My dearest friend and lab manager Heather, your meticulous training and ... Daniel, Dave, Elias, Sarah, Jacob and Fatima who shared my rants and raves, I.
Glial changes in atypical parkinsonian syndromes
Yun Ju Christine Song
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
August 2008
University of New South Wales Department of Anatomy
Supervisors: Professor Glenda M Halliday Dr Yue Huang 1
Acknowledgements It feels surreal realising that I have come to the end of this unbelievably challenging but rewarding journey. I could not have come this far without the amazing help and guidance from so many important people and I am deeply grateful to everybody who has contributed to the steps I have taken in completing this journey.
To my supervisor Glenda Halliday, thank you so much for all your encouragement and guidance. I have continuously learnt so much from you and there have been countless times where I have been in constant awe of your drive, passion and knowledge about these movement disorders. Thank you for all your teachings and enthusiasm for my work, and believing that I could achieve this. I could not have come this far without such a first-class supervisor.
To my co-supervisor Yue Huang, your passion for MSA pushed me to search for more and you always helped me to think from a different perspective. I have learnt many things from you and I thank you for your support.
My dearest friend and lab manager Heather, your meticulous training and perfectionism in the lab and has helped me to achieve this much. Thank you for your technical guidance, I have the deepest regard for you, both as a teacher and friend. Karen, my second lab manager, thanks for being so helpful and supportive right to the very end. You have become such a dear friend and I appreciate all that you have done for me. Heidi, I owe so much to you, thank you for your patience and turning my images into artwork. I thank you Farid, Claire and Emma for all your words of wisdom and Fabs for all your technical support.
To my collaborators Professor Poul Henning Jensen and Dr Paul Lockhart, your enthusiasm and interest in these glial proteins is what started this project and I thank you for your guidance and encouragement. I was so fortunate to do part of my PhD at the Queen Square Brain Bank. I owe so much to Professor Tamas 2
Revesz who taught me the beauty of neuropathology. Spending endless hours on the microscope with you and your encouragement and teachings has contributed so much to this journey, thank you. Dr Janice Holton, thank you for your ongoing support and faith in me as a researcher. Thank you to my friend and supervisor Dr Tammaryn Lashley, you are an amazing super-woman that had the answer to everything! To my dearest friends at Queen Square, Sudi, Kate, Hil, Perdi, Dan and Idris, your friendships made my UK experience so much more rewarding. I will always cherish your love and care, thank you!
I am so grateful for my dear friends Jo, Fran and Lol (fellow PhD buddy) who gave me hugs, smiles and cups of tea when needed most. My study room pals Daniel, Dave, Elias, Sarah, Jacob and Fatima who shared my rants and raves, I thank you all. To my support team Nancy, Jo, Narae, Julie, Danbi, Beks, Jen and Kristy, you girls have helped me maintain my sanity through the past years. Special thanks to Beks, Jen and Kristy for your amazing proof-reading skills and Nancy for being my special shoulder to cry on. I am so grateful for all your prayers and endless encouragement, and keeping me focused on the unseen, your friendships have been invaluable to this journey.
My family have put up with so much throughout this journey, I am so deeply grateful. To my mum and brother for believing in me, embracing my breakdowns and your constant prayers and words of comfort has helped me to persevere and continue in running the race. To Jamie, you have been so patient and understanding, I owe alot to you. Thank you for putting up with my tears and struggles, you helped me reach the finish line and I thank you dearly. To my Lord, you showed me that all things are possible through you. Thank you for your grace and guiding me till the very end. I give this and all glory to you.
This doctorate was funded by the Australian Government, GlaxoSmithKline Australia and Parkinson’s NSW and a personal thank you to all the brain donors and the Australian Brain Donor Programs. 3
Abstract Idiopathic Parkinson’s disease (PD) and the atypical parkinsonian syndromes progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) have substantial overlap in clinical features, with parkinsonian and cerebellar phenotypes identified. Pathologically, reactive glial changes occur in overlapping pathways in these disorders. Recent findings that glia concentrate proteins associated with genetic forms of PD, suggest a more significant role for these cells in the pathogenesis of these disorders.
This study is the first to comparatively assess glial abnormalities in PD, PSP and MSA. Cases were clinically and pathologically characterised to establish correlates to prominent glial changes. The three main types of glia (astrocytes, oligodendroglia and microglia) were characterised by morphological features and protein expression (using immunohistochemical techniques) for correlations with disease indices. Tissue features measured included subregional volumes, nigral cell loss, characteristic disease inclusions, and the densities of glia. Using these techniques, the parkin co-regulated gene protein was found to be a novel constituent protein in protoplasmic astrocytes, facilitating the assessment of glial subtypes.
The data show that distinct glial abnormalities associate with each parkinsonian syndrome. The most marked differences were observed in the astrocytic 4
reaction in each disorder. In PD, protoplasmic astrocytes accumulated nonfibrillar D-synuclein and degenerated over time, relating to a loss of levodopa responsiveness. In PSP, there was a marked protoplasmic astrogliosis (with these reactive glia strongly expressing parkin) that related to PSP-prominent clinical symptoms. In MSA, there was a marked fibrous astrogliosis and a loss of protoplasmic astrocytes in association with early changes in constituent myelin and oligodendroglial proteins. In contrast, similar microglial activation was observed across all disorders, with phagocytes concentrating in the substantia nigra, while non-phagocytic reactive microglia expressed parkin and associated with inclusion formation in each disorder. Overall, the glial reactions were considered to be either contributing to or ameliorating (neuroprotective) the neurodegenerative processes, and the timing of these reactions assessed with respect to indices of disease progression. The novel findings of this thesis show that glial abnormalities are prominent but distinct, and occur early in these parkinsonian syndromes. Suggestions on how these findings may translate into future therapeutic targets are given.
5
Table of Contents
CHAPTER 1: INTRODUCTION ............................................................... 22 1.1 Parkinson’s disease and atypical parkinsonian disorders .......... 22 1.1.1 Clinical features and diagnosis ....................................................... 22 1.1.2 Overlapping phenotypes in parkinsonian disorders ........................ 25 1.1.3 Genetics.......................................................................................... 27 1.1.4 Core pathologies differentiating Parkinson’s disease, progressive supranuclear palsy and multiple system atrophy ..................................... 31 1.1.5 Pathological staging and differences between clinical phenotypes. 35 1.1.5.1 Clinicopathological correlates: parkinsonian subtype ........ 38 1.1.5.1 Clinicopathological correlates: cerebellar subtype ............ 41 1.1.6 Summary......................................................................................... 43 1.2 Cell specific protein changes in Parkinson’s disease and atypical parkinsonian disorders ......................................................................... 44 1.2.1 Abnormal protein expression in neurons......................................... 44 1.2.2 Abnormal protein expression in astrocytes ..................................... 48 1.2.3 Abnormal protein expression in oligodendroglia ............................. 49 1.2.4 Abnormal protein expression in microglia ....................................... 52 1.2.5 Summary......................................................................................... 53 1.3 Study objectives .............................................................................. 54 1.4 Major hypotheses ............................................................................ 55
CHAPTER 2: COMMON MATERIALS AND METHODS ........................ 56 2.1 Sources of tissue ............................................................................. 56 2.2 Case ascertainment and clinical information ................................ 58 2.3 Standard preparation of brain tissue ............................................. 61 2.4 General immunohistochemical methods ....................................... 62 2.4.1 Peroxidase immunohistochemistry (counterstained with 6
cresyl violet) ............................................................................................. 62 2.4.2 Double-labelling immunofluorescence ............................................ 65 2.4.3 Combined peroxidase and immunofluorescence labelling .............. 66
CHAPTER 3: PATHOLOGICAL STAGING OF PROGRESSIVE SUPRANUCLEAR PALSY ...................................................................... 68 3.1 Introduction ...................................................................................... 68 3.1.1 Aim.................................................................................................. 71 3.2 Methods ............................................................................................ 71 3.2.1 Cases used ..................................................................................... 71 3.2.1.1 Cases for method development ........................................ 72 3.2.1.2 Cases for scheme validation ............................................. 72 3.2.1.3 Cases for clinical correlations............................................ 73 3.2.2 Development of a pathological staging scheme for progressive supranuclear palsy................................................................................... 74 3.2.2.1 Regional tissue sampling .................................................. 74 3.2.2.2 Feature identification and severity ratings ......................... 75 3.2.3 Correlations between tau stage and clinically relevant indices ....... 85 3.3 Results .............................................................................................. 85 3.3.1 Analysis of cohorts.......................................................................... 85 3.3.2 Analysis of variability in pathological features and the use of this variability in developing the scheme ........................................................ 86 3.3.3 The development of a staging scheme using the variability in pathological features and clinical correlates ............................................ 90 3.3.4 The pathological staging of progressive supranuclear palsy severity .................................................................................................... 94 3.3.5 Analysis and validation of the pathological staging of progressive supranuclear palsy................................................................................... 95 3.3.6 Correlations between progressive supranuclear palsy pathological severity, pathological tau burden and clinical indices............................... 96 3.4 Discussion........................................................................................ 96 7
3.4.1 Comparison with current diagnostic techniques for progressive supranuclear palsy................................................................................... 99 3.4.2 Accuracy and limitations of the staging scheme ........................... 100 3.4.3 Comparison with staging schemes for other parkinsonian and neurodegenerative disorders ................................................................. 101
CHAPTER 4: CLINICOPATHOLOGICAL CHARACTERISATION OF CASES FOR COMPARISON.............................................................................. 103 4.1 Introduction .................................................................................... 103 4.1.1 Aims and hypothesis ..................................................................... 103 4.2 Methods .......................................................................................... 104 4.2.1 Assessment of regional atrophy.................................................... 104 4.2.2 Assessment of pathological staging and severity of core pathologies ............................................................................................ 105 4.3 Results ............................................................................................ 112 4.3.1 Controls......................................................................................... 112 4.3.2 Parkinson’s disease diagnosis and characterisation..................... 114 4.3.2.1 Case demographics and clinical progression .................. 114 4.3.2.2 Pathology and atrophy .................................................... 114 4.3.3 Progressive supranuclear palsy diagnosis and characterisation... 117 4.3.3.1 Case demographics and clinical progression .................. 117 4.3.3.2 Pathology and atrophy .................................................... 118 4.3.4 Multiple system atrophy diagnosis and characterisation ............... 120 4.3.4.1 Case demographics and clinical progression .................. 120 4.3.4.2 Pathology and atrophy .................................................... 122 4.3.5 Cohort and disease comparisons.................................................. 125 4.3.5.1 Demographic cohort comparisons................................... 125 4.3.5.2 Clinical comparisons ....................................................... 126 4.3.5.3 Pathological comparisons of disease severity................. 132 4.3.5.4 Clinicopathological comparisons ..................................... 134 4.4 Discussion...................................................................................... 135 8
CHAPTER 5: PATHOLOGICAL CHANGES IN ASTROCYTES ........... 141 5.1 Introduction .................................................................................... 141 5.1.1 Specific hypotheses to be tested .................................................. 146 5.2 Methods .......................................................................................... 146 5.2.1 Case selection .............................................................................. 146 5.2.2 Tissue preparation ........................................................................ 146 5.2.3 Analyses ....................................................................................... 148 5.3 Results ............................................................................................ 149 5.3.1 Pathological changes in astrocytes in Parkinson’s disease, multiple system atrophy and progressive supranuclear palsy ............................. 149 5.3.2 Astrocytic parkin and PACRG in Parkinson’s disease, multiple system atrophy and progressive supranuclear palsy ......................................... 154 5.3.3 Types of astrocytes affected in Parkinson’s disease, multiple system atrophy and progressive supranuclear palsy ......................................... 156 5.3.4 Clinical correlations....................................................................... 161 5.4 Discussion...................................................................................... 161 5.4.1 Astrocytic subtypes affected in Parkinson’s disease, multiple system atrophy and progressive supranuclear palsy ......................................... 162 5.4.2 Parkin expression in protoplasmic astrocytes in Parkinson’s disease and progressive supranuclear palsy ...................................................... 164 5.4.3 The severity of these degenerative changes and their relationships........................................................................................... 167 5.4.4 Conclusions .................................................................................. 169
