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Publications in Frontotemporal Lobar Degeneration by NOMIS researchers

Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS

NOMIS Researcher(s)

Published in

August 1, 2020

Expansion of a (G4C2)n repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1007/s00401-020-02176-0

Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD

NOMIS Researcher(s)

Published in

April 15, 2020

The C9orf72 repeat expansion causes amyotrophic lateral sclerosis and frontotemporal dementia, but the poor correlation between C9orf72-specific pathology and TDP-43 pathology linked to neurodegeneration hinders targeted therapeutic development. Here, we addressed the role of the aggregating dipeptide repeat proteins resulting from unconventional translation of the repeat in all reading frames. Poly-GA promoted cytoplasmic mislocalization and aggregation of TDP-43 non-cell-autonomously, and anti-GA antibodies ameliorated TDP-43 mislocalization in both donor and receiver cells. Cell-to-cell transmission of poly-GA inhibited proteasome function in neighboring cells. Importantly, proteasome inhibition led to the accumulation of TDP-43 ubiquitinated within the nuclear localization signal (NLS) at lysine 95. Mutagenesis of this ubiquitination site completely blocked poly-GA-dependent mislocalization of TDP-43. Boosting proteasome function with rolipram reduced both poly-GA and TDP-43 aggregation. Our data from cell lines, primary neurons, transgenic mice, and patient tissue suggest that poly-GA promotes TDP-43 aggregation by inhibiting the proteasome cell-autonomously and non-cell-autonomously, which can be prevented by inhibiting poly-GA transmission with antibodies or boosting proteasome activity with rolipram.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.15252/embj.2019102811

Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins

NOMIS Researcher(s)

Published in

March 10, 2020

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms. Sterneckert and colleagues generated isogenic induced pluripotent stem cell lines and demonstrated that MNs with hexanucleotide repeat expansion (HRE) in C9ORF72 show reduced axonal trafficking, which is not recapitulated by knocking out C9ORF72 in MNs from healthy individuals. In contrast, knocking out C9ORF72 exacerbated phenotypes in MNs with HRE, suggesting that both gain- and loss-of-function mechanisms contribute to C9ORF72-ALS.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.stemcr.2020.01.010

Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model

NOMIS Researcher(s)

Published in

February 7, 2020

The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non-canonical translation of the expanded repeat results in abundant poly-GA inclusion pathology throughout the CNS. (GA)149-CFP expression in mice triggers motor deficits and neuroinflammation. Since poly-GA is transmitted between cells, we investigated the therapeutic potential of anti-GA antibodies by vaccinating (GA)149-CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA)10 conjugates and pre-aggregated carrier-free (GA)15. Only ovalbumin-(GA)10 immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA)10 immunization largely rescued the motor function in (GA)149-CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA)10-immunized poly-GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP-43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.15252/emmm.201910919

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

NOMIS Researcher(s)

Published in

January 17, 2020

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress-induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R-like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal-regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

Research field(s)
Health Sciences, Biomedical Research, Biochemistry & Molecular Biology

DOI
10.1074/jbc.RA119.010617

Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3

NOMIS Researcher(s)

Published in

January 1, 2020

Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine–arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine–alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1007/s00401-019-02082-0

Phenotypes and malignancy risk of different FUS mutations in genetic amyotrophic lateral sclerosis

NOMIS Researcher(s)

Published in

December 1, 2019

Objective: Mutations in Fused in Sarcoma (FUS or TLS) are the fourth most prevalent in Western European familial amyotrophic lateral sclerosis (ALS) populations and have been associated with causing both early and very late disease onset. FUS aggregation, DNA repair deficiency, and genomic instability are contributors to the pathophysiology of FUS-ALS, but their clinical significance per se and their influence on the clinical variability have yet to be sufficiently investigated. The aim of this study was to analyze genotype–phenotype correlations and malignancy rates in a newly compiled FUS-ALS cohort. Methods: We cross-sectionally reviewed FUS-ALS patient histories in a multicenter cohort with 36 novel cases and did a meta-analysis of published FUS-ALS cases reporting the largest genotype–phenotype correlation of FUS-ALS. Results: The age of onset (median 39 years, range 11–80) was positively correlated with the disease duration. C-terminal domain mutations were found in 90%. Among all, P525L and truncating/ frameshift mutations most frequently caused juvenile onset, rapid disease progression, and atypical ALS often associated with negative family history while the R521 mutation site was associated with late disease onset and pure spinal phenotype. Malignancies were found in one of 40 patients. Interpretation: We report the largest genotype–phenotype correlation of FUS-ALS, which enables a careful prediction of the clinical course in newly diagnosed patients. In this cohort, FUS-ALS patients did not have an increased risk for malignant diseases.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1002/acn3.50930

Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death

NOMIS Researcher(s)

Published in

April 17, 2019

TDP-43 aggregation is the major hallmark of multiple neurodegenerative diseases, including ALS and FTD. Gasset-Rosa et al. demonstrate that transient stress induces long-lasting cytoplasmic TDP-43 de-mixing independent of stress granules, driving nuclear import defects, nuclear TDP-43 clearance, and cell death.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1016/j.neuron.2019.02.038

