- Email: [email protected]
O-GLCNACYLATION AMELIORATES ISCHEMIA-INDUCED CEREBRAL INJURY
P1048
Poster Presentations: Wednesday, July 27, 2016
Figure 1. In vivo NADH fluorescence imaging of AD mice reveals chronic tissue hypoxia and cellular metabolic shift. A) Characteristic NADH pattern after reduction of oxygen supply. Arrowheads pointing to a vein; asterisks at arteries B) Two-photon imaging under the same conditions shows numerous cells with increased signal (green). C) A subset of those cells stain positive for the astrocyte marker SR101 (red). Arrowheads at cells double positive for NADH and SR101. D) Cortex also shows heavy CAA (blue) and tissue plaques (blue, asterisks).
angiopathy (CAA), leading to the death of smooth muscle cells, cerebral hypoperfusion and inadequate oxygen supply. These vascular events could also contribute to metabolic alterations in glucose homeostasis. High resolution in vivo study of the dynamic vascular and metabolic events may reveal which tissue regions and cell populations are affected and cast light on the mechanisms that contribute to AD pathogenesis. Methods: We used fluorescence imaging of nicotinamide adenine dinucleotide (NADH) as an intrinsic marker for cellular metabolic states and tissue oxygen supply in vivo. We resolved the tissue boundaries of NADH fluorescence in the cortex of transgenic AD mice (B6C3.Tg(APPswe-PSEN1de9), n¼4, 12-24 months old) and observed NADH pattern relative to vessels during hyperoxia and normoxia. We then used in vivo two-photon fluorescence microscopy together with cell-type specific labeling to determine the cellular origin of the intrinsic signal and the locality of CAA. Results: Reduction of oxygen supply from hyperoxia to normoxia produced no detectable changes in controls, however AD mice showed characteristic NADH pattern (Figure 1A), indicative of reduced oxygen gradient and rise in glycolysis in tissues further away from the arterial oxygen supply. Areas around capillary beds showed decreased NADH signal. Twophoton imaging under the same conditions revealed numerous cells with increased signal (Figure 1B) and only some of those cells stained positive for the astrocyte marker Sulforhodamine-101 (Figure 1C). All AD mice had CAA and tissue plaques seen with Methoxy-X04 staining (Figure 1D) and there appeared to be no association of the NADH signal with the plaques location. Conclusions: In agreement with previous findings, double transgenic AD mice display chronic tissue hypoxia. Our preliminary results also indicate that under those conditions a subset of cells may adapt by up-regulating glycolysis to overcome the deficient oxidative phosphorylation. The population of cells with increased NADH signal is likely a combination of neurons and glia. This work can lead to new strategies that target metabolic pathways to halt AD progression.
P4-094
MEASURING THE INTERACTION BETWEEN PUTATIVE PATHOLOGICAL STATUS IN ALZHEIMER’S DISEASE PATHOLOGY AND CARDIOVASCULAR RISKS IN AMNESTIC MCI INDIVIDUALS STRATIFIED BY APOE GENOTYPE
Yuliya Bodryzlova1, Olivier Potvin2, Simon Duchesne3, 1Institut Universitaire en Sante Mental de Montreal, Montreal, QC, Canada; 2 Universite Laval, Quebec city, QC, Canada; 3Institut Universitaire en Sante Mentale de Quebec, Quebec, QC, Canada. Contact e-mail: yuliya. [email protected]
