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Information for Researchers | ||
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DetailsThis page contains the titles of the projects carried out using tissue supplied by the Tissue Bank, together with any associated follow-up reports and publications.
TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR1
TR2 Publications (also for TR23 and 29): Eblan MJ, Walker JM, Sidransky E. The glucocerebrosidase gene and Parkinson's disease in Ashkenazi Jews. N Engl J Med. 2005 Feb 17;352(7):728-31
TR3 Publications (also for TR11, 30, 32, 33, 36, 37, 38 and 44):
TR4
TR5 |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR6
TR7
TR8
Introduction Human post-mortem brain tissue is one of the most valuable sources available for research into human neurological disorders. The success of experiments and reliability of data obtained for research purposes will depend on the quality of intracellular material obtained from the post-mortem tissue.
Experiment RNA was extracted from all the samples using the Qiagen lipid tissue kit and the protocol previously optimised for the network. RNA was quantified using the Nanodrop spectrophotometer and quality assessed using the Agilent Bioanalyser (28S: 18S ratio). The data obtained was analysed against predetermined variables. As the information supplied by a majority of the participants was limited to age, gender, PM delay, freezer interval, CSF pH and disease type, these were used as the main factors against which the quality of RNA was analysed. The statistical analysis of the data was carried out with advice and support from the Imperial College Statistical Advisory Service.
Results
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR10 The project was a preliminary investigation into the involvement of alpha-synuclein and oxidative stress in olfactory bulb pathology in PD. The project yielded very interesting preliminary results which highlighted a correlation with both oxidative stress and synuclein pathology in the olfactory bulbs and PD. The areas of synuclein and oxidative stress pathology in the bulbs were not correlated. This study warrants further investigation and we hope to request further tissue for this purpose. Summary: The aim of this project was to compare the distribution of Lewy pathology, activated microglia and oxidative stress within olfactory bulbs in Parkinson's disease and investigate whether they occur in the same regions. Immunohistochemical techniques were used to stain LBs and LNs, activated microglia and a marker of oxidative damage in serial sections of the olfactory bulbs and tracts of 6 control and 6 PD patients. 20 regions within each bulb were analysed for each feature and the amount of staining compared. A positive linear correlation was found when comparing the amount of Lewy pathology with both activated microglia and oxidative stress in the bulbs as a whole, indicating that they progress proportionally. However, there was no evidence that these features occurred in the same areas within individual bulbs. This corroborates the evidence that these factors are linked in the disease mechanism. However, the lack of specific co-localisation implies that a more generalised process is occurring. It is suggested that oxidative stress is a widespread phenomenon, while Lewy pathology may be specific to certain regions or cell types within the bulb. Localized areas of activated microglia may be linked to these pathological features by secreting soluble substances that enhance oxidative stress and affect the bulb as a whole.
TR11 Publications: See TR3
TR12 Tissue from the caudate and putamen of PD16 and the temporal cortex of PD10 has been used to explore and optimise immunohistochemical techniques pertinent to our research on glia and alpha-synuclein in PD. We have observed increased numbers of oligodendrocytes in PD cases and hoped to find an immunohistochemical method for labelling these cells for quantification. We were unable to replicate reports of oligodendrocyte-specific transferrin immunohistochemistry (Wakabayashi et al. Acta Neuropathologica (2000) 99:14-20), and have yet to find a reliable method to immunohistochemically label oligodendrocyte cell bodies in order to differentiate them from other cell types. We had excellent results in labelling myelin, however, using myelin basic protein and two different antibodies recognising myelin oligodendrocyte glycoprotein, and then turned to optimising double immunohistochemistry with MBP and alpha-synuclein, using a variety of chromogens in peroxidase immunohistochemistry and also fluorescence. We originally hoped to use tissue requested from the substantia nigra of PD16 for electron microscoscopy and immunohistochemistry in order to better localise an interesting pattern of peri-axonal alpha-synuclein deposition we observed in that brain area. We have some very promising results from double fluorescent immunohistochemistry using myelin basic protein and alpha-synuclein, which suggest that the peri-axonal deposition we observed is localised to the myelin sheath. Unfortunately, we did not have good results at the EM level. Peroxidase immunohistochemistry left artifactual electron density in the myelin sheath, our primary area of interest. We are currently evaluating how we can further develop our immunofluorescent results for publication. |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR13