The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity

NOMIS Researcher(s)

Published in

February 7, 2019

FUS is an RNA binding protein associated with several neurodegenerative diseases, for which mode of nucleic acid binding has been elusive. Loughlin et al. solved the solution structure of FUS bound to RNA, revealing a sequence-specific recognition for a GGU motif and an unusual shape recognition of a stem loop by two separate domains.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.molcel.2018.11.012

Review: Neuropathology of non-tau frontotemporal lobar degeneration

NOMIS Researcher(s)

Published in

February 1, 2019

Frontotemporal dementia (FTD) is a heterogeneous clinical syndrome associated with frontotemporal lobar degeneration (FTLD) as a relatively consistent neuropathological hallmark feature. However, the discoveries in the past decade of many of the relevant pathological proteins aggregating in human FTD brains in addition to several new FTD causing gene mutations underlined that FTD is a diverse condition on the neuropathological and genetic basis. This resulted in a novel molecular classification of these conditions based on the predominant protein abnormality and allows most cases of FTD to be placed now into one of three broad molecular subgroups; FTLD with tau, TAR DNA-binding protein 43 or FET protein accumulation (FTLD-tau, FTLD-TDP and FTLD-FET respectively). This review will provide an overview of the molecular neuropathology of non-tau FTLD, insights into disease mechanisms gained from the study of human post mortem tissue as well as discussion of current controversies in the field.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1111/nan.12526

FUS (fused in sarcoma) is a component of the cellular response to topoisomerase I–induced DNA breakage and transcriptional stress

NOMIS Researcher(s)

Published in

January 1, 2019

FUS (fused in sarcoma) plays a key role in several steps of RNA metabolism, and dominant mutations in this protein are associated with neurodegenerative diseases. Here, we show that FUS is a component of the cellular response to topoisomerase I (TOP1)–induced DNA breakage; relocalising to the nucleolus in response to RNA polymerase II (Pol II) stalling at sites of TOP1-induced DNA breaks. This relocalisation is rapid and dynamic, reversing following the removal of TOP1-induced breaks and coinciding with the recovery of global transcription. Importantly, FUS relocalisation following TOP1-induced DNA breakage is associated with increased FUS binding at sites of RNA polymerase I transcription in ribosomal DNA and reduced FUS binding at sites of RNA Pol II transcription, suggesting that FUS relocates from sites of stalled RNA Pol II either to regulate pre-mRNA processing during transcriptional stress or to modulate ribosomal RNA biogenesis. Importantly, FUS-mutant patient fibroblasts are hypersensitive to TOP1-induced DNA breakage, highlighting the possible relevance of these findings to neurodegeneration.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.26508/lsa.201800222

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation

NOMIS Researcher(s)

Published in

December 1, 2018

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1038/s41467-017-02299-1

Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

NOMIS Researcher(s)

Published in

October 12, 2018

TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193– 414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/ 410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43’s nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.

Research field(s)
Health Sciences, Biomedical Research, Biochemistry & Molecular Biology

DOI
10.1074/jbc.RA118.003440

Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers

NOMIS Researcher(s)

Published in

August 3, 2018

Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1186/s40478-018-0579-0

Hypertonic Stress Causes Cytoplasmic Translocation of Neuronal, but Not Astrocytic, FUS due to Impaired Transportin Function

NOMIS Researcher(s)

Published in

July 24, 2018

The primarily nuclear RNA-binding protein FUS (fused in sarcoma) forms pathological cytoplasmic inclusions in a subset of early-onset amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. In response to cellular stress, FUS is recruited to cytoplasmic stress granules, which are hypothesized to act as precursors of pathological inclusions. We monitored the stress-induced nucleocytoplasmic shuttling of endogenous FUS in an ex vivo mouse CNS model and human neural networks. We found that hyperosmolar, but not oxidative, stress induced robust cytoplasmic translocation of neuronal FUS, with transient nuclear clearance and loss of function. Surprisingly, this reaction is independent of stress granule formation and the molecular pathways activated by hyperosmolarity. Instead, it represents a mechanism mediated by cytoplasmic redistribution of Transportin 1/2 and is potentiated by transcriptional inhibition. Importantly, astrocytes, which remain unaffected in ALS/FTD-FUS, are spared from this stress reaction that may signify the initial event in the development of FUS pathology.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.celrep.2018.06.094

Age-dependent neurodegeneration and organelle transport deficiencies in mutant TDP43 patient-derived neurons are independent of TDP43 aggregation

NOMIS Researcher(s)

