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1.  Mild Parkinsonian Signs in the Elderly – Is There an Association with PD? Crossectional Findings in 992 Individuals 
PLoS ONE  2014;9(3):e92878.
Background
Mild parkinsonian signs (MPS) are common in the elderly population, and have been associated with vascular diseases, mild cognitive impairment and dementia; however their relation to Parkinson's disease (PD) is unclear. Hypothesizing that individuals with MPS may reflect a pre-stage of PD, i.e. a stage in which the nigrostriatal system is already affected although to a milder degree than at the time of PD diagnosis, aim of this study was to evaluate the similarities between MPS and PD.
Methods
The TREND study is a prospective cross-sectional cohort study in individuals >50 years with biennial assessments designed to identify markers for an earlier diagnosis of Parkinson's and Alzheimer's disease. For this substudy 992 individuals were included for analyses (892 controls, 73 MPS individuals, 27 PD patients). Parameters defining risk of PD (sex, age, positive family history), prodromal markers (hyposmia, REM sleep behavior disorder, depression and autonomic failure) as well as quantitative fine motor, axial motor and cognitive parameters were compared between the three cohorts.
Results
As expected, PD patients differed from controls with regard to 12 of 15 of the assessed parameters. MPS individuals differed significantly from controls in 12 of the PD-associated parameters, but differed from PD only in 5 parameters.
Conclusion
This study shows that individuals with MPS share many prodromal and clinical markers of PD with PD patients, implying that either a common dynamic process or similar constitutional factors occur in MPS individuals and PD patients.
doi:10.1371/journal.pone.0092878
PMCID: PMC3968033  PMID: 24675747
2.  LRRK2: Cause, Risk, and Mechanism 
Journal of Parkinson's disease  2013;3(2):85-103.
In 2004 it was first shown that mutations in LRRK2 can cause Parkinson's disease. This initial discovery was quickly followed by the observation that a single particular mutation is a relatively common cause of Parkinson's disease across varied populations. Further genetic investigation has revealed a variety of genetic ties to Parkinson's disease across this gene. These include common alleles with quite broad effects on risk, likely through both alterations at the protein sequence level, and in the context of expression. A great deal of functional characterization of LRRK2 and disease-causing mutations in this protein has occurred over the last 9 years, and considerable progress has been made. Particular attention has been paid to the kinase activity of LRRK2 as a therapeutic target, and while it is no means certain that this is viable target it is likely that this hypothesis will be tested in clinical trials sooner rather than later. We believe that the future goals for LRRK2 research are, while challenging, relatively clear and that the next 10 years of research promises to be perhaps more exciting than the last.
doi:10.3233/JPD-130192
PMCID: PMC3952583  PMID: 23938341
LRRK2; associated phenotype; disease risk; biology; future challenges; Parkinson's disease; parkinsonism; genetics
3.  The Parkinson’s disease genes Fbxo7 and Parkin interact to mediate mitophagy 
Nature neuroscience  2013;16(9):10.1038/nn.3489.
Compelling evidence indicates that two autosomal recessive Parkinson’s disease genes, PINK1 (PARK6) and Parkin (PARK2), co-operate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain containing protein Fbxo7 (PARK15) also cause early onset autosomal recessive Parkinson’s disease by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and plays a role in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression show deficiencies in Parkin mitochondrial translocation, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin supporting a functional relationship between the two proteins. Parkinson’s disease-causing mutations in Fbxo7 interfere with this process, emphasising the importance of mitochondrial dysfunction in Parkinson’s disease pathogenesis.
doi:10.1038/nn.3489
PMCID: PMC3827746  PMID: 23933751
Fbxo7; Parkin; PINK1; mitofusin 1; mitophagy; Drosophila; Parkinson’s disease
4.  No Association of Four Candidate Genetic Variants in MnSOD and SYNIII with Parkinson's Disease in Two Chinese Populations 
PLoS ONE  2014;9(2):e88050.
Background
The manganese superoxide dismutase (MnSOD) gene, which encodes a chief reactive oxygen species (ROS) scavenging enzyme, has been reported to be associated with the risk of developing sporadic Parkinson's disease (PD) in some Asian races and the synapsin III (SYN3) gene with some neuropsychiatric diseases. Objective: To explore the associations between the MnSOD and SYN III variations and PD in two Chinese populations from mainland China and Singapore.
