP mice. *p<0.05 compared to control and ^@p< 0.05 compared to MPTP, n = 4-6, per group.

Figure 2:. Active calpain formation, CD4+ cell expansion, and calpain/CD4 co-expression in the spleen of DSP-6-OHDA rat model of PD.

(A) Western blot analysis of frozen spleen samples from PD-like lesions induced in rats by injections with DSP-4 (50 mg/kg, i.p.) /6-OHDA (5μg/μl, 2μl/site) or saline. Calpain activation (arrows) is detected in frozen spleens from DSP-4/6-OHDA rats as compared to saline controls. (B) Quantitation of m-calpain bands by ImageJ software. Statistical analysis was performed by the student’s t test. *p<0.05. (C) Frozen spleen samples from DSP-4/6-OHDA injected rats were also analyzed by IHC for calpain and CD4+ T cells. Magnification, 20X. (D) Quantitative analysis of fluorescence intensity showed a slight elevation of m-calpain (calpain-2) and a significant increase in CD4+ T cell population (p<0.05) in DSP-4/6-OHDA rats.

Figure 3:. Calpeptin (Cp) attenuates gliosis and inflammatory signals, reducing α-synuclein aggregation in chronic MPTP mice.

(A) The effects of Cp against microglial activation were examined by IHC. Cp pre-treatment attenuated MPTP-induced increases in OX42 immunoreactivity (OX42-IR) in SC of MPTP mice. (B) Quantitation of OX42-IR showing a significant increase in fluorescence intensity in MPTP mice and attenuation of OX42-IR by calpeptin. *p<0.05, compared to control; #p<0.05, compared to MPTP. (C) Immunofluorescent staining of SN using antibodies to tyrosine hydroxylase (TH) (green) and α-synuclein (red). (D) Immunofluorescent staining of SC samples with neuron-specific NeuN antibody (green), and α-synuclein antibody (red). An increased aggregation of α-synuclein in SC was observed in chronic MPTP mice compared to probenecid+Cp controls; Cp treatment reduced α-synuclein aggregation. Representative image from n=4 for controls, and n=5-6 for MPTP with or without Cp.

Figure 4:. Effects of calpeptin on axonal motor protein in MPTP mice.

Immunofluorescent staining of cervical spinal cord samples from untreated control mice, MPTP-injected mice, and MPTP mice treated with calpeptin. (A) In control cervical SC, axonal motor protein kinesin is vividly distributed. Upper panel: the distribution of kinesin (red), pan-axonal marker (SMI312; green), and overlay (yellow). Expanded view of kinesin staining in dorsal horn (DH), interneuron (IN), and ventral horn (VH). (B) Acute MPTP regimen caused disintegration of kinesin, which was significantly attenuated by calpeptin post-treatment. (C) Values (fluorescence intensity) obtained from panel B were normalized and plotted. Statistical analysis was performed using one-way ANOVA. n = 3-4, Magnification, 200x.

Figure 5:. Inhibition of activated CD4+/CD8+ T cells by calpeptin in subacute MPTP mice.

(A) Splenocytes obtained from subacute MPTP mice, calpeptin treated subacute MPTP mice, and control untreated mice were stimulated with PMA (100nM) plus ionomycin (1μg/ml) in media for 72 hours (n=3-4). Cells were washed, stained with antibodies against CD4 and CD8 markers, and analyzed by flow cytometry. (B) Cells were also stained with CD25-PE showing that calpeptin attenuated activated T cells. Data are representative of at least three separate experiments. These data suggest that CD4+ T cell are expanded in MPTP mice, and calpeptin treatment attenuates the proliferation of T cells.

Figure 6:. Detection of cytotoxic CD4+ T and CD8+ cells in subacute MPTP mice, which were differentially modulated by inhibition of calpain.

Splenocytes obtained from chronic MPTP mice, calpeptin treated subacute MPTP mice, and untreated control mice (n=3-5) were stained with FITC, PE, and APC labeled antibodies against CD4, CD8, and granzyme B. Gated CD4+ T cells (A) and CD8+ T cells (B) were subjected to intracellular staining and analyzed by flow cytometry for detection of granzyme B. (C-D) Quantitative analyses of granzyme B positive (GZMB+) CD4+ and CD8+ T cells. Data suggest that cytotoxic CD4+ T cells are generated in subacute MPTP mice, and calpain inhibition attenuated these cytotoxic CD4+ T cells in subacute MPTP mice. *p<0.05, ns=not significant.

Figure 7:. Spleen samples from a rat model of PD neurodegeneration were analyzed by immunohistochemistry for CD4 and granzyme B.

Briefly, PD-like lesion rats received a systemic injection of noradrenergic neurotoxin DSP-4, followed one week later by bilateral intra-striatal injection of dopaminergic neurotoxin 6-hydroxydopamine as described in the methods. (A) After 21 days, rats were euthanized, and spleen samples were analyzed for cytotoxic CD4+ T cells (granzyme B and perforin) by immunohistochemistry. Non-lesion spleen samples were used as a control. Nuclei were also visualized using DAPI (Blue). (B) Quantitative analysis of fluorescence intensity suggests that granzyme B-producing CD4+ T cells are increased in PD-like disease in rats (n=3-4). Statistical analysis was performed using one-way ANOVA. Data are representative of at least three separate experiments. *p<0.05, **p<0.01.

Figure 8:. Detection of perforin producing cytotoxic CD4+ T cells in subacute MPTP mice, which was attenuated by inhibition of calpain.

Splenocytes obtained from subacute MPTP mice, calpeptin treated subacute MPTP mice, and untreated control mice (n=3-5) were stained with FITC, PE, and APC labeled antibodies against CD4, perforin and CD8. (A-B) Gated CD4 and CD8 T cells were analyzed by flow cytometry for detection of perforin as described. (C-D) Quantitative analyses of granzyme B positive (GZMB+) CD4+ and CD8+ T cells. Data suggest that perforin producing CD4+ T cells are generated in subacute MPTP mice, and calpain inhibition attenuated cytotoxic CD4+ T cells in these MPTP mice. *p<0.05, ns=not significant. Calcium-activated neutral protease, calpain, plays a role in T cell activation. A subset of CD4+ T cells was upregulated in rat and mouse models of PD. The CD4+ cytotoxic T cell subtype was attenuated with calpain inhibition.