Alteration of cholesterol homeostasis in the Huntington's disease brain

196 Pages·2017·5.45 MB·English

by  Fabian Kreilaus

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University of Wollongong Thesis Collection
University of Wollongong Thesis Collections
1954-2016
2015
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Fabian Kreilaus
University of Wollongong
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Kreilaus, Fabian, Alteration of cholesterol homeostasis in the Huntington's disease brain, Doctor of
Philosophy thesis, School of Biological Sciences, University of Wollongong, 2015. https://ro.uow.edu.au/
theses/4627
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Alteration of cholesterol homeostasis
in the Huntington’s disease brain

A thesis submitted in fulfilment of the requirements for the award of the degree
Doctor of Philosophy
from
The University of Wollongong
by
Fabian Kreilaus
(B Biotech. Hons.)

Biological Sciences
2015
Certification
I, Fabian Kreilaus, declare that this thesis, submitted in fulfilment of the requirements for the
award of Doctor of Philosophy, in the Department of Biological Sciences, University of
Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The
document has not been submitted for qualifications at any other academic institution.

Fabian Kreilaus
1st Sep. 2015

ii
Acknowledgements

There are several people I would like to acknowledge for assistance and support through my
candidature. My supervisors Andrew Jenner and Brett Garner for constantly providing excellent
advice and encouragement during all aspects of my research. Henry and Kalani for taking the
time to share their experience and knowledge in the laboratory. This has helped my work run
very smoothly and was much appreciated. Special thanks to Adena for assisting me in many
weeks of tissue collection, where your skilled animal dissections provided me with excellent
brain tissue samples. Also for giving me the most clear-cut advice in terms of laboratory work
and thesis writing, and providing constant emotional support in the last 3 years. I would also like
to acknowledge: the UoW statistical consulting service for providing advice on statistical
analysis; Anthony Hannan for providing R6/1 mice that made research into this animal model
possible; Catriona McLean and the Victorian Brain Bank, supported by The Florey Institute of
Neuroscience and Mental Health, The Alfred and the Victorian Forensic Institute of Medicine
and funded in part by Australia’s National Health & Medical Research Council, Parkinson’s
Victoria and MND Victoria, for providing human post mortem brain tissue.

iii
Publications from this thesis

Fabian Kreilaus, Adena S. Spiro, Catriona A. McLean, Brett Garner, and Andrew M. Jenner, (2015)
Evidence for altered cholesterol metabolism in Huntington’s disease post-mortem brain tissue,
Neuropathology and Applied Neurobiology, doi: 10.1111/nan.12286
Fabian Kreilaus, Adena S. Spiro, Anthony J. Hannan, Brett Garner, and Andrew M. Jenner,
(2015) Brain cholesterol synthesis and metabolism is progressively disturbed in the R6/1
mouse model of Huntington’s disease: A targeted GC-MS/MS sterol analysis, Journal of
Huntington’s Disease 4:305-318
Manuscript in preparation
Fabian Kreilaus, Adena S. Spiro, Anthony J. Hannan, Brett Garner, and Andrew M. Jenner,
Therapeutic effects of anthocyanins and environmental enrichment in R6/1 Huntington’s
disease mice

Conference poster presentations
Fabian Kreilaus, Adena S. Spiro, Andrew M. Jenner, Altered brain cholesterol oxidation and
synthesis in the R6/1 mouse model of Huntington’s disease, Joint Meeting of the Societies for
Free Radical Research of Australasia and Japan in Sydney (2013)
Fabian Kreilaus, Catriona A. McLean, Andrew M. Jenner, Altered cholesterol metabolism and
increased cholesterol peroxidation in human post mortem Huntington’s disease brain,
Australasian Neuroscience Society meeting (2014)
Fabian Kreilaus, Adena S. Spiro, Catriona A. McLean, Andrew M. Jenner, Brain cholesterol
synthesis and metabolism is altered in human Huntington’s disease brain, Australian Lipid
Meeting (2014)
Fabian Kreilaus, Adena S. Spiro, Catriona A. McLean, Andrew M. Jenner, Human Huntington’s
disease brain exhibits cholesterol synthetic and metabolic alterations, Australian Society for
Medical Research NSW annual scientific meeting (2015)

