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273 Pages·2012·5.05 MB·English

The Pennsylvania State University

The Graduate School

College of Medicine

HFE GENE VARIANTS AND DYSREGULATION OF
CHOLESTEROL AND IRON METABOLISM IN
ALZHEIMER DISEASE AND CANCER

A Dissertation in
Molecular Medicine
by

Fatima Ghazanfar Ali-Rahmani

© 2012 Fatima Ali-Rahmani

Submitted in Partial Fulfillment
of the Requirements
for the Degree of

Doctor of Philosophy
December 2012
The dissertation of Fatima Ali-Rahmani was reviewed and approved* by the
following:

James R. Connor
Distinguished Professor of Neurosurgery, Neural & Behavioral Sciences, and Pediatrics
Vice Chair of Neurosurgery
Dissertation Advisor
Chair of Committee

Cara-Lynne Schengrund
Emeritus Professor of Biochemistry and Molecular Biology

Ian Simpson
Professor of Neural & Behavioral Sciences

Patricia S. Grigson
Professor of Neural & Behavioral Sciences

Jong K. Yun
Associate Professor of Pharmacology
Co-chair of Intercollege Graduate Degree Program in Molecular Toxicology

Charles Lang
Distinguished Professor of Cellular and Molecular Physiology, and Surgery
Co-Chair, Intercollege Graduate Degree Program in Molecular Medicine
*Signatures are on file in the Graduate School
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ABSTRACT
Cholesterol and iron are essential for a number of cellular processes. Disruption of both
cholesterol and iron metabolism have been independently reported to contribute to
several diseases including Alzheimer disease (AD) and cancer. AD is a debilitating
disease characterized by progressive neuronal loss and memory impairment. Although
the underlying cause(s) of AD are not completely understood, iron accumulation and the
resultant oxidative stress are well established contributors to the development of AD.
Normal iron uptake is mediated by HFE (high iron, also called hemochromatosis protein).
H63D and C282Y are two common variants of the HFE protein that are implicated as
disease modifying risk factors for AD and several cancers, respectively. The main
objective of this thesis was to determine whether a relationship exists between iron and
cholesterol metabolism that could be of relevance for understanding the underlying
mechanism of diseases such as AD and cancer. This goal was achieved by studying the
effect(s) on cholesterol metabolism of two specific variants of HFE (H63D and C282Y)
known to affect iron metabolism.
The H63D variant of HFE, associated with dysregulation of iron homeostasis, has been
implicated as a risk factor for Alzheimer disease (AD). Apolipoprotein epsilon 4
(APOE4), a known risk factor for AD, is associated with disruption of cholesterol
metabolism. Recent studies showed that individuals with both the APOE4 allele and the
H63D variant of HFE had a much greater risk and earlier onset of AD than individuals
carrying either the APOE4 allele or H63D variant of HFE alone. Therefore, the
hypothesis that H63D-HFE is associated with development of cognitive impairment by
disrupting cholesterol metabolism thereby contributing to increased neurodegeneration,
and increased memory deficits was investigated. The effect(s) of H63D-HFE on
expression of cholesterol was studied using SH-SY5Y human neuroblastoma cells
transfected to stably express either wild type (WT-) or H63D-HFE. The ~50% reduction
in cholesterol content in cells expressing H63D-HFE was accompanied by a significant
decrease in expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR), and
a significant increase in expression of cholesterol hydroxylase (CYP46A1). Consistent
with in vitro studies, H67D-HFE (homologous to human H63D-HFE) knock-in mice,
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showed a genotype and age dependent decline in brain cholesterol relative to WT-HFE
