The Australian and New Zealand Society for Neuropathology
28th Annual Scientific Meeting, May 24th, 2008
Education Centre, Royal Brisbane Hospital, Brisbane.
Final Program
8:30-9:00am
Registration
9:00-10:30am
Slide Seminar (Presenters: Dr Urankar, Prof Blumbergs (2 cases), Dr Lau, Dr Grogan, Dr Robertson, Dr
Buckland, Dr Koszyca (2 cases).
10:30-11:00am
Morning Tea
11:00am-12:00pm
Invited Speaker – Manuel Graeber (UK).
“Functional genomics of Parkinson's disease”
12:00-1:00pm
12:00-12:15
Oral Presentations I.
Greg Sutherland: Do polymorphisms in the familial parkinsonism genes contribute to risk for sporadic
Parkinson’s disease?
12:15-12:30
12:30-12:45
12:45-1:00
George Mellick: Genetic risks for PD: Combinations and interactions.
Glenda Halliday: Myelin and oligodendroglial cell changes in multiple system atrophy.
Yue Huang: LRRK2 in early stage of glial inclusion of multiple system atrophy.
1:00-2:00pm
Lunch break
2:00-3:15pm
Oral Presentations II. The Bill Evans Memorial Young Investigator Award.
Smriti Agarwal: Gephyrin in Alzheimer’s disease.
Sarah Christen: Alzheimer’s disease and its genetic background: Potential association with single nucleotide
2:00-2:15
2:15-2:30
polymorphisms.
2:30-2:45
2:45-3:00
3:00-3:15
Florian Gebhardt: Housekeeper gene analysis in Alzheimer’s disease.
Dustin Proctor: Quantification of PSD-95 and SAP-102 mRNA and protein levels in Alzheimer’s disease.
Rudi Tannenberg: Markers of synaptic pathology in the alcoholic and Alzheimer brain.
3:15-3:30pm
Afternoon tea
3:30-5:00pm
Oral Presentations III.
Michael Buckland: FISH analysis of gliomas: the St Vincent’s Sydney experience.
Lorel Adams: The Australian MND DNA Bank: A continent-wide gene-environment resource.
Zhao Cai: Aquaporin-1 and peripheral nerves.
Guy Barry: Distinct glial populations regulate hippocampal morphogenesis.
Peter Blumbergs: TDP-43 pathology in frontotemporal lobar degeneration (FTLD) with motor neurone
3:30-3:45
3:45-4:00
4:00-4:15
4:15-4:30
4:30-4:45
disease (MND).
4:45-5:00
Naomi Etheridge: Synaptic proteome changes in the alcoholic brain.
CLOSE OF SCIENTIFIC PART OF THE MEETING.
5:00-5:30pm
ANZSNP Business Meeting.
(Includes announcement of the winner of the “Bill Evans Memorial Award”).
~7:00pm
ANZSNP Dinner. “Freestyle Tout restaurant”, 1000 Ann Street, Fortitude Valley. Located in
the Emporium complex. Freestyle Tout is on the corner as you enter Emporium off Ann St.
1
Invited International Speaker
Title: “Functional genomics of Parkinson's disease.”
Author:
Manuel B. Graeber MD PhD FRCPath
Abstract: Parkinson’s disease (PD) is one of the most common nervous system disorders but no therapy has been
proven to halt or even slow disease progression. A molecular therapeutic rationale based on a better understanding of
the system biology of this complex disorder is now becoming available through genetic research. There are presently
eight established PD genes, DJ1, GIGYF2, HTRA2, LRRK2, PARKIN, PINK1, SNCA, and UCHL1. In addition, the
ATP13A2 gene is of increasing interest.
In this lecture, I will review the functional connections between known and new PD genes and how knowledge of their
pathway interactions may lead to novel diagnostic and therapeutic approaches. Functional genomic studies predict the
existence of a shared pathway in both familial and sporadic cases of PD which implicates dysfunction of the
ubiquitin‐ proteasomal system, alpha‐ synuclein aggregation, oxidative stress and mitochondria. There are emerging
links to diabetes and to cancer which are intriguing. “Inflammatory” mechanisms may play a role in PD pathogenesis
but more studies especially on glial reactivity as well as primary gliodegeneration are required to clarify this point. I
will also highlight connections to traditional knowledge on PD, and I will review the history of the disease.
Brain tissue based investigations for which neuropathological expertise is of great advantage are essential for the cellbased mapping of the vast amount of new information which is becoming available through high-throughput analyses
such as expression microarrays. Detailed knowledge of the neurocytological phenome (morphonome) builds upon
traditional clinico-neuropathological findings and represents an asset which holds significant potential not only for the
development of molecular neuropathology but for the neurosciences at large. An examination of transcriptomic
changes in PD in relation to the cellular neuropathology of the Parkinsonian nigra will be used to illustrate these points.
Oral Presentations I
Title: “Do polymorphisms in the familial parkinsonism genes contribute to risk for sporadic Parkinson’s disease?”
Authors:
G.T. Sutherland1, G.M. Halliday2, P.A. Silburn1,5, F.L. Mastaglia3, D.B. Rowe4, R.S. Boyle6, J.D.
