Publications by authors named "Tania F Gendron"

87 Publications

A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks.

ACS Chem Neurosci 2021 Oct 22. Epub 2021 Oct 22.

Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States.

The hexanucleotide repeat expansion GGGGCC [r(GC)] within intron 1 of causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(GC) RNA gain- and loss-of-function mechanisms. The r(GC) RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(GC). Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(GC) hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.
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http://dx.doi.org/10.1021/acschemneuro.1c00470DOI Listing
October 2021

A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation.

Nat Commun 2021 Oct 15;12(1):6025. Epub 2021 Oct 15.

University of Chicago Medical Center, 5841S. Maryland Avenue, Chicago, IL, 60637, USA.

A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.
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http://dx.doi.org/10.1038/s41467-021-26303-xDOI Listing
October 2021

Serum neurofilament light protein correlates with unfavorable clinical outcomes in hospitalized patients with COVID-19.

Sci Transl Med 2021 07 15;13(602). Epub 2021 Jun 15.

Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA.

Brain imaging studies of patients with COVID-19 show evidence of macro- and microhemorrhagic lesions, multifocal white matter hyperintensities, and lesions consistent with posterior reversible leukoencephalopathy. Imaging studies, however, are subject to selection bias, and prospective studies are challenging to scale. Here, we evaluated whether serum neurofilament light chain (NFL), a neuroaxonal injury marker, could predict the extent of neuronal damage in a cohort of 142 hospitalized patients with COVID-19. NFL was elevated in the serum of patients with COVID-19 compared to healthy controls, including those without overt neurological manifestations. Higher NFL serum concentrations were associated with worse clinical outcomes. In 100 hospitalized patients with COVID-19 treated with remdesivir, a trend toward lower NFL serum concentrations was observed. These data suggest that patients with COVID-19 may experience neuroaxonal injury and may be at risk for long-term neurological sequelae. Neuroaxonal injury should be considered as an outcome in acute pharmacotherapeutic trials for COVID-19.
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http://dx.doi.org/10.1126/scitranslmed.abi7643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432951PMC
July 2021

Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases.

Brain 2021 05;144(4):1082-1088

Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA.

To examine the length of a hexanucleotide expansion in C9orf72, which represents the most frequent genetic cause of frontotemporal lobar degeneration and motor neuron disease, we employed a targeted amplification-free long-read sequencing technology: No-Amp sequencing. In our cross-sectional study, we assessed cerebellar tissue from 28 well-characterized C9orf72 expansion carriers. We obtained 3507 on-target circular consensus sequencing reads, of which 814 bridged the C9orf72 repeat expansion (23%). Importantly, we observed a significant correlation between expansion sizes obtained using No-Amp sequencing and Southern blotting (P = 5.0 × 10-4). Interestingly, we also detected a significant survival advantage for individuals with smaller expansions (P = 0.004). Additionally, we uncovered that smaller expansions were significantly associated with higher levels of C9orf72 transcripts containing intron 1b (P = 0.003), poly(GP) proteins (P = 1.3 × 10- 5), and poly(GA) proteins (P = 0.005). Thorough examination of the composition of the expansion revealed that its GC content was extremely high (median: 100%) and that it was mainly composed of GGGGCC repeats (median: 96%), suggesting that expanded C9orf72 repeats are quite pure. Taken together, our findings demonstrate that No-Amp sequencing is a powerful tool that enables the discovery of relevant clinicopathological associations, highlighting the important role played by the cerebellar size of the expanded repeat in C9orf72-linked diseases.
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http://dx.doi.org/10.1093/brain/awab006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105038PMC
May 2021

Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial.

Stem Cell Res Ther 2021 03 18;12(1):187. Epub 2021 Mar 18.

Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

Background: Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases.

Methods: In this study, we characterized changes in morphology, metabolism, and gene expression occurring in human adipose-derived MSCs cultured in human (hCSF) or artificial CSF (aCSF) as well as examined relevant protein levels in the CSF of subjects treated with MSCs for amyotrophic lateral sclerosis (ALS).

Results: Our results demonstrated that, under intrathecal-like conditions, MSCs retained their morphology, though they became quiescent. Large-scale transcriptomic analysis of MSCs revealed a distinct gene expression profile for cells cultured in aCSF. The aCSF culture environment induced expression of genes related to angiogenesis and immunomodulation. In addition, MSCs in aCSF expressed genes encoding nutritional growth factors to expression levels at or above those of control cells. Furthermore, we observed a dose-dependent increase in growth factors and immunomodulatory cytokines in CSF from subjects with ALS treated intrathecally with autologous MSCs.