Methods
We recruited 2342 subjects including 1200 sporadic PD patients and 1142 healthy controls from two independent Asian countries. Using a case-control methodology, we genotyped the single nucleotide polymorphisms (SNP) in MnSOD (rs4880) and SYN III (rs3788470, rs3827336, rs5998557) to explore the associations with risk of PD.
Results
The results showed the genotype distributions and minor allele frequencies (MAF) of MnSOD (rs4880) and SYN III (rs3788470, rs3827336, rs5998557) were not significantly different between PD patients and healthy controls in mainland China and Singapore, as well as in merged populations.
Conclusions
The variations of MnSOD (rs4880) and SYN III (rs3788470, rs3827336, rs5998557) were not major risk factors for PD among Chinese, at least in our study populations.
doi:10.1371/journal.pone.0088050
PMCID: PMC3935830  PMID: 24586301
5.  Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons 
PLoS ONE  2014;9(2):e87388.
Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson’s disease (PD), in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2), representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models.
doi:10.1371/journal.pone.0087388
PMCID: PMC3931621  PMID: 24586273
6.  Comparable Autoantibody Serum Levels against Amyloid- and Inflammation-Associated Proteins in Parkinson’s Disease Patients and Controls 
PLoS ONE  2014;9(2):e88604.
Naturally occurring autoantibodies (NAbs) against a number of potentially disease-associated cellular proteins, including Amyloid-beta1–42 (Abeta1–42), Alpha-synuclein (Asyn), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and S100 calcium binding protein B (S100B) have been suggested to be associated with neurodegenerative disorders, in particular Alzheimer’s (AD) and Parkinson’s disease (PD). Whereas the (reduced) occurrence of specific NAbs in AD is widely accepted, previous literature examining the relation of these NAb titres between PD patients and controls, as well as comparing these levels with demographic and clinical parameters in PD patients have produced inconsistent findings. We therefore aimed, in a cross-sectional approach, to determine serum titres of the above NAbs in a cohort of 93 PD patients (31 of them demented) and 194 controls. Levels were correlated with demographic and clinical variables, cerebrospinal fluid Abeta1–42, total tau and phospho-tau levels, as well as with single nucleotide polymorphisms (SNPs) of genes which either have been reported to influence the immune system, the amyloid cascade or the occurrence of PD (ApoE, GSK3B, HLA-DRA, HSPA5, SNCA, and STK39). The investigated NAb titres were neither significantly associated with the occurrence of PD, nor with demographic and clinical parameters, neurodegenerative markers or genetic variables. These results argue against a major potential of blood-borne parameters of the adaptive immune system to serve as trait or state markers in PD.
doi:10.1371/journal.pone.0088604
PMCID: PMC3931625  PMID: 24586351
7.  Mitochondrial DNA and karyotypic data confirm the presence of Mus indutus and Mus minutoides (Mammalia, Rodentia, Muridae, Nannomys) in Botswana 
ZooKeys  2013;35-51.
We use a combination of cytochrome b sequence data and karyological evidence to confirm the presence of Mus indutus and Mus minutoides in Botswana. Our data include sampling from five localities from across the country, including one site in northwestern Botswana where both species were captured in syntopy. Additionally, we find evidence for two mitochondrial lineages of M. minutoides in northwestern Botswana that differ by 5% in sequence variation. Also, we report that M. minutoides in Botswana have the 2n=34 karyotype with the presence of a (X.1) sex-autosome translocation.
doi:10.3897/zookeys.359.6247
PMCID: PMC3867174  PMID: 24363588
Africa; rodent; distribution; karyotype; sex-autosome translocation; cytochrome b
8.  Inhibition of LRRK2 kinase activity stimulates macroautophagy☆ 
Biochimica et Biophysica Acta  2013;1833(12):2900-2910.
Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.
