- Email: [email protected]
ESTROGEN REGULATION OF MITOCHONDRIAL RESPIRATION IS CELL TYPE AND ER SUBTYPE SPECIFIC
Poster Presentations: Monday, July 17, 2017
in maintaining mitochondrial morphology, regulating membrane potential, and mediating apoptosis while directly or indirectly interacting with APP and other AD-associated proteins (PINK1) and small molecules (IL6, IL10).
brain. Additionally, we are able to predict the sensitivity of those relationships to changes in activity brought about, for example, by drugs targeting the appropriate proteins expressed by those genes. P2-150
P2-149
A TESTABLE MATHEMATICAL MODEL FOR ALZHEIMER’S DISEASE USING BIOCHEMICAL SYSTEMS THEORY (BST) PREDICTS THAT MITOCHONDRIAL DYSFUNCTION IS LINKED TO CHANGES IN EXPRESSION OF FIVE GENES IMPACTING MITOCHONDRIAL ROS AND APOPTOSIS
Randolph A. Coleman1, Ceyda Durmaz1, Elena Gavrila2, Morgan Shelton2, Frank J. Castora3, 1College of William and Mary, Williamsburg, VA, USA; 2College of William & Mary, Williamsburg, VA, USA; 3Eastern Virginia Medical School, Norfolk, VA, USA. Contact e-mail: [email protected] Background: Abnormal mitochondrial function has become recog-
nized as a critical component in the pathogenesis of a variety of neurodegenerative diseases, including AD. We have recently found abnormal expression of several genes critical to mitochondrial biogenesis in AD brains. Using this subset of mitochondrial genes, we have begun to build a mathematical model of AD using Biochemical System Theory (BST). Through the development and application of appropriate differential equations, the flux of various metabolites and small molecules will be simulated and used to generate a testable model of mitochondrial involvement in AD pathogenesis. Methods: Human Mitochondrial Biogenesis and Human Alzheimer Disease RT2 Profiler PCR Arrays were used to assess expression of 168 mitochondrial function and AD genes in two control and five age- and gender-matched AD brains. These gene expression changes served as the starting point for MatLab-based mathematical analysis and BST dynamic computer simulations. Results: Our preliminary PCR array analysis identifies significant expression changes in genes involved with maintaining mitochondrial morphology or regulating mitochondrial membrane potential. Combining these data with the mathematical modeling of these interactions using BST, we have generated a tentative working model shown here. Conclusions: Our preliminary model depicts and predicts the relationships of HSPD1, DNM1L, CDKN2A, MFN2, and BCL2L1 to the processes of mitochondrial dysfunction and neuronal apoptosis in the AD
P665
DUAL EFFECT OF CHOLESTEROL ON Ab-INDUCED MITOPHAGY
Vicente Roca, Elisabet Barbero-Camps, Cristina de Dios, Isabel Bartolessis, Carmen Romero, Anna Colell, Instituto de Investigaciones Biomedicas de Barcelona (IIBB-CSIC), Barcelona, Spain. Contact e-mail: [email protected] Background: Abnormal mitophagy, resulting in accumulation of
dysfunctional mitochondria, is closely associated to several neurodegenerative diseases. However, the molecular mechanisms that produce this alteration still remain unclear. Evidence indicate that changes in cellular cholesterol content and trafficking can contribute to neuronal degeneration. Particularly, we have previously described that an increase in mitochondrial cholesterol exhacerbates the oxidative stress induced by amyloid-beta (Ab) peptides. The aim of this work was to evaluate the regulatory effect of cholesterol in mitophagy. Methods: We used APP/PS1 transgenic mice that overexpress the sterol regulatory element binding protein 2 (SREBP-2), a transcription factor that induces cholesterol biosynthesis. These mice display high brain cholesterol levels (total and mitochondrial) associated with depleted mitochondrial glutathione (GSH). Results: Western blot analysis showed increased levels of PINK1 and LC3 in mitochondria from APP/PS1/SREBP-2 mice. Enhanced presence of