Background: In a previous study (Bodryzlova et al.; Alz Dementia 10(4):P151) we demonstrated how 75% of MCI to AD conversion cases could be explained by the interaction between Alzheimer’s disease pathology (ADP) and cardiovascular risk factors (CVR). However, there were limitations to this study, namely our using Mini-Mental State Examination (MMSE) results as a proxy of ADP, and how both exposures could have been overestimated due our using odds ratios instead of relative risk (RR). Further, we did not take apolipoprotein E genotype (APOE) status into account. The objective of the current study was to overcome these limitations by calculating the fraction of conversion cases attributable to the interaction (FAI) between ADP and CVR by APOE strata, using conversion probability as an association measure in groups exposed to ADP only (P01), CVR only (P10), both (P11) or none (P00). Methods: We used data on MCIs from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) 1 dataset (total 404 subjects). We stratified participants according to their APOE genotype into either allele 4 non-carriers, heterozygote, or homozygote carriers. The ADP proxy was chosen as the dichotomized score on either the Wechsler Logical Memory delayed recall test, the Rey Auditory Verbal Learning test, the Alzheimer’s Disease Assessment Scale test, the MMSE, or the MMSE delayed recall. The CVR proxy was a dichotomized sum of normative levels in blood arterial tension, cholesterol and triglycerides (0 – normative level, 1 – moderate increase, 2 – considerable increase) compared to cognitively healthy controls in ADNI. We calculated the FAI as P11/(P11P10-P01+P00). Results: We found no conversion cases attributable to the interaction between ADP and CVR in APOE4 non-carriers and heterozygote subjects. In homozygote carriers the FAI varied according to the proxy chosen: for the MMSE it reached 68% (p¼0,004); the Wechsler 66% (p¼0,009); the Assessment Scale 58% (p¼0,02); and the Rey Auditory 62% (p¼0,02). The MMSE delayed recall was not significant (47%, p¼0.12). Conclusions: More than half of all conversion cases in the APOE e4 homozygote carriers seemed due to the interaction between ADP and CVR. Further, this was evident using a multitude of tests assessing functions other than episodic memory. P4-095
O-GLCNACYLATION AMELIORATES ISCHEMIAINDUCED CEREBRAL INJURY
Cheng-Xin Gong1,2, Jin-hua Gu1,2, Jianhua Shi1,2, Chunling Dai1, Yang Zhao1, Yanxing Chen1,3, Qian Yu1, Fei Liu2,4, Khalid Iqbal1, 1New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA; 2Nantong University, Nantong, China; 3The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 4New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA. Contact e-mail: [email protected] Background: O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins with b-N-acetylglucosamine (GlcNAc). It regulates many biological processes and also serves as a nutrient and stress sensor. Recent studies suggest that O-GlcNAcylation may be protective against acute heart ischemia. This study is aimed to investigate whether O-GlcNAcylation is altered during ischemia-induced brain injury, which is associated with the development of Alzheimer’s disease, and whether it is neuroprotective against cerebral ischemia. Methods: We investigated the time-dependent alteration of O-GlcNAcylation and the related key proteins by Western blots and immunohistochemistry in the ischemic brain tissue of mice after permanent and transient middle cerebral artery occlusion (MCAO). Postmortem human brain tissue with ischemic damage was also studied immunohistochemically.
