Alpha-1-microglobulin for use in the treatment of mitochondria-related diseases

Abstract

The present invention relates to alpha-1-microglobulin for use in the treatment of a mitochondria-related disease.

Claims

1. A method for treating a mitochondria disease or disorder comprising administering alpha-1-microglobulin (A1M) to a subject in need thereof, wherein the A1M is a peptide having an amino acid sequence selected from the group consisting of (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:1 and comprises residues corresponding to Y22, C34, K92, K118, K130, Y132, L180, I181, P182, and R183 of SEQ ID NO:1; and (b) an amino acid sequence that is at least 80% identical to SEQ ID NO:2, and comprises residues corresponding to Y40, C52, K110, K136, K148, Y150, L198, I199, P200, and R201 of SEQ ID NO:2; wherein the A1M is the only therapeutic agent administered to the subject to treat the mitochondria disease or disorder; and wherein the mitochondria disease or disorder is a Respiratory Chain Deficiency involving Complex I defects or Respiratory Chain Disorder involving Complex I defects.

2. The method according to claim 1, wherein the subject is a child or young adult.

3. The method according to claim 1, wherein the method is for treating one or more Respiratory Chain Deficiency-associated conditions or Respiratory Chain Disorders involving Complex I defects selected from the group consisting of Alpers disease (Progressive Infantile Poliodystrophy), Friedreich's ataxia, KSS, Leigh Disease or Syndrome, Leber's hereditary optic neuropathy (LHON), Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes (MELAS), Myoclonic Epilepsy and Ragged-Red Fiber Disease (MERRF), and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP).

4. The method according to claim 1, wherein the subject is a woman.

5. The method according to claim 1, wherein the method is for treating damage or dysfunction of retina or ocular diseases associated with mitochondrial defect(s) or dysfunction(s).

6. The method according to claim 1, wherein the subject is a human.

7. The method according to claim 1, wherein the method is for treating Friedreich's ataxia.

8. A method for reducing the risk of one or more conditions selected from the group consisting of a mitochondrial defect, a mitochondria disease or disorder, a drug-induced mitochondria side-effect or an environmentally induced mitochondria effect, comprising administering alpha-1-microglobulin (A1M) to a subject in need thereof, wherein the A1M is a peptide having an amino acid sequence selected from the group consisting of (a) an amino acid sequence that is at least 80% identical to SEQ ID NO:1 and comprises residues corresponding to Y22, C34, K92, K118, K130, Y132, L180, I181, P182, and R183 of SEQ ID NO:1; and (b) an amino acid sequence that is at least 80% identical to SEQ ID NO:2, and comprises residues corresponding to Y40, C52, K110, K136, K148, Y150, L198, I199, P200, and R201 of SEQ ID NO:2; wherein the A1M is the only therapeutic agent administered to the subject; and wherein the subject is suffering from a Respiratory Chain Deficiency involving Complex I defects or Respiratory Chain Disorder involving Complex I defects.

9. The method according to claim 8, wherein the subject is a human.

10. The method according to claim 8, wherein the method is for reducing the risk of one or more Respiratory Chain Deficiency-associated conditions or Respiratory Chain Disorders involving Complex I defects selected from Alpers disease (Progressive Infantile Poliodystrophy), Friedreich's ataxia, KSS, Leigh Disease or Syndrome, Leber's hereditary optic neuropathy (LHON), Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes (MELAS), Myoclonic Epilepsy and Ragged-Red Fiber Disease (MERRF), and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP).

11. The method according to claim 8, wherein the method is for reducing the risk of Friedreich's ataxia.

12. The method according to claim 8, wherein the method is for treating damage or dysfunction of retina or ocular diseases associated with mitochondrial defect(s) or dysfunction(s).

