The Australian and New Zealand Society for Neuropathology
Annual Scientific Meeting, May 19th, 2007
Prince of Wales Medical Research Institute, Sydney.
Program
8:30-9:15am
Registration
9:15-11:15am
Slide Seminar
11:15-11:35am
Morning Tea
11:35am-12:30pm
Invited Speaker – Raad Shakir (UK).
“Neurocysticercosis Clinical and Management Issues”
12:30-1:30pm
Lunch break
1:30-2:00pm
Oral Presentation I
Peter Patrikios:”Remyelination in multiple sclerosis lesions”.
2:00-2:45pm
Oral Presentations II - Bill Evans Memorial Young Investigator Award
2:00-2:15
Lolita Warden: Inflammation and tau are associated with oligomeric A in Alzheimer’s disease brain tissue.
2:15-2:30
Natasha Luquin: Looking for SOD1 somatic mutations in ALS
2:30-2:45
Kashem Abul: Differential Protein Expression in Alcoholic Postmortem Human Brain
2:45-3:00pm
Afternoon tea
3:00-4:30pm
Oral Presentations III
3:00-3:15
Zhao Cai: Novel fibroblastic onion bulbs in a demyelinating avian peripheral neuropathy produced by riboflavin
deficiency.
3:15-3:30
Nina Sundqvist: Post mortem psychiatric diagnosis: A reliability study.
3:30-3:45
Emma Schofield: Predicting tissue degeneration in progressive supranuclear palsy: perfusion changes identify
in vivo and postmortem regional volume losses.
3:45-4:00
Cindy Kersaitis: Immunoglobulin in frontotemporal dementia
4:00-4:15
Julia Morahan: Brain chromosomal copy number differences in sporadic MND
4:15-4:30
Yue Huang: P25 exocytosis in multiple system atrophy
CLOSE OF SCIENTIFIC PART OF THE MEETING
4:40-5:10pm
ANZSNP Business Meeting
(Includes the announcement of the winner of the “Bill Evans Memorial Award”).
6:30 for 7:00pm
ANZSNP Dinner
“Flavours of Thai” Restaurant
39 Perouse Raod, Randwick (Within walking distance of POWMRI)
Cost ~$30 per head plus drinks and tips (BYO available).
1
Title: Remyelination in multiple sclerosis lesions.
Authors: Patrikios P 1, Stadelmann C 2, Kutzelnigg A 1, Rauschka H 3, Schmidbauer M 3, Laursen H 4, Sorensen
PS 5, Brück W 2, Lucchinetti C 6, Lassmann H 1.
1: Center for Brain Research, Medical University of Vienna, Austria.
2: Department of Neuropathology, University of Göttingen, Germany.
3: Department of Neurology, Municipal Hospital Lainz, Vienna, Austria.
4: Laboratory of Neuropathology, Rigshospitalet, Copenhagen, Denmark.
5: Department of Neurology, University of Copenhagen, Denmark.
6: Department of Neurology, Mayo Clinic, Rochester, Minnesota, USA.
Abstract: Although spontaneous remyelination does occur in multiple sclerosis (MS) lesions, its extent within a broad
MS population is presently unknown. A systematic analysis was performed of the incidence and distribution of
completely remyelinated MS lesions (so-called shadow plaques) or partially remyelinated lesions (shadow plaque
areas) in 51 autopsies of MS patients with different clinical courses and disease durations. The extent of remyelination
was variable between cases. In 20% of the cases, the extent of remyelination was extensive with 60 to 96% of the total
lesion area remyelinated. Extensive remyelination was found not only in patients with relapsing MS, but also in a
subset of patients with primary and secondary progressive disease. Older age at death and longer disease duration were
associated with significantly more remyelination. No correlation was found between the extent of remyelination and
gender nor age at disease onset. Thus the variable and patient-dependent extent of remyelination observed in MS must
be considered in the design of future clinical trials aimed to promote CNS repair. In vivo imaging methods of
remyelination are urgently required.