controls and similar changes in expression of proteins regulating cholesterol metabolism.
The reduction in cholesterol in brains of H67D-HFE mice was accompanied by
a significant decrease in expression of synapse and myelin management proteins,
significant increase in caspase-3 expression, and cortical and hippocampal
neurodegeneration in the brains of 18 and 24 month old mice. Performance on learning
and memory tasks (novel object recognition and Barnes maze) indicated that H67D-HFE
mice had poorer cognitive function than WT-HFE mice. Magnetic resonance imaging of
24 month-old mice showed a greater reduction in the volume of brains in H67D-HFE
mice. Combined these data indicate that the H63D/H67D variant contributes to the
disease process in AD by altering cholesterol metabolism as well as that of iron and that
these changes are accompanied by neuronal loss, brain atrophy, and memory impairment
which are symptoms of AD. The results also provide a possible explanation for why
expression of both APOE4 and H63D-HFE is associated with earlier onset of AD.
Disruptions in the maintenance of iron and cholesterol metabolism have also been
implicated in several cancers. Therefore, in the second part of this work, the effects of
C282Y-HFE on lipid (cholesterol and sphingolipid) metabolism and its possible
relevance to cancer phenotype was also studied. Expression of C282Y-HFE altered
transcription of a number of enzymes involved in cholesterol and sphingolipid
metabolism. More specifically it was associated with significant changes in the
expression of genes involved in cholesterol metabolism were consistent with significant
elevation in total cholesterol content of C282Y-HFE cells. Of particular interest was the
finding that expression of the enzyme sphingosine kinase 1(SphK) and its pro-survival
metabolite, sphingosine-1-phosphate (S1P) was elevated. These effects may contribute to
the increased risk of colorectal, breast, prostate, ovarian, and brain tumors reported for
carriers of C282Y-HFE and explain why it is considered protective against Alzheimer’s
disease while H63D-HFE is a risk factor for neurodegenerative diseases. Changes in the
gene expression seen for proteins involved in cholesterol metabolism, indicate that
increased uptake of cholesterol is the likely cause for the elevation in cellular cholesterol
content seen in C282Y-HFE expressing cells. Since simvastatin is an inhibitor of
cholesterol synthesis and has been proposed to be beneficial for the treatment of certain
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cancers, its effect on the viability of C282Y-HFE cells was ascertained. A higher
concentration of simvastatin was needed to decrease survival of cells expressing C282Y-
HFE than WT-HFE. Because of the significant increase of SphK in C282Y cells, the
effect of a SphK inhibitor (SKI) on cell survival was monitored. Again, WT-HFE cells
were more sensitive to this treatment than C282Y-HFE cells, reflecting the drug resistant
phenotype of C282Y-HFE cells. Treatment of cells with both simvastatin and SKI
revealed a small effect on survival. Collectively, these studies indicated that C282Y-HFE
markedly affected both cholesterol and sphingolipid metabolism and support the
possibility that inhibition of both cholesterol synthesis/uptake and as well as synthesis of
S1P as a potential therapeutic strategy for treatment of cancer in carriers of C282Y-HFE.
Taken together, the studies with both genes variants of HFE provide evidence for the role
of HFE in lipid metabolism, in addition to iron management. These data argue for
stratification of patients in clinical trials based on HFE genotype to better understand
disease mechanism and evaluate therapeutic efficacy of treatments.

