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J. Biol. Chem., Vol. 282, Issue 39, 28904-28914, September 28, 2007

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Enhanced Lysosomal Pathology Caused by -Synuclein Mutants Linked to Dementia with Lewy Bodies^*

Jianshe Wei¹, Masayo Fujita¹, Masaaki Nakai¹, Masaaki Waragai, Kazuhiko Watabe, Hiroyasu Akatsu^¶, Edward Rockenstein^||, Eliezer Masliah^||, and Makoto Hashimoto²

From the Laboratory for Chemistry and Metabolism and the Laboratory for Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan, the ^¶Choju Medical Institute, Fukushimura Hospital, Aichi 441-8124, Japan, and the ^||Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0624

Two missense mutations (P123H and V70M) of -synuclein (-syn), the homologue of -syn, have been recently identified in dementia with Lewy bodies. However, the mechanism through which these mutations influence the pathogenesis of dementia with Lewy bodies is unclear. To investigate the role of the -syn mutations in neurodegeneration, each mutant was stably transfected into B103 neuroblastoma cells. Cells overexpressing mutated -syn had eosinophilic cytoplasmic inclusion bodies immunopositive for mutant -syn, and electron microscopy revealed that these cells were abundant in various cytoplasmic membranous inclusions resembling the histopathology of lysosomal storage disease. Consistent with these findings, the inclusion bodies were immunopositive for lysosomal markers, including cathepsin B, LAMP-2, GM2 ganglioside, and ATP13A2, which has recently been linked to PARK9. Notably, formation of these lysosomal inclusions was greatly stimulated by co-expression of -syn, was dependent on the phosphorylation of -syn at Ser-129, and was more efficient with the A53T familial mutant of -syn compared with wild type. Furthermore, the inclusion formation in cells overexpressing mutant -syn and transfected with -syn was significantly suppressed by treatment with autophagy-lysosomal inhibitors, which were associated with impaired clearance of syn proteins and enhanced apoptosis, indicating that formation of lysosomal inclusions might be protective. Collectively, the results demonstrated unambiguously that overexpression of -syn mutants (P123H and V70M) in neuroblastoma cells results in an enhanced lysosomal pathology. We suggest that these missense mutations of -syn might play a causative role in stimulating neurodegeneration.

Received for publication, May 4, 2007 , and in revised form, July 23, 2007.

^* This work was supported in part by a grant-in-aid for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and a project grant from Tokyo Metropolitan Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, references, and Figs. S1-S3.

¹ These authors equally contributed to this work.

² To whom correspondence should be addressed. Tel.: 81-42-325-3881; Fax: 81-42-321-8678; E-mail address: mhashimoto{at}tmin.ac.jp.

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