5,7
O’Sullivan , T. Ly3, S.D. Wilton3,, and G.D. Mellick1,6,7.
1
Eskitis Institute, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, QLD 4111, Australia, 2 Prince of Wales
Medical Research Institute, Sydney, NSW 2031, Australia, 3 Centre for Neuromuscular and Neurological Disorders, University of Western
Australia, Perth, WA 6009, Australia, 4 Department of Neurology, Royal North Shore Hospital, Sydney, NSW 2065 Australia,
5
The School of Medicine, University of Queensland, Brisbane, QLD 4072 Australia, 6Department of Neurology, Princess Alexandra Hospital,
Brisbane, QLD 4102, Australia, 7 Department of Neurology, Royal Brisbane and Women’s Hospital, Brisbane, QLD 4029, Australia.
Abstract: Recent whole genome association studies provided little evidence that polymorphisms at the familial
parkinsonism loci influence the risk for PD. However these studies are not designed to detect the types of subtle effects
that common variants may impose. Here we use an alternative targeted candidate gene approach to examine common
variation in 11 genes related to familial parkinsonism. PD cases (n=331) and unaffected control subjects (n=296) were
recruited from three specialist movement disorder clinics in Brisbane, Australia and the Australian Electoral Roll.
Common genetic variables (76 SNPs and 1 STR) were assessed in all subjects and haplotype, genotype and allele
associations explored. We observed no associations of sufficient magnitude to survive strict statistical corrections for
multiple comparisons. However modest associations (uncorrected p < 0.05) were observed for common variants around
SNCA, UCHL1, MAPT, and LRRK2. No associations were seen for PRKN, PINK1, GBA, ATP13A2, HTRA2, NR4A2
and DJ1. Our findings suggest that the individual effects of common genetic variables around these PD-related loci do
not contribute substantially to PD risk in Australians.
2
Oral Presentations I
Title: “Genetic risks for PD: Combinations and interactions”.
Authors:
G.D. Mellick and G.T. Sutherland
Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan QLD 4111, Australia
Abstract: The ways in which genetics contribute to sporadic Parkinson’s disease (PD) remain unclear. The most
prevalent hypothesis contends that common genetic variability acts in combination with environmental factors on a
background of physiological ageing to modify risk. However, most risk-factor studies neglect to consider these
combinatorial or interactive effects. This has important implications for the discovery of genetic risk factors and the
interpretation of previously published results. We have investigated the interactions between 56 haplotype tagging
SNPs (from 11 parkinsonism-related genes) and two risk altering environmental variables (namely, cigarette smoking
and pesticide exposure). The data-set used for analysis was generated from PD cases (n=331) and unaffected control
subjects (n=296) recruited as part of the Australian Parkinson’s Project. Two-way interactions (gene x environment)
were assessed using binary logistic regression adjusted for age, gender, smoking status (positive for doses >22 pack
years), pesticide exposure (positive for cumulative doses of herbicide, pesticide or fungicide > 28 days) and the main
effects of the individual SNP genotypes. We observed evidence for eight gene x environment interactions that alter PD
risk by 2-fold or greater, although all effects were modest (odds ratios for interaction ranged from 2.00 – 2.80).
Notably, none of the SNPs involved with these interactions showed individual association with PD in the absence of
exposure. The candidate interactions revealed in this preliminary analysis should be followed-up in adequately powered
replication case-control samples.
Oral Presentations I
Title: “Myelin and oligodendroglial cell changes in multiple system atrophy”.
Authors:
Y.J.C. Song1, D.M.S. Lundvig2, Y. Huang1, W.P. Gai3, P.C. Blumbergs4, P. Højrup5, D. Otzen6, P.H.
Jensen2 and G.M. Halliday1
1
Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia, 2Institute of Medical Biochemistry, University of Aarhus, DK8000 Aarhus-C, Denmark, 3Departments of Human Physiology & 4Medicine, Flinders University School of Medicine, Bedford Park, Adelaide,
SA 5042, Australia, 5Institute of Molecular Biology, University of Southern Denmark, DK-5230 Odense M. Denmark, 6Department of Life
Science, Aalborg University, DK-9000, Aalborg, Denmark.
Abstract: P25 is a constituent oligodendroglial protein that can induce aggregation of -synuclein and accumulates
in oligodendroglial cell bodies containing fibrillized a-synuclein in the neurodegenerative disease multiple system
atrophy (MSA). We demonstrate biochemically that p25
myelin basic protein (MBP), and in situ immunohistochemistry revealed MBP and p25 colocalizes in myelin in
normal human brains. Analysis of MSA cases reveals dramatic changes in p25 and MBP over the course of the
disease. In situ immunohistochemistry revealed a cellular redistribution of p25 immunoreactivity from the myelin to
the oligodendroglial cell soma, with no overall change in p25 protein concentration using immunoblotting.
