- Email: [email protected]
P4-190 Translational regulation on BACE expression
Poster Session P4: Molecular Mechanisms of Neurodegeneration - Enzyme Activities Proteasomes are multi-subunit complexes that selectively degrade intracellular proteins. Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitylated proteins by the proteasome. The ubiquitin-proteasome pathway has been implicated in the pathogenesis of neurodegenerative disorders. In this study, we found that treatment with proteasome inhibitors resulted in an increase in APP C99 levels, suggesting that APP processing at the 13-secretase site is affected by the Ubiquitin-Proteasome pathway. To investigate if the degradation of BACE is mediated by the proteasome pathway, cells stably transfected with BACE were treated with lactacystin. We found that BACE protein degradation was inhibited by lactacystin in a time- and dose-dependent manner. Non-proteasome protease inhibitors have no effect on BACE degradation. Furthermore, lactacystin increases APP C99 production and A[3 generation. Our data indicates that BACE protein and APP processing is regulated by the proteasome pathway.
V•fi•
IDENTIFICATION OF A NOVEL TRANSLATIONAL
ISOFORM OF INSULIN-DEGRADING ENZYME
Malcolm Leissring*, Wesley Farris, Xining Wu, Dennis J. Selkoe. Brigham
& Women's Hospital, Boston, MA, USA. Contact e-mail: mleissring @rics. bwh. harvard.edu
Background: We showed previously that Alzheimer-type pathology in APP transgenic mice can be reduced > 50% by a mere 1-fold increase in the expression of insulin-degrading enzyme (IDE) in neurons. Because only 12% of all IDE protein is believed to be localized to subcellular compartments in which A[3 is present, subtle alterations in the subcellular localization of IDE could effect large changes in the economy of brain A~. IDE mRNA contains an in-frame initiation codon 123 nucleotides upstream of the canonical translation start site, raising the possibility that an IDE isoform translated from this upstream initiation codon might represent a species that interacts with A[3. Objectives: To determine whether IDE translated from the upstream initiation codon might be localized to compartments relevant to A[~ degradation. Methods: Stable cell lines expressing IDE constructs beginning obligatorily either at the first or the second ATG were generated and fractionated by differential centrifugation. Corresponding IDE-EGFP fusion constructs were transiently expressed in CHO cells and visualized by confocal microscopy together with fluorescent subcellular organelle markers. Western blots were performed to detect endogenous IDE in mitochondria purified from HEK-293 cells or rat liver by differential centrifugation and/or flotation through a discontinuous iodixanol gradient. Results: IDE protein translated beginning at the upstream ATG (MetlIDE) contained an N-terminal sorting sequence that is predicted to be a mitochondrial presequence. Fusion of this N-terminal sequence to enhanced green fluorescent protein (EGFP) targeted this normally cytosolic protein exclusively to mitochondria. Full-length Metl-IDE consmlcts also were directed to mitochondiia-enriched fractions in stable CHO cell lines. Endogenous IDE protein was detected in mitochondria purified by various methods, where it was protected from digestion by trypsin and migrated at a size consistent with removal of the N-terminal targeting sequence upon transport into the mitochondrion. Conclusions: Our results identify a novel translational isoform of IDE present within mitochondria.
•
T R A N S L A T I O N A L R E G U L A T I O N O F BACE
EXPRESSION Sven Lammich*, Susanne Sch6bel, Ann-Katrin Zimmer, Stefan F. Lichtenthaler, Christian Haass. Ludwig Maximilians Universitiit,
Miinchen, Germany. Contact e-mail: slammich @reed.uni-muenchen.de
Background: The secretase BACE1 was identified as an amyloid precursor protein (APP) cleaving enzyme, which catalyzes the first step in the formation of the A[3 peptide that is deposited in the brain of Alzheimer's disease patients. BACE1 is mainly expressed in brain and pancreas and at lower levels in most other tissues. Its expression is up-regulated in Alzheimer's disease by so far unknown mechanisms. The 5 r untranslated region (YUTR) of BACE1 belongs to the class of the long 5'UTRs similar
$529
to many regulatory genes. It is 446 bp long, has a GC-content of 77% and contains 3 upstream open reading frames (uORFs). Furthermore the 51UTR is highly conserved in humans, mice and rats. Objective(s): Since the expression of genes with such long 51UTRs may be regulated by translational control, we analyzed whether BACE1 expression is translationally controlled by its 5rUTR. Methods: Several cell lines including HEK293, COS7 and H4 cells were transiently transfected with plasmids coding for BACE without YUTR, 51UTR-BACE or BACE variants with a truncated or mutated 5'UTR. Protein expression levels were compared to mRNA levels. Results: We demonstrate that the 5'UTR of BACE1 regulates the rate of BACE1 translation. In the presence of the 5'UTR we observed more than 90% reduction of BACE1 protein levels in HEK293, COS7 and H4 cells and a similar reduction of BACE1 activity in vitro, mRNA levels were not affected, demonstrating that the 5'UTR repressed the translation but not the transcription of BACE1. The 3/UTR did not affect BACE1 expression. An extensive mutagenesis analysis predicts that a GC-rich sequence close to the BACEI start codon forms a stable stem-loop structure, which may prevent the ribosome from efficiently translating the BACEI mRNA. Conclusions: The YUTR of the BACE mRNA acts as a strong suppressor of BACE1 translation and consequently BACE1 activity.