Conclusions: Overall, our results suggest that MSCs injected into the intrathecal space in ongoing clinical trials remain viable and may provide a therapeutic benefit to patients.
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http://dx.doi.org/10.1186/s13287-021-02241-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7977179PMC
March 2021

Structural Features of Small Molecules Targeting the RNA Repeat Expansion That Causes Genetically Defined ALS/FTD.

ACS Chem Biol 2020 12 16;15(12):3112-3123. Epub 2020 Nov 16.

Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States.

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r(GC)] within the gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r(GC) RNA gain-of-function mechanisms. Herein, we identified a benzimidazole derivative, CB096, that specifically binds to a repeating 1 × 1 GG internal loop structure, 5'CG/3'GC, that is formed when r(GC) folds. Structure-activity relationship (SAR) studies and molecular dynamics (MD) simulations were used to define the molecular interactions formed between CB096 and r(GC) that results in the rescue of disease-associated pathways. Overall, this study reveals a unique structural feature within r(GC) that can be exploited for the development of lead medicines and chemical probes.
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http://dx.doi.org/10.1021/acschembio.0c00049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788565PMC
December 2020

Plasma neurofilament light predicts mortality in patients with stroke.

Sci Transl Med 2020 11;12(569)

Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA.

Given the heterogeneity of stroke brain injury, there is a clear need for a biomarker that determines the degree of neuroaxonal injury across stroke types. We evaluated whether blood neurofilament light (NFL) would fulfill this purpose for patients with acute cerebral infarction (ACI; = 227), aneurysmal subarachnoid hemorrhage (aSAH; = 58), or nontraumatic intracerebral hemorrhage (ICH; = 29). We additionally validated our findings in two independent cohorts of patients with ICH ( = 96 and = 54) given the scarcity of blood biomarker studies for this deadliest stroke type. Compared to healthy individuals ( = 79 and = 48 for the discovery and validation cohorts, respectively), NFL was higher for all stroke types. NFL associated with radiographic markers of brain tissue damage. It correlated with the extent of early ischemic injury in patients with ACI, hemorrhage severity in patients with aSAH, and intracranial hemorrhage volume in patients with ICH. In all patients, NFL independently correlated with scores from the NIH Stroke Scale, the modified Rankin Scale, and the Mini-Mental State Examination at blood draw, which respectively assess neurological, functional, and cognitive status. Furthermore, higher NFL concentrations independently associated with 3- or 6-month functional disability and higher all-cause mortality. These data support NFL as a uniform method to estimate neuroaxonal injury and forecast mortality regardless of stroke mechanism. As a prognostic biomarker, blood NFL has the potential to assist with planning supportive and rehabilitation services and improving clinical trial efficiency for stroke therapeutics and devices.
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http://dx.doi.org/10.1126/scitranslmed.aay1913DOI Listing
November 2020

Sensitive ELISA-based detection method for the mitophagy marker p-S65-Ub in human cells, autopsy brain, and blood samples.

Autophagy 2021 Sep 28;17(9):2613-2628. Epub 2020 Oct 28.

Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.

Mitochondrial dysfunction is an early, imminent event in neurodegenerative disorders including Parkinson disease (PD) and Alzheimer disease (AD). The enzymatic pair PINK1 and PRKN/Parkin recognize and transiently label damaged mitochondria with ubiquitin (Ub) phosphorylated at Ser65 (p-S65-Ub) as a signal for degradation via the autophagy-lysosome system (mitophagy). Despite its discovery in cell culture several years ago, robust and quantitative detection of altered mitophagy has remained challenging. Here we developed a sandwich ELISA targeting p-S65-Ub with the goal to assess mitophagy levels in mouse brain and in human clinical and pathological samples. We characterized five total Ub and four p-S65-Ub antibodies by several techniques and found significant differences in their ability to recognize phosphorylated Ub. The most sensitive antibody pair detected recombinant p-S65-Ub chains in the femtomolar to low picomolar range depending on the poly-Ub chain linkage. Importantly, this ELISA was able to assess very low baseline mitophagy levels in unstressed human cells and in brains from wild-type and knockout mice as well as elevated p-S65-Ub levels in autopsied frontal cortex from AD patients vs. control cases. Moreover, the assay allowed detection of p-S65-Ub in blood plasma and was able to discriminate between mutation carriers and controls. In summary, we developed a robust and sensitive tool to measure mitophagy levels in cells, tissue, and body fluids. Our data strongly support the idea that the stress-activated PINK1-PRKN mitophagy pathway is constitutively active in mice and humans under unstimulated, physiological and elevated in diseased, pathological conditions.: Ab: antibody; AD: Alzheimer disease; AP: alkaline phosphatase; CV: coefficient of variation; ECL: electrochemiluminescence; KO: knockout; LoB: Limit of Blank; LoD: Limit of Detection; LoQ: Limit of Quantification; MSD: meso scale discovery; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated ubiquitin at serine 65; Std.Dev.: standard deviation; Ub: ubiquitin; WT: wild type.
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http://dx.doi.org/10.1080/15548627.2020.1834712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496550PMC
September 2021

