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1.  C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits 
Science (New York, N.Y.)  2015;348(6239):1151-1154.
The major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis is a G4C2 repeat expansion in C9ORF72. Efforts to combat neurodegeneration associated with “c9FTD/ALS” are hindered by a lack of animal models recapitulating disease features. We developed a mouse model to mimic both neuropathological and clinical c9FTD/ALS phenotypes. We expressed (G4C2)66 throughout the murine central nervous system by means of somatic brain transgenesis mediated by adeno-associated virus. Brains of 6-month-old mice contained nuclear RNA foci, inclusions of poly(Gly-Pro), poly(Gly-Ala), and poly(Gly-Arg) dipeptide repeat proteins, as well as TDP-43 pathology. These mouse brains also exhibited cortical neuron and cerebellar Purkinje cell loss, astrogliosis, and decreased weight. (G4C2)66 mice also developed behavioral abnormalities similar to clinical symptoms of c9FTD/ALS patients, including hyperactivity, anxiety, antisocial behavior, and motor deficits.
doi:10.1126/science.aaa9344
PMCID: PMC4692360  PMID: 25977373
2.  Ataxin-2 as potential disease modifier in C9ORF72 expansion carriers 
Neurobiology of aging  2014;35(10):2421.e13-2421.e17.
Repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are an important cause of both motor neuron disease (MND) and frontotemporal dementia (FTD). Currently, little is known about factors that could account for the phenotypic heterogeneity detected in C9ORF72 expansion carriers. In this study, we investigated four genes that could represent genetic modifiers: ataxin-2 (ATXN2), non-imprinted in Prader-Willi/Angelman syndrome 1 (NIPA1), survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2). Assessment of these genes, in a unique cohort of 331 C9ORF72 expansion carriers and 376 controls, revealed that intermediate repeat lengths in ATXN2 possibly act as disease modifier in C9ORF72 expansion carriers; no evidence was provided for a potential role of NIPA1, SMN1 or SMN2. The effects of intermediate ATXN2 repeats were most profound in probands with MND or FTD/MND (2.1% versus 0% in controls, P=0.013), whereas the frequency in probands with FTD was identical to controls. Though intermediate ATXN2 repeats were already known to be associated with MND risk, previous reports did not focus on individuals with clear pathogenic mutations, such as repeat expansions in C9ORF72. Based on our present findings, we postulate that intermediate ATXN2 repeat lengths may render C9ORF72 expansion carriers more susceptible to the development of MND; further studies are needed, however, to validate our findings.
doi:10.1016/j.neurobiolaging.2014.04.016
PMCID: PMC4105839  PMID: 24866401
C9ORF72; ataxin-2; ATXN2; motor neuron disease; amyotrophic lateral sclerosis; frontotemporal dementia; disease modifier
3.  Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers 
Acta Neuropathologica  2015;130(4):559-573.
Clinical and neuropathological characteristics associated with G4C2 repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, are highly variable. To gain insight on the molecular basis for the heterogeneity among C9ORF72 mutation carriers, we evaluated associations between features of disease and levels of two abundantly expressed “c9RAN proteins” produced by repeat-associated non-ATG (RAN) translation of the expanded repeat. For these studies, we took a departure from traditional immunohistochemical approaches and instead employed immunoassays to quantitatively measure poly(GP) and poly(GA) levels in cerebellum, frontal cortex, motor cortex, and/or hippocampus from 55 C9ORF72 mutation carriers [12 patients with ALS, 24 with frontotemporal lobar degeneration (FTLD) and 19 with FTLD with motor neuron disease (FTLD-MND)]. We additionally investigated associations between levels of poly(GP) or poly(GA) and cognitive impairment in 15 C9ORF72 ALS patients for whom neuropsychological data were available. Among the neuroanatomical regions investigated, poly(GP) levels were highest in the cerebellum. In this same region, associations between poly(GP) and both neuropathological and clinical features were detected. Specifically, cerebellar poly(GP) levels were significantly lower in patients with ALS compared to patients with FTLD or FTLD-MND. Furthermore, cerebellar poly(GP) associated with cognitive score in our cohort of 15 patients. In the cerebellum, poly(GA) levels similarly trended lower in the ALS subgroup compared to FTLD or FTLD-MND subgroups, but no association between cerebellar poly(GA) and cognitive score was detected. Both cerebellar poly(GP) and poly(GA) associated with C9ORF72 variant 3 mRNA expression, but not variant 1 expression, repeat size, disease onset, or survival after onset. Overall, these data indicate that cerebellar abnormalities, as evidenced by poly(GP) accumulation, associate with neuropathological and clinical phenotypes, in particular cognitive impairment, of C9ORF72 mutation carriers.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-015-1474-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-015-1474-4