mitochondria in autophagosomes was also observed by overexpression of SREBP-2; but, confocal analyses failed to show colocalization between mitochondrial and lysosomal markers. In SHY5Y cells, cholesterol enrichment induced mitochondrial accumulation of PINK1 and the following PARK2 recruitment, resulting in increased mitochondrial levels of p62 and LC3 after carbonyl cyanide m-chlorophenylhydrazine (CCCP) exposure. High cholesterol promoted the nuclear clustering of fragmented mitochondria after incubation with Ab or CCCP. Moreover, selective depletion of mGSH enhanced Ab-induced mitophagy by increasing mitochondrial ROS generation. Conversely, inhibition of ROS by mitoTEMPO although prevented Ab-induced mitochondrial fragmentation in control cells failed to block mitochondrial fission and collapse in cholesterol-enriched cells. Conclusions: Overall our data suggest that high mitochondrial cholesterol stimulates the recruitment of the autophagy machinery to damaged mitochondria but mitophagy resolution is ultimately impeded by the increased cellular cholesterol content. P2-151
ESTROGEN REGULATION OF MITOCHONDRIAL RESPIRATION IS CELL TYPE AND ER SUBTYPE SPECIFIC
Yiwei Wang1, Roberta Diaz Brinton1,2, 1University of Southern California, Los Angeles, CA, USA; 2University of Arizona, Tucson, AZ, USA. Contact e-mail: [email protected] Background: Late onset AD has an approximately 20-year prodro-
mal period, during which brain glucose hypometabolism can be detected in at risk groups and is predictive of disease progression. Loss of estrogen during the perimenopausal transition is associated with deficits in brain glucose metabolism and mitochondrial function, which may explain the two-fold greater lifetime risk of LOAD in females than males. In postmenopausal women, estrogen replacement therapy can improve brain glucose metabolism and recognition, and in ovariectomized rodents, estrogen treatment preserved mitochondrial respiration capacity, supporting the role of E2
P666
Poster Presentations: Monday, July 17, 2017
as a master regulator of brain bioenergetics via mitochondrial function. However, the underlying mechanism is not fully elucidated. Interestingly, in neuronal cells, estrogen receptor beta (ERb), but not estrogen receptor alpha (ERa), was demonstrated to co-localize with mitochondria, and has a functional role in ATP production, suggesting potential differential contributions of ERa and ERb to regulation of mitochondrial bioenergetics. Methods: To identify cell type specific responses to estrogen treatment, we used rat embryonic primary neurons and astrocytes. To identify the contribution of ER subtypes, cells were treated with either ERa selective antagonist (MPP), or ERb selective antagonist (PHTPP) prior to E2 treatment. Cellular respiratory capacity was determined by XF24 metabolic flux analyzer, and Oxygen consumption rate (OCR) was used as an indicator for mitochondrial oxidative phosphorylation. To determine whether ERs regulate mitochondrial respiration via regulation of transcription of mitochondrial genome, gene expression of the 13 mitochondrial protein encoding genes will be determined by real-time rtPCR. Ongoing analysis also explores how mitochondrial genetic variances affect treatment effect of selective estrogen modulators on perimenopausal symptoms. Results: Estrogen increased both ATP production and maximum respiration in neurons, but only ATP production in astrocytes. ERa antagonist prevented E2 mediated enhancement of mitochondrial respiration in astrocytes but not neurons, whereas ERb antagonist abolished E2 mediated enhancement of mitochondrial respiration in both cell types. Conclusions: E2 regulation of mitochondrial respiration is cell type and ER subtype specific, possibly through differential regulation of transcription of the mitochondrial genome. Acknowledgement: This work was supported by NIA R01 AG032236 to RDB; NIA P01AG026572 to RDB, Project 1 to RDB & EC.
