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1.  Phosphorylation Status of Thymidine Kinase 1 Following Antiproliferative Drug Treatment Mediates 3′-Deoxy-3′-[18F]-Fluorothymidine Cellular Retention 
PLoS ONE  2014;9(7):e101366.
Background
3′-Deoxy-3′-[18F]-fluorothymidine ([18F]FLT) is being investigated as a Positron Emission Tomography (PET) proliferation biomarker. The mechanism of cellular [18F]FLT retention has been assigned primarily to alteration of the strict transcriptionally regulated S-phase expression of thymidine kinase 1 (TK1). This, however, does not explain how anticancer agents acting primarily through G2/M arrest affect [18F]FLT uptake. We investigated alternative mechanisms of [18F]FLT cellular retention involving post-translational modification of TK1 during mitosis.
Methods
[18F]FLT cellular retention was assessed in cell lines having different TK1 expression. Drug-induced phosphorylation of TK1 protein was evaluated by MnCl2-phos-tag gel electrophoresis and correlated with [18F]FLT cellular retention. We further elaborated the amino acid residues involved in TK1 phosphorylation by transient transfection of FLAG-pCMV2 plasmids encoding wild type or mutant variants of TK1 into TK1 negative cells.
Results
Baseline [18F]FLT cellular retention and TK1 protein expression were associated. S-phase and G2/M phase arrest caused greater than two-fold reduction in [18F]FLT cellular retention in colon cancer HCT116 cells (p<0.001). G2/M cell cycle arrest increased TK1 phosphorylation as measured by induction of at least one phosphorylated form of the protein on MnCl2-phos-tag gels. Changes in [18F]FLT cellular retention reflected TK1 phosphorylation and not expression of total protein, in keeping with the impact of phosphorylation on enzyme catalytic activity. Both Ser13 and Ser231 were shown to be involved in the TK1 phosphorylation-modulated [18F]FLT cellular retention; although the data suggested involvement of other amino-acid residues.
Conclusion
We have defined a regulatory role of TK1 phosphorylation in mediating [18F]FLT cellular retention and hence reporting of antiproliferative activity, with implications especially for drugs that induce a G2/M cell cycle arrest.
doi:10.1371/journal.pone.0101366
PMCID: PMC4086825  PMID: 25003822
2.  Development, appraisal, validation and implementation of a consensus protocol for the assessment of cerebral amyloid angiopathy in post-mortem brain tissue 
In a collaboration involving 11 groups with research interests in cerebral amyloid angiopathy (CAA), we used a two-stage process to develop and in turn validate a new consensus protocol and scoring scheme for the assessment of CAA and associated vasculopathic abnormalities in post-mortem brain tissue. Stage one used an iterative Delphi-style survey to develop the consensus protocol. The resultant scoring scheme was tested on a series of digital images and paraffin sections that were circulated blind to a number of scorers. The scoring scheme and choice of staining methods were refined by open-forum discussion. The agreed protocol scored parenchymal and meningeal CAA on a 0-3 scale, capillary CAA as present/absent and vasculopathy on 0-2 scale, in the 4 cortical lobes that were scored separately. A further assessment involving three centres was then undertaken. Neuropathologists in three centres (Bristol, Oxford and Sheffield) independently scored sections from 75 cases (25 from each centre) and high inter-rater reliability was demonstrated. Stage two used the results of the three-centre assessment to validate the protocol by investigating previously described associations between APOE genotype (previously determined), and both CAA and vasculopathy. Association of capillary CAA with or without arteriolar CAA with APOE ε4 was confirmed. However APOE ε2 was also found to be a strong risk factor for the development of CAA, not only in AD but also in elderly non-demented controls. Further validation of this protocol and scoring scheme is encouraged, to aid its wider adoption to facilitate collaborative and replication studies of CAA.
PMCID: PMC3986608  PMID: 24754000
Angiopathy; amyloid; dementia; Delphi; validation; APOE; CAA; consensus; parenchymal; meningeal
3.  A Multicenter Study of Glucocerebrosidase Mutations in Dementia With Lewy Bodies 
JAMA neurology  2013;70(6):10.1001/jamaneurol.2013.1925.
Importance
While mutations in glucocerebrosidase (GBA1) are associated with an increased risk for Parkinson disease (PD), it is important to establish whether such mutations are also a common risk factor for other Lewy body disorders.
Objective
To establish whether GBA1 mutations are a risk factor for dementia with Lewy bodies (DLB).
Design
We compared genotype data on patients and controls from 11 centers. Data concerning demographics, age at onset, disease duration, and clinical and pathological features were collected when available. We conducted pooled analyses using logistic regression to investigate GBA1 mutation carrier status as predicting DLB or PD with dementia status, using common control subjects as a reference group. Random-effects meta-analyses were conducted to account for additional heterogeneity.
Setting
Eleven centers from sites around the world performing genotyping.
