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1.  Cross-region reduction in 5-hydroxymethylcytosine in Alzheimer’s disease brain 
Neurobiology of aging  2014;35(8):1850-1854.
Epigenetic processes play a key role in the central nervous system and altered levels of 5-methylcytosine have been associated with a number of neurological phenotypes, including Alzheimer’s disease (AD). Recently three additional cytosine modifications have been identified (5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine), which are thought to be intermediate steps in the demethylation of 5-methylcytosine to unmodified cytosine. Little is known about the frequency of these modifications in the human brain during health or disease. In this study we used immunofluorescence to confirm the presence of each modification in human brain and investigate their cross-tissue abundance in Alzheimer’s disease patients and elderly control samples. We identify a significant AD-associated decrease in global 5-hydroxymethylcytosine in entorhinal cortex and cerebellum, and differences in 5-formylcytosine levels between brain regions. Our study further implicates a role for epigenetic alterations in AD.
doi:10.1016/j.neurobiolaging.2014.02.002
PMCID: PMC4066184  PMID: 24679604
Epigenetics; DNA methylation; Brain; 5-methylcytosine; 5-mC; 5-hydroxymethylcytosine; 5-hmC; 5-formylcytosine; 5-fC; 5-carboxylcytosine; 5-caC; Alzheimer’s disease
2.  Dipeptide repeat protein inclusions are rare in the spinal cord and almost absent from motor neurons in C9ORF72 mutant amyotrophic lateral sclerosis and are unlikely to cause their degeneration 
Introduction
Cytoplasmic TDP-43 inclusions are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar dementia (FTLD). The G4C2 repeat mutation in C9ORF72 is the most common cause of ALS and FTLD in which, in addition to TDP-43 inclusions, five different di-peptide repeat (DPR) proteins have been identified. Di-peptide repeat proteins are translated in a non-canonical fashion from sense and antisense transcripts of the G4C2 repeat (GP, GA, GR, PA, PR). DPR inclusions are abundant in the cerebellum, as well as in the frontal and temporal lobes of ALS and FTLD patients and some are neurotoxic in a range of cellular and animal models, implying that DPR aggregation directly contributes to disease pathogenesis. Here we sought to quantify inclusions for each DPR and TDP-43 in ALS cases with and without the C9ORF72 mutation. We characterised the abundance of DPRs and their cellular location and compared this to cytoplasmic TDP-43 inclusions in order to explore the role of each inclusion in lower motor neuron degeneration.
Results
Spinal cord sections from ten cases positive for the C9ORF72 repeat expansion (ALS-C9+ve) and five cases that were not were probed by double immunofluorescence staining for individual DPRs and TDP-43. Inclusions immunoreactive for each of the DPRs were present in the spinal cord but they were rare or very rare in abundance (in descending order of frequency: GA, GP, GR, PA and PR). TDP-43 cytoplasmic inclusions were 45- to 750-fold more frequent than any DPR, and fewer than 4 % of DPR inclusions colocalized with TDP-43 inclusions. In motor neurons, a single cytoplasmic DPR inclusion was detected (0.1 %) in contrast to the 34 % of motor neurons that contained cytoplasmic TDP-43 inclusions. Furthermore, the number of TDP-43 inclusions in ALS cases with and without the C9ORF72 mutation was nearly identical.
Conclusions
For all other neurodegenerative diseases, the neurotoxic protein aggregates are detected in the affected population of neurons. TDP-43 cytoplasmic aggregation is the dominant feature of ALS spinal cord pathology irrespective of C9ORF72 mutation status. The near absence of DPR inclusions in spinal cord motor neurons challenges their contribution to lower motor neuron degeneration in ALS-C9+ve cases.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-015-0218-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s40478-015-0218-y
PMCID: PMC4479315  PMID: 26108573
C9ORF72; Dipeptide repeat; TDP-43; Motor neuron; Amyotrophic lateral sclerosis
3.  LRRK2 exonic variants and risk of multiple system atrophy 
Neurology  2014;83(24):2256-2261.