Description

LEGEND TO FIGURES

(1) FIG. 1. Binding of A1M to intact and apoptotic cells. A. HCQ.4 cells were cultured on hamster anti-mouse CD3 antibody (4 g/ml) coated plastics for 18 hours. For analysis of A1M binding, 110.sup.6 cells were incubated with 1 mg/ml A1M, washed, incubated with mouse anti-A1M antibodies, washed and finally incubated with FITC-conjugated goat anti-mouse IgG (GAM-FITC). 10 000 cells were analyzed for A1M binding (open peak). Background was set by cells incubated with BSA in the first step (shaded peak). Binding to apoptotic cells (right histogram) was compared to untreated cells (left histogram). B. The binding of A1M was correlated to the uptake of PI by culturing HCQ.4 cells in the presence of 5% ethanol or 10% DMSO for 15 hours. For the flow cytometry analysis, A1M was incubated with cells, followed by mouse anti-A1M antibodies and FITC-conjugated goat anti-mouse IgG. Before analysis, cells were also stained by PI to detect dead cells. C. Binding of A1M to apoptotic cells of the pre-B-cell line 70Z/3 was analyzed by flow cytometry. The cells were induced to apoptosis by the benzamide drug declopramide (3-CPA) for 15 hours and analyzed for A1M binding by incubation of biotinylated A1M, followed by SAPE. The background was set by cells incubated with SAPE only (shaded peak). D. K562 cells incubated with 20 M and 0.25 mg/ml A1M for 2 h and subjected to staining with mouse anti-A1M antibodies followed by goat anti-mouse IgG F(ab).sub.2-fragments (Alexa Fluor 594; red). Cells were mounted using ProLong Gold AntiFade Reagent with DAPI and visual inspection and recording was performed. The picture is representative for three separate experiments. Sizebar is 10 M. E. The specificity of the A1M binding was determined by a competitive cell-binding assay. HCQ.4 cells, induced to apoptosis by cross-linking with anti-CD3 for 18 hours (right histogram), were compared to untreated HCQ.4 cells (left histogram). 110.sup.6 cells/sample were mixed with 1 g/ml of .sup.126I-A1M (1), .sup.125I-A1M plus addition of 2.5 mg/ml of unlabeled A1M (2), ovalbumin (3), BSA (4) or AGP (5). The cells were incubated for 30 minutes at 4 C. centrifuged on a sucrose-gradient to separate unbound protein, tubes were then frozen and cell-pellet cut off and counted in a -counter. The results are presented as mean values of a triplicate from one experimentSEM. Statistical comparison between groups was made using Student's t test. *** P<0.001.

(2) FIG. 2. Time-studies of the binding of A1M to apoptotic cells. A. Apoptosis was induced in HCQ.4 cells by cultivation on anti-CD3 coated plastics. Samples were taken at different time-points after induction (0, 1, 2, 4, 8 and 16 hours). The cells were stained with FITC-conjugated A1M (0.1 mg/ml) and PI. 10 000 cells were analyzed. B. Binding of A1M to pre-B-cells, induced to apoptosis by incubation with the benzamide 3-CPA, were analyzed by flow cytometry and correlated to binding of annexin V and 7AAD uptake. The apoptotic 70Z/3 cells were incubated with biotinylated A1M (0.025 mg/ml) followed by SAPE, annexin V and 7AAD. 10 000 cells were analyzed and gated for 7AAD negative (left diagram) and 7AAD positive cells (right diagram) respectively.

(3) FIG. 3. Binding of A1M to mitochondria analyzed by confocal microscopy and transmission electron microscopy. A. K562 cells incubated with medium only (left) or 0.25 mg/ml A1M (right) for 2 h were washed and incubated with Mito-Tracker (red) for 15 minutes, and washed in fresh medium. After washing, cells were then stained with monoclonal mouse anti-A1M (BN 11.3) at 5 g/ml followed by goat anti-mouse IgG F(ab).sub.2 fragments (Alexa Fluor 488; green). Cells were mounted using ProLong Gold AntiFade Reagent with DAPI (blue) and visual inspection and recording was performed using confocal microscopy. The picture is representative of three separate experiments. Scale bar indicates 5 m. B. An overview of human primary keratinocytes incubated for 20 hours at RT with 10 M A1M. Mitochondrial structures are highlighted with arrows and shown in higher magnification in (C). Immunolabeling of human primary keratinocyte thin sections with gold-labeled anti-A1M was performed and shown to correlate to mitochondria. This is highlighted with arrowheads (C). The samples were prepared and observed as described in Materials and Methods. Scale bar in (B) indicates 2 m and in (C) 0.1 m.

(4) FIG. 4. Binding of A1M to mitochondria analyzed by .sup.125I-A1M binding. A. The specificity of A1M-binding to mitochondria was investigated by mixing approximately 2.5 g/ml of .sup.125I-A1M with 0.5 mg purified mitochondria in the presence or absence of 1.0 mg/ml of unlabeled protein (A1M or AGP) in PBS+4% BSA. The mixtures were incubated at 4 C. for 30 minutes, centrifuged on a sucrose-gradient to separate unbound protein, tubes were then frozen and cell-pellet cut off and counted in a -counter. Each point represents the meanSEM of three determinations. Statistical comparison between groups was made using Student's t test. ***P<0.001. B. The specificity of A1M-binding to mitochondria was further investigated using BN-PAGE and Western blotting. Five g mitochondrial membrane proteins from 2 separate individuals were separated on a BN-PAGE 4-16% Bis-Tris gel and blotted to a PVDF membrane. After blocking, the membranes were incubated with antibodies against subunit NDUFV1 of Complex I, Core I of Complex III, mouse A1M, or stained with Coomassie. C. The Complex I association was also investigated by immunoprecipitation of freshly prepared mitochondria with antibodies against Complex I. Following the immunoprecipitation, bound and eluted proteins were separated on 12% SDS-PAGE and blotted to PVDF membrane. After blocking, the membranes were incubated with antibodies against mouse A1M. Left lane, mitochondrial starting material (SM) and right lane, bound and eluted material (IP).

