- Email: [email protected]
Analysis of the synapse-rich brain proteome reveals potential markers of asymptomatic Alzheimer's disease
Oral Sessions: O2-09: Biomarkers: Differential Diagnosis and Co-morbidities Conclusions: These studies provide an insight to the mechanism of tau immunotherapy and its efficacy based on an epitope and a pool of tau being targeted.
O2-08-05
ELUCIDATING ALZHEIMER’S DISEASE NEURONAL DEATH BY THE ‘CALPAINCATHEPSIN HYPOTHESIS’
Tetsumori Yamashima, Kanazawa University Graduate School of Medicine, Kanazawa, Japan. Contact e-mail: yamashim@med. kanazawa-u.ac.jp Background: Alzheimer’s disease (AD) is characterized by slowly progressive neuronal death, but its molecular cascade remains elusive for over 100 years. Since accumulation of autophagic vacuoles (granulo-vacuolar degenerations) represents one of the histological hallmarks of degenerating neurons in AD, a causal link between autophagy failure and neuronal death is probable. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) as a molecular chaperone for damaged proteins and a guardian of lysosomal integrity. The aim of this study is at elucidating the underlying mechanism of AD by comparing ischemic neuronal death of monkeys and degenerative neuronal death of AD patients. Methods: Morphologic, immunoblotting, and proteomics analyses of both the ischemic monkey (Macaca fuscata, body weight of 5-10 Kg, n¼20) and human AD brains were done. The ischemic monkey data in the author’s laboratory were compared to the human AD patient data of the literature. The main topics are whether an agerelated decrease in lysosomal and autophagic activities has a causal connection to programmed neuronal necrosis in sporadic AD, and how genetic factors such as apolipoprotein E (APOE) and presenilin 1 can facilitate lysosomal de stabilization. Results: Both monkey and human brains exhibited evidence of transient or continual perturbed Ca 2+ homeostasis, calpain activation, and oxidative stresses upon Hsp70.1. Membrane lipids such as linoleic and arachidonic acids are vulnerable to the cumulative oxidative stresses, generating a toxic peroxidation product ’hydroxynonenal’ that carbonylated Hsp70.1. In the monkey brain, both the carbonylation and calpain-mediated cleavage occurred at Arg469 (i.e., key site) of Hsp70.1. This caused decrease of acid sphingomyelinase activity and accumulation of lysosomal sphingomyelin. Electron microscopic data of the AD neurons showed evidence of lysosomal destabilization and multilamellar lysosomal lipidosis. Taken together, impairments of lysosomal autophagy and stabilization should be driven by the calpain-mediated cleavage of carbonylated Hsp70.1, and this causes lysosomal permeabilization and/or rupture with the resultant release of the cell degradation enzyme, cathepsins (calpaincathepsin hypothesis: Yamashima T., J Neurochem. 120:477-494, 2012). Conclusions: Artificial or age-dependent oxidative stresses affects the autophagic-lysosomal system via Hsp70.1 carbonylation and cleavage. Alzheimer’s neuronal death conceivably occurs by the similar calpain-cathepsin cascade with ischemic neuronal death.
O2-08-06
P331
ANALYSIS OF THE SYNAPSE-RICH BRAIN PROTEOME REVEALS POTENTIAL MARKERS OF ASYMPTOMATIC ALZHEIMER’S DISEASE
Lenora Higginbotham1, Laura Donovan1, Duc Duong1, Eric Dammer1, Aimee Schantz2, Thomas Montine2, James Lah3, Allan Levey1, Nicholas Seyfried1, 1Emory University School of Medicine, Atlanta, Georgia, United States; 2University of Washington, Seattle, Washington, United States; 3Emory University, Atlanta, Georgia, United States. Contact e-mail: [email protected] Background: It is established that the neuropathological changes of Alzheimer’s disease (AD) begin years prior to the onset of symptoms. Advancements in neuroimaging and cerebral spinal fluid analysis have confirmed that approximately one-third of clinically asymptomatic older individuals demonstrate accumulation of beta-amyloid peptide up to a decade before the development of even subtle signs of cognitive impairment. Currently, our understanding of these early pathological events and the molecular transition from this asymptomatic phase to clinically evident disease is extremely limited. Analyzing the brain proteome in this preclinical stage will not only enhance our understanding of early AD progression, but also facilitate more accurate classification of at-risk asymptomatic individuals. By means of an unbiased proteomics approach, this study aimed to identify proteins differentially expressed in asymptomatic AD that could better classify disease progression. Methods: Using quantitative liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) we comparatively analyzed the synapse-rich proteomes of post-mortem frontal cortex samples of 18 individuals separated into the following cohorts (n¼6): i) cognitively normal individuals without AD pathology, ii) cognitively normal individuals with moderate to severe AD pathology (AsymAD), and iii) cognitively