Toward allele-specific targeting therapy and pharmacodynamic marker for spinocerebellar ataxia type 3.

Sci Transl Med 2020 10;12(566)

Department of Pathology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan.

Spinocerebellar ataxia type 3 (SCA3), caused by a CAG repeat expansion in the ataxin-3 gene (), is characterized by neuronal polyglutamine (polyQ) ATXN3 protein aggregates. Although there is no cure for SCA3, gene-silencing approaches to reduce toxic polyQ ATXN3 showed promise in preclinical models. However, a major limitation in translating putative treatments for this rare disease to the clinic is the lack of pharmacodynamic markers for use in clinical trials. Here, we developed an immunoassay that readily detects polyQ ATXN3 proteins in human biological fluids and discriminates patients with SCA3 from healthy controls and individuals with other ataxias. We show that polyQ ATXN3 serves as a marker of target engagement in human fibroblasts, which may bode well for its use in clinical trials. Last, we identified a single-nucleotide polymorphism that strongly associates with the expanded allele, thus providing an exciting drug target to abrogate detrimental events initiated by mutant ATXN3. Gene-silencing strategies for several repeat diseases are well under way, and our results are expected to improve clinical trial preparedness for SCA3 therapies.
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http://dx.doi.org/10.1126/scitranslmed.abb7086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927160PMC
October 2020

poly(GR) aggregation induces TDP-43 proteinopathy.

Sci Transl Med 2020 09;12(559)

Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA.

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by GC repeat expansions in the gene (c9FTD/ALS). Providing mechanistic insight into the link between mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded GC repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR) mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating GC repeat-expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that GC-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.
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http://dx.doi.org/10.1126/scitranslmed.abb3774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989020PMC
September 2020

Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia.

J Clin Invest 2020 11;130(11):6080-6092

Center for Genomics of Neurodegenerative Disease, and.

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.
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http://dx.doi.org/10.1172/JCI139741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598060PMC
November 2020

Chimeric Peptide Species Contribute to Divergent Dipeptide Repeat Pathology in c9ALS/FTD and SCA36.

Neuron 2020 07 5;107(2):292-305.e6. Epub 2020 May 5.

Department of Neurology, Emory University, Atlanta, GA 30322, USA.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.
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http://dx.doi.org/10.1016/j.neuron.2020.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138626PMC
July 2020

Hexanucleotide Repeat Expansions in c9FTD/ALS and SCA36 Confer Selective Patterns of Neurodegeneration In Vivo.

Cell Rep 2020 05;31(5):107616

Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

A GC hexanucleotide repeat expansion in an intron of C9orf72 is the most common cause of frontal temporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). A remarkably similar intronic TGC repeat expansion is associated with spinocerebellar ataxia 36 (SCA36). Both expansions are widely expressed, form RNA foci, and can undergo repeat-associated non-ATG (RAN) translation to form similar dipeptide repeat proteins (DPRs). Yet, these diseases result in the degeneration of distinct subsets of neurons. We show that the expression of these repeat expansions in mice is sufficient to recapitulate the unique features of each disease, including this selective neuronal vulnerability. Furthermore, only the GC repeat induces the formation of aberrant stress granules and pTDP-43 inclusions. Overall, our results demonstrate that the pathomechanisms responsible for each disease are intrinsic to the individual repeat sequence, highlighting the importance of sequence-specific RNA-mediated toxicity in each disorder.
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http://dx.doi.org/10.1016/j.celrep.2020.107616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480900PMC
May 2020

Reduced C9ORF72 function exacerbates gain of toxicity from ALS/FTD-causing repeat expansion in C9orf72.

