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Contact Program Director/Principal Investigator (Last, First, Middle):
Petersen, Ronald, C
3. PROGRAM DIRECTOR / PRINCIPAL INVESTIGATOR
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Petersen, Ronald, C
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Although the İ4 allele of the apolipoprotein E (APOE4) and İ2 allele (APOE2) are risk and protective factors,
respectively, for Alzheimer’s disease, APOE is also associated with longevity; several cross-sectional
studies comparing centenarians and younger adults showed that higher frequency of APOE2 in
centenarians abd lower frequency of APOE4. Despite such interesting observations, fewer longitudinal
studies exist demonstrating its effects on longevity. Moreover, it remains unclear whether APOE effects on
longevity are mediated by affecting cognitive decline or AD neuropathology, and whether there are
sex-dependent effects. Mechanistically, studies on APOE-associated cholesterol during aging in human are
scarce. Through accessing comprehensive longitudinal NACC clinical records and a large collection of
plasma/CSF samples from Mayo Clinic Study of Aging, we will address how APOE contributes to longevity.
Specific Aim 1.Determine the effects of APOE on longevity, depending on cardiovascular health, cognitive
decline, and Alzheimer’s neuropathology in NACC cohorts: By reviewing prospective clinical records of
NACC cohorts, we will assess whether APOE affect longevity in these cohorts, and how gender, cognitive
decline, and cardiovascular disease influence this effect. Moreover, by retrospectively analyzing the
relationship between APOE-mediated longevity and AD neuropathology, we well determine whether the
effects of APOE on longevity are mediated through reducing AD neuropathology.
Specific Aim 2. Address the mechanism underlying APOE-regulated longevity by examining the association
among aging-related biomarkers and apoE-associated cholesterol: By using human plasma and CSF, we
will assess changes of levels of biomarkers associated with apoE-cholesterol metabolism during aging. By
analyzing levels of apoE, cholesterols, apoE-associated cholesterols, and other APOE or aging-related
molecules in human plasma and CSF, we will determine the relationship between apoE-cholesterol
metabolism and aging, and address how APOE genotype contributes to longevity.
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Though several studies have observed that APOE is associated with longevity, its effects are not fully
elucidated. By reviewing comprehensive records of large NACC data and performing biochemical analysis
of clinical samples from Mayo Clinic, we aim to understand the precise mechanism of APOE-mediated
longevity and apply obtained knowledge to promote healthy aging in our aging society.
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Human Embryonic Stem Cells
No
Yes
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DETAILED BUDGET FOR INITIAL BUDGET PERIOD
DIRECT COSTS ONLY
)520
7+528*+
07/01/2016
06/30/2017
/LVW3(56211(/(Applicant organization only)
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BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD
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Personnel
Mitsuru Shinohara, Ph.D., Senior Research Fellow (25% effort) has been working in Dr. Bu’s laboratory for the
past three years. He has extensive experience in working with biochemistry, cell biology, pharmacology,
animal experiments, neuropathology, and epidemiology as shown in previous publications, and generated all
of the preliminary results for this project those presented in the preliminary studies. Dr. Shinohara will be
working on the experiments for proposed studies investigating effects of APOE genotype on longevity.
Supplies
We are requesting a total of $9,412 for supplies for this project. Funds are requested for lab supplies including
ELISA and enzymatic assays.
Travel
We are requesting a total of $3,000 for travel for this project. Funds are requested for presenting the results in
some representative scientific conferences in the field of APOE, longevity, or aging.
3+65HY$SSURYHG7KURXJK20%1R
3DJH 5
Form Page 5
Program Director/Principal Investigator (Last, First, Middle):
Shinohara, Mitsuru
BIOGRAPHICAL SKETCH
NAME
POSITION TITLE
Shinohara, Mitsuru
Research Associate
Department of Neuroscience
eRA COMMONS USER NAME (credential, e.g., agency login)
MSHINOHARA
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and
residency training if applicable.)
DEGREE
INSTITUTION AND LOCATION
MM/YY
FIELD OF STUDY
(if applicable)
The University of Tokyo, Faculty of
Pharmaceutical Sciences, Tokyo, Japan
Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Japan
Graduate School of Medicine, Osaka University,
Osaka, Japan
Washington University School of Medicine, St.
Louis, MO
Mayo Clinic College of Medicine, Jacksonville, FL.
B.S.
03/04
Pharmaceutics
M.S.
03/06
Medical Pharmaceutics
Ph.D.
07/10
Internal Medicine
Postdoctoral
10/10
Neuroscience
Postdoctoral
11/10-
Neuroscience
A. Personal Statement
APOE plays important roles in the pathogenesis of Alzheimer’s disease as well as longevity, and thus has
been studied by many researchers. However, its effects have not yet been fully elucidated.
I have been studied Alzheimer’s disease, APOE and aging for over 10 years. I strongly believe that extensive
efforts, combining my broad background; biochemistry, cell biology, animal experiments, neuroanatomy, clinical
neuropathology, and epidemiology related with aging, Alzheimer’s disease and APOE with elaborate
experimental designs should elucidate pathogenic mechanisms of APOE-mediated longevity and thus provide a
novel knowledge to achieve longevity and healthy aging.
B. Positions and Honors
Positions and Employment
2004- 2006 Graduate student, Graduate School of Pharmaceutical Sciences, The University of Tokyo
2006- 2010 Postgraduate student, Graduate School of Medicine, Osaka University
2009- 2010 Pharmacist, Greenmedic pharmacy, Osaka, Japan
2010
Post-doctoral Fellow, Department of Pediatrics, Washington University School of Medicine
2010Post-doctoral Fellow, Department of Neuroscience, Mayo Clinic College of Medicine
Other Experience and Professional Memberships
2005Member, Japanese Biochemical Society
2009Member, Society for Neuroscience
2009Member, Japan Neuroscience Society
2009Member, Japan Geriatrics Society
2009Member, Japan Society for Dementia Research
2009Member, Japan Society of Gene Therapy
Honors
2002- 2010 The Japan Scholarship Foundation (Tokyo, Japan)
2010
Research Fellowship, Japan Heart Foundation (Tokyo, Japan)
2010
Research Fellowship, The Naito Foundation (Tokyo, Japan)
2011
Young Investigator Scholarship, Alzheimer’s Drug Discovery Foundation (San Diego)
2012
ADRC Pilot Grant Award, Mayo Clinic Alzheimer’s Disease Research Center (Rochester)
2012
Young Investigator Scholarship, Alzheimer’s Drug Discovery Foundation (New Jersey)
PHS 398/2590 (Rev. 06/09)
Page
1
Biographical Sketch Format Page
Program Director/Principal Investigator (Last, First, Middle):
2012
2013
2013
2014
Shinohara, Mitsuru
Young Investigator Award, Japanese Society for Vascular Cognitive Impairment (Tokyo, Japan)
Travel Grant Award, the 7th Human Amyloid Imaging meeting (Miami)
Travel Grant Award, the 36th Molecular Biology Society of Japan (Kobe, Japan)
Travel Grant Award, the Keystone Symposia meeting on Alzheimer’s disease (Keystone)
C. Publication
 Full length, peer-reviewed, original articles
1. M. Tachibana, M. Shinohara, Y. Yamazaki, C. C. Liu, J. Rogers, G. Bu, T. Kanekiyo. Rescuing effects of
RXR agonist bexarotene on aging-related synapse loss depend on neuronal LRP1. Experimental
Neurology, 2015, Epub ahead or print.
2. C. Cook, S. S. Kang, Y. Carlomagno, W. L. Lin, M. Yue, A. Kurti, M. Shinohara, K. Jansen-West, E.
Perkerson, M. Castanedes-Casey, L. Rousseau, V. Phillips, G. Bu, D. W. Dickson, L. Petrucelli. J. D. Fryer.
Tau deposition drives neuropathological, inflammatory and behavioral abnormalities independently of
neuronal loss in a novel mouse model. Human Molecular Genetics, 24(21): 6198-6212, 2015.
3. N. Takasugi, T. Sasaki, M. Shinohara, T. Iwatsubo, T. Tomita. Synthetic ceramide analogues increase
amyloid-β 42 production by modulating γ-secretase activity. Biochemical and Biophysical Research
Communications, 457(2): 194-199, 2015.
4. C. S. Casey, Y. Atagi, Y. Yamazaki, M. Shinohara, M. Tachibana, Y. Fu, G. Bu, T. Kanekiyo.
Apolipoprotein E inhibits cerebrovascular pericyte mobility through a RhoA protein-mediated pathway.