Participants
Seven hundred twenty-one cases met diagnostic criteria for DLB and 151 had PD with dementia. We compared these cases with 1962 controls from the same centers matched for age, sex, and ethnicity.
Main Outcome Measures
Frequency of GBA1 mutations in cases and controls.
Results
We found a significant association between GBA1 mutation carrier status and DLB, with an odds ratio of 8.28 (95% CI, 4.78–14.88). The odds ratio for PD with dementia was 6.48 (95% CI, 2.53–15.37). The mean age at diagnosis of DLB was earlier in GBA1 mutation carriers than in noncarriers (63.5 vs 68.9 years; P<.001), with higher disease severity scores.
Conclusions and Relevance
Mutations in GBA1 are a significant risk factor for DLB. GBA1 mutations likely play an even larger role in the genetic etiology of DLB than in PD, providing insight into the role of glucocerebrosidase in Lewy body disease.
doi:10.1001/jamaneurol.2013.1925
PMCID: PMC3841974  PMID: 23588557
5.  Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72 
Background
Cases of Frontotemporal Lobar Degeneration (FTLD) and Motor Neurone Disease (MND) associated with expansions in C9ORF72 gene are characterised pathologically by the presence of TDP-43 negative, but p62 positive, inclusions in granule cells of the cerebellum and in cells of dentate gyrus and area CA4 of the hippocampus.
Results
We screened 84 cases of pathologically confirmed FTLD and 23 cases of MND for the presence of p62 positive inclusions in these three brain regions, and identified 13 positive cases of FTLD and 3 of MND. All cases demonstrated expansions in C9ORF72 by Southern blotting where frozen tissues were available. The p62 positive inclusions in both cerebellum and hippocampus were immunostained by antibodies to dipeptide repeat proteins (DPR), poly Gly-Ala (poly-GA), poly Gly-Pro (poly-GP) and poly Gly-Arg (poly-GR), these arising from a putative non-ATG initiated (RAN) sense translation of the GGGGCC expansion. There was also some slight, but variable, immunostaining with poly-AP antibody implying some antisense translation might also occur, though the relative paucity of immunostaining could reflect poor antigen avidity on the part of the antisense antibodies. Of the FTLD cases with DPR, 6 showed TDP-43 type A and 6 had TDP-43 type B histology; one had FTLD-tau with the pathology of corticobasal degeneration. There were no qualitative or quantitative differences in the pattern of immunostaining with antibodies to DPR, or p62, proteins between TDP-43 type A and type B cases. Ratings for frequency of inclusions immunostained by these poly-GA, poly-GP and poly-GR antibodies broadly correlated with those for immunolabelled by p62 antibody in all three regions.
Conclusion
We conclude that DPR are a major component of p62 positive inclusions in FTLD and MND.
doi:10.1186/2051-5960-1-68
PMCID: PMC3893586  PMID: 24252525
Frontotemporal lobar degeneration; C9ORF72; p62 inclusions; Dipeptide repeat proteins
6.  Nuclear Carrier and RNA Binding Proteins in Frontotemporal Lobar Degeneration associated with Fused in Sarcoma (FUS) pathological changes 
Neuropathology and applied neurobiology  2012;10.1111/j.1365-2990.2012.01274.x.
Aims
We aimed to investigate the role of the nuclear carrier and binding proteins, transportin-1 (TRN1) and transportin-2 (TRN2), TATA-binding protein-associated factor 15 (TAF15) and Ewing’s Sarcoma protein (EWS) in inclusion body formation in cases of Frontotemporal Lobar Degeneration (FTLD) associated with Fused in Sarcoma protein (FTLD-FUS).
Methods
Eight cases of FTLD-FUS (5 cases of atypical FTLD-U (aFTLD-U), 2 of Neuronal Intermediate Filament Inclusion Body Disease (NIFID) and 1 of Basophilic Inclusion Body Disease (BIBD)) were immunostained for FUS, TRN1, TRN2, TAF15 and EWS. 10 cases of FTLD associated with TDP-43 inclusions served as reference cases.
Results
The inclusion bodies in FTLD-FUS contained TRN1 and TAF15 and, to a lesser extent, EWS, but not TRN2. The patterns of immunostaining for TRN1 and TAF15 were very similar to that of FUS. None of these proteins was associated with tau or TDP-43 aggregations in FTLD.