Table of contents

Certification................................................................................................................................ ii
Acknowledgements ................................................................................................................... iii
Publications from this thesis ..................................................................................................... iv
Conference poster presentations ............................................................................................. iv
iv
List of figures ............................................................................................................................. ix
List of tables .............................................................................................................................. xi
List of appendix tables .............................................................................................................. xi
Abbreviations ........................................................................................................................... xii
Abstract ................................................................................................................................... xiv
Chapter 1 Introduction ................................................................................................................. 1
1.1 Cholesterol .......................................................................................................................... 2
1.1.1 Cholesterol in cell membranes .................................................................................... 2
1.1.2 Cholesterol in lipid rafts ............................................................................................... 3
1.2 Cholesterol in the brain ...................................................................................................... 3
1.2.1 Synthesis of cholesterol in the brain ............................................................................ 4
1.2.2 Cholesterol metabolism in the brain ........................................................................... 7
1.2.2.1 Formation of 24(S)-hydroxycholesterol ................................................................ 7
1.2.2.2 Possible regulation of brain cholesterol homeostasis by 24(S)-
hydroxycholesterol ........................................................................................................... 9
1.2.2.3 27-Hydroxycholesterol .......................................................................................... 9
1.2.3 Toxicity of cholesterol metabolites ............................................................................ 11
1.3 Cholesterol oxidation products ......................................................................................... 12
1.4 Phytosterols ...................................................................................................................... 13
1.5 Cholesterol and neurodegenerative disease .................................................................... 15
1.5.1 Huntington's disease .................................................................................................. 15
1.5.2 Symptoms .................................................................................................................. 16
1.5.3 Neuropathology ......................................................................................................... 16
1.5.4 Huntingtin protein ..................................................................................................... 17
1.5.5 Toxicity of mutant huntingtin .................................................................................... 17
1.5.6 Mouse models of Huntington’s disease ..................................................................... 18
1.5.7 Cellular and fly models of Huntington’s disease ........................................................ 20
1.5.8 Alteration of cholesterol homeostasis in Huntington's disease ................................ 20
1.5.9 Alterations of cholesterol biosynthesis in Huntington's disease ............................... 21
1.5.10 Cholesterol levels in Huntington's disease .............................................................. 22
1.5.11 Cholesterol metabolic alterations in Huntington's disease ..................................... 22
1.5.12 Proposed mechanisms altering cholesterol homeostasis in Huntington's disease . 23
1.6 Therapeutics in Huntington's disease mouse models ...................................................... 24
1.6.1 Environmental enrichment and Huntington's disease .............................................. 24
v
1.6.2 Flavonoid supplementation ....................................................................................... 25
1.6.2.1 Anthocyanins and Huntington's disease ............................................................. 25
1.7 Biological markers of neurodegeneration ........................................................................ 27
1.8 Aims................................................................................................................................... 28
Chapter 2 General materials and methods ................................................................................. 30
2.1 Methods of sterol analysis ................................................................................................ 31
2.1.1 Triple Quadrupole GC-MS .......................................................................................... 32
2.2 Materials ........................................................................................................................... 33
2.3 Methods ............................................................................................................................ 34
2.3.1 Mice............................................................................................................................ 34
2.3.2 RotaRod ...................................................................................................................... 34
2.3.3 Hind paw clasping ...................................................................................................... 35
2.3.4 Tissue collection ......................................................................................................... 35
2.3.5 Lipid extraction .......................................................................................................... 35
2.3.6 Triple quadrupole GC-MS sterol analysis ................................................................... 36
Chapter 3 Association of cholesterol metabolism with Huntington's disease progression in R6/1
transgenic mice ........................................................................................................................... 40
3.2 Materials and Methods ..................................................................................................... 42
3.3 Results ............................................................................................................................... 43
3.3.1 Physical phenotype .................................................................................................... 43
3.3.1.1 Weight loss in R6/1 mouse ................................................................................. 43
3.3.1.2 Hind paw clasping phenotype ............................................................................. 45
3.3.1.3 Motor performance ............................................................................................ 45
3.3.1.4 Brain mass ........................................................................................................... 45
3.3.2 Sterol analysis of R6/1 brain tissue ............................................................................ 49
3.3.2.1 Cholesterol synthetic precursors ........................................................................ 49
3.3.2.2 Cholesterol synthetic precursors during ageing ................................................. 50
3.3.2.3 Cholesterol .......................................................................................................... 53
3.3.2.4 Cholesterol metabolites ...................................................................................... 54
3.3.2.5 Cholesterol oxidation products ........................................................................... 57
3.3.2.6 Phytosterols ........................................................................................................ 57
3.4 Discussion .......................................................................................................................... 58
3.4.1 Cholesterol synthesis and metabolism ...................................................................... 59
3.4.1.1 Cholesterol synthetic precursors ........................................................................ 59
3.4.1.2 Age related changes to cholesterol synthetic precursors ................................... 63
