NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose

“Pilot to determine the relationship between obesity-related cytokines and

cerebrospinal fluid markers of neurodegenerative disease”

A. Clinical Rationale

Alzheimer’s disease (AD) is a common and devastating neurological disease that affects between 6-8%
of adults over age 65. Alzheimer disease is a progressive, neurodegenerative disease and the clinical
syndrome of dementia that results leads to irreversible decline in cognition, function, behavior and
eventually premature death. The neuropathologic hallmarks of AD are tau-protein associated
neurofibrillary tangles (NFT) and increased presence of amyloid plaques composed of insoluble beta-
amyloid. The resultant dementia syndrome of AD is clinically diagnosed based upon impaired memory
and cognition that impairs function and the clinical syndrome often presents years after the
development of the neuropathologic features of AD. Current treatments for AD slow the loss of function
and symptoms of the disease, however available treatments do not delay the neuropathologic
progression. There are more than 5.3 million persons in the U.S. with AD and the prevalence of AD is
projected to increase significantly over the next 20 years due to the aging of the U.S. population. There
is increasing interest in developing primary prevention strategies to delay the onset and halt the
progression to cognitive impairment and clinical dementia due to AD. To develop these preventive
strategies, there is a need to identify higher risk individuals for clinical trials and biomarkers of presence
and/or markers of risk of disease progression that can aid in the development of therapeutic and
preventive strategies. Biomarkers of AD have been limited to cerebrospinal fluid (CSF) biomarkers to
detect tau protein and beta-amyloid 42 and imaging techniques using amyloid markers (PIB), PET, MRI
or SPECT, however a recent meta-analysis of biomarkers showed that memory impairment was a more
accurate predictor of disease than CSF or imaging. Further work is needed and the identification of risk
factors of disease and serum biomarkers of AD would be of interest for the development of clinical
studies to determine risk reduction strategies. A risk profile for AD may also be used to target
therapeutic interventions and develop primary and secondary prevention strategies to reduce or delay
the incidence of dementia. Recent projections indicate that preventive interventions with even a small
effect size could reduce dementia prevalence by 100,000 cases in 10 years, resulting in a significant
public health impact. The proposed pilot study will provide data to assist in the design of a longitudinal,
cohort study of AD risk factors.
There is increasing interest in modifiable risk factors, such as obesity in AD prevention. Overweight
and obesity in mid- and late-life have been related to risk of dementia and AD however, not all studies
have found the same relationship depending upon gender, time of weight measurement (mid versus
late-life). Recently, the Health, Aging and Body Composition study showed that increasing levels of
total fat mass (adiposity), waist circumference and subcutaneous abdominal fat are strongly associated
with worsening cognitive function in men. In women, the same relationship was not seen and there
was an inverse relationship seen with a trend toward less cognitive decline in women with higher
adiposity that was not explained by sex hormone differences. The effect of obesity on cognition and risk
of dementia is not well understood and further study to determine the direct and indirect effects of
obesity on cognition is warranted. We have two previous data analyses of the Women’s Health
Initiative (WHI) and WHI Memory Study (WHIMS) (pending publication) showing effects of obesity on
cognitive decline and incident cognitive impairment in postmenopausal women that suggest that obesity
as a risk factor for AD should be further investigated for future risk and prevention population studies
(Tables 1 and Figure 2). The proposed pilot study will provide preliminary data on the effects of obesity
on CSF biomarkers of AD, and determine biomarker selection for a planned longitudinal cohort study of
the effect of obesity on AD risk.
Obesity and adiposity are associated with inflammatory cytokines, endogenous hormone production,
neurotrophic factors and cytokines directly related to visceral and subcutaneous adiposity known as
adipocytokines. Several of these cytokines, hormones and neurotrophic factors have been associated

