Pathological histone acetylation in Parkinson's disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition

Abstract

Parkinson's disease (PD) is associated with degeneration of nigrostriatal neurons due to intracytoplasmic inclusions composed predominantly of a synaptic protein called α-synuclein. Accumulations of α-synuclein are thought to 'mask' acetylation sites on histone proteins, inhibiting the action of histone acetyltransferase (HAT) enzymes in their equilibrium with histone deacetylases (HDACs), thus deregulating the dynamic control of gene transcription. It is therefore hypothesised that the misbalance in the actions of HATs/HDACs in neurodegeneration can be rectified with the use of HDAC inhibitors, limiting the deregulation of transcription and aiding neuronal homeostasis and neuroprotection in disorders such as PD. Here we quantify histone acetylation in the Substantia Nigra pars compacta (SNpc) in the brains of control, early and late stage PD cases to determine if histone acetylation is a function of disease progression. PD development is associated with Braak-dependent increases in histone acetylation. Concurrently, we show that as expected disease progression is associated with reduced markers of dopaminergic neurons and increased markers of activated microglia. We go on to demonstrate that in vitro, degenerating dopaminergic neurons exhibit histone hypoacetylation whereas activated microglia exhibit histone hyperacetylation. This suggests that the disease-dependent increase in histone acetylation observed in human PD cases is likely a combination of the contributions of both degenerating dopaminergic neurons and infiltrating activated microglia. The HDAC SIRT 2 has become increasingly implicated as a novel target for mediation of neuroprotection in PD: the neuronal and microglial specific effects of its inhibition however remain unclear. We demonstrate that SIRT 2 expression in the SNpc of PD brains remains relatively unchanged from controls and that SIRT 2 inhibition, via AGK2 treatment of neuronal and microglial cultures, results in neuroprotection of dopaminergic neurons and reduced activation of microglial cells. Taken together, here we demonstrate that histone acetylation is disease-dependently altered in PD, likely due the effects of dopaminergic neurodegeneration and microglial infiltration; yet SIRT 2 remains relatively unaltered with disease. Given the stable nature of SIRT 2 expression with disease and the effects of SIRT 2 inhibitor treatment on degenerating dopaminergic neurons and activated microglia detected in vitro, SIRT 2 inhibitors warrant further investigation as potential therapeutics for the treatment of the PD.

Keywords: Histone deacetylase inhibitor; Microglia; Neurodegeneration; Neuroprotection; Parkinson’s disease; SIRT 2.

Fig. 1

Histone Acetylation and Parkinson’s Disease Progression. Histone acetylation (AcH3-Lys9) was quantified in the Substantia Nigra pars compacta (SNpc) of each control, each early, and each late PD case relative to β-Actin using Western blot analysis. (A) Bar graph illustrating level of AcH3-Lys9 observed in each group of cases. (B) Representative blot of data presented in (A). (C) Graphical representation of correlation between Braak Stage of human cases and AcH3-Lys9 observed in the SNpc of that case. N = 8-12 per group (see supplementary Table 1). Statistical significance indicated with asterisks: *p < .05. Abbreviations: ePD, early Parkinson’s disease; lPD, late Parkinson’s disease.

Fig. 2

Relative mRNA Expression of TH, HLA-DPα1 and SIRT 2 in Control and PD SNpc Tissue Samples. (A) mRNA expression levels of cellular markers TH and HLA-DPα1 in the SNpc of control and PD cases, relative to a housekeeping gene expression, quantified using qRT-PCR. (B) mRNA expression levels of the HDAC SIRT 2 in the same cases. N = 8-12 per group (see supplementary Table 1). Statistical significance indicated with asterisks: *p < .05. Abbreviations: TH, tyrosine hydroxylase; HLA-PDα, Human Leukocyte Antigen DPα1, SIRT 2, Silent Information Regulator 2; ePD, early Parkinson’s disease; lPD, late Parkinson’s disease.

Fig. 3

Histone Acetylation Levels in Degenerating Dopaminergic Neuron and Microglial Cell Cultures. (A) Degeneration of dopaminergic neuronal cultures induced by lactacystin was confirmed using cell viability assays (MTS, neutral red, and Bradford assays). (B) Activation of microglial cultures induced by LPS was confirmed using assays for TNFα and NO. Histone acetylation (AcH3-Lys) was quantified in degenerating N27 dopaminergic neuronal cultures and N9 microglial cultures relative to β-Actin using Western blot analysis. (C) Level of AcH3-Lys9 observed with each treatment. (D) Representative blot of data presented in (C). N = 3 independent replicates. Statistical significance indicated with asterisks: *p < .05, ***p < .001.

Fig. 4

Neuroprotective Effects of SIRT 2 Inhibition via AGK2 Treatment of N27Cells. Cells were pre-treated with AGK2 (or vehicle) for 48 h prior to addition of lactacystin (0.75 μM or vehicle) for a further 24 h. (A) MTS, (B) neutral red, and (C) Bradford assays were then performed for quantification of AGK2 induced neuroprotection against lactacystin toxicity. Red bars indicate cells which received vehicle in place of both AGK2 and lactacystin. Blue bars indicate cells which received vehicle in place of AGK2 treatment but subsequently received lactacystin treatment. Green bars indicate cells treated with AGK2 and which subsequently received lactacystin treatment. N = 3 independent replicates. Statistical significance indicated with asterisks: *p < .05, ***p < .001.

Fig. 5

Anti-Inflammatory Effects of SIRT 2 Inhibition via AGK2 Treatment of N9Cells. Cells were pre-treated with AGK2 (or vehicle) for 24 h prior to addition of LPS (125 ng/ml or vehicle) for a further 24 h. (A) Griess Assay and (B) TNFα ELISAs were then performed on the medium for quantification of AGK2 induced reduction of LPS induced microglial activation. Red bars indicate cells which received vehicle in place of both AGK2 and LPS. Blue bars indicate cells which received vehicle in place of AGK2 treatment but subsequently received LPS treatment. Green bars indicate cells treated with AGK2 and which received subsequently received LPS treatment. (C) Cell viability assays of each treatment of N9 cells. N = 3 independent replicates. Statistical significance indicated with asterisks: *p < .05, **p < .01, ***p < .001.