vi
3.4.1.3 Cholesterol levels in the R6/1 mouse ................................................................. 63
3.4.1.4 Age related alteration to cholesterol levels in the brain .................................... 65
3.4.1.5 Cholesterol metabolite changes in ageing and Huntington’s disease ................ 66
3.4.1.6 Cholesterol oxidation product changes in ageing and HD .................................. 68
3.4.1.7 Phytosterol changes during ageing and HD ........................................................ 69
3.4.2 Physical phenotypic changes in the R6/1 mouse ....................................................... 69
3.4.2.1 Weight loss .......................................................................................................... 69
3.4.2.2 Hind paw clasping phenotype ............................................................................. 70
3.4.2.3 Motor performance ............................................................................................ 70
3.4.2.4 Brain mass ........................................................................................................... 72
3.4.3 Correlation of phenotype and sterol changes during HD progression ...................... 72
3.4.4 Conclusion .................................................................................................................. 73
Chapter 4 The effect of environmental enrichment on cholesterol homeostasis and motor
phenotype in the R6/1 HD mouse model ................................................................................... 75
4.1 Introduction ...................................................................................................................... 76
4.2 Materials and Methods ..................................................................................................... 77
4.3 Results ............................................................................................................................... 79
4.3.1 Phenotype .................................................................................................................. 79
4.3.1.1 Weight loss .......................................................................................................... 79
4.3.1.2 Hind paw clasping ............................................................................................... 81
4.3.1.3 RotaRod ............................................................................................................... 83
4.3.1.4 Brain mass ........................................................................................................... 83
4.3.2 Sterol analysis of R6/1 brain tissue ............................................................................ 85
4.3.2.1 Cholesterol oxidation products ........................................................................... 85
4.3.2.2 Cholesterol synthetic precursors, metabolic products and phytosterols ........... 85
4.4 Discussion .......................................................................................................................... 88
4.4.1 Motor performance ................................................................................................... 88
4.4.2 Mechanisms of environmental enrichment changes in the brain ............................. 89
4.4.3 Cholesterol synthesis and metabolism ...................................................................... 91
4.4.4 Cholesterol oxidation products .................................................................................. 92
4.4.5 Weight loss and brain mass ....................................................................................... 93
4.4.6 Conclusion .................................................................................................................. 94
Chapter 5 Berry extract supplementation in the R6/1 transgenic mouse model of Huntington's
disease ........................................................................................................................................ 95
5.1 Introduction ...................................................................................................................... 96
vii
5.2 Materials and Methods ..................................................................................................... 97
5.3 Results ............................................................................................................................... 99
5.3.1 Phenotype .................................................................................................................. 99
5.3.1.1 Body weight ........................................................................................................ 99
5.3.1.2 Hind paw clasping ............................................................................................... 99
5.3.1.3 RotaRod ............................................................................................................. 102
5.3.1.4 Brain mass ......................................................................................................... 102
5.3.2 Sterol analysis of R6/1 brain tissue .......................................................................... 104
5.4 Discussion ........................................................................................................................ 105
5.4.1 Motor performance in R6/1 transgenic mice and possible protective mechanisms of
dietary phenolics in the brain ........................................................................................... 106
5.4.2 Cholesterol homeostasis and BE supplementation in R6/1 mice ............................ 110
5.4.3 The influence of BE on hind paw clasping in R6/1 mouse ....................................... 111
5.4.4 Body weight loss and brain mass of R6/1 mice receiving a BE diet ......................... 112
5.4.5 Therapeutic intervention and bioavailability of phytochemicals ............................ 113
5.4.6 Conclusion ................................................................................................................ 114
Chapter 6 Cholesterol metabolism in Huntington's disease post-mortem brain tissue .......... 115
6.1 Introduction .................................................................................................................... 116
6.2 Materials and methods ................................................................................................... 117
6.3 Results ............................................................................................................................. 120
6.3.1 Sterol analysis .......................................................................................................... 120
6.3.1.1 Cholesterol metabolites .................................................................................... 120
6.3.1.2 Cholesterol oxidation products ......................................................................... 120
6.3.1.3 Cholesterol synthetic precursors ...................................................................... 120
6.3.2 Cholesterol metabolic and synthetic enzymes ........................................................ 121
6.4 Discussion ........................................................................................................................ 124
6.4.1 Alteration of cholesterol metabolism in human Huntington’s disease brain .......... 124
6.4.2 Alteration of cholesterol synthesis in human Huntington’s disease brain .............. 126
6.4.3 Cholesterol oxidation products in human Huntington’s disease brain ................... 128
6.4.4 Consequences of altered cholesterol homeostasis in human Huntington’s disease
brain .................................................................................................................................. 128
6.4.5 Conclusion ................................................................................................................ 129
Chapter 7 General discussion ................................................................................................... 130
7.1 Cholesterol homeostasis alterations in Huntington's disease ........................................ 131
7.2 Brain cholesterol synthesis and metabolism in human and mouse models of HD ........ 131
viii
7.3 Localisation of cholesterol synthetic and metabolic changes in Huntington's disease .. 136
7.4 Sex differences in Huntington's disease ......................................................................... 136
7.5 Future directions studying cholesterol homeostasis in HD ............................................ 137
7.6 Conclusion ....................................................................................................................... 139
References ............................................................................................................................ 141
Appendices ............................................................................................................................ 168