Poster Presentations: Wednesday, July 27, 2016
The role of O-GlcNAcylation in cerebral ischemia-reperfusion injury was investigated by modulation of brain O-GlcNAcylation level pharmacologically and with a molecular approach before induction of MCAO. Results: We found an initial large elevation (1-4 hours after ischemia) and then marked decline of protein O-GlcNAcylation during cerebral ischemia. If reperfusion occurred two hours after ischemia, the elevation of O-GlcNAcylation lasted longer. The transient elevation of brain O-GlcNAcylation during the early phase of ischemia was neuroprotective and helped ameliorate cerebral ischemia-reperfusion injury. Interference of the transient elevation of O-GlcNAcylation aggravated the ischemiainduced brain damage, motor deficits and mortality. Conclusions: This study reveals a novel regulation of cerebral ischemia-reperfusion injury by O-GlcNAcylation and also provides a possible new therapeutic strategy, i.e., to reduce the cerebral damage and improve the clinical outcome of ischemic stroke and dementia associated with cerebral ischemia by increasing O-GlcNAcylation. P4-096
PICALM MEDIATES AUTOPHAGIC ABETA CLEARANCE AND TOXICITY MITIGATION IN PERICYTES
Zhen Zhao, University of Southern California, Los Angeles, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the most common form of dementia in the elderly, manifesting the occurrence of amyloid plaques, neurofibrillary tangles and the destruction of the neurovascular unit. Vascular factors are considered as a major risk factor for dementia due to AD. Recent studies have demonstrated pericytes degenerate in AD and potentially play a key role in pathogenesis. However the cellular and molecular mechanisms underlying pericyte degeneration during AD pathogenesis remain elusive. Methods: We examined: i) Abeta accumulation and pericyte degeneration during AD pathogenesis in post mortem AD brain tissues using immunohistochemistry; ii) internalization of soluble Abeta and clearance of aggregated Amyloid oligomers via autophagy in primary brain pericytes using in vitro culture models; iii) the role of PICALM in mediating the clearance of oligomeric Abeta via autophagy in pericytes in vitro. Results: Our study showed that: a) pericytes internalize soluble Abeta species via LRP1 mediated endocytosis with the assistance of PICALM; b) pericytes clear aggregated Abeta oligomers via PICALM dependent autophagic pathway; c) PICALM-mediated autophagic clearance of intracellular Abeta protects pericytes from Abeta toxicity and cell death. Conclusions: The data suggest that PICALM-mediated autophagy of Abeta regulates its clearance and is a protective mechanism in pericytes in AD. P4-097
STALLED BLOOD FLOW IN BRAIN CAPILLARIES IS RESPONSIBLE FOR REDUCED CORTICAL PERFUSION IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Jean C. Cruz Hernandez, Calvin Kersbergen, Victorine Muse, Iryna Ivasyk, Oliver Bracko, Mohammad Haft-Javaherian, Yiming Kang, Joan Zhou, Jeffrey D. Beverly, Elizabeth Slack, Gabriel Otte, Thom P. Santisakultarm, Costantino Iadecola, Nozomi Nishimura, Chris B. Schaffer, Cornell University, Ithaca, NY, USA. Contact e-mail: [email protected] Background: A reduction of w30% in cerebral blood flow is observed
in patients and animal models of Alzheimer’s disease (AD). Although this hypoperfusion likely contributes to cognitive impairment and disease progression, no physiological explanation for this phenomenon has emerged. We observed that leukocytes often plug capillaries in
P1049
the brains of AD mouse models, effectively stopping blood flow in the smallest brain blood vessels. Here, we tested the hypothesis that these leukocytes plugs contribute to the blood flow reduction. Methods: Using in vivo two-photon excited fluorescence imaging in transgenic AD mice (APP/PS1), we quantify the number of flowing and stalled cortical capillaries and measure blood flow speed in penetrating arterioles before and after treatment with antibodies that reduce the incidence of capillary plugging. Results: We found that 1.6% of brain capillaries had stalled blood flow in AD mice, as compared to 0.3% in age-matched, littermate controls (AD n¼28 and wild-type n¼8; p<0.0001, Mann-Whitney). This capillary stalling occurred early in disease progression, before deposition of amyloid plaques. We determined that the majority of the capillary stalls were caused by neutrophils that were firmly adhered to the endothelium of brain capillaries. Administration of antibodies against Ly-6G, a neutrophil receptor implicated in their activation, led to a w60% reduction in the number of stalled capillaries within minutes, while isotype control antibodies did not impact the stalls (anti-Ly-6G n¼ 9 and isotype n¼ 6; p¼0.0004; Mann-Whitney; Fig. 1a). Blood flow in penetrating arterioles increased by a median of w30% after anti-Ly-6G treatment, while isotype antibodies led to no change in flow speed (anti-Ly-6G n¼3 and isotype n¼3; p<0.0001, Mann-Whitney; Fig.1b). Conclusions: Brain hypoperfusion likely contributes to cognitive deficits in
Poster Presentations: Wednesday, July 27, 2016
Figure 1. In vivo NADH fluorescence imaging of AD mice reveals chronic tissue hypoxia and cellular metabolic shift. A) Characteristic NADH pattern after reduction of oxygen supply. Arrowheads pointing to a vein; asterisks at arteries B) Two-photon imaging under the same conditions shows numerous cells with increased signal (green). C) A subset of those cells stain positive for the astrocyte marker SR101 (red). Arrowheads at cells double positive for NADH and SR101. D) Cortex also shows heavy CAA (blue) and tissue plaques (blue, asterisks).