Nat Neurosci 2020 05 13;23(5):615-624. Epub 2020 Apr 13.

Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA.

Hexanucleotide expansions in C9orf72, which encodes a predicted guanine exchange factor, are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although repeat expansion has been established to generate toxic products, mRNAs encoding the C9ORF72 protein are also reduced in affected individuals. In this study, we tested how C9ORF72 protein levels affected repeat-mediated toxicity. In somatic transgenic mice expressing 66 GGGGCC repeats, inactivation of one or both endogenous C9orf72 alleles provoked or accelerated, respectively, early death. In mice expressing a C9orf72 transgene with 450 repeats that did not encode the C9ORF72 protein, inactivation of one or both endogenous C9orf72 alleles exacerbated cognitive deficits, hippocampal neuron loss, glial activation and accumulation of dipeptide-repeat proteins from translation of repeat-containing RNAs. Reduced C9ORF72 was shown to suppress repeat-mediated elevation in autophagy. These efforts support a disease mechanism in ALS/FTD resulting from reduced C9ORF72, which can lead to autophagy deficits, synergizing with repeat-dependent gain of toxicity.
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http://dx.doi.org/10.1038/s41593-020-0619-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384305PMC
May 2020

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD.

Mol Neurodegener 2020 02 24;15(1):13. Epub 2020 Feb 24.

Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Biomedical Research Building Room 413, Florida, Miami, 33136, USA.

Background: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.

Methods And Results: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.

Conclusions: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.
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http://dx.doi.org/10.1186/s13024-020-00365-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041170PMC
February 2020

Nucleocytoplasmic Proteomic Analysis Uncovers eRF1 and Nonsense-Mediated Decay as Modifiers of ALS/FTD C9orf72 Toxicity.

Neuron 2020 04 13;106(1):90-107.e13. Epub 2020 Feb 13.

The Ken & Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift toward a more cytosolic proteome in C9-HRE cells. Among these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient induced pluripotent stem cell (iPSC) neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS and/or FTD.
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http://dx.doi.org/10.1016/j.neuron.2020.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272217PMC
April 2020

Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD.

Nat Commun 2019 11 29;10(1):5466. Epub 2019 Nov 29.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

Expanded GGGGCC (GC) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded GC repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded GC repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded GC repeats in human C9ORF72-ALS/FTD neurons.
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http://dx.doi.org/10.1038/s41467-019-13477-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884579PMC
November 2019

Extensive transcriptomic study emphasizes importance of vesicular transport in C9orf72 expansion carriers.

Acta Neuropathol Commun 2019 10 8;7(1):150. Epub 2019 Oct 8.

Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.

The majority of the clinico-pathological variability observed in patients harboring a repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) remains unexplained. This expansion, which represents the most common genetic cause of frontotemporal lobar degeneration (FTLD) and motor neuron disease (MND), results in a loss of C9orf72 expression and the generation of RNA foci and dipeptide repeat (DPR) proteins. The C9orf72 protein itself plays a role in vesicular transport, serving as a guanine nucleotide exchange factor that regulates GTPases. To further elucidate the mechanisms underlying C9orf72-related diseases and to identify potential disease modifiers, we performed an extensive RNA sequencing study. We included individuals for whom frontal cortex tissue was available: FTLD and FTLD/MND patients with (n = 34) or without (n = 44) an expanded C9orf72 repeat as well as control subjects (n = 24). In total, 6706 genes were differentially expressed between these groups (false discovery rate [FDR] < 0.05). The top gene was C9orf72 (FDR = 1.41E-14), which was roughly two-fold lower in C9orf72 expansion carriers than in (disease) controls. Co-expression analysis revealed groups of correlated genes (modules) that were enriched for processes such as protein folding, RNA splicing, synaptic signaling, metabolism, and Golgi vesicle transport. Within our cohort of C9orf72 expansion carriers, machine learning uncovered interesting candidates associated with clinico-pathological features, including age at onset (vascular endothelial growth factor A [VEGFA]), C9orf72 expansion size (cyclin dependent kinase like 1 [CDKL1]), DPR protein levels (eukaryotic elongation factor 2 kinase [EEF2K]), and survival after onset (small G protein signaling modulator 3 [SGSM3]). Given the fact that we detected a module involved in vesicular transport in addition to a GTPase activator (SGSM3) as a potential modifier, our findings seem to suggest that the presence of a C9orf72 repeat expansion might hamper vesicular transport and that genes affecting this process may modify the phenotype of C9orf72-linked diseases.
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http://dx.doi.org/10.1186/s40478-019-0797-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781370PMC
October 2019

RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats.

