Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease

Journal name:
Nature Cell Biology
Volume:
13,
Pages:
568–579
Year published:
DOI:
doi:10.1038/ncb2217
Received
Accepted
Published online
Corrected online

Abstract

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is increasingly implicated in various neurodegenerative diseases. Deregulated Cdk5 activity has been associated with neuronal death, but the underlying mechanisms are not well understood. Here we report an unexpected role for Cdk5 in the regulation of induced autophagy in neurons. We have identified endophilin B1 (EndoB1) as a Cdk5 substrate, and show that Cdk5-mediated phosphorylation of EndoB1 is required for autophagy induction in starved neurons. Furthermore, phosphorylation of EndoB1 facilitates EndoB1 dimerization and recruitment of UVRAG (UV radiation resistance-associated gene). More importantly, Cdk5-mediated phosphorylation of EndoB1 is essential for autophagy induction and neuronal loss in models of Parkinson’s disease. Our findings not only establish Cdk5 as a critical regulator of autophagy induction, but also reveal a role for Cdk5 and EndoB1 in the pathophysiology of Parkinson’s disease through modulating autophagy.

At a glance

Figures

  1. EndoB1 is a substrate of Cdk5/p35.
    Figure 1: EndoB1 is a substrate of Cdk5/p35.

    (a) p35 interacts with EndoB1 in a GST pulldown assay. (b,c) EndoB1 co-immunoprecipitates (IP) with p35 in COS-7 cell lysates expressing p35 and full-length EndoB1 (b) and adult rat brain lysate (c). (d) Putative Cdk5 phosphorylation site(s) in the EndoB1 sequence. Two proline-directed threonine residues (arrows) are present with a Cdk5 consensus site found at Thr 145 (underlined). (e) Dose-dependent phosphorylation of EndoB1 by Cdk5/p35 in an in vitro kinase assay. (f) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay. (g) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in COS-7 cells. (h) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5+/+, but not Cdk5−/− mouse brains. Quantification of p-Thr145-EndoB1 level is shown in the right panel. Data are means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8.

  2. Cdk5-mediated phosphorylation of EndoB1 is required for starvation-induced autophagy in neurons.
    Figure 2: Cdk5-mediated phosphorylation of EndoB1 is required for starvation-induced autophagy in neurons.

    (a) Left: starvation increases the level of RFP–LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP–LC3 puncta. Right: pretreatment with 10mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown. (b) Knockdown of EndoB1 abolishes the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP–LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=3. (c) Starvation enhances Cdk5 activity (top) and increases the level of Thr 145-phosphorylated EndoB1 in neurons (top and bottom). Histone H1 was used as a Cdk5 substrate. Data are means ± s.e.m.; n=3. (d,e) Pretreatment with Ros (d) or knockdown of Cdk5 (e) attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for d and n=8 for e. DMSO, dimethylsulphoxide. (f) Overexpression of EndoB1T145A significantly reduces the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP–LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=6. Uncropped images of blots are shown in Supplementary Fig. S8.

  3. Thr 145 phosphorylation of EndoB1 does not affect its lipid binding and co-localization with Atg5.
    Figure 3: Thr 145 phosphorylation of EndoB1 does not affect its lipid binding and co-localization with Atg5.

    (a) Left: lipid binding of EndoB1 is inhibited by mutating the five lysine/arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid (5E mutant). Right: purified GST protein was included as a negative control. S, supernatant fraction; P, pellet fraction that contains the lipid-bound proteins. (b,c) The 5E mutant of EndoB1 fails to dimerize (b) and interact with UVRAG (c) in 293T cells. (d) Overexpression of the 5E mutant of EndoB1 blocks starvation-induced autophagy in cortical neurons. Data are means ± s.e.m.; n=3. (e) Mutation of Thr 145 to alanine (T145A) or glutamic acid (T145E) has negligible effect on the lipid-binding property of EndoB1. Conversely, the 5E mutant is less able to bind lipid. The right panel shows quantified data as means ± s.e.m.; n=3. (f) Starvation increases co-localization of GFP–EndoB1 (green) with Atg5 (red) in neurons. The T145A mutation of GFP–EndoB1 does not affect its co-localization with Atg5-positive entities. Arrows denote GFP–EndoB1- and Atg5- positive vesicles. The right panel shows quantified data as means ± s.e.m.; n=9. Uncropped images of blots are shown in Supplementary Fig. S8.

  4. Cdk5-mediated Thr 145 phosphorylation of EndoB1 regulates its dimerization and interaction with UVRAG/Beclin 1.
    Figure 4: Cdk5-mediated Thr 145 phosphorylation of EndoB1 regulates its dimerization and interaction with UVRAG/Beclin 1.

    (a) Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells. Samples were run on the same gel and spliced together to create the image shown. (b) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells. (c) Diminished interaction between EndoB1 and UVRAG in Cdk5−/− mouse brain lysates. (d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) and Beclin 1 (e) in 293T cells. (f,g) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (f), or immunoprecipitation followed by immunoblotting (g). Uncropped images of blots are shown in Supplementary Fig. S8.

  5. Cdk5-mediated Thr 145 phosphorylation of EndoB1 is involved in autophagy triggered by MPTP.
    Figure 5: Cdk5-mediated Thr 145 phosphorylation of EndoB1 is involved in autophagy triggered by MPTP.