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D I F F E R E N T I A L E X P R E S S I O N O F CHOLESTEROL
24-HYDROXYLASE AND CHOLESTEROL 27-HYDROXYLASE IN ALZHEIMER'S DISEASE
Benjamin Wolozin .1 , James Brown 1, Simone Silberman 1, Catherine Theisler 1, Debra Yager 1, Janet Crawley 2, Debra Magnuson I , Allison Reiss 3 , Jack M. Lee 1, Christopher Eckman 4. 1Loyola University
Medical Center, Maywood, IL, USA; 2Washington University in Sr. Louis, St. Louis, MO, USA; 3New York University, New York, NY, USA; 4Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected]
Background: Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (Cyp46) and cholesterol 27-hydroxylase (Cyp27). Recent studies show an association between the Cyp46 and Alzheimer's disease. The expression of Cyp46 and Cyp27 in Alzheimer's and control brain has not been investigated. In addition, although prior studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols has yet to be examined. Objective(s): To understand the role of cholesterol oxidation in AD, we compared the actions of 24 and 27 hydroxycholesterol on the processing of APP and analyzed the distribution of the two cholesterol hydroxylases. Methods: The distribution of Cyp46 and Cyp27 was examined by immunohistochemistry and immunoblotting of Alzheimer and control brain. The regulation of APP processing was examined by immunoblot and ELISA in primary neurons, astrocytes and CHO cells. Results: Immunocytochemical studies show that Cyp46 and Cyp27 are expressed in neurons and some astrocytes in the normal brain; in addition, Cyp27 is present in oligodendrocytes. In AD, Cyp46 shows prominent expression in astrocytes and around amyloid plaques. In contrast, in AD Cyp27 expression decreases in neurons, is not apparent around amyloid plaques but increases in oligodendrocytes. Both oxysterols inhibit production of A~ in neurons to a much greater extent than in CHO cells or astrocytes, even after supplementation with retinoic acid. In addition, 24 hydroxycholesterol is approximately 1000-fold more potent than 27 hydroxycholesterol, with the IC50 of 24 hydroxycholesterol for inhibiting A[3 secretion being about 1 nM. Oxysterols also selectively inhibited both protein kinase C activity and APP secretion following stimulation of protein kinase C in neurons but not in astrocytes or CHO cells. Conclusions: The selective expression of Cyp46 around neuritic plaques and potent inhibition of APP processing in neurons by 24 hydroxycholesterol points to a potentially important role for this enzyme in the pathophysiology of AD.
V•fi•
IDENTIFICATION OF A NOVEL TRANSLATIONAL
ISOFORM OF INSULIN-DEGRADING ENZYME
Malcolm Leissring*, Wesley Farris, Xining Wu, Dennis J. Selkoe. Brigham
& Women's Hospital, Boston, MA, USA. Contact e-mail: mleissring @rics. bwh. harvard.edu
Background: We showed previously that Alzheimer-type pathology in APP transgenic mice can be reduced > 50% by a mere 1-fold increase in the expression of insulin-degrading enzyme (IDE) in neurons. Because only 12% of all IDE protein is believed to be localized to subcellular compartments in which A[3 is present, subtle alterations in the subcellular localization of IDE could effect large changes in the economy of brain A~. IDE mRNA contains an in-frame initiation codon 123 nucleotides upstream of the canonical translation start site, raising the possibility that an IDE isoform translated from this upstream initiation codon might represent a species that interacts with A[3. Objectives: To determine whether IDE translated from the upstream initiation codon might be localized to compartments relevant to A[~ degradation. Methods: Stable cell lines expressing IDE constructs beginning obligatorily either at the first or the second ATG were generated and fractionated by differential centrifugation. Corresponding IDE-EGFP fusion constructs were transiently expressed in CHO cells and visualized by confocal microscopy together with fluorescent subcellular organelle markers. Western blots were performed to detect endogenous IDE in mitochondria purified from HEK-293 cells or rat liver by differential centrifugation and/or flotation through a discontinuous iodixanol gradient. Results: IDE protein translated beginning at the upstream ATG (MetlIDE) contained an N-terminal sorting sequence that is predicted to be a mitochondrial presequence. Fusion of this N-terminal sequence to enhanced green fluorescent protein (EGFP) targeted this normally cytosolic protein exclusively to mitochondria. Full-length Metl-IDE consmlcts also were directed to mitochondiia-enriched fractions in stable CHO cell lines. Endogenous IDE protein was detected in mitochondria purified by various methods, where it was protected from digestion by trypsin and migrated at a size consistent with removal of the N-terminal targeting sequence upon transport into the mitochondrion. Conclusions: Our results identify a novel translational isoform of IDE present within mitochondria.