Nat Neurosci 2019 09 29;22(9):1383-1388. Epub 2019 Jul 29.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.
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http://dx.doi.org/10.1038/s41593-019-0455-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713615PMC
September 2019

Aberrant deposition of stress granule-resident proteins linked to C9orf72-associated TDP-43 proteinopathy.

Mol Neurodegener 2019 02 15;14(1). Epub 2019 Feb 15.

Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Rd, Jacksonville, FL, 32224, USA.

Background: A GC hexanucleotide repeat expansion in the noncoding region of C9orf72 is the major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). Putative disease mechanisms underlying c9FTD/ALS include toxicity from sense GC and antisense GC repeat-containing RNA, and from dipeptide repeat (DPR) proteins unconventionally translated from these RNA products.

Methods: Intracerebroventricular injections with adeno-associated virus (AAV) encoding 2 or 149 GC repeats were performed on postnatal day 0, followed by assessment of behavioral and neuropathological phenotypes.

Results: Relative to control mice, gliosis and neurodegeneration accompanied by cognitive and motor deficits were observed in (GC) mice by 6 months of age. Recapitulating key pathological hallmarks, we also demonstrate that sense and antisense RNA foci, inclusions of poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA) DPR proteins, and inclusions of endogenous phosphorylated TDP-43 (pTDP-43) developed in (GC) mice but not control (GC) mice. Notably, proteins that play a role in the regulation of stress granules - RNA-protein assemblies that form in response to translational inhibition and that have been implicated in c9FTD/ALS pathogenesis - were mislocalized in (GC) mice as early as 3 months of age. Specifically, we observed the abnormal deposition of stress granule components within inclusions immunopositive for poly(GR) and pTDP-43, as well as evidence of nucleocytoplasmic transport defects.

Conclusions: Our in vivo model of c9FTD/ALS is the first to robustly recapitulate hallmark features derived from both sense and antisense C9orf72 repeat-associated transcripts complete with neurodegeneration and behavioral impairments. More importantly, the early appearance of persistent pathological stress granules prior to significant pTDP-43 deposition implicates an aberrant stress granule response as a key disease mechanism driving TDP-43 proteinopathy in c9FTD/ALS.
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http://dx.doi.org/10.1186/s13024-019-0310-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377782PMC
February 2019

Heterochromatin anomalies and double-stranded RNA accumulation underlie poly(PR) toxicity.

Science 2019 02;363(6428)

Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, and Chan Zuckerberg Biohub, San Francisco, CA, USA.

How hexanucleotide GGGGCC (GC) repeat expansions in cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded GC repeats. The expression of green fluorescent protein-conjugated (PR) (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of -associated FTD and ALS.
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http://dx.doi.org/10.1126/science.aav2606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524780PMC
February 2019

Biomarkers for Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Associated With Hexanucleotide Expansion Mutations in .

Front Neurol 2018 5;9:1063. Epub 2018 Dec 5.

Department of Neuroscience, Mayo Clinic, Jacksonville, FL, United States.

Now that genetic testing can identify persons at risk for developing amyotrophic lateral sclerosis (ALS) many decades before symptoms begin, there is a critical need for biomarkers that signal the onset and progression of degeneration. The search for candidate disease biomarkers in patients with mutations in the gene has included imaging, physiology, and biofluid measurements. In cross-sectional imaging studies, C9+ ALS patients display diffuse reductions of gray and white matter integrity compared to ALS patients without mutations. This structural imaging signature overlaps with frontotemporal dementia (FTD), reflecting the frequent co-occurrence of cognitive impairment, even frank FTD, in C9+ ALS patients. Changes in functional connectivity occur as critical components of the networks associated with cognition and behavior degenerate. In presymptomatic C9+carriers, subtle differences in volumes of subcortical structures and functional connectivity can be detected, often decades before the typical family age of symptom onset. Dipeptide repeat proteins produced by the repeat expansion mutation are also measurable in the cerebrospinal fluid (CSF) of presymptomatic gene carriers, possibly throughout their lives. In contrast, a rise in the level of neurofilament proteins in the CSF appears to presage the onset of degeneration in presymptomatic carriers in one longitudinal study. Cross-sectional studies indicate that neurofilament protein levels may provide prognostic information for survival in C9+ ALS patients. Longitudinal studies will be needed to validate the candidate biomarkers discussed here. Understanding how these candidate biomarkers change over time is critical if they are to be used in future therapeutic decisions.
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http://dx.doi.org/10.3389/fneur.2018.01063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289985PMC
December 2018