Title: Inflammation and tau are associated with oligomeric A in Alzheimer’s disease brain tissue.
Authors: Warden LA, Halliday GM, Shepherd CE.
Prince of Wales Medical Research Institute, Barker Street, Randwick, Sydney 2031, Australia
Abstract: Inflammatory glia surround insoluble, fibrillar A deposits, known as senile plaques, in Alzheimer’s
Disease (AD). Whilst senile plaques and neurofibrillary tangles, consisting of insoluble tau, are considered
neuropathological hallmarks of AD, inflammation is the only reliable correlate of the neuronal cell loss that underlies
the dementia. Therefore, identifying potent stimulators of inflammation in AD represents an important area of research.
Recent in vitro studies have implicated soluble A oligomers in this inflammatory process, however, in vivo studies to
determine their association with plaque pathology and inflammation in human AD post mortem brain tissue has not
been described. Ten-micron formalin-fixed, paraffin-embedded sections from the temporal cortex of 8 AD and 8
control cases were immunhistochemically stained using antibodies against protein oligomers (A11), A (IE8; reactive
to all A species), phosphorylated tau (AT8) and microglia (HLA-DR). Double-labelling immunofluorescence was also
carried out using the following antibody combinations - A11 and IE8, A11 and AT8, A11 and HLA-DR. Confocal
microscopy confirmed A and oligomer protein co-localisation in AD plaques (8–64% of total plaques). In controls, no
oligomer protein staining was observed in the diffuse, amorphic A plaques associated with normal ageing.
Quantitative analysis of AD cases revealed a positive correlation between cored plaques containing inflammation and
oligomer protein staining in plaques (R2=0.95, P<0.000). Double immunofluorescence with phosphorylated tau and
HLA-DR revealed that plaques immunoreactive for oligomeric protein were consistently associated with tau-positive
neurites and inflammatory microglia. Collectively, this work illustrates that diffuse, non-oligomeric A deposits are not
disease related, and that only those plaques containing oligomeric protein associate with the degenerative inflammatory
and neuritic pathology characteristic of AD. This data therefore provides further evidence to suggest that oligomeric
forms of A underpin AD pathogenesis.
2
Title: Looking for SOD1 somatic mutations in ALS.
Authors:
Luquin, N 1,2, Trent, RJ 2, Yu, B 2 & Pamphlett, R 1
1
Dept of Pathology, The University of Sydney, Australia
2
Dept of Molecular and Clinical Genetics, The University of Sydney, Australia
Abstract. Introduction: A somatic mutation arising in an embryonic progenitor cell could cause a sporadic disease
that manifests in later life. In such cases the mutation would be found preferentially in the most affected tissue. Most
amyotrophic lateral sclerosis patients are sporadic (SALS). In familial ALS, mutations can be found in the gene for
superoxide dismutase 1 (SOD1). We therefore looked for brain-selective SOD1 mutations in patients with SALS.
Method: Blood DNA was extracted during life from 6 patients with SALS (4 male, 2 female, mean age 59 y SD ± 11
y). After death, DNA was extracted from the frontal cortex of the same patients. Primer sets for PCR amplification
spanned 1 kb upstream of all five SOD1 exons, all four introns and 1 kb downstream. Amplified fragments were
sequenced and compared to the GenBank reference sequence. Results: No mutations were found in any of the 5 exons
of SOD1 in brain DNA. Two rare non-coding variations (a DNA variant in intron 2 and a 3 bp downstream deletion)
were identified in both brain and blood DNA, indicating they were not brain-only somatic mutations. The positions of
these variations suggest they are unlikely to affect splicing. Conclusion: This method of comparing paired blood-brain
DNA samples permits searching for somatic mutations. No brain-specific SOD1 mutations were found in this pilot
study. However, a number of other SALS candidate genes could be examined by this method.
Title: Differential Protein Expression in Alcoholic Postmortem Human Brain.