Conclusion
Data suggest that FUS, TRN1 and TAF15 may participate in a functional pathway in an interdependent way, and imply that the function of TDP-43 may not necessarily be in parallel with, or complementary to, that of FUS, despite each protein sharing many similar structural elements.
doi:10.1111/j.1365-2990.2012.01274.x
PMCID: PMC3479345  PMID: 22497712
Frontotemporal Lobar degeneration; Fused in Sarcoma; TDP-43; transportins; TATA-binding protein-associated factor 15; Ewing’s sarcoma protein
7.  Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer 
Eccles, Suzanne A | Aboagye, Eric O | Ali, Simak | Anderson, Annie S | Armes, Jo | Berditchevski, Fedor | Blaydes, Jeremy P | Brennan, Keith | Brown, Nicola J | Bryant, Helen E | Bundred, Nigel J | Burchell, Joy M | Campbell, Anna M | Carroll, Jason S | Clarke, Robert B | Coles, Charlotte E | Cook, Gary JR | Cox, Angela | Curtin, Nicola J | Dekker, Lodewijk V | dos Santos Silva, Isabel | Duffy, Stephen W | Easton, Douglas F | Eccles, Diana M | Edwards, Dylan R | Edwards, Joanne | Evans, D Gareth | Fenlon, Deborah F | Flanagan, James M | Foster, Claire | Gallagher, William M | Garcia-Closas, Montserrat | Gee, Julia M W | Gescher, Andy J | Goh, Vicky | Groves, Ashley M | Harvey, Amanda J | Harvie, Michelle | Hennessy, Bryan T | Hiscox, Stephen | Holen, Ingunn | Howell, Sacha J | Howell, Anthony | Hubbard, Gill | Hulbert-Williams, Nick | Hunter, Myra S | Jasani, Bharat | Jones, Louise J | Key, Timothy J | Kirwan, Cliona C | Kong, Anthony | Kunkler, Ian H | Langdon, Simon P | Leach, Martin O | Mann, David J | Marshall, John F | Martin, Lesley Ann | Martin, Stewart G | Macdougall, Jennifer E | Miles, David W | Miller, William R | Morris, Joanna R | Moss, Sue M | Mullan, Paul | Natrajan, Rachel | O’Connor, James PB | O’Connor, Rosemary | Palmieri, Carlo | Pharoah, Paul D P | Rakha, Emad A | Reed, Elizabeth | Robinson, Simon P | Sahai, Erik | Saxton, John M | Schmid, Peter | Smalley, Matthew J | Speirs, Valerie | Stein, Robert | Stingl, John | Streuli, Charles H | Tutt, Andrew N J | Velikova, Galina | Walker, Rosemary A | Watson, Christine J | Williams, Kaye J | Young, Leonie S | Thompson, Alastair M
Introduction
Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.
Methods
More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.
Results
The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.
Conclusions
With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.
doi:10.1186/bcr3493
PMCID: PMC3907091  PMID: 24286369
8.  A longitudinal study on α-synuclein in blood plasma as a biomarker for Parkinson's disease 
Scientific Reports  2013;3:2540.
There have been no longitudinal studies on α-synuclein as a potential biomarker for the progression of Parkinson's disease (PD). Here, blood plasma ‘total α-synuclein’ and ‘Ser-129 phosphorylated α-synuclein’ were assayed at 4–6 monthly intervals from a cohort of 189 newly-diagnosed patients with PD. For log-transformed data, plasma total α-synuclein levels increased with time for up to 20 yrs after the appearance of initial symptoms (p = 0.012), whereas phosphorylated α-synuclein remained constant over this same period. The mean level of phosphorylated α-synuclein, but not of total α-synuclein, was higher in the PD plasma samples taken at first visit than in single samples taken from a group of 91 healthy controls (p = 0.012). Overall, we conclude that the plasma level of phosphorylated α-synuclein has potential value as a diagnostic tool, whereas the level of total α-synuclein could act as a surrogate marker for the progression of PD.
doi:10.1038/srep02540
PMCID: PMC3756331  PMID: 23985836
9.  Extensive deamidation at asparagine residue 279 accounts for weak immunoreactivity of tau with RD4 antibody in Alzheimer’s disease brain 
Background
Intracytoplasmic inclusions composed of filamentous tau proteins are defining characteristics of neurodegenerative tauopathies, but it remains unclear why different tau isoforms accumulate in different diseases and how they induce abnormal filamentous structures and pathologies. Two tau isoform-specific antibodies, RD3 and RD4, are widely used for immunohistochemical and biochemical studies of tau species in diseased brains.
Results
Here, we show that extensive irreversible post-translational deamidation takes place at asparagine residue 279 (N279) in the RD4 epitope of tau in Alzheimer’s disease (AD), but not corticobasal degeneration (CBD) or progressive supranuclear palsy (PSP), and this modification abrogates the immunoreactivity to RD4. An antiserum raised against deamidated RD4 peptide specifically recognized 4R tau isoforms, regardless of deamidation, and strongly stained tau in AD brain. We also found that mutant tau with N279D substitution showed reduced ability to bind to microtubules and to promote microtubule assembly.
Conclusion
The biochemical and structural differences of tau in AD from that in 4R tauopathies found in this study may therefore have implications for prion-like propagation of tau.
doi:10.1186/2051-5960-1-54
PMCID: PMC3893535  PMID: 24252707
Alzheimer’s disease; Tau; Deamidation; Aging; Microtubule
10.  Pathological assessments for the presence of hexanucleotide repeat expansions in C9ORF72 in Alzheimer’s disease 
Background
We have sought histological evidence, using TDP-43 and p62 immunohistochemistry, for the presence of expansions in C9ORF72 among 200 patients with pathologically confirmed AD.
