Nitrated alpha-synuclein and microglial neuroregulatory activities

Abstract

Microglial neuroinflammatory responses affect the onset and progression of Parkinson's disease (PD). We posit that such neuroinflammatory responses are, in part, mediated by microglial interactions with nitrated and aggregated alpha-synuclein (alpha-syn) released from Lewy bodies as a consequence of dopaminergic neuronal degeneration. As disease progresses, secretions from alpha-syn-activated microglia can engage neighboring glial cells in a cycle of autocrine and paracrine amplification of neurotoxic immune products. Such pathogenic processes affect the balance between a microglial neurotrophic and neurotoxic signature. We now report that microglia secrete both neurotoxic and neuroprotective factors after exposure to nitrated alpha-syn (N-alpha-syn). Proteomic (surface enhanced laser desorption-time of flight, 1D sodium dodecyl sulfate electrophoresis, and liquid chromatography-tandem mass spectrometry) and limited metabolomic profiling demonstrated that N-alpha-syn-activated microglia secrete inflammatory, regulatory, redox-active, enzymatic, and cytoskeletal proteins. Increased extracellular glutamate and cysteine and diminished intracellular glutathione and secreted exosomal proteins were also demonstrated. Increased redox-active proteins suggest regulatory microglial responses to N-alpha-syn. These were linked to discontinuous cystatin expression, cathepsin activity, and nuclear factor-kappa B activation. Inhibition of cathepsin B attenuated, in part, N-alpha-syn microglial neurotoxicity. These data support multifaceted microglia functions in PD-associated neurodegeneration.

Figure 1

SELDI-TOF, 1D SDS PAGE, and Western blot analyses of supernatant fluids obtained from N-α-syn-activated microglial. Representative SELDI-TOF spectral analysis (region 10–20 kDa) of untreated-control (top panel) and N-α-syn-stimulated microglia (bottom panel) at 16 h post-stimulation, shown in A. Marked by an asterisk are upregulated and uniquely expressed peaks corresponding in molecular weight within 1% of mass tolerance to proteins identified by LC-MS/MS. These include calcyclin (10,051 Da), thioredoxin (11,544 Da), calvasculin (11,721 Da), calmodulin (16,706 Da), and TNF-α (17,907 Da). Bands were excised from 1D SDS PAGE gel, digested by trypsin, and sequenced by LC-MS/MS. Lanes are supernatant fluids obtained from control (unstimulated) microglial = [Lanes 1–3] and supernatants from N-α-syn-activated microglia [Lanes 4–6] collected 8 h, 16 h, and 24 h post-stimulation, respectively. (B). Representative Western blots of supernatant fluids from control and N-α-syn-stimulated microglia 16 h post-stimulation for proteins identified by LC-MS/MS (C).

Figure 2

N-α-Syn activated microglial metabolic responses. Microglia were stimulated with N-α-syn over the course of 24h and culture supernatants were collected at specified times following stimulation for analysis of extracellular metabolites, shown in A. Analysis of intracellular metabolites following engagement with N-α-syn is shown in B. Mean metabolite concentrations are presented as values ± SEM, and are representative of three separate experiments, (*P < .001, v. unstimulated control).

Figure 3

Cathepsin B expression and functional activity following N-α-syn-microglial activation. Comparative analysis of cystatin B and cathepsin B protein expression in microglia at several time points during stimulation with aggregated N-α-syn by western blot, shown in A. Mean density of protein bands (± SEM) were normalized to GAPDH expression on the same blot (*P < 0.05 compared to unstimulated microglia at 0h). (B) Cathepsin B enzymatic activity prior to stimulation (0 h) and at 2 h, 4 h, 8 h, and 24 h after stimulation with N-α-syn. Enzymatic activity is visualized with the red fluorogenic substrate, CV-(RR)₂ and nuclei are stained blue with Hoechst dye. Fluorescence intensities were determined by ImageQuant and are presented as mean ± SEM for n = 3 fields for 4 replicates (P < 0.05 compared to mean at 0 h).

Figure 4

Effect of Cathepsin B inhibition on N-α-syn-mediated cytotoxicity. Supernatants from N-α-syn stimulated microglia induced significant DA cell death. Whereas, inhibition of cathepsin B activity by either the cell impermeable inhibitor CA-074 or the cell-permeable inhibitor CA-074 Me resulted in partial protection from N-α-syn mediated DA cell death, shown. Values are shown as mean dead DA cells ± SEM for n = 6 replicates per treatment paradigm.

Figure 5

Nuclear translocation of NF-κB subunits. Cytosol (top) and nuclear (bottom) fractions were prepared from microglia stimulated with N-α-syn for subsequent time-points and assessed for expression of NF-κB subunits NFκB1/ p50 and RelA/p65 by western blot (A). Mean density of protein bands for NFκB1/p50 (B) and RelA/p65 (C) by western blot were normalized to GAPDH in the same sample. Values are shown mean ± SEM for n = 3 replicates per time point (*P < 0.01 compared to 0h).