Highlights
•Inhibiting the kinase activity of LRRK2 induces autophagy•This induction is independent of an impact on the translational targets of mTORC1•Inhibition of LRRK2 kinase activity results in a translation dependent increase in p62 levels
doi:10.1016/j.bbamcr.2013.07.020
PMCID: PMC3898616  PMID: 23916833
LRRK2, Leucine Rich Repeat Kinase 2; mTOR, Mammalian target of rapamycin; ROC, Ras of Complex Proteins; COR, C-terminal of ROC domain; SDS, Sodium dodecyl sulphate; EDTA, Ethylene di-ammonium tetra acetic acid; DPBS, Dulbecco's phosphate buffered saline; DMSO, Dimethylsulfoxide; LRRK2; Macroautophagy; Parkinson's disease; LC3; p62; WIPI2
9.  Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation☆ 
Highlights
•Mutations in the ROC, COR and Kinase domain of LRRK2 alter the autophagic response to starvation.•LC3-I/II ratio following starvation is altered by mutations, as well as p62 and WIPI2 positive puncta.•This occurs independently of any alteration in downstream targets of mTORC1.
LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.
doi:10.1016/j.bbrc.2013.10.159
PMCID: PMC3858825  PMID: 24211199
LRRK2, leucine rich repeat kinase 2; ROC, ras of complex proteins; COR, C-terminal of ROC; PD, Parkinson’s disease; ICC, Immunocytochemistry; LRRK2; Parkinson’s disease; Autophagy; Lysosomes; Signaling pathways
10.  The Diagnostic Value of CSF α-Synuclein in the Differential Diagnosis of Dementia with Lewy Bodies vs. Normal Subjects and Patients with Alzheimer’s Disease 
PLoS ONE  2013;8(11):e81654.
The detection of α-synuclein (α-syn) in the cerebrospinal fluid (CSF) of patients with synucleinopathy has yielded promising but inconclusive results. The aim of the present study was to determine the diagnostic value of α-syn as a biological marker for Dementia with Lewy bodies (DLB) vs. normal subjects and patients with Alzheimer’s disease (AD), after strict control of several recognized confounders. Sixteen patients with DLB, 18 patients with AD and 22 age- and sex-matched normal controls (CTRL) were recruited. The levels of total α-syn in CSF were measured using a novel enzyme-linked immunosorbent assay. There was a significant increase of CSF α-syn levels in DLB patients as compared to the CTRL and AD groups (P= 0.049 and 0.01 respectively). ROC analysis revealed that increased α-syn was 81.8% specific for the discrimination of DLB vs. CTRL and 90% vs. AD. However, sensitivity was lower (56.2 % and 50% respectively). These findings provide evidence for a possible diagnostic role of α-syn as a surrogate biomarker for DLB.
doi:10.1371/journal.pone.0081654
PMCID: PMC3840054  PMID: 24282614
11.  Rare variants in LRRK1 and Parkinson's disease 
Neurogenetics  2013;15:49-57.
Approximately 20 % of individuals with Parkinson's disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset PD to identify 15 potentially causal variants. Segregation analysis and frequency assessment in 862 PD cases and 1,014 ethnically matched controls highlighted variants in EEF1D and LRRK1 as the best candidates. Mutation screening of the coding regions of these genes in 862 cases and 1,014 controls revealed several novel non-synonymous variants in both genes in cases and controls. An in silico multi-model bioinformatics analysis was used to prioritize identified variants in LRRK1 for functional follow-up. However, protein expression, subcellular localization, and cell viability were not affected by the identified variants. Although it has yet to be proven conclusively that variants in LRRK1 are indeed causative of PD, our data strengthen a possible role for LRRK1 in addition to LRRK2 in the genetic underpinnings of PD but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.
Electronic supplementary material
The online version of this article (doi:10.1007/s10048-013-0383-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s10048-013-0383-8
PMCID: PMC3968516  PMID: 24241507
Parkinson's disease; LRRK1; EEF1D; Exome sequencing
12.  LRRK2 Affects Vesicle Trafficking, Neurotransmitter Extracellular Level and Membrane Receptor Localization 
PLoS ONE  2013;8(10):e77198.
The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.
doi:10.1371/journal.pone.0077198
PMCID: PMC3805556  PMID: 24167564
13.  Divergent α-synuclein solubility and aggregation properties in G2019S LRRK2 Parkinson's disease brains with Lewy Body pathology compared to idiopathic cases☆ 
Neurobiology of Disease  2013;58(100):183-190.
Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). The most prevalent LRRK2 mutation is the G2019S coding change, located in the kinase domain of this complex multi-domain protein. The majority of G2019S autopsy cases feature typical Lewy Body pathology with a clinical phenotype almost indistinguishable from idiopathic PD (iPD). Here we have investigated the biochemical characteristics of α-synuclein in G2019S LRRK2 PD post-mortem material, in comparison to pathology-matched iPD. Immunohistochemistry with pS129 α-synuclein antibody showed that the medulla is heavily affected with pathology in G2019S PD whilst the basal ganglia (BG), limbic and frontal cortical regions demonstrated comparable pathology scores between G2019S PD and iPD. Significantly lower levels of the highly aggregated α-synuclein species in urea–SDS fractions were observed in G2019S cases compared to iPD in the BG and limbic cortex. Our data, albeit from a small number of cases, highlight a difference in the biochemical properties of aggregated α-synuclein in G2019S linked PD compared to iPD, despite a similar histopathological presentation. This divergence in solubility is most notable in the basal ganglia, a region that is affected preclinically and is damaged before overt dopaminergic cell death.
Highlights
•We compared α-synuclein biochemistry from LRRK2 G2019S and idiopathic PD brains.•We used four G2019S PD post-mortem brains and pathology matched idiopathic PD cases.•G2019S PD and idiopathic PD cases show comparable Limbic stage Lewy Body pathology.•Minimal SDS-insoluble α-synuclein seen in G2019S PD in contrast to idiopathic PD•We propose a divergent nature of α-synuclein pathogenic species in G2019S PD.
doi:10.1016/j.nbd.2013.05.017
PMCID: PMC3752970  PMID: 23747310
LRRK2; G2019S; α-Synuclein; Differential solubility; Immunohistochemistry
14.  Fine-Mapping, Gene Expression and Splicing Analysis of the Disease Associated LRRK2 Locus 
PLoS ONE  2013;8(8):e70724.
Association studies have identified several signals at the LRRK2 locus for Parkinson's disease (PD), Crohn's disease (CD) and leprosy. However, little is known about the molecular mechanisms mediating these effects. To further characterize this locus, we fine-mapped the risk association in 5,802 PD and 5,556 controls using a dense genotyping array (ImmunoChip). Using samples from 134 post-mortem control adult human brains (UK Human Brain Expression Consortium), where up to ten brain regions were available per individual, we studied the regional variation, splicing and regulation of LRRK2. We found convincing evidence for a common variant PD association located outside of the LRRK2 protein coding region (rs117762348, A>G, P = 2.56×10−8, case/control MAF 0.083/0.074, odds ratio 0.86 for the minor allele with 95% confidence interval [0.80–0.91]). We show that mRNA expression levels are highest in cortical regions and lowest in cerebellum. We find an exon quantitative trait locus (QTL) in brain samples that localizes to exons 32–33 and investigate the molecular basis of this eQTL using RNA-Seq data in n = 8 brain samples. The genotype underlying this eQTL is in strong linkage disequilibrium with the CD associated non-synonymous SNP rs3761863 (M2397T). We found two additional QTLs in liver and monocyte samples but none of these explained the common variant PD association at rs117762348. Our results characterize the LRRK2 locus, and highlight the importance and difficulties of fine-mapping and integration of multiple datasets to delineate pathogenic variants and thus develop an understanding of disease mechanisms.
doi:10.1371/journal.pone.0070724
PMCID: PMC3742662  PMID: 23967090
15.  Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src 
PLoS ONE  2013;8(7):e71035.
c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.
doi:10.1371/journal.pone.0071035
PMCID: PMC3724807  PMID: 23923048
16.  α-Synuclein mutations cluster around a putative protein loop☆ 
Neuroscience Letters  2013;546(100):67-70.
Highlights
•We map all five missense SNCA mutations on the proposed α-synuclein protein models.•4 mutations cluster around the protein loop linking the two legs of the hairpin.•4 mutations cluster around the point of hairpin convergence for tetramer formation.
With the recent identification of two new pathogenic mutations in α-synuclein, we map the five known pathogenic mutations onto the best available models of the protein structure. We show that four of the five mutations map to a potential fold in the protein with the exception being the A30P mutation in which the substitution would be expected to have a profound effect on protein structure. We discuss this localisation in terms of the proposed mechanisms for mutation pathogenicity.
doi:10.1016/j.neulet.2013.04.058
PMCID: PMC3694303  PMID: 23669636
α-Synuclein; SNCA; Genetics; Parkinson's disease
17.  Loss of β-Glucocerebrosidase Activity Does Not Affect Alpha-Synuclein Levels or Lysosomal Function in Neuronal Cells 
PLoS ONE  2013;8(4):e60674.