CHAPTER 6: PATHOLOGICAL CHANGES IN OLIGODENDROGLIA ........................................................................... 170 6.1 Introduction .................................................................................... 170 6.1.1 Hypotheses to be tested ............................................................... 173 6.2 Methods .......................................................................................... 173 6.2.1 Case and tissue preparation ......................................................... 173 9
6.2.2 Protein extractions and western blotting for myelin basic protein and p25D................................................................................................ 174 6.2.3 Immunohistochemistry to assess the distribution of myelinassociated proteins ................................................................................ 176 6.2.4 Assessment of axon diameter, fiber diameter and calculation of g ratios ................................................................................................... 178 6.2.5 Immunohistochemistry for the assessment of morphological changes in oligodendroglia-associated with GCI formation ................... 179 6.2.6 Assessment of oligodendroglia changes associated with GCI and coiled bodies .......................................................................................... 181 6.2.7 Correlations with clinical indices ................................................... 182 6.3 Results ............................................................................................ 182 6.3.1 Comparative changes in the amount of myelin-associated proteins .................................................................................................. 182 6.3.2 Comparative changes in the distribution of myelin-associated proteins .................................................................................................. 185 6.3.3 Affects of myelin changes on axons.............................................. 189 6.3.4 Oligodendroglial cell body changes in multiple system atrophy .... 192 6.3.5 Oligodendroglial changes associated with GCI............................. 193 6.3.6 Oligodendroglial changes associated with coiled bodies .............. 198 6.3.7 Correlations with clinical indices ................................................... 200 6.4 Discussion...................................................................................... 201 6.4.1 Conclusions .................................................................................. 206
CHAPTER 7: MICROGLIAL ACTIVATION ........................................... 208 7.1 Introduction .................................................................................... 208 7.1.1 Hypotheses to be tested ............................................................... 210 7.2 Methods .......................................................................................... 211 7.2.1 Cases and tissue preparation ....................................................... 211 7.2.3 Analyses ....................................................................................... 211 7.3 Results ............................................................................................ 215 10
7.3.1 Microglial activation in Parkinson’s disease, multiple system atrophy and progressive supranuclear palsy ...................................................... 215 7.3.2 Microglial parkin in Parkinson’s disease, multiple system atrophy and progressive supranuclear palsy ............................................................. 220 7.4 Discussion...................................................................................... 224 7.4.1 Conclusions .................................................................................. 231
CHAPTER 8: DISCUSSION .................................................................. 233
REFERENCES ...................................................................................... 244
11
List of Tables
Tables
Pages
Chapter 2 2.1
General case demographics for control, PD, PSP and MSA
58
2.2
Antigen retrieval methods for different primary antibodies
64
Chapter 3 3.1
Cohort characteristics
73
3.2
Feature assessment in brain regions of interest
85
3.3
Regional severity data for each pathological feature in cohort I
88
3.4
Development scheme 1 of PSP pathological staging based on feature assessment
3.5
Development scheme 2 of PSP pathological staging based on feature assessment
3.6
92
93
Refinement of the PSP pathological staging scheme with the closest two out of three variables required
94
Chapter 4 4.1
Demographic, volumetric and pathological variables for controls
113
4.2
Demographic, volumetric and pathological variables for PD
116 12
4.3
Demographic, volumetric and pathological variables for PSP
119
4.4
Demographic, volumetric and pathological variables for MSA
124
4.5
Clinical features for PD, PSP and MSA
131
4.6
Comparisons of regional atrophy in PD, PSP and MSA
133
Chapter 5 5.1
Regional severity gradings for nigral cell loss, pathological inclusion formation, and increased astrocytic GFAP, PACRG and parkin
151
Chapter 6 6.1
Group averages of the proportion of myelin proteins assessed densitometrically and standardised to GAPDH in each case
6.2
Semi-quantitative assessment of myelin protein immunoreactivity within pontine fiber tracts
6.3
185
189
P25D and D-synuclein associated oligodendroglial cell size measurements in Pm2
193
Chapter 7 7.1
Regional severity gradings for nigral cell loss, pathological inclusion formation, and increased HLA-DR-immunoreactive microglial activation
7.2
218
Mean and standard deviations of parkin-immunoreactive structures (per mm2) in controls, PD, PSP and MSA
224
13
List of Figures
Figures
Pages
Chapter 1 1.1
Core pathological features of PD, MSA and PSP
34
1.2
Braak staging of pathological PD
36
1.3
Neuronal and glial inclusions in PD, MSA and PSP
47
Chapter 3 3.1
Minimal neuron loss in the pons in PSP
77
3.2
Representative grades of neuron loss in PSP
78
3.3
Representative grades of NFT deposition in PSP
81
3.4
Minimal tufted astrocytes in the substantia nigra of PSP
82
3.5
Representative grades of tufted astrocytes in the caudate nucleus of PSP
3.6
83
The severity of the identified hierarchical pathologies in regions of interest
91
Chapter 4 4.1
Representative grades of neuronal loss in the substantia nigra
4.2
Representative grades of NFT as a core pathological feature of
108
14
PSP 4.3
109
Representative grades of GCI as a core pathological feature of MSA
110
Chapter 5 5.1
Astrocytes in control and PD
152
5.2
Astrocytic changes in MSA and PSP
153
5.3
Astrocytic PACRG in control and PD
157
5.4
Astrocytic PACRG and parkin in PSP
158
5.5
Astrocytic PACRG, parkin and tau in PSP
159
5.6
PACRG immunoreactivity in ApoD-immunoreactive protoplasmic astrocytes
160
Chapter 6 6.1
Western blots of controls, MSA, PD and PSP human brain extracts
6.2
184
In situ protein localisation in myelinated fiber bundles and oligodendroglia in controls
187
6.3
Changes in location of myelin-associated proteins in MSA
188
6.4
Loss of p25D without axonal degeneration in MSA
191
6.5
Control and MSA oligodendroglia
197
6.6
Representative grades of coiled bodies in PSP
199
15
Chapter 7 7.1
Different types and representative grades of HLA-DR-immunoreactive activated microglia
7.2
7.3
213
HLA-DR-immunoreactive phagocytic and non-phagocytic microglia
217
Parkin-immunoreactive activated microglia
222
16
List of abbreviations
ABDP
Australian Brain Donors Programs
ApoD
Apolipoprotein D
AR-JP
Autosomal recessive-juvenile parkinsonism
CB
Coiled bodies
CDR
Clinical dementia rating
CNPase
2’3’-cyclicnucleotide 3’-phosphodiesterase
CNS
Central nervous system
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
GCI
Glial cytoplasmic inclusions
GFAP
Glial fibrillary acidic protein
H&Y
Hoehn and Yahr
HLA-DR
Human leukocyte antigen DR-1
IHC
Immunohistochemistry
LB
Lewy bodies
L-dopa
Levodopa
LRRK2
Leucine-rich repeat kinase 2
MAPT
Microtubule associated protein tau
MBP
Myelin basic protein
MPTP
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MSA
Multiple system atrophy
17
MSA-C
Multiple system atrophy-cerebellar
MSA-P
Multiple system atrophy-parkinsonism
NCI
Neuronal cytoplasmic inclusions
NF
Neurofilament
NFT
Neurofibrillary tangles
NII
Neuronal intranuclear inclusions
NINDS-SPSP
National Institute of Neurological Disorders and Stroke and Society for Progressive Supranuclear Palsy
OPCA
Olivopontocerebellar atrophy
PACRG
Parkin co-regulated gene
PAGE
Polyacrylamide gel electrophoresis
PD
Parkinson’s disease
PET
Positron emission tomography
PINK1
Phosphatase and tensin homolog deleted on chromosome 10 induced kinase 1
PMD
Post-mortem delay
PNLA
Pallido-nigro-luysial atrophy
POWMRI TRC
Prince of Wales Medical Research Institute Tissue Resource Centre
PSP
Progressive supranuclear palsy
PSP-P
Progressive supranuclear palsy-parkinsonism
PSP-RS
Progressive supranuclear palsy-Richardson’s syndrome
QSBB
Queen Square Brain Bank
SABB
South Australian Brain Bank 18
SCP
Superior cerebellar peduncle
SD
Standard deviation
SDS
Sodium dodecyl sulfate
SN
Substantia nigra
SND
Striatonigral degeneration
STN
Subthalamic nucleus
TA
Tufted astrocytes
TESPA
3-aminopropyltrimethoxysilane
WB
Western blotting
19
List of publications arising from this thesis
Publications JM Taylor, YJC Song, Y Huang, MJ Farrer, M Delatycki, GM Halliday, PJ Lockhart. “PACRG is regulated by the ubiquitin-proteasomal system and is present in the pathological features of Parkinsonian diseases.” Neurobiology of Disease 2007 Aug; 27 (2): 238-47
YJC Song, D Lundvig, Y Huang, WP Gai, PC Blumbergs, P Horjup, D Otzen, GM Halliday, PH Jensen. “P25D relocalizes from myelin to cytoplasmic inclusions in multiple system atrophy.” American Journal of Pathology 2007 Oct; 171 (4): 1291-303
Y Huang, YJC Song, K Murphy, JL Holton, T Lashley, T Revesz, WP Gai, GM Halliday. “LRRK2 and parkin immunoreactivity in multiple system atrophy inclusions.” Acta Neuropathologica 2008 Dec; 1: 16 (6): 639-46
Abstracts YJC Song, Y Huang, WP Gai, PH Jensen, GM Halliday. “Selective p25D loss in multiple system atrophy.” 20
Proceedings of the Australian Neuroscience Society, January 2005.
YJC Song, Y Huang, WP Gai, PH Jensen, GM Halliday. “P25D relocalizes from myelin to cytoplasmic inclusions in multiple system atrophy.” Parkinsonism and Related Disorders 2007, 13 Supplementary. (2): 63
YJC Song, Y Huang, WP Gai, PH Jensen, GM Halliday. “Correlations between striatal oligodendroglial abnormalities and neuron loss in multiple system atrophy.” Movement Disorders 2008, 23 Supplementary. (1): S266
21
Chapter 1: Introduction
1.1 PARKINSON’S DISEASE AND ATYPICAL PARKINSONIAN DISORDERS 1.1.1 Clinical features and diagnosis Idiopathic Parkinson’s disease (PD) occurs in about 1% of people over 60 years of age (Nussbaum and Ellis, 2003). A clinical diagnosis of PD is given when the patient has the typical features of bradykinesia, resting tremor and/or extrapyramidal rigidity and the diagnosis is made more certain if there is a good response to levodopa (L-dopa) (Brooks, 2002; Gelb et al., 1999; Hald and Lotharius, 2005; Hoehn and Yahr, 1998). A diagnosis of definite PD is given based on the confirmation of substantia nigra (SN) cell loss and typical Lewy bodies (LB) at autopsy (Gibb and Lees, 1988; Jellinger, 2003). Disorders that present symptoms of rapidly progressing parkinsonism, poor or temporary response to L-dopa or other associated features such as early falls and postural instability are defined as atypical parkinsonian syndromes (Tolosa et al., 2004). Progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) are defined as sporadic neurodegenerative diseases that vary from typical PD in their clinicopathological presentations, and are thus classified as atypical parkinsonian disorders.
22
PSP is a disorder of unknown etiology with a prevalence of about 6.4 per 100,000 people (Schrag et al., 1999). Diagnostic criteria for PSP are based on consensus statements from the Scientific Issues Committee Task Force Appraisal and the National Institute of Neurological Disorders and Stroke and Society for PSP (NINDS-SPSP). For a possible diagnosis, either vertical supranuclear palsy or both postural instability with falls within a year of symptom onset and slowing of vertical saccades must occur. A probable diagnosis includes features of vertical supranuclear palsy and prominent postural instability with falls within the first year of symptom onset. A definite diagnosis of PSP is given when criteria for possible or probable PSP are met with the pathological confirmation of subcortical neurofibrillary tangles (NFT) at autopsy (Litvan et al., 1996a; Litvan et al., 2003).
MSA is also a disorder of unknown etiology with a prevalence of about 4-4.5 per 100,000 people (Schrag et al., 1999; Wenning et al., 2004). Diagnostic criteria for MSA are based on four clinical domains, which include autonomic and urinary dysfunction, parkinsonism, cerebellar dysfunction and corticospinal tract dysfunction (Litvan et al., 2003; Schrag et al., 1999; Wenning et al., 2004). A possible diagnosis is given when one domain feature occurs in addition to two features from other domains. A probable diagnosis is given when an autonomic and urinary dysfunction feature occurs with either cerebellar dysfunction or a poor response to L-dopa. A definite diagnosis is given at autopsy following pathological confirmation of widespread glial cytoplasmic inclusions (GCI) (Trojanowski and Revesz, 2007; Wenning et al., 2004). 23
It has become increasingly recognised that the clinical diversity observed in PD, PSP and MSA patients causes misdiagnosis (Collins et al., 1995; Dickson et al., 1999a). In particular, there is substantial clinical overlap across these parkinsonian syndromes in early disease stages (Poewe and Wenning, 2002; Wenning et al., 2004; Wenning et al., 2003), with a proportion of PSP and MSA patients continuing to have parkinsonian phenotypes throughout their course (Gilman et al., 1998; Gilman et al., 1999; Wenning et al., 1994; Williams et al., 2005; Williams et al., 2007a). 32% of PSP patients present with dominant parkinsonism (PSP-Parkinsonism or PSP-P) (Williams et al., 2005) and a larger proportion of MSA patients (80%) also present with dominant parkinsonian features (MSA-P) (Gilman et al., 1999; Wenning et al., 2004; Wenning and Jellinger, 2005). The more typical clinical features described previously to discriminate PSP occur in up to 54% of PSP patients, now classified as PSPRichardson’s syndrome (PSP-RS) (Williams et al., 2005), whereas only 20% of MSA patients present with features consistent with a probable diagnosis, now classified as MSA-C (Gilman et al., 1999; Wenning et al., 2004; Wenning and Jellinger, 2005). These data show that two major clinical phenotypes occur across these disorders, a parkinsonian phenotype (PD, PSP-P, MSA-P) and a cerebellar (MSA-C)/Richardson’s syndrome phenotype (PSP-RS). Few studies have assessed these phenotypes across these disorders to determine any pathological correlations or etiological similarities.
24
1.1.2 Overlapping phenotypes in parkinsonian disorders As described above, resting tremor, rigidity, bradykinesia, postural instability and L-dopa response are core features of PD (Hald and Lotharius, 2005), but are also commonly identified features in atypical parkinsonian syndromes. In addition, 20-30% of PD patients have atypical parkinsonian syndromes at autopsy (Poewe and Wenning, 2002). 5% of PSP-P patients are misdiagnosed with PD, whereas approximately 50% of MSA-P patients are misdiagnosed with PD (Litvan et al., 1997). Parkinsonism (progressive akinesia and rigidity) is the primary feature of all these conditions, with jerky postural tremor and tremor at rest as additional prominent presentations even in PSP-P and MSA-P (Wenning et al., 2004; Williams et al., 2005).