Results
We noted TDP-43 pathological changes in hippocampus and temporal cortex in 45 (22.5%) of these patients, but did not detect any cases where p62 positive changes in hippocampus and cerebellum, considered pathognomic for C9ORF72 expansions, were present.
Conclusion
We conclude that expansions in C9ORF72 associated with AD are a rare occurrence, and in those instances in the literature where these have been reported, the presence of AD may in fact be coincidental and unrelated to the expansion.
doi:10.1186/2051-5960-1-50
PMCID: PMC3893394  PMID: 24252571
Alzheimer’s disease; Genetics; C9ORF72
11.  Effect of Angle on Flow-Induced Vibrations of Pinniped Vibrissae 
PLoS ONE  2013;8(7):e69872.
Two types of vibrissal surface structures, undulated and smooth, exist among pinnipeds. Most Phocidae have vibrissae with undulated surfaces, while Otariidae, Odobenidae, and a few phocid species possess vibrissae with smooth surfaces. Variations in cross-sectional profile and orientation of the vibrissae also exist between pinniped species. These factors may influence the way that the vibrissae behave when exposed to water flow. This study investigated the effect that vibrissal surface structure and orientation have on flow-induced vibrations of pinniped vibrissae. Laser vibrometry was used to record vibrations along the whisker shaft from the undulated vibrissae of harbor seals (Phoca vitulina) and northern elephant seals (Mirounga angustirostris) and the smooth vibrissae of California sea lions (Zalophus californianus). Vibrations along the whisker shaft were measured in a flume tank, at three orientations (0°, 45°, 90°) to the water flow. The results show that vibration frequency and velocity ranges were similar for both undulated and smooth vibrissae. Angle of orientation, rather than surface structure, had the greatest effect on flow-induced vibrations. Vibration velocity was up to 60 times higher when the wide, flat aspect of the whisker faced into the flow (90°), compared to when the thin edge faced into the flow (0°). Vibration frequency was also dependent on angle of orientation. Peak frequencies were measured up to 270 Hz and were highest at the 0° orientation for all whiskers. Furthermore, CT scanning was used to quantify the three-dimensional structure of pinniped vibrissae that may influence flow interactions. The CT data provide evidence that all vibrissae are flattened in cross-section to some extent and that differences exist in the orientation of this profile with respect to the major curvature of the hair shaft. These data support the hypothesis that a compressed cross-sectional profile may play a key role in reducing self-noise of the vibrissae.
doi:10.1371/journal.pone.0069872
PMCID: PMC3724740  PMID: 23922834
12.  Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases 
Bioorganic & medicinal chemistry  2012;20(14):4371-4376.
Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-β. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.
doi:10.1016/j.bmc.2012.05.040
PMCID: PMC3389297  PMID: 22705189
Arylstibonic acids; protein tyrosine phosphatase; Cdc25 inhibitor
13.  A multi-centre study of ACE and the risk of late-onset Alzheimer’s disease 
A key pathological feature of late-onset Alzheimer’s disease (LOAD) is the abnormal extracellular accumulation of the amyloid beta (Aβ) peptide. Thus altered Aβ degradation could be a major contributor to the development of LOAD. Variants in the gene encoding the Aβ-degrading enzyme, angiotensin-1 converting enzyme (ACE) therefore represent plausible candidates for association with LOAD pathology and risk. Following Alzgene meta-analyses of all published case-control studies, the ACE variants rs4291 and rs1800764 showed significant association with LOAD risk. Furthermore ACE haplotypes are associated with both plasma ACE levels and LOAD risk. We tested three ACE variants (rs4291, rs4343 and rs1800764) for association with LOAD in ten Caucasian case-control populations (n=8,212). No association was found using multiple logistic models (all p>0.09). We found no population heterogeneity (all p>0.38) or evidence for association with LOAD risk following meta-analysis of the ten populations for rs4343 (OR=1.00), rs4291 (OR=0.97) or rs1800764 (OR=0.99). Although we found no haplotypic association in our complete dataset (p=0.51), a significant global haplotypic p-value was observed in one population (p=0.007) due to an association of the H3 haplotype (OR=0.72, p=0.02) and a trend towards an association of H4 (OR=1.38, p=0.09) and H7 (OR=2.07, p=0.08) although these did not survive Bonferroni correction. Previously reported associations of ACE variants with LOAD will be diminished following this study. At best, ACE variants have modest effect sizes, which are likely part of a complex interaction between genetic, phenotypic and pharmacological effects that would be undetected in traditional case-control studies.
doi:10.3233/JAD-2011-101914
PMCID: PMC3655234  PMID: 21297258
Alzheimer Disease; Late Onset; Angiotensin-1 Converting Enzyme; Haplotype; Heterogeneity; Meta-Analysis
14.  The Head Tracks and Gaze Predicts: How the World’s Best Batters Hit a Ball 
PLoS ONE  2013;8(3):e58289.