To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson’s disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme β-glucocerebrosidase (GCase), caused by mutations in the β-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD.
doi:10.1371/journal.pone.0060674
PMCID: PMC3620326  PMID: 23580063
18.  Genetic Loci Associated with Alzheimer’s Disease and Cerebrospinal Fluid Biomarkers in a Finnish Case-Control Cohort 
PLoS ONE  2013;8(4):e59676.
Objectives
To understand the relation between risk genes for Alzheimer’s disease (AD) and their influence on biomarkers for AD, we examined the association of AD in the Finnish cohort with single nucleotide polymorphisms (SNPs) from top AlzGene loci, genome-wide association studies (GWAS), and candidate gene studies; and tested the correlation between these SNPs and AD markers Aβ1–42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF).
Methods
We tested 25 SNPs for genetic association with clinical AD in our cohort comprised of 890 AD patients and 701-age matched healthy controls using logistic regression. For the correlational study with biomarkers, we tested 36 SNPs in a subset of 222 AD patients with available CSF using mixed models. Statistical analyses were adjusted for age, gender and APOE status. False discovery rate for multiple testing was applied. All participants were from academic hospital and research institutions in Finland.
Results
APOE-ε4, CLU rs11136000, and MS4A4A rs2304933 correlated with significantly decreased Aβ1–42 (corrected p<0.05). At an uncorrected p<0.05, PPP3R1 rs1868402 and MAPT rs2435211 were related with increased t-tau; while SORL1 rs73595277 and MAPT rs16940758, with increased p-tau. Only TOMM40 rs2075650 showed association with clinical AD after adjusting for APOE-ε4 (p = 0.007), but not after multiple test correction (p>0.05).
Conclusions
We provide evidence that APOE-ε4, CLU and MS4A4A, which have been identified in GWAS to be associated with AD, also significantly reduced CSF Aβ1–42 in AD. None of the other AlzGene and GWAS loci showed significant effects on CSF tau. The effects of other SNPs on CSF biomarkers and clinical AD diagnosis did not reach statistical significance. Our findings suggest that APOE-ε4, CLU and MS4A4A influence both AD risk and CSF Aβ1–42.
doi:10.1371/journal.pone.0059676
PMCID: PMC3616106  PMID: 23573206
19.  α-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson’s disease and multiple system atrophy? 
Acta Neuropathologica  2013;125(5):753-769.
We report a British family with young-onset Parkinson’s disease (PD) and a G51D SNCA mutation that segregates with the disease. Family history was consistent with autosomal dominant inheritance as both the father and sister of the proband developed levodopa-responsive parkinsonism with onset in their late thirties. Clinical features show similarity to those seen in families with SNCA triplication and to cases of A53T SNCA mutation. Post-mortem brain examination of the proband revealed atrophy affecting frontal and temporal lobes in addition to the caudate, putamen, globus pallidus and amygdala. There was severe loss of pigmentation in the substantia nigra and pallor of the locus coeruleus. Neuronal loss was most marked in frontal and temporal cortices, hippocampal CA2/3 subregions, substantia nigra, locus coeruleus and dorsal motor nucleus of the vagus. The cellular pathology included widespread and frequent neuronal α-synuclein immunoreactive inclusions of variable morphology and oligodendroglial inclusions similar to the glial cytoplasmic inclusions of multiple system atrophy (MSA). Both inclusion types were ubiquitin and p62 positive and were labelled with phosphorylation-dependent anti-α-synuclein antibodies In addition, TDP-43 immunoreactive inclusions were observed in limbic regions and in the striatum. Together the data show clinical and neuropathological similarities to both the A53T SNCA mutation and multiplication cases. The cellular neuropathological features of this case share some characteristics of both PD and MSA with additional unique striatal and neocortical pathology. Greater understanding of the disease mechanism underlying the G51D mutation could aid in understanding of α-synuclein biology and its impact on disease phenotype.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1096-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-013-1096-7
PMCID: PMC3681325  PMID: 23404372
Parkinson’s disease; Multiple system atrophy; α-Synuclein; SNCA
20.  A Geographic Information Systems (GIS)-based analysis of modern South African rodent distributions, habitat use, and environmental tolerances 
Ecology and Evolution  2012;2(11):2881-2894.