Concomittantly, an approximately 80% reduction in the concentration of full-length MBP protein was revealed by
immunoblotting along with the presence of immunoreactivity for MBP degradation products in oligodendroglia. The
oligodendroglial cell bodies in MSA displayed an enlargement along with the relocalization of p25 and this was
enhanced following the deposition of -synuclein in the glial cytoplasmic inclusions. Overall the data indicate that
changes in the cellular interactions between MBP and p25 occurs early in MSA and contributes to abnormalities in
myelin and subsequent -synuclein aggregation and the ensuing neuronal degeneration that characterizes this disease.
3
Oral Presentations I
Title: “LRRK2 in early stage of glial inclusions of Multiple System Atrophy”.
Authors:
1
2
Y. Huang1, K. Murphy1, W.P. Gai2 and G. Halliday1
Prince of Wales Medical Research Institute and University of New South Wales, NSW, Australia.
Department of Human Physiology, School of Medicine, Flinders University, Adelaide, Australia
Abstract: Background: Multiple system atrophy (MSA) is characterised by the aberrant accumulation alpha-synuclein
in glial cytoplasmic inclusions (GCI) and neuronal loss in affected regions. There are two clinical subtypes, cerebellar
ataxia predominant (MSA-C) and parkinsonian predominant (MSA-P) with pathological severity graded according
subtype1. Dorsolateral basal ganglia are among the first affected regions in both subtypes. LRRK2 is the protein
product of a recently identified Parkinson’s disease causative gene LRRK2, and probably a component of Lewy bodies2.
Aim: To investigate whether LRRK2 is also a component of GCI, we examined LRRK2 expression in the basal ganglia
of 12 MSA and 4 control cases. Methods: Paraffin-embedded basal ganglia tissue blocks from prospectively followed
brain donors were sectioned at 10µm and immunohistochemically stained for alpha-synuclein (mAb42, 1:100, mouse,
BD Transduction Labs) and LRRK2 (LB955, 1:500, rabbit, Abgent) using peroxidase visualization. The study was
approved by institutional human ethics committees. Co-localisation of LRRK2 and alpha-synuclein in GCIs was
examined using fluorescence microscopy at 200x magnification. Semi-quantitative grading of neuronal loss and GCI
load were semi-quantified by two researchers (kappa=0.9). Results: LRRK2 was detected in GCIs and co-localised
with alpha-synuclein in MSA, but not in glia in control cases. The proportion of GCI immunopositive for LRRK2 was
highest in early MSA stages (90-100% in stages I and II, N=8 cases), reducing at later stages (20-30% in stage III, N=4
cases). Conclusion: LRRK2 is localized in GCIs of MSA cases at early disease stages. This suggests LRRK2 may
contribute in the initial stage of GCI formation, similar to that suggested for early brainstem Lewy body formation2.
1. Jellinger KA, Seppi K, Wenning GK. Grading of neuropathology in multiple system atrophy: Proposal for a novel scale. Mov. Disord.
2005;20 Suppl. 12:S29-36.
2. Alegre-Abarrategui J, Ansorge O, Esiri M, Wade-Martins R. LRRK2 is a component of granular alpha-synuclein pathology in the brainstem
of Parkinson's disease. Neuropathol. Appl. Neurobiol. 2007;doi:10.1111/j.1365-2990.2007.00888.x
Bill Evans Memorial Award Entrant
Title: “Gephyrin in Alzheimer’s disease”.
Authors:
S. Agarwal, R.K. Tannenberg and P.R. Dodd.
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia, 4072
Abstract: 'Gephyrin,' derived from the Greek word meaning ‘bridge’, binds inhibitory glycine and GABA A receptors to
subsynaptic cytoskeletal elements, serving as an important receptor-microtubule linker for the assembly and
stabilization of inhibitory post-synaptic terminals in the central nervous system. Excitotoxicity, or neuronal loss due to
excessive excitatory neurotransmission, depends on the balance of excitatory and inhibitory factors, and has been
postulated by several research groups as a crucial factor contributing to synaptic loss in Alzheimer’s disease (AD).
Aberrant gephyrin levels might contribute to disease pathology by altering the normal inhibitory modulation of
excitatory impulses. We assayed gephyrin protein levels in two AD susceptible areas (i.e., hippocampus and inferior
temporal cortex) and a spared area (occipital cortex) in autopsy brain tissues from normal subjects (n=15) and AD
patients (n=15). Quantification of the protein levels was done against known concentrations of a recombinantly
expressed truncated gephyrin protein containing the immunogenic epitope. Gephyrin abundance was significantly
reduced (P < 0.01) in AD. Area-wise analysis showed that gephyrin levels were reduced in both spared and susceptible
regions, indicating a global phenomenon. When samples were categorized on an index of pathological severity, i.e.,
amyloid plaques, neurofibrillary tangles, neuronal loss and gliosis, gephyrin levels decreased with increasing severity
until a moderate index was reached, and then increased, suggesting that higher gephyrin levels might compensate for
excitotoxic damage in late stages of the disease. AD Males showed a more pronounced reduction in gephyrin levels
than AD females, c.f. same-sex controls. A major splice variant of gephyrin was detected in all cases and in all three
brain areas. This is the first study of gephyrin expression in AD.
Supported by Alzheimer’s Association (USA) under grant #IIRG-07-59015.