(5) FIG. 5. The specificity of A1M-binding to mitochondria was investigated using BN-PAGE, SDS-PAGE and Western blotting. Freshly isolated mitochondria were suspended in PBS, pelleted by centrifugation and dissolved to a concentration of 5 mg/ml in MB2 buffer. Mitochondrial membrane proteins were solubilized by incubation with 0.5-4.0 g digitonin/g protein for 5 min on ice. Samples were centrifuged, the supernatant was collected and SBG was added to a final concentration of 4.5%. A. Five g mitochondrial membrane proteins from 2 separate individuals (S1 and S2) were then separated on a BN-PAGE 4-16% Bis-Tris gel and blotted to a PVDF membrane. After blocking, the membranes were incubated with antibodies against subunit NDUFV1 (left) of Complex I, A1M (middle) and subunit Core I of Complex III. B. Trypsin treated (0-100 U Trypsin) isolated mitochondrial proteins (15 pg/lane) were separated on 12% SDS-PAGE and transferred to a PVDF membrane. After blocking, the membrane was incubated with antibodies against A1M.

(6) FIG. 6. A1M protects mitochondrial structure. Human primary keratinocytes were incubated for 20 hours at RT with culture medium only (A), 20 M heme (B) or 20 M heme+0.25 mg/ml A1M (C). Mitochondrial structures are highlighted with arrows and depicted in details (zoomed pictures). The samples were prepared and observed as described in Materials and Methods. Scale bar indicates 2 m (overview) and 0.5 m (zoomed picture).

(7) FIG. 7. Human primary keratinocytes were incubated for 20 hours at RT with culture medium only (A), 250 M H.sub.2O.sub.2 (B) or 250 M H.sub.2O.sub.2+0.25 mg/ml A1M (C). Mitochondrial structures are highlighted with arrows and depicted in details (zoomed pictures). The samples were prepared and observed as described in Materials and Methods. Scale bar indicates 2 m (overview) and 0.5 m (zoomed picture).

(8) FIG. 8. A1M protects mitochondrial function. The effect of A1M on mitochondrial function was investigated by measuring ATP-production of purified mitochondria exposed to heme or H.sub.2O.sub.2. Mitochondria were incubated with 1-20 M heme, with or without 0.25 mg/ml A1M (A) or 20-250 M H.sub.2O.sub.2 with or without 0.25 mg/ml A1M (B) for 30 minutes. Mitochondria were collected by centrifugation and ATP-production was measured using a luminescence assay kit. ATP levels were normalized to the corresponding sample protein. Each point represents the meanSEM of three determinations. Statistical comparison between groups was made using Student's t test. *P<0.05.

(9) FIG. 9. Sequence listing of A1M

(10) FIG. 10. Realtime PCR quantitation of mitochondrial rRNA (A) and cellular A1M, HO1 and SOD mRNA (B) during retina culture under mild and stress conditions. Each t-value corresponds to the amount of RNA in stressed conditions relative to the RNA amount in mild conditions, determined by realtime PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase. Each bar is the mean of duplicate measurements of triplicate cultures.

EXPERIMENTAL

Example 1

(11) Materials and Methods

(12) Proteins and Antibodies

(13) Human monomeric plasma A1M was isolated by anti-A1M affinity chromatography and Sephacryl S-300 gel-chromatography, as described previously (48). Recombinant human A1M, containing an N-terminal His-tag, was purified from the culture medium of baculovirus-infected insect cells (48) or expressed in E. coli and purified and refolded as described (20) with the addition of an ion-exchange chromatography purification step (32). Human serum .sub.1-acid glycoprotein (AGP) and ovalbumin were purchased from Sigma-Aldrich Co. (St. Louis, Mo., USA) and bovine serum albumin (BSA) was from Roche Diagnostics Scandinavia AB (Bromma, Sweden). Hemin (Ferriprotoporphyrin IX chloride) was purchased from Porphyrin Products, Inc. (Logan, Utah) and a 10 mM stock solution was prepared fresh by dissolving in dimethyl sulphoxide (DMSO; Sigma-Aldrich). H.sub.2O.sub.2 was from Acros Organics (Geel, Belgium). Mouse monoclonal antibodies against human A1M (BN11.3) were raised as described (29). Rabbit polyclonal anti-mouse A1M antibodies (Sven; IgG-fraction) were prepared by immunizing a rabbit with His-tagged mouse A1M expressed in baculovirus-infected insect cells (41). The hamster anti-mouse CD 3 antibody 145.2C11 was kindly provided by Dr. Rikard Holmdahl, Lund University. Fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (GAM-FITC) and phycoerythrein-conjugated streptavidin (SAPE) were purchased from DAKO A/S (Glostrup, Denmark), 7-amino actinomycin D (7 AAD) was from Sigma-Aldrich Co. and annexin V-FITC was from Trevigen Inc. (Gaithersburg, Md., USA).

(14) Cell Culture

(15) A mouse CD4+ T cell hybridoma cell line (HCQ.4), a murine pre-B-cell line (70Z/3), a human erythroid cell line (K562) and human primary keratinocytes (Cambrex Biologics, Karlskoga, Sweden) were employed for studies on A1M binding to cells and mitochondria. Cells were cultivated as described previously (25,28,37) and processed and analyzed as described below.