impaired individuals with moderate to severe AD pathology. Results: In total we identified and quantified over 40,000 peptides from 3,230 proteins. Hierarchical cluster analysis based on the overall pattern of protein expression was able to stratify certain AsymAD cases that had attributes similar to symptomatic AD cases. Pathway analysis of these AsymAD cases revealed a severe loss of synaptic proteins and an increase in proteins directly involved in oxidative stress. Interestingly, all AsymAD cases that clustered with AD cases carried at least one APOE-ε4 allele providing strong preliminary evidence of a genotype specific effect on the synaptic proteome. Conclusions: These results support an emerging hypothesis that AsymAD is characterized by differential protein signatures representing specific functional mechanisms which involve synaptic loss and oxidative stress, which are likely to involve known AD risk factors. ORAL SESSIONS: O2-09 BIOMARKERS: DIFFERENTIAL DIAGNOSIS AND CO-MORBIDITIES O2-09-01
ASSESSMENT OF RETINAL MICROVASCULATURE MAY AID DEMENTIA SUBTYPE DIFFERENTIATION
Yi-Ting Ong1, Carol Cheung2, Mohammad Kamran Ikram1, Saima Hilal3, Philip Yap4, Dennis Seow5, Tien Yin Wong6, Christopher Li-Hsian Chen3, 1 National University of Singapore, Singapore, Singapore; 2Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Singapore; 3 National University Health System, Singapore, Singapore; 4Khoo Teck Puat Hospital, Singapore, Singapore, Singapore; 5Singapore General Hospital, Singapore, Singapore, Singapore; 6Singapore Eye Research Institute, Singapore, Singapore. Contact e-mail: [email protected] Background: Retinal vascular changes, which are markers of microvascular pathology, have been linked to dementia and cognitive impairment. In this study, we examine the association of quantitative retinal microvascular parameters with subtypes of dementia to determine if assessment of retinal vasculature may enhance differentiation of subtypes. Methods: This is a prospectively-planned case-control study of clinically diagnosed dementia cases recruited from tertiary hospitals in Singapore. Cases were subtyped as Alzheimer’s disease (AD) and vascular dementia (VaD) based on
O2-08-05
ELUCIDATING ALZHEIMER’S DISEASE NEURONAL DEATH BY THE ‘CALPAINCATHEPSIN HYPOTHESIS’
Tetsumori Yamashima, Kanazawa University Graduate School of Medicine, Kanazawa, Japan. Contact e-mail: yamashim@med. kanazawa-u.ac.jp Background: Alzheimer’s disease (AD) is characterized by slowly progressive neuronal death, but its molecular cascade remains elusive for over 100 years. Since accumulation of autophagic vacuoles (granulo-vacuolar degenerations) represents one of the histological hallmarks of degenerating neurons in AD, a causal link between autophagy failure and neuronal death is probable. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) as a molecular chaperone for damaged proteins and a guardian of lysosomal integrity. The aim of this study is at elucidating the underlying mechanism of AD by comparing ischemic neuronal death of monkeys and degenerative neuronal death of AD patients. Methods: Morphologic, immunoblotting, and proteomics analyses of both the ischemic monkey (Macaca fuscata, body weight of 5-10 Kg, n¼20) and human AD brains were done. The ischemic monkey data in the author’s laboratory were compared to the human AD patient data of the literature. The main topics are whether an agerelated decrease in lysosomal and autophagic activities has a causal connection to programmed neuronal necrosis in sporadic AD, and how genetic factors such as apolipoprotein E (APOE) and presenilin 1 can facilitate lysosomal de stabilization. Results: Both monkey and human brains exhibited evidence of transient or continual perturbed Ca 2+ homeostasis, calpain activation, and oxidative stresses upon Hsp70.1. Membrane lipids such as linoleic and arachidonic acids are vulnerable to the cumulative oxidative stresses, generating a toxic peroxidation product ’hydroxynonenal’ that carbonylated Hsp70.1. In the monkey brain, both the carbonylation and calpain-mediated cleavage occurred at Arg469 (i.e., key site) of Hsp70.1. This caused decrease of acid sphingomyelinase activity and accumulation of lysosomal sphingomyelin. Electron microscopic data of the AD neurons showed evidence of lysosomal destabilization and multilamellar lysosomal lipidosis. Taken together, impairments of lysosomal autophagy and stabilization should be driven by the calpain-mediated cleavage of carbonylated Hsp70.1, and this causes lysosomal permeabilization and/or rupture with the resultant release of the cell degradation enzyme, cathepsins (calpaincathepsin hypothesis: Yamashima T., J Neurochem. 120:477-494, 2012). Conclusions: Artificial or age-dependent oxidative stresses affects the autophagic-lysosomal system via Hsp70.1 carbonylation and cleavage. Alzheimer’s neuronal death conceivably occurs by the similar calpain-cathepsin cascade with ischemic neuronal death.