Goals of this study were to: (1) develop distributional maps of modern rodent genera throughout the countries of South Africa, Lesotho, and Swaziland by georeferencing museum specimens; (2) assess habitat preferences for genera by cross-referencing locality position with South African vegetation; and (3) identify mean annual precipitation and temperature range where the genera are located. Conterminous South Africa including the countries of Lesotho and Swaziland Digital databases of rodent museum specimens housed in the Ditsong National Museum of Natural History, South Africa (DM), and the Division of Mammals, National Museum of Natural History, Smithsonian Institution, United States (NMNH), were acquired and then sorted into a subset of specimens with associated coordinate data. The coordinate data were then used to develop distributional maps for the rodent genera present within the study area. Percent habitat occupation and descriptive statistics for six climatic variables were then determined for each genus by cross-referencing locality positions with vegetation and climatic maps. This report presents a series of maps illustrating the distribution of 35 rodent genera based on 19,471 geo-referenced specimens obtained from two major collections. Inferred habitat use by taxon is provided for both locality and specimen percent occurrence at three hierarchical habitat levels: biome, bioregion, and vegetation unit. Descriptive statistics for six climatic variables are also provided for each genus based on locality and specimen percent incidence. As rodent faunas are commonly used in paleoenvironmental reconstructions, an accurate assessment of rodent environmental tolerance ranges is necessary before confidence can be placed in an actualistic model. While the data presented here represent only a subset of the modern geographic distributions for many of the taxa examined, a wide range of environmental regimes are observed, suggesting that more research is necessary in order to accurately reconstruct an environmental signature when these taxa are found in the fossil record.
doi:10.1002/ece3.384
PMCID: PMC3501638  PMID: 23170221
Distribution maps; environmental tolerances; GIS; rodents; South Africa; vegetation
21.  Radiating Amyloid Fibril Formation on the Surface of Lipid Membranes through Unit-Assembly of Oligomeric Species of α-Synuclein 
PLoS ONE  2012;7(10):e47580.
Background
Lewy body in the substantia nigra is a cardinal pathological feature of Parkinson's disease. Despite enormous efforts, the cause-and-effect relationship between Lewy body formation and the disorder is yet to be explicitly unveiled.
Methodology/Principal Findings
Here, we showed that radiating amyloid fibrils (RAFs) were instantly developed on the surface of synthetic lipid membranes from the β-sheet free oligomeric species of α-synuclein through a unit-assembly process. The burgeoning RAFs were successfully matured by feeding them with additional oligomers, which led to concomitant dramatic shrinkage and disintegration of the membranes by pulling off lipid molecules to the extending fibrils. Mitochondria and lysosomes were demonstrated to be disrupted by the oligomeric α-synuclein via membrane-dependent fibril formation.
Conclusion
The physical structure formation of amyloid fibrils, therefore, could be considered as detrimental to the cells by affecting membrane integrity of the intracellular organelles, which might be a molecular cause for the neuronal degeneration observed in Parkinson's disease.
doi:10.1371/journal.pone.0047580
PMCID: PMC3471876  PMID: 23077644
22.  Evaluation of Parkinson Disease Risk Variants as Expression-QTLs 
PLoS ONE  2012;7(10):e46199.
The recent Parkinson Disease GWAS Consortium meta-analysis and replication study reports association at several previously confirmed risk loci SNCA, MAPT, GAK/DGKQ, and HLA and identified a novel risk locus at RIT2. To further explore functional consequences of these associations, we investigated modification of gene expression in prefrontal cortex brain samples of pathologically confirmed PD cases (N = 26) and controls (N = 24) by 67 associated SNPs in these 5 loci. Association between the eSNPs and expression was evaluated using a 2-degrees of freedom test of both association and difference in association between cases and controls, adjusted for relevant covariates. SNPs at each of the 5 loci were tested for cis-acting effects on all probes within 250 kb of each locus. Trans-effects of the SNPs on the 39,122 probes passing all QC on the microarray were also examined. From the analysis of cis-acting SNP effects, several SNPs in the MAPT region show significant association to multiple nearby probes, including two strongly correlated probes targeting the gene LOC644246 and the duplicated genes LRRC37A and LRRC37A2, and a third uncorrelated probe targeting the gene DCAKD. Significant cis-associations were also observed between SNPs and two probes targeting genes in the HLA region on chromosome 6. Expanding the association study to examine trans effects revealed an additional 23 SNP-probe associations reaching statistical significance (p<2.8×10−8) including SNPs from the SNCA, MAPT and RIT2 regions. These findings provide additional context for the interpretation of PD associated SNPs identified in recent GWAS as well as potential insight into the mechanisms underlying the observed SNP associations.