The Journal of Biological Chemistry, 290(22): 14208-14217
5. M. Shinohara, S. Fujioka, M. E. Murray, A. Wojtas, M. Baker, A. Rovelet-Lecrux, R. Rademakers, P. Das,
J. E. Parisi, N. R. Graff-Radford, R. C. Petersen, D. W. Dickson, G. Bu. Regional distribution of synaptic
markers and APP correlate with distinct clinicopathological features in sporadic and familial Alzheimer’s
disease. Brain, accepted for publication.
6. T. Kanekiyo, J. R. Cirrito, C. C. Liu, M. Shinohara, J. Li, D. R. Schler, M. Shinohara, D. M. Holtzman, G.
Bu. Neuronal Clearance of Amyloid-β by Endocytic Receptor LRP1. Journal of Neuroscience,
33(49):19276-19283, 2013.
7. M. Shinohara, R. C. Petersen, D. W. Dickson, G. Bu. Brain Regional Correlation of Amyloid-β with
Synapses and Apolipoprotein E in Non-Demented Individuals: Potential Mechanisms underlying
Regional Vulnerability to Amyloid-β Accumulation. Acta Neuropathologica, 125(4): 535-547, 2013
8. J. Li, T. Kanekiyo, M. Shinohara, Y. Zhang, MJ. Ladu, H. Xu, G. Bu. Differential Regulation of Amyloid-β
Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms. The Journal of
Biological Chemistry, 287 (53): 44593-44601, 2012.
9. T. Kanekiyo, C. C. Liu, M. Shinohara, J. Li, G. Bu. LRP1 in Brain Vascular Smooth Muscle Cells Mediates
Local Clearance of Alzheimer’s Amyloid-β. Journal of Neuroscience, 32 (46): 16458-16465, 2012.
10. N. Sato, M. Okochi, M. Shinohara, G. Thinakaran, S. Takeda, A. Fukumori, M. Noma, A. Tolia, M. Takeda,
R. Morishita. Detection of differential regulation of Amyloid Precursor Protein/Presenilin 1 interaction
during Aβ 40/42 production by study using fusion constructs. PLoS One, 7 (11): e48551, 2012
11. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Shimamura, T. Yamashita, Y. Uchiyama,
H. Rakugi, R. Morishita. Reduction of Brain β-Amyloid (Aβ) by Fluvastatin, a Hydroxymethylglutaryl-CoA
Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments
(APP-CTFs) and Aβ Clearance. The Journal of Biological Chemistry, 285 (29): 22091-22102, 2010
12. S. Takeda, N. Sato, K. Uchio-Yamada, K. Sawada, T. Kunieda, D. Takeuchi, H. Kurinami, M. Shinohara,
H. Rakugi, R. Morishita. Diabetes accelerated memory dysfunction via cerebrovascular inflammation and
Aβ deposition in an Alzheimer mouse model with diabetes. Proc Natl Acad Sci U S A, 13 (107):
7036-7041, 2010
13. S. Takeda, N. Sato, D. Takeuchi, H. Kurinami, M. Shinohara, K. Niisato, M. Kano, T. Ogihara, H. Rakugi,
R. Morishita. Angiotensin Receptor Blocker Prevented β-Amyloid-Induced Cognitive Impairment
Associated with Recovery of Neurovascular Coupling. Hypertension, 54: 1345-52, 2009
14. S. Takeda, N. Sato, K. Niisato, D. Takeuchi, H. Kurinami, M. Shinohara, H. Rakugi, M. Kano, R.
Morishita. Validation of Aβ1-40 Administration into Mouse Cerebroventricles as an Animal Model for
Alzheimer Disease. Brain Research, 1280: 137-147, 2009
PHS 398/2590 (Rev. 06/09)
Page
2
Biographical Sketch Format Page
Program Director/Principal Investigator (Last, First, Middle):
Shinohara, Mitsuru
15. S. Takeda, N. Sato, K. Uchio-Yamada, K. Sawada, T. Kunieda, D. Takeuchi, H. Kurinami, M. Shinohara,
H. Rakugi, R. Morishita. Elevation of Plasma β-amyloid Level by Glucose Loading in Alzheimer Mouse
Models. Biochemical and Biophysical Research Communications, 385: 193-197, 2009
16. H. Kurinami, N. Sato, M. Shinohara, D. Takeuchi, S. Takeda, M. Shimamura, T. Ogihara, R. Morishita.
Prevention of amyloid β-induced memory impairment by fluvastatin, associated with the decrease in
amyloid β accumulation and oxidative stress in amyloid β injection mouse model. International Journal
of Molecular Medicine, 21(5): 531-537, 2008.
17. D. Takeuchi, N. Sato, M. Shimamura, H. Kurinami, S. Takeda, M. Shinohara, S. Suzuki, M. Kojima, T.
Ogihara, R. Morishita. Alleviation of Aβ-induced cognitive impairment by ultrasound-mediated gene
transfer of HGF in a mouse model. Gene Therapy, 15: 561-571, 2008.
18. N. Isoo, C. Sato, H. Miyashita, M. Shinohara, N. Takasugi, Y. Morohashi, S. Tsuji, T. Tomita, T.
Iwatsubo. Aβ42 overproduction associated with structural changes in the catalytic pore of γ-secretase:
common effects of Pen-2 amino terminal elongation and fenofibrate. The Journal of Biological
Chemistry, 282: 12388-12396, 2007.
 Editorials and review articles
1. M. Shinohara, N. Sato, H. Kurinami, T. Hamasaki, A. Chatterjee, H. Rakugi, R. Morishita. Possible
Modification of Alzheimer’s Disease by Statins in Midlife: Interactions with Genetic and Non-Genetic
Risk Factors. Frontier in Aging Neuroscience, 6: 71, 2014
2. M. Shinohara, G. Bu. What can we learn from regional vulnerability to Aβ accumulation in non-demented
individuals? Neurodegenerative disease management, 3 (3): 187-189, 2013
3. N. Sato, M. Shinohara, H. Rakugi, R. Morishita. Dual effects of statins on Aβ metabolism; upregulation of
the degradation of APP-CTF and A β clearance. Neurodegenerative disease, 10 (1-4): 305-308, 2012
4. N. Sato, M. Shinohara, H. Kurinami, M. Shimamura, R. Morishita. Anti-aging against brain – The effects
of statins (Japanese title). Anti-aging Science, 10 (3): 2011
5. M. Shinohara, N. Sato, R. Morishita. Statins and Alzheimer’s disease (Japanese title). Cardiovascular
Frontier, 2 (1): 40-46, 2011
D. Presentation at scientific conference
 Presented as an oral speaker
1. M. Shinohara, DW. Dickson, G. Bu. Brain Regional Distribution of Amyloid-β and Related Molecules in
Non-demented Individuals. AD/PD 2013 (The 11th International Conference On Alzheimer’s &
Parkinson’s Diseases), Florence, Italy
2. M. Shinohara, N. Sato, H. Kurinami, M. Shimamura, H. Rakugi, R. Morishita. Reduction of Brain Aβ by
Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of
APP-CTFs and Aβ Clearance (Japanese title). 3rd General Meeting of the Japanese Society for
Vascular Cognitive Impairment 2012, Tokyo, Japan,
3. M. Shinohara, N. Sato, H. Rakugi, R. Morishita. The effect of hypertension on β-Amyloid peptides in
animal models (Japanese title). 3rd General Meeting of the Japanese Society for Vascular Cognitive
Impairment 2012, Tokyo, Japan
4. M. Shinohara, N. Sato, H. Rakugi, R. Morishita. The plausible mechanism of the regional vulnerability to
β-Amyloid accumulation; animal study (Japanese title). 3rd General Meeting of the Japanese Society
for Vascular Cognitive Impairment 2012, Tokyo, Japan
5. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Noma, H. Rakugi, R. Morishita. The
effect of fluvastatin (an HMG-CoA reductase inhibitor) on Aβ metabolism in brain (Japanese title). The
28th Annual Meeting of Japan Society for Dementia Research 2009, Sendai, Japan
6. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Noma, H. Rakugi, R. Morishita.
Fluvastatin reduces Aβ levels in brain by upregulating APP-CTFs degradation and Aβ clearance.
Neuroscience 2009, Chicago, 211.12
7. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Noma, H. Rakugi, R. Morishita.