angiopathy (CAA), leading to the death of smooth muscle cells, cerebral hypoperfusion and inadequate oxygen supply. These vascular events could also contribute to metabolic alterations in glucose homeostasis. High resolution in vivo study of the dynamic vascular and metabolic events may reveal which tissue regions and cell populations are affected and cast light on the mechanisms that contribute to AD pathogenesis. Methods: We used fluorescence imaging of nicotinamide adenine dinucleotide (NADH) as an intrinsic marker for cellular metabolic states and tissue oxygen supply in vivo. We resolved the tissue boundaries of NADH fluorescence in the cortex of transgenic AD mice (B6C3.Tg(APPswe-PSEN1de9), n¼4, 12-24 months old) and observed NADH pattern relative to vessels during hyperoxia and normoxia. We then used in vivo two-photon fluorescence microscopy together with cell-type specific labeling to determine the cellular origin of the intrinsic signal and the locality of CAA. Results: Reduction of oxygen supply from hyperoxia to normoxia produced no detectable changes in controls, however AD mice showed characteristic NADH pattern (Figure 1A), indicative of reduced oxygen gradient and rise in glycolysis in tissues further away from the arterial oxygen supply. Areas around capillary beds showed decreased NADH signal. Twophoton imaging under the same conditions revealed numerous cells with increased signal (Figure 1B) and only some of those cells stained positive for the astrocyte marker Sulforhodamine-101 (Figure 1C). All AD mice had CAA and tissue plaques seen with Methoxy-X04 staining (Figure 1D) and there appeared to be no association of the NADH signal with the plaques location. Conclusions: In agreement with previous findings, double transgenic AD mice display chronic tissue hypoxia. Our preliminary results also indicate that under those conditions a subset of cells may adapt by up-regulating glycolysis to overcome the deficient oxidative phosphorylation. The population of cells with increased NADH signal is likely a combination of neurons and glia. This work can lead to new strategies that target metabolic pathways to halt AD progression.
P4-094
MEASURING THE INTERACTION BETWEEN PUTATIVE PATHOLOGICAL STATUS IN ALZHEIMER’S DISEASE PATHOLOGY AND CARDIOVASCULAR RISKS IN AMNESTIC MCI INDIVIDUALS STRATIFIED BY APOE GENOTYPE
Yuliya Bodryzlova1, Olivier Potvin2, Simon Duchesne3, 1Institut Universitaire en Sante Mental de Montreal, Montreal, QC, Canada; 2 Universite Laval, Quebec city, QC, Canada; 3Institut Universitaire en Sante Mentale de Quebec, Quebec, QC, Canada. Contact e-mail: yuliya. [email protected]
Background: In a previous study (Bodryzlova et al.; Alz Dementia 10(4):P151) we demonstrated how 75% of MCI to AD conversion cases could be explained by the interaction between Alzheimer’s disease pathology (ADP) and cardiovascular risk factors (CVR). However, there were limitations to this study, namely our using Mini-Mental State Examination (MMSE) results as a proxy of ADP, and how both exposures could have been overestimated due our using odds ratios instead of relative risk (RR). Further, we did not take apolipoprotein E genotype (APOE) status into account. The objective of the current study was to overcome these limitations by calculating the fraction of conversion cases attributable to the interaction (FAI) between ADP and CVR by APOE strata, using conversion probability as an association measure in groups exposed to ADP only (P01), CVR only (P10), both (P11) or none (P00). Methods: We used data on MCIs from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) 1 dataset (total 404 subjects). We stratified participants according to their APOE genotype into either allele 4 non-carriers, heterozygote, or homozygote carriers. The ADP proxy was chosen as the dichotomized score on either the Wechsler Logical Memory delayed recall test, the Rey Auditory Verbal Learning test, the Alzheimer’s Disease Assessment Scale test, the MMSE, or the MMSE delayed recall. The CVR proxy was a dichotomized sum of normative levels in blood arterial tension, cholesterol and triglycerides (0 – normative level, 1 – moderate increase, 2 – considerable increase) compared to cognitively healthy controls in ADNI. We calculated the FAI as P11/(P11P10-P01+P00). Results: We found no conversion cases attributable to the interaction between ADP and CVR in APOE4 non-carriers and heterozygote subjects. In homozygote carriers the FAI varied according to the proxy chosen: for the MMSE it reached 68% (p¼0,004); the Wechsler 66% (p¼0,009); the Assessment Scale 58% (p¼0,02); and the Rey Auditory 62% (p¼0,02). The MMSE delayed recall was not significant (47%, p¼0.12). Conclusions: More than half of all conversion cases in the APOE e4 homozygote carriers seemed due to the interaction between ADP and CVR. Further, this was evident using a multitude of tests assessing functions other than episodic memory. P4-095
O-GLCNACYLATION AMELIORATES ISCHEMIAINDUCED CEREBRAL INJURY
Cheng-Xin Gong1,2, Jin-hua Gu1,2, Jianhua Shi1,2, Chunling Dai1, Yang Zhao1, Yanxing Chen1,3, Qian Yu1, Fei Liu2,4, Khalid Iqbal1, 1New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA; 2Nantong University, Nantong, China; 3The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 4New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA. Contact e-mail: [email protected] Background: O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins with b-N-acetylglucosamine (GlcNAc). It regulates many biological processes and also serves as a nutrient and stress sensor. Recent studies suggest that O-GlcNAcylation may be protective against acute heart ischemia. This study is aimed to investigate whether O-GlcNAcylation is altered during ischemia-induced brain injury, which is associated with the development of Alzheimer’s disease, and whether it is neuroprotective against cerebral ischemia. Methods: We investigated the time-dependent alteration of O-GlcNAcylation and the related key proteins by Western blots and immunohistochemistry in the ischemic brain tissue of mice after permanent and transient middle cerebral artery occlusion (MCAO). Postmortem human brain tissue with ischemic damage was also studied immunohistochemically.
