Abstract
Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.
Highlights
␣-Synuclein is a major component of Lewy bodies (LBs)2 found in Parkinson disease (PD), dementia with LB, Alzheimer disease (AD), and multiple system atropy [1]
In these neurodegenerative disorders, LBs are characterized by fibrillar, cytoplasmic ␣-synuclein aggregates within selective populations of neurons and glial cells [2]. ␣-Synuclein inclusion formation is clearly involved in the pathogenic process of PD. ␣-Synuclein was first identified as a partial fragment in AD amyloid plaques [41], and subsequently three missense mutations in the ␣-synuclein gene were reported in early onset familial PD of some kindred [3, 4]
Materials—Peroxidase-conjugated anti-rabbit and antimouse IgGs were purchased from Zymed Laboratories Inc. (San Francisco, CA); Dulbecco’s modified Eagle’s medium, fetal bovine serum, Lipofectamine Plus reagent, and cell culture reagents were from Invitrogen; glutathione-Sepharose 4B and Protein A-Sepharose were from Amersham Biosciences; ECL reagents and [␥-32P]ATP were from PerkinElmer Life Sciences; anti-␣-synuclein IgG was from AbCam; anti-Dual specificity tyrosine-regulated kinase-1A (Dyrk1A) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and anti-phosphoserine/phosphothreonine and anti-tyrosine antibodies were from Sigma
Summary
Materials—Peroxidase-conjugated anti-rabbit and antimouse IgGs were purchased from Zymed Laboratories Inc. (San Francisco, CA); Dulbecco’s modified Eagle’s medium, fetal bovine serum, Lipofectamine Plus reagent, and cell culture reagents were from Invitrogen; glutathione-Sepharose 4B and Protein A-Sepharose were from Amersham Biosciences; ECL reagents and [␥-32P]ATP were from PerkinElmer Life Sciences; anti-␣-synuclein IgG was from AbCam; anti-Dyrk1A antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and anti-phosphoserine/phosphothreonine and anti-tyrosine antibodies were from Sigma. Cells were incubated at 4 °C for 24 h with the appropriate primary antibody and diluted in phosphate-buffered saline containing 1% bovine serum albumin. After washing with phosphate-buffered saline, either rhodamine- or fluorescein isothiocyanate-coupled secondary antibodies were added and incubated for 2 h at room temperature. Samples were centrifuged at 100,000 ϫ g for 10 min, and supernatants were removed and diluted 10 times with Tris-buffered saline. These dilutions were analyzed by their absorbance at 280 nm. For electron microscopy of ␣-synuclein aggregates, the samples were prepared as described previously [23]. Dyrk1A siRNA duplexes were transfected into cells using the Lipofectamine Plus reagent according to the manufacturer’s instructions
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