Fluvastatin reduces Aβ levels in brain by upregulating APP-CTFs degradation and Aβ clearance. The
32th Annual Meeting of the Japan Neuroscience Society 2009, Nagoya, Japan
PHS 398/2590 (Rev. 06/09)
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Program Director/Principal Investigator (Last, First, Middle):
Shinohara, Mitsuru
8. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Noma, R. Morishita. The protective
effect of fluvastatin, an HMG-CoA reductase inhibitor, on the Aβ brain accumulation. Japan Society of
Gene Therapy 2009, Osaka, Japan
9. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Noma, H. Rakugi, R. Morishita. The
effect of fluvastatin (an HMG-CoA reductase inhibitor) on Aβ metabolism for the development of
disease-modifying therapy for Alzheimer’s disease (Japanese title). The 51st Annual Meeting of the
Japan Geriatrics Society 2009, Yokohama, Japan
10. M. Shinohara, M. Niimura, C. Chijiiwa, N. Isoo, Y. Takahashi, Y. Morohashi, T. Tomita, T. Iwatsubo.
Search for modulators of γ-secretase activity by RNAi screening in Drosophila cells. The 78th Annual
meeting of the Japanese Biochemical Society 2005, Kobe, Japan
 Presented as a poster presenter
1. M. Shinohara, T. Kanekiyo, J. Fryer, G. Bu. APOE2 protects against age-related memory decline: a
clinical and pre-clinical evaluation Neuroscience 2015, Chicago
2. M. Shinohara, DW. Dickson, G. Bu. Regional distribution of Amyloid-β, soluble APP and synaptic
markers in human brains. The 38th Annual meeting of the Japan Neuroscience Society 2015, Kobe,
Japan
3. M. Shinohara, S. Fujioka, ME. Murray, RC. Petersen, DW. Dickson, G. Bu. Regional Distribution of
syanpses and Amyloid Precursor Protein Correlate with Distinct Clinicopathological Features in Sporadic
and Familial Alzheimer’s Disease. Keystone Symposia on Alzheimer’s Disease 2014, Keystone, CO
4. M. Shinohara, S. Fujioka, ME. Murray, RC. Petersen, DW. Dickson, G. Bu. Regional Distribution of
Synapse and Amyloid Precursor Protein respectively correlate with the distinct clinicopathological
features in Sporadic and Familial Alzheimer’s Disease. The 36th Annual Meeting of the Molecular
Biology Society of Japan 2013, Kobe, Japan
5. M. Shinohara, DW. Dickson, G. Bu. Brain Regional Distribution of Amyloid-β and Related Molecules in
Non-demented Individuals. Human Amyloid Imaging 2013, Miami, FL
6. M. Shinohara, DW. Dickson, G. Bu. Brain Regional Distribution of Amyloid-β and Related Molecules in
Non-demented Individuals. 13th International Conference on Alzheimer’s Drug Discovery 2012,
Jersey City, NJ
7. M. Shinohara, DW. Dickson, G. Bu. Brain regional distribution of apoE and Aβ in cognitively normal
persons. Alzheimer’s Association International Conference 2012, Vancouver, Canada
8. M. Shinohara, DW. Dickson, G. Bu. Brain regional distribution of Aβ, apoE, apoE receptors and other
Aβ-related molecules in non-demented individuals. 3rd ApoE, ApoE receptors, and
Neurodegeneration 2012, Jacksonville, FL
9. M. Shinohara, N. Sato, G. Bu, R. Morishita. Reduction of Brain β-Amyloid (Aβ) by Fluvastatin, a
Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor
Protein C-terminal Fragments (APP-CTFs) and Aβ Clearance. 5th Drug Discovery For
Neurodegeneration 2011, San Diego
10. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, M. Noma, H. Rakugi, R. Morishita.
Fluvastatin (an HMG-CoA reductase inhibitor) reduces Aβ levels in brain by upregulating APP-CTFs
degradation and Aβ clearance. International Conference on Molecular Neurodegeneration 2009,
Xiamen, China.
11. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, F. Nakagami, R. Morishita. The protective
effect of fluvastatin, an HMG-CoA reductase inhibitor, on the Aβ brain accumulation. Neuroscience
2008, Washington DC, 138.11/J7.
12. M. Shinohara, N. Sato, H. Kurinami, D. Takeuchi, S. Takeda, F. Nakagami, M. Noma, R. Morishita. The
analysis of the Aβ clearance from brain by Brain efflux systems. (Japanese title) The 27th Annual
Meeting of Japan Society for Dementia Research 2008, Maebashi, Japan
13. M. Shinohara, N. Sato, M. Shimamura, H. Kurinami, D. Takeuchi, S. Takeda, M. Ishimoto, H. Hamada,
T. Ogihara, R. Morishita. Delayed postischemic treatment with fluvastatin improved cognitive impairment
after stroke in rats. (Japanese title). The 25th Annual Meeting of Japan Society for Dementia
Research 2006, Hiroshima, Japan
PHS 398/2590 (Rev. 06/09)
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Program Director/Principal Investigator (Last, First, Middle):
Shinohara, Mitsuru
14. M. Shinohara, M. Niimura, C. Chijiiwa, N. Isoo, Y. Takahashi, Y. Morohashi, T. Tomita, T. Iwatsubo.
Search for modulators of γ-secretase activity by RNAi screening in Drosophila cells. The 78th Annual
meeting of the Japanese Biochemical Society 2005, Kobe, Japan
E. Research Support
Current Research Support
Title:
PI:
Agency:
Type:
Period:
Goal:
Synaptic regulation of Abeta metabolism and secreted markers
Shinohara, M.
BrightFocus Foundation
Postdoctoral Fellowship
7/01/14 - 6/30/16
To clarify the role of synaptic regulation of Abeta metabolism, and identify associated biomarkers
Complete Research Support
Title:
PI:
Agency:
Type:
Period:
Goal:
ApoE & ApoE receptors in Alzheimer’s disease patients
Shinohara, M.
Mayo Clinic Alzheimer’s Disease Research Center
Postdoctoral Fellowship
5/01/12 - 4/30/13
To clarify the potential changes of ApoE and ApoE receptors levels in the brains of Alzheimer’s
disease patient by using novel ELISA systems
Title:
PI:
Agency:
Type:
Period:
Goal:
ApoE, ApoE receptors and Alzheimer’s disease
Shinohara, M.
Japan Heart Association
Postdoctoral Fellowship
8/01/10 - 7/31/11
To identify the roles of APOE and APOE receptors in the pathogenesis of Alzheimer’s disease
Title:
PI:
Agency:
Type:
Period:
Goal:
ApoE, ApoE receptors and Alzheimer’s disease
Shinohara, M.
The Naito Foundation
Postdoctoral Fellowship
8/01/10 - 7/31/11
To identify the roles of APOE and APOE receptors in the pathogenesis of Alzheimer’s disease
PHS 398/2590 (Rev. 06/09)
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5
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OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)
BIOGRAPHICAL SKETCH
Provide the following information for the Senior/key personnel and other significant contributors.
Follow this format for each person. DO NOT EXCEED FIVE PAGES.
NAME: Bu,
Guojun
eRA COMMONS USER NAME (credential, e.g., agency login): BUGUOJUN
POSITION TITLE: Professor
of Neuroscience
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency
training if applicable. Add/delete rows as necessary.)
INSTITUTION AND LOCATION
Beijing Normal University, Beijing, China
Virginia Tech, Blacksburg, Virginia
Washington University, St. Louis, Missouri
DEGREE
(if applicable)
Completion Date
MM/YYYY
B.S.
07/1985
Biology
Ph.D.
09/1990
Biochemistry
Postdoctoral
09/1994
Cell Biology
FIELD OF STUDY
A. Personal Statement
My lab has been studying the brain functions of apoE and apoE receptors, as well as their roles in the
pathogenesis of AD and other neurodegenerative diseases. We have used both in vitro and in vivo systems to
address how apoE isoforms and LRP1 function in regulating Aβ metabolism, synaptic functions and behaviors.
I have a broad background in biochemistry, molecular biology, cell biology and neuroscience, with specific
training and expertise in the key research areas for this application. My research in AD began in 1993 when
apoE4 was discovered as a strong risk factor for AD and later for CAA. Since I established my own laboratory
in 1995, I have focused on dissecting the biological and pathological functions of apoE and apoE receptors
with particular emphasis on their roles in the pathogenesis of AD and related dementia. My laboratory uses
biochemical and molecular tools, as well as cellular and animal models to address both Aβ-dependent and Aβindependent pathogenic pathways for AD. With >200 publications in this research area, h-Index of 60 and an
average citation of 42, we have made major impacts in the field and accumulated all the experience, expertise
and experimental tools to study the pathogenic pathways and potential therapeutic strategies for AD.