Authors: Kashem MA, Harper C, Matsumoto I
Discipline of Pathology, University of Sydney, NSW 2006, Australia.
Abstract: Background: Ethanol is an addictive drug regulates different metabolic pathways and induced cognitive
dysfunction in the brain. Neuroimaging analysis revealed that alcohol-induced brain damage appears to be regionspecific and major dysmorphology has been observed in the prefrontal cortex and the white matter (WM) particularly
in the corpus callosum (CC). Recent diffusion tensor imaging (DTI) analysis indicated that microstructural degradation
is prominent in the genu followed by the body and the splenium. Molecular mechanisms underlying these structural
changes are largely unknown. Methods: Human postmortem 25 genu and 23 splenium tissues samples were provided
by NSW Tissue Resource Centre. Proteins were extracted from samples [(12 control for genu and 9 control for
splenium); 7 uncomplicated alcoholic and 6 alcoholic complicated with hepatic cirrhosis] and separated by 2-D gel
electrophoresis. Gel images were analysed by Nonlinear Phoretix 2D Expression software. Digested proteins were
analysed by MALDI-TOF. The spectra were searched against the NCBI protein databases using the MASCOT search
engine (http://www.matrixscience.com/). Protein identification was made based on MOWSE score, pI, MW and
sequence coverage. Results: Two separate experiments were conducted using the tissues of 2 sub-regions of the CC,
the genu and splenium. The relative expression of 43 proteins in the splenium and 50 proteins in the genu was
identified respectively and, among the identified proteins 21 were found to be overlapped within the sub-regions. Of
the identified splenium and genu proteins, 46 % (20 proteins) and 44% (22 proteins) were specifically regulated in only
alcoholics complicated with hepatic cirrhosis cases respectively. Conclusion: Hepatic cirrhosis has synergetic effect on
brain protein expression. Identified proteins were involved in a number of metabolic pathways, including lipid
peroxidation, oxidative stress, energy and vitamin cascade pathways, signaling and apoptosis and, interaction of these
pathways are the primary factors for brain damage.
This Study is Supported by Australian Brewers Foundation and NSW BioFirst Award and MAK is an UPA award holder.
3
Title: Novel fibroblastic onion bulbs in a demyelinating avian peripheral neuropathy produced by riboflavin
deficiency.
Authors: Cai Z, Blumbergs PC, Finnie JW, Manavis J, Thompson PD.
Hanson Institute Centre for Neurological Diseases, Institute of Medical and Veterinary Science, Adelaide, Australia.
Department of Neurology and University Department of Medicine, Royal Adelaide Hospital, Australia.
Abstract: The finding of novel fibroblastic onion bulb-like structures in peripheral nerves is reported for the first time
in avian riboflavin deficiency. Day old broiler meat chickens were fed a riboflavin deficient diet (1.8mg/kg) and were
killed on postnatal days 6, 11, 16, 21 and 31, while control chickens were fed a conventional diet containing 5.0mg/kg
riboflavin. The fibroblastic onion-bulb like structures were found in sciatic and brachial nerves from days 11 onwards
and consisted of long cytoplasmic processes of hypertrophied fibroblasts surrounding demyelinated, remyelinated and
normally myelinated axons. The fibroblast cytoplasmic processes often enveloped more than one nerve fibre to produce
a unique compound-like onion bulb structure. These onion bulb-like structures occurred early in the course of
segmental demyelination at the same time as tomacula formation and became increasingly more prominent in the later
stages of demyelination and remyelination. The molecular basis of formation of these unique structures requires further
study as to the basis of the attraction of the fibroblast processes to nerve fibres associated with myelinating Schwann
cells. The model may also be useful in investigating the role of endoneurial fibroblasts in endoneurial fibrosis as the
early fibroblastic response in the onion bulbs is distinct from the more usual fibroblastic deposition of collagen in endstage peripheral nerve disease.