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NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose

with cognitive function in both humans and transgenic mouse models of AD. There are several
possible biologic mechanisms that may explain the link between obesity and adiposity to cognitive
impairment. Adipose tissue hormones that cross the blood-brain barrier may influence brain function
and memory either directly or indirectly through known neuropathologic mechanisms. Specifically,
plasminogen activator inhibitor-1(PAI-1) and tissue plasminogen activator(tPA) have been implicated in
Alzheimer’s disease in recent studies of the plasmin proteolysis cascade which found associations
between plasmin generation and amyloid beta(Abeta). Two recent publications have shown enhanced
Abeta catabolism that is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1). PAI-1 in
human CSF has been reported as a marker of neurological disease. Drs. Kerwin and Vaughan have
preliminary (unpublished) serum data showing an association between PAI-1 and body mass index and
central fat mass and an association between serum adiponectin and visual memory function in a non-
demented cohort of elderly women that warrants further investigation of CSF associations. Adiponectin
is an adipocyte-derived secretory factor that is produced by adipocytes and effects energy
homeostasis. Serum levels of adiponectin are related to visceral and subcutaneous adipose tissue.
Gender differences in CSF adiponectin levels have been reported in the literature and may help to
explain the gender differences seen on the relationship of obesity and cognitive function and dementia.
Leptin is a circulating adipocytokine hormone linked to eating behavior through its action in the
hypothalamus and striatal brain regions and more recently leptin has been associated with age-related
cognitive decline. Leptins’ role in cognitive function may be due to regulation of some types of
hippocampal activity. A recent study of the relationship between serum leptin levels and cognitive
function in non-demented, older adults showed that higher leptin levels were associated with poorer
performance on cognitive tests of executive function after adjustment for demographic and medical
conditions. Studies investigating inflammatory marker levels in AD patients have found an association
with elevated levels of interleukin-6(IL-6) secretion compared to healthy controls.

ApolipoproteinE4 is the major apolipoprotein in the brain and APOEε4 is the most common genetic risk
factor for late-onset AD and suggests that cholesterol may play a role in the pathogenesis of AD in the
brain. A recent case-control study reported that obesity and APOEε4 status independently increase risk
of AD. In the proposed study, all participants have APOEε status determined upon enrollment in the
CNADC Clinical Core and the results are in the CNADC Clinical Core data base for this analysis.

The purpose of the proposed study is to begin to investigate the relationship between serum and CSF
levels of obesity-related cytokines, such as PAI-1, leptin, adiponectin, IL6, APOE allele and effect on
CSF tau protein and beta-amyloid 42, biomarkers of neurologic disease in humans .
Hypothesis:
We hypothesize that obesity-related cytokines in the serum and CSF will be related to CSF tau
protein and beta-amyloid levels in elderly participants without dementia.

Specific Aims:
1. To determine the relationship between serum and CSF obesity-related cytokine levels (PAI-1,
leptin,adiponectin, IL6) and CSF levels of total tau protein, phosphorylated tau protein, beta-amyloid 42.

2. To determine the effect of APOE genetic allele status on levels of obesity-related cytokines in the
serum and CSF.

B. Research Methods

The Northwestern University, Cognitive Neurology and Alzheimer Disease Center (CNADC) is one of
thirty-two Alzheimer's Disease Core Centers (ADCC) across the United States funded by the National
Institute on Aging, and one of three state Alzheimer's centers funded by the Illinois Department of
Public Health. The CNADC Clinical Core consists of over 400 persons with and without dementia, all
registrants are followed annually in the CNADC Neurobehavioral Clinic. Clinical Core registrants