(16) Induction of Apoptosis

(17) Apoptosis was induced in the T cell hybridoma by three different treatments: Cells were incubated on anti-CD3 antibody coated plastics (4 g/ml) (43), or incubated in medium supplemented with either 5% ethanol or 10% of DMSO (24). The cells were incubated in a CO.sub.2-incubator at 37 C. for various times. Apoptosis was detected as DNA-fragmentation by agarose-gel electrophoresis (described below) and cell viability was measured by trypan blue exclusion. In the pre-B-cell line 70Z/3, apoptosis was induced by the benzamide-drug declopramide (3-CPA, Oxigene Inc.) as described in (25).

(18) Agarose Electrophoresis

(19) To detect DNA fragmentation, approximately 110.sup.6 cells were lysed, proteinase K- and RNAse A-treated and analyzed by agarose electrophoresis.

(20) Labeling of A1M

(21) For analysis of A1M-binding to cells, A1M was biotinylated, FITC conjugated or .sup.125I radiolabeled. A1M was biotinylated with long arm-biotin N hydroxysuccinimide (Vector Laboratories Inc., Burlingame, Calif., USA) (9) and diluted to a concentration of 0.2 mg/ml. A1M was FITC-conjugated as described previously (13) by FITC adsorbed on Celite (Calbiochem Corp, San Diego, Calif., USA). A1M was labeled with .sup.125I using the chloramine T method (16). The specific radioactivity obtained was around 0.1-0.2 MBq/g.

(22) Flow Cytometry

(23) A1M-binding to cells was analyzed by flow cytometry. Approximately 110.sup.6 cells were analyzed for A1M-binding in one of three different ways: 1. The cells were incubated with 1 mg/ml of plasma or recombinant insect cell-A1M, followed by 10 g/ml of monoclonal mouse anti-A1M (BN 11.3) and GAM-FITC (diluted 20 times). 2. The cells were incubated with 10 g/ml biotinylated-A1M followed by SAPE (diluted according to the manufacturer's recommendations). 3. The cells were incubated with 0.1 mg/ml FITC-conjugated A1M. All incubations were performed in PBS+1 mg/ml of BSA for 10 minutes at RT. Between the incubations, the cells were washed 2-3 times in PBS. To detect leaking cells, cells were incubated with propidium iodide (PI; Invitrogen Inc.) or 7AAD (according to manufacturers' instructions). To detect apoptotic 70Z/3 cells, cells were also incubated with FITC-conjugated annexin V in a Ca.sup.2+-containing buffer (according to the manufacturers' instructions). All analyses were performed using a Becton Dickinson FACSorter and the Cell Quest software package.

(24) Fluorescence and Confocal Microscopy

(25) K562 cells were washed and re-suspended in culture medium to 0.5-4.010.sup.6 cells/ml and incubated with or without A1M as indicated in the figure legends. Cells were then either incubated with Mito-Tracker (Invitrogen Inc.) for 15 minutes at 37 C. and washed in fresh medium (FIG. 3A) or directly washed in fresh medium (FIG. 1C) After washing, staining of the cells was performed by re-suspending in ice-cold Na-medium (5.4 mM KCl; 1.2 mM KH.sub.2PO.sub.4; 0.8 mM MgSO.sub.4; 5.6 mM D-glucose; 127 mM NaCl; 10 mM Hepes; 1.8 mM CaCl.sub.2; pH 7.3), fixation with 1% BD CellFIX on ice for 15 min and at RT for 45 min. Cells were washed in blocking solution (Na-medium; 1% BSA; 5% goat serum) followed by permeabilization in 0.02% Triton-X and blocking in 1% BSA, 5% goat serum, 0.2% Tween-20 for 1 hour at RT. The cells were then stained at 4 C. over-night with monoclonal mouse anti-A1M (BN 11.3) at 5 g/ml. Subsequently, goat anti-mouse IgG F(ab).sub.2 fragments (Alexa Fluor 594; Invitrogen Inc.), was applied for 1 h at RT. Cells were mounted using ProLong Gold AntiFade Reagent with DAPI. For fluorescence microscopy, visual inspection and recording of images were performed using a Nikon Eclipse TE300 inverted fluorescence microscope equipped with a Hamamatsu C4742-95 cooled CCD camera, using a Plan Apochromat 100 objective. For confocal microscopy, analyses of cells and fluorescent markers were performed using an epi-fluorescence microscope (Nikon Eclipse TE300) and a confocal laser scanning microscope (Zeiss LSM 510 Meta). The epi-fluorescens microscope was equipped with the appropriate filter combinations to selectively visualize the used fluorophores. Analyses were made using a Plan Apochromat 100 lens, and the image data was collected with a Hamamatsu C4742-95 CCD camera. To analyze intracellular labeling and co-labeling in subcellular structures, confocal scanning of optical sections were recorded through the cells. For excitation of the fluorophores, the 405 nm laser line was used for DAPI (diode laser 405-30), the 488 nm laser line was used for Alexa Fluor 488 (Argon laser), and the 561 nm laser line was used for Mito-Tracker (DPSS 561-10). The individual fluorophore emission wavelengths were detected using the following filters: bandpass 420-480 nm for DAPI, bandpass 505-550 nm for Alexa Fluor 488, and longpass 575 nm for Mitotracker. The pinhole for detection of Alexa Fluor 488 (488 nm excitation) was set to correspond to 1 (one) Airy unit, and the pinholes for the other detection channels were then adjusted to give optical sections of the same thickness, i.e. to ensure comparisons of the corresponding confocal volumes. Laser power and detection settings (gain and offset) were optimized for the individual channels, giving a detection range from highly saturated pixels of larger structures to non-saturated pixels of small structures. The different fluorophores were sequentially scanned, i.e. with optimal settings for one fluorophore in each channel, at 512512 or 10241024 frame size. To determine cellular morphology, differential interpherence contrast (DIC) images were obtained using the 405 nm laser as transmitted light. The spatial relation between the Alexa Fluor 488 fluorescence (green) and Mito-Tracker fluorescence (red) was determined via merging of the optical sections from the individually scanned channels (yellow when co-localized), confirmed via analyses of merged images using the LSM Zen software (Profile, data not shown).