Hitters in fast ball-sports do not align their gaze with the ball throughout ball-flight; rather, they use predictive eye movement strategies that contribute towards their level of interceptive skill. Existing studies claim that (i) baseball and cricket batters cannot track the ball because it moves too quickly to be tracked by the eyes, and that consequently (ii) batters do not – and possibly cannot – watch the ball at the moment they hit it. However, to date no studies have examined the gaze of truly elite batters. We examined the eye and head movements of two of the world’s best cricket batters and found both claims do not apply to these batters. Remarkably, the batters coupled the rotation of their head to the movement of the ball, ensuring the ball remained in a consistent direction relative to their head. To this end, the ball could be followed if the batters simply moved their head and kept their eyes still. Instead of doing so, we show the elite batters used distinctive eye movement strategies, usually relying on two predictive saccades to anticipate (i) the location of ball-bounce, and (ii) the location of bat-ball contact, ensuring they could direct their gaze towards the ball as they hit it. These specific head and eye movement strategies play important functional roles in contributing towards interceptive expertise.
doi:10.1371/journal.pone.0058289
PMCID: PMC3596397  PMID: 23516460
15.  Prion-like spreading of pathological α-synuclein in brain 
Brain  2013;136(4):1128-1138.
α-Synuclein is the major component of filamentous inclusions that constitute the defining characteristic of neurodegenerative α-synucleinopathies. However, the molecular mechanisms underlying α-synuclein accumulation and spread are unclear. Here we show that intracerebral injections of sarkosyl-insoluble α-synuclein from brains of patients with dementia with Lewy bodies induced hyperphosphorylated α-synuclein pathology in wild-type mice. Furthermore, injection of fibrils of recombinant human and mouse α-synuclein efficiently induced similar α-synuclein pathologies in wild-type mice. C57BL/6J mice injected with α-synuclein fibrils developed abundant Lewy body/Lewy neurite-like pathology, whereas mice injected with soluble α-synuclein did not. Immunoblot analysis demonstrated that endogenous mouse α-synuclein started to accumulate 3 months after inoculation, while injected human α-synuclein fibrils disappeared in about a week. These results indicate that α-synuclein fibrils have prion-like properties and inoculation into wild-type brain induces α-synuclein pathology in vivo. This is a new mouse model of sporadic α-synucleinopathy and should be useful for elucidating progression mechanisms and evaluating disease-modifying therapy.
doi:10.1093/brain/awt037
PMCID: PMC3613715  PMID: 23466394
α-synuclein; Lewy bodies; Parkinson’s disease; propagation
16.  Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations 
Brain  2012;135(3):693-708.
The identification of a hexanucleotide repeat expansion in the C9ORF72 gene as the cause of chromosome 9-linked frontotemporal dementia and motor neuron disease offers the opportunity for greater understanding of the relationship between these disorders and other clinical forms of frontotemporal lobar degeneration. In this study, we screened a cohort of 398 patients with frontotemporal dementia, progressive non-fluent aphasia, semantic dementia or mixture of these syndromes for mutations in the C9ORF72 gene. Motor neuron disease was present in 55 patients (14%). We identified 32 patients with C9ORF72 mutations, representing 8% of the cohort. The patients’ clinical phenotype at presentation varied: nine patients had frontotemporal dementia with motor neuron disease, 19 had frontotemporal dementia alone, one had mixed semantic dementia with frontal features and three had progressive non-fluent aphasia. There was, as expected, a significant association between C9ORF72 mutations and presence of motor neuron disease. Nevertheless, 46 patients, including 22 familial, had motor neuron disease but no mutation in C9ORF72. Thirty-eight per cent of the patients with C9ORF72 mutations presented with psychosis, with a further 28% exhibiting paranoid, deluded or irrational thinking, whereas <4% of non-mutation bearers presented similarly. The presence of psychosis dramatically increased the odds that patients carried the mutation. Mutation bearers showed a low incidence of motor stereotypies, and relatively high incidence of complex repetitive behaviours, largely linked to patients’ delusions. They also showed a lower incidence of acquired sweet food preference than patients without C9ORF72 mutations. Post-mortem pathology in five patients revealed transactive response DNA-binding protein 43 pathology, type A in one patient and type B in three. However, one patient had corticobasal degeneration pathology. The findings indicate that C9ORF72 mutations cause some but not all cases of frontotemporal dementia with motor neuron disease. Other mutations remain to be discovered. C9ORF72 mutations are associated with variable clinical presentations and pathology. Nevertheless, the findings highlight a powerful association between C9ORF72 mutations and psychosis and suggest that the behavioural characteristics of patients with C9ORF72 mutations are qualitatively distinct. Mutations in the C9ORF72 gene may be a major cause not only of frontotemporal dementia with motor neuron disease but also of late onset psychosis.