Objective:
The aim of this study was to evaluate the association between common exonic variants in the leucine-rich repeat kinase 2 (LRRK2) gene and risk of multiple system atrophy (MSA).
Methods:
One series from the United States (92 patients with pathologically confirmed MSA, 416 controls) and a second series from the United Kingdom (85 patients with pathologically confirmed MSA, 352 controls) were included in this case-control study. We supplemented these data with those of 53 patients from the United States with clinically probable or possible MSA. Seventeen common LRRK2 exonic variants were genotyped and assessed for association with MSA.
Results:
In the combined series of 177 patients with pathologically confirmed MSA and 768 controls, there was a significant association between LRRK2 p.M2397T and MSA (odds ratio [OR] = 0.60, p = 0.002). This protective effect was observed more strongly in the US series (OR = 0.46, p = 0.0008) than the UK series (OR = 0.82, p = 0.41). We observed other noteworthy associations with MSA for p.G1624G (OR = 0.63, p = 0.006) and p.N2081D (OR = 0.15, p = 0.010). The p.G1624G-M2397T haplotype was significantly associated with MSA in the US series (p < 0.0001) and combined series (p = 0.003) but not the UK series (p = 0.67). Results were consistent when additionally including the US patients with clinical MSA, where the strongest single-variant association was again observed for p.M2397T (OR = 0.59, p = 0.0005).
Conclusions:
These findings provide evidence that LRRK2 exonic variants may contribute to susceptibility to MSA. Validation in other series and meta-analytic studies will be important.
doi:10.1212/WNL.0000000000001078
PMCID: PMC4277668  PMID: 25378673
4.  Cross-tissue methylomic profiling strongly implicates a role for cortex-specific deregulation of ANK1 in Alzheimer’s disease neuropathology 
Nature neuroscience  2014;17(9):1164-1170.
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by progressive neuropathology and cognitive decline. We describe a cross-tissue analysis of methylomic variation in AD using samples from three independent human post-mortem brain cohorts. We identify a differentially methylated region in the ankyrin 1 (ANK1) gene that is associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as significantly hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex) but not in the cerebellum, a region largely protected from neurodegeneration in AD, nor whole blood obtained pre-mortem, from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents the first epigenome-wide association study (EWAS) of AD employing a sequential replication design across multiple tissues, and highlights the power of this approach for identifying methylomic variation associated with complex disease.
doi:10.1038/nn.3782
PMCID: PMC4410018  PMID: 25129077
5.  Epigenomic and transcriptomic signatures of a Klinefelter syndrome (47,XXY) karyotype in the brain 
Epigenetics  2014;9(4):587-599.
Klinefelter syndrome (KS) is the most common sex-chromosome aneuploidy in humans. Most affected individuals carry one extra X-chromosome (47,XXY karyotype) and the condition presents with a heterogeneous mix of reproductive, physical and psychiatric phenotypes. Although the mechanism(s) by which the supernumerary X-chromosome determines these features of KS are poorly understood, skewed X-chromosome inactivation (XCI), gene-dosage dysregulation, and the parental origin of the extra X-chromosome have all been implicated, suggesting an important role for epigenetic processes. We assessed genomic, methylomic and transcriptomic variation in matched prefrontal cortex and cerebellum samples identifying an individual with a 47,XXY karyotype who was comorbid for schizophrenia and had a notably reduced cerebellum mass compared with other individuals in the study (n = 49). We examined methylomic and transcriptomic differences in this individual relative to female and male samples with 46,XX or 46,XY karyotypes, respectively, and identified numerous locus-specific differences in DNA methylation and gene expression, with many differences being autosomal and tissue-specific. Furthermore, global DNA methylation, assessed via the interrogation of LINE-1 and Alu repetitive elements, was significantly altered in the 47,XXY patient in a tissue-specific manner with extreme hypomethylation detected in the prefrontal cortex and extreme hypermethylation in the cerebellum. This study provides the first detailed molecular characterization of the prefrontal cortex and cerebellum from an individual with a 47,XXY karyotype, identifying widespread tissue-specific epigenomic and transcriptomic alterations in the brain.