Prolonged L-dopa responsiveness is considered one of the main differentiating features for PD (Forno, 1996), although a proportion of patients with atypical syndromes can also respond to L-dopa. Approximately 30-40% of PSP and MSA patients initially respond to L-dopa, however less than 5% of patients express a marked response after 5 years of treatment (Berciano et al., 2002; Birdi et al., 2002; Jankovic, 1984; Nieforth and Golbe, 1993; Wenning et al., 2004; Williams et al., 2005). The PSP-P and MSA-P subtypes are likely to have higher response rates to L-dopa (Constantinescu et al., 2007), with a moderate initial response to L-dopa required for the diagnosis of PSP-P (Williams et al., 2005). Although the majority of PD patients still respond to L-dopa at 5 years, wearing off and atypical side affects are often problematic, affecting 50% of 25
patients by 5 years and almost all patients by 10 years (Hely et al., 2000; Hely et al., 1994). The similar and prominent initial response to L-dopa in PD, MSA and PSP (in particular MSA-P and PSP-P) suggests a similar pathological change at this time, followed by differentiating pathologies.
Less recognised is the potential similarities between PSP-RS and MSA-C. This is due to the recency of the concept of PSP-RS (Williams et al., 2005). In particular, a proportion of PSP and MSA cases present with the dominant atypical symptoms of early falls, with oculomotor and cerebellar symptoms and other features, like dysarthria and dysphagia, can not distinguish between them (Muller et al., 2001). Early falls are thought to be characteristic for PSP, in particular PSP-RS (Wenning et al., 1999; Williams et al., 2006). However, early falls within 3 years of disease onset has also been identified as a “red flag” warning feature of MSA (Muller et al., 2000; Wenning et al., 1999). The overlap of this cerebellar clinical phenotype may explain that 18% of PSP cases are misdiagnosed as MSA (Litvan et al., 1997). Early cerebellar features, such as ataxia, postural instability and falls, dominate the clinical picture in these cases. Supranuclear vertical down-gaze palsy, axial dystonia and cognitive impairment are features that can help differentiate PSP from PD and MSA (Colosimo et al., 1995). While these clinical features can be helpful, like vertical supranuclear gaze palsy, the signature sign for PSP, eye signs may also occur in MSA (Wenning et al., 2004). In MSA-C, cerebellar ataxia is the main clinical presentation and may include other clinical symptoms such as scanning dysarthria and cerebellar oculomotor disturbances (Wenning et al., 2004). MSA26
C may therefore be difficult to differentiate from PSP-RS (Dickson et al., 1999a). Despite this clinical overlap, there have been no comparative pathological studies of PSP-RS and MSA-C. In particular, it is not clear if the initial focus and cell types involved are the same, and there are limited studies on the pathological
progression
of
these
phenotypes
either
independently
or
comparatively.
1.1.3 Genetics Historically, PD was thought to be less genetically influenced than other neurodegenerative conditions, with substantial evidence for the involvement of environmental toxicants (Morris, 2005). However, this has not been sustained, with thirteen pathogenic genetic loci established from families with PD and eight genes identified, five of which are found in multiple families with characteristic PD. The five genes are D-synuclein (PARK1), parkin (PARK2), PTEN induced kinase 1 (PINK1) (PARK6), DJ-1 (PARK7) and leucine-rich repeat kinase 2 (LRRK2) (PARK8) (Schiesling et al., 2008). The expression of some of these genetic proteins in human brain tissue has been described. In particular, Dsynuclein, parkin, PINK1, DJ-1 and LRRK2 are expressed in neurons and glia in controls and patients with PD, PSP and MSA (Bandopadhyay et al., 2004; Gandhi et al., 2006; Higashi et al., 2007; Miklossy et al., 2006; Neumann et al., 2004; Spillantini et al., 1998; Wakabayashi et al., 1998; Wenning and Jellinger, 2005; Zarate-Lagunes et al., 2001).
27
D-Synuclein was the first PD gene discovered and is found in LB in PD (McInerney-Leo et al., 2005; Spillantini et al., 1997). D-Synuclein is a soluble synaptic protein in the normal human brain (Tu et al., 1998), primarily localised to synaptic terminals (Jakes et al., 1994). D-Synuclein mutations are commonly used in mouse models, not only to model PD but other parkinsonian disorders such as MSA (McInerney-Leo et al., 2005). Similar to PD, D-synuclein is the major protein localised in the inclusions of MSA but there have been no Dsynuclein gene mutations identified in MSA, further confirming the non-familial and sporadic presentation of this disease (Wenning and Jellinger, 2005; Wenning et al., 1993). However, there has been one report of an autosomal dominant inheritance within an MSA family (Wullner et al., 2004). Genetic studies have been carried out in effort to analyse the potential risk factors and inheritability involved in MSA. It has been hypothesised that there are different subsets of genetic susceptibility factors that are different between MSA-P and MSA-C (Ozawa, 2006). It may be that genes associated with parkinsonism may be linked to MSA-P and genes associated with ataxia could be linked to MSA-C. Genetic risk factors may therefore also differ depending on the clinical subtype. Understanding important proteins involved in the pathology of specific clinical MSA subtypes may aid in elucidating the potential genetic components in relation to clinical presentations.
Parkin is a widely expressed ubiquitin E3 ligase, implicating the ubiquitinmediated protein degradation pathway in neurodegeneration (Casarejos et al.,
28
2006; Klein et al., 2006; Shimura et al., 1999; Vercammen et al., 2006). Expression of parkin is neuroprotective and mutations in or loss of parkin protein lead to a hereditary form of PD, called autosomal recessive-juvenile parkinsonism (AR-JP) (Perez and Palmiter, 2005). Several neuropathological studies on cases with AR-JP showed a loss of dopaminergic neurons in the SN and the absence of LB (the pathological hallmark for PD), although there have been few neuropathological studies on parkin mutations (Schiesling et al., 2008). A gene closely affiliated with parkin called the parkin co-regulated gene (PACRG) has also been identified (West et al., 2003). This gene is transcriptionally co-regulated with the parkin gene by a shared bi-directional promoter, and despite its close association with parkin, mutations of PACRG are not associated with early-onset parkinsonism (Deng et al., 2005). However, deletion of the shared promoter has been linked to early-onset parkinsonism (Lesage et al., 2007).
The PINK1 gene was first identified in a large Italian family with an autosomal recessive form of PD (Valente et al., 2004). PINK1 encodes a 581 amino acid protein and thought to have a neuroprotective role (Gandhi et al., 2006; Petit et al., 2005; Valente et al., 2004). To date, there have only been four brains of patients with heterozygous PINK1 mutations that have had neuropathological analyses performed (Gandhi et al., 2006). The pathology in these patients is typical of idiopathic PD with substantial SN cell loss in the presence of LB (Schiesling et al., 2008). Similar to PINK1, mutations in the DJ-1 gene have also been shown to cause early-onset autosomal recessive PD (Bonifati et al., 29
2003a; Bonifati et al., 2003b). This protein is thought to be associated with oxidative stress (Mitsumoto and Nakagawa, 2001). To date, there have been no studies on the neuropathology of patients with DJ-1 mutations (Schiesling et al., 2008).
Mutations in the LRRK2 gene are currently the most frequent genetic cause of idiopathic and familial PD, with more than 50 variants identified as responsible for approximately 2-10% of idiopathic and 10-40% of familial PD (Berg et al., 2005; Di Fonzo et al., 2005; Mata et al., 2006; Schiesling et al., 2008). However, LRRK2 is distinct from the other PD-related genes in that the clinical phenotype can vary widely (atypical parkinsonism and dementia phenotypes) even within one family (Zimprich, Biskup, Leitner, Farrer et al 2004; Funayama, Hasegawa, Kowa et al 2002). In addition to the heterogeneity in clinical phenotype, the pathology in these patients can also be highly variable, including brainstem LB, diffuse LB disease pathology and pure nigral degeneration without distinctive histopathology and PSP-like pathology (Zimprich et al., 2004).
PSP has been described as a sporadic tauopathy, as there has been no direct causative genes identified, however there is a strong genetic association with the microtubule associated protein tau (MAPT) gene (Albers and Augood, 2001; Morris et al., 1999). An extended 5’-MAPT haplotype, Haplotype A, has been noted in 98% of PSP cases and has been recognised as a good indicator for sporadic PSP (Higgins et al., 2000). However, this haplotype has also been identified in 33% of controls, suggesting that there may be additional 30
environmental and genetic factors associated with the disease (Albers and Augood, 2001). Haplotype H1 was found to have a strong affiliation with PSP (Pittman et al., 2004), although recent subtyping of PSP into clinical phenotypes has found that haplotype H1 has a stronger affiliation for PSP-RS than PSP-P (Williams et al., 2005). Additionally, there have been reports of families with PSP (Pastor and Tolosa, 2002; Rojo et al., 1999). A silent mutation in exon 10 of the MAPT gene has been discovered in an Australian family member with PSP-like pathology (Stanford et al., 2000). Another mutation in the MAPT gene was also found to cause a tauopathy with dementia and supranuclear palsy (Delisle et al., 1999). These MAPT gene mutations increase the inclusion of exon 10 in the mature messenger RNA thus causing a surplus of 4-repeat tau over the 3-repeat tau isoforms, a feature found in PSP (Goedert et al., 1998; Mailliot et al., 1998).
1.1.4 Core pathologies differentiating PD, PSP and MSA SN cell loss is a key pathological feature of both typical (PD) and atypical (PSP and MSA) parkinsonian syndromes. However, cell loss in PSP and MSA is more extensive, affecting other basal ganglia, cerebellar and spinal structures (Bak et al., 2005). The definitive diagnosis of different parkinsonian disorders is based on the type and distribution of pathological inclusions. In PD, the core LB inclusion is present in neurons (Figure 1.1A) and consists primarily of the Dsynuclein protein. PD is therefore categorised as an D-synucleinopathy (Geddes,
2005).
In
D-synucleinopathies,
insoluble
D-synuclein
is
phosphorylated, nitrated and often ubiquitinated (Duda et al., 2000a). Like PD,
31
D-synuclein is the dominant abnormal protein expressed in the core GCI inclusions found in oligodendroglia in MSA (Figure 1.1B). However in LB, filaments are orientated in a radiating fashion in the periphery of the inclusions and less heavily coated by amorphous material (Forno, 1996). Whereas in GCI, the filaments are heavily coated by amorphous material and are arranged in parallel bundles towards the processes of the oligodendroglia (Gai et al., 2003). MSA is also classified as a D-synucleinopathy. Previously, three pathological forms of MSA were identified as either striatonigral degeneration (SND) (Adams et al., 1964), olivopontocerebellar atrophy (OPCA) (Geary et al., 1956) or ShyDrager syndrome (Shy and Drager, 1960). The first description of GCI in oligodendroglia (Papp et al., 1989) helped to unify MSA pathologically, although MSA stands out among other neurodegenerative diseases in that its core inclusion pathology (the GCI) are localised in oligodendroglia rather than neurons.
The neuronal and glial pathologies found in PSP are composed of hyperphosphorylated tau, classifying PSP as a tauopathy (Gibb et al., 2004). Tau is a microtubule associated protein related to axonal transport and in the normal brain is located predominantly in the axons (Komori, 1999). Tau proteins support the assembly and stabilise microtubules in a polymerised state. In the brain there are 6 tau isoforms generated by alternative splicing of exons 2, 3 and 10 in the MAPT gene (Tolosa et al., 2004). The accumulation of selective hyperphosphorylated tau isoforms that contain four microtubule binding domains
32
(4 repeat tau, 64 and 69kDa in molecular weight) is observed in PSP, and this accumulation of hyperphosphorylated tau disrupts microtubule function and interferes with axonal transport (Komori, 1999). AT8 is a robust immunoreaction marker commonly used to identify hyperphosphorylated tau protein (Braak et al., 2006). PSP is also a distinct disorder in that it is associated with abnormal tau deposition in three main types of cells – neurons, astrocytes and oligodendroglia (Takahashi et al., 2002). Neuronal NFT (Figure 1.1C) are the core inclusion used for the pathological diagnosis of PSP (Hauw et al., 1994; Litvan et al., 1996b). In addition to NFT in neurons, glial inclusions are also considered important pathological markers for PSP (Burn and Lees, 2002). In PSP, glial inclusions are in the form of tufted astrocytes (TA) and coiled bodies (CB) in the oligodendroglia. TA contain hyperphosphorylated tau and are considered part of the
degenerative
process
in
PSP
(Togo
and
Dickson,
2002).
33
Figure 1.1 Core pathological features of PD, MSA and PSP
Representative brightfield photomicrographs of 5Pm thick, paraffin-embedded sections taken at x400 magnification, immunoperoxidase labelled and counterstained
with
cresyl
violet.
Scale
in
C
is
equivalent
for
all
photomicrographs. A
A dopaminergic neuron in the substantia nigra of a PD case containing an D-synuclein-immunoreactive Lewy body (LB).
B
D-Synuclein-immunoreactive glial cytoplasmic inclusions (GCI) in the pons of a MSA case.
C
Tau (AT8)-immunoreactive neurofibrillary tangles (NFT) in the pons of a PSP case.
