| pmid | 11007892 | ||||||||
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| title | The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress. | ||||||||
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| journal | J Neurosci | ||||||||
| year | 2000 | ||||||||
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| full_text_extraction_method | html | ||||||||
| pmcid | PMC6772756 | ||||||||
| doi | 10.1523/JNEUROSCI.20-19-07345.2000 |
The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress.
Authors: Greeve I, Hermans-Borgmeyer I, Brellinger C, Kasper D, Gomez-Isla T, Behl C, Levkau B, Nitsch RM Journal: J Neurosci (2000) DOI: 10.1523/JNEUROSCI.20-19-07345.2000 PMC: PMC6772756
- J Neurosci. 2000 Oct 1;20(19):7345-52. doi: 10.1523/JNEUROSCI.20-19-07345.2000.
The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress.
Greeve I(1), Hermans-Borgmeyer I, Brellinger C, Kasper D, Gomez-Isla T, Behl C, Levkau B, Nitsch RM.
Author information: (1)Center for Molecular Neurobiology Hamburg, University of Hamburg, 20246 Hamburg, Germany. isabell.greeve@zmnh.uni-hamburg.de
In Alzheimer's disease (AD) brains, selected populations of neurons degenerate heavily, whereas others are frequently spared from degeneration. To address the cellular basis for this selective vulnerability of neurons in distinct brain regions, we compared gene expression between the severely affected inferior temporal lobes and the mostly unaffected fronto-parietal cortices by using an mRNA differential display. We identified seladin-1, a novel gene, which was downregulated in large pyramidal neurons in vulnerable regions in AD but not control brains. Seladin-1 is a human homolog of the DIMINUTO/DWARF1 gene described in plants and Caenorhabditis elegans. Its sequence shares similarities with flavin-adenin-dinucleotide (FAD)-dependent oxidoreductases. In human control brain, seladin-1 was highly expressed in almost all neurons. In PC12 cell clones that were selected for resistance against AD-associated amyloid-beta peptide (Abeta)-induced toxicity, both mRNA and protein levels of seladin-1 were approximately threefold higher as compared with the non-resistant wild-type cells. Functional expression of seladin-1 in human neuroglioma H4 cells resulted in the inhibition of caspase 3 activation after either Abeta-mediated toxicity or oxidative stress and protected the cells from apoptotic cell death. In apoptotic cells, however, endogenous seladin-1 was cleaved to a 40 kDa derivative in a caspase-dependent manner. These results establish that seladin-1 is an important factor for the protection of cells against Abeta toxicity and oxidative stress, and they suggest that seladin-1 may be involved in the regulation of cell survival and death. Decreased expression of seladin-1 in specific neurons may be a cause for selective vulnerability in AD.
DOI: 10.1523/JNEUROSCI.20-19-07345.2000 PMCID: PMC6772756 PMID: 11007892 [Indexed for MEDLINE]
Abstract
In Alzheimer's disease (AD) brains, selected populations of neurons degenerate heavily, whereas others are frequently spared from degeneration. To address the cellular basis for this selective vulnerability of neurons in distinct brain regions, we compared gene expression between the severely affected inferior temporal lobes and the mostly unaffected fronto-parietal cortices by using an mRNA differential display. We identified seladin-1, a novel gene, which was downregulated in large pyramidal neurons in vulnerable regions in AD but not control brains. Seladin-1 is a human homolog of the DIMINUTO/DWARF1 gene described in plants and Caenorhabditis elegans . Its sequence shares similarities with flavin-adenin-dinucleotide (FAD)-dependent oxidoreductases. In human control brain, seladin-1 was highly expressed in almost all neurons. In PC12 cell clones that were selected for resistance against AD-associated amyloid-β peptide (Aβ)-induced toxicity, both mRNA and protein levels of seladin-1 were approximately threefold higher as compared with the non-resistant wild-type cells. Functional expression of seladin-1 in human neuroglioma H4 cells resulted in the inhibition of caspase 3 activation after either Aβ-mediated toxicity or oxidative stress and protected the cells from apoptotic cell death. In apoptotic cells, however, endogenous seladin-1 was cleaved to a 40 kDa derivative in a caspase-dependent manner. These results establish that seladin-1 is an important factor for the protection of cells against Aβ toxicity and oxidative stress, and they suggest that seladin-1 may be involved in the regulation of cell survival and death. Decreased expression of seladin-1 in specific neurons may be a cause for selective vulnerability in AD.