PMCID: PMC4575385  PMID: 26350237
Amyotrophic lateral sclerosis; C9ORF72 repeat expansion; c9RAN proteins; Cognition; Dipeptide repeat proteins; Frontotemporal dementia; Frontotemporal lobar degeneration; Neuropathological diagnosis; Repeat-associated non-ATG translation
4.  A novel, efficient, randomized selection trial comparing combinations of drug therapy for ALS 
Combining agents with different mechanisms of action may be necessary for meaningful results in treating ALS. The combinations of minocycline-creatine and celecoxib-creatine have additive effects in the murine model. New trial designs are needed to efficiently screen the growing number of potential neuroprotective agents. Our objective was to assess two drug combinations in ALS using a novel phase II trial design. We conducted a randomized, double-blind selection trial in sequential pools of 60 patients. Participants received minocycline (100 mg)-creatine (10 g) twice daily or celecoxib (400 mg)-creatine (10 g) twice daily for six months. The primary objective was treatment selection based on which combination best slowed deterioration in the ALS Functional Rating Scale-Revised (ALSFRS-R); the trial could be stopped after one pool if the difference between the two arms was adequately large. At trial conclusion, each arm was compared to a historical control group in a futility analysis. Safety measures were also examined. After the first patient pool, the mean six-month decline in ALSFRS-R was 5.27 (SD=5.54) in the celecoxib-creatine group and 6.47 (SD=9.14) in the minocycline-creatine group. The corresponding decline was 5.82 (SD=6.77) in the historical controls. The difference between the two sample means exceeded the stopping criterion. The null hypothesis of superiority was not rejected in the futility analysis. Skin rash occurred more frequently in the celecoxib-creatine group. In conclusion, the celecoxib-creatine combination was selected as preferable to the minocycline-creatine combination for further evaluation. This phase II design was efficient, leading to treatment selection after just 60 patients, and can be used in other phase II trials to assess different agents.
doi:10.1080/17482960802195632
PMCID: PMC4354803  PMID: 18608093
Amyotrophic lateral sclerosis; ALS; minocycline; celecoxib; creatine; clinical trial; neuroprotection; combination therapy; selection trial
5.  TMEM106B protects C9ORF72 expansion carriers against frontotemporal dementia 
Acta neuropathologica  2014;127(3):397-406.
Variants in transmembrane protein 106 B (TMEM106B) modify the disease penetrance of frontotemporal dementia (FTD) in carriers of progranulin (GRN) mutations. We investigated whether TMEM106B is also a genetic modifier of disease in carriers of chromosome 9 open reading frame 72 (C9ORF72) expansions. We assessed the genotype of 325 C9ORF72 expansion carriers (cohort 1), 586 FTD patients lacking C9ORF72 expansions (with or without motor neuron disease [MND]; cohort 2), and a total of 1,302 controls for TMEM106B variants (rs3173615 and rs1990622) using MassArray iPLEX and Taqman genotyping assays. For our primary analysis, we focused on functional variant rs3173615, and employed a recessive genotypic model. In cohort 1, patients with C9ORF72 expansions showed a significantly reduced frequency of carriers homozygous for the minor allele as compared to controls (11.9% versus 19.1%, odds ratio (OR): 0.57, p=0.014; same direction as carriers of GRN mutations). The strongest evidence was provided by FTD patients (OR: 0.33, p=0.009) followed by FTD/MND patients (OR: 0.38, p=0.017), whereas no significant difference was observed in MND patients (OR: 0.85, p=0.55). In cohort 2, the frequency of carriers homozygous for the minor allele was not significantly reduced in patients as compared to controls (OR: 0.77, p=0.079); however, a significant reduction was observed when focusing on those patients with frontotemporal lobar degeneration and TAR DNA-binding protein 43 inclusions (FTLD-TDP; OR: 0.26, p<0.001).
Our study identifies TMEM106B as the first genetic factor modifying disease presentation in C9ORF72 expansion carriers. Homozygosity for the minor allele protects carriers from developing FTD, but not from developing MND; similar effects are seen in FTLD-TDP patients with yet unknown genetic causes. These new findings show that the protective effects of TMEM106B are not confined to carriers of GRN mutations, and might be relevant for prognostic testing, and as a promising therapeutic target for the entire spectrum of FTLD-TDP.
doi:10.1007/s00401-013-1240-4
PMCID: PMC3944829  PMID: 24385136
C9ORF72; TMEM106B; frontotemporal dementia; motor neuron disease; amyotrophic lateral sclerosis; disease modifier
6.  Associations of repeat sizes with clinical and pathological characteristics in C9ORF72 expansion carriers (Xpansize-72): a cross-sectional cohort study 
Lancet neurology  2013;12(10):10.1016/S1474-4422(13)70210-2.