doi:10.1093/brain/awr355
PMCID: PMC3286329  PMID: 22300873
frontotemporal lobar degeneration; clinical characteristics; motor neuron disease; psychosis; neuropathology
17.  Correlation of Alzheimer Disease Neuropathologic Changes With Cognitive Status: A Review of the Literature 
Clinicopathologic correlation studies are critically important for the field of Alzheimer disease (AD) research. Studies on human subjects with autopsy confirmation entail numerous potential biases that affect both their general applicability and the validity of the correlations. Many sources of data variability can weaken the apparent correlation between cognitive status and AD neuropathologic changes. Indeed, most persons in advanced old age have significant non-AD brain lesions that may alter cognition independently of AD. Worldwide research efforts have evaluated thousands of human subjects to assess the causes of cognitive impairment in the elderly, and these studies have been interpreted in different ways. We review the literature focusing on the correlation of AD neuropathologic changes (i.e. β-amyloid plaques and neurofibrillary tangles) with cognitive impairment. We discuss the various patterns of brain changes that have been observed in elderly individuals to provide a perspective for understanding AD clinicopathologic correlation and conclude that evidence from many independent research centers strongly supports the existence of a specific disease, as defined by the presence of Aβ plaques and neurofibrillary tangles. Although Aβ plaques may play a key role in AD pathogenesis, the severity of cognitive impairment correlates best with the burden of neocortical neurofibrillary tangles.
doi:10.1097/NEN.0b013e31825018f7
PMCID: PMC3560290  PMID: 22487856
Aging; Alzheimer disease; Amyloid; Dementia; Epidemiology; Neuropathology; MAPT; Neurofibrillary tangles
18.  Vascular histomolecular analysis by sequential endoarterial biopsy in a shunt model of pulmonary hypertension 
Pulmonary Circulation  2013;3(1):50-57.
The molecular mechanisms of pulmonary arterial hypertension (PAH) remain ill-defined. The aims of this study were to obtain sequential endoarterial biopsy samples in a surgical porcine model of PAH and assess changes in histology and mRNA expression during the disease progression. Differentially expressed genes were then analyzed as potential pharmacological targets. Four Yucatan micro-pigs underwent surgical anastomosis of the left pulmonary artery to the descending aorta. Endovascular samples were obtained with a biopsy catheter at baseline (before surgery) and from the left lung 7, 60, and 180 days after surgery. RNA was isolated from biopsy samples, amplified and analyzed. Dysregulated genes were linked to drugs with potential to treat or prevent PAH. With the development of PAH in our model, we identified changes in histology and in the expression of several genes with known or investigational inhibitors and several novel genes for PAH. Gene dysregulation displayed time-related variations during disease progression. Endoarterial biopsy provides a new method of assessing pulmonary vascular histology and gene expression in PAH. This analysis could identify novel applications for existing and new PAH drugs. The detection of stage- and disease-specific variation in gene expression could lead to individualized therapies.
doi:10.4103/2045-8932.109913
PMCID: PMC3641740  PMID: 23662174
endoarterial biopsy; pulmonary hypertension; vascular histomolecular analysis
19.  Neurophysiological studies may provide a misleading picture of how perceptual-motor interactions are coordinated 
i-Perception  2013;4(1):78-80.
Neurophysiological measurement techniques like fMRI and TMS are increasingly being used to examine the perceptual-motor processes underpinning the ability to anticipate the actions of others. Crucially, these techniques invariably restrict the experimental task that can be used and consequently limit the degree to which the findings can be generalised. These limitations are discussed based on a recent paper by Tomeo et al. (2012) who sought to examine responses to fooling actions by using TMS on participants who passively observed spliced video clips where bodily information was, and was not, linked to the action outcome. We outline two particular concerns with this approach. First, spliced video clips that show physically impossible actions are unlikely to simulate a “fooling” action. Second, it is difficult to make meaningful inferences about perceptual-motor expertise from experiments where participants cannot move. Taken together, we argue that wider generalisations based on these findings may provide a misunderstanding of the phenomenon such a study is designed to explore.
doi:10.1068/i0569ic
PMCID: PMC3690417  PMID: 23799189
anticipation; transcranial magnetic stimulation; motor; action; vision; football
20.  Next generation sequencing of CLU, PICALM and CR1: pitfalls and potential solutions 
CLU, PICALM and CR1 were identified as genetic risk factors for late onset Alzheimer’s disease (AD) in two large genome wide association studies (GWAS) published in 2009, but the variants that convey this alteration in disease risk, and how the genes relate to AD pathology is yet to be discovered. A next generation sequencing (NGS) project was conducted targeting CLU, CR1 and PICALM, in 96 AD samples (8 pools of 12), in an attempt to discover rare variants within these AD associated genes. Inclusion of repetitive regions in the design of the SureSelect capture lead to significant issues in alignment of the data, leading to poor specificity and a lower than expected depth of coverage. A strong positive correlation (0.964, p<0.001) was seen between NGS and 1000 genome project frequency estimates. Of the ~170 “novel” variants detected in the genes, seven SNPs, all of which were present in multiple sample pools, were selected for validation by Sanger sequencing. Two SNPs were successfully validated by this method, and shown to be genuine variants, while five failed validation. These spurious SNP calls occurred as a result of the presence of small indels and mononucleotide repeats, indicating such features should be regarded with caution, and validation via an independent method is important for NGS variant calls.