4
Bill Evans Memorial Award Entrant
Title: “Alzheimer’s disease and its genetic background: Potential association with single nucleotide polymorphisms”.
Authors:
S. Christen, R.K. Tannenberg and P.R. Dodd.
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia, 4072
Abstract: Besides environmental factors, Alzheimer’s disease (AD) – the predominant form of dementia worldwide –
is thought to be additionally influenced by genetic factors. In the past, various studies could show an association of
genes such as APP or APOE with AD. In our experiments, we examined single nucleotide polymorphisms (SNPs) in
three other genes: A2M, APOC1 and TNF. Genomic DNA was extracted from neuropathologically characterised postmortem brain tissue and amplified via polymerase chain reaction (PCR). A subsequent restriction fragment length
polymorphism (RFLP) analysis was employed to determine the respective genotypes of the samples. We then
compared the genotype and allele frequencies of control cases with the frequencies we found in AD cases, cases that
had been diagnosed with other neurodegenerative diseases, and cases that showed signs of both AD and other
neurodegenerative diseases together. The A2M locus was chosen as the A2M protease inhibitor seems to be able to bind
Aβ, a protein that is abundantly found in plaques in the brains of AD patients. We examined a SNP in codon 1000
(rs669), a position close to the active site of the molecule. According to our data, this SNP is not associated with AD,
but showed a non-significant trend towards an involvement of the G allele in non-AD neurodegenerative diseases. The
second locus, a 4 bp insertion/deletion in the APOC1 promoter (rs11568822) was investigated as the apoC1 protein
interacts with the apoE protein which is encoded by the APOE, the major AD risk gene. We found a strong association
of the allele containing the insertion (H2) as well as the H2/H2 genotype with AD and other neurodegenerative
diseases. As inflammation is also thought to play a role in the progression of AD, we studied two additional SNPs in
the promoter region of TNFα (position -308: rs1800629; position - 850: rs1799724), the gene encoding the
proinflammatory cytokine TNFα. It was found that the -308 locus did not show a significant association with AD,
despite an overrepresentation of the G allele and G/G genotype in all but control cases. The -850 T allele however was
found to be significantly more frequent in AD cases, thus representing a potential risk factor. In our future studies, we
hope to corroborate our present findings with greater case numbers. Furthermore we will include other factors such as
age, sex or APOE genotype into our statistical analyses.
Supported by Alzheimer’s Association (USA) under grant #IIRG-07-59015.
Bill Evans Memorial Award Entrant
Title: “Housekeeper gene analysis in Alzheimer’s disease”.
Authors:
F.M. Gebhardt, H.A. Walton, R.K. Tannenberg and P.R. Dodd
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia, 4072
Abstract: Alzheimer’s Disease (AD) is the commonest form of dementia and accounts for 50% to 70% of all dementia
cases. Metabolism in the AD brain appears to be disordered compared with matched controls and is a key contributor to
the pathogenesis of AD. The loss of neurons or synapses in AD correlates well with the cognitive impairment of the
disease and might account for the reduced levels of neuronal or synaptic gene products. Quantitative real-time RT-PCR
(qRT-PCR) is a powerful and reliable tool for gene expression studies. Multiple genes must be validated for use as a
stably expressed internal housekeeper gene. For many applications it has been shown that the use of multiple
housekeeper genes increase the accuracy of normalisation. The aim of this study was to develop a qRT-PCR assay for
nine putative reference genes in control and Alzheimer’s disease (AD) brains to determine which housekeeper is the
most stable for this particular disease state. These genes included, -actin, 18s ribosomal RNA, cyclophilin,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT), TATA-box-binding
protein (TBP), enolase 2, and ribosomal protein L13 (RPL13). Absolute mRNA levels of these genes from four
different brain regions of 12 controls, 9 AD and 11 AD/Lewy Body disease (LBD) cases were tested. Inferior temporal
and inferior frontal cortices were included as brain regions relatively susceptible to AD damage, and posterior motor
and occipital cortices were sampled as areas relatively spared. Frequently used housekeeper genes, such as GAPDH
and -actin, had an overall reduced expression level in AD and AD/LBD cases in comparison to controls. RPL13 and
18s were the most stably expressed genes overall. Statistical analysis was used to examine whether expression values of
all genes are influenced by RNA quality, brain weight, disease state and the post-mortem interval. Testing for RNA
quality should be included in all data processing and samples should be matched for RNA quality. Synaptophysin and
glial fibrillary acidic protein (GFAP) were used as controls since their levels in AD is well reported. We confirmed that
synaptophysin expression was down-regulated in AD cases whereas GFAP showed an increase.
5
Bill Evans Memorial Award Entrant
Title: “Quantification of PSD-95 and SAP-102 mRNA and protein levels in Alzheimer’s disease”.
Authors:
D.T. Proctor and P.R. Dodd.
