Essential Role of E2-25K/Hip-2 in Mediating Amyloid-β Neurotoxicity Sungmin Song, So-Young Kim, Yeon-Mi Hong, Dong-Gyu Jo, Joo-Yong Lee, Sang Mi Shim, Chul-Woong Chung, Soo Jung Seo, Yung Joon Yoo, Jae-Young Koh, Min Chul Lee, Allan J Yates, Hidenori Ichijo, Yong-Keun Jung  Molecular Cell  Volume 12, Issue 3, Pages 553-563 (September 2003) DOI: 10.1016/j.molcel.2003.08.005

Figure 1 Increased Expression of E2-25K/Hip-2 by Aβ1-42 and in Alzheimer's Disease (A) Northern blot analysis. B103 cells were treated with 5 μM Aβ1-42 for 48 hr and total RNAs were subjected to Northern blotting with E2-25K/Hip-2. β-actin served as an internal control. (B) Quantitative RT-PCR analysis. Total RNA was purified from mouse cortical neurons exposed to 5 μM Aβ1-42 for 1 day. Expression of E2-25K/Hip-2 and E3 ligases interacting with E2-25K/Hip-2 (dinG, HSD4, and RNF3) was examined using RT-PCR analysis described in Experimental Procedures. (C and D) Aβ1-42-specific induction of E2-25K/Hip-2 protein in primary-cultured neurons. Mouse cortical neurons were incubated with 5 μM Aβ1-42 for 48 hr, 1 μg/ml tunicamycin (Tuni.), 1 μM thapsigargin (Tg.), or 0.2 μM staurosporine (Stau.) for 12 hr. Expression level of E2-25K/Hip-2 was analyzed with Western blotting (C) and immunostaining (D) using anti-E2-25K/Hip-2 and anti-tau (PHF-1) antibodies. (E) Immunostaining of E2-25K/Hip-2 in Swedish mutant APP Tg2576 mice. Cerebral cortexes obtained from Tg2576 mice and littermates were stained with Congo-red for the senile plaque (asterisk) and anti-E2-25K/Hip-2 antibody (arrow). (F) Increase of E2-25K/Hip-2 in the brain of AD patients. Sections of cerebral cortex derived from AD patient (upper panel) and age-matched control (lower panel) were stained with anti-Aβ (4G8) and anti-E2-25K/Hip-2 antibodies. Amyloid plaque (asterisk) and E2-25K/Hip-2 immunoreactivity (arrow) were detected in AD patients. Arrowheads indicate weak Aβ-positive signals colocalized with E2-25K/Hip-2. High power view is in small box (upper panel). Molecular Cell 2003 12, 553-563DOI: (10.1016/j.molcel.2003.08.005)

