Stress granule recruitment and deposition of proteins of the FET family and TDP-43 in ALS and FTD

Neurodegenerative diseases such as Alzheimer´s disease, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are defined by progressive and selective loss of neurons. With increasing age the risk of developing a neurodegenerative disease exponentially rises. To date these diseases are untreatable, imposing a significant medical, social and financial burden onto our ageing society. Typical features of neurodegenerative diseases are abnormal aggregation of a disease characterizing protein and its deposition in pathological inclusions. A unifying feature in the majority of ALS cases and several subtypes of FTD is the pathological deposition of the TAR DNA-binding protein of 43kDa (TDP-43) or the Fused in Sarcoma (FUS) protein. Furthermore, stress granule (SG) marker proteins are consistently detected in FUS inclusions, suggesting that SGs might be involved in the formation of FUS inclusions. However, whether pathologic TDP-43 inclusions contain SG marker proteins is still controversially discussed. In this thesis I demonstrate that cytosolically mislocalized full-length TDP-43 is recruited into SGs, whereas C-terminal fragments of TDP-43 (TDP-CTFs) fail to localize to SGs. In accordance with these cell culture data, spinal cord inclusions in ALS and FTD patients contain full-length TDP-43 and SG marker proteins. In contrast, hippocampal inclusions are enriched for TDP-CTFs but are SG marker-negative. Thus, the protein composition of TDP-43 inclusions determines whether SG marker proteins are co-deposited in TDP-43 inclusions or not. By analyzing the prerequisites for SG recruitment of TDP-43 and FUS, I demonstrate that cytosolic mislocalization of TDP-43 and FUS is required for their localization in SGs. Additionally, I found that both proteins have the same requirements for SG recruitment, as their main RNA-binding domain and a glycine-rich domain are essential for SG localization. A detailed analysis of the protein composition of FUS inclusions in ALS and FTD cases unveiled that FUS inclusions in FTD cases contain not only FUS, but all FET (FUS, Ewing sarcoma protein (EWS), TATA binding protein-associated factor 15 (TAF15)) family proteins. Here, I provide evidence that this cytosolic deposition of FET proteins can be mimicked in cultured cells by inhibition of Transportin-mediated nuclear import, which causes cytosolic mislocalization of all FET proteins and recruitment of these proteins in SGs. In contrast to FTD cases, FUS inclusions in ALS cases contain only FUS, but not EWS and TAF15. In line with that, I show that ALS-associated FUS mutations result in cytosolic mislocalization of FUS that is upon subsequent cellular stress sequestered into SGs. These SGs then contain only FUS but not EWS or TAF15, demonstrating that mutant FUS is unable to co-sequester EWS or TAF15. In addition, I contributed to two studies that revealed that nuclear import defects are involved in the pathogenesis of ALS and FTD. ALS associated FUS mutations are frequently located within the proline-tyrosine nuclear localization signal (PY-NLS) of FUS and thus disrupt Transportin-mediated nuclear import and cause cytosolic mislocalization of FUS. EWS and TAF15 also contain a PY-NLS and thus are imported into the nucleus via Transportin. This interaction between Transportin and FET proteins can be modulated by arginine methylation that reduces Transportin binding. In FTD patients with FUS inclusions, this post-translational modification seems to be defective, as FUS inclusions in these cases contain hypomethylated FUS. Taken together, these data provide evidence that nuclear import defects and sequestration of FUS and TDP-43 in SGs are consecutive steps in the pathogenesis of ALS and several subtypes of FTD.Neurodegenerative Erkrankungen wie die Alzheimer-Erkrankung, die Amyotrophe Lateralsklerose (ALS) und die Frontotemporale Demenz (FTD) sind durch den progressiven und selektiven Verlust von Neuronen gekennzeichnet. Mit steigendem Alter nimmt das Risiko eine neurodegenerative Erkrankung zu entwickeln exponentiell zu. Bislang gelten diese Krankheiten als nicht behandelbar, was eine signifikante medizinische, soziale und finanzielle Belastung für unsere alternde Gesellschaft darstellt. Typische Charakteristika neurodegenerativer Erkrankungen sind die abnormale Aggregation eines Krankheits-assoziierten Proteins, sowie dessen Anhäufung in pathologischen Ablagerungen. Gemeinsames Merkmal der meisten ALS Fälle und