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First published online 12 May 2009
doi: 10.1242/jcs.042226

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Omi is a mammalian heat-shock protein that selectively binds and detoxifies oligomeric amyloid-β

¹ Laboratory of Genetics, Department of Microbiology and Immunology and Institute for Medical Sciences, Chonbuk National University Medical School, Chonju 561-712, South Korea
² The Geriatric Research Education and Clinical Center, Veteran's Affairs Medical Center, Bedford, MA 01730, USA
³ Department of Neurology, Boston University School of Medicine, Boston, MA 02118, USA
⁴ Research Division, Jinis Biopharmaceuticals, Chonju 561-360, South Korea
⁵ Signal Transduction Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK

View larger version (62K): [in this window] [in a new window]   Fig. 1. Omi localizes to mitochondria and the ER in neuronal cells. (A-D) Omi localized both within mitochondria and the ER in NT2 and K269 cells. The cells were stained with an anti-Omi antibody together with the subcellular-organelle markers MitoTracker Red or PDI (an ER marker). Images were visualized using a confocal microscope (LSM510, Carl Zeiss). (E-H) Omi localized within both mitochondria and the ER, but not in lysosomes or the Golgi complex in primary mouse cortical neurons. The primary mouse cortical neurons transiently expressing GFP-tagged Omi were stained with the subcellular-organelle markers MitoTracker Red, ERTracker Red or LysoTracker Red. For Golgi staining, a DsRed-Monomer-Golgi fusion protein was coexpressed with GFP-tagged Omi. Images were visualized using a confocal microscope (TCS SP5, Leica). Scale bars: 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Omi binds preferentially to oligomeric Aβ rather than to APP. (A) Omi and Aβ colocalized in NT2N cells, which are neuronally differentiated human NT2 cells. Primary rabbit anti-Omi and mouse anti-Aβ (specific to Aβ C-terminal and no cross-reactivity to APP) antibodies, in addition to secondary goat anti-rabbit IgG-FITC and horse anti-mouse IgG–Texas-Red antibodies, were used to detect intracellular Omi (green) and Aβ (red), respectively. Images were visualized using a confocal microscope (LSM510, Carl Zeiss). Scale bars: 50 µm. (B) Omi and Aβ colocalized in Tg2576 mouse brain tissues. Primary mouse anti-Omi and rabbit anti-Aβ (specific to Aβ C-terminal and no cross-reactivity to APP) antibodies, in addition to secondary Alexa-Fluor-488-conjugated goat anti-mouse IgG and Alexa-Fluor-568-conjugated goat anti-rabbit IgG antibodies, were used to detect intracellular Omi (green) and Aβ (red), respectively. Confocal images were obtained with a Carl Zeiss LSM510 Meta microscope. Scale bars: 20 µm. (C) Both monomeric and oligomeric forms of Aβ were found in Tg2576 mouse brain mitochondria, and Omi was also located in the mitochondria. WT, wild-type littermate; FL-Omi, full-length Omi without processing into mature Omi; Cyt C, the mitochondrial protein cytochrome C. Arrow indicates oligomeric Aβ. Asterisks indicate non-specific bands from the mouse brain mitochondria. (D,E) Omi and Aβ formed complexes inside K269 cells and Tg2576 mouse brain mitochondria. Omi-Aβ complexes were detected by reciprocal immunoprecipitation of K269 cell protein extracts and Tg2576 mouse brain mitochondria with either anti-Omi or anti-Aβ (6E10). rIgG and mIgG, nonimmune rabbit and mouse IgG, respectively. (F) Omi bound selectively to oligomeric Aβ in a dose-dependent fashion in an immunoblot binding assay. The same amounts of reverse Aβ (Aβ42-1), monomeric Aβ or oligomeric Aβ were spotted on the membranes as indicated. The upper immunoblot was initially hybridized with the Omi protein and then probed with an anti-Omi antibody. The lower immunoblots were directly detected with an anti-oligomer (A11) antibody specific to the oligomeric Aβ forms or an anti-Aβ (6E10) antibody that recognizes both monomeric and oligomeric Aβ. One representative experiment out of three independent experiments is shown.