Figure 2 Proapoptotic Function of E2-25K/Hip-2 in Aβ1-42 Neurotoxicity (A) Resistance of B103AS-E2 stable cells to Aβ toxicity. B103 cells transfected with pcDNA3 (Mock) or pAS-E2-25K/Hip-2 (AS) were incubated with 800 μM G418 for 10 days and subjected to immunoblotting with anti-E2-25K/Hip-2 antibody (left upper panel). The cells were exposed to 5 μM Aβ1-42 for 48 hr and examined for the expression of E2-25K/Hip-2 (left lower panel) and cell viability (right panel). (B) Selective resistance of the individual B103AS-E2 cell line to Aβ toxicity. Four B103 cell lines permanently transfected with pcDNA3 (C) or pAS-E2-25K/Hip-2 (#1, #3, #4, and #7) were selected and analyzed for the expression level of E2-25K/Hip-2 with Western blotting (lower panels) and exposed to 5 μM Aβ1-42 for 48 hr (upper panel). (C) Lack of resistance to staurosporine- or tunicamycin-triggered cell death. Two B103AS-E2 stable cell lines (#1 and #7) were incubated for 16 hr with 0.2 μM staurosporine (Stau.) or 1 μg/ml tunicamycin (Tuni.). Cell viability was assessed by tryphane blue exclusion assay. All values represent the means ± SD from four independent experiments. (D) Proapoptotic effect of E2-25K/Hip-2. B103 cells transfected with pDsRed (RFP) and either empty vector (Mock) or pE2-25K/Hip-2 for 48 hr were stained with Annexin V (Ann. V) (left panel). Several neuronal (B103, SK-N-BE2[C]) and nonneuronal cell lines (HEK293 [293], HeLa, and Hep3B) were transfected with pDsRed (RFP) and either empty vector (Mock) or pE2-25K/Hip-2 for 48 hr and examined for cell viability based on the morphology of the RFP-positive cells under the fluorescence microscope (right panel). Bars depict the means ± SD (n = 4). (E) Mutation analysis of E2-25K/Hip-2 toxicity. B103 cells were transiently transfected with pDsRed (RFP) and either empty vector (Mock), pE2-25K/Hip-2, pE2-25K/Hip-2 (Δtail), pE2-25K/Hip-2 (S86Y), or pE2-25K/Hip-2 (C92S) for 48 hr. Cell viability was determined as described in Figure 2D. (F) Inhibitory effect of E2-25K/Hip-2 mutants on Aβ1-42 neurotoxicity. B103 cells were transfected with the indicated plasmids for 24 hr and subsequently exposed to Aβ1-42 for 48 hr. Molecular Cell 2003 12, 553-563DOI: (10.1016/j.molcel.2003.08.005)

Figure 3 Contribution of E2-25K/Hip-2 to Aβ1-42-Induced Inhibition of Proteasome Activity and Accumulation of Ubiquitin Conjugates (A) Inhibition of proteasome activity by Aβ1-42. B103 cells were transfected with pGFP-degron (pGFPu), CL1, for 24 hr and then treated with 5 μM Aβ1-42. After 48 hr, GFPu-positive cells were counted under fluorescence microscope, and the relative ratio of expression is represented as the means ± SD (n = 4) (upper panel). Proteasome activity was measured in B103 cells exposed to 5 μM Aβ1-42 using fluorogenic substrate, Suc-LLVY-Amc (lower panel). *p < 0.0005 as compared with control. (B) E2-25K/Hip-2-mediated inhibition of proteasome activity by Aβ1-42. Control B103 and two B103AS-E2 cell lines (#1 and #7) were transiently transfected with pGFPu for 24 hr and incubated with 0.1 μM MG132 or 5 μM Aβ1-42 for additional 48 hr. GFPu expression was analyzed with Western blotting using anti-GFP antibody (upper panel). GFP was included for transfection efficiency (middle panel). B103 cells stably expressing pGFPu (B103-GFPu) were transfected with pcDNA3 (Mock), pE2-25K/Hip-2, pE2-25K/Hip-2 (Δtail), pE2-25K/Hip-2 (S86Y), pE2-25K/Hip-2 (C92S), or pubcH5b (E2D) for 60 hr, and the GFPu-positive cells were scored (n = 3) (lower panel). (C and D) Promotion of Aβ and E2-25K/Hip-2 neurotoxicity by proteasome inhibitor MG132. B103 cells were transfected with pE2-25K/Hip-2 (C) or incubated with Aβ1-42 for 1 day (D) in the presence or absence of 0.1 μM MG132. Cell viability was then assessed as described in Figure 2D and by trypan blue assay. (E) E2-25K/Hip-2-mediated accumulation of ubiquitin conjugates in Aβ1-42-treated neuronal cells. Mouse cortical neurons were incubated with 5 μM Aβ1-42, 1 μg/ml tunicamycin (Tuni.), 1 μM thapsigargin (Tg.), or 0.2 μM staurosporine (Stau.) (left panel). The stable B103 cells transfected with pcDNA3 (Mock) or pAS-E2-25K/Hip-2 were exposed to 5 μM Aβ1-42 (right panel). Ubiquitin and ubiquitin conjugates were detected with Western blot analysis using anti-ubiquitin antibody: asterisk, nonspecific; Ub, ubiquitin; Ub-Ub, dimeric ubiquitin; Ub-conj., ubiquitin conjugates. (F) Accumulation of ubiquitin conjugates by forced expression of E2-25K/Hip-2. B103 cells transiently transfected with pE2-25K/Hip-2 were analyzed for ubiquitin conjugates as described in (E). Asterisk, nonspecific; Ub, ubiquitin; Ub-conj., ubiquitin conjugates. Molecular Cell 2003 12, 553-563DOI: (10.1016/j.molcel.2003.08.005)

