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Publications in Health Sciences by NOMIS researchers

Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris

NOMIS Researcher(s)

Published in

October 18, 2018

Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3′-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

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

DOI
10.1038/s41586-018-0590-4

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

Variation at the TRIM11 locus modifies progressive supranuclear palsy phenotype

NOMIS Researcher(s)

Published in

October 1, 2018

Objective: The basis for clinical variation related to underlying progressive supranuclear palsy (PSP) pathology is unknown. We performed a genome-wide association study (GWAS) to identify genetic determinants of PSP phenotype. Methods: Two independent pathological and clinically diagnosed PSP cohorts were genotyped and phenotyped to create Richardson syndrome (RS) and non-RS groups. We carried out separate logistic regression GWASs to compare RS and non-RS groups and then combined datasets to carry out a whole cohort analysis (RS = 367, non-RS = 130). We validated our findings in a third cohort by referring to data from 100 deeply phenotyped cases from a recent GWAS. We assessed the expression/coexpression patterns of our identified genes and used our data to carry out gene-based association testing. Results: Our lead single nucleotide polymorphism (SNP), rs564309, showed an association signal in both cohorts, reaching genome-wide significance in our whole cohort analysis (odds ratio = 5.5, 95% confidence interval = 3.2–10.0, p = 1.7 × 10−9). rs564309 is an intronic variant of the tripartite motif-containing protein 11 (TRIM11) gene, a component of the ubiquitin proteasome system (UPS). In our third cohort, minor allele frequencies of surrogate SNPs in high linkage disequilibrium with rs564309 replicated our findings. Gene-based association testing confirmed an association signal at TRIM11. We found that TRIM11 is predominantly expressed neuronally, in the cerebellum and basal ganglia. Interpretation: Our study suggests that the TRIM11 locus is a genetic modifier of PSP phenotype and potentially adds further evidence for the UPS having a key role in tau pathology, therefore representing a target for disease-modifying therapies. Ann Neurol 2018;84:485–496.

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

DOI
10.1002/ana.25308

Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen

NOMIS Researcher(s)

Published in

September 20, 2018

Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism. Defense strategies that are cooperative rather than antagonistic can promote asymptomatic infection and select for reduced virulence in a bacterial pathogen.

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

DOI
10.1016/j.cell.2018.07.016

Glioblastoma treatments: An account of recent industrial developments

NOMIS Researcher(s)

Published in

September 13, 2018

The different drugs and medical devices, which are commercialized or under industrial development for glioblastoma treatment, are reviewed. Their different modes of action are analyzed with a distinction being made between the effects of radiation, the targeting of specific parts of glioma cells, and immunotherapy. Most of them are still at a too early stage of development to firmly conclude about their efficacy. Optune, which triggers antitumor activity by blocking the mitosis of glioma cells under the application of an alternating electric field, seems to be the only recently developed therapy with some efficacy reported on a large number of GBM patients. The need for early GBM diagnosis is emphasized since it could enable the treatment of GBM tumors of small sizes, possibly easier to eradicate than larger tumors. Ways to improve clinical protocols by strengthening preclinical studies using of a broader range of different animal and tumor models are also underlined. Issues related with efficient drug delivery and crossing of blood brain barrier are discussed. Finally societal and economic aspects are described with a presentation of the orphan drug status that can accelerate the development of GBM therapies, patents protecting various GBM treatments, the different actors tackling GBM disease, the cost of GBM treatments, GBM market figures, and a financial analysis of the different companies involved in the development of GBM therapies.

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

DOI
10.3389/fphar.2018.00879

Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

NOMIS Researcher(s)

Published in

September 4, 2018

Background: Heterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear. Methods: mRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins. Results: We demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN. Conclusions: The present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells.

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

DOI
10.1186/s13024-018-0281-5

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

Non-political anger shifts political preferences towards stronger leaders

NOMIS Researcher(s)

Published in

August 1, 2018

Past research has shown that anger is associated with support for confrontational and punitive responses during crises, and notably with the endorsement of authoritarian ideologies. One important question is whether it is anger generated specifically in a political context that explains the association between anger and specific political preferences or whether any feeling of anger would be associated with changes in political attitudes. Here, we tested the effect of non-politically motivated incidental anger on the preference for strong leaders. In line with past research, we predicted that anger would increase preferences for strong leaders. Across two experiments, we exposed participants to an anger induction task. Before and after this experimental manipulation, we measured participants’ political leader preferences by asking them to choose between the faces of two leaders they would vote for in a hypothetical election. The level of self-reported anger predicted the probability of choosing more dominant-looking and less trustworthy-looking leaders after the induction, suggesting that even non-political incidental anger increases preferences for strong leaders.