List of figures

Figure 1.1 Chemical structure of cholesterol………………………………………………………………………… 2
Figure 1.2 Simplified pathway showing cholesterol synthesis, metabolism and free radical
oxidation relevant to this thesis ………………………………………………………………………………………….... 5
Figure 1.3 Chemical structure of 24(S)-hydroxycholesterol………………………………………………….. 8
Figure 1.4 A hypothesised mechanism of cholesterol regulation between neurons and
astrocytes…………………………………………………………………………………………………………………….…..….. 10
Figure 1.5 Chemical structure of 27-hydroxycholesterol……………………………………………………….11
Figure 1.6 The major movements of 24(S)-hydroxycholesterol and 27-hydroxycholesterol
across the blood-brain barrier……………………………………………………………………………………………….12
Figure 1.7 Chemical structure of 7β-hydroxycholesterol and 7-ketocholesterol …………………. 13
Figure 1.8 The chemical structure of common phytosterols; campesterol, β-sitosterol,
stigmasterol and brassicasterol …………………………………………………………………………………........... 14
Figure 1.9 The basic chemical structure of an anthocyanidin …………………………………………….. 26
Figure 1.10 The chemical structure of anthocyanins predominantly found in berries
(blueberry, blackberry, black currant) ……………………………………………………………………….………… 27
Figure 2.1 Typical chromatograms of sterol compounds analysed by triple quadrupole GC-
MS……………………………………………………………………………………………………………………………………..38
Figure 2.2 Typical chromatograms of sterol compounds analysed by triple quadrupole GC-
MS……………………………………………………………………………………………………………………………………. 39
Figure 3.1 Weight loss in R6/1 mice during HD progression ..……………………………………………... 44
Figure 3.2 Hind paw clasping phenotype in R6/1 mice………………………………………………………… 46
Figure 3.3 RotaRod motor performance of R6/1 mice …………………………………………………….... 47
ix