34
A
B
C
50μm
1.1.5 Pathological staging and differences between clinical phenotypes The severity of D-synuclein-immunopositive LB and Lewy neurites are graded to pathologically stage the progression of PD (Braak et al., 2003; Braak et al., 2004) (Figure 1.2). The staging scheme is divided into 6 stages dependent on the progression of regions affected. Stages 1 and 2 reflects a confinement of pathology to olfactory regions, the medulla oblongata and pontine tegmentum, with stage 3 including regions affected in the lower and upper midbrain, and the anteromedial temporal mesocortex additionally affected in stage 4. End-stages 5 and 6 include all areas affected in stages 1 to 4 with additional neocortical areas affected. Therefore, the core LB pathology in PD can be used to stage the progression of pathology in PD.
The progression of pathology in some tauopathies can be staged based on the degrees of progressive atrophy divided into four stages (Broe et al., 2003). Stage 1 reflects mild atrophy in the orbital and superior medial frontal cortices and hippocampus. Stage 2 progresses to include atrophy of other anterior frontal regions, temporal cortices and basal ganglia. Stage 3 involves all remaining tissue in coronal slices and stage 4 reflects overall marked atrophy in all areas. However, only limited atrophy is observed in PSP and this staging scheme for atrophy cannot be applied to PSP (Schofield et al., 2005).
35
Figure 1.2 Braak staging of pathological PD
A schematic diagram of the pathological staging of PD based on the deposition of Lewy bodies and Lewy neurites, courtesy of Heiko Braak. A
Pathological Braak stages 1 and 2 initially affecting the medulla oblongata and pontine tegmentum.
B
In addition to pathology in stages 1 and 2, pathological Braak stages 3 and 4 also affects the midbrain, basal prosencephalon and mesocortex.
C
In addition to the pathological regions affected in stages 1-4, the neocortex and premotor areas including primary sensory areas and the primary motor field are affected in stages 5 and 6.
36
Parkinson’s disease stage 1&2 A Braak Preclinical Parkinson’s disease
B
C
Braak Parkinson’s disease stage 3&4 Clinical Parkinson’s disease
Braak Parkinson’s disease stage 5&6 Cognitive impairment
Courtesy of Heiko Braak
The division of clinical subtypes in PSP and MSA has lead to the establishment of novel pathological grading systems for PSP and MSA that differ in the regions examined and the pathologies assessed, although there is some overlap. The clinical subtypes in PSP can be distinguished based on the recently published pathological tau severity scale (Williams et al., 2007a). A 12-tiered grading system has been established based on grading the severity of CB and associated threads in the SN, caudate nucleus and dentate nucleus. Four severity grades (grades 1-4) of CB and threads were used to evaluate the 3 selected regions. The grades for all 3 regions were summed to provide a total score ranging from a minimum of 0 to a maximum of 12. PSP-RS was consistently graded higher than PSP-P, as no PSP-P case was scored higher than 5. In addition, tau burden was topographically more restricted in PSP-P, with tau burden significantly greater in PSP-RS in all regions except for the putamen and SN. More recently, the differentiation of PSP clinical subtypes based on this pathological tau severity scheme (Williams et al., 2007a) was validated in another cohort to confirm the distinguishable PSP clinical subtypes based on tau burden (Jellinger, 2008).
In MSA, the pathological grading system is based on rating atrophy, neuronal loss, gliosis and GCI, separating MSA-P and MSA-C (Jellinger et al., 2005). MSA-P and MSA-C can be divided into four grades ranging from grade 0 to 3. Briefly, MSA-P grade 1 represents similarities to minimal change MSA, whereby the SN is significantly degenerated and the striatum preserved with minimal GCI and gliosis in the posterior putamen. MSA-P grade 2 features neuronal loss, 37
gliosis and GCI in the SN and putamen. MSA-P grade 3 reflects greater GCI severity in the SN and putamen than grade 1 and 2, and additional neuronal loss in the caudate nucleus and globus pallidus (Jellinger et al., 2005; Wenning et al., 2002). In contrast, MSA-C grade 1 reflects widespread GCI with minimal atrophy, neuronal loss and gliosis, except for neuronal loss in the SN. MSA-C grade 2 represents widespread GCI, minimal atrophy, mild neuronal loss and gliosis in the pons and inferior olives but a variable severity of neuronal loss in the SN. MSA-C grade 3 features substantial atrophy of the cerebellum and pons, Purkinje cell loss and demyelination in the cerebellum, neuron and fiber loss in the pons and severe atrophy and gliosis in the inferior olives of the medulla. At grade 3, the SN often includes severe neuronal loss and gliosis, and GCI severity can decrease with LB accumulation (Jellinger et al., 2005). It is also important to note that in addition to pure MSA-P or MSA-C grades, cases can present with varying combinations (Jellinger et al., 2005; Ozawa et al., 2004).
1.1.5.1 Clinicopathological correlates: parkinsonian subtype The progressive loss of dopaminergic neurons in the SN is a key pathological feature in PD leading to the clinical and pharmacological abnormalities observed in the disease (Schulz and Falkenburger, 2004). However, all atypical parkinsonian syndromes have this feature, which may explain their initial early response to L-dopa (Berciano et al., 2002; Williams et al., 2005). Imaging studies have shown that midbrain atrophy (hummingbird sign) is worse in PSP than MSA-P and PD (Cosottini et al., 2007; Groschel et al., 2006; Kato et al., 2003; Yekhlef et al., 2003). The SN is a consistently affected region in PSP 38
(Fearnley and Lees, 1991; Halliday et al., 2000; Oyanagi et al., 2001), but whether this is more prominent in PSP-P is yet to be determined. In MSA-P, the degree of degeneration in the SN correlates with the severity of parkinsonism (Jellinger et al., 2005; Wenning et al., 2002), with the severity of akinesia correlating with the severity of cell loss in the putamen and SN, while more severe rigidity correlates with greater SN loss (Wenning et al., 1997). For MSAP, the L-dopa response is transient due to prominent subsequent putaminal degeneration (90% have loss of putamen at autopsy). Only 5% of patients with MSA-P have long-term benefits from L-dopa (Burn and Jaros, 2001). In contrast in PSP-P, putamen loss is not a key pathological feature and all PSP-P patients have substantive benefit from L-dopa (Williams et al., 2005).
Despite the clinical similarities between these parkinsonian conditions, both PSP-P and MSA-P have additional and more global regions of degeneration, although the timing of this additional degeneration has not been identified. Due to the recent division of PSP subtypes, patterns of degeneration have not been well studied for the different clinical groups. The major study defining pathological PSP subtypes used tau burden as a marker of disease severity (Williams et al., 2007a). In this study, gaze palsy was absent from PSP-P cases along with less severe tau burden (Williams et al., 2007a; Williams et al., 2008). Regions of degeneration correlating with parkinsonian symptoms in PSP are the thalamus (Henderson et al., 2000), striatum (Burn and Lees, 2002; Schulz et al., 1999; Warren et al., 2005), globus pallidus (Hardman and Halliday, 1999) as well as the SN (Halliday et al., 2000; Hardman et al., 1997; Kluin et al., 2001). In 39
particular, degeneration of the external globus pallidus appears to be related to increased thalamic inhibition through the basal ganglia output nuclei, as observed in PD (Hardman and Halliday, 1999).
Regional changes have been confirmed at autopsy for MSA-P patients. These patients present, with an overall discolouration and atrophy of the putamen (Konagaya et al., 1994; Schulz et al., 1999), increased hypointensities in or slitlike hyperintense bands lateral to the putamen (Konagaya et al., 1994; Schulz et al., 1999), with neuronal loss largely confined to the striatum and midbrain (Kume et al., 1993). Detailed studies of the striatum and SN reported marked loss in the caudal putamen and SN, supporting a region-specific cell loss (Kume et al., 1993; Sato et al., 2007; Wenning et al., 1997).
In the majority of MSA cases, neuronal loss is quite marked at autopsy, independent of clinical subtype. Regions affected include the basal ganglia, brainstem and cerebellar regions. Several semi-quantitative studies have shown neuron loss in the caudate, putamen, pallidum, SN, brainstem reticular formation, pontine base and cranial motor nuclei (Papp and Lantos, 1994; Tsuchiya et al., 2000; Wenning et al., 1997; Wenning et al., 1996). Quantitative studies (Benarroch et al., 2003; Benarroch et al., 2001; Benarroch et al., 1998) have demonstrated the selective neuronal loss in subnuclei of the pons and medulla oblongata, including one study correlating catecholaminergic neuron loss in the medulla with autonomic failure in MSA. Autonomic failure is observed in both clinical subtypes of MSA. Most studies have not correlated these 40
pathological changes with atrophy and have not compared degenerative changes across the parkinsonian phenotypes PD, PSP-P and MSA-P.
1.1.5.2 Clinicopathological correlates: cerebellar subtype In PSP, atrophy of the superior cerebellar peduncle (SCP) appears to differentiate PSP from other parkinsonian conditions (Blain et al., 2006; Kataoka et al., 2008; Paviour et al., 2005; Price et al., 2004; Quattrone et al., 2008). The SCP is involved in the cerebellar output pathway from the dentate nucleus targeting the ventrolateral nucleus of the thalamus and the red nucleus (Nolte, 1999). Comparatively, there is striking atrophy of the cerebellum, middle cerebellar peduncles, pontine basis and olivary nuclei in vivo in MSA-C (Wenning and Jellinger, 2005), with T2-weighted images showing signal hyperintensities known as the “hot cross bun” signal within the pons and middle cerebellar peduncles in some cases (Scherfler et al., 2005; Schrag et al., 1998). The middle and inferior cerebellar peduncles are associated with the cerebellar input pathways from the pons and spinal cord (Nolte, 1999). This suggests that different cerebellar systems (superior versus middle and inferior cerebellar systems) are involved in PSP-RS compared with MSA-C. Possible differences in cerebellar pathway involvement in PSP-RS and MSA-C may implicate different cerebellar motor presentations and pathology, however this needs to be further studied in detail.
Despite the cerebellar focus observed in these atypical parkinsonian syndromes, degeneration in the cerebellar system has not been well studied pathologically in 41
either PSP-RS or MSA-C. Neuronal loss is observed in the pons (Hirsch et al., 1987; Kasashima and Oda, 2003; Malessa et al., 1991; Zweig et al., 1987) and cerebellar dentate nucleus (Matsumoto et al., 1998) in PSP. In contrast, clinical correlations to histopathology link cerebellar ataxia with neuronal loss in the inferior olives and cerebellar cortex in MSA-C (Wenning et al., 1997) and may be related to the degeneration of cerebellar input pathways. It will be important to determine if this pattern of degeneration is limited to these clinical subtypes, as well as to determine the type and timing of pathological degeneration within these cerebellar pathways.
As discussed previously, both PSP-RS and MSA-C subtypes can have significant eye signs, particularly as these diseases progress. The pathological substrate for these clinical features has not been determined for MSA-C. However, studies have shown that the severity of tau deposition and neuronal loss in the SN pars reticulata (Halliday et al., 2000), mesencephalon (Juncos et al., 1991) and the pontine nucleus raphe interpositus (Revesz et al., 1996) are increased in PSP patients with gaze palsy. Cerebellar pathways involved in the motor control of eye movements may be disrupted in both PSP-RS and MSA-C and correlates to specific eye signs may help differentiate these phenotypes.
Although substantive cortical atrophy is not a feature of these atypical parkinsonian conditions, mild frontal lobe atrophy is well known to correlate with both the severity of NFT pathology and behavioural changes in PSP-RS (Cordato et al., 1997; Cordato et al., 2002). In MSA-C, supratentorial volume 42
loss has been found in the orbitofrontal and mid-frontal regions as well as in temporomesial and insular areas of both hemispheres (Brenneis et al., 2005). However, there are limited studies on correlations to the clinical symptoms of both PSP-RS and MSA-C with atrophy and neuronal loss.
1.1.6 Summary In summary, the clinical subtypes of PSP-P and MSA-P present similar clinical symptoms,
closely
mimicking
PD.
In
contrast,
PSP-RS
and
MSA-C
pathologically differ from PD but are similar to each other in their clinical presentations. In PD, PSP-P and MSA-P, the SN is a region that is consistently affected with correlates to parkinsonian features. The atrophy of the putamen is marked in MSA-P, whereas clinicopathological correlates to PSP-P have not been well characterised. The middle cerebellar peduncles appear to be affected in MSA-C, whereas in PSP, the atrophy of the SCP is used as a differentiating marker. Histopathological studies in cerebellar regions have shown correlates to eye signs in PSP-RS and ataxia in MSA-C, however these cerebellar clinical subtypes have not been well studied. Despite the similarities in clinical features and pathways affected, there have been no comparative studies of pathological comparisons across these disorders. The similarities in the PSP and MSA clinical subtypes and their differentiation based on glial pathology suggest that the cell types in particular pathways may be related to specific clinical subtypes. The similar and more global regions/pathways affected warrants further investigation
of
the
glial
changes
across
these
clinically
overlapping
parkinsonian disorders. 43
1.2
CELL
SPECIFIC
PROTEIN
CHANGES
IN
PD
AND
ATYPICAL
PARKINSONIAN DISORDERS 1.2.1 Abnormal protein expression in neurons Both PD and MSA are defined as D-synucleinopathies with neuronal D-synuclein inclusions featured in both disorders. In PD, the core pathological feature is localised in neurons in the form of D-synuclein-immunoreactive LB (Figure 1.3A). The distribution of LB in PD is in both subcortical and cortical regions, as well as in the periphery (Hughes et al., 1993; Ohama and Ikuta, 1976; Takahashi and Wakabayashi, 2005). In contrast, D-synuclein-immunoreactive inclusions in neurons are less prominently observed in MSA (Burn and Jaros, 2001). This is also in contrast to the core D-synuclein-immunoreactive GCI pathology observed in MSA. Both neuronal cytoplasmic inclusions (NCI) and neuronal intranuclear inclusions (NII) occur in MSA (Figure 1.3B), largely in the pontine and olivary nuclei (Nishie et al., 2004; Papp and Lantos, 1992)
The deposition of D-synuclein has not been studied in detail in PSP, but there are reports of D-synuclein-immunopositive LB in pathologically proven PSP cases (Fearnley et al., 1991; Gearing et al., 1994; Judkins et al., 2002; Mori et al., 2002; Tsuboi et al., 2003b). These rare reports of PSP and PD combined cases present a predominance of LB in neurons in the SN and dorsal motor nucleus, and at times in the cerebral cortex. In addition, another study has shown that the anterior olfactory nucleus of PSP patients have mild D-synuclein 44
pathology (Tsuboi et al., 2003b). In addition to neuronal inclusions, one report has shown that the C-terminus of D-synuclein was immunoreactive in glial inclusions of PSP following pre-treatment with proteinase K (Takeda et al., 1998).