1. Bu G (2009). Apolipoprotein E and its receptors in Alzheimer's disease: pathways, pathogenesis and
therapy. Nat Rev Neurosci 10:333-344. PMCID: PMC2908393.
2. Liu C-C, Kanekiyo T, Xu H, Bu G (2013). Apolipoprotein E and Alzheimer’s disease: risk, mechanisms, and
therapy. Nat Rev Neurol 9:106-118. PMCID: PMC3726719.
3. Kanekiyo T, Xu H, Bu G (2014). ApoE and Aβ in Alzheimer's disease: accidental encounters or partners?
Neuron 81:740-754. PMCID: PMC3983361.
B. Positions and Honors
Positions and Employment
1995-2000
Assistant Professor of Neuroscience and Cell Biology, Washington University School of
Medicine, St. Louis, MO
2001-2006
Associate Professor of Neuroscience and Cell Biology, Washington University School of
Medicine, St. Louis, MO
2007-2010
Professor of Neuroscience and Cell Biology, Washington University School of Medicine, St.
Louis, MO
2007-2010
Unit Leader, Pathobiology Research Unit, Department of Pediatrics, Washington University
School of Medicine, St. Louis, MO
2010-present Professor and Consultant, Department of Neuroscience, Mayo Clinic, Jacksonville, FL
2010-present Director, Program on Synaptic Biology and Memory, Mayo Clinic, Jacksonville, FL
2011-present Director, Mayo Clinic Stem Cell Lab, Mayo Clinic, Jacksonville, FL
2014-present Associate Director, Mayo Clinic Alzheimer’s Disease Research Center (ADRC), Mayo Clinic,
Rochester, MN and Jacksonville, FL
2015-present Eugene and Marcia Applebaum Professor in Neuroscience, Department of Neuroscience, Mayo
Clinic, Jacksonville, FL
Other Experience and Professional Memberships
1994Member, American Society for Cell Biology
1996Member, Society for Neuroscience
2004NIH Peer Review Committees: Cell Death in Neurodegeneration (CDIN) Study Section, 20072011; Ad hoc member for several others since 2004.
2008-2013
Editorial board member, Journal of Biological Chemistry
2008-2011
Editorial board member, Journal of Lipid Research
2006Editor-in-Chief, Molecular Neurodegeneration
Honors
1988
1993-1994
1995-1998
2001-2004
2008-2011
2015
Anderson Award, Outstanding Graduate Student, Department of Biochemistry, Virginia Tech
NIH National Research Service Award
Faculty Scholar Award, Alzheimer’s Association
Established Investigator of the American Heart Association
Zenith Fellows Award, Alzheimer’s Association
Investigator of the Year, Mayo Clinic
C. Contribution to Science
1. Brain Aβ and ApoE Metabolism by LRP1, a Major Receptor for ApoE and Aβ: While overproduction of
Aβ due to mutations in the APP and PS1/PS2 genes is the primary cause of early-onset AD, increasing
evidence indicates that impaired brain Aβ clearance, perhaps in conjunction with other pathogenic insults,
is likely the major driver for late-onset AD, which accounts for the vast majority of AD cases. Our work
using conditional knockout mouse models crossed with amyloid model mice have clearly demonstrated that
LRP1, a major receptor for both apoE and Aβ, in several brain cell types plays essential roles in brain Aβ
clearance. These studies not only demonstrated a critical role of LRP1 in Aβ clearance but also proved that
various brain cell types, including neurons and glia in the parenchyma, and smooth muscle cells and
pericytes along the vasculature, participate in brain Aβ clearance. In an Aβ-independent manner, our work
also showed that neuronal deletion of LRP1 leads to impaired brain lipid metabolism and age-dependent,
progressive loss of synapses and eventual neurodegeneration. These cell biological and in vivo works,
published in Neuron, Journal of Neuroscience, and other journals, demonstrated critical physiological and
pathological pathways by which LRP1 regulates the metabolism and functions of Aβ and apoE.
a. Liu Q, Zerbinatti CV, Zhang J, Hoe H-S, Wang B, Cole SL, Herz J, Muglia L, Bu G (2007). Amyloid
precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein
receptor LRP1. Neuron 56:66-78. PMCID: PMC2045076.
b. Kanekiyo T, Zhang J, Liu Q, Liu C-C, Zhang L, Bu G (2011). Heparan sulphate proteoglycan and
the low-density lipoprotein receptor-related protein 1 constitute major pathways for neuronal
amyloid-β uptake. J Neurosci 31:1644-1651. PMCID: PMC3272839.
c. Kanekiyo T, Liu C-C, Shinohara M, Li J, Bu G (2012). Local clearance of Alzheimer's amyloid-β by
LRP1-mediated lysosomal degradation pathway in brain vascular smooth muscle cells. J Neurosci
32:16458-16465. PMCID: PMC3508699.
d. Kanekiyo T, Cirrito JR, Liu C-C, Shinohara M, Li J, Schuler DR, Shinohara M, Holtzman DM, Bu G
(2013). Neuronal clearance of amyloid-β by endocytic receptor LRP1. J Neurosci 33:19276-19283.
PMCID: PMC3850043.
2. LRP-mediated Signaling: Neuronal Survival, Body Homeostasis, and Rejuvenating: Understanding of
why neurons die in neurodegeneration is essential for developing therapeutic strategies to slow or prevent
neurodegeneration in AD. We do know that Aβ aggregates alone are not sufficient as little
neurodegeneration is found in the preclinical AD brains when amyloid plaques are abundant. In a paper
published in 2014 in Neuron, we showed that the Wnt signaling pathway, critical for cell viability, is
significantly impaired in AD brains. Using conditional knockout mouse model deleting an essential Wnt
signaling receptor LRP6 in adult brains, we showed that a reduction of neuronal Wnt signaling leads to
synaptic loss and memory impairments. Interestingly, a loss of neuronal LRP6-mediated Wnt signaling also
leads to increased brain Aβ and amyloid plaques, demonstrating that Wnt signaling in neurons not only
supports synaptic functions and neuronal survival but also keeps brain Aβ levels in check. In the
proceeding work published in PNAS, Structure and other journals, we have also discovered a molecular
chaperone for LRP6 called Mesd and firmly established cellular trafficking and signaling pathways of LRP6,
which mediates canonical Wnt signaling. Other LRP-mediated signaling pathways include LRP1-regulated
leptin signaling in the hypothalamus that regulates body homeostasis and LRP1-regulated insulin signaling
that promotes neuronal survival and cerebral glucose metabolism.
a. Liu Q, Trotter J, Zhang J, Peters MM, Cheng H, Bao J, Han X, Weeber EJ, Bu G (2010). Impaired
brain lipid metabolism in lipoprotein receptor-deficient mice leads to progressive, age-dependent
synapse loss and neurodegeneration. J Neurosci 30:17068-17078. PMCID: PMC3146802.
b. Liu Q, Zhang J, Zerbinatti C, Zhan Y, Kolber BJ, Herz J, Muglia LJ, Bu G (2011). Lipoprotein
receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous
system. PLoS Biol 9(1):e1000575. PMCID: PMC3019112.
c. Liu CC, Tsai CW, Deak F, Rogers J, Penuliar M, Sung YM, Maher JN, Fu Y, Li X, Xu H, Estus S,
Hoe HS, Fryer JD, Kanekiyo T, Bu G (2014). Deficiency in LRP6-mediated Wnt signaling
contributes to synaptic abnormalities and amyloid pathology in Alzheimer's disease. Neuron 84:6377. PMCID: PMC4199382.
d. Liu CC, Hu J, Tsai C-W, Yue M, Melrose HL, Kanekiyo T, Bu G (2015). Neuronal LRP1 regulates
glucose metabolism and insulin signaling in the brain. J Neurosci. In press.
