Molecular Neurobiology

, Volume 53, Issue 10, pp 6620–6634 | Cite as

ENC1 Modulates the Aggregation and Neurotoxicity of Mutant Huntingtin Through p62 Under ER Stress

  • Huikyong Lee
  • Hye-Hyun Ahn
  • WonJae Lee
  • Yumin Oh
  • Hyunwoo Choi
  • Sang Mi Shim
  • Jaekyoon Shin
  • Yong-Keun JungEmail author


Huntington’s disease (HD) is a devastating neurodegenerative disorder, which is caused by the expression and aggregation of polyQ-expanded mutant huntingtin protein (mtHTT). While toxic mtHTT aggregates are primarily eliminated through autophagy, autophagy dysfunction is often observed in HD pathogenesis. Here, we show that ectodermal-neural cortex 1 (ENC1), a novel binding partner of sequestosome 1 (p62), negatively regulates autophagy under endoplasmic reticulum (ER) stress. We found that ER stress significantly increases the expression of ENC1 via inositol-requiring enzyme 1 (IRE1)-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) pathway. Ectopic expression of ENC1 alone induces the accumulation of detergent-resistant mtHTT aggregates and downregulation of ENC1 alleviates ER stress-induced mtHTT aggregation. Simultaneously, ER stress-induced impairment of autophagy flux is ameliorated by downregulation of ENC1. From immunoprecipitation and immunocytochemical assays, we found that ENC1 binds to p62 through its BTB and C-terminal Kelch (BACK) domain and this interaction is enhanced under ER stress. In particular, ENC1 preferentially interacts with the phosphorylated p62 at Ser403 during ER stress. Interestingly, ENC1 colocalizes with mtHTT aggregates and its C-terminal Kelch domain is required for interfering with the access of p62 to ubiquitinated mtHTT aggregates, thus inhibiting cargo recognition of p62. Accordingly, knockdown of ENC1 expression enhances colocalization of p62 with mtHTT aggregates. Consequently, ENC1 knockdown relieves death of neuronal cells expressing mtHTT under ER stress. These results suggest that ENC1 interacts with the phosphorylated p62 to impair autophagic degradation of mtHTT aggregates and affects cargo recognition failure under ER stress, leading to the accumulation and neurotoxicity of mtHTT aggregates.


Aggregation Autophagy ENC1 p62/SQSTM1 ER stress Huntington’s disease 



Dulbecco’s modified Eagle’s medium


Ectodermal-neural cortex 1


Fetal bovine serum


Huntington’s disease


Kelch-like ECH-associated protein 1


Lysosome-associated membrane protein 2


Mutant huntingtin


Mouse embryonic fibroblasts


Mammalian target of rapamycin


NF-E2-related factor 2




Secretory carrier membrane protein 5


Sequestosome 1


Unfolded protein response


Ubiquitin-proteasome system



This work was supported by the Global Research Laboratory (NRF-2010-00341) and a CRI grant (NRF-2013R1A2A1A01016896) funded by the Ministry of Education, Science and Technology in Korea. We thank Dr. N. Nukina (RIKEN Brain Science Institute, Japan) for monoclonal phospho-p62 (S403) antibody and Dr. M.E. MacDonald (Harvard University, USA) for STHdhQ7/7 and STHdhQ111/111 cells.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12035_2015_9557_Fig9_ESM.jpg (403 kb)
Fig. S1

Overexpression of ENC1 increases total mtHTT. HEK293T cells were co-transfected with pHTTex120Q-GFP and pcDNA, pENC1 or pIRE1 for 24 h. Cells were harvested and analyzed by Western blotting. Arrowhead indicates specific signal detected by anti-IRE1 antibody and asterisk shows non-specific signal (JPG 402 kb)

12035_2015_9557_Fig10_ESM.jpg (452 kb)
Fig. S2

ENC1 does not affect wtHTT aggregation. a and b Effects of ENC1 overexpression on the aggregation of wtHTT. AF5 cells were co-transfected with pHTTex18Q-GFP (wtHTT) and pcDNA, pIRE1 or pENC1 for the indicated times and examined for the aggregation under fluorescence microscope. Percentages of aggregation were determined by counting the cells showing dot patterns among total GFP-positive cells. Bars represent the mean values ± S.D. (n = 3) (A). HEK293T cells were co-transfected with pHTTex18Q-GFP and pcDNA, pIRE1 or pENC1 for 24 h. Cell lysates were separated into soluble and insoluble fractions and analyzed by Western blotting (B) (JPG 451 kb)

12035_2015_9557_Fig11_ESM.jpg (527 kb)
Fig. S3

ENC1 level affects basal autophagy. SH-SY5Y cells were co-transfected with pmCherry-GFP-LC3 and pcDNA (Mock), pENC1 or pSuper-shENC1 for 24 h and then observed for LC3 dots with GFP or mCherry signals under confocal microscope. Scale bars: 10 μm (JPG 526 kb)