In PSP, NFT is the core neuronal pathological feature and can be either globose or flame-shaped in morphology (Figure 1.3C) (Tellez-Nagel and Wisniewski, 1973). NFT are tau-immunoreactive, with the 4R isoform predominating. The distribution of NFT in PSP concentrates in the basal ganglia, diencephalon and brainstem (Dickson et al., 1999a; Dickson et al., 2007). The current neuropathologic criteria for PSP requires a high degree of NFT and neuropil threads in at least three of the following regions: pallidum, subthalamic nucleus (STN), SN, or pontine basis. In addition to this, a low to high density of these pathological features is required in any one or more of the following regions: the striatum, oculomotor complex, medulla or dentate nucleus.
In addition to D-synuclein, a number of other proteins associated with genetic forms of PD are normally expressed in neurons. The dominant constituent expression of parkin is thought to be neuronal (Zarate-Lagunes et al., 2001) and there are varying reports of parkin expression in LB in typical PD (Schlossmacher et al., 2002; Zarate-Lagunes et al., 2001). PINK1 protein is expressed in mitochondria in neurons and in glial cells and is unchanged in PD, although 5-10% of brainstem LB also contains PINK1 immunoreactivity (Gandhi
45
et al., 2006). In contrast, the normal expression of DJ-1 is weak in neurons, although NFT in PSP are DJ-1 immunopositive (Rizzu et al., 2004). D-Synucleinimmunopositive LB in PD do not contain DJ-1 (Rizzu et al., 2004). Similarly, LRRK2 is not a major component of LB in idiopathic PD, however it is expressed in neurons of the dopaminergic nigrostriatal pathway (Higashi et al., 2007; Melrose et al., 2007). LRRK2 is also expressed in neuronal inclusions in a number of neurodegenerative conditions including Alzheimer’s disease, Pick’s disease, amyotrophic lateral sclerosis and Huntington’s disease (Miklossy et al., 2006). Therefore, LRRK2 is strongly associated with pathological inclusions in several neurodegenerative conditions and is not confined to the core pathologies associated with parkinsonian conditions.
46
Figure 1.3 Neuronal and glial inclusions in PD, MSA and PSP
Representative brightfield photomicrographs of 5Pm thick, paraffin-embedded sections immunoperoxidase labelled with D-synuclein or tau (AT8) and counterstained with cresyl violet. Scale in D is equivalent for photomicrographs A-C. Scale in G is equivalent for photomicrographs E & F. A
An D-synuclein-immunoreactive Lewy body (LB) in a dopaminergic neuron of the substantia nigra in a PD case.
B
An D-synuclein-immunoreactive neuronal cytoplasmic inclusion (NCI) in the pons of a MSA case.
C
A tau (AT8)-immunoreactive neurofibrillary tangle (NFT) adjacent to a normal neuron in the pons of a PSP case.
D
A tau (AT8)-immunoreactive tufted astrocyte (TA) in the motor cortex of a PSP case.
E
D-Synuclein-immunoreactive glial cytoplasmic inclusions (GCI) in the pons of a MSA case.
F
A tau (AT8)-immunoreactive coiled body adjacent to a normal neuron in the pons of a MSA case.
G
HLA-DR-immunoreactive activated microglia in the pons of a MSA case.
47
A
B
C
D
25μm
E
F
G
50μm
1.2.2 Abnormal protein expression in astrocytes Astrocytes provide essential support to neurons, including roles in synapse formation, maintenance and plasticity, and regulation of cerebral blood flow (Lobsiger and Cleveland, 2007). The importance of astrocytes has begun to emerge with the growing knowledge that they are involved in almost every task the brain performs (Ransom et al., 2003). In neurodegenerative diseases, astrocytic changes are associated with the upregulation of glial fibrillary acidic protein (GFAP), an intermediate filament found in all astrocytes (Teismann and Schulz, 2004). Astrogliosis is observed across all parkinsonian disorders and in addition some of these disorders accumulate inclusions in astrocytes.
In PD, reactive astrogliosis is thought to be minimal (Hirsch et al., 2005; Mirza et al., 2000), however, GFAP expression of astrocytes is observed primarily in the SN (Hirsch et al., 2005; Vila et al., 2001). In addition, D-synucleinimmunoreactive astrocytes are also observed in PD and shown to correlate with neuronal inclusion pathology (Braak et al., 2007; Wakabayashi et al., 2000). In contrast to PD, although severe astrogliosis is commonly observed in MSA (Ozawa et al., 2004), there have been no reports of D-synuclein-immunoreactive astrocytic inclusions. In PSP, increased reactive astrogliosis is observed with additional tau-immunoreactive TA (Figure 1.3D). These TA are thought to contribute to degeneration, rather than participating in a reactive process in PSP (Togo and Dickson, 2002).
48
Many of the pathogenic proteins associated with genetic forms of PD (parkin, PINK1 and DJ-1) are normally expressed in glia in the human brain (Bandopadhyay et al., 2004; Gandhi et al., 2006; Neumann et al., 2004; ZarateLagunes et al., 2001). Parkin immunoreactivity is observed in glial cells (ZarateLagunes et al., 2001) and in a large proportion of GFAP-negative astrocytes in rat brain tissue (D'Agata et al., 2000). In PSP and MSA, PINK1 protein is present in upregulated reactive astrocytes (Gandhi et al., 2006). Similarly, DJ-1 immunoreactivity is also observed in reactive astrocytes in PD, PSP and MSA (Bandopadhyay et al., 2004; Neumann et al., 2004). In addition to DJ-1 immunoreactivity in reactive astrocytes, tau-immunoreactive TA in PSP also express DJ-1 (Kumaran et al., 2007).
1.2.3 Abnormal protein expression in oligodendroglia The distinguishing and core pathological feature in MSA is the D-synucleinimmunoreactive GCI in oligodendroglia (Figure 1.3E) (Papp et al., 1989; Trojanowski and Revesz, 2007). However, some reports have also shown Dsynuclein-immunoreactive oligodendroglia in PD (Campbell et al., 2001; Wakabayashi et al., 2000). GCI are described as non-membrane bound cytoplasmic inclusions composed of loosely aggregated filaments/tubular structures (Burn and Jaros, 2001). A large number of GCI are identified using Dsynuclein in MSA (Burn and Jaros, 2001; Wakabayashi et al., 1998) even though D-synuclein is a synaptic protein not normally found in oligodendroglia (Culvenor et al., 2002; Duda et al., 2000b; Richter-Landsberg, 2000). No
49
increase in D-synuclein mRNA occurs in MSA (Miller et al., 2005; Ozawa et al., 2001). This suggests D-synuclein aggregation within these oligodendroglia may result from an imbalance of multi-protein interactions (Gai et al., 1999).
CB are argyrophilic and tau-immunoreactive (Arima et al., 1997) (Figure 1.3F) occurring in oligodendroglia as fine bundles of filaments around a nucleus and extending into the proximal part of the cell process (Chin and Goldman, 1996). CB are commonly found in the precentral cortex, internal capsule, pencil fibers in the lenticular nuclei, midbrain and the tegmentum of the pons and medulla oblongata (Komori, 1999). As described above, tau-immunoreactive CB and associated threads have also been used to grade tau burden and differentiate PSP subtypes (Williams et al., 2007a) with the degree of CB thought to decline with disease duration (Josephs et al., 2006; Williams et al., 2007a). However, CB is also thought to be strongly associated with regions of severe neuronal loss (Jin et al., 2006). Tau immunoreactivity has also been observed in oligodendroglial GCI in MSA (Cairns et al., 1997; Giasson et al., 2003; Takeda et al., 1997). One study has reported the expression of insoluble PINK1 in GCI in MSA (Murakami et al., 2007). The oligodendroglial pathologies of GCI in MSA and CB in PSP are also immunoreactive for DJ-1 (Kumaran et al., 2007; Neumann et al., 2004).
There have been very few studies on oligodendroglia-associated changes in PD and although PSP or MSA are not recognised as gross demyelinating diseases
50
using routine myelin stains, the significant involvement of oligodendroglia in both disorders suggests some disruption to this cell type. In PSP, the dentate hilus and SCP are regions that are associated with demyelination (Ishizawa and Dickson,
2001).
Such
changes
have
been
associated
with
grumose
degeneration (abnormalities in Purkinje cell synapses) in the dentate nucleus (Cruz-Sanchez et al., 1992; Ishizawa and Dickson, 2001; Suyama et al., 1997). Myelin disruption has been observed in the pons, caudate, putamen and globus pallidus of MSA cases using antibodies sensitive to abnormalities in myelin basic protein (MBP) (Matsuo et al., 1998). Few other studies have been performed identifying disease effects on myelin in MSA and PSP. In particular, there have been no studies on changes in constituent oligodendroglial proteins in these disorders. In MSA, GCI are thought to represent the primary change and neuronal pathology is considered secondary to oligodendroglial pathology (Armstrong et al., 2006; Wenning and Jellinger, 2005). Furthermore, the implementation of CB as a measure of tau burden in PSP subtyping also suggests the importance of oligodendroglial pathology in PSP. Therefore, it would be of interest to identify changes in constituent oligodendroglial and myelin proteins to determine if such changes occur in both PSP and MSA.
51
1.2.4 Abnormal protein expression in microglia Microglia comprise 10-20% of the total glial cell population and are evenly distributed throughout the normal brain, with their processes close together, but not touching each other (Gerhard et al., 2003). Using their processes, resting microglia constantly evaluate their surroundings under normal conditions, however with the slightest alteration in their micro-environment or as a consequence of pathological changes, they rapidly activate and express a morphological change (Hald and Lotharius, 2005). Microglial activation as a response to tissue injury is a feature of many neurodegenerative disorders, whereby the cell surface expression of the Human Leukocyte Antigen DR-1 (HLA-DR) can be used as a marker for tissue degeneration as activated microglia respond to tissue injury (Figure 1.3G) (Gerhard et al., 2003).
In PD, similar to astrocytic activation, microglial activation occurs with the loss of dopaminergic neurons (Hirsch et al., 2005). Animal models and in vitro culture studies of PD are indirect, but they do illustrate that the dopaminergic cells in the SN are highly vulnerable to inflammatory attack and that microglial cells can be activated to mount such an attack (McGeer and McGeer, 2008). 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) models of PD have been used to produce a sustained microglial inflammatory reaction in the SN, although LB are not formed (Barcia et al., 2004; McGeer et al., 2003). Furthermore, studies have shown that D-synuclein is an inflammatory stimulant for microglia that contribute
52
to the death of dopaminergic cells (Esposito et al., 2007; Gao et al., 2003; Lee et al., 2005; McLaughlin et al., 2006; Shavali et al., 2006; Zhang et al., 2005).
The pathological changes in microglia have been less studied in post-mortem brain tissue of the atypical parkinsonian disorders PSP and MSA. However, the use of the in vivo activated microglial marker [11C] (R)-PK11195 positron emission tomography (PET) in parkinsonian disorders have shown a pattern of increased microglial activation in regions of neuropathological alterations in PD, PSP and MSA (Gerhard et al., 2003; Gerhard et al., 2006). In PSP, tau pathology correlates with microglial activation in all regions except for the brainstem, suggesting that in the brainstem, microglial activation is not related to tau deposition alone (Ishizawa, Dickson et al 2001). In MSA, the transformation of microglia into HLA-DR-immunopositive antigen presenting cells correlates with GCI burden (Ishizawa et al., 2004). The suggested role of microglial activation in these neurodegenerative conditions is to arbitrate neuronal loss (Gerhard et al., 2003).
1.2.5 Summary The abnormal protein expression in neurons differs across parkinsonian disorders, however, some similarities occur in the pathological changes in glia. The role of glia has long been underestimated, with accumulating evidence suggesting that glial cells may represent a primary target of degenerative disease processes (Ransom, Behar, Nedergaard 2003; Croisier & Graeber 2006). It is still unclear how glial abnormalities facilitate degeneration in these 53
disorders and in particular, the earliest intracellular abnormalities associated with these conditions have not been identified and therefore, the mechanism of degeneration remains poorly understood. To determine early changes, it may be useful in initially studying the constituent glial proteins and how they are associated with other core pathological features observed in these disorders. Many of the pathogenic proteins associated with genetic forms of PD appear to be expressed in glial cells and even in some of the glial inclusions observed in parkinsonian disorders. Despite this knowledge, there have been no comparative studies of how these glial proteins are affected in different parkinsonian disorders. The prominent glial-related inflammatory processes and inclusion pathology in PD, PSP and MSA warrants further studies of these glial changes across these parkinsonian disorders.