MATERIALS AND METHODS
Brain tissues. The following AD brains were obtained from the Alzheimer's disease tissue resource center at the Massachusetts General Hospital: brain A93-80 (postmortem time interval 3 hr, male, 87 years; regions: sensorimotor cortex, inferior temporal cortex, and hippocampus), brain A95-34 (postmortem time interval 4 hr, female, 92 years; regions: frontal cortex, inferior temporal cortex, and hippocampus), brain A95-267 (postmortem time interval 3 hr, female, 65 years; regions: frontal cortex and inferior temporal cortex), and brain A45-271 (postmortem time interval 4 hr, female, 63 years; regions: frontal cortex and inferior temporal cortex). The following brains were obtained from the Kathleen Price Bryan brain bank from Duke University (Durham, NC): brain 803 (Braak IV, postmortem time interval 3:30 hr, male, 72 years; regions: frontal cortex and inferior temporal cortex, midfrontal cortex and superior temporal cortex), brain 765 (Braak V, postmortem time interval 1:30 hr, male, 62 years; regions: frontal cortex and inferior temporal cortex, midfrontal cortex and superior temporal cortex), brain 673 (normal control, postmortem time interval 1:10 hr, female, 80 years; regions: frontal cortex and inferior temporal cortex), brain A95-006 (normal control, postmortem time interval 2:35 hr, female, 92 years; regions: midfrontal cortex and superior temporal cortex), brain A98-046 (normal control, postmortem time interval 2 hr, male, 83 years; regions: midfrontal cortex and superior temporal cortex), brain A96-263 (normal control, postmortem time interval 2:22 hr, female, 78 years; regions: midfrontal cortex and superior temporal cortex).
Differential display and reverse Northern analysis. The differential display (DD) screen was done exactly as described ( von der Kammer et al., 1998 , 1999 ). Reverse Northern blotting was performed as described ( Van Gelder et al., 1990 ; Poirier et al., 1997 ). Briefly, 2 μg of total RNA from each brain region were reverse-transcribed using the Superscript Plasmid System for cDNA Synthesis (Life Technologies, Karlsruhe, Germany). RNA amplification was performed with the whole cDNA reaction using the Ampliscribe T7 Transcription kit (Epicenter, Biozym, Hessisch Oldendorf, Germany). [ 32 P]-labeled cDNA probes were made with the amplified RNA using Superscript Preamplification System (Life Technologies) and 0.5 mCi per reaction of [α 32 P]-dCTP (3000 Ci/mmol) (Amersham Pharmacia, Freiburg, Germany). The cloned cDNAs identified in the DD screen were spotted identically on two nylon membranes (Schleicher und Schüll, Dassel, Germany) and were hybridized with radiolabeled cDNA (10 7 cpm/ml) from either frontal or temporal cortex of AD brains. Blots were analyzed by autoradiography, and signal intensities relative to β-actin were determined by densitometry.
Cloning and sequencing of seladin-1. The full-length cDNA for seladin-1 was isolated from a human brain library provided by the Resource Center Primary Database (RZPD) of the Human Genome Project. The cDNA sequence was confirmed by automated DNA sequence analysis using an ABI 377 sequencer.
Northern blot analysis. RNA from brain tissues or cells was extracted with Trizol (Life Technologies), separated on 0.8% formaldehyde-agarose gels, and blotted on Hybond-N+ nylon membranes (Amersham Pharmacia). Northern blots were hybridized with [ 32 P]-labeled cDNA of seladin-1 (nt 1–3505) and [ 32 P]-labeled cDNA of human β -actin as control (Clontech, Heidelberg, Germany) in ExpressHyb solution (Clontech) at 68°C. Northern blot filters of distinct brain regions and peripheral tissues were obtained from Clontech.