The Hairpin Form of r(GC) in c9ALS/FTD Is Repeat-Associated Non-ATG Translated and a Target for Bioactive Small Molecules.

Cell Chem Biol 2019 02 29;26(2):179-190.e12. Epub 2018 Nov 29.

Departments of Chemistry and Neuroscience, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA. Electronic address:

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded GC repeat [(GC)] in C9ORF72. ALS/FTD-associated toxicity has been traced to the RNA transcribed from the repeat expansion [r(GC)], which sequesters RNA-binding proteins (RBPs) and undergoes repeat-associated non-ATG (RAN) translation to generate toxic dipeptide repeats. Using in vitro and cell-based assays, we identified a small molecule (4) that selectively bound r(GC), prevented sequestration of an RBP, and inhibited RAN translation. Indeed, biophysical characterization showed that 4 selectively bound the hairpin form of r(GC), and nuclear magnetic resonance spectroscopy studies and molecular dynamics simulations defined this molecular recognition event. Cellular imaging revealed that 4 localized to r(GC) cytoplasmic foci, the putative sites of RAN translation. Collectively, these studies highlight that the hairpin structure of r(GC) is a therapeutically relevant target and small molecules that bind it can ameliorate c9ALS/FTD-associated toxicity.
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http://dx.doi.org/10.1016/j.chembiol.2018.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386614PMC
February 2019

Amyotrophic Lateral Sclerosis: An Update for 2018.

Mayo Clin Proc 2018 11 4;93(11):1617-1628. Epub 2018 Jul 4.

Department of Neurology, Mayo Clinic, Rochester, MN.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons and other neuronal cells, leading to severe disability and eventually death from ventilatory failure. It has a prevalence of 5 in 100,000, with an incidence of 1.7 per 100,000, reflecting short average survival. The pathogenesis is incompletely understood, but defects of RNA processing and protein clearance may be fundamental. Repeat expansions in the chromosome 9 open reading frame 72 gene (C9orf72) are the most common known genetic cause of ALS and are seen in approximately 40% of patients with a family history and approximately 10% of those without. No environmental risk factors are proved to be causative, but many have been proposed, including military service. The diagnosis of ALS rests on a history of painless progressive weakness coupled with examination findings of upper and lower motor dysfunction. No diagnostic test is yet available, but electromyography and genetic tests can support the diagnosis. Care for patients is best provided by a multidisciplinary team, and most interventions are directed at managing symptoms. Two medications with modest benefits have Food and Drug Administration approval for the treatment of ALS: riluzole, a glutamate receptor antagonist, and, new in 2017, edaravone, a free radical scavenger. Many other encouraging treatment strategies are being explored in clinical trials for ALS; herein we review stem cell and antisense oligonucleotide gene therapies.
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http://dx.doi.org/10.1016/j.mayocp.2018.04.007DOI Listing
November 2018

Long-read sequencing across the C9orf72 'GGGGCC' repeat expansion: implications for clinical use and genetic discovery efforts in human disease.

Mol Neurodegener 2018 08 21;13(1):46. Epub 2018 Aug 21.

Department of Neuroscience, Mayo Clinic, Jacksonville, FL, 32224, USA.

Background: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 'GGGGCC' (GC) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences' (PacBio) and Oxford Nanopore Technologies' (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing GC repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 GC repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.

Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained > 800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual's repeat region was > 99% GC content, though we cannot rule out small interruptions.

Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-GC content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.
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http://dx.doi.org/10.1186/s13024-018-0274-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102925PMC
August 2018

Poly-GR dipeptide repeat polymers correlate with neurodegeneration and Clinicopathological subtypes in C9ORF72-related brain disease.

Acta Neuropathol Commun 2018 07 20;6(1):63. Epub 2018 Jul 20.

Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.

