Cytoplasmic aggregates of phosphorylated extracellular signal-regulated protein kinases in Lewy body diseases

Abstract

A better understanding of cellular mechanisms that occur in Parkinson's disease and related Lewy body diseases is essential for development of new therapies. We previously found that 6-hydroxydopamine (6-OHDA) elicits sustained extracellular signal-regulated kinase (ERK) activation that contributes to neuronal cell death in vitro. As subcellular localization of activated kinases affect accessibility to downstream targets, we examined spatial patterns of ERK phosphorylation in 6-OHDA-treated cells and in human postmortem tissues representing the full spectrum of Lewy body diseases. All diseased human cases exhibited striking granular cytoplasmic aggregates of phospho-ERK (P-ERK) in the substantia nigra (involving 28 +/- 2% of neurons), which were largely absent in control cases (0.3 +/- 0.3%). Double-labeling studies and examination of preclinical cases suggested that these P-ERK alterations could occur relatively early in the disease process. Development of granular cytoplasmic P-ERK staining in 6-OHDA-treated cells was blocked by neuroprotective doses of catalase, supporting a role for oxidants in eliciting neurotoxic patterns of ERK activation. Evidence of nuclear translocation was not observed in degenerating neurons. Moreover, granular cytoplasmic P-ERK was associated with alterations in the distribution of downstream targets such as P-RSK1, but not of P-Elk-1, suggesting functional diversion of ERK-signaling pathways in Lewy body diseases.

Figure 1.

Abnormal ERK distribution in human Lewy body diseases. A–G: Midbrain sections from PD (A), age-matched control (B), or DLB (C, E–G) patients were stained for P-ERK using antibodies from Calbiochem (A–C), Sigma (E, F), and Promega (G). Note abnormal cytoplasmic distribution of P-ERK (coarse red granules of varying sizes) in endogenously pigmented substantia nigra neurons (fine brown uniform granules). D: Nonimmune rabbit serum was used as a negative staining control. H–J: Representative images from PD (H), DLB (I), and control (J) patients stained for T-ERK. Note discrete T-ERK granules in the diseased cases (H and I, arrows) against a background of mottled cytoplasmic staining. K–M: Double-label confocal immunofluorescence study of a PD case showing association (yellow) of P-ERK granules (green) at the periphery of ubiquitinated (red) Lewy bodies. N–P: Double-label confocal immunofluorescence study of a DLB case showing association and partial co-localization (yellow) of P-ERK granules (green) with abnormal α-synuclein aggregates (red). Scale bars, 50 μm.

Figure 2.

Distribution of P-ERK and P-RSK abnormalities in control, preclinical, and diseased cases. A: Percentage of pigmented substantia nigra neurons with granular cytoplasmic P-ERK immunoreactivity is shown for control (Cont), preclinical (pc), PD, and DLB groups. *, P < 0.05 compared to control; ^†, P < 0.05 compared to preclinical. B: Bar graph showing proportion of P-ERK-positive neurons that are overtly abnormal by routine histology (white bar), histologically unremarkable with early α-synucleinopathy (gray bar), or lacking either histological or α-synuclein pathology (black bar) was assessed as described in the text. Note that 20 to 25% of P-ERK-positive neurons do not manifest other pathology in PD and DLB cases (black bar), and this fraction is increased to 41% in preclinical cases. None of the rare P-ERK-positive neurons observed in the control cases showed abnormal α-synuclein positivity, but ∼10% did show pigment loss. C: The percentage of pigmented substantia nigra neurons with cytoplasmic P-RSK immunoreactivity was determined and the mean ± SEM determined for each group. *, P < 0.05 compared to control.

Figure 3.

Distribution of P-ERK-positive neurons by substantia nigra region. Preclinical (A, B) and PD/DLB (C, D) cases were analyzed for neuronal P-ERK positivity in medial (M) versus lateral (L) regions of the substantia nigra as described in Materials and Methods. The lateral region was further subdivided into ventrolateral (VL) and dorsal (D) groups. The data are expressed as either the total number of P-ERK-positive neurons in each region (A, C), or as the percentage of neurons in each region with P-ERK granules (B, D). *, P < 0.05 compared to medial region; ^†, P < 0.05 compared to dorsal group.