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NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose

undergo apolipoprotein genetic allele determination upon enrollment and annual neuropsychological
testing and interviews. The registrants agree to be contacted for invitations to studies of AD and other
dementias. The current study will draw upon registrants without clinical dementia and will include 30
men and 30 women over the age of 60 and enrolled in the CNADC Clinical Core. Male participants will
be invited to participate in the study based upon age and no history of clinical dementia. Female
participants will be selected based upon dual participation in both the CNADC Clinical Core and also
the Northwestern University WHI Ancillary Study by Dr. Sandy Weintraub, the Cognitive Change in
Women Study (CCW). The women enrolled in both CCW and CNADC are selected due to plans for
inclusion in a follow-up longitudinal study of the CCW that is pending funding applications by Dr.
Kerwin. Participants that respond to the invitation to participate will be scheduled an appointment at the
CNADC Neurobehavioral Clinic and consented to participate in this study. At the study visit, standard
measurements of height, weight, waist and hip circumference and serum and CSF will be obtained.
Standard lumbar puncture will be performed under regional anesthesia, performed and supervised by
the CNADC clinical faculty. CSF will be divided into two samples and labeled with a unique identifier,
one 2 mL sample will be collected in a polypropylene transfer tube supplied by the ADMark
Alzeheimer’s Evaluation kit and shipped to Athena Diagnostics, Inc. Measurements of phosphorylated-
tau protein, total-tau protein and AΒ42 peptide in CSF will be determined by ELISA based analysis.
Athena Diagnostics provides a complete report and analysis of results within 7-14 days of receipt of
sample. The remaining CSF volume will be frozen and stored for analysis with the serum samples in
Dr. Vaughan’s lab. Veni-punture will be performed by trained research technicians in the CNADC and
a 5 ml blood sample will be transported for centrifuge in Dr. Vaughan’s lab. Body mass index (BMI) will
be determined and used for data analysis. APOE genetic allele type and cognitive function from annual
testing will be available from the CNADC Clinical Core database will be used for data analysis. The
proposed pilot study will provide preliminary data on the obesity-related cytokines and CSF to assist in
the design of the larger, longitudinal study.

C. Budget Justification

The pilot study application is to pay for the costs of a clinically available CSF analysis service provided
by Athena Diagnostics. The Athena Diagnostics CSF analysis for tau and beta amyloid was chosen
because it is routinely used in the CNADC Neurobehavioral Clinic for AD diagnosis and provides
clinical relevance for possible inclusion in a future, longitudinal cohort study. The costs of the serum
and CSF cytokine analyses will be covered by start-up funds for research related expenses that Dr.
Kerwin has available through the Department of Medicine and will be carried out in Dr. Douglas
Vaughan’s lab. Dr. Kerwin is a new faculty member, hired as Assistant Professor in the clinician
researcher track commencing July 1, 2009. Dr. Kerwin has Departmental support consisting of 80
percent protected research time and research-related start up funds for an initial 2 year period or until
successful application of an RO1 by Dr. Kerwin. The Athena Diagnostics CSF analysis is a crucial part
of Dr. Kerwin’s preliminary work, but the costs of the analysis is prohibitive and exceeds currently
available research funds. This pilot grant funding of $50,000 will allow Dr. Kerwin to obtain important
preliminary data that will be used for a future, planned longitudinal cohort project design and funding
applications.

D. Timeline

0 Enrollment of 30 men and 30 women from the CNADC will begin March 1, 2010 pending IRB
approval. The 30 women will be selected based upon enrollment in the CNADC Clinical Core registry
and also have participated in an ancillary study of the WHI run by Dr. Sandra Weintraub called the
Cognitive Change in Women Study (CCW). These women are selected because they will also be
enrolled in a future, longitudinal follow-up of this cohort that is currently pending funding applications fro

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NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose

July 1, 2010. The 30 male participants will be invited for participation from the CNADC Core registry
based upon age. The CNADC Clinical Core registry participants are followed annually with follow-up
visits and cognitive testing and have agreed to be contacted for participation in studies of AD. The
relationship with the CNADC will facilitate contacting and enrolling the 60 participants for this pilot study
and because the CNADC Clinical Core registry has over 400 active registrants, we do not anticipate
enrollment issues, including refusals or inability to obtain the CSF sample. The Athena kits will be
purchased and stocked in the CNADC Neurobehavioral Clinic and sent out following each participant
evaluation and results stored in a database. We anticipate that enrollment will be completed by
September 30, 2010 and data analysis completed by February 28. 2011. If Dr. Kerwin is successful in
obtaining a Paul Beeson Career Development award at the first submission, the larger, longitudinal
cohort study will begin July 1, 2010 and this pilot cohort will be consented and included in the larger,
longitudinal study.