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Interaction of Omi with oligomeric Aβ leads to mutual detoxification. (A-C) Oligomeric Aβ reduced the apoptotic activity of Omi. (A) Caspase-3 activation by Omi was weakened by pre-incubation with oligomeric Aβ (lane 8 versus 6). Lane 1, cell extracts alone (as a non-activating procaspase-3 control); lane 2, caspase-3 activation induced by adding cyt-c/dATP into the cell extracts; lanes 3-5, cyt-c/dATP-activated cell extracts plus control peptide, monomeric Aβ and oligomeric Aβ (indicated as C, M and O, respectively); lanes 6-8, cyt-c/dATP-activated cell extracts plus Omi pre-incubated with C, M and O. The active caspase-3 levels in replicate experiments were quantified by densitometry and expressed as the band intensity relative to lane 2 (right panel). ***P<0.001, lane 6 versus 2, lane 8 versus 6. A two-tailed Student's t-test was used (mean ± s.d., n=3). (B) Formation of active Omi was affected by pre-incubation with oligomeric Aβ. Lanes 1-3, Omi incubated with C, M or O. (C) The proteolytic activity of Omi was inhibited by pre-incubation with oligomeric Aβ in a dose-dependent pattern. Lane 1, XIAP; lane 2, XIAP plus Omi; lanes 3-11, XIAP plus Omi pre-incubated with C, M or O at the indicated concentrations. (D-G) Omi reduced Aβ neurotoxicity by disaggregating toxic oligomeric Aβ. (D) Exogenous Omi reduced the neurotoxicity of oligomeric Aβ, increasing the viability of NT2 cells. ***P<0.001, versus 0 nM Omi. (E) The reduction of endogenous Omi augmented the neurotoxicity of oligomeric Aβ. *P<0.05; ***P<0.001, versus 0 nM siRNA-Omi. A two-tailed Student's t-test was used (mean ± s.d., n=4). (F) Omi disaggregated the toxic oligomeric Aβ, especially the trimeric and tetrameric forms of Aβ. Omi (0-0.5 µM) was incubated with 0.5 µM (lanes 1-3) or 1 µM (lanes 4-6) of oligomeric Aβ for 1 hour. (G) Omi prevented the formation of toxic oligomeric Aβ. Monomeric Aβ (1 µM) was incubated without (lane 1) or with (lane 2) 0.5 µM of Omi under oligomerization conditions for 24 hours. One representative experiment out of three independent experiments is shown.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Omi expression is correlated with Aβ secretion. (A-D) Omi expression was upregulated along with neurogenesis and neuronal maturation. (A) Images of retinoic acid (RA)-induced NT2 differentiation and postmitotic NT2N process formation (visualized by a Nikon ECLIPSE TE2000-S microscope). Scale bars: 20 µm. (B) Aβ production increased along with neuronal differentiation in NT2/RA and NT2N cells, and Omi showed a similar expression pattern. Extracellular Aβ was immunoprecipitated by addition of the 6E10 antibody to media conditioned for 24 hours from NT2/RA and NT2N cells at the indicated time points, and then analyzed by an immunoblot assay. (C) Omi expression was upregulated along with neuronal differentiation (NT2/RA) and neuronal process formation (NT2N), but was not upregulated in a somatic cell line (EcR293/RA). Whole-cell lysates were used to analyze the expression levels of Omi by a western blot assay. (D) Omi expression was upregulated along with the development of brain tissues (cortex and cerebellum), whereas constant levels of expression were maintained in somatic tissues (liver). One representative experiment out of three independent experiments is shown. (E-H) Omi expression affected cellular Aβ release, thus reducing Aβ-induced stress. The conditioned growth media were collected and analyzed for levels of secreted Aβ with both a quantitative IP/WB assay (E,G) and ELISA (F,H). Aβ levels in replicate experiments of IP/WB assay (E and G, left panel) were quantified by densitometry and expressed as the band intensity relative to the siRNA-Ctrl (E, right panel) or vector control (G, right panel). (E,F) The reduction of Omi in K269 cells clearly increased Aβ secretion. The level of Omi was reduced by Omi siRNA (66 nM) when compared with controls (siRNA-Ctrl) (E). (G,H) Overexpression of Omi in K269 cells clearly reduced Aβ secretion. *P<0.05; **P<0.01; ***P<0.001, versus siRNA-Ctrl or the vector control. A two-tailed Student's t-test was used (mean ± s.d., n=3 or 4). One representative experiment out of three independent experiments is shown.

View larger version (20K): [in this window] [in a new window]   Fig. 5. A direct interaction between Omi and intracellular Aβ leads to the reduction of Aβ stress both in vitro and in vivo. (A) Overexpression of inactivated OmiS306A clearly reduced Aβ secretion. Extracellular Aβ was immunoprecipitated from the conditioned growth media using the 6E10 antibody, and then analyzed by a western blot assay. FL-Omi, full-length Omi without processing into mature Omi. (B) The absence of wild-type Omi caused the accumulation of Aβ in the brain of MND2 and Omi-knockout (KO) mice. The total Aβ in transgenic mice hemi-brains was extracted by 70% formic acid, followed by neutralization with 2 M Tris base and analysis with an ELISA kit specific for mouse Aβ40 or Aβ42. Sample Aβ values were normalized to the protein concentration of the brain extracts and expressed as pg of Aβ per mg of protein. *P<0.05; **P<0.01; ***P<0.001. A two-tailed Student's t-test was used (mean ± s.d., n=4 of wild-type mice, and n=3 of MND2 or Omi-KO mice).

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