Address for correspondence:
Professor Peter C. Blumbergs, Hanson Institute Centre for Neurological Diseases, Institute of Medical and Veterinary
Science, Adelaide, SA 5000, Australia.
Tel: +61 8 8222 3679; Fax: +61 8 8222 3392, Email: peter.blumbergs@imvs.sa.gov.au
Title: Post mortem psychiatric diagnosis: A reliability study.
Authors: Sundqvist N 1,2., Sheedy D 2, Garrick T 2.
1
Schizophrenia Research Institute, Sydney, Australia, 2Discipline of Pathology, University of Sydney, Australia
Abstract: Introduction The validity of post-mortem human brain research relies upon accurate clinical and
psychopathological diagnosis. Current literature reveals few instances where standardised diagnostic assessment tools
such as the Diagnostic Instrument for Brain Studies (DIBS) have been utilised1,2. The DIBS is a semi-structured
instrument designed for post-mortem psychiatric assessment using medical documentation and informants where
possible. This instrument enables diagnosis at a sub-syndrome and symptom-based level, providing comprehensive
information for prospective research, while increasing the reliability of clinical diagnosis 3. Aims The primary aim is to
investigate the degree of concordance between predominant ante-mortem psychiatric diagnoses indicated in medical
records, and post-mortem diagnoses derived through structured diagnostic instruments such as the DIBS and the Item
Group Checklist (IGCL) of the Schedules for Clinical Assessment in Neuropsychiatry (SCAN)4. Methods Fifty-eight
subjects (42 males, 16 females) from the New South Wales Tissue Resource Centre database, all of whom had been
diagnosed with a mental illness during their lifetime, were included in the study. The predominant ante-mortem
psychiatric diagnosis was compared to its corresponding post-mortem diagnosis obtained through structured case
reviews to which either the DIBS or the IGCL of the SCAN were applied. Demographic variables such as age of onset,
and alcohol or drug use were also examined. Results Comparison of diagnoses obtained produced an overall kappa
coefficient of 0.66. Kappa coefficients for the schizophrenia cohort were 0.61, 0.35 for the schizoaffective cohort, 0.95
for the major depressive disorder cohort and 0.70 for the bipolar disorder cohort. Discussion Although the inter-rater
reliability in this sample is moderate to excellent, there is sufficient disagreement, particularly in the schizoaffective
cohort, to suggest the value of applying a standardised and structured assessment to psychiatric diagnosis. A
standardised approach would enhance both diagnostic accuracy and prospective validity of tissue-based research. This
study also highlights the importance of accurate and detailed medical record keeping at a symptom-based level across
all mental health professions. In their absence, the reliability of both ante-mortem and post-mortem diagnosis is
compromised, ultimately jeopardising research outcomes.
Acknowledgement This work was supported by the Schizophrenia Research Institute, utilising infrastructure funding from NSW Health.
References 1. Hill et al, The Diagnostic Instrument for Brain Studies, Version 3 (Mental Health Research Institute, Melbourne, 2001). 2. Hill
et al. Am J Psychiatry 153, 533-537 (1996). 3. Keks et al. In Using CNS Tissue in Psychiatric Research (ed. Dean, Kleinman & Hyde) 19-37
(Harwood Academic Publishers, Amsterdam, 1999). 4. World Health Organisation. SCAN: Schedules for Clinical Assessment in
Neuropsychiatry (World Health Organisation, Geneva, 1992).
4
Title: Predicting tissue degeneration in progressive supranuclear palsy: perfusion changes identify in vivo and
postmortem regional volume losses.