(26) Yeast 2-Hybrid System

(27) A GAL4-based yeast 2-hybrid system was used to search for A1M-interacting cellular proteins. DNA encoding the A1M-part (amino acids 1-183) of the A1M-bikunin gene (AMBP) was amplified by PCR using a pCR-Script construct as a template. The fragment was completely sequenced and ligated into the yeast 2-hybrid vector pBD-GAL4 Cam phagemid vector (Stratagene, La Jolla, Calif., USA). The recombinant vector was then transformed into the S. cerevisiae yeast host strain YRG-2 (Stratagene). Growth and maintenance of the yeast strains and 2-hybrid assays were performed using standard protocols as recommended by Stratagene and www.umanitoba.ca/faculties/medicine/units/biochem/gietz. Approximately 7.510.sup.8 YRG-2 carrying the bait plasmid, pBD-GAL4-A1M was transformed with 15-20 g of a human leukocyte MATCHMAKER cDNA library (Clontech Laboratories, Inc., Palo Alto, Calif., USA). The resulting approximately 210.sup.6 transformants were analyzed by histidine prototrophy assay and -gal colony lift assay. Recombinant library plasmids from the His.sup.+LacZ.sup.+ transformants were isolated and retested in direct 2-hybrid assays together with the A1M bait plasmid as well as with bait plasmids encoding unrelated proteins. Plasmids resulting in activation of the reporter genes together with A1M-encoding bait plasmid, but not with the bait plasmids encoding unrelated proteins were regarded as true positives. The DNA sequence of the inserts was determined using the vector primers pAD5:5-tccagattacgctagcttgggtggtcatatg-3 (SEQ ID NO: 6) and pAD3:5-gtgaacttgcggggtttttcagtatctacga-3 (SEQ ID NO: 7). One of the inserts was sequenced completely by Innovagen AB (Lund, Sweden).

(28) Mitochondria Preparation from Mouse Liver Tissue

(29) Mouse liver tissue was collected in ice cold isolation buffer (320 mM Sucrose, 10 mM Trizma Base, 2 mM EGTA) and subsequently homogenized in 2 ml homogenization buffer (isolation buffer supplemented with 1% BSA). Mitochondria were prepared from homogenates by sequential centrifugation including density purification on 19% Percoll. The protein concentration of mitochondrial preparations was determined using Nanodrop and isolated mitochondria were used without freezing.

(30) Competitive Cell- and Mitochondria-Binding Assay.

(31) The specificity of A1M-binding to cells and mitochondria was investigated by a competitive cell-binding assay as described (3,49). Apoptosis was induced in HCQ.4 cells by anti-CD3 cross-linking for 15-18 hours. The cells were harvested and compared to normal cells in the binding assay. An affinity constant for the binding was calculated using a Scatchard plot of the data.

(32) Immunocapture of Complex I

(33) Immunoprecipitation of Complex I was performed on freshly prepared mitochondria using the Complex I Immunocapture Kit (MitoSciences). Following the immunoprecipitation, bound proteins were eluted using SDS-buffer and subsequently analyzed using SDS-PAGE and Western blotting.

(34) Isolation of Respiratory Chain Complexes and Supercomplexes

(35) Freshly isolated, non-frozen mitochondrial pellets were suspended in PBS supplemented with Complete Mini Protease inhibitor. Mitochondria were pelleted for 5 min at 5000g and subsequently dissolved to a concentration of 5 mg/ml in MB2 buffer (1.75 M aminocaproic acid, 7.5 mM Bis-Tris pH 7.0, +2 mM EGTA pH 8.0). Mitochondrial membrane proteins were solubilized by incubation with 0.5% digitonin for 5 min on ice. Samples were centrifuged for 30 min at 13000g, the supernatant was collected and the protein concentration measured as before. Finally, SBG (750 mM aminocaproic acid, 5% Serva Blue G) was added to a final concentration of 4.5%.