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia, 4072
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterised by global cognitive
decline involving memory, behaviour and reasoning associated with the deterioration of metabolic and neuronal
processing and synaptic loss. Many factors have been identified which contribute to the pathology of AD including the
specific loss of glutamatergic synapses in localised areas of the brain via glutamate-evoked excitotoxicity, possibly
from the over-stimulation of NMDA receptors. Previous studies on human AD and control post-mortem brain tissue
conducted in the lab have demonstrated a reduction in mRNA transcript and protein levels of specific NMDA receptor
subtypes in pathologically susceptible brain regions, whereas areas less affected by neuronal loss in AD showed
comparable NMDA mRNA levels to those in controls. This suggests that neurons expressing specific NMDA receptor
subtypes are more susceptible to death from glutamate excitotoxicity. The overall expression, activity and stability of
NMDA receptors on the post-synaptic membrane can be regulated by a number of processes including interactions with
the cytoplasmic scaffolding proteins PSD-95 and SAP-102. PSD-95 and SAP-102 bind NMDA receptor subunits in a
phosphorylation-dependent manner. They modulate the receptor by increasing the expression and functional activity of
the channel through interactions with numerous intracellular proteins, kinases and phosphatases. This study aimed to
detect changes in PSD-95 and SAP-102 mRNA and protein levels which could be responsible for the corresponding
decreases in NMDA receptor subunits observed in AD. This was achieved by measuring mRNA transcript levels of the
scaffold proteins using absolute quantification via Real-Time PCR. Precise levels of protein were quantified against
recombinant PSD-95 and SAP-102 truncated standards by western blotting techniques. Studies were conducted using
two AD-susceptible regions, the hippocampus (HIPP) and inferior temporal cortex (ITC) and a region spared from
marked cell loss, the occipital cortex (OCC) from 15 pathologically confirmed AD cases and 15 controls. We found a
reduction in the level of PSD-95 and SAP-102 mRNA levels in the HIPP and ITC of AD cases compared to controls.
There was no significant difference in the OCC between AD cases and controls. Preliminary protein experiments
showed a similar trend with the mRNA data. The data suggests a possible mechanism for the reduction in NMDA
receptor subunit expression observed in AD. This research provides further understanding to the excitotoxic pathology
of AD at the molecular level, which could lead to more specific and effective targets for drug treatment.
Bill Evans Memorial Award Entrant
Title: “Markers of synaptic pathology in the Alcoholic and Alzheimer brain”.
Authors:
R.K. Tannenberg and P.R. Dodd.
School of Molecular and Microbial Sciences, University of Queensland, St Lucia 4072.
Abstract: Neurodegenerative diseases such as Alzheimer disease (AD) and chronic alcoholic brain damage show
regionally selective loss of neurones and synapses. The loss of synapses in particular could manifest in the cognitive
decline associated with both disorders. N-cadherin is a synaptic junction protein that bridges pre- and postsynaptic
excitatory terminals via b-catenin-mediated attachment to the cytoskeleton. Brain tissue from pathologically confirmed
AD, alcoholic and control cases was obtained at autopsy with informed written consent and frozen at -80C in 0.32 M
sucrose. Areas susceptible to AD pathology, hippocampus and inferior temporal cortex, and the relatively spared area,
occipital cortex were investigated. The frontal cortex, which shows the greatest neuronal loss in chronic alcoholism was
compared with controls. Levels of N-cadherin and b-catenin were measured by “in-gel” immunodetection on crude
membrane preparations of frozen human brain tissue. Despite the marked loss of synapses and synaptic proteins
reported in AD, we observed a significant elevation in both N-cadherin (P < 0.05) and b-catenin (P < 0.01) levels in
AD cases (n = 15) compared with controls (n = 15). Both N-cadherin and b-catenin levels were significantly higher in
the hippocampus of AD cases than in controls, but only N-cadherin levels were significantly raised in the AD occipital
cortex. b-catenin levels were also markedly higher (P < 0.01) in the frontal cortex of alcoholic cases (n = 9) than in
controls (n = 9) whereas the levels of N-cadherin were unchanged. Cases were also scored for the severity of AD
pathology accounting for neuronal loss, tangle and plaque load, and gliosis. A score of 0 indicated no pathology; 1,
mild pathology; 2, moderate pathology; and 3, severe pathology, for each area studied. There was a positive correlation
toward rising levels of N-cadherin and b-catenin with increasing severity. These results demonstrate that different
populations of synapses are altered in an area-specific manner in AD and alcoholism which shows a different type of
synaptic pathology.
Supported by Alzheimer’s Association (USA) under grant #IIRG-07-59015.
6
Oral Presentations III
Title: “FISH analysis of gliomas: the St Vincent’s Sydney experience”
Authors:
M.E. Buckland, B. Brown, C. Elhage, S. O’Toole and A. Morey
Department of Anatomical Pathology, St Vincent’s Hospital, Darlinghurst, NSW, 2010.
Abstract: Knowledge of 1p and 19q allelic status is fast becoming a standard part of glioma assessment as it imparts
prognostic information and may guide therapeutic decisions. Assessment of EGFR gene amplification helps identify
small cell glioblastomas and may also be prognostically relevant. Since 2004, the paraffin FISH laboratory at St
Vincent’s Hospital, Sydney, has analysed over 250 gliomas for losses of chromosomal arms 1p and 19q and EGFR
amplification. These cases have derived from 11 separate institutions in NSW, the ACT, and New Zealand, referred by
over 20 separate pathologists. This presentation details the relative frequencies of 1p and/or 19q loss and EGFR
amplification in glioma subtypes, including specific assessment of the problematic ‘oligoastrocytoma’. Heterogeneous
EGFR gene amplification in glioblastomas is also discussed. Finally, the rationale and practicality of additional
molecular tests in glioma diagnosis will be presented.