Summary
Background
Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are currently the major genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND). Presently, it is unknown whether expansion size affects disease severity or phenotypes.
Methods
We performed a cross-sectional Southern blot characterization study (Xpansize-72) in a cohort of subjects obtained at the Mayo Clinic and Banner Sun Health Research Institute. All subjects carried GGGGCC repeat expansions in C9ORF72, and high quality DNA was available from the frontal cortex, cerebellum and/or blood. Southern blotting techniques and densitometry were employed to estimate the repeat size of the most abundant expansion species. Comparisons of repeat sizes between tissues were made using Wilcoxon rank sum and Wilcoxon signed rank tests, and between disease subgroups using Kruskal-Wallis rank sum tests. The association of repeat size with age at onset and age at collection was evaluated using a Spearman’s test of correlation; whereas the association between repeat size and survival after disease onset was examined using Cox proportional hazards regression models.
Findings
Our cohort consisted of 84 C9ORF72 expansion carriers, including FTD patients (n=35), FTD/MND patients (n=16), MND patients (n=30), and unaffected subjects (n=3). We focused our analysis on three major tissue subgroups: frontal cortex (41 subjects [21 FTD, 11 FTD/MND, 9 MND]), cerebellum (40 subjects [20 FTD, 12 FTD/MND, 8 MND]), and blood (50 subjects [15 FTD, 9 FTD/MND, 23 MND, 3 unaffected expansion carriers]). Repeat lengths in the cerebellum were significantly smaller (median 12·3 kb [~1667 repeat units], IQR 11·1–14·3) than in the frontal cortex (median 33·8 kb [~5250 repeat units], IQR 23·5–44·9, p<0·0001), or in blood (median 18·6 kb [~2717 repeat units], IQR 13·9–28·1, p=0·0002). Within these tissues, there was no significant difference in repeat length between disease subgroups (cerebellum p=0·96, frontal cortex p=0·27, blood p=0·10). In the frontal cortex of FTD patients, repeat length correlated with age at onset (r=0·63, p=0·003) and age at collection (r=0·58, p=0·006); this correlation was not detected in the cerebellum or blood. Finally, only in the cerebellum, survival after disease onset was poorer in patients from our overall cohort with repeat lengths greater than 1467 repeat units (25th percentile, HR 3·27, 95% CI 1·34–7·95, p=0·009): the median survival was 4·8 years (IQR 3·0–7·4) in the group with longer expansions versus 7·4 years (IQR 6·3–10·9) in the group with smaller expansions.
Interpretation
Substantial variation in repeat size is observed between cerebellum, frontal cortex, and blood; relatively long repeat sizes in the cerebellum confer an important survival disadvantage. Our findings indicate that expansion size does affect disease severity, which could be relevant for genetic counseling.
doi:10.1016/S1474-4422(13)70210-2
PMCID: PMC3879782  PMID: 24011653
7.  Genetic modifiers in carriers of repeat expansions in the C9ORF72 gene 
Background
Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are causative for frontotemporal dementia (FTD) and motor neuron disease (MND). Substantial phenotypic heterogeneity has been described in patients with these expansions. We set out to identify genetic modifiers of disease risk, age at onset, and survival after onset that may contribute to this clinical variability.
Results
We examined a cohort of 330 C9ORF72 expansion carriers and 374 controls. In these individuals, we assessed variants previously implicated in FTD and/or MND; 36 variants were included in our analysis. After adjustment for multiple testing, our analysis revealed three variants significantly associated with age at onset (rs7018487 [UBAP1; p-value = 0.003], rs6052771 [PRNP; p-value = 0.003], and rs7403881 [MT-Ie; p-value = 0.003]), and six variants significantly associated with survival after onset (rs5848 [GRN; p-value = 0.001], rs7403881 [MT-Ie; p-value = 0.001], rs13268953 [ELP3; p-value = 0.003], the epsilon 4 allele [APOE; p-value = 0.004], rs12608932 [UNC13A; p-value = 0.003], and rs1800435 [ALAD; p-value = 0.003]).
Conclusions
Variants identified through this study were previously reported to be involved in FTD and/or MND, but we are the first to describe their effects as potential disease modifiers in the presence of a clear pathogenic mutation (i.e. C9ORF72 repeat expansion). Although validation of our findings is necessary, these variants highlight the importance of protein degradation, antioxidant defense and RNA-processing pathways, and additionally, they are promising targets for the development of therapeutic strategies and prognostic tests.