Figure 4.

Frozen substantia nigra from two control cases (C), one PD case (P), two DLB cases (D), and one preclinical case (pc) were homogenized and the soluble fraction was subjected to immunoblot analysis (A) and in vitro kinase activity (B) as described in Materials and Methods. Shown is a representative immunoblot using the Calbiochem antibody for P-ERK (top), with reprobing for T-ERK (bottom). The same pattern of P-ERK staining was observed using the E10 monoclonal antibody. Kinase activity is expressed in arbitrary units based on phosphorylation of a recombinant ERK substrate.

Figure 5.

Relationship of P-ERK granules to α-synuclein and P-RSK staining. A–C: Double-label immunohistochemical stains for P-ERK and P-RSK revealed abnormal P-ERK (red) in otherwise morphologically normal neurons without evidence of abnormal α-synuclein immunoreactivity (A); neurons with both P-ERK (red) and abnormal synuclein inclusions (brown) (B); and neurons with advanced synucleinopathy (brown), but no P-ERK immunoreactivity (C). Cresyl violet was used to convert endogenous pigment to a gray-green color. D–F: Abnormal cytoplasmic P-RSK1 staining in PD/DLB cases. Substantia nigra from normal control (D) and PD (E, F) cases were stained for P-RSK1 (red). The substantia nigra neurons of PD/DLB cases showed significantly increased levels of coarse, granular cytoplasmic staining. Note involvement of pale bodies (E), and the halo region of Lewy bodies (F), a distribution similar to that of P-ERK. G–I: Confocal immunofluorescent microscopy showing co-localization (yellow) of a subset of P-ERK granules (green) with P-RSK1 (red). Scale bars, 50 μm.

Figure 6.

P-ERK- and P-RSK-staining patterns in B65 cells. A–F: Immunofluorescent labeling of P-ERK (green) in B65 cells treated for 6 hours with ascorbate vehicle (A), 500 μmol/L of 6-OHDA (B, C), 6-OHDA plus 25 U/ml of catalase (D), or 6-OHDA plus heat-inactivated catalase (E). F: Negative control: 6-OHDA-treated cells stained with E10 antibody absorbed with immunizing peptide. A, B, and F show merged image with red propidium iodide-stained nuclei. G–J: Immunofluorescent labeling of P-RSK (red) in B65 cells treated for 6 hours with ascorbate vehicle (G) or 500 μmol/L of 6-OHDA (H). I: Double-label confocal immunofluorescence study of P-ERK (green) and P-RSK (red) in 6-OHDA-treated cells demonstrating scattered co-localization (yellow). J: Orthogonal image analysis of Z-sectioned series confirming co-localization of P-ERK and P-RSK signals in a subset of granules. Scale bars: 30 μm (C, E); 20 μm (H).

Figure 7.

Subcellular fractionation of 6-OHDA elicited P-ERK and P-RSK. B65 cells were treated with 500 μmol/L of 6-OHDA for 6 hours. Cell lysates obtained using 0.1% Triton X-100 (L) were compared to cytoplasmic (C) and nuclear (N) extracts. Blots were analyzed for P-ERK (E10 monoclonal) and P-RSK, and stripped and reprobed for T-ERK, RSK1, nuclear marker Lamin A, and cytoplasmic marker actin.

Figure 8.

Catalase confers protection from 6-OHDA and inhibits ERK phosphorylation. A: B65 cells were treated with 6-OHDA (filled squares), 6-OHDA with 5 U/ml of active catalase (open circles), and 6-OHDA with heat-inactivated catalase (open triangles). Viability was assessed using the MTS metabolic assay, normalized to vehicle-treated cells, and expressed as mean ± SEM. Phase microscopic images of B65 cells treated with ascorbate (the vehicle for 6-OHDA) (B), 1 mmol/L 6-OHDA (C), 6-OHDA plus 25 U/ml catalase (D), or 6-OHDA plus heat-inactivated catalase (E). F: Cells were treated with ascorbate or 500 μmol/L of 6-OHDA for 22 hours in the absence or presence of 5 U/ml of catalase and analyzed for ERK phosphorylation. Blots were stripped and reprobed for T-ERK.