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TABLE OF CONTENTS

List of Figures ……………………………………………………………………………ix
List of Tables ………………………………………………………………………….....xi
List of Abbreviations…………………………………………………………………….xii
Acknowledgments...………………………………………………………………........xvii
Chapter 1: Iron, cholesterol, and Alzheimer’s disease……………………………….1
Introduction ……………………………………………………………………………...18
Genetic Risk factors ……………………………………………………………………..20
Mechanisms implicated in AD…………………………………………………………...21
1. Amyloid Hypothesis……………………………………………………………. 22
2. Tau Pathology…………………………………………………………………... 23
3. Mitochondrial Dysfunction …………………………………………………….. 24
4. Oxidative Stress………………………………………………………………… 25
5. Synaptic Faliure ……………………………………………………………….. 26
6. Metal Toxicity………………………………………………………………….. 27
- Iron ………………………………………………………………………… 27
- Iron in AD ………………………………………………………………….. 28
- Iron, HFE, and AD …………………………………………………………. 30
- HFE gene …………………………………………………………………... 31
- Structure of HFE protein……………………………………………………. 32
- Function of HFE ………………………………………………………….. 33
- HFE expression …………………………………………………...………...36
- H63D-HFE and AD………………………………………………………… 36
7. Lipid dyshomeostasis ……………………………………………………………38
Lipid rafts ………………………………………………………………………..38
i) Sphingolipids ………………………………………………………..41
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-Role of sphingolipids in AD…………………………………… 42
ii) Cholesterol …………………………………………………………..44
- Cholesterol metabolism and function in circulation …………...44
- Cholesterol metabolism in the brain …………………………...45
- Brain vs. plasma cholesterol …………………………………...49
- Role of cholesterol in the brain………………………………... 49
- Cholesterol homeostasis and AD pathology …………………...51
- Cholesterol in aging brain ……………………………………...51
- ApoE functions and isoform-specific effects relevant to AD
pathology ………………………………………………………………………………...53
Rationale for thesis ……………………………………………………………………...55
References ………………………………………………………………………………58
Chapter 2: Iron, cholesterol, and Alzheimer’s disease…………………………...…101
Introduction …………………………………………………………………………….101
Experimental Procedures……………………………………………………………… 103
Results ………………………………………………………………………………….111
Discussion ……………………………………………………………………………...121
Figure and legends…………………………………………………………………….. 130
References ……………………………………………………………………………...143
Chapter 3: Effect of HFE variants on sphingolipid expression by SH-SY5Y human
neuroblastoma cells…………………………………………………………………... 155
Experimental Procedures……………………………………………………………….157
Results ………………………………………………………………………………….160
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Discussion……………………………………………………………………………... 162
Figures…………………………………………………………………………………. 165
References ……………………………………………………………………………...173
Chapter 4: C282Y increases cellular proliferation by inducing cholesterol
accumulation…………………………………………………………………………..178
Introduction ……………………………………………………………………………179
Role of iron in cancer …………………………………………………………………..179
C282Y in cancer ……………………………………………………………………….181
Role of cholesterol in cancer …………………………………………………………..182
Methods and materials …………………………………………………………………184
Results ………………………………………………………………………………….185
Discussion ……………………………………………………………………………...188
Tables and figures…………………………………………………………………… 193
References …………………………………………………………………………….199
Chapter 5 Summary and Future perspectives……………………………………. 212
Introduction …………………………………………………………………………..212
Is there a link between iron and cholesterol metabolism? ……………………………215
How does H63D-HFE affect interactions between glial and neuronal cells? ………...219
Potential biomarkers for AD progression ……………………………………………..222
Is it iron accumulation or expression of the HFE variants that is the culprit? ………...224
Therapeutic implications: Is statin therapy appropriate for AD? ……………………...226
References ……………………………………………………………………………..228
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Appendix: Cholesterol, GM1, and Autism
Introduction ……………………………………………………………………………236
Methods………………………………………………………………………………... 238
Results ………………………………………………………………………………….239
Discussion……………………………………………………………………………... 240
Figures ………………………………………………………………………………….244
References……………………………………………………………………………. 247

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List of Figures
Figure 1.1: Role of HFE and its variants in iron regulation ……………………………..35
Figure 1.2: Cholesterol synthesis pathway……………………………………………... 45
Figure 1.3: Mechanism of cholesterol transport between astrocytes and neurons in the
brain ……………………………………………………………………………………..48
Figure 1.4: Schematic to show redistribution of membrane lipids to allow cholesterol
efflux via 24S-HC………………………………………………………………………. 48
Figure 2.1: Effect of expression of H63D-HFE on cholesterol and cholesterol
metabolizing enzymes …..………………………………………………………….......132
Figure 2.2: Effect of cholesterol manipulation on survival of HFE-variant
cells……………………………………………………………………………………. 133
Figure 2.3: Effect of expression of H67D-HFE on expression of cholesterol and
cholesterol metabolizing enzymes in brains of mice ……………………….………….134
Figure 2.4: H67D-HFE alters expression of synaptic proteins ………………………...135
Figure 2.5: Effect of H67D-HFE on expression of proteins involved in myelin
maintenance ………………………………………………………………………….136
Figure 2.6: H67D-HFE induces neurodegeneration..………………………………...137
Figure 2.7: Total brain volume (gray and white matter) of 22 month old WT- and H67D-
HFE expressing mice………………………………………………………................138
Figure 2.8: Effect of expression of H67D-HFE on learning and memory-
Latency measures……………………………………………………………………... 139
Figure 2.9: Effect of expression of H67D-HFE on learning and memory-
Total Distance & errors…………………………………………………………………140
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