Authors:
Schofield EC 1, Cordato NJ 2, McCredie RL 3, Macdonald V 1, Kril JJ 4, Halliday GM 1
1
Prince of Wales Medical Research Institute and The University of New South Wales; 2Department of Geriatric
Medicine, Westmead Hospital and The University of Sydney; 3Department of Medical Physics and Department of
Nuclear Medicine and Ultrasound, Westmead Hospital; 4Department of Pathology, The University of Sydney
Abstract: Progressive supranuclear palsy (PSP) is a movement disorder which is associated with less severe brain
atrophy than neurodegenerative dementias, but can similarly result in substantial cognitive dysfunction. Studies to date
have shown that the greatest tissue degeneration in PSP concentrates in the midbrain. The present study, firstly
examines global degeneration in PSP (as additional volume loss has been shown to occur in the cortex) and secondly
compares in vivo perfusion change and volume loss with volume loss at postmortem so as to examine the progression
of tissue degeneration. Methods: Participants fulfilling clinical criteria for PSP, at approximately mid-stage disease,
were recruited by Dr Cordato through Westmead Hospital, (approved by the South West Area Health Service ethics
committee). The pattern and severity of global perfusion and volume differences between PSP (n=17, mean age=706
years, mean duration=41years) and age-matched controls (n=23) was assessed on a voxel level from SPECT (single
photon emission computed tomography) and magnetic resonance images respectively. Statistical analysis was
performed using ‘SPM2’ software on a ‘Matlab version 7’ platform. Regional volume loss was also examined in 24
autopsy-confirmed PSP cases relative to 22 controls selected from the Australian Brain Donor Program’s POWMRI
Tissue Resource Centre where formalin-fixed brain tissue is routinely processed into 3mm coronal slices and
photographed. Using these photographs and a published point-counting technique, 48 cortical and subcortical grey
matter regions of interest were delineated and their volumes calculated. Three individuals were examined in both PSP
cohorts, providing additional experimental validation. Results: Postmortem volumetry identified eight regions of
interest with significant volume loss in the PSP group. Most severely affected were the pallidum (internal
segment=50% loss), parietal cortex (supramarginal gyrus=36%), cingulate gyrus (motor region=33%), inferior frontal
gyrus (21%) and thalamus (19%). Voxel-based morphometry showed that these affected regions (excepting the
pallidum) were also foci of perfusion changes at mid-stage disease. The cingulate and inferior frontal gyri and the
thalamus were hypoperfused and additionally were atrophic at mid-stage disease (p<0.05). Interestingly, the
supramarginal gyrus showed no early indication of atrophy, but had increased perfusion at mid-stage disease.
Conclusions: In restricted cortical and subcortical brain regions, tissue degeneration at end-stage PSP is comparably
severe. Cingulate, inferior frontal and thalamic tissue atrophy begin early in the disease course with concurrent
hypoperfusion identified by SPECT imaging. Hyperperfusion in the parietal lobe was predictive of atrophy at endstage PSP. This suggests that regional increases in blood flow at mid-disease stage may prove useful for identifying
brain tissue vulnerable to neurodegeneration in PSP.
Title: Immunoglobulin in frontotemporal dementia.
Authors:
Kersaitis C 1,2, Halliday GM 3, Kril JJ 1,4.
1
Discipline of Pathology, The University of Sydney, NSW,
2
School of Biomolecular and Health Sciences, University of Western Sydney, NSW,
3
Prince of Wales Medical Research Institute, Randwick NSW,
4
Discipline of Medicine, The University of Sydney, Sydney NSW
Abstract: The pathogenesis of Frontotemporal dementia (FTD) has not been fully elucidated, and several mechanisms
including immune responses have been implicated. The inflammatory response in FTD does not involve leukocyte
infiltration, although microglial upregulation is a key feature. The microglial activation may be a response to
complement activation in addition to other injurious stimuli. Various complement proteins have been identified in some
cases of FTD, and the absence of Factor B suggests activation of the classical complement pathway. This study
evaluates the microglial response and immunoglobulin presence in 18 cases of FTD. Our findings indicate that an
inflammatory response is present from early stage FTD, and that secondary inflammatory events may also be triggered
by humoral mechanisms. The humoral response was more pronounced in cases of FTD without tau deposition, and is
associated with significantly greater inflammation. This suggests that humoral immunity contributes to the
inflammatory processes in the progression of FTD.