In situ hybridization. In situ hybridization of human brain sections was performed as described ( Hartmann et al., 1995 ; Susens et al., 1997 ). The following tissue specimens were used: 765; 803; A93-80; A95-34; A96-263; A95-006; and A98-046.
Fragments of 650 and 900 bp of the seladin-1 open reading frame (ORF) were amplified by PCR using the following primer pairs: 1s (nt 76–99: GCG CTT ACC GCG CGG CGC CGC ACC), 1as (nt 749–726: GAC CAG GGT ACG GCA TAG AAC AGG) and 3s (nt 803–826: AGA AGT ACG TCA AGC TGC GTT TCG), 3as (nt: 1749–1726: TTC TCT TTG AAA GTG TGG ATC TAG). PCR products were cloned into pGEM-Teasy (Promega, Heidelberg, Germany), excised with Eco RI, and cloned into pBluescript KS+. [ 35 S]-UTP-labeled antisense and sense riboprobes were generated on Not I and Cla I linearized plasmids with T3 and T7-Polymerase by using the Ambion Maxiscript kit (Ambion, AMS Biotechnology, Wiesbaden, Germany). Hybridized sections were dipped in NTB-2 photographic emulsion (Kodak, Stuttgart, Germany), exposed for 4 weeks, and counterstained with Giemsa. All brain sections were hybridized with identical antisense or sense probes, respectively and processed in parallel. For quantification of neuronal grain density, neuronal grains were automatically counted in three fields from temporal and frontal cortices of three AD and three normal control brains, respectively, that contained on average identical amounts of neurons (∼50 per field). The fields were chosen by an investigator blinded to diagnosis and brain region. Statistically significant differences were calculated by applying Student's t test.
Cell culture and stable cell lines. H4 cells were cultured in DMEM medium (Life Technologies), supplemented with 10% calf serum, or in OptiMEM medium (Life Technologies), supplemented with 2 m m CaCl 2 . PC12 cells were cultured in DMEM medium, supplemented with 10% calf serum, 5% horse serum, 1% sodium pyruvate, and 1% penicillin–streptomycin (Life Technologies). Human umbilical vein endothelial cells (HUVECs) were cultured in RPMI 1640 medium (Life Technologies), supplemented with 15% calf serum, 3% endothelial cell growth supplement, and 50 μg/ml heparin. The seladin-1 open reading frame was amplified by PCR using the primers sel- Nhe (CCT AGC TAG CGG GCC AGG CGC GGA GCT GGC GGC) and sel- Kpn (GCG GTA CCG TGT GCC TGG CGG CCT TGC AGA TCT TGT C). The sequence was confirmed by DNA sequence analysis. The seladin-1 ORF was cloned at the Nhe / Kpn I site of pEGFP-N1 (Clontech). H4 cells were stably transfected with the empty vector pEGFP-N1 or with the seladin-1-enhanced green fluorescence protein (EGFP) fusion construct using DOTAP transfection reagent (Roche, Mannheim, Germany). Stably expressing cells were selected under 500 μg/ml G418 (Roche) and cloned. Expression of EGFP or the fusion protein was confirmed in several selected clones by fluorescence microscopy.
Immunfluorescence. H4 human neuroglioma cells that stably express the seladin-1-EGFP fusion construct were grown on coverslips and fixed with 4% paraformaldehyde in PBS or treated for 45 min with 250 n m of the red fluorescent mitochondrial stain MitoTracker red CM-H 2 Xros (Molecular Probes, MoBiTec, Göttingen, Germany) or with 500 n m M of the red fluorescence Golgi marker Bodipy TR ceramide (Molecular Probes, MoBiTec) before fixation. The cells that were not prestained with the MitoTracker or Bodipy probe were fixed, permeabilized in 0.2% Triton X-100 in PBS, blocked overnight at 4°C in 5% low-fat milk, 0.1% Triton X-100 in PBS, and incubated for 2 hr at room temperature with a monoclonal antibody against mouse disulfide isomerase (anti-PDImAb; StressGen Biotechnologies Corp., Biomol, Hamburg, Germany), a marker for the endoplasmic reticulum. After washing, the cells were incubated for 1 hr with an anti-mouse IgG, CY3-labeled secondary antibody (Amersham Pharmacia). Subsequently, cells were visualized by confocal laser scanning microscopy.