Research field(s)
Health Sciences, Psychology & Cognitive Sciences, Social Psychology

DOI
10.1038/s41598-022-15765-8

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

Exosomal secretion of α-synuclein as protective mechanism after upstream blockage of macroautophagy

NOMIS Researcher(s)

Published in

July 1, 2018

Accumulation of pathological α-synuclein aggregates plays a major role in Parkinson’s disease. Macroautophagy is a mechanism to degrade intracellular protein aggregates by wrapping them into autophagosomes, followed by fusion with lysosomes. We had previously shown that pharmacological activation of macroautophagy protects against α-synuclein-induced toxicity in human neurons. Here, we hypothesized that inhibition of macroautophagy would aggravate α-synuclein-induced cell death. Unexpectedly, inhibition of autophagosome formation by silencing of ATG5 protected from α-synuclein-induced toxicity. Therefore, we studied alternative cellular mechanisms to compensate for the loss of macroautophagy. ATG5 silencing did not affect the ubiquitin-proteasome system, chaperone systems, chaperone-mediated autophagy, or the unfolded protein response. However, ATG5 silencing increased the secretion of α-synuclein via exosomes. Blocking exosomal secretion exacerbated α-synuclein-induced cell death. We conclude that exosomal secretion of α-synuclein is increased after impaired formation of autophagosomes to reduce the intracellular α-synuclein burden. This compensatory mechanism prevents α-synuclein-induced neuronal cell death.

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

DOI
10.1038/s41419-018-0816-2

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

Afferent cardiac signals modulate attentional engagement to low spatial frequency fearful faces

NOMIS Researcher(s)

Published in

July 1, 2018

Despite the growing consensus that the continuous dynamic cortical representations of internal bodily states shape the subjective experience of emotions, physiological arousal is typically considered only a consequence and rarely a determinant of the emotional experience. Recent experimental approaches study how afferent autonomic signals from the heart modulate the processing of sensory information by focussing on the phasic properties of arterial baroreceptor firing that is active during cardiac systole and quiescent during cardiac diastole. For example, baroreceptor activation has been shown to enhance the processing of threat-signalling stimuli. Here, we investigate the role of cardiac afferent signals in the rapid engagement and disengagement of attention to fear stimuli. In an adapted version of the emotional attentional cueing paradigm, we timed the presentation of cues, either fearful or neutral faces, to coincide with the different phases of the cardiac cycle. Moreover, we presented cues with different spatial frequency ranges to investigate how these interoceptive signals influence the processing of visual information. Results revealed a selective enhancement of attentional engagement to low spatial frequency fearful faces presented during cardiac systole relative to diastole. No cardiac cycle effects were observed to high spatial frequency nor broad spatial frequency cues. These findings expand our mechanistic understanding of how body–brain interactions may impact the visual processing of fearful stimuli and contribute to the increased attentional capture of threat signals.

Research field(s)
Health Sciences, Psychology & Cognitive Sciences, Experimental Psychology

DOI
10.1016/j.cortex.2017.06.016

A novel CHCHD10 mutation implicates a Mia40-dependent mitochondrial import deficit in ALS

NOMIS Researcher(s)

Published in

June 1, 2018

CHCHD10 mutations are linked to amyotrophic lateral sclerosis, but their mode of action is unclear. In a 29-year-old patient with rapid disease progression, we discovered a novel mutation (Q108P) in a conserved residue within the coiled-coil-helix-coiled-coil-helix (CHCH) domain. The aggressive clinical phenotype prompted us to probe its pathogenicity. Unlike the wild-type protein, mitochondrial import of CHCHD10 Q108P was blocked nearly completely resulting in diffuse cytoplasmic localization and reduced stability. Other CHCHD10 variants reported in patients showed impaired mitochondrial import (C122R) or clustering within mitochondria (especially G66V and E127K) often associated with reduced expression. Truncation experiments suggest mitochondrial import of CHCHD10 is mediated by the CHCH domain rather than the proposed N-terminal mitochondrial targeting signal. Knockdown of Mia40, which introduces disulfide bonds into CHCH domain proteins, blocked mitochondrial import of CHCHD10. Overexpression of Mia40 rescued mitochondrial import of CHCHD10 Q108P by enhancing disulfide-bond formation. Since reduction in CHCHD10 inhibits respiration, mutations in its CHCH domain may cause aggressive disease by impairing mitochondrial import. Our data suggest Mia40 upregulation as a potential therapeutic salvage pathway.