5
Title: Brain chromosomal copy number differences in sporadic MND.
Authors:
Morahan JM, Yu B, Trent RJ, Pamphlett R
Departments of Pathology and Molecular and Clinical Genetics, The University of Sydney
Telephone: int+(61-2) 9351-3318 Fax: int+(61-2) 9351-3429 Email: rogerp@med.usyd.edu.au
Abstract: Introduction. Sporadic and familial forms of motor neuron disease are clinically and pathologically
indistinguishable, and genetic mechanisms are suspected to underlie both forms of the disease. Chromosome copy
number changes are involved in several neurological diseases and may preferentially involve the central nervous
system. In an attempt to see if copy number changes in the brain could lead to sporadic MND (SMND) we compared
brain and blood DNA copy number across the genome in SMND and control subjects. Methods. Copy numbers across
the whole genome were analysed in 7 SMND patients and 7 matched controls using Affymetrix 500K arrays, which
can detect multiplications or deletions of copy number down to 1 kb in size. Copy numbers were compared between (1)
frontal cortex DNA in patients and controls, and (2) frontal cortex and blood DNA in SMND patients (to look for
somatic differences). Results. (1) Five regions of copy number difference were identified in SMND brains compared
with control brains. None of these regions however was found in more than one patient or contained candidate genes.
(2) Thirteen regions of copy number difference were identified in SMND brains that were not found in a matched blood
sample. One of these regions was found in multiple samples but did not contain any candidate genes. Regions of
increased copy number, identified in single samples, contained candidate genes such as a tubulin gene, a neurotrophic
factor, four calcium-related genes and five genes specifically expressed in the nervous system. Discussion. In this pilot
study, no candidate genes were identified in SMND brains compared to controls. Copy number differences in patient
brains, not present in blood, included several candidate genes that could be involved in SMND. Larger numbers of
cases are needed to confirm these findings.
Title: P25a exocytosis in multiple system atrophy.
Authors: Huang Y 1, Song CYJ 1, McCann H 1, Gai WP 2, Jensen PH 3, Halliday G 1
1
Prince of Wales Medical Research Institute and University of New South Wales, Australia;
2
Physiology department, Flinders University, SA, Australia;
3
Institute of Medical Biochemistry, University of Aarhus, Denmark
Abstract: P25a and aba-synuclein protofibrils and glial cytoplasmic inclusion (GCI) formation in
multiple system atrophy (MSA), where GCI load associates with progressive neuronal loss. The aim of this study was
to identify any oligodendroglia characteristics associated with neuronal degeneration. Brain tissue from 12 MSA
patients and 12 controls without neurological disease was obtained from the institutionally approved Prince of Wales
Medical Research Institute Tissue Resource Centre. Ten micron paraffin-embedded tissue blocks of the pons and
putamen were serially sectioned and routinely stained with haematoxylin and eosin or peroxidase
immunohistochemistry for -synuclein, p25 and ab. Triple
labelling using -synuclein immunoflourescence, p25 immunofluorescence and thioflavin S histofluorescence
(detects -sheet conformation) was evaluated using a confocal microscope. The number of -synuclein, ab -crystallin
and p25 immunoreactive GCIs and neuron loss was graded at 200x magnification in serial sections. Similar numbers
a-synuclein and ab -crystallin, suggesting colocalisation (100% of GCI labelled),
whereas less GCIs contained p25 (average 75% of GCI). The proportion of p25-positive GCIs negatively correlated
with the severity of neuronal loss using Spearman rank tests. Within and near oligodendroglia with a-synuclein-positive
GCI, p25a immunoreactive (but a-synuclein- and thioflavin S-negative) protein aggregates were found in vesicles that
occasionally contained a-synuclein-immunoreactivity on the vesicle membrane. As neuronal loss directly relates to a
reduction in p25a-immunoreactivity in GCI-containing oligodendroglia, the mechanism for p25a clearance is important
to determine, and could include vesicular trafficking possibly via exocytosis.
6