Electronic supplementary material
The online version of this article (doi:10.1186/1750-1326-9-38) contains supplementary material, which is available to authorized users.
doi:10.1186/1750-1326-9-38
PMCID: PMC4190282  PMID: 25239657
C9ORF72; Frontotemporal dementia; Motor neuron disease; Genetic modifier; Repeat expansion
8.  Profilin-1 mutations are rare in patients with amyotrophic lateral sclerosis and frontotemporal dementia 
Objective
Mutations in profilin-1 (PFN1) have recently been identified in patients with amyotrophic lateral sclerosis (ALS). Because of the considerable overlap between ALS and the common subtype of frontotemporal dementia, which is characterized by transactive response DNA-binding protein 43 pathology (FTLD-TDP), we tested cohorts of ALS and FTLD-TDP patients for PFN1 mutations.
Methods
DNA was obtained from 342 ALS patients and 141 FTLD-TDP patients at our outpatient clinic and brain bank for neurodegenerative diseases at the Mayo Clinic Florida, Jacksonville, USA. We screened these patients for mutations in coding regions of PFN1 by Sanger sequencing. Subsequently, we used TaqMan genotyping assays to investigate the identified variant in 1167 control subjects.
Results
One variant, p.E117G, was detected in 1 ALS patient, 1 FTLD-TDP patient, and 2 control subjects. The mutation frequency of patients versus control subjects was not significantly different (p-value = 0.36). Moreover, PFN1 and TDP-43 staining of autopsy material did not differ between patients with or without this variant.
Conclusion
The p.E117G variant appears to represent a benign polymorphism. PFN1 mutations, in general, are rare in ALS and FTLD-TDP patients.
doi:10.3109/21678421.2013.787630
PMCID: PMC3923463  PMID: 23634771
Amyotrophic lateral sclerosis; frontotemporal dementia; profilin-1; TDP-43; genetics
9.  Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress 
Acta Neuropathologica  2014;128(4):505-524.
The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the “c9RAN proteins” thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-014-1336-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-014-1336-5
PMCID: PMC4159567  PMID: 25173361
Amyotrophic lateral sclerosis; C9ORF72; Expanded repeat; Frontotemporal dementia; Repeat-associated non-ATG translation; Poly(GA) proteins; Proteasome activity; ER stress
10.  Clinicopathologic variability of the GRN A9D mutation, including amyotrophic lateral sclerosis 
Neurology  2013;80(19):1771-1777.
Objective:
We examined the clinical and pathologic phenotypes of GRN mutation carriers with the pathogenic A9D (g.26C>A) missense mutation.
Methods:
Three patients with GRN A9D mutations were evaluated clinically and came to autopsy with subsequent neuropathologic examination.
Results:
The clinical diagnoses of patients with GRN A9D mutations were amyotrophic lateral sclerosis, atypical extrapyramidal disorder, and behavioral variant frontotemporal dementia. Immunohistochemistry for TAR DNA-binding protein 43 (TDP-43) revealed variability in morphology and distribution of pathology. One patient had notable involvement of motor neurons in the spinal cord as well as type B TDP-43, whereas 2 other patients had type A TDP-43.
Conclusions:
The clinical presentation of the GRN A9D missense mutation is not restricted to behavioral variant frontotemporal dementia and may include aphasia, extrapyramidal features, and, notably, amyotrophic lateral sclerosis.
doi:10.1212/WNL.0b013e3182919059
PMCID: PMC3719429  PMID: 23596077
11.  C9ORF72 repeat expansions in cases with previously identified pathogenic mutations 
Neurology  2013;81(15):1332-1341.
Objective:
To identify potential genetic modifiers contributing to the phenotypic variability that is detected in patients with repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), we investigated the frequency of these expansions in a cohort of 334 subjects previously found to carry mutations in genes known to be associated with a spectrum of neurodegenerative diseases.
Methods:
A 2-step protocol, with a fluorescent PCR and a repeat-primed PCR, was used to determine the presence of hexanucleotide expansions in C9ORF72. For one double mutant, we performed Southern blots to assess expansion sizes, and immunohistochemistry to characterize neuropathology.
Results:
We detected C9ORF72 repeat expansions in 4 of 334 subjects (1.2% [or 1.8% of 217 families]). All these subjects had behavioral phenotypes and also harbored well-known pathogenic mutations in either progranulin (GRN: p.C466LfsX46, p.R493X, p.C31LfsX35) or microtubule-associated protein tau (MAPT: p.P301L). Southern blotting of one double mutant with a p.C466LfsX46 GRN mutation demonstrated a long repeat expansion in brain (>3,000 repeats), and immunohistochemistry showed mixed neuropathology with characteristics of both C9ORF72 expansions and GRN mutations.