Aims of Project
The study was carried out by a 4th year medical student from Keele University, under the supervision of Professor Caroline Sewry Inclusion body myositis (IBM) is one of the most common muscle problems in adults. It usually presents after the age of 50 years and leads to severe disability. The name is derived from the vacuoles, filamentous inclusions and inflammation that characterise a muscle biopsy. Patients rarely respond to immunosuppression but accurate diagnosis is important so that unnecessary use of steroids and their accompanying side effects can be avoided. More appropriate therapy can then be selected. Many of the proteins seen in the brain in Alzheimer disease occur in muscle in IBM but it is not known if they can be detected in the absence of the classical features of vacuoles and filamentous inclusions. This project aimed to identify which abnormal proteins in muscle are the most reliable markers for the diagnosis of IBM and if they can be identified in the absence of the classical vacuoles and filaments. Muscle biopsies from 33 patients with, or suspected of having, IBM at Charing Cross Hospital, the Radcliffe Infirmary, Oxford and the Robert Jones & Agnes Hunt Orthopaedic Hospital, Oswestry were studied. All samples had been taken for diagnostic purposes and the study was an audit to determine which are the best diagnostic markers for assessment of the biopsies. Comparisons were made with control muscle samples from patients with cerebral palsy, previously obtained with informed consent and with ethical approval (01/74/RJ) and one positive control sample of frozen brain from the Parkinson's Disease Tissue Bank at Charing Cross. The techniques used were staining with haematoxylin and eosin, with the modified Gomori trichrome technique with Congo Red and immunohistochemical labelling of frozen sections with commercially available antibodies to phosphorylated tau (SMI 310), ubiquitin,anti-alzheimer precursor protein and beta-amyloid. Results showed that vacuoles were visible with histological stains in 14 muscle biopsies of IBM cases and that the most useful antibodies are to phosphorylated tau and ubiquitin. Accumulation of proteins was found in the absence of typical vacuoles seen with histological stains. Congo red viewed with fluorescent optics is also useful but the various published methods produced variable results. The frozen brain sample was an essential positive control but even this gave variable results with Congo red in comparison with a fixed paraffin embedded sample of kidney containing kidney.
Conclusions
TR14 |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR15 Report (also for TR19 and 22): Our initial tissue request for this project was for tissue sections from the substantia nigra for alpha-synuclein and microglial MHC class II immunohistochemistry. Immunoreactivity was scored using semi-quantitative severity ratings and, where possible, sections already stained by the PDSTB for diagnostic purposes were used in order to conserve tissue. We found a highly significant correlation between alpha-synuclein and MHCII imunoreactivity in the substantia nigra of PD cases, although we found no clinical correlates to the level of immunoreactivity. Our second tissue request was for an additional section from the same cases, for immunohistochemistry for CD68. While MHCII is expressed in microglia in all stages of activation, CD68 is only expressed in macrophages (the end-stage of microglial activation, in which they are essentially indistinguishable from blood-borne macrophages). We found that CD68 did not correlate to levels of alpha-synuclein or MHCII immunoreactivity, but did find a significant correlation between CD68 and disease duration, with cases showing high levels of CD68 having a shorter disease course. This work has been accepted for publication by the Journal of Neuroinflammation. A provisional .pdf of the manuscript can be accessed at http://www.jneuroinflammation.com/content/pdf/1742-2094-2-14.pdf, or a paper copy can be provided upon request.
Publications: Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB. Microglial inflammation in the Parkinsonian substantia nigra: relationship to alpha-synuclein deposition. J Neuroinflammation. 2005 Jun 3;2:14.