O2-08-06
P331
ANALYSIS OF THE SYNAPSE-RICH BRAIN PROTEOME REVEALS POTENTIAL MARKERS OF ASYMPTOMATIC ALZHEIMER’S DISEASE
Lenora Higginbotham1, Laura Donovan1, Duc Duong1, Eric Dammer1, Aimee Schantz2, Thomas Montine2, James Lah3, Allan Levey1, Nicholas Seyfried1, 1Emory University School of Medicine, Atlanta, Georgia, United States; 2University of Washington, Seattle, Washington, United States; 3Emory University, Atlanta, Georgia, United States. Contact e-mail: [email protected] Background: It is established that the neuropathological changes of Alzheimer’s disease (AD) begin years prior to the onset of symptoms. Advancements in neuroimaging and cerebral spinal fluid analysis have confirmed that approximately one-third of clinically asymptomatic older individuals demonstrate accumulation of beta-amyloid peptide up to a decade before the development of even subtle signs of cognitive impairment. Currently, our understanding of these early pathological events and the molecular transition from this asymptomatic phase to clinically evident disease is extremely limited. Analyzing the brain proteome in this preclinical stage will not only enhance our understanding of early AD progression, but also facilitate more accurate classification of at-risk asymptomatic individuals. By means of an unbiased proteomics approach, this study aimed to identify proteins differentially expressed in asymptomatic AD that could better classify disease progression. Methods: Using quantitative liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) we comparatively analyzed the synapse-rich proteomes of post-mortem frontal cortex samples of 18 individuals separated into the following cohorts (n¼6): i) cognitively normal individuals without AD pathology, ii) cognitively normal individuals with moderate to severe AD pathology (AsymAD), and iii) cognitively impaired individuals with moderate to severe AD pathology. Results: In total we identified and quantified over 40,000 peptides from 3,230 proteins. Hierarchical cluster analysis based on the overall pattern of protein expression was able to stratify certain AsymAD cases that had attributes similar to symptomatic AD cases. Pathway analysis of these AsymAD cases revealed a severe loss of synaptic proteins and an increase in proteins directly involved in oxidative stress. Interestingly, all AsymAD cases that clustered with AD cases carried at least one APOE-ε4 allele providing strong preliminary evidence of a genotype specific effect on the synaptic proteome. Conclusions: These results support an emerging hypothesis that AsymAD is characterized by differential protein signatures representing specific functional mechanisms which involve synaptic loss and oxidative stress, which are likely to involve known AD risk factors. ORAL SESSIONS: O2-09 BIOMARKERS: DIFFERENTIAL DIAGNOSIS AND CO-MORBIDITIES O2-09-01
ASSESSMENT OF RETINAL MICROVASCULATURE MAY AID DEMENTIA SUBTYPE DIFFERENTIATION
Yi-Ting Ong1, Carol Cheung2, Mohammad Kamran Ikram1, Saima Hilal3, Philip Yap4, Dennis Seow5, Tien Yin Wong6, Christopher Li-Hsian Chen3, 1 National University of Singapore, Singapore, Singapore; 2Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Singapore; 3 National University Health System, Singapore, Singapore; 4Khoo Teck Puat Hospital, Singapore, Singapore, Singapore; 5Singapore General Hospital, Singapore, Singapore, Singapore; 6Singapore Eye Research Institute, Singapore, Singapore. Contact e-mail: [email protected] Background: Retinal vascular changes, which are markers of microvascular pathology, have been linked to dementia and cognitive impairment. In this study, we examine the association of quantitative retinal microvascular parameters with subtypes of dementia to determine if assessment of retinal vasculature may enhance differentiation of subtypes. Methods: This is a prospectively-planned case-control study of clinically diagnosed dementia cases recruited from tertiary hospitals in Singapore. Cases were subtyped as Alzheimer’s disease (AD) and vascular dementia (VaD) based on