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The expression of T regulatory cells (Foxp3), regulatory (interleukin [IL]-10 and transforming growth factor beta [TGF-β]) and proinflammatory (tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1β) cytokines was quantified using real time polymerase chain reaction (qRT-PCR) in the liver of sheep during early stages of infection with Fasciola hepatica (1, 3, 9, and 18 days post-infection [dpi]). Portal fibrosis was also evaluated by Masson’s trichrome stain as well as the number of Foxp3+ cells by immunohistochemistry. Animals were divided into three groups: (a) group 1 was immunized with recombinant cathepsin L1 from F. hepatica (FhCL1) in Montanide adjuvant and infected; (b) group 2 was uniquely infected with F. hepatica; and (c) group 3 was the control group, unimmunized and uninfected. An overexpression of regulatory cytokines of groups 1 and 2 was found in all time points tested in comparison with group 3, particularly at 18 dpi. A significant increase of the number of Foxp3+ lymphocytes in groups 1 and 2 was found at 9 and 18 dpi relative to group 3. A progressive increase in portal fibrosis was found in groups 1 and 2 in comparison with group 3. In this regard, group 1 showed smaller areas of fibrosis than group 2. There was a significant positive correlation between Foxp3 and IL-10 expression (by immunohistochemistry and qRT-PCR) just as between portal fibrosis and TGF-β gene expression. The expression of proinflammatory cytokines increased gradually during the experience. These findings suggest the induction of a regulatory phenotype by the parasite that would allow its survival at early stages of the disease when it is more vulnerable.

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Parkinson’s disease is characterized by the intracellular accumulation of α-synuclein which has been linked to early dopaminergic axonal degeneration. Identifying druggable targets that can promote axonal growth in cells overexpressing α-synuclein is important in order to develop strategies for early intervention. Class-IIa histone deacetylases (HDACs) have previously emerged as druggable targets, however, it is not known which specific class-IIa HDACs should be targeted to promote neurite growth in dopaminergic neurons. To provide insight into this, we used gene co-expression analysis to identify which, if any, of the class-IIa HDACs had a positive correlation with markers of dopaminergic neurons in the human substantia nigra. This revealed that two histone deacetylases, HDAC5 and HDAC9, are co-expressed with TH, GIRK2 and ALDH1A1 in the human SN. We further found that HDAC5 and HDAC9 are expressed in dopaminergic neurons in the adult mouse substantia nigra. We show that siRNAs targeting HDAC5 or HDAC9 can promote neurite growth in SH-SY5Y cells, and that their pharmacological inhibition, using the drug MC1568, promoted neurite growth in cultured rat dopaminergic neurons. Moreover, MC1568 treatment upregulated the expression of the neurotrophic factor, BMP2, and its downstream transcription factor, SMAD1. In addition, MC1568 or siRNAs targeting HDAC5 or HDAC9 led to an increase in Smad-dependent GFP expression in a reporter assay. Furthermore, MC1568 treatment of cultured rat dopaminergic neurons increased cellular levels of phosphorylated Smad1, which was prevented by the BMP receptor inhibitor, dorsomorphin. Dorsomorphin treatment prevented the neurite growth-promoting effects of siRNAs targeting HDAC5, as did overexpression of dominant-negative Smad4 or of the inhibitory Smad7, demonstrating a functional link to BMP signaling. Supplementation with BMP2 prevented the neurite growth-inhibitory effects of nuclear-restricted HDAC5. Finally, we report that siRNAs targeting HDAC5 or HDAC9 promoted neurite growth in cells overexpressing wild-type or A53T-α-synuclein and that MC1568 protected cultured rat dopaminergic neurons against the neurotoxin, MPP+. These findings establish HDAC5 and HDAC9 as novel regulators of BMP-Smad signaling, that additionally may be therapeutic targets worthy of further exploration in iPSC-derived human DA neurons and in vivo models of Parkinson’s disease.