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Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration.
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from patients with sporadic ALS and familial ALS with GGGGCC repeat expansion in the C9orf72 gene, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS-FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability
CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint. Nat Cell Biol
Here we show that suppression of synthesis of the microtubule motor CENP-E (centromere-associated protein E), a component of the kinetochore corona fibres of mammalian centromeres, yields chromosomes that are chronically mono-orientated, with spindles that are flattened along the plane of the substrate. Despite apparently normal microtubule numbers and the continued presence at kinetochores of other microtubule motors, spindle poles fragment in the absence of CENP-E, which implicates this protein in delivery of components from kinetochores to poles. CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signalling cascade, as depletion of this motor leads to profound checkpoint activation, whereas immunoprecipitation reveals a nearly stoichiometric association of CENP-E with the checkpoint kinase BubR1 during mitosis. hromosome movements during mitosis are orchestrated primarily by the interaction of spindle microtubules with the kinetochore 1 , the site for attachment of spindle microtubules to the centromere. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore has an active function in chromosomal segregation through microtubule motors located at or near i
Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers.
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
Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease
Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.
Significance Immunotherapy has been proposed for neurodegenerative disorders including Alzheimer’s or Parkinson’s diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G4C2)149 mice
ALS-causative mutations in FUS/TLS confer gain- and loss-of-function by altered association with SMN and U1-snRNP
The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease ALS, is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and dysregulate its function, including loss of Gems and altered levels of small nuclear RNAs (snRNAs). The same mutants are found to have reduced association with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, these studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function
ACC/AHA/SCAI/AMA–Convened PCPI/NCQA 2013 Performance Measures for Adults Undergoing Percutaneous Coronary Intervention A Report of the American College of Cardiology/American Heart Association Task Force on Performance Measures, the Society for Cardiovascular Angiography and Interventions, the American Medical Association–Convened Physician Consortium for Performance Improvement, and the National Committee for Quality Assurance
Journal of the American College of Cardiology Ó 2014 by the American College of Cardiology Foundation, American Heart Association, Inc., American Medical Association, and National Committee for Quality Assurance Published by Elsevier Inc. Vol. 63, No. 7, 2014 ISSN 0735-1097/$36.00 http://dx.doi.org/10.1016/j.jacc.2013.12.003 PERFORMANCE MEASURES ACC/AHA/SCAI/AMA–Convened PCPI/NCQA 2013 Performance Measures for Adults Undergoing Percutaneous Coronary Intervention A Report of the American College of Cardiology/American Heart Association Task Force on Performance Measures, the Society for Cardiovascular Angiography and Interventions, the American Medical Association–Convened Physician Consortium for Performance Improvement, and the National Committee for Quality Assurance Developed in Collaboration With the American Association of Cardiovascular and Pulmonary Rehabilitation and Mended Hearts Endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation and Mended Hearts WRITING COMMITTEE MEMBERS Brahmajee K. Nallamothu, MD, MPH, FACC, FAHA, Co-Chair*; Carl L. Tommaso, MD, FACC, FAHA, FSCAI, Co-Chairy; H. Vernon Anderson, MD, FACC, FAHA, FSCAI*; Jeffrey L. Anderson, MD, FACC, FAHA, MACP*; Joseph C. Cleveland, J R , MDz; R. Adams Dudley, MD, MBA; Peter Louis Duffy, MD, MMM, FACC, FSCAIy; David P. Faxon, MD, FACC, FAHA*; Hitinder S. Gurm, MD, FACC; Lawrence A. Hamilton, Neil C. Jensen, MHA, MBA; Richard A. Josephson, MD, MS, FACC, FAHA, FAACVPRx; David J. Malenka, MD, FACC, FAHA*; Calin V. Maniu, MD, FACC, FAHA, FSCAIy; Kevin W. McCabe, MD; James D. Mortimer, Manesh R. Patel, MD, FACC*; Stephen D. Persell, MD, MPH; John S. Rumsfeld, MD, PhD, FACC, FAHAjj; Kendrick A. Shunk, MD, PhD, FACC, FAHA, FSCAI*; Sidney C. Smith, J R , MD, FACC, FAHA, FACP{; Stephen J. Stanko, MBA, BA, AA#; Brook Watts, MD, MS *ACC/AHA Representative. ySociety of Cardiovascular Angiography and Interventions Representative. zSociety of Thoracic Surgeons Representative. xAmerican Association of Cardiovascular and Pulmonary Rehabilitation Representative. kACC/AHA Task Force on Performance Measures Liaison. {National Heart Lung and Blood Institute Representative. #Mended Hearts Representative. The measure specifications were approved by the American College of Cardiology Board of Trustees, American Heart Association Science Advisory and Coordinating Committee, in January 2013 and the American Medical Association–Physician Consortium for Performance Improvement in February 2013. This document was approved by the American College of Cardiology Board of Trustees and the American Heart Association Science Advisory and Coordinating Committee in October 2013, and the Society of Cardiovascular Angiography and Interventions in December 2013. The American College of Cardiology requests that this document be cited as follows: Nallamothu BK, Tommaso CL, Anderson HV, Anderson JL, Cleveland JC, Dudley RA, Duffy PL, Faxon DP, Gurm HS, Hamilton LA, Jensen NC, Josephson RA, Malenka DJ, Maniu CV, McCabe KW, Mortimer JD, Patel MR, Persell SD, Rumsfeld JS, Shunk KA, Smith SC, Stanko SJ, Watts B. ACC/AHA/SCAI/AMA–Convened PCPI/NCQA 2013 perfor- mance measures for adults undergoing percutaneous coronary intervention: a report of the American College of Cardiology/American Heart Association Task Force on Performance Measures, the Society for Cardiovascular Angiography and Interventions, the American Medical Association–Convened Physician Consortium for Performance Improvement, and the National Committee for Quality Assurance. J Am Coll Cardiol 2014;63:722–45. This article has been copublished in Circulation. Copies: This document is available on the World Wide Web sites of the American College of Cardiology (www.cardiosource.org) and the American Heart Asso- ciation (http://my.americanheart.org). For copies of this document, please contact Elsevier Inc. Reprint Department, fax (212) 633-3820, e-mail [email protected]. Permissions: Multiple copies, modification, alteration, enhancement, and/or distribution of this document are not permitted without the express permission of the American College of Cardiology. Requests may be completed online via the Elsevier site (http://www.elsevier.com/authors/obtaining- permission-to-re-use-elsevier-material). This Physician Performance Measurement Set (PPMS) and related data specifications were developed by the Physician Consortium for Performance Improvement (the Consortium), including the American College of Cardiology (ACC), the American Heart Association (AHA), and the American Medical Association (AMA), to facilitate quality-improvement activities by physicians. The performance measures contained in this PPMS are not clinical guidelines, do not establish a standard of medical care, and have not been tested for all potential applications. Although copyrighted, they can be reproduced and distributed, without modification, for noncommercial purposesdfor example, use by health care pro
Widespread occurrence of non-canonical transcription termination by human RNA polymerase III
Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNAs
Chromosomal Instability by Inefficient Mps1 Auto-Activation Due to a Weakened Mitotic Checkpoint and Lagging Chromosomes
BACKGROUND: Chromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase. PRINCIPAL FINDINGS: Here we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively. CONCLUSIONS/SIGNIFICANCE: We propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability
Exome-wide Rare Variant Analysis Identifies TUBA4A Mutations Associated with Familial ALS
Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis
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