Subcellular fractionation and enzyme assays. Subcellular organelle fractionation and enzyme assays were performed as described ( Graham et al., 1994 ). Briefly, 10 confluent tissue culture plates of H4 cells were washed three times with ice-cold PBS and incubated for 15 min in 0.2 m sucrose. The cells were homogenized with 25 strokes of a Kontes homogenizer in medium A [8% (w/v) sucrose, 20 m m Tricine-NaOH, pH 7.4, 1 m m EDTA]. The homogenate was centrifuged at 1000 × g for 10 min, and the supernatant was recentrifuged at 17,000 × g for 15 min. The 17,000 × g pellet (P2) was resuspended with a Dounce homogenizer in 1 ml of medium A and subjected to an Iodixanol gradient (Optiprep, Nycomed Pharma, Oslo, Norway). To generate the Iodixanol gradients, 5 vol of Optiprep solution were diluted to a working solution of 50% by the addition of 1 vol of 8% (w/v) sucrose, 120 m m Tricine-NaOH, pH 7.4, 6 m m EDTA. The working solution was diluted to 10 and 30% gradient medium by addition of medium A. Three milliliters of P2 in medium A adjusted to 35% Iodixanol by addition of 50% working solution were layered under the gradient consisting of 5 ml of high-density medium (30%) followed by 5 ml of low-density medium (10%). The gradient was centrifuged at 52,000 × g av for 1.5 hr using a Beckman SW60 rotor in a Beckman L7–55 ultracentrifuge. The gradients were fractionated into 26 fractions of 0.5 ml each by upward displacement using a gradient unloader (Nycomed Pharma, Oslo, Norway). All procedures were performed at 4°C. Each gradient fraction was directly assayed for mitochondrial succinate-dehydrogenase, and for endoplasmic reticulum NADPH-cytochrome c reductase activities exactly as described by Nycomed Pharma. Proteins of each fraction were separated by 10% SDS-PAGE and subjected to immunoblotting. Briefly, the Immobilon membranes (Millipore GmbH, Eschborn, Germany) were blocked in 5% low-fat milk for 4 hr at 4°C and incubated overnight at 4°C with a rabbit polyclonal peptide-specific seladin-1 antiserum (1:1000 diluted) or with a mouse monoclonal anti-Golgi 58K antibody (1:1000 diluted) (Sigma Aldrich, Munich, Germany). Peroxidase-coupled goat anti-rabbit (Vector, Alexis Biochemicals, Grünberg, Germany) or goat anti-mouse antibodies (Amersham Pharmacia) were used as secondary antibodies. Immunoblots were visualized by enhanced chemiluminescence (Amersham Pharmacia).
Flow cytometry. Ten and 16 hr after incubation of three seladin-1-EGFP clones (A6, B1, B5) and three EGFP-control clones (B5, B6, C1) in OptiMEM medium containing 200 μ m H 2 O 2 , the cells remaining attached to the culture dish as well as the cells in the supernatant were harvested and stained with 7-amino-actinomycin D (ViaProbe, PharMingen, Becton Dickinson GmbH, Heidelberg, Germany) to distinguish viable from dead cells. Only membranes of dead and damaged cells are permeable to this DNA dye and stain positive. Live/dead counts were performed by using a FACSCalibur (Becton Dickinson) fluorescence-activated cell sorter. Cells (10 5 per clone) were counted in three independent experiments. Statistically significant differences were determined by using Student's t test.