(36) Blue Native PAGE, SDS-PAGE and Western Blotting

(37) Five g mitochondrial membrane proteins were separated on a BN-PAGE 4-16% Bis-Tris gel (Invitrogen Inc.) either stained with Coomassie Brilliant Blue or blotted to a PVDF membrane (Immobilon, Millipore, Bedford, Mass., USA) using Iblot equipment (Invitrogen Inc.). Complex I-immunoprecipitated proteins were separated on a 12% SDS-PAGE and transferred to a PVDF membrane. After blocking over-night at 4 C. the membranes were incubated with antibodies against subunit NDUFV1 of Complex I (Sigma) or mouse A1M. Primary antibodies were detected by incubation with HRP-coupled goat anti-mouse (DAKO) or goat anti-rabbit (DAKO).

(38) Transmission Electron Microscopy (TEM)

(39) Human keratinocytes (about 1 million cells), incubated for 20 hours at RT with 20 M heme, with or without 10 M A1M, were pelleted by centrifugation and subsequently fixed for 1 hour at RT and then overnight at 4 C. in 2.5% glutaraldehyde in 0.15 M sodium cacodylate, pH 7.4 (cacodylate buffer). Samples were then washed with cacodylate buffer and post-fixed for 1 hour at RT in 1% osmium tetroxide in cacodylate buffer, dehydrated in a graded series of ethanol, and then embedded in Epon 812 using acetone as an intermediate solvent. Specimens were sectioned with a diamond knife into 50-70 nm-thick ultrathin sections on an LKB ultramicrotome. The ultrathin sections were stained with uranyl acetate and lead citrate. Specimens were observed in a JEOL JEM 1230 electron microscope operated at 80 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera. Immunolabeling of thin sections with gold-labeled anti-A1M (BN11.3) were performed as described previously (39) with the modification that Aurion-BSA was used as a blocking agent. Samples were finally stained with uranyl acetate and lead citrate and observed in a Jeol JEM 1230 electron microscope, operated at 80 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 charge-coupled device camera.

(40) ATP Assay

(41) Cellular ATP production was measured using a luminescence assay kit (Promega, Madison, Wis.), based on the ATP-dependent activity of luciferase. ATP levels were normalized to the corresponding sample protein content.

(42) Statistical Analysis

(43) Statistical analysis was performed using Origin 8 software. Student's t-test was used for statistical evaluation and was considered significant when P<0.05.

(44) Results

(45) Specific Binding of A1M to Damaged Cells

(46) Binding of A1M to apoptotic and healthy cells was analyzed by flow cytometry and compared to untreated cells. First, apoptosis was induced in murine T cell hybridomas (HCQ.4) by cross-linking of the CD3 molecule with immobilized anti-mouse CD3 antibodies (FIGS. 1A, E), or by incubation with 5% ethanol or 10% DMSO (FIG. 1B). These treatments resulted in DNA fragmentation and uptake of trypan blue after 15-18 hours (not shown). A weak binding of A1M could be detected to untreated cells (FIG. 1A, left panel). An additional stronger binding could be detected to cells cross-linked with anti-CD3 (FIG. 1A, right panel) or treated with ethanol or DMSO (FIG. 1B). The binding could be correlated to PI uptake, i.e. only cells that could incorporate PI displayed the stronger binding of A1M (FIG. 1B). Flow cytometry of a murine pre-B-cell line, induced to apoptosis using the drug 3-CPA, and incubated with A1M followed by anti-A1M, showed similar results (FIG. 1C), indicating that the binding to apoptotic cells is not restricted to T cells.

(47) In order to further characterize the A1M binding to damaged cells, the binding was studied using fluorescence microscopy of the human erythroid cell line K562 (FIG. 1D) and the promyelocytic cell line HL 60 (not shown) induced to apoptosis by addition of heme, and incubated with A1M followed by anti-A1M. As illustrated in the figure, two different types of staining could be seen, a weak granular staining to the cell surface of most cells and a more pronounced, intracellular and uniform staining to a subset (approximately 6%) of the cells. Similar results were obtained with the HL 60 cells (not shown). These results indicate that the strong binding of A1M to apoptotic cells is mainly intracellular, which was confirmed by confocal microscopy (see below; FIG. 3A).

(48) To investigate the specificity of the binding, a competitive cell-binding assay was performed on HCQ.4 cells, induced to apoptosis by CD3 cross-linking and compared to normal untreated cells. .sup.125I-labeled A1M and an excess of unlabeled A1M, ovalbumin, BSA or AGP were added to the cells (FIG. 1E). More A1M was bound to apoptotic cells (FIG. 1E, left) compared to untreated cells (right). Excess of unlabeled A1M blocked the .sup.125I-A1M binding to the same basal level for apoptotic cells as for untreated cells. The reduction was found to be significant (p<0.001). None of the unlabeled control proteins could significantly reduce the binding of .sup.125I-A1M to untreated cells, thus indicating a specific binding of A1M. To the apoptotic cells, there was a small, significant reduction by the control proteins (p<0.05). This small reduction may be due to an increased unspecific background binding to exposed intracellular structures. Accordingly, the results indicate a specific stronger binding of A1M to apoptotic cells. From a Scatchard plot an affinity constant for the A1M binding to apoptotic HCQ.4 cells could be determined to 110.sup.6 M.sup.1. The viability of these cells was 25% according to trypan blue exclusion (not shown).