Frontotemporal lobar degeneration (FTLD) is heterogeneous in clinical presentation, neuropathological characteristics and genetics. An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of both FTLD and motor neuron disease (MND). Dipeptide repeat polymers (DPR) are generated through repeat-associated non-ATG translation, and they aggregate in neuronal inclusions with a distribution distinct from that of TDP-43 pathology. Recent studies from animal and cell culture models suggest that DPR might be toxic, but that toxicity may differ for specific DPR. Arginine containing DPR (poly-GR and poly-PR) have the greatest toxicity and are less frequent than other DPR (poly-GP, poly-GA). A unique feature of arginine-containing DPR is their potential for post-translational modification by methyl-transferases, which produces methylarginine DPR. In this report, we explored the relationship of DPR and methylarginine to markers of neurodegeneration using quantitative digital microscopic methods in 40 patients with C9ORF72 mutations and one of three different clinicopathologic phenotypes, FTLD, FTLD-MND or MND. We find that density and distribution of poly-GR inclusions are different from poly-GA and poly-GP inclusions. We also demonstrate colocalization of poly-GR with asymmetrical dimethylarginine (aDMA) immunoreactivity in regions with neurodegeneration. Differences in aDMA were also noted by clinical phenotype. FTLD-MND had the highest burden of poly-GR pathology compared to FTLD and MND, while FTLD-MND had higher burden of aDMA than FTLD. The results suggest that poly-GR pathology is associated with toxicity and neurodegeneration. It remains to be determined if dimethylarginine modification of poly-GR could contribute to its toxicity.
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http://dx.doi.org/10.1186/s40478-018-0564-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054740PMC
July 2018

Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis.

Nat Med 2018 08 25;24(8):1136-1142. Epub 2018 Jun 25.

Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.

The major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a C9orf72 GC repeat expansion. Proposed mechanisms by which the expansion causes c9FTD/ALS include toxicity from repeat-containing RNA and from dipeptide repeat proteins translated from these transcripts. To investigate the contribution of poly(GR) dipeptide repeat proteins to c9FTD/ALS pathogenesis in a mammalian in vivo model, we generated mice that expressed GFP-(GR) in the brain. GFP-(GR) mice developed age-dependent neurodegeneration, brain atrophy, and motor and memory deficits through the accumulation of diffuse, cytoplasmic poly(GR). Poly(GR) co-localized with ribosomal subunits and the translation initiation factor eIF3η in GFP-(GR) mice and, of importance, in c9FTD/ALS patients. Combined with the differential expression of ribosome-associated genes in GFP-(GR) mice, these findings demonstrate poly(GR)-mediated ribosomal distress. Indeed, poly(GR) inhibited canonical and non-canonical protein translation in HEK293T cells, and also induced the formation of stress granules and delayed their disassembly. These data suggest that poly(GR) contributes to c9FTD/ALS by impairing protein translation and stress granule dynamics, consequently causing chronic cellular stress and preventing cells from mounting an effective stress response. Decreasing poly(GR) and/or interrupting interactions between poly(GR) and ribosomal and stress granule-associated proteins may thus represent potential therapeutic strategies to restore homeostasis.
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http://dx.doi.org/10.1038/s41591-018-0071-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520050PMC
August 2018

Loss of Tmem106b is unable to ameliorate frontotemporal dementia-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity.

Acta Neuropathol Commun 2018 05 31;6(1):42. Epub 2018 May 31.

Department of Neuroscience, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.

Loss-of-function mutations in progranulin (GRN) and a non-coding (GGGGCC) hexanucleotide repeat expansions in C9ORF72 are the two most common genetic causes of frontotemporal lobar degeneration with aggregates of TAR DNA binding protein 43 (FTLD-TDP). TMEM106B encodes a type II transmembrane protein with unknown function. Genetic variants in TMEM106B associated with reduced TMEM106B levels have been identified as disease modifiers in individuals with GRN mutations and C9ORF72 expansions. Recently, loss of Tmem106b has been reported to protect the FTLD-like phenotypes in Grn-/- mice. Here, we generated Tmem106b-/- mice and examined whether loss of Tmem106b could rescue FTLD-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity. Our results showed that neither partial nor complete loss of Tmem106b was able to rescue behavioral deficits induced by the expression of (GGGGCC) repeats (66R). Loss of Tmem106b also failed to ameliorate 66R-induced RNA foci, dipeptide repeat protein formation and pTDP-43 pathological burden. We further found that complete loss of Tmem106b increased astrogliosis, even in the absence of 66R, and failed to rescue 66R-induced neuronal cell loss, whereas partial loss of Tmem106b significantly rescued the neuronal cell loss but not neuroinflammation induced by 66R. Finally, we showed that overexpression of 66R did not alter expression of Tmem106b and other lysosomal genes in vivo, and subsequent analyses in vitro found that transiently knocking down C9ORF72, but not overexpression of 66R, significantly increased TMEM106B and other lysosomal proteins. In summary, reducing Tmem106b levels failed to rescue FTLD-like phenotypes in a mouse model mimicking the toxic gain-of-functions associated with overexpression of 66R. Combined with the observation that loss of C9ORF72 and not 66R overexpression was associated with increased levels of TMEM106B, this work suggests that the protective TMEM106B haplotype may exert its effect in expansion carriers by counteracting lysosomal dysfunction resulting from a loss of C9ORF72.
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http://dx.doi.org/10.1186/s40478-018-0545-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984311PMC
May 2018