bestimmter Untergruppen von FTD sind pathologische Ablagerungen, die hauptsächlich das TAR DNA-binding protein of 43kDa (TDP-43) oder das Fused in Sarcoma (FUS) Protein enthalten. In FUS Ablagerungen werden stets auch Markerproteine für Stress-Körnchen (engl. stress granules, SG) detektiert, was darauf schließen lässt, dass SGs an der Bildung von FUS Ablagerungen beteiligt sein könnten. Bei pathologischen TDP-43 Ablagerungen ist hingegen immer noch umstritten ob diese SG Markerproteine enthalten. In der vorliegenden Arbeit konnte ich zeigen, dass zytosolisch mislokalisiertes, unfragmentiertes TDP-43 in SGs rekrutiert wird, wohingegen C-terminale Fragmente von TDP-43 (TDP-CTFs) nicht in SGs lokalisieren. Diese Ergebnisse stimmen mit den Beobachtungen in ALS und FTD Patienten überein, wo TDP-43 Ablagerungen im Rückenmark unfragmentiertes TDP-43 und SG Markerproteine enthalten. Im Gegensatz dazu sind hippocampale Ablagerungen mit TDP-CTFs angereichert, enthalten jedoch keine SG Marker. Die Proteinzusammensetzung der TDP-43 Ablagerungen bestimmt also, ob SG Markerproteine darin abgelagert werden oder nicht. Bei der Bestimmung von Voraussetzungen für die Rekrutierung von TDP-43 und FUS in SGs konnte ich feststellen, dass eine zytosolische Umverteilung notwendig ist, damit TDP-43 und FUS in SGs sequestriert werden können. Des Weiteren konnte ich zeigen, dass beide Proteine ihre Haupt-RNA-bindende Domäne, sowie die Glycin-reiche Domäne für die Lokalisierung in SGs benötigen. Eine detaillierte Analyse der Proteinzusammensetzung von FUS Ablagerungen in ALS und FTD hat aufgedeckt, dass FUS Ablagerungen in FTD-Patienten nicht nur FUS, sondern alle FET (FUS, Ewing sarcoma protein (EWS), TATA binding protein-associated factor 15 (TAF15)) Familienproteine beinhalten. Ich konnte zeigen, dass diese cytosolische Ablagerung von FET Proteinen in Zellkultur durch eine Hemmung des Transportin-vermittelten Kerntransports nachgestellt werden kann, da dies zur zytosolischen Anhäufung aller FET Proteine und deren Rekrutierung in SGs führt. Im Gegensatz zu FTD Fällen enthalten FUS Ablagerungen in ALS nur FUS, nicht aber EWS und TAF15. In Zellkultur-Experimenten konnte ich zeigen, dass ALS-assoziierte FUS Mutationen zur zytosolischen Umverteilung von FUS führen, welches dann durch nachfolgenden zellulären Stress in SGs rekrutiert wird. Diese SGs enthalten FUS, jedoch nicht EWS oder TAF15, was beweist, dass mutiertes FUS nicht wildtypisches EWS oder TAF15 sequestrieren kann. Darüber hinaus habe ich an zwei Publikationen mitgearbeitet, in denen gezeigt wurde, dass Defekte im Kernimport an der Pathogenese von ALS und FTD beteiligt sind. ALS-assoziierte FUS Mutationen sind häufig im Prolin-Tyrosin Kernlokalisierungs-Signal (PY-NLS) lokalisiert und zerstören so den Transportin-vermittelten Kernimport und führen zur zytosolischen Misslokalisierung von FUS. EWS und TAF15 enthalten ebenfalls ein PY-NLS und werden daher über Transportin in den Kern importiert. Die Interaktion zwischen Transportin und den FET Proteinen kann durch Arginin-Methylierung moduliert werden, welche die Transportin-Bindung reduziert. In FTD Patienten mit FUS Ablagerungen scheint diese post-translationale Modifikation gestört zu sein, da FUS Ablagerungen in diesen Fällen hypomethyliertes FUS enthalten. Diese Daten liefern Beweise dafür, dass Defekte im Kernimport und die Sequestrierung von FUS und TDP-43 in SGs aufeinanderfolgende Schritte in der Pathogenese von ALS und verschiedenen Varianten von FTD sind

Stress granule recruitment and deposition of proteins of the FET family and TDP-43 in ALS and FTD

Neurodegenerative diseases such as Alzheimer´s disease, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are defined by progressive and selective loss of neurons. With increasing age the risk of developing a neurodegenerative disease exponentially rises. To date these diseases are untreatable, imposing a significant medical, social and financial burden onto our ageing society. Typical features of neurodegenerative diseases are abnormal aggregation of a disease characterizing protein and its deposition in pathological inclusions. A unifying feature in the majority of ALS cases and several subtypes of FTD is the pathological deposition of the TAR DNA-binding protein of 43kDa (TDP-43) or the Fused in Sarcoma (FUS) protein. Furthermore, stress granule (SG) marker proteins are consistently detected in FUS inclusions, suggesting that SGs might be involved in the formation of FUS inclusions. However, whether pathologic TDP-43 inclusions contain SG