Trypan blue and Hoechst dye staining. Three seladin-1-EGFP clones (A6, B1, B5) and three EGFP-control clones (B5, B6, C1) were grown to 50% confluence on coverslips in 24-well plates and treated either for 20 hr with 200 μ m H 2 O 2 or for 18 hr with 10 μ m Aβ 25–35 using OptiMEM, 2 m m CaCl 2 . Cells on one plate were stained with trypan blue. The trypan blue-positive and -negative cells were counted in five 20× fields of each well, and the percentage of trypan blue-positive cells for each clone was calculated. Cells on the second plate were fixed in 4% paraformaldehyde and stained with 1 μg/ml Hoechst dye 33342 (Molecular Probes, MoBiTec) after permeabilization of cell membranes with 0.1% Triton X-100. Hoechst-stained cells were visualized under epifluorescence illumination (350 nm excitation, 461 nm emission) with a 40× oil immersion objective. Two hundred cells were counted per well, and the percentage for apoptotic cells with condensed and fragmented nuclei in each well was determined. Statistically significant differences were determined by using Student's t test.
Caspase 3 activity. Caspase 3 activity was measured in cell lysates of three seladin-1-EGFP clones (A6, B1, B5) and three EGFP-control clones (B5, B6, C1) plated to identical densities by using the caspase 3 assay kit (PharMingen, Becton Dickinson GmbH). After exposure to 200 μ m H 2 O 2 for 2 and 4 hr or to 25 μ m Aβ 25–35 for 4 hr, cells were washed briefly in PBS and lysed in 100 μl 10 m m Tris-HCl, pH 7.5, 10 m m NaH 2 PO 4 /NaHPO 4 , pH 7.5, 130 m m NaCl, 1% Triton-X-100, 10 n m NaPPi. Lysates (100 μg of protein) were incubated in 200 μl HEPES buffer for 1 hr at 37°C with 5 μg of the caspase 3 fluorogenic substrate Ac-DEVD-AMC or with 5 μg Ac-DEVD-AMC in the presence of 0.5 μg of the caspase 3 aldehyde inhibitor Ac-DEVD-CHO in a 96-multiwell plate. AMC liberated from Ac-DEVD by caspase cleavage was measured on a spectrofluorometer (Spectramax Gemini, Molecular Devices, Munich, Germany) at excitation wavelength of 380 nm and an emission wavelength spectrum from 420 to 460 nm. The means (±SEM) of caspase 3 activity of three independent experiments, each with three seladin-1-EGFP and three EGFP-control clones, are given in relative fluorescence units (RFUs). Statistically significant differences were calculated applying Student's t test.
Generation of seladin-1 antisera. For immunizations of rabbits, a peptide consisting of amino acid residues 203–218 of seladin-1 was synthesized and coupled to keyhole limpet hemocyanin at the C terminus: H2N-TPS ENS DLF YAV PWS C-CONH2 (Eurogentec, Seraing, Belgium). The rabbit immune serum was purified on HiTrap affinity columns (Amersham Pharmacia) to which the peptide was coupled according to the instructions of the manufacturer.
Protein analysis. For Western blotting, human brain tissues 673, 765, and 803 were used. Brain tissues were homogenized with mortar and pestle in liquid nitrogen and subsequently with a Dounce homogenizer in 20% glycerol, 2% SDS, 125 m m NaCl, 0.075 m DTT, 1% Triton X-100. Protein concentrations were measured with a Bradford assay, and 25 μg of protein of each brain extract were separated by 10% SDS-PAGE. Proteins were transferred to Immobilon membranes (Millipore GmbH) that were subsequently blocked in 5% low-fat milk overnight at 4°C and incubated overnight at 4°C with a 1:200 dilution of the seladin-1-specific, affinity-purified antiserum, followed by 1 hr incubation with 1:30,000 of a goat anti-rabbit peroxidase-conjugated secondary antibody at room temperature (Vector, Alexis Biochemicals). To control for equal loading, blots were reprobed with a rabbit polyclonal anti-actin antibody (Sigma Aldrich). Immunoblots were visualized by enhanced chemiluminescence (Amersham Pharmacia). PC12 cells were extracted in 10 m m Tris/HCl, 10 m m NaH 2 PO 4 /NaHPO 4 , pH 7.5, 130 m m NaCl, 1% Triton X-100, 10 m m sodium pyrophosphate. Twenty micrograms of protein of the extracts were separated by 10% SDS-PAGE, transferred to Immobilon membrane (Millipore GmbH), and incubated with the seladin-1-specific antibody as described above. HUVEC cells were lysed in 50 m m Tris/HCl, pH 7.4, 250 m m NaCl, 0.5% NP40, 10% glycerol, 5 m m EDTA, 50 m m NaF, 0.5 m m Na 3 VO 4 , 10 m m glycerophosphate, 0.5 m m PMSF, 5 mg/ml leupeptin and aprotinin, and were subjected to 10% SDS-PAGE. Films were scanned and analyzed by using the NIH image software.