doi:10.4161/epi.27806
PMCID: PMC4121369  PMID: 24476718
Klinefelter syndrome; DNA methylation; gene expression; 47,XXY; prefrontal cortex; cerebellum
6.  Novel mutations support a role for Profilin 1 in the pathogenesis of ALS 
Neurobiology of Aging  2015;36(3):1602.e17-1602.e27.
Mutations in the gene encoding profilin 1 (PFN1) have recently been shown to cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. We sequenced the PFN1 gene in a cohort of ALS patients (n = 485) and detected 2 novel variants (A20T and Q139L), as well as 4 cases with the previously identified E117G rare variant (∼ 1.2%). A case-control meta-analysis of all published E117G ALS+/− frontotemporal dementia cases including those identified in this report was significant p = 0.001, odds ratio = 3.26 (95% confidence interval, 1.6–6.7), demonstrating this variant to be a susceptibility allele. Postmortem tissue from available patients displayed classic TAR DNA-binding protein 43 pathology. In both transient transfections and in fibroblasts from a patient with the A20T change, we showed that this novel PFN1 mutation causes protein aggregation and the formation of insoluble high molecular weight species which is a hallmark of ALS pathology. Our findings show that PFN1 is a rare cause of ALS and adds further weight to the underlying genetic heterogeneity of this disease.
doi:10.1016/j.neurobiolaging.2014.10.032
PMCID: PMC4357530  PMID: 25499087
Amyotrophic lateral sclerosis; Profilin 1; TDP-43 proteinopathy
7.  Evidence that the presynaptic vesicle protein CSPalpha is a key player in synaptic degeneration and protection in Alzheimer’s disease 
Molecular Brain  2015;8:6.
Background
In Alzheimer’s disease synapse loss precedes neuronal loss and correlates best with impaired memory formation. However, the mechanisms underlying synaptic degeneration in Alzheimer’s disease are not well known. Further, it is unclear why synapses in AD cerebellum are protected from degeneration. Our recent work on the cyclin-dependent kinase 5 activator p25 suggested that expression of the multifunctional presynaptic molecule cysteine string protein alpha (CSPalpha) may be affected in Alzheimer’s disease.
Results
Using western blots and immunohistochemistry, we found that CSPalpha expression is reduced in hippocampus and superior temporal gyrus in Alzheimer’s disease. Reduced CSPalpha expression occurred before synaptophysin levels drop, suggesting that it contributes to the initial stages of synaptic degeneration. Surprisingly, we also found that CSPalpha expression is upregulated in cerebellum in Alzheimer’s disease. This CSPalpha upregulation reached the same level as in young, healthy cerebellum. We tested the idea whether CSPalpha upregulation might be neuroprotective, using htau mice, a model of tauopathy that expresses the entire wild-type human tau gene in the absence of mouse tau. In htau mice CSPalpha expression was found to be elevated at times when neuronal loss did not occur.
Conclusion
Our findings provide evidence that the presynaptic vesicle protein CSPalpha is a key player in synaptic degeneration and protection in Alzheimer’s disease. In the forebrain CSPalpha expression is reduced early in the disease and this may contribute to the initial stages of synaptic degeneration. In the cerebellum CSPalpha expression is upregulated to young, healthy levels and this may protect cerebellar synapses and neurons to survive. Accordingly, CSPalpha upregulation also occurs in a mouse model of tauopathy only at time when neuronal loss does not take place.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-015-0096-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-015-0096-z
PMCID: PMC4314762  PMID: 25631211
Alzheimer’s disease; Cerebellum; Cysteine string protein; Hippocampus; Synapses; Neuroprotection
8.  Gamma-synuclein pathology in amyotrophic lateral sclerosis 
Objective
The prominent histopathological feature of the amyotrophic lateral sclerosis (ALS) is the presence of intracellular inclusions in degenerating neurons and their axons. The appearance and localization of these pathological structures depend on an aggregated protein that forms their scaffold. We investigated if γ-synuclein, an aggregation-prone protein highly expressed in healthy motor neurons, and predominantly localized in their axons and synaptic terminals is involved in ALS pathology.