    (a) MPTP administration decreases the numbers of tyrosine hydroxylase (TH)- and cresyl violet (CV)-positive neurons in the substantia nigra. Representative images of tyrosine hydroxylase-stained brain sections are shown. Right panels show quantified data as means ± s.e.m.; n=9 for tyrosine hydroxylase and n=3 for cresyl violet. (b) MPTP administration upregulates the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the midbrain. Lower panels show quantified data as means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 inhibits MPP+-induced autophagy in cultured neurons. Data are means ± s.e.m.; n=5. (d) Cdk5 mediates the MPP+-induced EndoB1 phosphorylation at Thr 145 and the MPP+-induced increase in the LC3-II level in neurons. (e) Overexpression of EndoB1T145A attenuates MPP+-induced autophagy in neurons. Data are means ± s.e.m.; n=7. (f) MPTP fails to increase LC3-II and Thr 145-phosphorylated EndoB1 levels in the midbrain of p35−/− mice. The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated. Right panels show quantified data as means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8.

  6. Thr 145 phosphorylation of EndoB1 by Cdk5 contributes to [alpha]-synucleinA53T mutant-induced autophagy.
    Figure 6: Thr 145 phosphorylation of EndoB1 by Cdk5 contributes to α-synucleinA53T mutant-induced autophagy.

    (a) Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice. Right panels show quantified data as means ± s.e.m.; n=3. (b) Overexpression of α-synucleinA53T mutant upregulates LC3-II level and Thr 145-phosphorylated EndoB1 in cultured neurons. Lower panel shows quantified data as means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 inhibits α-synucleinA53T mutant-induced autophagy induction in neurons. Data are means ± s.e.m.; n=7. (d) Cdk5 mediates α-synucleinA53T mutant-induced Thr 145 phosphorylation of EndoB1 and the increase of LC3-II in neurons. (e) Overexpression of EndoB1T145A attenuates α-synucleinA53T mutant-induced autophagy in neurons. Data are means ± s.e.m.; n=7. Uncropped images of blots are shown in Supplementary Fig. S8.

  7. Cdk5-mediated Thr 145 phosphorylation of EndoB1 is required for MPP+- and [alpha]-synucleinA53T mutant-induced neuronal death.
    Figure 7: Cdk5-mediated Thr 145 phosphorylation of EndoB1 is required for MPP+- and α-synucleinA53T mutant-induced neuronal death.

    (a) Pretreatment with 3-MA, but not z-VAD-fmk, significantly reduces MPP+-induced neuronal death. Data are means ± s.e.m.; n=6. (b) Knockdown of Atg5 expression abrogates MPP+-induced neuronal loss, whereas it reduces neuronal survival in untreated cells. Data are means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 expression abolishes MPP+-induced neuronal death. Data are means ± s.e.m.; n=8. (d) Overexpression of EndoB1T145A inhibits MPP+-induced neuronal death. Data are means ± s.e.m.; n=5. (e) Pretreatment with 3-MA, but not z-VAD-fmk, reduces α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6. (f) Knockdown of Atg5 expression abolishes α-synucleinA53T mutant-induced neuronal death, whereas it reduces neuronal survival in vector-transfected cells. Data are means ± s.e.m.; n=3. (g) Knockdown of EndoB1 or Cdk5 expression abolishes α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=4. (h) Expression of EndoB1T145A inhibits α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.

  8. Proposed model for the role of Cdk5 and EndoB1 in induced autophagy and neuronal death.
    Figure 8: Proposed model for the role of Cdk5 and EndoB1 in induced autophagy and neuronal death.

    Cdk5-mediated Thr 145 phosphorylation of EndoB1 is required for induced autophagy and cell death in neurons. Starvation, treatment with MPTP/MPP+ and overexpression of the α-synucleinA53T mutant increase the level of EndoB1 Thr 145 phosphorylation by Cdk5/p35 in neurons. Phosphorylation of EndoB1 promotes its dimerization and recruitment of the UVRAG/Beclin 1 complex to induce autophagy.

Change history

Corrected online 21 April 2011
In the version of this article initially published online and in print, there were some errors in Fig. 3b. These errors have been corrected in the HTML and PDF versions of the article.

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Affiliations

  1. Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    • Alan S. L. Wong,
    • Rebecca H. K. Lee,
    • Anthony Y. Cheung,
    • Zelda H. Cheung &
    • Nancy Y. Ip
  2. Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    • Alan S. L. Wong,
    • Rebecca H. K. Lee,
    • Anthony Y. Cheung,
    • Zelda H. Cheung &
    • Nancy Y. Ip
  3. State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    • Alan S. L. Wong,
    • Rebecca H. K. Lee,
    • Anthony Y. Cheung,
    • Zelda H. Cheung &
    • Nancy Y. Ip
  4. Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

    • Patrick K. Yeung &
    • Sookja K. Chung
  5. Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

    • Sookja K. Chung

Contributions

A.S.L.W., R.H.K.L., A.Y.C. and P.K.Y. carried out the experiments. A.S.L.W., R.H.K.L., A.Y.C., P.K.Y., Z.H.C. and N.Y.I. planned the studies, designed the experiments and interpreted the results. S.K.C., Z.H.C. and N.Y.I. provided reagents and resources. A.S.L.W., Z.H.C. and N.Y.I. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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