3. Life Cycle of LRP1, From Ligand Identification to Biogenesis and Trafficking: LRP1 at 600 kDa is one
of the largest cell surface receptors, binding over 30 structurally and functionally distinct ligands. LRP1 also
adopts a complex structure including >100 disulfide bonds in the extracellular domains and multiple
trafficking signals within its cytoplasmic tail. Thus, how cells make such a large and complex receptor and
how such a “valuable” receptor is used by cells became important questions in addressing the biological
functions of LRP1. Our work, published in the EMBO J and several JBC papers, first demonstrated that a
molecular chaperone termed receptor-associated protein (RAP) is essential for LRP1 folding in the ER and
subsequent trafficking to the cell surface. We then showed that LRP1, using multiple endocytosis signals,
has the fastest known endocytosis rate with a half time of internalization at <0.5 min. In another EMBO J
paper, we discovered that an adaptor protein called sorting nexin 17 that binds to a recycling motif in the
LRP1 cytoplasmic tail and promotes its recycling. Thus, LRP1 as a major metabolic receptor has the
fastest endocytosis rate and also efficient recycling supported by a specific adaptor protein. With a job of
transporting a large number of ligands, LRP1, analogous to the function of an escalator, transports many
ligands into the cells in the most efficient fashion. Such systematic work addressing the biogenesis,
trafficking and processing of LRP1 has allowed subsequent studies on the biological function and
pathological dysfunction for LRP1.
a. Bu G, Williams S, Strickland DK, Schwartz AL (1992). Low-density lipoprotein receptor-related
protein/α2-macroglobulin receptor is a hepatic receptor for tissue-type plasminogen activator. Proc
Natl Acad Sci USA 89:7427-7431.
b. Bu G, Geuze HJ, Strous GJ, Schwartz AL (1995). 39-kDa receptor-associated protein is an ER
resident protein and molecular chaperone for LDL receptor-related protein. EMBO J 14:2269-2280.
c. Li Y, Marzolo, MP, van Kerkhof P, Strous GJ, Bu G (2000). The YXXL motif, but not the two NPXY
motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related
protein. J Biol Chem 275:17187-17194.
d. Van Kerkhof P, Lee J, McCormick L, Tetrault E, Lu W, Schoenfish M, Oorschot V, Strous GJ,
Klumperman J, Bu G (2005). Sorting nexin 17 facilitates LRP recycling in the early endosome.
EMBO J 24:2851-2861. PMCID: PMC1187941.
4. Lessons from Human Brains: Clinical and pathological studies have and will continue to teach us about
the relevance of our studies using model systems in human brains. Our work at Mayo Clinic also includes
close collaborations with neurologists and neuropathologists. In collaboration with Drs. Dennis Dickson and
Ron Petersen, we analyzed the brain regional distributions of various AD-related molecules in brain
samples from non-demented individuals. We found that while the levels of synaptic markers positively
correlate with those of soluble Aβ, the levels of apoE negatively correlate with those of insoluble Aβ. These
results support a function of synaptic activity in promoting Aβ generation and an important role of apoE in
promoting Aβ clearance. In a more recent study using brain samples from individuals with normal aging,
pathological aging, familial AD and sporadic AD, we discovered distinct pathological features between
familial and sporadic AD. Specifically, while synaptic activity correlates strongly with brain Aβ levels in
sporadic AD; such correlation is much weaker in familial AD. Rather, in familial AD, Aβ levels correlate the
best with those of APP and its processing products. These studies, published in Acta Neuropathologica
and Brain, have strong implications in both disease mechanisms and design of mechanism-based therapy.
Such work also exemplified the power of collaboration and team-based science, allowing AD studies going
from patients to bench, and then back to patients.
a. Shinohara M, Petersen RC, Dickson DW, Bu G (2013). Brain regional correlation of amyloid-β with
synapses and apolipoprotein E in non-demented individuals: potential mechanisms underlying
regional vulnerability to amyloid-β accumulation. Acta Neuropathol 125:535-547. PMCID:
PMC3612369.
b. Shinohara M, Fujioka S, Murray ME, Wojtas A, Baker M, Lecrux AR, Rademakers R, Das P, Parisi
JE, Graff-Radford NR, Petersen RC, Dickson DW, Bu G (2014). Regional distribution of synaptic
markers and amyloid precursor protein correlate with distinct clinicopathological features in sporadic
and familial Alzheimer’s disease. Brain 137:1533-1549. PMCID: PMC3999719.
5. TREM2 and APOE, Two Strong Genetic Risk Factors Linked in Neuroinflammation and AD: The
discovery of TREM2 as a strong risk factor for AD, along with other microglia-related genes including CD33
and APOE, has reignited the interests in addressing the roles of microglia and brain innate immunity in AD
etiology. As a significant contribution to the field, we have recently discovered that apoE is a ligand for
TREM2, thus establishing a functional relationship between the two strong genetic risk factors for AD. In
the process, we have also addressed the molecular and cellular mechanisms of TREM2 signaling including
a dissection on the function of TREM2 adaptor protein DAP12. These studies, published in the Journal of
Biological Chemistry, further demonstrated the critical roles of inflammation pathways in the CNS and AD.
a. Zhong L, Chen XF, Zhang ZL, Wang Z, Shi XZ, Xu K, Zhang YW, Xu H, Bu G. DAP12 stabilizes the
C-terminal fragment of the triggering receptor expressed on myeloid cells-2 (TREM2) and protects
against LPS-induced pro-inflammatory response. J Biol Chem 2015; 290:15866-15877. PMCID:
PMC4505493.
b. Atagi Y, Liu CC, Painter MM, Chen XF, Verbeeck C, Zheng H, Li X, Rademakers R, Kang SS, Xu H,
Younkin S, Das P, Fryer JD, Bu G. Apolipoprotein E is a ligand for triggering receptor expressed on
myeloid cells 2 (TREM2). J Biol Chem 2015; 290:26043-26050. PMCID: PMC4560063.
Complete List of Published Work in MyBibliography:
http://www.ncbi.nlm.nih.gov/sites/myncbi/guojun.bu.1/bibliography/40915021/public/?sort=date&directi
on=ascending
D. Research Support
Ongoing Research Support
NIH/NIA, R01-AG027924
Bu (PI)
04/01/06-05/31/17
“ApoE receptor pathways and abeta metabolism"
The major goal of this project is to define how apoE receptors LRP1 and HSPG regulate brain Aβ metabolism.
NIH/NINDS, P01-NS074969
Holtzman (PI)
05/15/12-04/30/17
Project 3, Bu (PI): "Lipoprotein receptor and synaptic regulation of Aβ metabolism"
The major goal of this project is to examine how synaptic activity regulates brain Aβ production and clearance
through lipoprotein receptors.
NIH/NIA, R01-AG046205
Bu (PI)
“ApoE isoform-specific therapy for Alzheimer disease”
09/30/13-05/31/18
The major goal of this project is to use mouse models to establish therapeutic strategies targeting apoE for AD
therapy.
NIH/NIA, RF1-AG051504
Bu/Taner (MPI)
09/30/15-08/31/20
“Integrative translational discovery of vascular risk factors in aging and dementia”
The goal of this project is to use genetic, systems-based, and functional approaches to discover and
characterize pathways that represent risks for vascular diseases and AD.
NIH/NIA, R01-AG035355
Bu (PI)
09/01/10-05/31/20
“ApoE and LRP1 in brain insulin signaling and glucose metabolism”
The goal of this project is to address the effects of apoE isoforms and apoE receptor LRP1 in brain insulin
signaling and glucose metabolism using cellular and animal models.
NIH/NIA, P50-AG016574
Petersen (PI)
05/01/14-04/30/19
Project 3 Bu (PI): “ApoE isoforms in brain vasculature and CAA”.
The goal of this project is to investigate how different isoforms of apoE regulate brain vascular functions and
the pathogenesis of CAA.
Completed Research Support
NIH/NIA, P01-AG030128
LaDu (PI)
08/15/09-06/30/14
Project 3, Bu (PI): “LRP and APP processing in neurodegeneration”
The major goal of this project is to analyze the mechanism and regulation of LRP and APP proteolytic
processing in normal and Alzheimer’s disease brains.
Alzheimer’s Association Zenith Fellows Award (Bu)
09/01/08-08/31/11
Title: LRP and apoE isoforms in brain synaptic functions
The major goal of this grant is to study the roles of apoE isoforms and apoE receptor LRP1 in synaptic
functions under normal physiological and AD pathological conditions.