Methods
Immunostaining of histological sections and sequential protein extraction from postmortem neural samples followed by immunoblotting.
Results
Immunohistochemical screening revealed a subset of sporadic (9 of 31) and familial (8 of 23) ALS cases with a novel type of pathology characterized by the accumulation of γ-synuclein in distinct profiles within the dorsolateral column. Sequential fractionation of proteins from the spinal cord tissues revealed detergent-insoluble γ-synuclein species specifically in the dorsolateral corticospinal tracts of a ALS patient with γ-synuclein-positive profiles in this region. These profiles are negative for protein markers commonly found in pathological inclusions in the spinal cord of ALS patients and most probably represent degenerated axons of upper motor neurons that have lost their neurofilaments. A subset of these profiles was found in association with phagocytic cells positive for Mac-2/Galectin-3. A smaller subset of studied ALS cases (4 of 54) contained large cytoplasmic inclusions in the cell body of remaining spinal motor neurons.
Interpretation
Our observations suggest that pathological aggregation of γ-synuclein might contribute to the pathogenesis of ALS.
doi:10.1002/acn3.143
PMCID: PMC4301672  PMID: 25642432
9.  Genetic analysis implicates APOE, SNCA and suggests lysosomal dysfunction in the etiology of dementia with Lewy bodies 
Human molecular genetics  2014;23(23):6139-6146.
Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson’s and Alzheimer’s diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.
doi:10.1093/hmg/ddu334
PMCID: PMC4222357  PMID: 24973356
10.  Investigating the role of rare coding variability in Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP) in late-onset Alzheimer's disease 
Neurobiology of Aging  2014;35(12):2881.e1-2881.e6.
The overlapping clinical and neuropathologic features between late-onset apparently sporadic Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and other neurodegenerative dementias (frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, and Creutzfeldt-Jakob disease) raise the question of whether shared genetic risk factors may explain the similar phenotype among these disparate disorders. To investigate this intriguing hypothesis, we analyzed rare coding variability in 6 Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP), in 141 LOAD patients and 179 elderly controls, neuropathologically proven, from the UK. In our cohort, 14 LOAD cases (10%) and 11 controls (6%) carry at least 1 rare variant in the genes studied. We report a novel variant in PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), absent in controls and both likely pathogenic. Our findings support previous studies, suggesting that (1) rare coding variability in PSEN1 and PSEN2 may influence the susceptibility for LOAD and (2) GRN, MAPT, and PRNP are not major contributors to LOAD. Thus, genetic screening is pivotal for the clinical differential diagnosis of these neurodegenerative dementias.
Highlights
•We have used exome sequencing to investigate rare coding variability in Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP) in a cohort composed of 141 late-onset sporadic Alzheimer's disease cases and 179 elderly controls, autopsy proven from the UK.•We report a novel mutation in PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), both likely pathogenic.•We conclude that PSEN1 and PSEN2 harbor susceptibility factors for sporadic Alzheimer's disease. By contrast, GRN, MAPT, and PRNP do not play a major role for the development of late-onset sporadic Alzheimer's disease.•Genetic screening is therefore pivotal for a clinical differential diagnosis of sporadic late-onset Alzheimer's disease and other neurodegenerative dementias (frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, and Creutzfeldt-Jakob disease).
doi:10.1016/j.neurobiolaging.2014.06.002
PMCID: PMC4236585  PMID: 25104557
Alzheimer's disease; Neurodegenerative dementia; APP; PSEN1; PSEN2; MAPT; GRN; PRNP; Exome sequencing
11.  Methylomic profiling of human brain tissue supports a neurodevelopmental origin for schizophrenia 
Genome Biology  2014;15(10):483.