TR16
The neuropathological diagnosis of Parkinson's disease is based on the identification of alpha synuclein deposits and particularly of Lewy bodies (LB) in the substantia nigra and other brain areas. Most studies of PD and synucleinopathies have been carried out with non-commercial antibodies and there is no standardised protocol available. In order to set a standardised protocol for identification of alpha-synuclein in brains donated to the Parkinson's Disease Society Tissue Bank and useful in the diagnostic practice of PD and other synucleinopathies we have compared the seven commercially available monoclonal (mAb) antibodies directed against human alpha-synuclein. We have used paraffin embedded brain tissue from the frontal lobe and nigra of patients with idiopathic PD and dementia with Lewy bodies and from a case of a patient with multiple sclerosis showing incidental LB in the substantia nigra. One normal brain was used as control cased. Our results showed that all mAbs tested were able to stain LB and neuropil threads but there was variability in the intensity and extent of immunoreactivity. Only the mAb supplied by Becton Dickinson, Alexis and Santa Cruz recognised alpha-synuclein deposits in glial cells and highlighted cytoplasmic granular positivity which likely represents a pre-stage of LB. Notably, the monoclonal supplied by Zymed showed an intense periaxonal deposition of alpha-synuclein. Variability of immunostaining has mainly been attributed to the immunogen or the epitopes recognised by respective mAbs and to the aggregation state of alpha-synuclein. Given the growing importance of detection of this molecule by means of immunohistochemical techniques, our results emphasize the need for a standardised protocol in the diagnosis of alpha-synucleinopathies. Publications (also for TR25): Croisier E, Mres DE, Deprez M, Goldring K, Dexter DT, Pearce RK, Graeber MB, Roncaroli F. Comparative study of commercially available anti-alpha-synuclein antibodies. Neuropathol Appl Neurobiol. 2006 Jun;32(3):351-6. |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR17
A previous study on standardisation of the immunohistochemical protocol for alpha-synucelin demonstrated that some antibodies and particularly the one supplied by Zymed (clone LB509) highlighted deposition of alpha-synuclein around axons. To prove that alpha-synuclein can accumulated in myelin sheaths, we have examined samples from one case of idiopathic Parkinson’s disease and one normal brain as control matched for age. Sections form the superior frontal gyrus and substantia nigra were stained with immunohistochemical reactions using the avidin-biotin complex/peroxidase method with antibody anti-alpha synuclein (Zymed, clone LB509) and anti-myelin basic protein. Double immunofluorescence with the two antibodies was also performed to demonstrate co-localization of the two proteins in myelin. Antigen unmasking was carried out according to manufacturer instructions. Electron microscopy was carried out with fragments of tissue taken from a section stained with anti-alpha synuclein and an experiment of protein analysis was attempted using Western-blot using fresh samples from the two cases. Immunoreactions showed co-localization of alpha-synuclein and myelin basic protein in axons of the white matter of centrum semiovale and cerebral peduncle. No alpha-synuclein immunolabelling was conversely found in the control brain. This suggested that alpha-synuclein can accumulate within myelin and that such an accumulation is more likely to be a feature of Parkinson’s disease. The sample used for electron microscopy demonstrated non-specific deposits of diaminobenzidine but no definite positivity due to considerable damage of tissue. To our knowledge, alpha synuclein deposition has never been reported in myelin of patients with Parkinson's disease. Publications: See TR15
TR18
TR19 Report and Publications: See TR15)
TR20
TR21
TR22 Report and Publications: See TR15
TR23 Publications: See TR2 |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR24
TR25 Report and Publications: See TR16
TR26
TR27 Traditionally, Parkinson's disease (PD) has been characterised as a hypokinetic disorder with the main clinical features of resting tremor, bradykinesia, rigidity and abnormalities of gait, balance and posture. Clinico-pathological investigations and animal experimental work have shown that the classical motor signs in PD are due to pathological changes in the substantia nigra pars compacta (SNc). However, non-motor symptoms are also commonly present with dementia and visual hallucinations (VHs) representing one of the most frequent complications with important clinical consequences for the quality of life of PD patients and caregivers. The prevalence of dementia in PD has been reported to be between 12 and 41% with PD patients having a six-fold risk for dementia than non-PD age-matched controls. Prevalence figures for VHs are in the range of 6-60%. Despite such a high frequency the specific anatomical and pathological correlates of dementia and VHs remain under scrutiny. alpha-Synuclein (aSN) pathology in limbic and neocortical regions has been considered causative for dementia and hallucinations in Parkinson's disease (PD). However, concomitant Alzheimer's disease (AD) changes have complicated such findings. Furthermore, the concurrent occurrence of multiple neuropsychiatric symptoms in PD (i.e. dementia with VHs, anxiety, confusion etc.) has caused a difficulty in attributing specific pathological changes to specific clinical symptoms. In order to address this problem, we designed a unique study whereby the selection of cases enabled us to isolate clinical features of interest and study their anatomical and pathological basis. The medical histories of 81 cases from the UK Parkinson’s Disease Society Tissue Bank (UKPDSTB) were retrospectively examined and a severity score for dementia and hallucinations attributed without prior knowledge of the neuropathological diagnosis. Subsequently, cases with confirmed PD were grouped on the basis of clinical phenotype representing extremes of the spectrum for dementia and hallucinations as follow: 1) cases with absent or mild dementia and hallucinations (n=9), 2) cases with high clinical scores for hallucinations and no dementia (n=5), 3) cases with high clinical scores for dementia and no hallucinations (n=4) and 4) cases with high scores for both dementia and hallucinations (n=12). Subsequently, aSN and AD-type pathology burden was examined in detail in eight brain regions that are affected in PD and which have shown to subserve cognitive functions. In our clinico-pathological study we have found that dementia and hallucinations relate to pathological changes in anatomical regions of the limbic system. Furthermore, we have shown that aSN pathology is the main pathological correlate of those deficits.