Poster Presentations: Wednesday, July 27, 2016
The role of O-GlcNAcylation in cerebral ischemia-reperfusion injury was investigated by modulation of brain O-GlcNAcylation level pharmacologically and with a molecular approach before induction of MCAO. Results: We found an initial large elevation (1-4 hours after ischemia) and then marked decline of protein O-GlcNAcylation during cerebral ischemia. If reperfusion occurred two hours after ischemia, the elevation of O-GlcNAcylation lasted longer. The transient elevation of brain O-GlcNAcylation during the early phase of ischemia was neuroprotective and helped ameliorate cerebral ischemia-reperfusion injury. Interference of the transient elevation of O-GlcNAcylation aggravated the ischemiainduced brain damage, motor deficits and mortality. Conclusions: This study reveals a novel regulation of cerebral ischemia-reperfusion injury by O-GlcNAcylation and also provides a possible new therapeutic strategy, i.e., to reduce the cerebral damage and improve the clinical outcome of ischemic stroke and dementia associated with cerebral ischemia by increasing O-GlcNAcylation. P4-096
PICALM MEDIATES AUTOPHAGIC ABETA CLEARANCE AND TOXICITY MITIGATION IN PERICYTES
Zhen Zhao, University of Southern California, Los Angeles, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the most common form of dementia in the elderly, manifesting the occurrence of amyloid plaques, neurofibrillary tangles and the destruction of the neurovascular unit. Vascular factors are considered as a major risk factor for dementia due to AD. Recent studies have demonstrated pericytes degenerate in AD and potentially play a key role in pathogenesis. However the cellular and molecular mechanisms underlying pericyte degeneration during AD pathogenesis remain elusive. Methods: We examined: i) Abeta accumulation and pericyte degeneration during AD pathogenesis in post mortem AD brain tissues using immunohistochemistry; ii) internalization of soluble Abeta and clearance of aggregated Amyloid oligomers via autophagy in primary brain pericytes using in vitro culture models; iii) the role of PICALM in mediating the clearance of oligomeric Abeta via autophagy in pericytes in vitro. Results: Our study showed that: a) pericytes internalize soluble Abeta species via LRP1 mediated endocytosis with the assistance of PICALM; b) pericytes clear aggregated Abeta oligomers via PICALM dependent autophagic pathway; c) PICALM-mediated autophagic clearance of intracellular Abeta protects pericytes from Abeta toxicity and cell death. Conclusions: The data suggest that PICALM-mediated autophagy of Abeta regulates its clearance and is a protective mechanism in pericytes in AD. P4-097
STALLED BLOOD FLOW IN BRAIN CAPILLARIES IS RESPONSIBLE FOR REDUCED CORTICAL PERFUSION IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Jean C. Cruz Hernandez, Calvin Kersbergen, Victorine Muse, Iryna Ivasyk, Oliver Bracko, Mohammad Haft-Javaherian, Yiming Kang, Joan Zhou, Jeffrey D. Beverly, Elizabeth Slack, Gabriel Otte, Thom P. Santisakultarm, Costantino Iadecola, Nozomi Nishimura, Chris B. Schaffer, Cornell University, Ithaca, NY, USA. Contact e-mail: [email protected] Background: A reduction of w30% in cerebral blood flow is observed
in patients and animal models of Alzheimer’s disease (AD). Although this hypoperfusion likely contributes to cognitive impairment and disease progression, no physiological explanation for this phenomenon has emerged. We observed that leukocytes often plug capillaries in
P1049
the brains of AD mouse models, effectively stopping blood flow in the smallest brain blood vessels. Here, we tested the hypothesis that these leukocytes plugs contribute to the blood flow reduction. Methods: Using in vivo two-photon excited fluorescence imaging in transgenic AD mice (APP/PS1), we quantify the number of flowing and stalled cortical capillaries and measure blood flow speed in penetrating arterioles before and after treatment with antibodies that reduce the incidence of capillary plugging. Results: We found that 1.6% of brain capillaries had stalled blood flow in AD mice, as compared to 0.3% in age-matched, littermate controls (AD n¼28 and wild-type n¼8; p<0.0001, Mann-Whitney). This capillary stalling occurred early in disease progression, before deposition of amyloid plaques. We determined that the majority of the capillary stalls were caused by neutrophils that were firmly adhered to the endothelium of brain capillaries. Administration of antibodies against Ly-6G, a neutrophil receptor implicated in their activation, led to a w60% reduction in the number of stalled capillaries within minutes, while isotype control antibodies did not impact the stalls (anti-Ly-6G n¼ 9 and isotype n¼ 6; p¼0.0004; Mann-Whitney; Fig. 1a). Blood flow in penetrating arterioles increased by a median of w30% after anti-Ly-6G treatment, while isotype antibodies led to no change in flow speed (anti-Ly-6G n¼3 and isotype n¼3; p<0.0001, Mann-Whitney; Fig.1b). Conclusions: Brain hypoperfusion likely contributes to cognitive deficits in