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

DOI
10.15252/emmm.201708558

Potential genetic modifiers of disease risk and age at onset in patients with frontotemporal lobar degeneration and GRN mutations: a genome-wide association study

NOMIS Researcher(s)

Published in

June 1, 2018

Background: Loss-of-function mutations in GRN cause frontotemporal lobar degeneration (FTLD). Patients with GRN mutations present with a uniform subtype of TAR DNA-binding protein 43 (TDP-43) pathology at autopsy (FTLD-TDP type A); however, age at onset and clinical presentation are variable, even within families. We aimed to identify potential genetic modifiers of disease onset and disease risk in GRN mutation carriers. Methods: The study was done in three stages: a discovery stage, a replication stage, and a meta-analysis of the discovery and replication data. In the discovery stage, genome-wide logistic and linear regression analyses were done to test the association of genetic variants with disease risk (case or control status) and age at onset in patients with a GRN mutation and controls free of neurodegenerative disorders. Suggestive loci (p<1 × 10 −5 ) were genotyped in a replication cohort of patients and controls, followed by a meta-analysis. The effect of genome-wide significant variants at the GFRA2 locus on expression of GFRA2 was assessed using mRNA expression studies in cerebellar tissue samples from the Mayo Clinic brain bank. The effect of the GFRA2 locus on progranulin concentrations was studied using previously generated ELISA-based expression data. Co-immunoprecipitation experiments in HEK293T cells were done to test for a direct interaction between GFRA2 and progranulin. Findings: Individuals were enrolled in the current study between Sept 16, 2014, and Oct 5, 2017. After quality control measures, statistical analyses in the discovery stage included 382 unrelated symptomatic GRN mutation carriers and 1146 controls free of neurodegenerative disorders collected from 34 research centres located in the USA, Canada, Australia, and Europe. In the replication stage, 210 patients (67 symptomatic GRN mutation carriers and 143 patients with FTLD without GRN mutations pathologically confirmed as FTLD-TDP type A) and 1798 controls free of neurodegenerative diseases were recruited from 26 sites, 20 of which overlapped with the discovery stage. No genome-wide significant association with age at onset was identified in the discovery or replication stages, or in the meta-analysis. However, in the case-control analysis, we replicated the previously reported TMEM106B association (rs1990622 meta-analysis odds ratio [OR] 0·54, 95% CI 0·46–0·63; p=3·54 × 10 −16 ), and identified a novel genome-wide significant locus at GFRA2 on chromosome 8p21.3 associated with disease risk (rs36196656 meta-analysis OR 1·49, 95% CI 1·30–1·71; p=1·58 × 10 −8 ). Expression analyses showed that the risk-associated allele at rs36196656 decreased GFRA2 mRNA concentrations in cerebellar tissue (p=0·04). No effect of rs36196656 on plasma and CSF progranulin concentrations was detected by ELISA; however, co-immunoprecipitation experiments in HEK293T cells did suggest a direct binding of progranulin and GFRA2. Interpretation: TMEM106B-related and GFRA2-related pathways might be future targets for treatments for FTLD, but the biological interaction between progranulin and these potential disease modifiers requires further study. TMEM106B and GFRA2 might also provide opportunities to select and stratify patients for future clinical trials and, when more is known about their potential effects, to inform genetic counselling, especially for asymptomatic individuals. Funding: National Institute on Aging, National Institute of Neurological Disorders and Stroke, Canadian Institutes of Health Research, Italian Ministry of Health, UK National Institute for Health Research, National Health and Medical Research Council of Australia, and the French National Research Agency.