3URJUDP'LUHFWRU3ULQFLSDO,QYHVWLJDWRU/DVW)LUVW0LGGOH
Petersen, Ronald, C
RESOURCES
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Our Mayo Clinic is conducting a longitudinal observational study, called Mayo Clinic Study of Aging. In this
comprehensive prospective study, cognitive status and other health information of more than 2000 subjects
with APOE genotype are periodically being followed-up (Dr. Ronald Petersen, Director). All subjects have
frozen plasma, and subset of them have frozen CSF. We can assess these samples under the approval of
Mayo Clinic IRB. Our laboratory is adequately equipped with advanced equipment/instruments to perform
statistical analyses and biochemical experiments in this proposed study, including statistical softwares (JMP,
SAS, and R), ELISAs (Multi-Channel Pipettes, Theremo Fisher Scientific Inc; 96 well automated Microplate
Washer and Dispenser connected with Plate stacker, BioTek; and Multi-mode Microplate Reader connected
with Plate stacker, BioTek) and Western blotting (Odyssey Infrared Imaging System, LI-COR Biosciences).
We also have expert biomecial statisticians (Dr. Julia Crook and Mr. Mike Heckman etc.) for consulting
statistical analyses, and animal facility to analyze mouse models in the potential future studies. Our facility
has several seminar series, and meetings with other researchers and clinicians studying Alzheimer’s disease.
Research fellows, including me, have periodical chances to present the research progress in front of them.
These environments are suitable to pursue the aims in this proposed study as well as further future
investigations.
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Checklist Form Page
200 First Street SW
Rochester, Minnesota 55905
507-284-2511
January 5, 2016
Ronald C. Petersen, Ph.D., M.D.
Cora Kanow Professor of
Alzheimer’s Disease Research
507-538-0487, Fax 507-538-6012
Walter A. Kukull, Director
National Alzheimer’s Coordinating Center
University of Washington
4311 11th Ave NE Ste 300
Seattle, WA 98105
Re: Application for Junior Investigator funding
Dear Bud:
I am pleased to write to you in support of the grant application titled “Effects of APOE genotype on
longevity,” which is being submitted by Mitsuru Shinohara for NACC funding. As the Director of the
Mayo Clinic Alzheimer’s Disease Research Center, I fully support this application and am pleased
that Mitsuru Shinohara is interested in engaging in a secondary analysis of the NACC data.
Mitsuru Shinohara is certified in the protection of human subjects, as mandated by DHHS, and has
completed our facility’s comprehensive requirements to conduct this research in a responsible
manner.
I am aware of the consortium grant policies established by the National Institutes of Health, and if
this project is approved by the NACC Steering Committee, I will accept responsibility for disbursing
the awarded funds to Mitsuru Shinohara and tracking project costs. If I may be of further assistance,
please feel free to contact me.
Yours sincerely,
Ronald C. Petersen, Ph.D., M.D.
Professor of Neurology
Distinguished Mayo Clinic Investigator
Cora Kanow Professor of Alzheimer’s Disease Research
Cadieux Director, Mayo Alzheimer’s Disease Research Center
Director, Mayo Clinic Study of Aging
rcp/djc
A. Research question/hypothesis and brief rationale
Given the rising rate of survival into advanced old age, achieving longevity and healthy aging have become
increasingly important in our society. The ε4 allele of the apolipoprotein E gene (APOE4) and the ε2 allele
(APOE2) are risk and protective factors, respectively, for sporadic late-onset Alzheimer’s disease. In addition,
APOE is also associated with longevity; several cross-sectional studies comparing centenarians and younger
adults showed higher frequency of APOE2 in centenarians and lower frequency of APOE4. Despite such
interesting observations, few longitudinal studies exist demonstrating APOE effects on longevity. Moreover, it
remains unclear whether APOE effects on longevity are mediated by affecting cognitive decline or AD
neuropathology, and whether there are sex-dependent effects. Mechanistically, studies on APOE-associated
cholesterol during aging in human are scarce. Through accessing comprehensive longitudinal NACC clinical
records and a large collection of plasma/CSF samples from Mayo Clinic Study of Aging, we will address how
APOE contributes to longevity.
Specific Aim 1.Determine the effects of APOE on longevity, depending on cardiovascular health, cognitive
decline, and Alzheimer’s neuropathology in NACC cohorts: By reviewing prospective clinical records of
NACC cohorts, we will assess whether APOE affects longevity in these cohorts, and how gender, cognitive
decline, and cardiovascular disease influence this effect. Moreover, by retrospectively analyzing the relationship
between APOE-mediated longevity and AD neuropathology, we will determine whether the effects of APOE on
longevity are mediated through reducing AD neuropathology.
Specific Aim 2. Address the mechanism underlying APOE-regulated longevity by examining the association
among aging-related biomarkers and apoE-associated cholesterol: By using human plasma and CSF, we will
assess changes of levels of biomarkers associated with apoE-cholesterol metabolism during aging. By analyzing
the levels of apoE, total cholesterol, apoE-associated cholesterol, and other APOE or aging-related molecules in
human plasma and CSF, we will determine the relationship between apoE-cholesterol metabolism and aging, and
address how APOE genotype contributes to longevity.
B. Background
APOE gene: Apolipoprotein E (apoE, protein; APOE, gene) is a polymorphic apolipoprotein synthesized
primarily by the astrocytes in the brain and by the liver in the periphery. ApoE transports cholesterol and other
lipids via apoE receptors. In the brain, apoE plays important roles in maintaining synaptic function and
controlling neuroinflammation. The APOE gene has three major allelic variants (ε2, ε3, and ε4), which code for
three isoforms of apoE protein (apoE2, apoE3 and apoE4). The three apoE isoforms differ from each other by
single amino acid substitutions at residues 112 and 158; apoE3 contains a cysteine and arginine at these positions,
apoE2 has two cysteines and apoE4 two arginines. The differences of these two amino acid residues influence the
activity of apoE in a isoform-specific manner in both healthy and pathological states [1]. Indeed, compared to the
common APOE3 allele, one APOE4 allele increases the risk of AD by 3-4 fold, whereas one APOE2 allele
reduces the risk by approximately half [2]. APOE is also associated with cardiovascular diseases, though
controversies exist regarding the effect of each APOE genotype [3]. Moreover, by reviewing NACC records and
performing animal studies, we recently observed that APOE affects cognitive decline during aging independently
of AD neuropathology, but is associated with apoE-cholesterol metabolism (manuscript in revision at Annals of
Neurology). These results indicate that APOE genotype significantly impact the risk for central and peripheral
diseases associated with aging, although the mechanisms underlying these risks are not clear.
APOE and longevity: APOE is a thoroughly studied gene that has been associated with longevity. Several crosssectional studies have shown a lower frequency of APOE4 allele and a higher frequency of APOE2 allele in the
elderly people [4-7]. However, these cross-sectional analyses have limitations as they assume the same allele
frequency between time and place [8]. More importantly, such analyses cannot distinguish the confounding effects
of APOE on cognitive impairment or cardiovascular diseases, which are also associated with earlier mortality. In
longitudinal analyses, APOE effects were sometimes conflicting likely due to the limitations of their study design
or number of enrolled subjects [9-11]. Thus, it remains unclear how APOE contributes to longevity. By reviewing
large comprehensive records of NACC Uniform Data Set (UDS), which longitudinally follows cognitive status,
cardiovascular health, and other health information until death in voluntary-recruited subjects with APOE
genotype, we will study the effects of APOE genotype on longevity by adjusting for these confounding factors.
APOE-associated cholesterol metabolism: Though some controversies exist, APOE4 is also associated with
lower levels of apoE, while APOE2 associates with higher levels of apoE, in CSF or plasma of cognitively normal
subjects [12-14]. Animal studies clearly show these associations [15]. Of note, apoE levels themselves in CSF or
plasma can be associated with a risk of dementia, independently of APOE genotype [12-14]. APOE genotype also
affects cholesterol levels in plasma and CSF in humans as well as animal models ([3] & Fig. 4 of section D).
Interestingly, several studies reported cholesterol levels in plasma/CSF showed an age-dependent decrease,
despite no change of triglycerides levels, suggesting that cholesterol can be a potential biomarkers for aging [16].
Moreover, several recent studies, including ours, showed that up-regulation of lipidation status of apoE can
benefit synaptic formation, cognitive function and inflammation of mouse models without amyloid accumulation
[17-19], indicating that apoE-associated cholesterol might be an important key to understand the effects of APOE
on cognitive decline as well as other health issues, possibly including longevity. In this proposed study, we will
determine the levels of apoE, cholesterol, apoE-associated cholesterol, and other APOE-related molecules in CSF
and plasma of subjects of Mayo Clinic Study of Aging.