E. Future Direction

Dr. Kerwin is under the mentorship of Sandra Weintraub, PhD, Marsel Mesulam, MD of the CNADC
and Douglas Vaughan, MD, Chairman of the Department of Medicine (DOM) and is currently applying
for career development awards and Alzheimer’s Association grants to obtain preliminary data for an
RO1 application in the next 2-3 years. Dr. Kerwin was recruited as a clinician researcher and has a
primary faculty appointment in the Department of Medicine and also is affiliated faculty with the CNADC
Neurobehavioral Clinic. Dr. Kerwin collaborates with faculty within Department of Medicine and
CNADC with mentorship and collaboration from Drs. Vaughan, Weintraub and Mesulam to develop a
career in cognitive aging and specifically obesity-related effects on cognition and dementia risk. The
proposed pilot study data will be used to develop a larger, prospective longitudinal study of these
factors and risk of subsequent cognitive decline and AD clinical disease. Dr. Kerwin is planning a
January 7, 2010 application to the Alzheimer’s Association and a January 14, 2010 application to the
Paul Beeson Career Development award. The proposed pilot study will provide valuable preliminary
data to assist in the design of a larger, longitudinal cohort study of AD risk factors and RO1 application.

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 NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose

FIGURES
Table 1: Relationship between BMI and risk of the composite endpoint of probable dementia and/or
MCI. Results from previous data analyses of the WHI Memory Study longitudinal data set showing an
increased risk of incident dementia related to BMI and waist-hip ratio, suggesting a relationship between
adiposity and body fat distribution and cognitive decline.

Adjustment for WHI treatment assignment and age

Interaction between BMI and waist-hip ratio: p= 0.002

Waist-Hip Waist-Hip
Body Mass Ratio <0.80 Ratio >0.80
Index
95% CI Overall p- Hazard 95% CI Overall p-
value Ratio value
<20.0 0.73 [0.32,1.64] 0.67 [0.09,4.98]
20.0-24.9 0.44 [0.24,0.81] 1.81 [1.12,2.93]
25.0-29.9 0.39 [0.21,0.74] 0.02 1.12 [0.71,1.78] <0.001
30.0-34.9 0.65 [0.32,1.30] 0.93 [0.56,1.53]
>35.0 1.00 1.00

Full covariate adjustment

Interaction between BMI and waist-hip ratio: p= 0.01

Waist-Hip Waist-Hip
Body Mass Ratio <0.80 Ratio >0.80
Index
Overall p- Hazard Overall p-
95% CI value Ratio 95% CI value
<20.0 1.43 [0.57,3.59] 1.26 [0.17,9.50]
20.0-24.9 0.76 [0.38,1.50] 2.20 [1.32,3.67]
25.0-29.9 0.51 [0.25,1.04] 0.07 1.27 [0.79,2.05] 0.001
30.0-34.9 0.75 [0.36,1.56] 0.94 [0.56,1.56]
>35.0 1.00 1.00

1.
2.
3.
4.

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NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose

Figure 1: Previous analysis of the WHI baseline data set showing the cross-sectional relationship
between BMI, waist-hip ratio and cognitive performance in non-demented women. This data suggests
an inverse relationship between BMI and cognitive performance in women with lower waist-hip ratio
and a protective effect
in women with higher waist-hip
ratio. This pilot and future longitudinal, cohort studies by Dr. Kerwin will
investigate the effect of obesity and dementia risk.

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NUCATS Institute CTI Pilot Grant Proposal Principal Investigator Kerwin, Diana Rose