Oral Presentations III
Title: “The Australian MND DNA bank: a continent-wide gene-environment resource”.
Authors:
L. Adams
MND DNA Bank, Department of Pathology, University of Sydney, NSW, 2006.
Abstract: Background. Current thinking is that sporadic MND (SMND) is a complex disorder most likely due to a
gene-environment interaction. Large numbers of cases and controls are needed for genetic association studies to
provide sufficient power for reproducible results. In addition, environmental exposures or demographic factors may be
risk factors for SMND. Until recently no DNA collections of SMND were available in the southern hemisphere. We
therefore set up an Australia-wide DNA and environmental data resource for MND researchers. Objectives. (1) To
collect blood DNA samples and environmental data from sufficient numbers of people with SMND and controls to
perform gene-environment studies. (2) To offer this material to both Australian and international researchers. Methods
Since 2000, blood samples have been collected from donors throughout Australia, both in large population centres and
in remote country areas. Donors fill out 8-page questionnaires on demographic and environmental factors. Clinical
information on MND donors is received from treating neurologists. Results. The Bank currently has 1,694 blood DNA
samples with matching questionnaires. Of these, 32% have SMND, 22% have SALS, and 63% are controls. The
proportion of donors from each state is: New South Wales 51%, South Australia 12%, Queensland 12%, Victoria 10%,
West Australia 7%, ACT 5% and Tasmania 2%. The great majority of donors are Caucasian in origin. Only 4 samples
are from indigenous Australians. Samples from the Bank have been used in a number of SMND genetic studies [1-5].
Discussion. This is the first attempt to collect DNA samples from people with SMND throughout a whole continent.
We estimate that we collect each year about 30% of all cases of SMND in Australia. Strategies are in place to collect
more samples from remote areas. The Bank could be particularly useful for replicating genetic or environmental studies
with a southern hemisphere population. Further requests for samples are anticipated as the number of donors increases
to our target of 1,000 SMND patients and 2,000 controls.
1. Sreedharan J, Blair IP, Tripathi VB, et al. Science 2008;319:1668-72.
2. Blair IP, Vance C, Durnall JC, et al. J Neurol Neurosurg Psychiatr Feb 2008 Epub.
3. Morahan JM, Yu B, Trent RJ, et al. Neurotoxicology 2007;28:532-40.
4. Morahan JM, Yu B, Trent RJ, et al. Am J Med Genet B Neuropsychiatr Genet 2007;144:885-90.
5. Morahan JM, Pamphlett R. Neuroepidemiology 2006;27:130-5.
7
Oral Presentations III
Title: “Aquaporin-1 and peripheral nerves”.
Authors:
Z. Cai, P.C. Blumbergs, J.W. Finnie, J. Manavis, P.D. Thompson and G. Sarvestani.
Hanson Centre for Neurological Diseases, Institute of Medical and Veterinary Sciences, University of Adelaide, Adelaide, SA.
Abstract: Aquaporins (AQPs) are a family of water channel proteins that contribute to water homeostasis in many
tissues. AQP1 has been reported in peripheral nerve but detailed information about its normal localization is lacking. In
the present study, immunoperoxidase and immunofluorescent methods were used to investigate the anatomical location
of AQP1 in human sural nerves. AQP1 was expressed in endothelial cells of microvessels in epineurium, perineurium
and endoneurium. AQP1 immunoreactivity was abundantly found in perineurium, showing multilayered discontinuous
linear staining of perineurial cells. Double labelling with AQP1 and axonal and Schwann cell markers demonstrated
coexpression of AQP1 in axons and Schwann cells. These findings suggest that AQP1 contributes to water homeostasis
in human sural nerves. Preliminary results of AQP1 expression in diabetic neuropathy, IgM anti-MAG neuropathy and
riboflavin deficient neuropathy are also discussed.
Bill Evans Memorial Award Entrant
Title: “Distinct glial populations regulate hippocampal morphogenesis”.
Authors:
G. Barry1, M. Piper1, C. Lindwall1, S. Mason1, R. Moldrich1, R. Gronostajski2 and L.J. Richards1
1
The University of Queensland, Queensland Brain Institute and The School of Biomedical Sciences, Brisbane, 4072, Australia. 2Department of
Biochemistry and the Program in Neuroscience and Center of Excellence in Bioinformatics and Life Sciences, State University of New York at
Buffalo, Buffalo, NY.
