Low levels of astroglial markers in Parkinson's disease: relationship to α-synuclein accumulation

Abstract

Although gliosis is a normal response to brain injury, reports on the extent of astrogliosis in the degenerating substantia nigra in Parkinson's disease (PD) are conflicting. It has also been recently suggested that accumulation of nigral α-synuclein in this disorder might suppress astrocyte activation which in turn could exacerbate the degenerative process. This study examined brain protein levels (intact protein, fragments, and aggregates, if any) of astroglial markers and their relationship to α-synuclein in PD and in the positive control parkinson-plus conditions multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). Autopsied brain homogenates of patients with PD (n=10), MSA (n=11), PSP (n=11) and matched controls (n=10) were examined for the astroglial markers glial fibrillary acidic protein (GFAP), vimentin, and heat shock protein-27 (Hsp27) by quantitative immunoblotting. As expected, both MSA (putamen>substantia nigra>caudate>frontal cortex) and PSP (substantia nigra>caudate>putamen, frontal cortex) showed widespread but regionally specific pattern of increased immunoreactivity of the markers, in particular for the partially proteolyzed fragments (all three) and aggregates (GFAP). In contrast, immunoreactivity of the three markers was largely normal in PD in brain regions examined with the exception of trends for variably increased levels of cleaved vimentin in substantia nigra and frontal cortex. In patients with PD, GFAP levels in the substantia nigra correlated inversely with α-synuclein accumulation whereas the opposite was true for MSA. Our biochemical findings of generally normal protein levels of astroglial markers in substantia nigra of PD, and negative correlation with α-synuclein concentration, are consistent with some recent neuropathology reports of mild astroglial response and with the speculation that astrogliosis might be suppressed in this disorder by excessive α-synuclein accumulation. Should astrogliosis protect, to some extent, the degenerating substantia nigra from damage, therapeutics aimed at normalization of astrocyte reaction in PD could be helpful.

Keywords: Astrogliosis; Glial fibrillary acidic protein; Heat shock protein-27; Multiple system atrophy; Parkinson’s disease; Progressive supranuclear palsy; Quantitative Western blot; Substantia nigra; Vimentin; α-synuclein.

Fig. 1

Substantia nigra: representative immunoblots of the astroglial markers including glial fibrillary acidic protein (GFAP), vimentin, and heat shock protein-27 (Hsp27) in controls and in patients with Parkinson's disease (PD), progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). Protein bands for the intact markers and the partially proteolyzed low molecular weight (LMW) species and truncated Hsp27 (tHsp27) are identified. The top half of the blots for GFAP was over-exposed to show high molecular weight (HMW) aggregates of GFAP. Note that GFAP immunoreactivity was markedly decreased versus controls in some cases of PD whereas was significantly enhanced in both MSA and PSP, which also showed marked increase in immunoreactivity for vimentin and Hsp27, in particular the LMW species. Also shown are blots of the control proteins including α-tubulin and neuron specific enolase (NSE).

Fig. 2

Substantia nigra. Scatter plots of levels (total, low molecular weight species [LMW] and high molecular weight aggregates [HMW]) of glial fibrillary acidic protein (GFAP), vimentin, and heat shock protein-27 (Hsp27) in controls and in patients with Parkinson's disease (PD), progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). The inset shows the enlarged graph for GFAP aggregates in controls and PD. ***p<0.001, **p<0.01, and *p<0.05, PD, PSP, or MSA vs. controls (Kruskal-Wallis ANOVA followed by Dunn's multiple comparison tests; #p<0.05, PD, PSP, or MSA vs. controls (Mann-Whitney U-test in cases that failed Dunn's multiple comparison correction). tHsp27 = truncated Hsp27.

Fig. 3

Correlations (Pearson) between the extent of dopamine loss (expressed as percentage loss) reported previously (Tong et al., 2010) in the caudate nucleus and levels of the low molecular weight species (LMW) of glial fibrillary acidic protein (GFAP), vimentin, and truncated heat shock protein-27 (tHsp27) in the substantia nigra of patients with Parkinson's disease (PD). The inset shows enlarged portion and linear correlation for vimentin without the outlier (arrow).

Fig. 4

Correlations (Pearson) between levels (total or low molecular weight species [LMW]) of glial fibrillary acidic protein (GFAP), vimentin, and truncated heat shock protein-27 (tHsp27) and that of α-synuclein (17 kDa intact or high molecular weight species [HMW]) accumulation in the membrane fraction (α-S_M) reported previously (Tong et al., 2010) in substantia nigra (A) of patients with Parkinson's disease (PD) and multiple system atrophy (MSA) and in putamen (B) of MSA. AU = arbitrary unit.

Fig. 5

Putamen, caudate and frontal cortex: representative immunoblots of the astroglial markers including glial fibrillary acidic protein (GFAP), vimentin, and heat shock protein-27 (Hsp27) in controls and in patients with Parkinson's disease (PD), progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). Protein bands for the intact markers and the partially proteolyzed low molecular weight (LMW) species and truncated Hsp27 (tHsp27) are identified. The top half of the blots for GFAP was over-exposed to show high molecular weight (HMW) aggregates of GFAP. Note markedly increased immunoreactivity (especially of the HMW and LMW species) in MSA putamen and in PSP/MSA caudate. Also shown are blots of the control proteins including α-tubulin and neuron specific enolase (NSE).

Fig. 6

Putamen. Scatter plots of levels (total, low molecular weight species [LMW] and high molecular weight [HMW] aggregates) of glial fibrillary acidic protein (GFAP), vimentin, and heat shock protein-27 (Hsp27) in controls and in patients with Parkinson's disease (PD), progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). The insets show enlarged graphs for vimentin and HMW GFAP in controls, PD and PSP. ***p<0.001, **p<0.01, and *p<0.05, PD, PSP, or MSA vs. controls (Kruskal-Wallis ANOVA followed by Dunn's multiple comparison tests; #p<0.05, PD, PSP, or MSA vs. controls (Mann-Whitney U-test in cases that failed Dunn's multiple comparison correction). tHsp27 = truncated Hsp27.