1.3 STUDY OBJECTIVES The current literature suggests that there is substantial clinical overlap between typical PD and the atypical parkinsonian disorders. The overlap in clinical phenotypes suggests that similar pathways and cell types are affected in these disorders. There has been growing evidence of the significant degree of overlap between neurodegenerative diseases (Feany & Dickson 1996; Armstrong, Cairns, Lantos 2001; Armstrong, Cairns, Lantos 2005; Galpern & Lang 2005; Armstrong, Cairns, Lantos 2008). In particular, Armstrong & colleagues have suggested that although there are differences in morphology and molecular diversity, pathological lesions often express similar spatial patterns in several neurodegenerative disorders, implying a degree of overlap between different 54
disorders and shared pathological mechanisms (Armstrong, Cairns, Lantos 2001). Despite this, there have been no comparative studies on the pathological changes in these overlapping parkinsonian disorders. Pathologically, the loss of dopaminergic neurons in the SN is a key feature observed in PD, PSP and MSA, although a comparison of glial changes in protein expression and deposition warrants further studies.
1.4 MAJOR HYPOTHESES
Clinical subtype is related to pathological regions and cell type/s affected rather than the type of inclusion pathology.
Astrocytic dysfunction occurs early in PSP or the cerebellar/Richardson’s syndrome phenotypes of PSP and MSA.
Oligodendroglial dysfunction occurs early in MSA or in the parkinsonian phenotypes of MSA and PSP.
Microglial activation occurs globally across all parkinsonian disorders and different forms of microglia relate to the pattern of pathological severity and degeneration.
55
Chapter 2: Common materials and methods
2.1 SOURCES OF TISSUE Cases were sourced from different brain banks, which used the same diagnostic criteria (refer to chapter 1). Controls were matched for age and were without neurological or neuropathological disease. All tissue, and clinical and pathological information was collected with appropriate consent from brain donors and their next-of-kin, and the collections were approved by appropriate institutional Human Ethics Committees. Cases were longitudinally assessed by clinicians associated with each brain bank.
The Prince of Wales Medical Research Institute Tissue Resource Centre (POWMRI TRC) provided fresh frozen and formalin-fixed brain tissue samples from longitudinally studied PD (n=10), PSP (n=17), MSA
(n=12) and age-
matched control (n=13) cases (Table 2.1) as requested through the Australian Brain Donor Programs (ABDP) coordinated by Associate Professor Jillian Kril. Neuropathological diagnoses for these cases were performed by Professor Glenda Halliday and Associate Professor Jillian Kril. The POWMRI TRC tissue was used for both immunohistochemistry (IHC) and western blotting (WB).
56
The Queen Square Brain Bank (QSBB) provided formalin-fixed brain tissue samples from prospectively-studied PD (n=5), PSP (n=19), MSA (n=17) and control (n=5) cases (Table 2.1) as requested through the QSBB coordinated by Professors Andrew Lees and Tamas Revesz. Neuropathological diagnoses for these cases were performed by Dr Janice Holton and Professor Tamas Revesz. The QSBB tissue was used for IHC.
As the supply of fresh frozen brain tissue for MSA cases was limited from these brain banks, a further request for fresh frozen MSA tissue (n=5) was granted from the South Australian Brain Bank (SABB) coordinated by Professor Peter Blumbergs. Neuropathological diagnoses for these cases were performed by Professor Peter Blumbergs. This tissue was used for WB.
57
Table 2.1 General case demographics for control, PD, PSP and MSA cases Case cohorts
Sex
Age at death
PMD
(M/F)
(Years, Mean r SD)
(Hours, Mean r SD)
Control POWMRI TRC cohort 1
4M : 4F
72 r 8
16 r 10
Control POWMRI TRC cohort 2
4M : 1F
71 r 7
14 r 6
Control QSBB TRC cohort 3
2M : 3F
78 r 14
64 r 29
PD POWMRI TRC cohort 4
6M : 2F
75 r 5
17 r 16
PD POWMRI TRC cohort 5
2M : 0F
83 r 7
4r4
PD QSBB cohort 6
4M : 1F
79 r 4
70 r 45
PSP POWMRI TRC cohort 7
12M : 3F
74 r 8
10 r 7
PSP POWMRI TRC cohort 8
2M : 0F
76 r 2
12 r 11
PSP QSBB cohort 9
12M : 7F
76 r 9
27 r 13
MSA POWMRI TRC cohort 10
7M : 5F
69 r 8
13 r 12
MSA QSBB cohort 11
9M : 8F
68 r 7
34 r 23
MSA SABB cohort 12
1M: 4F
72 r 7
10 r 7
PMD= post-mortem delay, M= male, F= female, SD= standard deviation, POWMRI TRC= Prince of Wales Medical Research Institute Tissue Resource Centre, QSBB= Queen Square Brain Bank, PD= Parkinson’s disease, PSP= progressive supranuclear palsy, MSA= multiple system atrophy, SABB= South Australian Brain Bank.
2.2 CASE ASCERTAINMENT AND CLINICAL INFORMATION Information regarding the following clinical features was provided for all prospectively-studied cases, with some exceptions. Standard clinical information 58
available for all cases included age at onset and disease duration (determined from the earliest clinical presentation as assessed by medical practitioners from patient and informant information).
The clinical indices chosen for correlation studies were based on dominant clinical features and key differentiating markers of PD, PSP or MSA. Akinesia and rigidity are considered to be the core clinical features observed across these parkinsonian disorders (Brooks, 2002; Poewe and Wenning, 2002). Whereas resting tremor, rigidity, bradykinesia and postural instability are the major clinical features in PD (Hald and Lotharius, 2005). The clinical diagnosis of PD is certain if subjects have good clinical improvement with L-dopa therapy (Gelb et al., 1999). Therefore, all these features were assessed to evaluate the parkinsonian signs in PD, PSP and MSA. The neurological examinations also involved Hoehn and Yahr (H&Y) staging (Hoehn and Yahr, 1998), which describes the progression of parkinsonian symptoms from stages 0 (no signs or symptoms) to 5 (severely affected). The last H&Y stage recorded for each patient was used for parkinsonism-associated clinical correlations.
Dysarthria refers to speech impairment and dysphagia is associated with swallowing difficulties. These features are more commonly associated with atypical parkinsonian disorders, in contrast to typical PD. In addition to these parkinsonian-related features, cognitive impairment is also commonly observed in neurodegenerative disorders, particularly with disease progression. The Clinical Dementia Rating (CDR) scale (Morris, 1993) for the severity of dementia 59
was assessed, and the last CDR stage prior to death was used for clinical correlations associated with cognitive dysfunction. The CDR consists of five stages ranging from 0, 0.5, 1, 2 and 3.
As described in chapter 1, a possible diagnosis of PSP is given when either slow vertical saccades, vertical supranuclear gaze palsy or postural instability with falls occurs within a year of onset of symmetric akinesia or rigidity (Burn and Lees, 2002). A probable PSP diagnosis includes all these symptoms with no evidence of other diseases that could explain them (Burn and Lees, 2002). Supporting features for the diagnosis of PSP include postural abnormalities, poor response to L-dopa therapy and early dysphagia or dysarthria (Litvan et al., 1996a; Litvan et al., 2003). Early falls, postural instability and eye signs were assessed as clinical indices related to PSP.
As described in Chapter 1, the Minneapolis Consensus Conference identified four clinical domains and a response to L-dopa to differentiate cases into possible and probable MSA in the absence of autopsy confirmation (Wenning et al., 2004) as 1) parkinsonism, 2) autonomic and urinary dysfunction, 3) cerebellar dysfunction and 4) corticospinal tract dysfunction. Therefore, autonomic dysfunction, postural hypotension and ataxia were primary features assessed to evaluate symptoms associated with MSA.
60
2.3 STANDARD PREPARATION OF BRAIN TISSUE Brains from donors were obtained with consent at autopsy from the POWMRI TRC, QSBB and the SABB. Brains were fixed for at least two weeks in 15% buffered formalin (39% aqueous solution of formaldehyde). Following fixation, the cerebellum and brainstem were separated from the cerebrum by sectioning through the cerebral peduncles. The weight and volume of the cerebrum was determined and the length of each hemisphere measured. The cerebrum was embedded in 3% agarose and cut into coronal slices. The cutting surface was perpendicular to the fronto-occipital axis and cut at 3mm intervals on a rotary slicer and examined macroscopically (Cullen and Halliday, 1998; Harding et al., 2002). Standard blocks and blocks from regions of interest were taken from coronal slices including the midbrain (taken at the transverse level including the red nucleus), the putamen (dorsolateral level) and the pontine basis (taken at the transverse level including the SCP).
Blocks of all tissue regions of interest for each case were paraffin-embedded using a Tissue Tek automatic processor (Sakura Inc., Torrance, USA). Blocks were processed through graded ethanols and xylene and then infiltrated with paraffin wax under vacuum pressure. Paraffin-embedded blocks were then cut at 5-8Pm on a rotary microtome (HM 325 Microm). To ensure good adhesion of all brain tissue sections, slides for mounting the tissue sections were coated with gelatin or 3-aminopropyltrimethoxysilane (TESPA) (Sigma, St Louis, USA). Gelatinised slides were coated with a 1% gelatin solution, then coated with a 4%
61
formaldehyde solution and dried to make ready for use. TESPA-coated slides were first coated with absolute ethanol for 10 seconds and then with TESPA for 5 minutes. The slides were then rinsed in dry acetone, followed by distilled water for 10 seconds each and then dried overnight in an oven at 40qC. Tissue sections were mounted onto gelatinised or TESPA-coated slides in preparation for IHC.
2.4 GENERAL IMMUNOHISTOCHEMICAL METHODS 2.4.1 Peroxidase immunohistochemistry (counterstained with cresyl violet) Standard protocols for peroxidase IHC were utilised to identify the proteins of interest in all cases, as previously described (Halliday et al., 2005b). Firstly, sections were deparaffinized and rehydrated in xylene (twice, 3 minutes each), 100% ethanol (twice, 3 minutes each), 95% ethanol (3 minutes), 70% ethanol (3 minutes) and distilled water (3 minutes). Appropriate antigen retrieval methods were utilised depending on the primary antibody used (Table 2.2), followed by incubation in a 3% hydrogen peroxide and 50% ethanol solution (40 minutes, room temperature) to quench endogenous peroxidases. Following two washes in distilled water and 0.1M tris buffer (pH 7.4), a blocking step was performed by incubating slides in a 20% normal horse serum solution (1 hour, room temperature). Primary antibody incubations varied depending on the antibody (Table 2.2) and were followed by three 0.1M tris buffer washes (5 minutes). Sections were then incubated in rabbit or mouse biotinylated secondary antibody (1:200; 30 minutes at 37qC; Vector Laboratories Inc., Burlingame,
62
USA), followed by three 0.1M tris washes. The tertiary antibody incubation required an avidin-biotin-peroxidase tertiary complex (1:500; 30 minutes at room temperature;
Vectastain,
Vector
Laboratories
Inc.,
Burlinghame,
USA).
Visualisation was achieved by incubation with 0.7% hydrogen peroxide in 0.15% diamniobenzidine tetrahydrochloride (Sigma, St Louis, USA) for 15 minutes at room temperature, followed by several 0.1M tris buffer and distilled water washes.
Following the completion of the peroxidase immunohistochemical procedure, sections were counterstained with cresyl violet (0.5%) or Mayer’s haemotoxylin to visualise Nissl substance. Sections were placed in cresyl violet for 30-60 seconds and then rinsed with water, and differentiated using 70% ethanol to obtain optimal histological intensity. Sections were then placed in 95% ethanol (1 minute), 100% ethanol (twice, for 3 minutes each) and xylene (twice, for 3 minutes each). Slides were then coverslipped using DPX neutral mounting medium (Ajax Finechem, Taren Point, Australia) to make ready for analysis.
63
Table 2.2 Antigen retrieval methods for different primary antibodies Primary antibodies
Antigen retrieval method
Primary
antibody
incubation time AT8,
GFAP
(anti- None
1 hour at 37qC
rabbit), EIII-tubulin D-Synuclein, GFAP 99% formic acid (3 minutes)
1 hour at 37qC
(anti-mouse) PACRG
99% formic acid (3 minutes)
Parkin
99% formic acid (10 minutes) and 1
1.5 hours at 37qC hour
at
pressure cooking
temperature
P25D
0.2M citrate buffer, pH6.0 (15 minutes)
Overnight at 4qC
MBP,
CNPase, 0.2M citrate buffer, pH 6.0 (5 minutes)
Overnight at 4qC
room
MAP1B Neurofilament Apolipoprotein degraded
0.2M citrate buffer, pH 6.0 (10 minutes) D, 4% aluminium chloride (3 minutes)
1 hour at 37qC Overnight at 4qC
MBP,
HLA-DR GFAP = glial fibrillary acidic protein, PACRG = parkin co-regulated gene, MBP = myelin basic protein, CNPase = 2’3’-cyclicnucleotide 3’phosphodiesterase, MAP1B = microtubule associated protein 1B, HLA-DR = human leukocyte antigen-DR1.