(49) As mentioned above, the A1M-binding cells internalized PI (FIG. 1B). This indicates that the A1M-binding occurred late in the apoptotic process after the cell membranes had started to leak. To confirm this result, time studies on the binding of A1M to HCQ.4 cells, induced to apoptosis by anti-CD3 cross-linking, were performed. Flow cytometry of samples taken at various time-points after induction shows that the PI uptake precedes the binding of A1M (FIG. 2A). The clear binding correlation was not seen to cells negative for PI uptake. The same result was obtained when the murine pre-B-cells were triple-stained with A1M, annexin V (marker for apoptosis) and 7AAD (marker dye for cell membrane permeability) (FIG. 2B). Only 7AAD positive cells showed a strong A1M binding, whereas cells positive for annexin V, but not for 7AAD, did not bind A1M.

(50) Identification of Intracellular A1M-Binding Proteins

(51) To search for cellular proteins interacting with A1M, the yeast 2-hybrid system was used. cDNA coding for A1M was used as a bait to search for A1M-interacting proteins in a human leukocyte library. Approximately 210.sup.6 transformants were analyzed for reporter gene activation. A total of 168 colonies survived on plates lacking histidine and 13 of them were also positive for 13-galactosidase. The His.sup.+LacZ.sup.+ recombinant library plasmids were isolated and tested in direct 2-hybrid assays with bait plasmids encoding only the bait protein as well as the protein fused to unrelated proteins. Eleven recombinant plasmids were shown to encode proteins that interacted with A1M, but not with the bait protein or other unrelated proteins fused to it. DNA sequencing of the inserts revealed that seven of them were a truncated form of the SDAP subunit (NDUFAB1) in mitochondrial Complex I, one was the complete sequence of the same subunit, one was a snRNA binding protein, one was N-acetylglucosamine kinase and one was a colon cancer antigen. All inserts were in frame in the prey vector (Table I).

(52) TABLE-US-00001 TABLE 1 A1M interacting proteins found in the yeast-two hybrid system. No. of Genebank Accession Protein colonies No. Bases No.* NADH dehydrogenase 7 NM_005003 142-670 8 kDa, SDAP subunit (NDUFAB1) NADH dehydrogenase 1 NM_005003 18-670 8 kDa, SDAP subunit (NDUFAB1) U6 snRNA-associated 1 AF182291 14-735 Sm-like protein (LSM5) N-acetylglucosamine 1 AJ242910 7-1187 kinase (NAGK) Serologically defined 1 AK001296 0-1441 colon cancer antigen 3, NY-CO-3 (SDCCAG3) *According to the base numbering of the Genebank Accession No. assigned in this table.

(53) Binding of A1M to Mitochondrial Complex I

(54) The results from the yeast 2-hybrid experiments thus suggest that a subunit of mitochondrial Complex I is a major A1M-binding intracellular protein. Binding to mitochondria, and to Complex I in particular, was therefore investigated in detail using several independent methods: confocal microscopy, EM, subcellular fractionation, and PAGE. Using a mitochondrial fluorescent probe (Mito-Tracker) and confocal microscopy we evaluated the subcellular localization of the intracellular A1M in K562 cells with or without addition of exogenous A1M (FIG. 3A). Analyzing cells without exogenously added A1M a very weak unspecific intracellular staining was observed (not shown). However, with the addition of exogenous A1M an intense, mitochondria-specific staining was observed. The subcellular localization of the bound A1M was also studied by Transmission EM (TEM) using primary human keratinocyte cultures (FIG. 3B-C). TEM of keratinocytes, containing exogenously added A1M and incubated with gold-labeled anti-A1M, showed a highly specific localization of A1M to the mitochondria (FIG. 3C).

(55) Confirmation of mitochondrial binding and verification of specificity was performed using purified mitochondria from mouse liver (FIG. 4A). .sup.125I-labeled A1M was incubated with the mitochondria, with or without an excess of unlabeled A1M or the control protein AGP. Excess of unlabeled A1M blocked the .sup.125I-A1M binding significantly at the two higher concentrations, whereas AGP at the highest concentration had no effect on the binding. Scatchard analysis of the binding data yielded an affinity constant of the binding at 1.210.sup.6 M.sup.1.