PMCID: PMC3508540  PMID: 23205178
Next generation sequencing; Alzheimer’s disease; genes; CLU; PICALM; CR1
21.  CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms 
PLoS Biology  2012;10(11):e1001419.
A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.
doi:10.1371/journal.pbio.1001419
PMCID: PMC3491025  PMID: 23139639
22.  A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD 
Renton, Alan E. | Majounie, Elisa | Waite, Adrian | Simón-Sánchez, Javier | Rollinson, Sara | Gibbs, J. Raphael | Schymick, Jennifer C. | Laaksovirta, Hannu | van Swieten, John C. | Myllykangas, Liisa | Kalimo, Hannu | Paetau, Anders | Abramzon, Yevgeniya | Remes, Anne M. | Kaganovich, Alice | Scholz, Sonja W. | Duckworth, Jamie | Ding, Jinhui | Harmer, Daniel W. | Hernandez, Dena G. | Johnson, Janel O. | Mok, Kin | Ryten, Mina | Trabzuni, Danyah | Guerreiro, Rita J. | Orrell, Richard W. | Neal, James | Murray, Alex | Pearson, Justin | Jansen, Iris E. | Sondervan, David | Seelaar, Harro | Blake, Derek | Young, Kate | Halliwell, Nicola | Callister, Janis | Toulson, Greg | Richardson, Anna | Gerhard, Alex | Snowden, Julie | Mann, David | Neary, David | Nalls, Michael A. | Peuralinna, Terhi | Jansson, Lilja | Isoviita, Veli-Matti | Kaivorinne, Anna-Lotta | Hölttä-Vuori, Maarit | Ikonen, Elina | Sulkava, Raimo | Benatar, Michael | Wuu, Joanne | Chiò, Adriano | Restagno, Gabriella | Borghero, Giuseppe | Sabatelli, Mario | Heckerman, David | Rogaeva, Ekaterina | Zinman, Lorne | Rothstein, Jeffrey | Sendtner, Michael | Drepper, Carsten | Eichler, Evan E. | Alkan, Can | Abdullaev, Zied | Pack, Svetlana D. | Dutra, Amalia | Pak, Evgenia | Hardy, John | Singleton, Andrew | Williams, Nigel M. | Heutink, Peter | Pickering-Brown, Stuart | Morris, Huw R. | Tienari, Pentti J. | Traynor, Bryan J.
Neuron  2011;72(2):257-268.
The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one third of familial ALS cases of outbred European descent making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.
doi:10.1016/j.neuron.2011.09.010
PMCID: PMC3200438  PMID: 21944779
23.  No consistent evidence for association between mtDNA variants and Alzheimer disease 
Hudson, G. | Sims, R. | Harold, D. | Chapman, J. | Hollingworth, P. | Gerrish, A. | Russo, G. | Hamshere, M. | Moskvina, V. | Jones, N. | Thomas, C. | Stretton, A. | Holmans, P.A. | O'Donovan, M.C. | Owen, M.J. | Williams, J. | Chinnery, P.F. | Harold, Denise | Abraham, Richard | Hollingworth, Paul | Sims, Rebecca | Gerrish, Amy | Chapman, Jade | Russo, Giancarlo | Hamshere, Marian | Pahwa, Jaspreet Singh | Moskvina, Valentina | Dowzell, Kimberley | Williams, Amy | Jones, Nicola | Thomas, Charlene | Stretton, Alexandra | Morgan, Angharad | Lovestone, Simon | Powell, John | Proitsi, Petroula | Lupton, Michelle K | Brayne, Carol | Rubinsztein, David C. | Gill, Michael | Lawlor, Brian | Lynch, Aoibhinn | Morgan, Kevin | Brown, Kristelle | Passmore, Peter | Craig, David | McGuinness, Bernadette | Todd, Stephen | Johnston, Janet | Holmes, Clive | Mann, David | Smith, A. David | Love, Seth | Kehoe, Patrick G. | Hardy, John | Mead, Simon | Fox, Nick | Rossor, Martin | Collinge, John | Maier, Wolfgang | Jessen, Frank | Heun, Reiner | Kölsch, Heike | Schürmann, Britta | van den Bussche, Hendrik | Heuser, Isabella | Kornhuber, Johannes | Wiltfang, Jens | Dichgans, Martin | Frölich, Lutz | Hampel, Harald | Hüll, Michael | Rujescu, Dan | Goate, Alison | Kauwe, John S.K. | Cruchaga, Carlos | Nowotny, Petra | Morris, John C. | Mayo, Kevin | Livingston, Gill | Bass, Nicholas J. | Gurling, Hugh | McQuillin, Andrew | Gwilliam, Rhian | Deloukas, Panagiotis | Holmans, Peter | O'Donovan, Michael | Owen, Michael J. | Williams, Julie
Neurology  2012;78(14):1038-1042.