Background
Schizophrenia is a severe neuropsychiatric disorder that is hypothesized to result from disturbances in early brain development. There is mounting evidence to support a role for developmentally regulated epigenetic variation in the molecular etiology of the disorder. Here, we describe a systematic study of schizophrenia-associated methylomic variation in the adult brain and its relationship to changes in DNA methylation across human fetal brain development.
Results
We profile methylomic variation in matched prefrontal cortex and cerebellum brain tissue from schizophrenia patients and controls, identifying disease-associated differential DNA methylation at multiple loci, particularly in the prefrontal cortex, and confirming these differences in an independent set of adult brain samples. Our data reveal discrete modules of co-methylated loci associated with schizophrenia that are enriched for genes involved in neurodevelopmental processes and include loci implicated by genetic studies of the disorder. Methylomic data from human fetal cortex samples, spanning 23 to 184 days post-conception, indicates that schizophrenia-associated differentially methylated positions are significantly enriched for loci at which DNA methylation is dynamically altered during human fetal brain development.
Conclusions
Our data support the hypothesis that schizophrenia has an important early neurodevelopmental component, and suggest that epigenetic mechanisms may mediate these effects.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0483-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0483-2
PMCID: PMC4262979  PMID: 25347937
12.  Exome sequencing identifies 2 novel presenilin 1 mutations (p.L166V and p.S230R) in British early-onset Alzheimer's disease☆ 
Neurobiology of Aging  2014;35(10):2422.e13-2422.e16.
Early-onset Alzheimer's disease (EOAD) represents 1%–2% of the Alzheimer's disease (AD) cases, and it is generally characterized by a positive family history and a rapidly progressive symptomatology. Rare coding and fully penetrant variants in amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are the only causative mutations reported for autosomal dominant AD. Thus, in this study we used exome sequencing data to rapidly screen rare coding variability in APP, PSEN1, and PSEN2, in a British cohort composed of 47 unrelated EOAD cases and 179 elderly controls, neuropathologically proven. We report 2 novel and likely pathogenic variants in PSEN1 (p.L166V and p.S230R). A comprehensive catalog of rare pathogenic variants in the AD Mendelian genes is pivotal for a premortem diagnosis of autosomal dominant EOAD and for the differential diagnosis with other early onset dementias such as frontotemporal dementia (FTD) and Creutzfeldt-Jakob disease (CJD).
doi:10.1016/j.neurobiolaging.2014.04.026
PMCID: PMC4099516  PMID: 24880964
Early-onset Alzheimer's disease; APP; PSEN1; PSEN2; British cohort
13.  Genetic analysis implicates APOE, SNCA and suggests lysosomal dysfunction in the etiology of dementia with Lewy bodies 
Human Molecular Genetics  2014;23(23):6139-6146.
Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson's and Alzheimer's diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.
