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Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitin–proteasome system in PC12D cells

  • Basic Neurosciences, Genetics and Immunology - Original Article
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Abstract

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson’s disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin–proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-40Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin–proteasome system in PC12D cells.

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Abbreviations

ChAT:

Choline acetyltransferase

DA:

Dopamine

NGF:

Nerve growth factor

OA:

Okadaic acid

PD:

Parkinson’s disease

PKA:

Cyclic AMP-dependent protein kinase (protein kinase A)

PP2A:

Protein phosphatase 2A

TH:

Tyrosine hydroxylase

p40-TH:

Tyrosine hydroxylase phosphorylated at 40Ser

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Acknowledgments

This work was supported by the Research Grant (18A-2) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare of Japan; by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; and by JST, CREST.

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Authors and Affiliations

  1. Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B7, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan

    Ichiro Kawahata, Hirofumi Tokuoka & Hiroshi Ichinose

  2. CNRS, Institut Alfred Fessard de Neurosciences, French National Research Centre, Gif sur Yvette, France

    Hasan Parvez

Authors
  1. Ichiro Kawahata
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  2. Hirofumi Tokuoka
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  3. Hasan Parvez
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  4. Hiroshi Ichinose
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Corresponding author

Correspondence to Hiroshi Ichinose.

Electronic supplementary material

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702_2009_304_MOESM1_ESM.tif

Supplement Fig. 1. Images with lower magnification for Fig. 1A. All the cells were differentiated with 50 ng/mL NGF for 7 days and exposed to 250 nM MG-132 for indicated hours. Scale bars, 10 μm (TIFF 593 kb)

702_2009_304_MOESM2_ESM.tif

Supplement Fig. 2. Effect of proteasome Inhibitor I (PSI) on p40-TH aggregate formation. PC12D cells are differentiated with 50 ng/ml NGF for 7 days and exposed to PSI for indicated hours, and immunostained with primary antibodies against p40-TH and ubiquitin. 20S proteasomal inhibition induced formation of p40-TH immunoreactive cytoplasmic aggregates, which were similar to MG-132 treated ones. Control, vehicle treatment (DMSO). Scale bar, 10 μm for all images (TIFF 763 kb)

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Kawahata, I., Tokuoka, H., Parvez, H. et al. Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitin–proteasome system in PC12D cells. J Neural Transm 116, 1571–1578 (2009). https://doi.org/10.1007/s00702-009-0304-z

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  • DOI: https://doi.org/10.1007/s00702-009-0304-z

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