doi:10.1371/journal.pone.0046199
PMCID: PMC3465315  PMID: 23071545
23.  Macaque Homologs of EBV and KSHV Show Uniquely Different Associations with Simian AIDS-related Lymphomas 
PLoS Pathogens  2012;8(10):e1002962.
Two gammaherpesviruses, Epstein-Barr virus (EBV) (Lymphocryptovirus genus) and Kaposi's sarcoma-associated herpesvirus (KSHV) (Rhadinovirus genus) have been implicated in the etiology of AIDS-associated lymphomas. Homologs of these viruses have been identified in macaques and other non-human primates. In order to assess the association of these viruses with non-human primate disease, archived lymphoma samples were screened for the presence of macaque lymphocryptovirus (LCV) homologs of EBV, and macaque rhadinoviruses belonging to the RV1 lineage of KSHV homologs or the more distant RV2 lineage of Old World primate rhadinoviruses. Viral loads were determined by QPCR and infected cells were identified by immunolabeling for different viral proteins. The lymphomas segregated into three groups. The first group (n = 6) was associated with SIV/SHIV infections, contained high levels of LCV (1–25 genomes/cell) and expressed the B-cell antigens CD20 or BLA.36. A strong EBNA-2 signal was detected in the nuclei of the neoplastic cells in one of the LCV-high lymphomas, indicative of a type III latency stage. None of the lymphomas in this group stained for the LCV viral capsid antigen (VCA) lytic marker. The second group (n = 5) was associated with D-type simian retrovirus-2 (SRV-2) infections, contained high levels of RV2 rhadinovirus (9–790 genomes/cell) and expressed the CD3 T-cell marker. The third group (n = 3) was associated with SIV/SHIV infections, contained high levels of RV2 rhadinovirus (2–260 genomes/cell) and was negative for both CD20 and CD3. In both the CD3-positive and CD3/CD20-negative lymphomas, the neoplastic cells stained strongly for markers of RV2 lytic replication. None of the lymphomas had detectable levels of retroperitoneal fibromatosis herpesvirus (RFHV), the macaque RV1 homolog of KSHV. Our data suggest etiological roles for both lymphocryptoviruses and RV2 rhadinoviruses in the development of simian AIDS-associated lymphomas and indicate that the virus-infected neoplastic lymphoid cells are derived from different lymphocyte lineages and differentiation stages.
Author Summary
The incidence of Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma increased in conjunction with the epidemic of HIV disease and AIDS. These malignancies are now known to be associated with secondary infections with a gammaherpesvirus; KS, with the Kaposi's sarcoma-associated herpesvirus (KSHV) and lymphoma, with both KSHV and Epstein-Barr virus (EBV). Similar AIDS-related malignancies have been observed in monkeys with simian AIDS and monkey gammaherpesviruses related to KSHV and EBV have been implicated in the development of disease. The study of monkey models of AIDS-related malignancies provides important approaches for understanding the role of gammaherpesviruses in AIDS-related tumorigenesis. Here we have used a combined molecular and immunological approach to identify, quantitate and localize infections of gammaherpesviruses in AIDS-associated lymphomas in macaques. We found high levels of macaque viruses related to EBV and KSHV in the tumor cells of distinct types of macaque lymphomas, suggesting that the virus-infected tumor cells belong to different lymphocyte lineages and differentiation stages.
doi:10.1371/journal.ppat.1002962
PMCID: PMC3464224  PMID: 23055934
24.  GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1 
Scientific Reports  2012;2:695.
The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.
doi:10.1038/srep00695
PMCID: PMC3458246  PMID: 23019516
25.  Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research 
PLoS ONE  2012;7(8):e43099.
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.
doi:10.1371/journal.pone.0043099
PMCID: PMC3428297  PMID: 22952635

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