Colocalization of 14-3-3 proteins with SOD1 in Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis cases and the animal model.

Yoko Okamoto,Yasuhiro Kawamoto,Ryosuke Takahashi,Shun Shimohama,Hirofumi Yamashita,Hidekazu Tomimoto,Asao Hirano,Masafumi Ihara,Miki Oono,Shinsuke Kato,Hidefumi Ito,Yoshitomo Shirakashi,Makoto Urushitani

doi:10.1371/journal.pone.0020427

Abstract

Background and PurposeCu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.MethodsWe examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.ResultsIn all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.DiscussionThese results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice.

Highlights

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the degeneration of motor neurons in the motor cortex, brainstem and spinal cord
The double immunofluorescent study showed 14-3-3 proteins were colocalized with SOD1 in LBHIs
Such distribution patterns were quite similar to those of the mutant SOD1-Tg mice. This is the first report that demonstrates a close relationship between 14-3-3 and SOD1 both in patients with familial amyotrophic lateral sclerosis (FALS) and mutant SOD1-Tg mice

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the degeneration of motor neurons in the motor cortex, brainstem and spinal cord. Transgenic mice carrying several copies of human mutant SOD1 genes show ALS-like symptoms such as progressive motor disturbances and neurogenic amyotrophy, and develop a pathology resembling ALS [4]. These Tg mice demonstrate atrophy of the motor neuronal system, vacuolar degeneration of the motor neurons, and ubiquitinated neuronal hyaline inclusions which contain SOD1 in their cell bodies and swollen processes [5]. Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice

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Conclusion