doi:10.1093/hmg/ddu334
PMCID: PMC4222357  PMID: 24973356
14.  Brain distribution of dipeptide repeat proteins in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72 
A hexanucleotide (GGGGCC) expansion in C9ORF72 gene is the most common genetic change seen in familial Frontotemporal Lobar Degeneration (FTLD) and familial Motor Neurone Disease (MND). Pathologically, expansion bearers show characteristic p62 positive, TDP-43 negative inclusion bodies within cerebellar and hippocampal neurons which also contain dipeptide repeat proteins (DPR) formed from sense and antisense RAN (repeat associated non ATG-initiated) translation of the expanded repeat region itself. ‘Inappropriate’ formation, and aggregation, of DPR might therefore confer neurotoxicity and influence clinical phenotype. Consequently, we compared the topographic brain distribution of DPR in 8 patients with Frontotemporal dementia (FTD), 6 with FTD + MND and 7 with MND alone (all 21 patients bearing expansions in C9ORF72) using a polyclonal antibody to poly-GA, and related this to the extent of TDP-43 pathology in key regions of cerebral cortex and hippocampus. There were no significant differences in either the pattern or severity of brain distribution of DPR between FTD, FTD + MND and MND groups, nor was there any relationship between the distribution of DPR and TDP-43 pathologies in expansion bearers. Likewise, there were no significant differences in the extent of TDP-43 pathology between FTLD patients bearing an expansion in C9ORF72 and non-bearers of the expansion. There were no association between the extent of DPR pathology and TMEM106B or APOE genotypes. However, there was a negative correlation between the extent of DPR pathology and age at onset. Present findings therefore suggest that although the presence and topographic distribution of DPR may be of diagnostic relevance in patients bearing expansion in C9ORF72 this has no bearing on the determination of clinical phenotype. Because TDP-43 pathologies are similar in bearers and non-bearers of the expansion, the expansion may act as a major genetic risk factor for FTLD and MND by rendering the brain highly vulnerable to those very same factors which generate FTLD and MND in sporadic disease.
Electronic supplementary material
The online version of this article (doi:10.1186/2051-5960-2-70) contains supplementary material, which is available to authorized users.
doi:10.1186/2051-5960-2-70
PMCID: PMC4229740  PMID: 24950788
Frontotemporal lobar degeneration; Motor neurone disease; C9ORF72; Dipeptide repeat proteins
15.  The C9ORF72 expansion mutation is a common cause of ALS+/−FTD in Europe and has a single founder 
A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/−FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/−FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/−FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10−8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.
doi:10.1038/ejhg.2012.98
PMCID: PMC3522204  PMID: 22692064
ALS; common founder; C9ORF72
16.  Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic 
Cell Reports  2013;5(5):1178-1186.
Summary
The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.
Graphical Abstract
Highlights
•Longer G4C2 transcripts form neurotoxic RNA foci in cells and zebrafish•Longer G4C2 foci sequester RNA binding proteins hnRNP-H, SC35, and SF2•G4C2 RNA foci and hnRNP-H knockdown similarly affect TARBP2 splicing•Seventy percent of G4C2 RNA foci in C9ORF72 ALS/FTD brains colocalize with hnRNP-H
In this study, Shaw and colleagues explore mechanisms underlying toxicity of the expanded G4C2 hexanucleotide intronic repeat in C9ORF72, the most common known cause of ALS and FTD. Pathologically expanded G4C2 RNA transcripts form intranuclear foci, sequester specific RNA-binding proteins, and are potently toxic in transfected cells and zebrafish embryos. One protein, hnRNP-H, is detected in 70% of foci in C9ORF72 brain tissues, and loss of hnRNP-H leads to aberrant RNA splicing that could contribute to neurodegeneration.
doi:10.1016/j.celrep.2013.10.049
PMCID: PMC3898469  PMID: 24290757
17.  Compromised paraspeckle formation as a pathogenic factor in FUSopathies 
Human Molecular Genetics  2013;23(9):2298-2312.
Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of paraspeckles, in an RNA-dependent manner and responds in the same way as other paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and paraspeckle formation in cultured cells, and FUS deficiency leads to loss of paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.
doi:10.1093/hmg/ddt622
PMCID: PMC3976330  PMID: 24334610
20.  Simulated surgical-type cerebral biopsies from post-mortem brains allows accurate neuropathological diagnoses in the majority of neurodegenerative disease groups 
Background
In theory, cerebral biopsies could provide the diagnosis in a significant proportion of patients with neurodegenerative diseases, however, there are considerable ethical barriers. Previous series of cerebral biopsies have shown variable diagnostic accuracy but have understandably suffered because of lack of post-mortem tissue with which to compare the diagnosis. To determine the accuracy of such biopsies in neurodegenerative disease we took small biopsy-sized samples of predominantly fresh post-mortem brain tissue from frontal and temporal lobes in 62 cases. These were processed as for a biopsy and stained for H&E, p62, tau, Aβ, α-synuclein, and TDP-43. The sections were assessed blind by 3 neuropathologists and the results compared with the final post-mortem diagnosis.