Apoptosis in HUVECs. Confluent HUVECs were deprived of growth factors for 12 hr. Cell lysates of control cells and surviving viable and apoptotic floaters were immunoblotted and analyzed with an antigen affinity-purified antibody against seladin-1. In addition, confluent HUVECs were deprived of growth factors for 16 hr in the absence or the presence of the cell-permeable caspase inhibitor ZVAD. Cell lysates including attached and floating cells were immunoblotted and analyzed with the seladin-1 antibody.
In vitro translation. The complete ORF of seladin-1 was amplified by PCR using the following primers: sel- Bam Hs (CGG GAT CCA TGG AGC CCG CCG TGT CGC TGG CC) and sel- Xho as (CCG CTC GAG CTC AGT GCC TGG CGG CCT TGC AG), and cloned in the Bam HI/ Xho I site of pcDNA3.0 (Invitrogen, Groningen, Netherlands). The sequence of the seladin-1 ORF was confirmed by DNA sequencing. In vitro transcription and translation of seladin-1 was performed in the presence of [ 35 S]-methionine (1000 Ci/mmol; Amersham Pharmacia) by using the TNT-coupled reticulocyte lysate system (Promega). Two microliters of the translation reaction (25 μl) were incubated at 37°C for 1 hr or 2.5 hr with 250 ng of recombinant caspases 3, 6, and 7 (provided by K. Orth and V. Dixit, University of Michigan, Ann Arbor, MI) in a total volume of 10 μl in 10 m m HEPES/KOH, pH 7.4, 2 m m EDTA, 5 m m DTT, 1% NP40, 5 μg/ml leupeptin and aprotinin, or with reaction buffer alone (control). The samples were separated on a 10% SDS-PAGE, and full-length seladin-1 as well as the resulting seladin-1 fragments were visualized by autoradiography.
DISCUSSION
In this study, we identified the novel gene seladin-1 that shares domain homologies with a gene family of FAD-dependent oxidoreductases. Seladin-1 was highly expressed in neuronal cells throughout mammalian brains, and its expression was low in neurons within selectively vulnerable regions of AD brains. Reduced levels of seladin-1 mRNA in affected areas of AD brains was related to reduced amounts of seladin-1 mRNA within remaining neurons and did not simply reflect neuronal cell loss. Reduced brain tissue levels of seladin-1 mRNA were paralleled by reduced levels of seladin-1 protein in affected regions. Reduced expression of seladin-1 in vulnerable brain regions in AD is in line with results of previous studies on antioxidant enzyme activities in AD brains that found lower activities of catalase and superoxide dismutase in temporal cortices from AD brains as compared with temporal cortices from non-demented normal controls ( Marcus et al., 1998 ). Differential activity of antioxidant enzymes in nerve cell populations may be one important cause for the selective resistance of specific cells against degeneration on toxic factors such as Aβ, which is distributed throughout the brain in both vulnerable and protected regions. In cell culture, overexpression of seladin-1 protected cells from apoptosis induced not only by oxidative stress but also by Aβ, and high expression of endogenous seladin-1 was associated with resistance against Aβ-induced toxicity. Resistance of cultured cells to Aβ toxicity was previously found to be attributable to the transcriptional activation of antioxidant enzymes, including glutathione peroxidase or catalase ( Behl et al., 1994 ; Sagara et al., 1996 , 1998 ). Our data extend that proposal by adding seladin-1, which may function in concert with these enzymes in protecting cells from oxidative stress and Aβ toxicity.