Figure 4 Regulation of JNK by E2-25K/Hip-2 during Aβ Neurotoxicity (A) Effect of JNK inhibitor on Aβ- and E2-25K/Hip-2-induced cell death. B103 cells were incubated for 48 hr with 5 μM Aβ1-42 (left panel) or transfected with pE2-25K/Hip-2 (right panel) in the presence or absence of 10 μM SP600125 (SP) or 25 μM PD98059 (PD). Bars depict the means (% of cell death) ± SD (n = 4). (B) Reduced activation of JNK in the Aβ1-42-treated B103AS-E2 cells. Control B103 (C) and B103AS-E2 stable cell lines (#1, #3, and #7) were incubated with 5 μM Aβ1-42 for 48 hr, and cell extracts were analyzed with Western blotting using anti-JNK, anti-phospho-JNK, or anti-tubulin antibody. (C) Lack of E2-25K/Hip-2-induced cell death in ASK1−/− cells. Wild-type and ASK1−/− EF cells were transiently transfected with pEGFP and either pE2-25K/Hip-2 or pCaspase-8 (Casp8). The viability was determined based on the morphology of cells showing green fluorescence under fluorescence microscope. Bars represent the means (% of cell death) ± SD (n = 7). Molecular Cell 2003 12, 553-563DOI: (10.1016/j.molcel.2003.08.005)

Figure 5 Functional Coordination of E2-25K/Hip-2 with UBB+1, a Ubiquitin Mutant Found in AD (A) Colocalization of UBB+1 and E2-25K/Hip-2 in the brains of AD patients. Sections of cerebral cortex derived from AD patients (right panel) and age-matched controls (left panel) were stained with anti-UBB+1 and anti-E2-25K/Hip-2 antibodies. (B) Quantitative determination of colocalization of UBB+1 and E2-25K/Hip-2 in the brain of AD patients. Cerebral cortexes of three AD patients (P1–P3) and a normal control (N) were analyzed with immunohistochemistry as shown in (A). The incidence of colocalization of UBB+1 and E2-25K/Hip-2 was determined in more than five regions of each brain section (the numbers of immunoreactive spots per patient > 100). (C) Functional interaction of E2-25K/Hip-2 with UBB+1. Left panel: control B103 (C) and B103AS-E2 cells (#1 and #7) were transfected with pEGFP, rtTA-M2, and either pTet-splice, pTet-splice-ubiquitin, or pTet-splice-UBB+1 in the presence of 1 μg/ml doxycycline for 1 day. Right panel: B103 (C) and B103AS-E2 (AS) cells were transfected with pEGFP, rtTA-M2, and either pTet-splice, pTet-splice-UBB+1, or pTet-splice-UBB+1(K48R) in the presence of 1 μg/ml doxycycline for 1 day. Cell viability (the means ± SD, n = 4) was determined based on the morphology of the GFP-positive cells under fluorescence microscope. Molecular Cell 2003 12, 553-563DOI: (10.1016/j.molcel.2003.08.005)

Figure 6 Schematic Diagram Showing the Proposed Role of E2-25K/Hip-2 in Aβ Neurotoxicity Induction of E2-25K/Hip-2 is required for neuronal cell death during Aβ toxicity and may cause inhibition of proteasome activity and concomitant accumulation of ubiquitin conjugates, which leads to the activation of ASK1/JNK. UBB+1 found in the brain of AD patients may be ubiquitinylated by E2-25K/Hip-2 for proteasome inhibition. Molecular Cell 2003 12, 553-563DOI: (10.1016/j.molcel.2003.08.005)