64
2.4.2 Double-labelling immunofluorescence Double-labelling
immunofluorescence
was
performed
using
previously
established methods (Baker et al., 2006), including appropriate antigen retrieval methods depending on the different primary antibodies used (Table 2.2). Paraffin sections were dewaxed through xylene, graded ethanols (twice in xylene, twice in 100% ethanol, 95% ethanol, 75% ethanol, for 3 minutes each) and lastly in distilled water (3 minutes). All appropriate antigen retrieval methods were carried out for both primary antibodies used for double-labelling immunofluorescence. Slides were then washed in distilled water and 0.1M tris buffer and placed in a solution of 3% hydrogen peroxide and 50% ethanol for 40 minutes at room temperature, then placed in a 20% normal horse serum solution for 1 hour at room temperature. Primary antibodies were then incubated at different times depending on the primary antibodies used (Table 2.2) and visualised with a cocktail of an anti-rabbit secondary antibody conjugated to Alexa 568 (1:250; Molecular Probes, Invitrogen, Carlsbad, US) and anti-mouse secondary antibody conjugated to Alexa 488 (1:400; Molecular Probes, Invitrogen, Carlsbad, USA) for 2 hours at room temperature. Slides were then washed in 0.1M tris (3 times in 5 minutes) and coverslipped with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, USA). To test the specificity and ensure non-cross reactivity of secondary fluorescent probes, a section without primary antibodies was included for each staining procedure as a negative control. In addition, a cocktail of the secondary antibodies were applied to sections with only one primary antibody incubated on each section.
65
2.4.3 Combined peroxidase and immunofluorescence labelling For the purpose of using same species-derived primary antibodies, a combined method of peroxidase visualisation and immunofluorescence was used. Firstly, the standard peroxidase protocol was performed as described above, with all appropriate antigen retrieval methods carried out for all primary antibodies included in the experimental procedure (Table 2.2) (Baker et al., 2006; Halliday et al., 2005b). Briefly, slides were incubated with the first primary antibody, and then incubated with a rabbit or mouse biotinylated secondary antibody (1:200, Vector Laboratories Inc., Burlingame, USA) for 30 minutes at 37qC. The tertiary complex involved the incubation with an avidin-biotin-peroxidase tertiary complex (1:500, Vectastain, Vector Laboratories, Burlinghame, USA) for 30 minutes at room temperature. Visualisation was achieved by incubation with 0.7% H2O2, in 0.15% diaminobenzidine tetrahydrochloride (Sigma, St Louis, USA) for 15 minutes at room temperature, followed by several 0.1M tris washes. This was then followed by the immunofluorescent labelling step, either using one fluorescent probe (method used in chapter 5) or a cocktail of two fluorescent probes (method used in chapter 6). Prior to the second primary antibody incubation, slides were incubated in a 20% normal horse serum solution for 1 hour at room temperature. Sections were then incubated with the second same species-derived primary antibody for 1 hour at 37qC. Following several 0.1M tris washes (3 times in 5 minutes) sections were incubated with one or a cocktail of secondary antibodies (anti-mouse conjugated to Alexa 568, Molecular Probes, Invitrogen, Carlsbad, USA or anti-rabbit conjugated to Alexa 488, Molecular
66
Probes, Invitrogen, Carlsbad, USA) for 2 hours at room temperature. Slides were then washed in 0.1M tris (3 times in 5 minutes) and coverslipped with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, USA). To ensure specificity of the immunohistochemical technique, a positive and negative (with no primary antibody incubation) control was included in each experiment.
67
Chapter 3: Pathological staging of Progressive Supranuclear Palsy
3.1 INTRODUCTION As described in chapter 1, PSP is a progressive neurodegenerative disorder with a variety of tau-immunoreactive neuropathologies occurring in association with fibrillary gliosis and neuronal loss predominantly in subcortical regions (Jin et al., 2006; Komori, 1999; Steele et al., 1964). Despite thorough knowledge of the distribution and type of pathology, the pathological progression of PSP has not been clearly defined. This emphasises the necessity of a severity scale for the progression of PSP based on tissue histopathology.
The most consistent types of histopathology in PSP are well described. The presence of intraneuronal fibrillised tau in the form of NFT establishes a definitive diagnosis for PSP (Hauw et al., 1994; Litvan et al., 1996b), with current criteria requiring a high degree of NFT and neuropil threads in at least three of the following regions: pallidum, STN, SN, or pontine basis and a low to high density of these pathological features in at least one of the following regions: striatum, oculomotor complex, medulla or dentate nucleus. This concentration has recently been confirmed using more modern IHC (Dickson et al., 2007; Williams et al., 2007a). Neuronal loss is thought to be variable, and tau68
immunopositive astrocytes or processes in these regions help to confirm the neuropathological diagnosis.
The two major clinical phenotypes, PSP-P and PSP-RS, relate to significantly different quantities and distributions of pathological tau (AT8-immunoreactive CB and threads), with more restricted and milder tau pathology occurring in PSP-P compared with PSP-RS (Williams et al 2007). As described in chapter 1, a histopathological grading system has been developed to assess pathology that can distinguish these clinical subtypes using overall tau burden in the SN, caudate and dentate nucleus (Williams et al., 2007a). A similar approach has been used to distinguish another rare clinical phenotype, pallido-nigro-luysial atrophy (PNLA), which has less pathological tau in the motor cortex, striatum, pontine nuclei and cerebellum (Ahmed et al., 2008). These grading systems enable the overall evaluation of pathological tau to distinguish clinical phenotypes of PSP. However, all cases have similar core pathological PSP features, and the progression of these core pathologies is still poorly understood.
An interesting finding in PSP is that, as the disease progresses over time, there is a negative rather than positive correlation between the burden of pathological tau in subcortical and cortical regions and disease duration (Halliday et al., 2000; Josephs et al., 2006; Rub et al., 2002; Williams et al., 2007a). This negative relationship, in association with the differences observed between clinical phenotypes, limits the usefulness of tau deposition alone as an indicator 69
of disease severity. Furthermore, in addition to tau pathology, Ahmed and colleagues also evaluated the degree of neuronal loss in the SN, STN and dentate nucleus. A correlation between neuronal loss and disease duration in these regions was not identified in either the PSP or PSP-PNLA groups (Ahmed et al., 2008). This suggests that the assessment of neuronal loss alone is also a poor indicator of disease severity and progression in PSP.
Most other neurodegenerative tauopathies can be staged by their progressive atrophy into 5 stages (0-4) (Broe et al., 2003). This staging scheme is not applicable for PSP due to the limited atrophy observed (refer to chapter 1) (Schofield et al., 2005), indicating a substantive difference in the consequence of the tau abnormality and regional deposition in this disorder. The staging scheme developed for MSA may be a useful template, as MSA also has two major phenotypes (MSA-P and MSA-C) with progression determined by grading several pathological features (Jellinger et al., 2005) including the assessment of atrophy, neuronal loss, astrogliosis and abnormal protein deposition in GCI. Using this concept for PSP, the STN, SN and globus pallidus are the most consistently affected and are suggested to be the earliest regions affected (Verny et al., 1996; Williams et al., 2007a). While it has been suggested that the pattern of NFT topography commences subcortically then spreads cortically in PSP (Armstrong et al., 2006; Armstrong et al., 2007b; Bergeron et al., 1997), this concept has been challenged by recent findings suggesting the motor cortices are affected early, followed by other subcortical pathways (cerebellum and pons, caudate nucleus) and finally extending into prefrontal and parietal 70
cortical regions (Williams et al., 2007a). The loss of neurons and atrophy in cerebellar relay pathways have been identified as contributors to early falls in PSP (Halliday et al., 2005a), with atrophy of the SCP potentially diagnostic for PSP (Paviour et al., 2005; Tsuboi et al., 2003a). The severity of neuronal loss in the SN has been correlated with the later clinical features of gaze palsy (Halliday et al., 2000) and dysarthria (Kluin et al., 2001). Establishing the pathological staging of PSP will assist in determining its pathogenesis.
3.1.1 Aim To establish a staging scheme, by grading different core pathological features of PSP.
3.2 METHODS 3.2.1 Cases Used In order to develop and validate a staging scheme for PSP that reflects disease progression over time, several cohorts are required, some of which need to include prospectively collected cases ranging from extremely early in the disease to end stage. As the fortuitous death of PSP cases at early disease stages are extremely rare, a collaborative approach between major collections of pathological specimens was instigated in order to assemble sufficient cases with adequate clinical follow-up at both early and late stages of disease analysis. Further validation of the staging scheme was performed on an independent sample. Therefore, several cohorts were used to 1) develop the scheme and 2) validate the scheme. Consent for all tissue samples was provided and approved 71
by the Human Ethics Committees associated with all institutions. Clinical details of age at death, age at onset and disease duration from the time of diagnosis was collated for all cases. Duration of clinical symptoms (early falls and slow or absent supranuclear gaze) were established from the first symptom onset through to death. Cases used were pure PSP cases and did not have any coexisting Alzheimer’s disease, therefore Braak NFT staging (Braak et al., 2006) was used to stage tangle pathology. Additionally, Williams’s PSP stages (Williams et al., 2007a) and widths of the SCP (Tsuboi et al., 2003a) were analysed for cases included in correlation studies.
3.2.1.1 Cases for method development 13 prospectively followed cases with clinical and pathologically diagnosed PSP from the ABDP processed through the POWMRI TRC (cohort I - POWMRI TRC, Table 3.1) were used for method development.
3.2.1.2 Cases for scheme validation To validate the PSP staging scheme, a separate cohort was used, which included 16 prospectively followed cases with clinical and pathological PSP (excluding those from cohort I) from the POWMRI TRC (cohort II - POWMRI TRC, Table 3.1).
72
Table 3.1 Cohort characteristics Cohort I
Cohort II
Cohort III
POWMRI
POWMRI
International
TRC
TRC
cases
Gender ratio M:F
10:3
10:6
15:6
PSP-P:PSP-RS ratio
3:10
0:16
5:16
Age at onset (y)
68± 9
63± 8
69± 9
Age at death (y)
75± 7
70± 6
74± 8
Post-mortem delay (h)
10± 7
18± 21
11± 7
Disease duration (y)
7± 3
7± 3
5± 4
Duration of falls (y)
4± 3
5± 2
3± 3
Duration of eye signs (y)
2± 1
3± 2
1± 1**
1235± 158
1171± 151
1242± 150
Braak neuritic stage (/6)
1± 1
2± 2
1± 1
Williams PSP stage (/12)
4± 2
-
4± 2
4.2± 0.6
3.8± 0.8
4.2± 0.6
Brain weight (g)
Width of SCP (mm)
POWMRI TRC= Prince of Wales Medical Research Institute Tissue Resource Centre, M= male, F= female, PSP-P= progressive supranuclear palsy – parkinsonism, PSPRS= progressive supranuclear palsy – Richardson’s syndrome, y= years, h= hours, g= grams, SCP= superior cerebellar peduncle. ** from both groups using one-way analysis of variance p STN (NFT) > pontine basis (NFT) > anterior caudate nucleus (TA) > dentate nucleus (tau burden) > motor cortex (TA) (see also Figure 3.2).
90
Figure 3.6 The severity of the identified hierarchical pathologies in regions of interest
100%
90%
80%
70%
60% none mild mod severe
50%
40%
30%
20%
10%
0%
SN_NL
STN_NL
STN_NFT
Pons_NFT
Caudate_TA
Dentate_Tau
MTR_TA
SN_NL= ventral substantia nigra neuron loss, STN_NL= subthalamic nucleus neuron loss, Pons_NFT= neurofibrillary tangles in the pontine basis, Caudate_TA= tufted astrocytes in the anterior caudate nucleus, Dentate_Tau= tau burden in the dentate nucleus, MTR_TA= tufted astrocytes in the motor cortex.
Based on this data, a severity scheme with six stages (1-6) was trialled (Table 3.4). As moderate to severe neuronal loss was consistently observed in the ventral SN, a moderate or severe degree of neuronal loss in this region was considered a mandatory feature.
91
Table 3.4 Development scheme 1 of PSP pathological staging based on feature assessment. Moderate to severe SN cell loss is a mandatory feature. STN neuron loss
Stage 1
1
0
0
0
Motor cortex TA 0
Stage 2
2
1
0/1
0/1
0/1
Stage 3
2/3
1/2
1/2
0/1
0/1
Stage 4
3
1/2
2
2
1/2
Stage 5
3
2
3
2
2
Stage 6
3
3
3
3
3
STAGE
STN NFT
Dentate nucleus Tau
Pons NFT / Caudate TA
STN= subthalamic nucleus, NFT= neurofibrillary tangles, TA= tufted astrocytes.
Applying this development scheme 1 (Table 3.4) to cohort I revealed that only 15% of the cases fitted into the stages identified. To determine which features could be used to optimise the scheme, each pathological feature was individually excluded and the proportion of cases that fitted into these variant schemes determined. Due to the most non-hierarchical pattern observed being in the dentate nucleus (as shown in Figure 3.6), the variant of the dentate was excluded and this significantly improved the proportion of cases that fitted the scheme (46%). Therefore, a new scheme (development scheme 2, Table 3.5) was applied with the exclusion of dentate tau burden. To improve the effectiveness of the scheme, further features were excluded for optimisation as explained below.
92
Table 3.5 Development scheme 2 of PSP pathological staging based on feature assessment. Moderate to severe SN cell loss is a mandatory feature.
Stage 1
STN neuron loss 1
0
0
Motor cortex TA 0
Stage 2
2
1
0/1
0/1
Stage 3
2/3
1/2
1/2
0/1
Stage 4
3
1/2
2
1/2
Stage 5
3
2
3
2
Stage 6
3
3
3
3
STAGE
Pons NFT / Caudate TA
STN NFT
STN= subthalamic nucleus, NFT= neurofibrillary tangles, TA= tufted astrocytes.