Abstract: Mice deficient in the transcription factor Nuclear Factor I B (Nfib) display abnormalities in lung maturation
and brain development including callosal agenesis and defects in the developing forebrain, including the hippocampal
formation. Especially prominent is the loss of the upper blade of the dentate gyrus. Nfib is highly expressed in the
developing dentate gyrus and hippocampus. In this study, we examined the hippocampal formation of Nfib-deficient
mice in detail to elucidate what developmental processes could be regulated by Nfib in this region. First, we examined
the cortical hem, a patterning centre responsible for of the early development of the hippocampus, and which can be
distinguished by Wnt2b expression. We show that Wnt2b expression is unaffected at E12.5 in Nfib-deficient mice
indicating the likely normal development of the cortical hem. To analyse the formation of the hippocampus in more
detail, we used immunohistochemistry to study specific cell populations of this structure. Antibodies against Tbr-1, a
neuron-specific transcription factor, calretinin, a calcium-binding protein expressed in interneurons, and Prox1, a
protein expressed in dentate granule cells, were used. No differences in the Nfib-deficient mice as compared to
wildtype at E13 and E14 were observed with Tbr-1 and calretinin. However, at E15 lamination defects in the
hippocampus are evident and the final destinations of neurons within the hippocampal structure, most notably the
dentate gyrus, are compromised. Prox1 labels dentate granule cells that migrate from the VZ to the dentate granule
layer from E15.5. In Nfib-deficient mice Prox1-positive granule cells migrating from a specialised region of the VZ do
not reach the dentate gyrus and appear stalled near the dentate anlage. We studied the radial glial scaffold as a potential
explanation for the migration defects and found that the differentiation of radial glia of the hippocampal formation is
affected in Nfib mutants. Whereas nestin-positive glia, encompassing all types of radial glia, are detected in the
hippocampal region during development, the more mature GFAP-positive glia are severely reduced in the dentate gyrus
and hippocampal ventricular zone of the Nfib-deficient mice. Notably, a GFAP-positive population of mature glia near
the fimbria, which is the first site of GFAP detection in normal development, are present in Nfib-deficient mice
compared to wildtype mice. Our results suggest that defects in the differentiation of a subset of radial glia in the dentate
gyrus prevents the ensuing migration of neurons to their appropriate final destinations and has a profound impact on the
morphology of this structure in mice. Therefore, Nfib is required for specific aspects of maturation of the hippocampal
development in the prenatal mouse embryo.
8
Oral Presentations III
Title: “TDP-43 pathology in frontotemporal lobar degeneration (FTLD) with motor neurone disease (MND)”.
Authors:
P. Blumbergs, J. Temlett, J. Manavis and P.D. Thompson.
Hanson Centre for Neurological Diseases, Institute of Medical and Veterinary Sciences and University of Adelaide, SA, 5000.
Abstract: Frontotemporal dementia is accompanied by motor neurone disease (FTD +MND) in 10% of cases. TDP43, a 43 KDa nuclear TAR DNA-binding protein, has been identified as the major disease protein in FTLD with
ubiquitinated inclusions (FTLD-U), FTLD-U with motor neurone disease (FTLD-U + MND) and amyotrophic lateral
sclerosis (ALS). Neuronal and glial TDP-43 histochemical abnormalities are described in a series of 5 cases with
sporadic FTLD-MND and 3 cases of familial FTD + MND from 2 separate families (mutation status currently
unknown). A spectrum of neuronal TDP-43 changes including neuronal skein-like inclusions, Lewy body-like
inclusions, pre-inclusions (granular/diffuse cytoplasmic staining with negative nuclear staining) and dystrophic neurites
were present in cerebral cortex (frontal and hippocampus) basal ganglia, brainstem and spinal cord in FTLD-MND but
not in control tissue. The TDP-43 immunopositive pre-inclusions were negative or only weakly ubiquitin
immunoreactive. TDP-43 immunostaining abnormalities were often present in the absence of ubiquitin abnormalities
and their widespread anatomical distribution supports the concept that FTLD-MND is a TDP-43 multisystem
proteinopathy.
Oral Presentations III
Title: “Synaptic proteome changes in the alcoholic brain”
Authors:
N. Etheridge1, J.M. Lewohl2, R.D. Mayfield3, R.A. Harris3 and P.R. Dodd1.
1, School of Molecular and Microbial Sciences, University of Queensland, St Lucia, Queensland, 4072, Australia
2, Genomics Research Centre and School of Medical Sciences, Griffith University, Southport, Queensland, 4222, Australia
3, University of Texas, Waggoner Center for Alcohol and Addiction Research, 1 University Station A4800, Austin, TX 78712-0159, USA
Abstract: Changes in mRNA and protein expression patterns are widely invoked to explain the pathogenesis of brain
disease, although evidence for this is limited. We undertook a synaptic proteome analysis of human alcoholic brain
with and without comorbid disease, to explore the molecular basis of morphological changes induced by alcoholism.
Expression patterns varied regionally and with disease state. Proteins altered in alcoholics were involved in vesicle
transport, energy metabolism, protein folding and trafficking, and signal transduction, all of which have the potential to
influence synaptic activity. Notably, we found evidence for synchronized control of post-transcriptional or posttranslational protein modification of certain proteins. For example, the vesicle recycling protein dynamin 1 was arrayed
in eight different isoforms on the 2D gel, six of which were down regulated in the alcoholic brain. In contrast, different
PTM isoforms of heat-shock-protein cognate 70 kDa (HSPA8) were oppositely regulated in the SFG and the OC. Our
results suggest that post-translational modification of protein isoforms is a key mechanism of the brain’s response to
alcoholism.