Despite its activity on increasing resistance to apoptosis, seladin-1 itself is apparently cleaved to p40 by apoptosis-related endoproteolytic cleavage at one of two possible motifs at positions 122–125 or 383–386. Our antibody recognizes an epitope in between these two motifs and therefore detects cleavage at either position. Together, our results suggest that seladin-1 is an integral component of the cellular machinery that protects neurons from oxidative stress. Once oxidative stress becomes overwhelming, seladin-1 is cleaved and presumably inactivated because the putative domain for non-covalent FAD binding is located within the caspase cleavage motifs. Dual roles in apoptosis are known for several negative regulators of apoptosis. These antiapoptotic proteins include Bcl 2, ICAD ( Cheng et al., 1997 ; Xue and Horvitz, 1997 ; Adams and Cory, 1998 ; Enari et al., 1998 ), and NfκB ( Levkau et al., 1999 ), all of which are cleaved during apoptosis by caspases and turn into proapoptotic stimuli ( Cheng et al., 1997 ; Xue and Horvitz, 1997 ; Adams and Cory, 1998 ; Enari et al., 1998 ; Levkau et al., 1999 ). It will be interesting to determine whether p40 also has pro-apoptotic functions.
Accumulating data underscore the importance of reactive oxygen species in the pathogenesis of neurodegenerative diseases, including AD ( Beal, 1996 ; Multhaup et al., 1996 ; Browne et al., 1999 ). Our data raise the possibility that seladin-1 protects large pyramidal neurons from Aβ-induced toxicity via a mechanism that involves increased resistance against oxidative stress. Seladin-1 thus may link oxidoreductase activity to apoptosis and neurodegeneration, similar to the recently reported protein ERAB or ABAD, an endoplasmic reticulum-associated amyloid β-peptide binding protein that participates in fatty acid β-oxidation and is known to mediate apoptosis ( He et al., 1998 ; Oppermann et al., 1999 ; Yan et al., 1999 ). In contrast to ERAB, which is mainly localized in mitochondria and at the cytosolic site of the ER, seladin-1 is predominantly localized within the ER, and to a lesser amount in Golgi complexes, suggesting that the ER/intermediate compartment (IC) is a possible site for interaction with Aβ, which is known to be produced in the ER/IC ( Xia et al., 1998 ; Soriano et al., 1999 ; Skovronsky et al., 2000 ). Whether seladin-1 interacts with Aβ in the ER/IC and modifies Aβ toxicity within cells will be addressed in further studies.
Despite the observed potential of seladin-1 for neuroprotection, its precise physiological function has yet to be established. In plants, the seladin-1 homolog “Diminuto/Dwarf1” is required for the synthesis of brassinosteroids, which are important plant sterols essential for normal plant growth and development ( McMorris, 1997 ; Klahre et al., 1998 ; Bishop et al., 1999 ). The diminuto/dwarf1 mutant is defective in synthesizing the early precursor of brassinolide, campesterol ( Klahre et al., 1998 ; Choe et al., 1999 ), leading to the accumulation of 24-methylencholesterol and resulting in severe growth defects in Arabidopsis thaliana ( Klahre et al., 1998 ; Choe et al., 1999 ). Analogous to the function of diminuto/dwarf1, seladin-1 may well be required for FAD-dependent oxidation of a specific metabolic intermediate necessary for cell growth and differentiation.
The initial cause for the loss of seladin-1 expression in selectively vulnerable regions of AD brains remains to be investigated, as well as possible genetic heterogeneity at this locus. Taken together, our results provide evidence for an involvement of seladin-1 in neurodegeneration and offer a novel therapeutic strategy for delaying—or preventing—neurodegeneration in AD and other neurodegenerative diseases.