Results
The agreement and sensitivity in most cases was good especially: controls; Alzheimer’s disease (AD); multiple system atrophy (MSA); frontotemporal lobar degeneration with TDP-43 positive inclusions and/or motor neurone disease (FTLD-TDP/MND); Huntington’s disease (HD); corticobasal degeneration (CBD) / microtubular associated protein tau mutation cases with CBD-like features (CBD/MAPT); and combined AD- Dementia with Lewy Bodies (AD-DLB) where the sensitivity on assessing both brain regions varied between 75-100%. There was poor sensitivity for progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) (both 0%), but moderate sensitivity for pure DLB (60%). The temporal lobe assessment was marginally more accurate than the frontal lobe but these were only slightly worse than both combined.
Conclusions
The study shows that with certain caveats the cerebral biopsy in life should be a viable method of accurately diagnosing many neurodegenerative diseases.
doi:10.1186/2051-5960-1-53
PMCID: PMC3893367  PMID: 24252649
Neurodegeneration; Biopsy; Dementia
21.  ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules 
Human Molecular Genetics  2013;22(13):2676-2688.
Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein–protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.
doi:10.1093/hmg/ddt117
PMCID: PMC3674807  PMID: 23474818
22.  Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight 
Epigenetics  2012;7(2):155-163.
IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ∼25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.
doi:10.4161/epi.7.2.18910
PMCID: PMC3335909  PMID: 22395465
epigenetic; DNA methylation; genomic imprinting; cerebellum; IGF2; H19; brain; expression; frontal cortex; genetic; single nucleotide polymorphism
23.  A comparative clinical, pathological, biochemical and genetic study of fused in sarcoma proteinopathies 
Brain  2011;134(9):2548-2564.
Neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration are rare diseases characterized by ubiquitin-positive inclusions lacking transactive response DNA-binding protein-43 and tau. Recently, mutations in the fused in sarcoma gene have been shown to cause familial amyotrophic lateral sclerosis and fused in sarcoma-positive neuronal inclusions have subsequently been demonstrated in neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration with ubiquitinated inclusions. Here we provide clinical, imaging, morphological findings, as well as genetic and biochemical data in 14 fused in sarcoma proteinopathy cases. In this cohort, the age of onset was variable but included cases of young-onset disease. Patients with atypical frontotemporal lobar degeneration with ubiquitinated inclusions all presented with behavioural variant frontotemporal dementia, while the clinical presentation in neuronal intermediate filament inclusion disease was more heterogeneous, including cases with motor neuron disease and extrapyramidal syndromes. Neuroimaging revealed atrophy of the frontal and anterior temporal lobes as well as the caudate in the cases with atypical frontotemporal lobar degeneration with ubiquitinated inclusions, but was more heterogeneous in the cases with neuronal intermediate filament inclusion disease, often being normal to visual inspection early on in the disease. The distribution and severity of fused in sarcoma-positive neuronal cytoplasmic inclusions, neuronal intranuclear inclusions and neurites were recorded and fused in sarcoma was biochemically analysed in both subgroups. Fused in sarcoma-positive neuronal cytoplasmic and intranuclear inclusions were found in the hippocampal granule cell layer in variable numbers. Cortical fused in sarcoma-positive neuronal cytoplasmic inclusions were often ‘Pick body-like’ in neuronal intermediate filament inclusion disease, and annular and crescent-shaped inclusions were seen in both conditions. Motor neurons contained variable numbers of compact, granular or skein-like cytoplasmic inclusions in all fused in sarcoma-positive cases in which brainstem and spinal cord motor neurons were available for study (five and four cases, respectively). No fused in sarcoma mutations were found in any cases. Biochemically, two major fused in sarcoma species were found and shown to be more insoluble in the atypical frontotemporal lobar degeneration with ubiquitinated inclusions subgroup compared with neuronal intermediate filament inclusion disease. There is considerable overlap and also significant differences in fused in sarcoma-positive pathology between the two subgroups, suggesting they may represent a spectrum of the same disease. The co-existence of fused in sarcoma-positive inclusions in both motor neurons and extramotor cerebral structures is a characteristic finding in sporadic fused in sarcoma proteinopathies, indicating a multisystem disorder.