P2-152
IRON AND APOLIPOPROTEIN E LINK IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE
Abdel Ali Belaidi, Scott Ayton, Ashley I. Bush, The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia. Contact e-mail: [email protected] Background: The ε4 allele of the apolipoprotein E (APOE) gene is
the greatest genetic risk factor for late onset Alzheimer’s Disease (AD). ApoE is a lipid transporting protein, but it is not clear that this function underlies the risk for AD. Methods: Using ferritin (the major iron binding protein) and APOE levels in CSF as an index in pre-clinical subjects (n¼91), we recently showed that individuals harboring the ε4 allele express a higher level of ferritin in their CSF, reporting elevated brain iron levels1, and CSF levels of ApoE are highly correlated with CSF levels of ferritin. Cell culture experiments using astrocytes knocked-in with the different ApoE isoforms, and neurons that were exposed to ApoE recombinant protein. Results: Cortical iron elevation has been identified in AD in post mortem studies, and by using MRI measures of iron. We also showed that elevated CSF ferritin predicted accelerated cognitively decline, especially in ε4 carriers2. These findings raise the possibility of an unexpected, causative role for ApoE in regulating brain iron content. We replicated these clinical findings in cell culture experiments, where ApoE markedly changed iron and ferritin in both astrocytes knockedin with the different ApoE isoforms, and neuronal cells exposed
to ApoE recombinant protein. Conclusions: These results validate our clinical observations and suggest that ApoE has an unexpected role in brain iron homeostasis, which might underlie how the ε4 allele confers risk for AD. References: 1-Ayton, S., N. G. Faux, A. I. Bush and I. Alzheimer’s Disease Neuroimaging (2015). “Ferritin levels in the cerebrospinal fluid predict Alzheimer’s disease outcomes and are regulated by APOE.” Nat Commun 6: 6760. 2-Ayton, S., N. G. Faux and A. I. Bush (2017). “Association of Cerebrospinal Fluid Ferritin Level With Preclinical Cognitive Decline in APOE-epsilon4 Carriers.” JAMA Neurol 74(1): 122-125.
P2-153
OSTEOPONTIN-DEPENDENT PHAGOCYTOSIS OF SYNAPTOTOXIC Ab BY INFILTRATING MONOCYTES AND MACROPHAGES
Altan Rentsendorj, Julia Sheyn, Dieu-Trang Fuchs, David A. Daley, Songlin Li, Hannah E. Schubloom, Nadav J. Hart, Keith L. Black, Yosef Koronyo, Koronyo-Hamaoui Maya, Cedars-Sinai Medical Center, Los Angeles, CA, USA. Contact e-mail: [email protected]
Background: Osteopontin (OPN) is a multifunctional matricellular glycoprotein, highly expressed by bone marrow (BM)-derived myelomonocytic cells and modulates immune cell migration, communication, and overall response. We previously demonstrated that glatiramer acetate (GA) immunization attenuates Alzheimer’s disease (AD)-related neuropathology and cognitive decline through enhanced macrophage recruitment to cerebral amyloid b-protein (Ab) plaque sites in transgenic (ADtg) mice. Yet, the immune mechanisms underlying GA neuroprotection are unclear. Methods: To explore the possible involvement of OPN in Ab clearance, and how GA may affect OPN expression levels in the brain, the expression of OPN in GA-immunized bigenic APPSWE/PS1dE9 mice was studied. Further, the extent by which GA impacts OPN expression in primary bone marrowderived macrophage cultures and their phagocytic capacity to uptake fibrillar Ab, were evaluated. Results: We found a substantial upregulation of OPN along with reduced amyloid Ab-plaques in AD-related brain regions: entorhinal cortex, cingulate cortex, and hippocampus of GA-immunized ADtg mice. Treatment combining GA and peripheral blood enrichment of CD115+ monocytes further increased OPN levels surrounding Ab plaques. OPN was predominantly expressed by infiltrating monocyte-derived macrophages involved in Ab-plaque uptake. Correlogram analysis indicated a linear correlation between osteopontin and macrophage infiltration, as well as inverse relations between osteopontin and Ab-plaque burden. In vitro studies, corroborating the in vivo findings, showed that GA directly upregulates osteopontin expression in bone marrowderived macrophages and further promotes an anti-inflammatory phenotype highly phagocytic of Ab. Osteopontin inhibition by siRNA or minocycline in macrophages impairs uptake of Ab142 fibrils and reverses the effects of GA on macrophage phenotype. Conclusions: This study demonstrates a novel role for OPN as an immune-modulator of the capacity of monocyte derived macrophages to resist synaptotoxic Ab associated with AD.