Poly(GP), neurofilament and grey matter deficits in expansion carriers.

Ann Clin Transl Neurol 2018 May 6;5(5):583-597. Epub 2018 Apr 6.

Division of Neurogeriatrics Department NVS Karolinska Institutet Center for Alzheimer Research Huddinge 14157 Sweden.

Objective: To evaluate poly(GP), a dipeptide repeat protein, and neurofilament light chain (NfL) as biomarkers in presymptomatic repeat expansion carriers and patients with associated frontotemporal dementia. Additionally, to investigate the relationship of poly(GP) with indicators of neurodegeneration as measured by NfL and grey matter volume.

Methods: We measured poly(GP) and NfL levels in cerebrospinal fluid (CSF) from 25 presymptomatic expansion carriers, 64 symptomatic expansion carriers with dementia, and 12 noncarriers. We explored associations with grey matter volumes using region of interest and voxel-wise analyses.

Results: Poly(GP) was present in expansion carriers and absent in noncarriers (specificity 100%, sensitivity 97%). Presymptomatic carriers had lower poly(GP) levels than symptomatic carriers. NfL levels were higher in symptomatic carriers than in presymptomatic carriers and healthy noncarriers. NfL was highest in patients with concomitant motor neuron disease, and correlated with disease severity and survival. Associations between poly(GP) levels and small grey matter regions emerged but did not survive multiple comparison correction, while higher NfL levels were associated with atrophy in frontotemporoparietal cortices and the thalamus.

Interpretation: This study of expansion carriers reveals that: (1) poly(GP) levels discriminate presymptomatic and symptomatic expansion carriers from noncarriers, but are not associated with indicators of neurodegeneration; and (2) NfL levels are associated with grey matter atrophy, disease severity, and shorter survival. Together, poly(GP) and NfL show promise as complementary biomarkers for clinical trials for associated frontotemporal dementia, with poly(GP) as a potential marker for target engagement and NfL as a marker of disease activity and progression.
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http://dx.doi.org/10.1002/acn3.559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945959PMC
May 2018

A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism.

Acta Neuropathol 2018 03 4;135(3):427-443. Epub 2018 Jan 4.

Department of Neurosciences, Experimental Neurology, KU Leuven-University of Leuven, 3000, Leuven, Belgium.

The exact mechanism underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) associated with the GGGGCC repeat expansion in C9orf72 is still unclear. Two gain-of-function mechanisms are possible: repeat RNA toxicity and dipeptide repeat protein (DPR) toxicity. We here dissected both possibilities using a zebrafish model for ALS. Expression of two DPRs, glycine-arginine and proline-arginine, induced a motor axonopathy. Similarly, expanded sense and antisense repeat RNA also induced a motor axonopathy and formed mainly cytoplasmic RNA foci. However, DPRs were not detected in these conditions. Moreover, stop codon-interrupted repeat RNA still induced a motor axonopathy and a synergistic role of low levels of DPRs was excluded. Altogether, these results show that repeat RNA toxicity is independent of DPR formation. This RNA toxicity, but not the DPR toxicity, was attenuated by the RNA-binding protein Pur-alpha and the autophagy-related protein p62. Our findings demonstrate that RNA toxicity, independent of DPR toxicity, can contribute to the pathogenesis of C9orf72-associated ALS/FTD.
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http://dx.doi.org/10.1007/s00401-017-1796-5DOI Listing
March 2018
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