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR28
TR29 Dementia is a very common secondary feature of Parkinson's disease (PD) but the basis for cognitive decline remains unclear. Most studies addressing this question have focused on the hippocampal formation, an area of the brain known to have a significant role in cognition and memory. Of particular interest are the observations that the CA2 sector of the hippocampus appears to show a differential susceptibility to pathological change. In an effort to characterize these changes further, we analyzed the CA2 sector in hippocampal sections taken from 21 PD cases (from the UKPD tissue bank) and 5 controls. Semi-quantitative assessment of ubiquitin, alpha-synuclein and tau immunostaining was performed and the results were analysed with respect to clinical variables including dementia and memory loss. Alpha-synuclein pathology, in the form of Lewy neurites, was most commonly seen in the CA2 sector of the hippocampus. Tau-immunoreactive neurites were also seen in this area in 17 of the cases but there was no statistically significant association between the two pathologies. However, there was a significant association between ubiquitin staining and the presence of both tau and alpha-synuclein neurites. The amount of alpha-synuclein pathology present in CA2 correlated with semi-quantitative measures of dementia, anxiety, confusion and memory loss, but not hallucinations. These results reinforce the importance of the CA2 sector in terms of the dementia associated with PD. The reasons for the selective susceptibility of this region remain obscure but recent electrophysiological data suggest that CA2 may be the target of direct input from the entorhinal cortex, in parallel to the well described perforant pathway and dentate gyrus-CA3-CA1 system. Further work is required to determine why this particular pathway should be more susceptible and what role it might have in normal memory function. Publications: See TR2
TR30 Publications: See TR3
TR31 We have performed immunostains for PBR, MCH class II antigen and GFAP in PD38, PD40 and PD65 and we have observed that the number of microglial cells expressing PBRs is substantially lower than the amount of microglial cells seen with MHC class II antigen. This suggests a down-regulation of PBR. The next step will be to examine three cases of idiopathic Parkinson’s disease with the same immunoreactions and we will request tissue for these experiments.
The preliminary results obtained with PD38, PD40 and PD65 have been included in this paper: |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR32 Whole genome analysis demonstrated a dysregulation in the mRNA expression of gamma-synuclein, a member of the synuclein family to which alpha-synuclein belongs, in Parkinson’s patients compared to control brains. As part of a 3 month MSc project conducted by Alex Slonimsky, this result was investigated; initially validating the changes in gamma-synulein mRNA in PD brain compared to controls using quantitative real-time PCR. As a follow up to this, a commercial antibody was found and purchased and tissue sections were requested (both snap-frozen and fixed wax-embedded sections) to investigate the pattern of gamma-synuclein protein expression. These tissue sections were used to optimise the antibody concentration for subsequent studies and establish if fixation significantly affected the immunoreactivity/expression of this protein in the substantia nigra. Publications: See TR3
TR33 TR33 and TR38 were made to confirm and extend findings from our on going global expression profiling experiments, funded by the UK Parkinson’s Disease Society (Moran et al., 2006; Duke et al., 2006). Our expression profiling experiments have identified many candidate genes that may be important in the neurodegenerative processes in PD substantia nigra. Interestingly, microarray experiments looking at expression in the PD superior frontal gyrus compared to control, show that there are some genes that are deregulated in the cortex as well as the nigra. Therefore, a number of transcriptional changes that we have identified are not exclusive to the substantia nigra. This strongly suggests that PD is "systemic", at least at the organ level, rather than representing a disease of the substantia nigra. To investigate this cortical deregulation in more detail, we requested samples of superior frontal gyrus were requested tissue from cases used in our microarray cohort, TR33, and from additional cases, TR38, to confirm and extend findings of our global expression profiling experiments. With the tissue obtained through these requests, we have performed quantative real-time polymerase chain reaction (qRT-PCR), in-situ hybridization (ISH), and immunohistochemistry (ICC) to examine gene and protein expression of known PD related genes, UCHL1, NURR1 and SNCA (Moran et al., 2006, and manuscripts in preparation). Furthermore, we have also investigated several other genes that were identified from our microarray analysis, HSP70, HSP40, HERP1 and HIP2 in greater detail, using qRT-PCR, ISH and/or ICC (manuscripts in preparation). These studies have helped us further define important PD-related pathways that were identified by our global expression profiling experiments and determine the extent to which these specific pathways show deregulation outside of the nigra. Publications: See TR3