in maintaining mitochondrial morphology, regulating membrane potential, and mediating apoptosis while directly or indirectly interacting with APP and other AD-associated proteins (PINK1) and small molecules (IL6, IL10).
brain. Additionally, we are able to predict the sensitivity of those relationships to changes in activity brought about, for example, by drugs targeting the appropriate proteins expressed by those genes. P2-150
P2-149
A TESTABLE MATHEMATICAL MODEL FOR ALZHEIMER’S DISEASE USING BIOCHEMICAL SYSTEMS THEORY (BST) PREDICTS THAT MITOCHONDRIAL DYSFUNCTION IS LINKED TO CHANGES IN EXPRESSION OF FIVE GENES IMPACTING MITOCHONDRIAL ROS AND APOPTOSIS
Randolph A. Coleman1, Ceyda Durmaz1, Elena Gavrila2, Morgan Shelton2, Frank J. Castora3, 1College of William and Mary, Williamsburg, VA, USA; 2College of William & Mary, Williamsburg, VA, USA; 3Eastern Virginia Medical School, Norfolk, VA, USA. Contact e-mail: [email protected] Background: Abnormal mitochondrial function has become recog-
nized as a critical component in the pathogenesis of a variety of neurodegenerative diseases, including AD. We have recently found abnormal expression of several genes critical to mitochondrial biogenesis in AD brains. Using this subset of mitochondrial genes, we have begun to build a mathematical model of AD using Biochemical System Theory (BST). Through the development and application of appropriate differential equations, the flux of various metabolites and small molecules will be simulated and used to generate a testable model of mitochondrial involvement in AD pathogenesis. Methods: Human Mitochondrial Biogenesis and Human Alzheimer Disease RT2 Profiler PCR Arrays were used to assess expression of 168 mitochondrial function and AD genes in two control and five age- and gender-matched AD brains. These gene expression changes served as the starting point for MatLab-based mathematical analysis and BST dynamic computer simulations. Results: Our preliminary PCR array analysis identifies significant expression changes in genes involved with maintaining mitochondrial morphology or regulating mitochondrial membrane potential. Combining these data with the mathematical modeling of these interactions using BST, we have generated a tentative working model shown here. Conclusions: Our preliminary model depicts and predicts the relationships of HSPD1, DNM1L, CDKN2A, MFN2, and BCL2L1 to the processes of mitochondrial dysfunction and neuronal apoptosis in the AD
P665
DUAL EFFECT OF CHOLESTEROL ON Ab-INDUCED MITOPHAGY
Vicente Roca, Elisabet Barbero-Camps, Cristina de Dios, Isabel Bartolessis, Carmen Romero, Anna Colell, Instituto de Investigaciones Biomedicas de Barcelona (IIBB-CSIC), Barcelona, Spain. Contact e-mail: [email protected] Background: Abnormal mitophagy, resulting in accumulation of
dysfunctional mitochondria, is closely associated to several neurodegenerative diseases. However, the molecular mechanisms that produce this alteration still remain unclear. Evidence indicate that changes in cellular cholesterol content and trafficking can contribute to neuronal degeneration. Particularly, we have previously described that an increase in mitochondrial cholesterol exhacerbates the oxidative stress induced by amyloid-beta (Ab) peptides. The aim of this work was to evaluate the regulatory effect of cholesterol in mitophagy. Methods: We used APP/PS1 transgenic mice that overexpress the sterol regulatory element binding protein 2 (SREBP-2), a transcription factor that induces cholesterol biosynthesis. These mice display high brain cholesterol levels (total and mitochondrial) associated with depleted mitochondrial glutathione (GSH). Results: Western blot analysis showed increased levels of PINK1 and LC3 in mitochondria from APP/PS1/SREBP-2 mice. Enhanced presence of mitochondria in autophagosomes was also observed by overexpression of