Objective:
Although several studies have described an association between Alzheimer disease (AD) and genetic variation of mitochondrial DNA (mtDNA), each has implicated different mtDNA variants, so the role of mtDNA in the etiology of AD remains uncertain.
Methods:
We tested 138 mtDNA variants for association with AD in a powerful sample of 4,133 AD case patients and 1,602 matched controls from 3 Caucasian populations. Of the total population, 3,250 case patients and 1,221 elderly controls met the quality control criteria and were included in the analysis.
Results:
In the largest study to date, we failed to replicate the published findings. Meta-analysis of the available data showed no evidence of an association with AD.
Conclusion:
The current evidence linking common mtDNA variations with AD is not compelling.
doi:10.1212/WNL.0b013e31824e8f1d
PMCID: PMC3317529  PMID: 22442439
24.  Mechanisms of disease in frontotemporal lobar degeneration: gain of function versus loss of function effects 
Acta neuropathologica  2012;124(3):373-382.
Frontotemporal lobar degeneration (FTLD) is clinically, pathologically and genetically heterogeneous. Three major proteins are implicated in its pathogenesis. About half of cases are characterized by depositions of the microtubule associated protein, tau (FTLD-tau). In most of the remaining cases, deposits of the transactive response (TAR) DNA-binding protein with Mw of 43 kDa, known as TDP-43 (FTLD-TDP), are seen. Lastly, about 5–10 % of cases are characterized by abnormal accumulations of a third protein, fused in sarcoma (FTLD-FUS). Depending on the protein concerned, the signature accumulations can take the form of inclusion bodies (neuronal cytoplasmic inclusions and neuronal intranuclear inclusions) or dystrophic neurites, in the cerebral cortex, hippocampus and subcortex. In some instances, glial cells are also affected by inclusion body formation. In motor neurone disease (MND), TDP-43 or FUS inclusions can present within motor neurons of the brain stem and spinal cord. This present paper attempts to critically examine the role of such proteins in the pathogenesis of FTLD and MND as to whether they might exert a direct pathogenetic effect (gain of function), or simply act as relatively innocent witnesses to a more fundamental loss of function effect. We conclude that although there is strong evidence for both gain and loss of function effects in respect of each of the proteins concerned, in reality, it is likely that each is a single face of either side of the coin, and that both will play separate, though complementary, roles in driving the damage which ultimately leads to the downfall of neurons and clinical expression of disease.
doi:10.1007/s00401-012-1030-4
PMCID: PMC3445027  PMID: 22878865
Frontotemporal lobar degeneration; Motor neurone disease; Microtubule associated protein; Tau; TDP-43; FUS; Gain of function; Loss of function
25.  Glomerular Expression of Kidney Injury Molecule-1 and Podocytopenia in Diabetic Glomerulopathy 
American Journal of Nephrology  2011;34(3):268-280.
Background/Aims
Studies have shown that kidney injury molecule-1 (KIM-1) is upregulated in damaged renal proximal tubules. In this study, we examined KIM-1 expression in glomerular epithelial cells in diabetic glomerulopathy.
Methods
Renal histology, immunostaining and Western blot for protein level, and real-time PCR for mRNA expression of KIM-1 and podocyte markers were evaluated in untreated or losartan-treated Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats.
Results
The diabetic rats showed an increased glomerular expression of KIM-1. KIM-1 staining was localized primarily in the hyperplastic parietal epithelium of Bowman's capsule in the early stages of diabetes with subsequent increase in KIM-1-positive cells in the glomerular tuft in the more advanced stages. The increase in glomerular KIM-1 was associated with a decrease in podocytes in Fa/Fa rats. Antiproteinuric treatment with losartan attenuated podocytopenia and decreased renal expression of KIM-1 in treated diabetic rats. In an in vitro study, albumin overload increased KIM-1 protein in the primary cultures of rat glomerular epithelial cells.
Conclusion
These results show that glomerular KIM-1 expression was increased, in proportion to the extent of proteinuria and podocytopenia in the diabetic animals, supporting that KIM-1 could be used as a potential biomarker for glomerular injury in proteinuric kidney disease.
doi:10.1159/000330187
PMCID: PMC3169370  PMID: 21822010
Albuminuria; Kidney injury molecule-1; Parietal epithelial cells; Podocytes; Glomerulopathy

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