TR34 Twenty sections of snap-frozen tissue from the cortex of PD29 were requested in order to assess the protein expression of the immunoglobulin G Fc-gamma receptor, CD32. Three monoclonal antibodies were tested on the sections. While the precise epitopes recognised by each antibody are not known, they have been demonstrated to have varying affinities for different types of myeloid cells. All three antibodies labelled peri-vascular cells in the PD cortex. We found that one antibody also labelled microglia, another also labelled astrocytes, and the third labelled both microglia and astrocytes. The differential staining of astrocytes and microglia with this panel of antibodies has interesting repercussions for the isoforms of CD32 they express. We followed up this initial study with a larger-scale comparison of PD cases with control tissue and continued to use the tissue supplied in this request as a positive control to ensure reproducibility in each immunohistochemical stain. |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR35 Tissue from the frontal, cingulate and visual cortex from 12 PD cases, 4 MSA cases and 2 control cases has been immunostained for tau, beta-amyloid, alpha-synuclein (ASN), Major Histocompatibility Complex Class II (MHCII) and CD68. The goal of this study has been to assess to what extent markers of microglial activation reflect pathological protein aggregation. Additional, clinico-pathological correlations between protein aggregation, microglial activation and clinical information including disease duration, severity and predominant type (hemi-tremulous or akinetic-rigid) of motor symptoms and drug treatment were also sought. Immunoreactivty was scored using semi-quantitative severity ratings. Clinical information was extracted from the clinical summaries corresponding to each case. We found that MHCII was related to ASN in the frontal and cingulate cortex when they were analysed separately, however MHCII expression remained constant in white matter tracts and primary visual cortex independent of protein aggregation. A general upregulation of MHCII in PD cases compared to controls suggests that disease-specific degeneration of the white matter may occur in PD independent of protein aggregation. CD68, a marker of phagocytosis, was found to be associated with tau but not ASN or beta-amyloid. No differences in microglial activation were observed between PD and MSA. Our results suggest that MHCII, a common marker of microglial activation in post-mortem and animal studies of microglia and disease, may not be the optimal indicator of microglial response in PD. MHCII expression is confounded by age and is not localised to areas of patho-anatomical relevance to PD. Previous studies have reported discrepant findings regarding the relationship between MHCII and protein aggregation. The present study, through the inclusion of primary visual cortex which is not thought to have any involvement in PD, will perhaps clarify those findings by highlighting the independence of MHCII from other detectable disease processes. We have also highlighted the possible importance of white matter in PD progression. This research will be included in a thesis currently in preparation, and a manuscript will subsequently be submitted for publication. Publications: See TR15
TR36 Publications: See TR3
TR37 Publications: See TR3
TR38 Publications: See TR3
TR39
TR40 |
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR41
TR42
TR43 The aim of this was to see if the pineal gland could be fixed and embedded for histological examination in the same way as neural tissue. This would enable future study on this structure where scientifically and clinically important. This tissue was taken last summer for processing as part of an MSc research project. This project was examining the changes in the SCN of patients with severe mental illness, and the SCN regulates the release of melatonin from the pineal gland. The pineal from PD119 was taken to see if it could be examined in concert with the hypothalamic target. However, due to time issues this part of the project was never performed. We hope to run this in July 2007 along with the tissue treatment for another student project. If the gland can be processed and examined then it will lead to another avenue of research into various disease states.
TR44 Publications: See TR3
TR45
TR46
TR47
TR48
TR49
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TR01-05 TR06-10 TR11-15 TR16-20 TR21-25 TR26-30
TR51
TR52
TR53
TR54
TR55
TR56
TR57
TR58
TR59
TR60 |
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