SREBP-2; but, confocal analyses failed to show colocalization between mitochondrial and lysosomal markers. In SHY5Y cells, cholesterol enrichment induced mitochondrial accumulation of PINK1 and the following PARK2 recruitment, resulting in increased mitochondrial levels of p62 and LC3 after carbonyl cyanide m-chlorophenylhydrazine (CCCP) exposure. High cholesterol promoted the nuclear clustering of fragmented mitochondria after incubation with Ab or CCCP. Moreover, selective depletion of mGSH enhanced Ab-induced mitophagy by increasing mitochondrial ROS generation. Conversely, inhibition of ROS by mitoTEMPO although prevented Ab-induced mitochondrial fragmentation in control cells failed to block mitochondrial fission and collapse in cholesterol-enriched cells. Conclusions: Overall our data suggest that high mitochondrial cholesterol stimulates the recruitment of the autophagy machinery to damaged mitochondria but mitophagy resolution is ultimately impeded by the increased cellular cholesterol content. P2-151
ESTROGEN REGULATION OF MITOCHONDRIAL RESPIRATION IS CELL TYPE AND ER SUBTYPE SPECIFIC
Yiwei Wang1, Roberta Diaz Brinton1,2, 1University of Southern California, Los Angeles, CA, USA; 2University of Arizona, Tucson, AZ, USA. Contact e-mail: [email protected] Background: Late onset AD has an approximately 20-year prodro-
mal period, during which brain glucose hypometabolism can be detected in at risk groups and is predictive of disease progression. Loss of estrogen during the perimenopausal transition is associated with deficits in brain glucose metabolism and mitochondrial function, which may explain the two-fold greater lifetime risk of LOAD in females than males. In postmenopausal women, estrogen replacement therapy can improve brain glucose metabolism and recognition, and in ovariectomized rodents, estrogen treatment preserved mitochondrial respiration capacity, supporting the role of E2
P666
Poster Presentations: Monday, July 17, 2017
as a master regulator of brain bioenergetics via mitochondrial function. However, the underlying mechanism is not fully elucidated. Interestingly, in neuronal cells, estrogen receptor beta (ERb), but not estrogen receptor alpha (ERa), was demonstrated to co-localize with mitochondria, and has a functional role in ATP production, suggesting potential differential contributions of ERa and ERb to regulation of mitochondrial bioenergetics. Methods: To identify cell type specific responses to estrogen treatment, we used rat embryonic primary neurons and astrocytes. To identify the contribution of ER subtypes, cells were treated with either ERa selective antagonist (MPP), or ERb selective antagonist (PHTPP) prior to E2 treatment. Cellular respiratory capacity was determined by XF24 metabolic flux analyzer, and Oxygen consumption rate (OCR) was used as an indicator for mitochondrial oxidative phosphorylation. To determine whether ERs regulate mitochondrial respiration via regulation of transcription of mitochondrial genome, gene expression of the 13 mitochondrial protein encoding genes will be determined by real-time rtPCR. Ongoing analysis also explores how mitochondrial genetic variances affect treatment effect of selective estrogen modulators on perimenopausal symptoms. Results: Estrogen increased both ATP production and maximum respiration in neurons, but only ATP production in astrocytes. ERa antagonist prevented E2 mediated enhancement of mitochondrial respiration in astrocytes but not neurons, whereas ERb antagonist abolished E2 mediated enhancement of mitochondrial respiration in both cell types. Conclusions: E2 regulation of mitochondrial respiration is cell type and ER subtype specific, possibly through differential regulation of transcription of the mitochondrial genome. Acknowledgement: This work was supported by NIA R01 AG032236 to RDB; NIA P01AG026572 to RDB, Project 1 to RDB & EC.