Exclusion of the motor cortex TA did not significantly change the proportion that could fit this variant scheme (54%). Exclusion of the pons NFT / caudate TA significantly improved the proportion of cases that could fit this variation of the scheme (77%). However, the most successful variation for PSP staging was to exclude the evaluation of NFT pathology in the STN. All thirteen cases could be staged using this variation. Therefore, a refined version of development schemes 1 and 2 was established for the pathological staging of PSP (Table 3.6).
93
Table 3.6 Refinement of the PSP pathological staging scheme with the closest two out of three variables required. Moderate to severe SN cell loss is a mandatory feature. Pons NFT/ Caudate tufted astrocytes
Motor cortex tufted astrocytes
STAGE
STN neuron loss
Stage 1
0
0
0
Stage 2
1
0/1
0
Stage 3
2
0/1
0/1
Stage 4
2/3
1/2
0/1
Stage 5
2/3
2/3
2/3
STN= subthalamic nucleus, NFT= neurofibrillary tangles.
3.3.4 The pathological staging of PSP severity Stage 1 represents loss of neurons in the SN without visible loss of subthalamic neurons. Due to the tau deposition in the SN, STN and globus pallidus not being used in this staging scheme, these cases still fulfil pathological diagnosis for PSP (Hauw et al., 1994; Litvan et al., 1996b).
Stage 2 represents moderate to severe loss of the SN, a mild loss of neurons in the STN, and none or mild pathology in the pons and/or caudate nucleus.
Stage 3 represents moderate to severe loss of the SN, moderate loss of neurons in the STN, and none or mild degree of tau pathology in the pons 94
and/or caudate nucleus. None or mild tau-immunoreactive TA in the motor cortex.
Stage 4 indicates moderate to severe loss of the SN, a moderate or severe loss of neurons in the STN, with a mild or moderate degree of tau pathology in the pons and/or caudate nucleus but mild or no pathology in the motor cortex.
Stage 5 is the most severe, with all regions moderately or severely affected. There is moderate to severe loss of the SN and STN, with the degree of tau pathology moderate to severe in all fields of view of the pons and/or caudate and the motor cortex.
3.3.5 Analysis and validation of the pathological staging of PSP For cohort I, common factor analysis of stage, disease duration, brain weight and SCP width revealed these variables were all related to a single dominant factor explaining 52% of the variance (loading for duration=0.75, pathological stage= 0.64, brain weight=-0.70, SCP width=-0.78). This suggests that the pathological stage in this cohort is positively related to disease duration and negatively related to brain weight and SCP width.
Cohort II contained only the PSP-RS phenotype and, as may then be expected, presented a greater disease severity overall (n=14, 88% at stages 4-5). However, common factor analysis of stage, disease duration, brain weight and SCP width revealed that stage, disease duration and brain weight were related 95
to a dominant factor explaining 38% of the variance (loading for duration=0.65, pathological stage=0.82, brain weight=-0.65). This confirms that pathological stage is positively related to disease duration and negatively related to brain weight.
3.3.6 Correlations between PSP pathological severity, pathological tau burden and clinical indices Half the cases in cohort III were selected to have non-end stage disease so that correlations with clinical variables could be more effectively tested. For the PSPRS group there was a bimodal distribution with 36% of cases having pathological stage 2 and 3 disease and 18% having stage 4 disease. The PSPP cases had a unimodal distribution with pathological stages 2-4. The clinical indices tested in this cohort were age at death, age at onset, disease duration, duration of falls, duration of eye signs, and SCP width. The pathological stage of disease positively correlated with disease duration (Rho=0.75, p0.89, all p values 0.05).
115
Table 4.2 Demographic, volumetric and pathological variables for PD Cohort 4
Cohort 5
Cohort 6
POWMRI TRC
POWMRI
QSBB (n=5)
(n=8)
TRC (n=2)
Technique assessed
IHC
WB
IHC
Included in chapters
4, 5, 6, 7
6
5, 7
Sex (M : F)
6M : 2F
2M : 0F
4M : 1F
Age at onset (Years, Mean r SD)
60 r 6
55 r 18
61 r 9
Age at death (Years, Mean r SD)
75 r 5
83 r 7
79 r 4
Disease duration (Years, Mean r SD)
15 r 5
28 r11
18 r 7
H&Y (Mean, range)
5.0 (4 – 5)
N/A
N/A
CDR (Mean, range)
0.5 (0 – 3)
N/A
N/A
PMD (Hours, Mean r SD)
17 r 16
4r4
70 r 45*
Brain volume (ml, Mean r SD)
1188 r 155
N/A
N/A
Braak LB stage (Mean, range)
5 (4-5)
6 (6)
N/A
Caudate volume (ml)
6r1
N/A
N/A
Putamen volume (ml)
7r9
N/A
N/A
Globus pallidus volume (ml)
3r1
N/A
N/A
Midbrain volume (ml)
6r2
N/A
N/A
Pons volume (ml)
11 r 3
N/A
N/A
SN neuronal loss
3 (2-3)
N/A
N/A
SN LB severity (Mean, range)
2 (0-3)
N/A
N/A
Putamen LB severity (Mean, range)
0 (0-1)
N/A
N/A
Pons LB severity (Mean, range)
1 (0-1)
N/A
N/A
POWMRI TRC= Prince of Wales Medical Research Institute Tissue Resource Centre, QSBB= Queen Square Brain Bank, IHC= immunohistochemistry, WB= western blotting, M= male, F= female, SD= standard deviation, H&Y= Hoehn & Yahr (Hoehn and Yahr, 1998), CDR= Clinical Dementia Rating (Morris, 1993), PMD= post-mortem delay, SN= substantia nigra, LB= Lewy bodies, N/A= not available, *significantly different.
116
4.3.3 PSP diagnosis and characterisation All PSP cases reached clinical criteria for either possible or probable PSP and were definitely diagnosed according to NINDS-SPSP guidelines by the presence of significant numbers of tau-immunoreactive NFT in basal ganglia and pontine nuclei with no other neurodegenerative pathologies of note (Litvan et al., 1996b).
4.3.3.1 Case demographics and clinical progression The POWMRI TRC cases with PSP consisted of 12 males and 3 female patients with an average age of onset of 67 years, average age at death of 74 years and an average disease duration of 7 years (Table 4.3). Their average H&Y score was 4 (Table 4.3), while their average CDR was 1 (highest score of 3 in two cases with the remaining cases ranging from 0-2, Table 4.3).
The QSBB PSP cases included 12 males and 7 females and the average age of onset and death was 69 and 76 years old, respectively (Table 4.3). The average disease duration was 7 years (Table 4.3). H&Y and CDR scores were not documented for the QSBB cohort.
As described in chapter 1, PSP cases can be further subtyped into a parkinsonian phenotype, PSP-P, and a more globally impaired PSP-RS phenotype (Williams et al., 2005). PSP-P is diagnosed when the patient has an asymmetric onset with resting tremor as the dominant clinical feature. These cases often have some benefit from L-dopa therapy. A diagnosis of PSP-RS is 117
given when there is an early onset of postural instability and falls often progressing to cognitive dysfunction. PSP-RS cases consistently have supranuclear vertical gaze palsy during their disease course. 71% of the POWMRI TRC PSP cases were of the PSP-RS subtype (12 cases, Table 4.3) and 29% were PSP-P cases (5 cases, Table 4.3), similar to previously described distributions of these phenotypes (54% PSP-RS versus 32% PSP-P, Williams et al 2005). The QSBB cohort had a more equal distribution of PSP phenotypes, 58% being PSP-RS and 42% PSP-P (Table 4.3).
4.3.3.2 Pathology and atrophy The pathological PSP staging scheme was not available for the QSBB cohort but was applied to the POWMRI cases (refer to chapter 3). The majority of cases (60%) had stage 4 disease and 13% of cases had stage 2, 20% of cases with stage 5 disease. The smallest proportion (7%) of cases had stage 3 disease.
The assessment of the volume of regional structures in PSP showed that the pons was largest, followed by the putamen and caudate nucleus, while the midbrain and globus pallidus were the smallest structures assessed (Table 4.3). The volume of the putamen and globus pallidus correlated (Rho=0.73, p=0.04), as did the volume of the caudate nucleus and whole brain (Rho=0.71, p=0.047). The caudate nucleus, putamen and globus pallidus volumes correlated with the volume of the midbrain (all Rho>0.77, all p values 0.05). There was no increase in GFAP immunoreactivity in the putamen in PD and no significant LB formation in this region (Table 1). In addition to increased reactivity, colocalisation studies revealed that approximately 45% of GFAP-immunoreactive astrocytes in the PD cases also contained D-synuclein immunoreactivity (Figure 5.1C-E).
In MSA, the putamen had the most marked reactive astrogliosis with an increase in GFAP immunoreactivity (Table 5.1) and changes in their morphology
149
(enlarged cell bodies and distorted processes, Figure 5.2A). Markedly reduced astrogliosis was observed in the SN and pons of the MSA cases examined, with only a mild increase in the severity of GFAP immunoreactivity (Table 1) and few morphological changes. The increased severity of GFAP-immunoreactive astrocytes in the putamen positively correlated with an increased number of Dsynuclein-immunoreactive GCI in the pons (Rho=0.73, p=0.007) and MSA pathological stage (Rho=0.67, p=0.02). In contrast to PD, and as previously identified (Wakabayashi et al., 2000), double-labelling immunofluorescence showed no deposition of D-synuclein in GFAP-immunopositive astrocytes in MSA cases.
150
Table 5.1 Regional severity gradings for nigral cell loss, pathological inclusion formation, and increased astrocytic GFAP, PACRG and parkin Mean severity grade (range)
Control
PD
MSA
PSP
Cell loss
0
3 (2 – 3)
3 (2 – 3)
3 (2 – 3)
Inclusion formation
0
2 (0 – 3)
1 (0 – 3)
2 (0 – 3)
GFAP increase
1 (0 – 1)
1 (0 – 3)
1 (0 – 3)
2 (1 – 3)
PACRG increase
0 (0 – 1)
1 (0 – 3)
0 (0 - 2)
2 (0 - 3)
Parkin increase
0
0
0
2 (0 - 3)
Inclusion formation
0
0 (0 – 1)
2 (1 – 3)
0 (0 -1)
GFAP increase
1 (0 – 2)
0 (0 – 1)
2 (0 – 3)
1 (0 – 2)
PACRG increase
1 (0 – 1)
1 (0 – 2)
1 (0 – 2)
2 (0 – 3)
Parkin increase
0
0
0
2 (1 - 3)
Inclusion formation
0
1 (0 – 1)
1 (0 – 3)
1 (0 – 3)
GFAP increase
1 (0 – 1)
2 (2 – 3)
1 (0 – 2)
1 (0 – 2)
PACRG increase
0 (0 – 1)
2 (1 – 2)
0 (0 – 1)
1 (0 – 2)
Parkin increase
0
0
0
1 (0 - 2)
Substantia nigra
Putamen
Pons
PD= Parkinson’s disease, MSA= multiple system atrophy, PSP= progressive supranuclear palsy, GFAP= glial fibrillary acidic protein, PACRG= parkin co-regulated gene.
151
Figure 5.1 Astrocytes in controls and PD
Representative photomicrographs of 5Pm thick, paraffin-embedded sections of control and PD cases. A&B Brighfield photomicrographs of typical stellate astrocytes with finely branching processes immunohistochemically labelled with GFAP and counterstained with cresyl violet in the putamen of a control (A) and PD case (B), were morphologically similar. Scale in B is equivalent for A. C-E
Immunofluorescent images of a typical stellate GFAP-immunoreactive (Alexa 568, red, C) astrocyte colocalising with D-synuclein (Alexa 488, green, D), shown in the merge image (E) in the pons of a PD case. Scale in E is equivalent for C and D.
152
A
B
20μm
C
D
E
20μm
Figure 5.2 Astrocytic changes in MSA and PSP
Representative photomicrographs of 5Pm thick, paraffin-embedded sections of the basal ganglia in MSA and PSP. A
A brightfield photomicrograph of astrocytes with enlarged cell bodies and distorted processes in MSA, immunoperoxidase labelled with GFAP and counterstained with cresyl violet. Scale in B is equivalent for A.
B
A brightfield photomicrograph of enlarged and stellate reactive astrocytes in PSP, immunoperoxidase labelled with GFAP and counterstained with cresyl violet.
C
An immunofluorescent merge image of a GFAP-immunopositive (Alexa 568, red) reactive astrocyte also immunopositive for tau (AT8) (Alexa 488, green) in PSP.
D
A
brightfield
photomicrograph
of
a
tufted
astrocyte
in
PSP,
immunohistochemically labelled with tau (AT8) counterstained with cresyl violet.
153
B
A
100μm
C
D
25μm
50μm
As expected in PSP, obvious reactive astrogliosis was observed, as evidenced by the increase in GFAP-immunoreactive astrocytes with enlarged stellate morphology (Figure 5.2B) in all regions examined (Table 1). The greatest increase in the number of GFAP-immunoreactive astrocytes was observed in the SN (moderate severity, Table 1). An increase in the severity of GFAPimmunoreactive astrocytes in both the SN and putamen positively correlated with increasing SN neuronal loss (Rho>0.53, p0.56, p
![Thesis Chapter 3[1]](https://m.moam.info/img/260x300/thesis-chapter-31_59b692801723dddbc635a089.jpg)