9
SLIDE SEMINAR
AUSTRALIAN AND NEW ZEALAND SOCIETY OF NEUROPATHOLOGY
ANNUAL MEETING, BRISBANE, May 24th, 2008.
CLINICAL CASE SUMMARIES
10
Case 1
EX08-088 (SS07J3144) Presented by Dr Kathy Urankar, John Tonge Centre Institute of Forensic Pathology,
Brisbane.
Male 78
Hx of hypertension, ischaemic heart disease, heart failure, asthma and three previous THRs. Presented with headaches
and visual disturbances and some limb paraesthesias. CT scan showed right parietal lobe lesion.
Case 2
EX08-089 (99-50173-N17) Presented by Professor Peter Blumbergs, Centre for Neurological Diseases, Hanson
Institute, Adelaide.
This 21 year-old woman was brought to a peripheral hospital by her parents in an agitated state with dysarthria. This
had been ongoing for two to three days, and had followed commencement of metoclopramide for a viral illness. The
viral illness had lasted about 10 days. CT scan demonstrated generalised cerebral oedema. A diagnosis of organic
brain syndrome possibly due to viral encephalitis or Maxolon induced neuroleptic malignant syndrome with a dystonic
reaction was made.
She was admitted to the Royal Adelaide Hospital following deterioration in clinical state. An MRI was normal. A
diagnosis of lymphocytic meningitis with an encephalopathic process was made with a possible diagnosis of cerebral
Whipple's disease. Fitting occurred with an oculogyric crisis. A follow up MRI was negative. A PEG tube was
inserted for reflux oesophagitis. A small bowel biopsy was normal with no evidence of Whipple's disease. The
diagnosis was therefore thought to be oculopalatopharyngiobranchial myoclonus secondary to post infective brain stem
encephalitis. She was not responding to spoken command and was ventilated. A spiking fever was thought to be due
to a urinary tract infection, and she suffered a right subclavian vein thrombosis with swelling of the right arm.
Continued high fevers were noted with negative microbiological assessment suggesting a cerebrally mediated event.
Cardiac arrest occurred four and a half months after onset of illness.
Case 3
EX08-090 (08-506944) Presented by Professor Peter Blumbergs, Centre for Neurological Diseases, Hanson
Institute, Adelaide.
49 year-old woman present with neuroimaging of an extra-axial cystic lesion of the left inferior cerebellum.
Case 4
EX08-091 (RB08P1558) Presented by Dr Queenie Lau, Gold Coast Hospital and Royal Brisbane & Women’s
Hospital.
M16, 4 year history of right forearm and hand weakness with increasing difficulty playing the trumpet. Examination
revealed significant wasting of the right forearm and hand muscles. He demonstrated weakness in the pronation of the
forearm, flexion of wrist and digits as well as thumb movements. No sensory deficit is identified. MRI showed a
diffuse fusiform swelling of the right median nerve throughout most of its course within the arm with mildly increased
T2 signal. The nerve was twice the size of the accompanying brachial artery.
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Case 5
EX08-092 (SP-07-4489 ) Presented by Dr Judith Grogan, University of Sydney.
2007: A 29 year old female was diagnosed with acromegaly after presenting with increasing hand and shoe sizes. She
was noted to have bilaterally restricted visual fields, worse in the inferior fields. There were no other neurological
findings.
MRI showed a large macroadenoma extending from the enlarged sella superiorly into the suprasellar cistern. There was
severe thinning and elevation of the optic chiasm.
The patient underwent an elective transphenoidal resection of the macroadenoma with subsequent resolution of the
visual field deficit.
Post-operative MRI showed residual adenoma.
2008: Repeat transphenoidal resection of the adenoma was completed with adequate clearance on post-operative MRI.
Case 6
EX08-093 (RB03P12961) Presented by Dr Tom Robertson, Royal Brisbane & Women’s Hospital.
M41. Acromegaly
Case 7
EX08-094 (Ref#SO-07-15531) Presented by Dr Michael Buckland, St Vincent’s Pathology, Darlinghurst.
70yr old female. Left arm weakness for several months. Initially diagnosed as CVA. MRI appearances suggest it is
slowly increasing in size. History of coeliac disease; ?lymphoma ? infection.
Case 8
EX08-095 (07-533614) Presented by Dr Barbara Koszyca, Centre for Neurological Diseases, Hanson Institute,
Adelaide.
Female 66 years
Six month history of symptoms related to 6 cm anterior cranial fossa tumour.
Case 9
EX08-096 (07-562422) Presented by Dr Barbara Koszyca, Centre for Neurological Diseases, Hanson Institute,
Adelaide.
Female 36 years
Mother of four, working full time describes a twelve month history of increase lethargy and generalized myalgia,
requiring time off from work and analgesics. Also complains of difficulty climbing stairs and getting up from a
squatting position. No significant family history. CK 996 and normal CRP.
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