Conclusions:
Our findings indicate that co-occurrence of 2 evidently pathogenic mutations could contribute to the pleiotropy that is detected in patients with C9ORF72 repeat expansions. These findings suggest that patients with known mutations should not be excluded from further studies, and that genetic counselors should be aware of this phenomenon when advising patients and their family members.
doi:10.1212/WNL.0b013e3182a8250c
PMCID: PMC3806926  PMID: 24027057
12.  TARDBP mutations in Parkinson’s disease 
Parkinsonism & related disorders  2012;19(3):312-315.
Mutations of the TARDBP gene encoding TDP-43 protein have been shown to cause amyotrophic lateral sclerosis and have been reported to present with clinical heterogeneity including parkinsonism. In addition, TDP-43 pathology has been observed across a spectrum of neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Herein we report the presence of a TDP-43 mutation in a patient with a clinical diagnosis of Parkinson’s disease. The TDP-43 p.N267S substitution has been previously implicated in both amyotrophic lateral sclerosis and behavioral variant frontotemporal dementia. Our findings widen the phenotypic presentation for the TDP-43 p.N267S substitution and support a possible role for rare TDP-43 mutations presenting with Parkinson’s disease.
doi:10.1016/j.parkreldis.2012.11.003
PMCID: PMC3582838  PMID: 23231971
TDP-43; amyotrophic lateral sclerosis; Parkinson’s disease
13.  Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS 
Neuron  2013;77(4):639-646.
Summary
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9ORF72 are the major genetic cause of FTD and ALS (c9FTD/ALS). The RNA structure of GGGGCC repeats renders these transcripts susceptible to an unconventional mechanism of translation – repeat-associated non-ATG (RAN) translation. Antibodies generated against putative GGGGCC repeat RAN translated peptides (anti-C9RANT) detected high molecular weight, insoluble material in brain homogenates, and neuronal inclusions throughout the central nervous system of c9FTD/ALS cases. C9RANT immunoreactivity was not found in other neurodegenerative diseases, including CAG repeat disorders, or in peripheral tissues of c9FTD/ALS. The specificity of C9RANT for c9FTD/ALS is a potential biomarker for this most common cause of FTD and ALS. These findings have significant implications for treatment strategies directed at RAN translated peptides and their aggregation, and the RNA structures necessary for their production.
doi:10.1016/j.neuron.2013.02.004
PMCID: PMC3593233  PMID: 23415312
14.  Tau Pathology in Frontotemporal Lobar Degeneration with C9ORF72 Hexanucleotide Repeat Expansion 
Acta neuropathologica  2012;125(2):289-302.
An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration associated with TDP-43 pathology (FTLD-TDP). In addition to TDP-43-positive neuronal and glial inclusions, C9ORF72-linked FTLD-TDP has characteristic TDP-43-negative neuronal cytoplasmic and intranuclear inclusions as well as dystrophic neurites in the hippocampus and cerebellum. These lesions are immunopositive for ubiquitin and ubiquitin-binding proteins, such as sequestosome-1/p62 and ubiquilin-2. Studies examining the frequency of the C9ORF72 mutation in clinically probable Alzheimer’s disease (AD) have found a small proportion of AD cases with the mutation. This prompted us to systematically explore the frequency of Alzheimer type pathology in a series of 17 FTLD-TDP cases with mutations in C9ORF72 (FTLD-C9ORF72). We identified 4 cases with sufficient Alzheimer type pathology to meet criteria for intermediate-to-high likelihood AD. We compared AD pathology in the 17 FTLD-C9ORF72 to 13 cases of FTLD-TDP linked to mutations in the gene for progranulin (FTLD-GRN) and 36 cases of sporadic FTLD (sFTLD). FTLD-C9ORF72 cases had higher Braak neurofibrillary tangle stage than FTLD-GRN. Increased tau pathology in FTLD-C9ORF72 was assessed with thioflavin-S fluorescent microscopy-based neurofibrillary tangle counts and with image analysis of tau burden in temporal cortex and hippocampus. FTLD-C9ORF72 had significantly more neurofibrillary tangles and higher tau burden compared with FTLD-GRN. The differences were most marked in limbic regions. On the other hand, sFTLD and FTLD-C9ORF72 had a similar burden of tau pathology. These results suggest FTLD-C9ORF72 has increased propensity for tau pathology compared to FTLD-GRN, but not sFTLD. The accumulation of tau as well as lesions immunoreactive for ubiquitin and ubiquitin binding proteins (p62 and ubiquilin-2) suggests that mutations in C9ORF72 may involve disrupted protein degradation that favors accumulation of multiple different proteins.
doi:10.1007/s00401-012-1048-7
PMCID: PMC3551994  PMID: 23053135
frontotemporal lobar degeneration; C9ORF72; ubiquitin; p62; ubiquilin-2; tau
15.  Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype 
Neurobiology of aging  2012;33(12):2950.e5-2950.e7.