Published in

July 1, 2018

TAR DNA-binding protein 43 (TDP43) plays a significant role in familiar and sporadic amyotrophic lateral sclerosis (ALS). The diverse postulated mechanisms by which TDP43 mutations cause the disease are not fully understood. Human wildtype and TDP43 S393L and G294V mutant spinal motor neuron cultures were differentiated from patient-derived iPSCs. Mutant hTDP43 and wildtype motor neuron cultures did not differ in neuron differentiation capacity during early maturation stage. During aging we detected a dramatic neurodegeneration including neuron loss and pathological neurofilament abnormalities only in TDP43 mutant cultures. Additionally mitochondria and lysosomes of aging spinal motor neurons revealed robust TDP43 mutation dependent abnormal phenotypes in size, shape, speed and motility which all appeared without TDP43 mislocalization or aggregation formation. Furthermore, D-sorbitol – known to induce stress granules and cytoplasmic mislocalization of TDP43 – rescued axonal trafficking phenotypes without signs of TDP43 mislocalization or aggregation formation. Our data indicate TDP43 mutation-dependent but cytosolic aggregation-independent mechanisms of motor neuron degeneration in TDP43 ALS.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1016/j.nbd.2018.03.010

Selective genetic overlap between amyotrophic lateral sclerosis and diseases of the frontotemporal dementia spectrum

NOMIS Researcher(s)

Published in

July 1, 2018

IMPORTANCE: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by loss of upper and lower motor neurons. Although novel ALS genetic variants have been identified, the shared genetic risk between ALS and other neurodegenerative disorders remains poorly understood. OBJECTIVES: To examine whether there are common genetic variants that determine the risk for ALS and other neurodegenerative diseases and to identify their functional pathways. DESIGN, SETTING, AND PARTICIPANTS: In this study conducted from December 1, 2016, to August 1, 2017, the genetic overlap between ALS, sporadic frontotemporal dementia (FTD), FTD with TDP-43 inclusions, Parkinson disease (PD), Alzheimer disease (AD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP) were systematically investigated in 124 876 cases and controls. No participants were excluded from this study. Diagnoses were established using consensus criteria. MAIN OUTCOMES AND MEASURES: The primary outcomes were a list of novel loci and their functional pathways in ALS, FTD, PSP, and ALS mouse models. RESULTS: Among 124 876 cases and controls, genome-wide conjunction analyses of ALS, FTD, PD, AD, CBD, and PSP revealed significant genetic overlap between ALS and FTD at known ALS loci: rs13302855 and rs3849942 (nearest gene, C9orf72; P = .03 for rs13302855 and P = .005 for rs3849942) and rs4239633 (nearest gene, UNC13A; P = .03). Significant genetic overlap was also found between ALS and PSP at rs7224296, which tags the MAPT H1 haplotype (nearest gene, NSF; P = .045). Shared risk genes were enriched for pathways involving neuronal function and development. At a conditional FDR P < .05, 22 novel ALS polymorphisms were found, including rs538622 (nearest gene, ERGIC1; P = .03 for ALS and FTD), which modifies BNIP1 expression in human brains (35 of 137 females; mean age, 59 years; P = .001). BNIP1 expression was significantly reduced in spinal cord motor neurons from patients with ALS (4 controls: mean age, 60.5 years, mean [SE] value, 3984 [760.8] arbitrary units [AU]; 7 patients with ALS: mean age, 56 years, mean [SE] value, 1999 [274.1] AU; P = .02), in an ALS mouse model (mean [SE] value, 13.75 [0.09] AU for 2 SOD1 WT mice and 11.45 [0.03] AU for 2 SOD1 G93A mice; P = .002) and in brains of patients with PSP (80 controls: 39 females; mean age, 82 years, mean [SE] value, 6.8 [0.2] AU; 84 patients with PSP: 33 females, mean age 74 years, mean [SE] value, 6.8 [0.1] AU; β = -0.19; P = .009) or FTD (11 controls: 4 females; mean age, 67 years; mean [SE] value, 6.74 [0.05] AU; 17 patients with FTD: 10 females; mean age, 69 years; mean [SE] value, 6.53 [0.04] AU; P = .005). CONCLUSIONS AND RELEVANCE: This study found novel genetic overlap between ALS and diseases of the FTD spectrum, that the MAPT H1 haplotype confers risk for ALS, and identified the mitophagy-associated, proapoptotic protein BNIP1 as an ALS risk gene. Together, these findings suggest that sporadic ALS may represent a selectively pleiotropic, polygenic disorder.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1001/jamaneurol.2018.0372

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation

NOMIS Researcher(s)

Published in

April 19, 2018

Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-β2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import—namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration. Phase separation of FUS is regulated by both arginine methylation and binding to the nuclear import receptor Transportin/Karyopherin-β2, and this control can be compromised in the neurodegenerative diseases FTD and ALS.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.cell.2018.03.004

Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy

NOMIS Researcher(s)

Published in

February 13, 2018

Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments. Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG) phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.stemcr.2017.12.018

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment

NOMIS Researcher(s)

Published in

February 8, 2018

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes. Neuronal poly-GA aggregates linked to amyotrophic lateral sclerosis and frontotemporal dementia selectively sequester proteasomes.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.cell.2017.12.030

Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity

NOMIS Researcher(s)

Published in

January 1, 2018

Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)149 and nucleolar (PR)175-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Over-expressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule–like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomes may chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.26508/lsa.201800070

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