doi:10.1093/brain/awr160
PMCID: PMC3170529  PMID: 21752791
frontotemporal lobar degeneration; FUS; clinical presentation; neuropathology; biochemistry
24.  Schizophrenia is associated with dysregulation of a Cdk5 activator that regulates synaptic protein expression and cognition 
Brain  2011;134(8):2408-2421.
Cyclin-dependent kinase 5 is activated by small subunits, of which p35 is the most abundant. The functions of cyclin-dependent kinase 5 signalling in cognition and cognitive disorders remains unclear. Here, we show that in schizophrenia, a disorder associated with impaired cognition, p35 expression is reduced in relevant brain regions. Additionally, the expression of septin 7 and OPA1, proteins downstream of truncated p35, is decreased in schizophrenia. Mimicking a reduction of p35 in heterozygous knockout mice is associated with cognitive endophenotypes. Furthermore, a reduction of p35 in mice results in protein changes similar to schizophrenia post-mortem brain. Hence, heterozygous p35 knockout mice model both cognitive endophenotypes and molecular changes reminiscent of schizophrenia. These changes correlate with reduced acetylation of the histone deacetylase 1 target site H3K18 in mice. This site has previously been shown to be affected by truncated p35. By restoring H3K18 acetylation with the clinically used specific histone deacetylase 1 inhibitor MS-275 both cognitive and molecular endophenotypes of schizophrenia can be rescued in p35 heterozygous knockout mice. In summary, we suggest that reduced p35 expression in schizophrenia has an impact on synaptic protein expression and cognition and that these deficits can be rescued, at least in part, by the inhibition of histone deacetylase 1.
doi:10.1093/brain/awr155
PMCID: PMC3155706  PMID: 21772061
animal models; brain; cognition; schizophrenia; signalling
25.  Functional annotation of the human brain methylome identifies tissue-specific epigenetic variation across brain and blood 
Genome Biology  2012;13(6):R43.
Background
Dynamic changes to the epigenome play a critical role in establishing and maintaining cellular phenotype during differentiation, but little is known about the normal methylomic differences that occur between functionally distinct areas of the brain. We characterized intra- and inter-individual methylomic variation across whole blood and multiple regions of the brain from multiple donors.
Results
Distinct tissue-specific patterns of DNA methylation were identified, with a highly significant over-representation of tissue-specific differentially methylated regions (TS-DMRs) observed at intragenic CpG islands and low CG density promoters. A large proportion of TS-DMRs were located near genes that are differentially expressed across brain regions. TS-DMRs were significantly enriched near genes involved in functional pathways related to neurodevelopment and neuronal differentiation, including BDNF, BMP4, CACNA1A, CACA1AF, EOMES, NGFR, NUMBL, PCDH9, SLIT1, SLITRK1 and SHANK3. Although between-tissue variation in DNA methylation was found to greatly exceed between-individual differences within any one tissue, we found that some inter-individual variation was reflected across brain and blood, indicating that peripheral tissues may have some utility in epidemiological studies of complex neurobiological phenotypes.
Conclusions
This study reinforces the importance of DNA methylation in regulating cellular phenotype across tissues, and highlights genomic patterns of epigenetic variation across functionally distinct regions of the brain, providing a resource for the epigenetics and neuroscience research communities.
doi:10.1186/gb-2012-13-6-r43
PMCID: PMC3446315  PMID: 22703893

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