Expansions of the non-coding GGGGCC hexanucleotide repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene were recently identified as the long sought-after cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) on chromosome 9p. In this study we aimed to determine whether the length of the normal - unexpanded - allele of the GGGGCC repeat in C9ORF72 plays a role in the presentation of disease or affects age at onset in C9ORF72 mutation carriers. We also studied whether the GGGGCC repeat length confers risk or affects age at onset in FTD and ALS patients without C9ORF72 repeat expansions. C9ORF72 genotyping was performed in 580 FTD, 995 ALS and 160 FTD-ALS patients and 1444 controls, leading to the identification of 211 patients with pathogenic C9ORF72 repeat expansions and an accurate quantification of the length of the normal alleles in all patients and controls. No meaningful association between the repeat length of the normal alleles of the GGGGCC repeat in C9ORF72 and disease phenotype or age at onset was observed in C9ORF72 mutation carriers or non-mutation carriers.
doi:10.1016/j.neurobiolaging.2012.07.005
PMCID: PMC3617405  PMID: 22840558
Amyotrophic lateral sclerosis; Frontotemporal Dementia; C9ORF72; Repeat-expansion disease; Association study
16.  Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood 
Acta Neuropathologica  2013;126(6):895-905.
Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause “c9FTD/ALS” has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1199-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-013-1199-1
PMCID: PMC3830740  PMID: 24166615
Amyotrophic lateral sclerosis; Frontotemporal dementia; C9orf72; Epigenetic modification; Repeat expansion; Histone methylation
17.  Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS 
Acta Neuropathologica  2013;126(6):829-844.
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. A hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of both diseases. The mechanisms by which this repeat expansion causes “c9FTD/ALS” are not definitively known, but RNA-mediated toxicity is a likely culprit. RNA transcripts of the expanded GGGGCC repeat form nuclear foci in c9FTD/ALS, and also undergo repeat-associated non-ATG (RAN) translation resulting in the production of three aggregation-prone proteins. The goal of this study was to examine whether antisense transcripts resulting from bidirectional transcription of the expanded repeat behave in a similar manner. We show that ectopic expression of (CCCCGG)66 in cultured cells results in foci formation. Using novel polyclonal antibodies for the detection of possible (CCCCGG)exp RAN proteins [poly(PR), poly(GP) and poly(PA)], we validated that (CCCCGG)66 is also subject to RAN translation in transfected cells. Of importance, foci composed of antisense transcripts are observed in the frontal cortex, spinal cord and cerebellum of c9FTD/ALS cases, and neuronal inclusions of poly(PR), poly(GP) and poly(PA) are present in various brain tissues in c9FTD/ALS, but not in other neurodegenerative diseases, including CAG repeat disorders. Of note, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell, suggesting these events represent two distinct ways in which the C9ORF72 repeat expansion may evoke neurotoxic effects. These findings provide mechanistic insight into the pathogenesis of c9FTD/ALS, and have significant implications for therapeutic strategies.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1192-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-013-1192-8
PMCID: PMC3830741  PMID: 24129584
Amyotrophic lateral sclerosis; Bidirectional transcription; C9ORF72; Expanded repeat; Frontotemporal dementia; Repeat-associated non-ATG translation; RNA foci
18.  Prion-like domain mutations in hnRNPs cause multisystem proteinopathy and ALS 
Nature  2013;495(7442):467-473.
Summary
Algorithms designed to identify canonical yeast prions predict that ~250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here, we define pathogenic mutations in PrLDs of hnRNPA2/B1 and hnRNPA1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Wild-type hnRNPA2 and hnRNPA1 display an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Importantly, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant ‘steric zipper’ motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs must be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.
doi:10.1038/nature11922
PMCID: PMC3756911  PMID: 23455423
20.  TREM2 in neurodegeneration: evidence for association of the p.R47H variant with frontotemporal dementia and Parkinson’s disease 
Background
A rare variant in the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) gene has been reported to be a genetic risk factor for Alzheimer’s disease by two independent groups (Odds ratio between 2.9-4.5). Given the key role of TREM2 in the effective phagocytosis of apoptotic neuronal cells by microglia, we hypothesized that dysfunction of TREM2 may play a more generalized role in neurodegeneration. With this in mind we set out to assess the genetic association of the Alzheimer’s disease-related risk variant in TREM2 (rs75932628, p.R47H) with other related neurodegenerative disorders.
