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Ann. N.Y. Acad. Sci. 991: 1–3 (2003). © 2003 New York Academy of Sciences.

Subcellular Compartmentalization of P-ERKs in the Lewy Body Disease Substantia Nigra

CHARLEEN T. CHU AND JIAN-HUI ZHU

Department of Pathology, Division of Neuropathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA

KEYWORDS: extracellular signal–related kinase (ERK); Lewy body disease;substantia nigra; mitochondria

Extracellular signal–regulated kinases (ERKs) are involved in regulating neuronal sur-vival, differentiation, and plasticity. Recent studies also indicate that ERKs can play adetrimental role in models of oxidative neuronal injury.1,2 Our previous studiesshowed discrete cytoplasmic accumulations of phophorylated ERKs (P-ERKs) inParkinson’s disease and other Lewy body diseases, and in 6-hydroxydopamine–treatedneuronal cell cultures.3 As the effects of ERK phosphorylation are critically dependentupon subcellular localization and access to downstream targets, we investigated thesubcellular distribution of P-ERKs in Lewy body disease using double-label confocalmicroscopy. The association of P-ERK granules with a subset of (sometimes enlarged)mitochondria suggests a potential interaction between mitochondrial function and theERK signaling pathway in degenerating dopaminergic neurons.

METHODS

Substantia nigra sections from a diffuse Lewy body disease patient were stainedfor P-ERKs as described previously3 and then incubated with organelle-specificantibodies and appropriate Cy3-conjugated secondary antibodies. Negative controlsincluded double labeling of sections substituting nonimmune mouse or rabbit IgGfor the primary antibodies. The slides were observed using a Zeiss laser scanningconfocal imaging system.

RESULTS AND DISCUSSION

Immunofluorescence staining showed coarse, granular, or vesicular-appearingaccumulations of P-ERKs in substantia nigra neurons. The immunoreactivity of theP-ERKs was confined to the cytoplasm and did not colocalize with nuclear stains.3

Address for correspondence: Charleen T. Chu, M.D., Ph.D., Division of Neuropathology,Room A-516, UPMC Presbyterian, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. Voice:412-647-3744; fax: 412-647-5602.

chu@np.awing.upmc.edu

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FIGURE 1. Double labeling of P-ERK (A) with the 60-kDa mitochondrial protein (B).In the overlap image (C), note that regions of colocalization (arrows) often appear as smallerpunctate areas within the P-ERK profile. Enlarged mitochondria occasionally appear to beassociated centrally within vesicular-appearing P-ERK profiles (star).

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3CHU & ZHU: SUBCELLULAR COMPARTMENTALIZATION OF P-ERKs

P-ERK granules were occasionally colocalized with the early endosome markerRab5, but not with markers of the lysosome (cathepsin D), 20S proteasome (β sub-unit), or endoplasmic reticulum (cytochrome P450 reductase). P-ERK immunoreac-tivity was more commonly co-localized with 60-kDa (FIG. 1) and 110-kDamitochondrial proteins, and with manganese superoxide dismutase. The areas ofcolocalization, verified by orthogonal analysis, usually appeared as smaller punctateareas within the P-ERK profile. A second type of association was also observed inwhich vesicular-appearing P-ERK accumulations appeared to envelop, rather thancolocalize with, enlarged mitochondria (FIG. 1, star).

Sustained activation of ERKs is associated with 6-OHDA toxicity in B65 (seeRef. 2) and SH-SY5Y cell lines (C.T. Chu, unpublished data), and MEK inhibitorsconfer significant protection. P-ERK staining is cytoplasmic, attaining a discretepunctate appearance following commitment to cell death.3 Moreover, substantia ni-gra neurons in patients with the full spectrum of Lewy body diseases displayunusual, discrete cytoplasmic, but not nuclear, accumulations of P-ERK immunore-activity.3 In this study, we found that the P-ERK granules can be associated with anearly endosomal marker, but were more commonly associated with mitochondrialmarkers.

Partial colocalization of a subset of P-ERK granules with an early endosomemarker, Rab5, may reflect physiologic recruitment and assembly of Ras-ERK sig-naling cascades on endosomal surfaces. Recent evidence that ERK1 can phosphory-late Rab51 further suggests the possibility for cross-talk between the Ras-ERKsignaling pathway and the endocytic machinery.4

Mitochondria are vulnerable to various insults and can undergo enlargement andstructural disorganization, associated with decreased membrane potential and re-duced ATP production. The association of P-ERK granules with a subset of (some-times enlarged) mitochondria suggests a potential interaction between mitochondrialfunction and the ERK signaling pathway in dopaminergic neurons. P-ERKs havebeen reported to form signaling modules with other MAP kinases in cardiac mito-chondria.5 Alternatively, it is possible that these structures reflect sequestration ofdamaged mitochondria.

REFERENCES

1. STANCIU, M., Y. WANG, R. KENTOR, et al. 2000. Persistent activation of ERK contrib-utes to glutamate-induced oxidative toxicity in a neuronal cell line and primary corti-cal neuron cultures. J. Biol. Chem. 275: 12200–12206.

2. KULICH, S.M. & C.T. CHU. 2001. Sustained extracellular signal-regulated kinase acti-vation by 6-hydroxydopamine: implications for Parkinson’s disease. J. Neurochem.77: 1058–1066.

3. ZHU, J.-H., S.M. KULICH, T.D. OURY & C.T. CHU. 2003. Cytoplasmic aggregates ofphosphorylated extracellular signal-regulated kinase in Lewy body diseases. Am. J.Pathol.: in press.

4. RIZZO, M.A., K. SHOME, S.C. WATKINS & G. ROMERO. 2000. The recruitment of Raf-1to membranes is mediated by direct interaction with phosphatidic acid and is inde-pendent of association with Ras. J. Biol. Chem. 275: 23911–23918.

5. BAINES, C.P., J. ZHANG, G.W. WANG, et al. 2002. Mitochondrial PKCepsilon andMAPK form signaling modules in the murine heart: enhanced mitochondrial PKCep-silon-MAPK interactions and differential MAPK activation in PKCepsilon-inducedcardioprotection. Circ. Res. 90: 390-397.

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