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

DOI
10.1016/S1474-4422(18)30126-1

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

The mitochondrial ATP synthase is a shared drug target for aging and dementia

NOMIS Researcher(s)

Published in

April 1, 2018

Aging is a major driving force underlying dementia, such as that caused by Alzheimer’s disease (AD). While the idea of targeting aging as a therapeutic strategy is not new, it remains unclear how closely aging and age-associated diseases are coupled at the molecular level. Here, we discover a novel molecular link between aging and dementia through the identification of the molecular target for the AD drug candidate J147. J147 was developed using a series of phenotypic screening assays mimicking disease toxicities associated with the aging brain. We have previously demonstrated the therapeutic efficacy of J147 in several mouse models of AD. Here, we identify the mitochondrial α-F 1 -ATP synthase (ATP5A) as a target for J147. By targeting ATP synthase, J147 causes an increase in intracellular calcium leading to sustained calcium/calmodulin-dependent protein kinase kinase β (CAMKK2)-dependent activation of the AMPK/mTOR pathway, a canonical longevity mechanism. Accordingly, modulation of mitochondrial processes by J147 prevents age-associated drift of the hippocampal transcriptome and plasma metabolome in mice and extends lifespan in drosophila. Our results link aging and age-associated dementia through ATP synthase, a molecular drug target that can potentially be exploited for the suppression of both. These findings demonstrate that novel screens for new AD drug candidates identify compounds that act on established aging pathways, suggesting an unexpectedly close molecular relationship between the two.

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

DOI
10.1111/acel.12715

Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells

NOMIS Researcher(s)

Published in

April 1, 2018

The term “nonsense-mediated mRNA decay” (NMD) originally described the degradation of mRNAs with premature translation–termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%–10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5–SMG7 or SMG5–PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5??–3?? exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.

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

DOI
10.1261/rna.063719.117

Fisetin Reduces the Impact of Aging on Behavior and Physiology in the Rapidly Aging SAMP8 Mouse

NOMIS Researcher(s)

Published in

March 2, 2018

Alzheimer’s disease (AD) is rarely addressed in the context of aging even though there is an overlap in pathology. We previously used a phenotypic screening platform based on old age-associated brain toxicities to identify the flavonol fisetin as a potential therapeutic for AD and other age-related neurodegenerative diseases. Based on earlier results with fisetin in transgenic AD mice, we hypothesized that fisetin would be effective against brain aging and cognitive dysfunction in rapidly aging senescence-accelerated prone 8 (SAMP8) mice, a model for sporadic AD and dementia. An integrative approach was used to correlate protein expression and metabolite levels in the brain with cognition. It was found that fisetin reduced cognitive deficits in old SAMP8 mice while restoring multiple markers associated with impaired synaptic function, stress, and inflammation. These results provide further evidence for the potential benefits of fisetin for the treatment of age-related neurodegenerative diseases.

Research field(s)
Health Sciences, Public Health & Health Services, Gerontology

DOI
10.1093/gerona/glx104

Clinical sequencing: From raw data to diagnosis with lifetime value

NOMIS Researcher(s)

Published in

March 1, 2018

High-throughput sequencing (HTS) has revolutionized genetics by enabling the detection of sequence variants at hitherto unprecedented large scale. Despite these advances, however, there are still remaining challenges in the complete coverage of targeted regions (genes, exome or genome) as well as in HTS data analysis and interpretation. Moreover, it is easy to get overwhelmed by the plethora of available methods and tools for HTS. Here, we review the step-by-step process from the generation of sequence data to molecular diagnosis of Mendelian diseases. Highlighting advantages and limitations, this review addresses the current state of (1) HTS technologies, considering targeted, whole-exome, and whole-genome sequencing on short- and long-read platforms; (2) read alignment, variant calling and interpretation; as well as (3) regulatory issues related to genetic counseling, reimbursement, and data storage.

Research field(s)
Health Sciences, Biomedical Research, Genetics & Heredity

DOI
10.1111/cge.13190

The emerging role of minor intron splicing in neurological disorders

NOMIS Researcher(s)

Published in

March 1, 2018

Pre-mRNA splicing is an essential step in eukaryotic gene expression. Mutations in cis-acting sequence elements within pre-mRNA molecules or trans-acting factors involved in pre-mRNA processing have both been linked to splicing dysfunction that give rise to a large number of human diseases. These mutations typically affect the major splicing pathway, which excises more than 99% of all introns in humans. However, approximately 700-800 human introns feature divergent intron consensus sequences at their 5′ and 3′ ends and are recognized by a separate pre-mRNA processing machinery denoted as the minor spliceosome. This spliceosome has been studied less than its major counterpart, but has received increasing attention during the last few years as a novel pathomechanistic player on the stage in neurodevelopmental and neurodegenerative diseases. Here, we review the current knowledge on minor spliceosome function and discuss its potential pathomechanistic role and impact in neurodegeneration.

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

DOI
10.15698/cst2018.03.126

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