C. Overall significance of the research
Life expectancy and the rate of survival into old age have risen dramatically throughout the past century in the
United States. Achieving longevity and healthy aging, which can be defined in a variety of ways, is thus gaining
prominence in the popular press and is of increasing concern to older adults and their families, from a public
health perspective. Though several studies have observed that APOE is associated with longevity, most of these
studies were based on cross-sectional designs, which has methodological limitations due to their questionable
assumptions that the relative allele frequency and risk of mortality of APOE do not interact with environmental
factors, which actually vary by time and place [8]. Moreover, it remains unclear whether APOE-associated
longevity is mediated through cognitive impairment or cardiovascular diseases, which are also associated with
earlier mortality. In addition, even if it is true, the detail mechanism underlying how APOE contributes to
longevity is still unclear. Indeed, by reviewing NACC records and analyzing animal models, we recently observed
that APOE2 protects against age-related cognitive decline independently of AD neuropathology, but associated
with apoE-cholesterol metabolism (manuscript in revision at Annals of Neurology). Thus, in the current proposal,
by using NACC UDS, which is a large and cumulative longitudinal prospective record of cognitive and other
health status in volunteer persons (n > 20,000), we will confirm the effects of APOE on longevity, and also assess
the relationships among APOE, cognitive status, cardiovascular diseases, and longevity. In addition, by reviewing
NACC neuropathology (NP) data, we will analyze how AD neuropathology affects APOE-regulated longevity.
Moreover, we will analyze potential changes of APOE-associated cholesterol metabolism during aging in human
plasma and CSF of subjects at Mayo Clinic Study of Aging to dissect the role of APOE-associated cholesterol
metabolism in longevity. These approaches combining comprehensive records of large NACC data and
biochemical analysis of Mayo Clinic cohorts are highly unique and have a potentially innovative impact on the
research regarding the role of apoE in longevity.
Our laboratory, myself in particular, has been investigating the effects of APOE on cognitive decline, AD
neuropathology, lipid metabolism, synaptic function, and neuroinflammation by analyzing human/animal samples
and reviewing NACC clinical records, as shown in several publications [17,20-22] and currently revised
manuscript at Annals of Neurology (see D. Methodological development and innovative strategies). Based on
results of this proposed study, our eventual goal is to understand the precise mechanism of APOE-regulated
longevity and apply obtained knowledge to promote healthy aging in the society.
D. Methodological development and innovative strategies
APOE effects on cognitive decline, and relationship with neuropathology: Recently, we have reviewed NACC
clinical records, and found out that APOE2 protects against cognitive decline during aging independently of
amyloid pathology. By reviewing subjects whose APOE genotypes were characterized (n=21,531), we used a
unique NACC variable “NACCAGED” in UDS records to define the onset of cognitive decline, which was
determined by clinicians after consulting with medical records, direct observation and subject/informant report.
This variable was used as a dependent variable, and gender, education and APOE genotype were used as
independent variables in the Cox proportional hazard model, where subjects who did not show any cognitive
decline until the last visit were rightcensored (33.7% of total subjects). In
this model, APOE4 accelerated
cognitive decline, while APOE2
subjects were protected from cognitive
decline, compared to control APOE3
homozygous subjects (Fig. 1A).
Moreover, we reviewed NACC NP
records and defined 492 individuals
who had minimal amyloid pathology
Fig. 1. APOE affects cognitive decline during aging in total NACC
(CERAD neuritic plaque score =0, and
subjects (A) and subjects with minimal amyloid pathology (B)
CERAD diffuse plaque = 0, 1 or
undefined). In these subjects, protective effects of APOE2 and
harmful effects of APOE4 were still observed, indicating that
APOE genotype-dependent effects on cognitive decline are
independent of Aβ accumulation (Fig. 1B). In addition, effects of
APOE genotype on cognitive decline scores (CDR sum of boxes
(SOB), and CDR memory) in elderly subjects (>70 years old at
death, n=2,163) were observed in the multiple linear regression
models by adjusting for amyloid accumulation as well as tau and
vascular pathology (Fig. 2). These results are currently in
revision for publication in the Annals of Neurology. These studies
demonstrate that we have mastered strong expertise to uniquely
Fig. 2. APOE affects CDR SOB (A) and memory
assess NACC UDS and neuropathology records to address the
(B) after adjustment for AD neuropathology
effects of APOE by using several statistical methods, which
should facilitate our analysis of NACC records in the proposed study.
Development of biochemical assays to
detect apoE-cholesterol metabolism
and other APOE or aging-associated
molecules: By using in-house developed
ELISA, we observed that APOE2 is
associated with higher apoE levels, while
APOE4 is associated with lower apoE
levels, compared to APOE3, in the
brains, CSF and plasma of mouse models
expressing human apoE2, apoE3, or
Fig. 3. ApoE levels in brain (A), CSF (B) and plasma (C) of apoE-TR mice
apoE4, under the control of the mouse
apoE promoter (target replacement or “TR” mice) (Fig. 3). These results are consistent with those from previous
studies [15]. Moreover, we observed that APOE2 is associated with higher cholesterol levels in CSF and plasma,
but with lower cholesterol levels in the brain parenchyma of these mice (Fig. 4). In addition to these biochemical
assays, we have recently established a
novel cholesterol assay that enables us to
measure cholesterol levels associated
with apoE protein [22]. In this assay,
apoE-lipid particles in biological
samples are initially incubated with antiapoE rabbit polyclonal antibodies
conjugated with biotin (Meridian).
Streptavidin beads are then used to
immunoprecipitate apoE-lipid particles.
Fig. 4. Cholesterol levels in brain (A), CSF (B) and plasma (C) of apoE-TR mice
Cholesterol levels associated with
immunoprecipitated apoE-lipid
particles are determined by Amplex®
cholesterol assay kit (Invitrogen).
We’re now in the process of
determining the levels of other lipids
associated with apoE particles. This
innovative technology should allow us
to address the specific roles of both
apoE and apoE-transported
cholesterol, as recent studies have
Fig. 5. Age- and APOE-dependent changes of IL1β (A), TNFα (B) and
shown that lipidation status of apoE can
MDA (malondialdehyde, C) levels in brain of apoE-TR mice
play critical roles more than apoE protein itself [17,18,23]. Moreover, by using in-house developed ELISAs and
commercially available biochemical assays, we have also analyzed several molecules associated with
inflammation or mitochondrial oxidative stress in mouse models, and observed that these levels showed agedependent changes as well as APOE-dependent changes (Fig. 5). In the proposed study, we will use these in-house
developed ELISAs or biochemical assays to determine the potential changes of apoE-cholesterol metabolism, and
other APOE or aging-related molecules in human CSF and plasma during aging.
E. Analytic approach
Specific Aim 1. Determine the effects
of APOE on longevity, depending on
cognitive decline, cardiovascular
health, and Alzheimer’s
neuropathology in NACC cohorts:
NACC UDS or MDS data record of
subjects with APOE genotype and
cognitive status will be obtained
through the help of Dr. Lilah M. Besser
at the University of Washington (Table
1). To assess changes over time in the
Table 1. NACC cohorts with cognitive status and APOE genotype
distribution of APOE genotypes, chisquare statistics will be used. Empirical
survival curves will be produced using the Kaplan-Meier method to estimate age-specific proportions of surviving
individuals with different APOE genotypes. We will use cox proportional hazard model with delayed entry to
assess the effects of APOE on mortality by adjusting for gender, education, cardiovascular disease and cognitive
status. We will also examine the interaction between APOE and these covariates. We will assess the effects of
APOE on mortality separately for gender, presence/absence of cardiovascular disease or dementia. For all Cox
regression models, we will test the proportional hazard assumption globally and for each covariate. To confirm
findings of Cox models, logistic regression models adjusting for age, gender, education, cardiovascular disease
and cognitive status will be used to evaluate the effects of APOE on mortality. In subsequent analyses, we will use
NP data records to address the relationship between APOE-mediated mortality and AD neuropathology. First, we
will analyze the effects of APOE on mortality in subjects without AD neuropathology by Cox regression models.
Second, we will analyze the effects of APOE on mortality by a logistic regression model adjusting for age, gender,
education, cardiovascular disease, cognitive status and AD neuropathology.
Specific Aim 2. Address the mechanism underlying APOE-regulated longevity by examining the association
among aging-related biomarkers and apoE-associated cholesterol. Human plasma and CSF samples from
subjects of Mayo Clinic Study of Aging will
be obtained through the support of Dr.