(56) To investigate if endogenous A1M is found in mitochondria associated with Complex I, mouse mitochondria were purified without freezing, solubilized, separated under non-denaturing conditions, and analyzed by Western blotting using antibodies against subunits of Complex I and III (denoted NDUFV1 and Core I, respectively) and against mouse A1M (FIG. 4B). The results show that A1M co-migrates with the major Complex I-containing band and a supercomplex-band containing both Complex I and III, whereas no co-migration was seen between A1M and the major Complex III-containing band. Taken together, this support a specific association between A1M and a Complex I subunit. However, a large fraction of A1M migrated at a position corresponding to approximately 350-400 kDa, suggesting that A1M is also associated with other, as yet unidentified, large structures in mitochondria. The blotting intensity of all bands decreased with increasing digitonin concentrations, suggesting that all bands seen in the gels results from non-covalent protein-protein interactions (FIG. 5A). The binding between A1M and Complex I was confirmed by anti-Complex I immunoprecipitation followed by blotting with anti-A1M (FIG. 4C). The results showed that the majority of mitochondria-associated A1M positive bands in the starting material (FIG. 4C, left) were precipitated. Also, a new band, not detectable in the starting material, was seen in the immunoprecipitate. Trypsin digestion of intact mitochondria before SDS-PAGE and blotting with anti-A1M did not decrease the amount of A1M found in the mitochondria, supporting a localization of A1M in the inner mitochondrial membrane (FIG. 5B).

(57) A1M Protects Mitochondrial Structure and Function

(58) Hypothesizing that the physiological role of mitochondrial-bound A1M is to confer protection of this organelle, we first employed TEM to investigate the impact of A1M on the structure of mitochondria in cells exposed to heme and H.sub.2O.sub.2 (FIGS. 6 and 7). TEM was performed on cultured human primary keratinocytes. Extensive destructive effects were seen by heme (FIG. 6B) and H.sub.2O.sub.2 (FIG. 7B), i.e. vast formation of vacuoles, structural des-organization of keratin fibres and swelling of the mitochondria (FIGS. 6B and 7B, zoomed in). These effects were counteracted by the addition of A1M, where a particular impact was seen on the mitochondrial swelling (FIGS. 6C and 7C, zoomed in). The results suggest that A1M protects and preserves cellular structures otherwise damaged and disintegrated.

(59) We next investigated the effects of A1M on mitochondrial function by measuring ATP-production of purified mitochondria exposed to heme or H.sub.2O.sub.2 (FIG. 8). A significant reduction in the rate of ATP-production was seen by 5 and 20 M heme (FIG. 8A). This reduction was reversed by A1M, and no reduction in ATP-production rate was seen by heme in any of the tested concentrations when 10 M A1M was present. Similar results were obtained using H.sub.2O.sub.2 (FIG. 8B). Thus, H.sub.2O.sub.2 significantly reduced the rate of ATP-production, but the effects were significantly reversed in the presence of 10 M A1M.

Example 2. Stress Conditions in Retina Cultures Induce Structural and Functional Damage of Mitochondria, Cellular Antioxidation Response and Cellular A1M Up-Regulation

(60) Methods

(61) Pig retinas were dissected and cultured in Petri dishes under mild and stress conditions in vitro as described for rat retinas (Cederlund M, Ghosh F, Arner K, Andreasson S, kerstrm B. Vitrous levels of oxidative stress biomarkers and the radical scavenger alpha-1-microglobulin/A1M in human rhegmatogenous retinal detachment. Graefe's Arch Clin Exp Ophtalmol (2013) 251: 725-732). After 2 h or 48 h, mRNA was isolated and quantitated, cDNA synthesized by reversed transcription and the amount of specific sequences quantitated by realtime PCR. The obtained amounts of each mRNA species in stressed cultures were normalized to mRNA from the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase and expressed in relation to the normalized genes in non-stressed conditions (Ct).

(62) Results

(63) The expression of mitochondria-specific ribosomal RNA (12S rRNA) was dramatically down regulated in retinas cultured 48h under stress conditions as compared to mild conditions (FIG. 10a), suggesting damage to mitochondrial structure and function. At the same time, the A1M-gene and the two antioxidation genes heme oxygenase 1 (HO1) and superoxide dismutase (SOD) were up regulated in stressed cultures as compared to mild cultures after both 2 h and 48 h (FIG. 10b), suggesting that retina cellular defense mechanisms including A1M are activated.

CONCLUSIONS

(64) Retinal stress during in vitro culture negatively affects retinal mitochondrial structure and function and upregulates antioxidation defense and A1M-expression. These results support a role of A1M in mitochondrial protection during retinal culture.

LIST OF ABBREVIATIONS

(65) Reactive oxygen species ROS

(66) Hemoglobin Hb

(67) Superoxide dismutase SOD

(68) Glutathione peroxidase GPx

(69) .sub.1-microglobulin A1M

(70) Violaxanthin-deepoxidase VDE

(71) Zeaxtanthin epoxidase ZDE

(72) .sub.1-acid glycoprotein AGP

(73) Bovine serum albumin BSA

(74) Dimethyl sulphoxide DMSO

(75) Fluorescein isothiocyanate-conjugated GAM-FITC goat anti-mouse immunoglobulin

(76) Phycoerythrein-conjugated streptavidin SAPE

(77) 7-amino actinomycin D 7 AAD

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