Activation of tyrosine kinase c-Abl contributes to α-synuclein–induced neurodegeneration
Saurav Brahmachari, … , Ted M. Dawson, Han Seok Ko
Saurav Brahmachari, … , Ted M. Dawson, Han Seok Ko
Published June 27, 2016
Citation Information: J Clin Invest. 2016;126(8):2970-2988. https://doi.org/10.1172/JCI85456.
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Activation of tyrosine kinase c-Abl contributes to α-synuclein–induced neurodegeneration

Abstract

Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein–induced neuropathology. In mice expressing a human α-synucleinopathy–associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein–induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies.

Authors

Saurav Brahmachari, Preston Ge, Su Hyun Lee, Donghoon Kim, Senthilkumar S. Karuppagounder, Manoj Kumar, Xiaobo Mao, Joo Ho Shin, Yunjong Lee, Olga Pletnikova, Juan C. Troncoso, Valina L. Dawson, Ted M. Dawson, Han Seok Ko

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Figure 6

c-Abl interacts with and phosphorylates α-synuclein.

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c-Abl interacts with and phosphorylates α-synuclein. (A) Coimmunoprecipi...
(A) Coimmunoprecipitation of myc-tagged α-syn (myc-α-syn) and GFP-tagged c-Abl (GFP-c-Abl) by anti-myc antibody in SH-SY5Y cells cotransfected with myc-α-syn and GFP-c-Abl or kinase-dead (KD) (lysine 290 arginine) version of c-Abl (GFP-c-Abl-KD) followed by IB. (B) Coimmunoprecipitation of α-syn and c-Abl by anti–α-syn antibody in the brain stem from nontransgenic mice followed by IB. Anti-IgG was used as a negative control. (C) Coimmunoprecipitation of α-syn and c-Abl by anti–α-syn antibody in the brain tissue lysates from WT, c-Abl knockout (c-AblKO), hA53Tα-syn, and c-AblKO hA53Tα-syn mice followed by IB. (D) In vitro kinase assay showing that c-Abl phosphorylates α-syn. Autoradiogram indicates that STI-571, a c-Abl kinase inhibitor, dose-dependently reduced phosphorylation of α-syn and c-Abl. Immunoblot in the bottom panel shows equivalent amount of α-syn used in the experiment. (E) c-Abl phosphorylates α-syn on tyrosine (Y) 39. Mass spectrometric analysis reveals 100% sequence coverage of α-syn, showing that all tyrosine residues were investigated for phosphorylation status. Phosphorylated Y39 is indicated in red; other tyrosines are indicated in green (top). α-Syn phosphorylated by c-Abl was separated by 2-DE followed by IB (bottom left). The arrows indicate tyrosine phosphorylation of α-syn. Both nonphosphorylated and phosphorylated α-syn were subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) to identify the phosphorylation site (bottom right). LC-MS/MS spectra of the nonphosphorylated peptide (EGVLYVGSK) and the phosphorylated peptide (EGVLpYVGSK) are compared, demonstrating that there is the 80-Da shift for the Y39 ion containing the phosphate moiety. The phosphorylated amino acid is preceded by a “p” and highlighted in red. (F) IP of myc-α-syn by anti-myc antibody in SH-SY5Y cells cotransfected with indicated plasmids followed by IB with anti-myc, anti-pY39 α-syn, anti-pTyr, or anti-pY125 α-syn antibodies. Inputs were immunoblotted with anti-GFP antibodies. All experiments were repeated at least 3 times.