Ronald C. Petersen at Mayo Clinic (also see
his support letter). This study is a
comprehensive longitudinal study for a large
cohort of middle aged-elderly individuals,
providing a very unique opportunity to
assess age-related changes (Table 2 & [24]).
We will focus on a subset of individuals to
reveal the relationship between APOE and
aging/longevity (n=~300), including three
APOE genotype (E2/2 or E2/E3, E3/E3, and
E4/E3 or E4/E4) and relatively younger and
Table 2. CSF/Plasma samples of Mayo Clinic Study of Aging
older subjects. We will measure levels of
apoE, cholesterol, apoE-associated cholesterol to address the potential changes of apoE-cholesterol metabolism
during aging as a function of APOE genotype (Table 3). Moreover, we will measure levels of mitochondrial or
oxidative stress markers, and inflammatory
markers that are reported to be associated with
aging (Table 3). We will assess the effects of
APOE genotype on the levels of these apoEcholesterol or aging-related molecules by
multiple regression analysis adjusting for age or
separating for different age categories if
interactions exist between APOE and age.
Conversely, we will also assess the effects of
aging on the levels of apoE-cholesterol or agingTable 3. APOE or aging-related molecules assessed in this study
related molecules by multiple regression analysis
adjusting for APOE or separating for each APOE
genotype if interactions exist between APOE and age. Potential interaction between these apoE-cholesterolrelated molecules and aging-related molecules will also be assessed by multivariate analyses and multiple
regression analyses. Gender, cognitive status, and cardiovascular health will always be considered for potential
confounding factors. By performing these analyses, we will address how apoE-cholesterol metabolism changes
during aging, and interacts with other aging-related pathways.
F.
Potential NACC data elements to be used
From UDS:
NACCID,VISITMO,VISITDAY,VISITYR,NACCVNUM,NACCDIED,NACCMOD,NACCYOD,NACCAUTP,N
ACCACTV,NACCNOVS,NACCDSMO,NACCSDY,NACCDSYR,NACCNURP,NACCNRMO,NACCNRDY,N
ACCNRYR,NACCFTD,NACCMDSS,NACCPAFF,NACCREAS,NACCREFR,BIRTHMO,BIRTHYR,SEX,HISP
ANIC,HISPOR,HISPORX,RACE,RACEX,RACESEC,RACESECX,PRIMLANG,PRIMLANX,EDUC,MARIST
AT,NACCLIVS,INDEPEND,RESIDENC,HANDED,TOBAC30,TOBAC100,SMOKYRS,PACKESPER,QUITS
MOK,ALCOCCAS,ALCFREQ,CVHATT,HATTMULT,HATTYEAR,CVAFIB,CVANGIO,CVBYPASS,CVPAC
DEF,CVPACE,CVCHF,CVANGINA,CVHALVE,CVOTHR,CVOTHX,CBSTROKE,STROKMUL,NACCSTYR,
CBTIA,TIAMULT,NACCTIYR,PD,PDYR,PDOTHR,PDOTHRYR,DIABETES,DIABETYPE,HYPERTEN,HYP
ERCHO,B12DEF,THROID,ARTHRIT,ARTHTYPE,ARTHTYPX,BPDIAS,HRATE,BPSYS,HYHYPER,HXSTR
OKE,HACHIN,CVDCOG,STROKCOG,CVDIMAG,CVDIMAG1,CVDIMAG2,CVDIMAG3,CVDIMAG4,CVD
IMAGX,MEMORY,ORIENT,JUDEMENT,COMMUN,HOMEHOBB,PERSCARE,CDRSUM,CDRGLOB,DEC
AGE,MMSECOMP,MMSELOC,MMSELAN,MMSELANX,MMSEVIS,MMSEHEAR,MMSEORDA,MMSEOR
LO,PENTAGON,NACCMMSE,LOGIMEM,MEMUNITS,MEMTIME,WHODIDDX,DXMETHOD,NORMGOC
,DEMENTED,NACCUDSD,AMNDEM,PCA,NACCPPA,NACCPPAG,NACCPPME,NACCBVFT,NACCLBDS,
MAMNDEM,NACCTMCI,IMAGLINF,IMAGLAC,IMAGMACH,IMAGMICH,IMAGMWMH,IMAGEWMH,N
ACCALZD,NACCALZP,PROBAD,PROBADIF,POSSAD,POSSADIF,NACCLBDE,NACCLBDP,PARK,CVD,C
VDIF,PREVSTK,STROKDEC,STKIMAG,INFNETW,INFWMH,VASC,VASCIF,VASCPS,VASCPSIF,STROKE,
STROKIF,HIV,HIVIF,ALCDEM,ALCDEMIF,ALCABUSE,CANCER,CANSITE,DIABET,MYOINF,CONGHRT
,AFIBRILL,HYPERT,ANGINA,HYPCHOL,VB12DEF,THYDIS,ARTH,ARTYPE,NACCAVST,NACCNVST,N
ACCAGE,NACCAGEB,NACCNIHR,NACCNINR,NACCNORM,NACCIDEM,NACCMCII,NACCETPR
From NP:
NACCiD,NACCADC,NPfoRMVeR,NPSeX,NACCDAGe,NACCMoD,NACCYoD,NACCiNT,NPPMiH,NPfiX,
NPfiXX,NPWBRWT,NPWBRf,NACCNBRN,NPGRCCA,NPGRLA,NPGRHA,NPGRSNH,NPGRLCH,NACCA
VAS,NPTAN,NPTANX,NPABAN,NPABANX,NPASAN,NPASANX,NPTDPAN,NPTDPANX,NPHiSMB,NPHi
SG,NPHiSSS,NPHiST,NPHiSo,NPHiSoX,NPTHAL,NACCBRAA,NACCNeUR,NPADNC,NACCDiff,NACCV
ASC,NACCAMY,NPLiNf,NPLAC,NPiNf,NACCiNf,NPHeM,NPHeMo,NPMiCRo,NPoLD,NACCMiCR,NPoL
DD,NACCHeM,NACCARTe,NPWMR,NPPATH,NACCNeC,NPPATH2,NPPATH3,NPPATH4,NPPATH5,NPPAT
H6,NPPATH7,NPPATH8,NPPATH9,NPPATH10,NPPATH11,NPPATH0,NPPATHoX,NPART,NPoANG,NACCLe
WY,NPLBoD,NPNLoSS,NPHiPSCL,NPSCL,NPfTDTAU,NACCPiCK,NPfTDT2,NACCCBD,NACCPRoG,NPf
TDT5,NPfTDT6,NPfTDT7,NPfTDT8,NPfTDT9,NPfTDT10,NPfRoNT,NPTAU,NPfTD,NPfTDTDP,NPALSMN
D,NPofTD,NPofTD1,NPofTD2,NPofTD3,NPofTD4,NPofTD5,NPfTDNo,NPfTDSPC,NPPDXA,NPPDXB,NAC
CPRio,NPPDXD,NPPDXe,NPPDXf,NPPDXG,NPPDXH,NPPDXi,NPPDXJ,NPPDXK,NPPDXL,NPPDXM,NP
PDXN,NACCDoWN,NPPDXP,NPPDXQ,NACCoTHP,NACCWRi1,NACCWRi2,NACCWRi3
From MDS:
MDB001,ADCID,PTID,ABSMO,ABSDAY,ABSYEAR,BIRTHMO,BIRTHDAY,BIRTHYR,SEX,RACE,HISPA
NIC,PRIMLANG,EDUC,MARISTAT,RESIDENC,FEVALMO,FEVALDAY,FEVALYR,MMSEFRST,REVALM
O,REVALDAY,REVALYR,MMSELAST,CLINDEM,NOTDEMCI,AGEDEM,CLDEMDX,CLDEMLEW,NONA
DDEM,STROKE,PDNODEM,DEP,DEL,VITALST,DEATHMO,DEATHDAY,DEATHYR,AUTOPSY,NPTHPRI
M,NPTHSECN,APOE,NPSYCH,MMSEMO,MMSEDAY,MMSEYR,CONTMO,CONTDAY,CONTYR,ACTIVE
G. Plans for publication
We will finish analyzing NACC data and obtain all biochemical data from human CSF and plasma during the first
six months of the funding period, and prepare and submit a manuscript to an appropriate peer-reviewed journal in
the remaining six months of the period for publication. If it is difficult to report our findings in one manuscript,
we will separate them into two or three and submit them to appropriate peer-reviewed journals within the funding
period for publication.
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