•
T R A N S L A T I O N A L R E G U L A T I O N O F BACE
EXPRESSION Sven Lammich*, Susanne Sch6bel, Ann-Katrin Zimmer, Stefan F. Lichtenthaler, Christian Haass. Ludwig Maximilians Universitiit,
Miinchen, Germany. Contact e-mail: slammich @reed.uni-muenchen.de
Background: The secretase BACE1 was identified as an amyloid precursor protein (APP) cleaving enzyme, which catalyzes the first step in the formation of the A[3 peptide that is deposited in the brain of Alzheimer's disease patients. BACE1 is mainly expressed in brain and pancreas and at lower levels in most other tissues. Its expression is up-regulated in Alzheimer's disease by so far unknown mechanisms. The 5 r untranslated region (YUTR) of BACE1 belongs to the class of the long 5'UTRs similar
$529
to many regulatory genes. It is 446 bp long, has a GC-content of 77% and contains 3 upstream open reading frames (uORFs). Furthermore the 51UTR is highly conserved in humans, mice and rats. Objective(s): Since the expression of genes with such long 51UTRs may be regulated by translational control, we analyzed whether BACE1 expression is translationally controlled by its 5rUTR. Methods: Several cell lines including HEK293, COS7 and H4 cells were transiently transfected with plasmids coding for BACE without YUTR, 51UTR-BACE or BACE variants with a truncated or mutated 5'UTR. Protein expression levels were compared to mRNA levels. Results: We demonstrate that the 5'UTR of BACE1 regulates the rate of BACE1 translation. In the presence of the 5'UTR we observed more than 90% reduction of BACE1 protein levels in HEK293, COS7 and H4 cells and a similar reduction of BACE1 activity in vitro, mRNA levels were not affected, demonstrating that the 5'UTR repressed the translation but not the transcription of BACE1. The 3/UTR did not affect BACE1 expression. An extensive mutagenesis analysis predicts that a GC-rich sequence close to the BACEI start codon forms a stable stem-loop structure, which may prevent the ribosome from efficiently translating the BACEI mRNA. Conclusions: The YUTR of the BACE mRNA acts as a strong suppressor of BACE1 translation and consequently BACE1 activity.
•i•
D I F F E R E N T I A L E X P R E S S I O N O F CHOLESTEROL
24-HYDROXYLASE AND CHOLESTEROL 27-HYDROXYLASE IN ALZHEIMER'S DISEASE
Benjamin Wolozin .1 , James Brown 1, Simone Silberman 1, Catherine Theisler 1, Debra Yager 1, Janet Crawley 2, Debra Magnuson I , Allison Reiss 3 , Jack M. Lee 1, Christopher Eckman 4. 1Loyola University
Medical Center, Maywood, IL, USA; 2Washington University in Sr. Louis, St. Louis, MO, USA; 3New York University, New York, NY, USA; 4Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected]
Background: Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (Cyp46) and cholesterol 27-hydroxylase (Cyp27). Recent studies show an association between the Cyp46 and Alzheimer's disease. The expression of Cyp46 and Cyp27 in Alzheimer's and control brain has not been investigated. In addition, although prior studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols has yet to be examined. Objective(s): To understand the role of cholesterol oxidation in AD, we compared the actions of 24 and 27 hydroxycholesterol on the processing of APP and analyzed the distribution of the two cholesterol hydroxylases. Methods: The distribution of Cyp46 and Cyp27 was examined by immunohistochemistry and immunoblotting of Alzheimer and control brain. The regulation of APP processing was examined by immunoblot and ELISA in primary neurons, astrocytes and CHO cells. Results: Immunocytochemical studies show that Cyp46 and Cyp27 are expressed in neurons and some astrocytes in the normal brain; in addition, Cyp27 is present in oligodendrocytes. In AD, Cyp46 shows prominent expression in astrocytes and around amyloid plaques. In contrast, in AD Cyp27 expression decreases in neurons, is not apparent around amyloid plaques but increases in oligodendrocytes. Both oxysterols inhibit production of A~ in neurons to a much greater extent than in CHO cells or astrocytes, even after supplementation with retinoic acid. In addition, 24 hydroxycholesterol is approximately 1000-fold more potent than 27 hydroxycholesterol, with the IC50 of 24 hydroxycholesterol for inhibiting A[3 secretion being about 1 nM. Oxysterols also selectively inhibited both protein kinase C activity and APP secretion following stimulation of protein kinase C in neurons but not in astrocytes or CHO cells. Conclusions: The selective expression of Cyp46 around neuritic plaques and potent inhibition of APP processing in neurons by 24 hydroxycholesterol points to a potentially important role for this enzyme in the pathophysiology of AD.