Results
The study included 609 patients with frontotemporal dementia, 765 with amyotrophic lateral sclerosis, 1493 with Parkinson’s disease, 772 with progressive supranuclear palsy, 448 with ischemic stroke and 1957 controls subjects free of neurodegenerative disease. A significant association was observed for the TREM2 p.R47H substitution in susceptibility to frontotemporal dementia (OR = 5.06; p-value = 0.001) and Parkinson’s disease (OR = 2.67; p-value = 0.026), while no evidence of association with risk of amyotrophic lateral sclerosis, progressive supranuclear palsy or ischemic stroke was observed.
Conclusions
Our results suggest that the TREM2 p.R47H substitution is a risk factor for frontotemporal dementia and Parkinson’s disease in addition to Alzheimer’s disease. These findings suggest a more general role for TREM2 dysfunction in neurodegeneration, which could be related to its role in the immune response.
doi:10.1186/1750-1326-8-19
PMCID: PMC3691612  PMID: 23800361
TREM2; Frontotemporal dementia; Parkinson disease; Genetic association
21.  Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72 
Acta Neuropathologica  2011;122(6):673-690.
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of a disease spectrum associated with TDP-43 pathology. Strong evidence supporting this is the existence of kindreds with family members affected by FTD, ALS or mixed features of FTD and ALS, referred to as FTD-MND. Some of these families have linkage to chromosome 9, with hexanucleotide expansion mutation in a noncoding region of C9ORF72. Discovery of the mutation defines c9FTD/ALS. Prior to discovery of mutations in C9ORF72, it was assumed that TDP-43 pathology in c9FTD/ALS was uniform. In this study, we examined the neuropathology and clinical features of 20 cases of c9FTD/ALS from a brain bank for neurodegenerative disorders. Included are six patients clinically diagnosed with ALS, eight FTD, one FTD-MND and four Alzheimer type dementia. Clinical information was unavailable for one patient. Pathologically, the cases all had TDP-43 pathology, but there were three major pathologic groups: ALS, FTLD-MND and FTLD-TDP. The ALS cases were morphologically similar to typical sporadic ALS with almost no extramotor TDP-43 pathology; all had oligodendroglial cytoplasmic inclusions. The FTLD-MND showed predominantly Mackenzie Type 3 TDP-43 pathology, and all had ALS-like pathology in motor neurons, but more extensive extramotor pathology, with oligodendroglial cytoplasmic inclusions and infrequent hippocampal sclerosis. The FTLD-TDP cases had several features similar to FTLD-TDP due to mutations in the gene for progranulin, including Mackenzie Type 1 TDP-43 pathology with neuronal intranuclear inclusions and hippocampal sclerosis. FTLD-TDP patients were older and some were thought to have Alzheimer type dementia. In addition to the FTD and ALS clinical presentations, the present study shows that c9FTD/ALS can have other presentations, possibly related to age of onset and presence of hippocampal sclerosis. Moreover, there is pathologic heterogeneity not only between ALS and FTLD, but within the FTLD group. Further studies are needed to address the molecular mechanism of clinical and pathological heterogeneity of c9FTD/ALS due to mutations in C9ORF72.
doi:10.1007/s00401-011-0907-y
PMCID: PMC3277860  PMID: 22083254
22.  Expanded GGGGCC hexanucleotide repeat in non-coding region of C9ORF72 causes chromosome 9p-linked frontotemporal dementia and amyotrophic lateral sclerosis 
Neuron  2011;72(2):245-256.
SUMMARY
Several families have been reported with autosomal dominant frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), genetically linked to chromosome 9p21. Here we report an expansion of a non-coding GGGGCC hexanucleotide repeat in the gene C9ORF72 that is strongly associated with disease in a large FTD/ALS kindred, previously reported to be conclusively linked to chromosome 9p. This same repeat expansion was identified in the majority of our families with a combined FTD/ALS phenotype and TDP-43 based pathology. Analysis of extended clinical series found the C9ORF72 repeat expansion to be the most common genetic abnormality in both familial FTD (11.7%) and familial ALS (22.5%). The repeat expansion leads to the loss of one alternatively spliced C9ORF72 transcript and to formation of nuclear RNA foci, suggesting multiple disease mechanisms. Our findings indicate that repeat expansion in C9ORF72 is a major cause of both FTD and ALS.
doi:10.1016/j.neuron.2011.09.011
PMCID: PMC3202986  PMID: 21944778
23.  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
24.  Ataxin-2 repeat-length variation and neurodegeneration 
Human Molecular Genetics  2011;20(16):3207-3212.
