Abstract The nuclear protein fused in sarcoma (FUS) is found in cytoplasmic inclusions in a subset of patients with the neurodegenerative disorder frontotemporal lobar degeneration (FTLD-FUS). FUS ...contains a methylated arginine–glycine–glycine domain that is required for transport into the nucleus. Recent findings have shown that this domain is hypomethylated in patients with FTLD-FUS. To determine whether the cause of hypomethylation is the result of mutations in protein N -arginine methyltransferases (PRMTs), we selected 3 candidate genes ( PRMT1 , PRMT3, and PRMT8 ) and performed complete sequencing analysis and real-time polymerase chain reaction mRNA expression analysis in 20 FTLD-FUS cases. No mutations or statistically significant changes in expression were observed in our patient samples, suggesting that defects in PRMTs are not the cause of FTLD-FUS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract Insertion and deletion variants (indels) within poly glycine tracts of fused in sarcoma (FUS) were initially reported as causative of disease in amyotrophic lateral sclerosis (ALS). ...Subsequent studies identified similar indels in controls and suggested that these indels may confer susceptibility to ALS. We aimed to elucidate the role of previously published and novel exonic indels in FUS in an extensive cohort of 630 ALS patients and 1063 controls. We detected indels in FUS exons 5, 6, 12, and 14 with similar frequencies in patients (0.95%) and controls (0.75%). Exonic indels in poly glycine tracts were also observed with similar frequencies. The largest indel (p.Gly138_Tyr143del) was observed in 1 control. In 1 patient, a 3 base pair deletion in exon 14 (p.Gly475del) was identified, however in vitro studies did not reveal abnormal localization of p.Gly475del mutant FUS. These findings suggest that not all exonic indels in FUS cause disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
OBJECTIVES To describe the clinical features of a Brazilian kindred with C9orf72 frontotemporal dementia–amyotrophic lateral sclerosis and compare them with other described families with C9orf72 and ...frontotemporal dementia–amyotrophic lateral sclerosis–causing mutations. DESIGN Report of a kindred. SETTING Dementia center at a university hospital. PATIENTS One kindred encompassing 3 generations. RESULTS The presence of a hexanucleotide (GGGGCC) expansion in C9orf72 was confirmed by repeat-primed polymerase chain reaction and Southern blot. The observed phenotypes were behavioral variant frontotemporal dementia and amyotrophic lateral sclerosis with dementia, with significant variability in age at onset and duration of disease. Parkinsonian features with focal dystonia, visual hallucinations, and more posterior atrophy on neuroimaging than is typical for frontotemporal dementia were seen. CONCLUSIONS Behavioral variant frontotemporal dementia due to C9orf72 expansion displays some phenotypic heterogeneity and may be associated with hallucinations, parkinsonism, focal dystonia, and posterior brain atrophy. Personality changes may precede the diagnosis of dementia by many years and may be a distinguishing feature of this mutation.
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 (GRNp.C466LfsX46, p.R493X, p.C31LfsX35) or microtubule-associated protein tau (MAPTp.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.
The discoveries of mutations in SNCA were seminal findings that resulted in the knowledge that alpha-synuclein (alphaS) is the major component of Parkinson's disease-associated Lewy bodies. Since the ...pathologic roles of these protein inclusions and SNCA mutations are not completely established, we characterized the aggregation properties of the recently identified SNCA mutations, H50Q and G51D, to provide novel insights. The properties of recombinant H50Q, G51D, and wild-type alphaS to polymerize and aggregate into amyloid were studied using (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene fluorometry, sedimentation analyses, electron microscopy, and atomic force microscopy. These studies showed that the H50Q mutation increases the rate of alphaS aggregation, whereas the G51D mutation has the opposite effect. However, H50Q and G51D alphaS could still be similarly induced to form intracellular aggregates from the exposure to exogenous amyloidogenic seeds under conditions that promote their cellular entry. Both mutant alphaS proteins, but especially G51D, promoted cellular toxicity under cellular stress conditions. These findings reveal that the novel pathogenic SNCA mutations, H50Q and G51D, have divergent effects on aggregation properties relative to the wild-type protein, with G51D alphaS demonstrating reduced aggregation despite presenting with earlier disease onset, suggesting that these mutants promote different mechanisms of alphaS pathogenesis. The alpha-synuclein (SNCA) mutations, H50Q and G51D, cause Parkinson's disease. We found that H50Q increases and G51D decreases the rate of alpha-synuclein aggregation in vitro, and cells over-expressing the mutant proteins show decreased viability when stressed, compared to wild type. G51D is the first SNCA mutation to show decreased alpha-synuclein aggregation, suggesting a distinct disease mechanism to other SNCA mutations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Pathological inclusions containing aggregated, highly phosphorylated (at serine129) alpha-synuclein (alphaS pSer129) are characteristic of a group of neurodegenerative diseases termed ...synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting alphaS inclusions in human tissue and experimental models of synucleinopathies. However, the generation of extensively specific pSer129 antibodies has been problematic, in some cases leading to the misinterpretation of alphaS inclusion pathology. One common issue is cross-reactivity to the low molecular mass neurofilament subunit (NFL) phosphorylated at Ser473. Here, we generated a series of monoclonal antibodies to the pSer129 alphaS and pSer473 NFL epitopes. We determined the relative abilities of the known alphaS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and alphaS, while using this information to characterize the specificity of the new antibodies. NFL can be phosphorylated by PLK1, 2 and 3 at Ser473; however CKII shows the highest phosphorylation efficiency and specificity for this site. Conversely, PLK3 is the most efficient kinase at phosphorylating alphaS at Ser129, but there is overlay in the ability of these kinases to phosphorylate both epitopes. Antibody 4F8, generated to the pSer473 NFL epitope, was relatively specific for phosphorylated NFL, however it could uniquely cross-react with pSer129 alphaS when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological alphaS inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 alphaS antibodies reacted with the pSer473 NFL epitope, but 2 clones (LS3-2C2 and LS4-2G12) did not. However, LS3-2C2 demonstrated cross-reactivity with other proteins. Our findings further demonstrate the difficulties in generating specific pSer129 alphaS antibodies, but highlights that the use of multiple antibodies, such as those generated here, can provide a sensitive and accurate assessment of alphaS pathology. Keywords: Monoclonal antibodies, alpha-synuclein, Neurofilament, Parkinson's disease, Phosphorylation
In order to further evaluate the parameters whereby intracerebral administration of recombinant alpha-synuclein (alphaS) induces pathological phenotypes in mice, we conducted a series of studies ...where alphaS fibrils were injected into the brains of M83 (A53T) and M47 (E46K) alphaS transgenic (Tg) mice, and non-transgenic (nTg) mice. Using multiple markers to assess alphaS inclusion formation, we find that injected fibrillar human alphaS induced widespread cerebral alphaS inclusion formation in the M83 Tg mice, but in both nTg and M47 Tg mice, induced alphaS inclusion pathology is largely restricted to the site of injection. Furthermore, mouse alphaS fibrils injected into nTg mice brains also resulted in inclusion pathology restricted to the site of injection with no evidence for spread. We find no compelling evidence for extensive spread of alphaS pathology within white matter tracts, and we attribute previous reports of white matter tract spreading to cross-reactivity of the alphaS pSer129/81A antibody with phosphorylated neurofilament subunit L. These studies suggest that, with the exception of the M83 Tg mice which appear to be uniquely susceptible to induction of inclusion pathology by exogenous forms of alphaS, there are significant barriers in mice to widespread induction of alphaS pathology following intracerebral administration of amyloidogenic alphaS.PUBLICATION ABSTRACT
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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