P2-152
IRON AND APOLIPOPROTEIN E LINK IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE
Abdel Ali Belaidi, Scott Ayton, Ashley I. Bush, The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia. Contact e-mail: [email protected] Background: The ε4 allele of the apolipoprotein E (APOE) gene is
the greatest genetic risk factor for late onset Alzheimer’s Disease (AD). ApoE is a lipid transporting protein, but it is not clear that this function underlies the risk for AD. Methods: Using ferritin (the major iron binding protein) and APOE levels in CSF as an index in pre-clinical subjects (n¼91), we recently showed that individuals harboring the ε4 allele express a higher level of ferritin in their CSF, reporting elevated brain iron levels1, and CSF levels of ApoE are highly correlated with CSF levels of ferritin. Cell culture experiments using astrocytes knocked-in with the different ApoE isoforms, and neurons that were exposed to ApoE recombinant protein. Results: Cortical iron elevation has been identified in AD in post mortem studies, and by using MRI measures of iron. We also showed that elevated CSF ferritin predicted accelerated cognitively decline, especially in ε4 carriers2. These findings raise the possibility of an unexpected, causative role for ApoE in regulating brain iron content. We replicated these clinical findings in cell culture experiments, where ApoE markedly changed iron and ferritin in both astrocytes knockedin with the different ApoE isoforms, and neuronal cells exposed
to ApoE recombinant protein. Conclusions: These results validate our clinical observations and suggest that ApoE has an unexpected role in brain iron homeostasis, which might underlie how the ε4 allele confers risk for AD. References: 1-Ayton, S., N. G. Faux, A. I. Bush and I. Alzheimer’s Disease Neuroimaging (2015). “Ferritin levels in the cerebrospinal fluid predict Alzheimer’s disease outcomes and are regulated by APOE.” Nat Commun 6: 6760. 2-Ayton, S., N. G. Faux and A. I. Bush (2017). “Association of Cerebrospinal Fluid Ferritin Level With Preclinical Cognitive Decline in APOE-epsilon4 Carriers.” JAMA Neurol 74(1): 122-125.
P2-153
OSTEOPONTIN-DEPENDENT PHAGOCYTOSIS OF SYNAPTOTOXIC Ab BY INFILTRATING MONOCYTES AND MACROPHAGES
Altan Rentsendorj, Julia Sheyn, Dieu-Trang Fuchs, David A. Daley, Songlin Li, Hannah E. Schubloom, Nadav J. Hart, Keith L. Black, Yosef Koronyo, Koronyo-Hamaoui Maya, Cedars-Sinai Medical Center, Los Angeles, CA, USA. Contact e-mail: [email protected]
Background: Osteopontin (OPN) is a multifunctional matricellular glycoprotein, highly expressed by bone marrow (BM)-derived myelomonocytic cells and modulates immune cell migration, communication, and overall response. We previously demonstrated that glatiramer acetate (GA) immunization attenuates Alzheimer’s disease (AD)-related neuropathology and cognitive decline through enhanced macrophage recruitment to cerebral amyloid b-protein (Ab) plaque sites in transgenic (ADtg) mice. Yet, the immune mechanisms underlying GA neuroprotection are unclear. Methods: To explore the possible involvement of OPN in Ab clearance, and how GA may affect OPN expression levels in the brain, the expression of OPN in GA-immunized bigenic APPSWE/PS1dE9 mice was studied. Further, the extent by which GA impacts OPN expression in primary bone marrowderived macrophage cultures and their phagocytic capacity to uptake fibrillar Ab, were evaluated. Results: We found a substantial upregulation of OPN along with reduced amyloid Ab-plaques in AD-related brain regions: entorhinal cortex, cingulate cortex, and hippocampus of GA-immunized ADtg mice. Treatment combining GA and peripheral blood enrichment of CD115+ monocytes further increased OPN levels surrounding Ab plaques. OPN was predominantly expressed by infiltrating monocyte-derived macrophages involved in Ab-plaque uptake. Correlogram analysis indicated a linear correlation between osteopontin and macrophage infiltration, as well as inverse relations between osteopontin and Ab-plaque burden. In vitro studies, corroborating the in vivo findings, showed that GA directly upregulates osteopontin expression in bone marrowderived macrophages and further promotes an anti-inflammatory phenotype highly phagocytic of Ab. Osteopontin inhibition by siRNA or minocycline in macrophages impairs uptake of Ab142 fibrils and reverses the effects of GA on macrophage phenotype. Conclusions: This study demonstrates a novel role for OPN as an immune-modulator of the capacity of monocyte derived macrophages to resist synaptotoxic Ab associated with AD.