12035_2015_9557_Fig12_ESM.jpg (603 kb)
Fig. S4

ER stress affects the level of ENC1 protein. a and b SH-SY5Y cells were incubated with 2 μg/ml tunicamycin (Tuni., A) or 0.5 μM brefeldin A (BFA, B) for the indicated times. Cell extracts were examined with Western blot analysis. C, HT22 cells were exposed to 0.5 μM thapsigargin (Tg) for the indicated times and then analyzed by Western blotting (JPG 603 kb)

12035_2015_9557_Fig13_ESM.jpg (490 kb)
Fig. S5

Degradation of ENC1 protein by proteasome. a Short half-life of ENC1 protein in SH-SY5Y cells. SH-SY5Y cells were incubated with 30 μg/ml cycloheximide (CHX) for the indicated times and examined with Western blot analysis. b ENC1 degradation is blocked by MG132. SH-SY5Y cells were pretreated with DMSO, 5 μM MG132 or 20 nM bafilomycin A1 (Baf.A1) for 1 h and then exposed to 30 μg/ml cycloheximide (CHX) for 6 h. Cells extracts were then subjected to Western blotting (JPG 489 kb)

12035_2015_9557_Fig14_ESM.jpg (480 kb)
Fig. S6

Interaction of ENC1 with phosphorylated p62 under ER stress. SH-SY5Y cells were incubated with 0.5 μM thapsigargin (Tg) for 24 h. Cell extracts were immunoprecipitated (IP) with mouse IgG or anti-p62 antibody. The immunoprecipitates were left untreated or treated with λ phosphatase (λ PPase) for 1 h and then analyzed by Western blotting. The arrowheads indicate specific signals detected by each antibody and the asterisks show heavy chains of immunoglobulins (right panels). Whole cell lysates (WCL) were proved by Western blotting (left panels) (JPG 479 kb)

12035_2015_9557_Fig15_ESM.jpg (748 kb)
Fig. S7

ENC1 impairs the access of p62 or LC3 to mtHTT aggregates. a Colocalization of p62 with ubiquitin-positive mtHTT aggregates. SH-SY5Y cells were transfected with pHTTex120Q-GFP (mtHTT-GFP) for 24 h and immunostained with anti-p62 (red) or anti-ubiquitin (blue) antibodies. Colocalization of their signals was examined under confocal microscope. The arrows indicate colocalization of mtHTT aggregates and/or the indicated proteins. b Increased colocalization between LC3 with mtHTT aggregates by ENC1 knockdown. SH-SY5Y/pSuper-Neo (Neo) and SH-SY5Y/pSuper-shENC1 (Mix) cells were transfected with pHTTex120Q-GFP for 24 h and immunostained with anti-ubiquitin (blue) or anti-LC3 (red) antibodies. Scale bars: 10 μm. c ENC1 down-regulation enhances the access of phosphorylated p62 (p-p62) to mtHTT aggregates. SH-SY5Y/Super-Neo (Neo) and SH-SY5Y/shENC1 (Mix) cells were transfected with pHTTex120Q-GFP and then incubated with DMSO or 0.5 μM thapsigargin (Tg) for 24 h. Cells were immunostained with p-p62 antibody and observed under confocal microscope. Scale bars: 10 μm (JPG 747 kb)

12035_2015_9557_Fig16_ESM.jpg (638 kb)
Fig. S8

ENC1 knockdown does not affect death of SH-SY5Y cells expressing wtHTT. SH-SY5Y/pSuper-Neo (SH/Neo) and SH-SY5Y/pSuper-shENC1 (SHshENC1 Mix, #1 and #2) cells were transfected with pHTTex18Q-GFP (wtHTT) for the indicated times. Cell death rates were determined under fluorescence microscope (left panel) and cell extracts were analyzed with Western blotting (right panel) (JPG 637 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Huikyong Lee
    • 1
  • Hye-Hyun Ahn
    • 1
  • WonJae Lee
    • 1
  • Yumin Oh
    • 1
    • 3
  • Hyunwoo Choi
    • 1
  • Sang Mi Shim
    • 1
  • Jaekyoon Shin
    • 2
  • Yong-Keun Jung
    • 1
    Email author
  1. 1.Global Research Laboratory, School of Biological Science/Bio-MAX InstituteSeoul National UniversitySeoulSouth Korea
  2. 2.Department of Molecular Cell BiologySungkyunkwan University School of Medicine and Samsung Biomedical Research InstituteSuwon-SiSouth Korea
  3. 3.Russell H. Morgan Department of Radiology and Radiological ScienceJohns Hopkins UniversityBaltimoreUSA

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