Expanded glutamine repeats of the ataxin-2 (ATXN2) protein cause spinocerebellar ataxia type 2 (SCA2), a rare neurodegenerative disorder. More recent studies have suggested that expanded ATXN2 repeats are a genetic risk factor for amyotrophic lateral sclerosis (ALS) via an RNA-dependent interaction with TDP-43. Given the phenotypic diversity observed in SCA2 patients, we set out to determine the polymorphic nature of the ATXN2 repeat length across a spectrum of neurodegenerative disorders. In this study, we genotyped the ATXN2 repeat in 3919 neurodegenerative disease patients and 4877 healthy controls and performed logistic regression analysis to determine the association of repeat length with the risk of disease. We confirmed the presence of a significantly higher number of expanded ATXN2 repeat carriers in ALS patients compared with healthy controls (OR = 5.57; P= 0.001; repeat length >30 units). Furthermore, we observed significant association of expanded ATXN2 repeats with the development of progressive supranuclear palsy (OR = 5.83; P= 0.004; repeat length >30 units). Although expanded repeat carriers were also identified in frontotemporal lobar degeneration, Alzheimer's and Parkinson's disease patients, these were not significantly more frequent than in controls. Of note, our study identified a number of healthy control individuals who harbor expanded repeat alleles (31–33 units), which suggests caution should be taken when attributing specific disease phenotypes to these repeat lengths. In conclusion, our findings confirm the role of ATXN2 as an important risk factor for ALS and support the hypothesis that expanded ATXN2 repeats may predispose to other neurodegenerative diseases, including progressive supranuclear palsy.
doi:10.1093/hmg/ddr227
PMCID: PMC3140823  PMID: 21610160
25.  Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72 
Brain  2012;135(3):765-783.
Numerous kindreds with familial frontotemporal dementia and/or amyotrophic lateral sclerosis have been linked to chromosome 9, and an expansion of the GGGGCC hexanucleotide repeat in the non-coding region of chromosome 9 open reading frame 72 has recently been identified as the pathogenic mechanism. We describe the key characteristics in the probands and their affected relatives who have been evaluated at Mayo Clinic Rochester or Mayo Clinic Florida in whom the hexanucleotide repeat expansion were found. Forty-three probands and 10 of their affected relatives with DNA available (total 53 subjects) were shown to carry the hexanucleotide repeat expansion. Thirty-six (84%) of the 43 probands had a familial disorder, whereas seven (16%) appeared to be sporadic. Among examined subjects from the 43 families (n = 63), the age of onset ranged from 33 to 72 years (median 52 years) and survival ranged from 1 to 17 years, with the age of onset <40 years in six (10%) and >60 in 19 (30%). Clinical diagnoses among examined subjects included behavioural variant frontotemporal dementia with or without parkinsonism (n = 30), amyotrophic lateral sclerosis (n = 18), frontotemporal dementia/amyotrophic lateral sclerosis with or without parkinsonism (n = 12), and other various syndromes (n = 3). Parkinsonism was present in 35% of examined subjects, all of whom had behavioural variant frontotemporal dementia or frontotemporal dementia/amyotrophic lateral sclerosis as the dominant clinical phenotype. No subject with a diagnosis of primary progressive aphasia was identified with this mutation. Incomplete penetrance was suggested in two kindreds, and the youngest generation had significantly earlier age of onset (>10 years) compared with the next oldest generation in 11 kindreds. Neuropsychological testing showed a profile of slowed processing speed, complex attention/executive dysfunction, and impairment in rapid word retrieval. Neuroimaging studies showed bilateral frontal abnormalities most consistently, with more variable degrees of parietal with or without temporal changes; no case had strikingly focal or asymmetric findings. Neuropathological examination of 14 patients revealed a range of transactive response DNA binding protein molecular weight 43 pathology (10 type A and four type B), as well as ubiquitin-positive cerebellar granular neuron inclusions in all but one case. Motor neuron degeneration was detected in nine patients, including five patients without ante-mortem signs of motor neuron disease. While variability exists, most cases with this mutation have a characteristic spectrum of demographic, clinical, neuropsychological, neuroimaging and especially neuropathological findings.
doi:10.1093/brain/aws004
PMCID: PMC3286335  PMID: 22366793
frontotemporal dementia; amyotrophic lateral sclerosis; motor neuron disease; TDP-43; neurogenetics; chromosome 9

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