marker proteins is still controversially discussed. In this thesis I demonstrate that cytosolically mislocalized full-length TDP-43 is recruited into SGs, whereas C-terminal fragments of TDP-43 (TDP-CTFs) fail to localize to SGs. In accordance with these cell culture data, spinal cord inclusions in ALS and FTD patients contain full-length TDP-43 and SG marker proteins. In contrast, hippocampal inclusions are enriched for TDP-CTFs but are SG marker-negative. Thus, the protein composition of TDP-43 inclusions determines whether SG marker proteins are co-deposited in TDP-43 inclusions or not. By analyzing the prerequisites for SG recruitment of TDP-43 and FUS, I demonstrate that cytosolic mislocalization of TDP-43 and FUS is required for their localization in SGs. Additionally, I found that both proteins have the same requirements for SG recruitment, as their main RNA-binding domain and a glycine-rich domain are essential for SG localization. A detailed analysis of the protein composition of FUS inclusions in ALS and FTD cases unveiled that FUS inclusions in FTD cases contain not only FUS, but all FET (FUS, Ewing sarcoma protein (EWS), TATA binding protein-associated factor 15 (TAF15)) family proteins. Here, I provide evidence that this cytosolic deposition of FET proteins can be mimicked in cultured cells by inhibition of Transportin-mediated nuclear import, which causes cytosolic mislocalization of all FET proteins and recruitment of these proteins in SGs. In contrast to FTD cases, FUS inclusions in ALS cases contain only FUS, but not EWS and TAF15. In line with that, I show that ALS-associated FUS mutations result in cytosolic mislocalization of FUS that is upon subsequent cellular stress sequestered into SGs. These SGs then contain only FUS but not EWS or TAF15, demonstrating that mutant FUS is unable to co-sequester EWS or TAF15. In addition, I contributed to two studies that revealed that nuclear import defects are involved in the pathogenesis of ALS and FTD. ALS associated FUS mutations are frequently located within the proline-tyrosine nuclear localization signal (PY-NLS) of FUS and thus disrupt Transportin-mediated nuclear import and cause cytosolic mislocalization of FUS. EWS and TAF15 also contain a PY-NLS and thus are imported into the nucleus via Transportin. This interaction between Transportin and FET proteins can be modulated by arginine methylation that reduces Transportin binding. In FTD patients with FUS inclusions, this post-translational modification seems to be defective, as FUS inclusions in these cases contain hypomethylated FUS. Taken together, these data provide evidence that nuclear import defects and sequestration of FUS and TDP-43 in SGs are consecutive steps in the pathogenesis of ALS and several subtypes of FTD.Neurodegenerative Erkrankungen wie die Alzheimer-Erkrankung, die Amyotrophe Lateralsklerose (ALS) und die Frontotemporale Demenz (FTD) sind durch den progressiven und selektiven Verlust von Neuronen gekennzeichnet. Mit steigendem Alter nimmt das Risiko eine neurodegenerative Erkrankung zu entwickeln exponentiell zu. Bislang gelten diese Krankheiten als nicht behandelbar, was eine signifikante medizinische, soziale und finanzielle Belastung für unsere alternde Gesellschaft darstellt. Typische Charakteristika neurodegenerativer Erkrankungen sind die abnormale Aggregation eines Krankheits-assoziierten Proteins, sowie dessen Anhäufung in pathologischen Ablagerungen. Gemeinsames Merkmal der meisten ALS Fälle und bestimmter Untergruppen von FTD sind pathologische Ablagerungen, die hauptsächlich das TAR DNA-binding protein of 43kDa (TDP-43) oder das Fused in Sarcoma (FUS) Protein enthalten. In FUS Ablagerungen werden stets auch Markerproteine für Stress-Körnchen (engl. stress granules, SG) detektiert, was darauf schließen lässt, dass SGs an der Bildung von FUS Ablagerungen beteiligt sein könnten. Bei pathologischen TDP-43 Ablagerungen ist hingegen immer noch umstritten ob diese SG Markerproteine enthalten. In der vorliegenden Arbeit konnte ich zeigen, dass zytosolisch mislokalisiertes, unfragmentiertes TDP-43 in SGs rekrutiert wird, wohingegen C-terminale Fragmente von TDP-43 (TDP-CTFs) nicht in SGs lokalisieren. Diese Ergebnisse stimmen mit den Beobachtungen in ALS und FTD Patienten überein, wo TDP-43 Ablagerungen im Rückenmark unfragmentiertes TDP-43 und SG Markerproteine enthalten. Im Gegensatz dazu sind hippocampale Ablagerungen mit TDP-CTFs angereichert, enthalten jedoch keine SG Marker. Die Proteinzusammensetzung der TDP-43 Ablagerungen bestimmt also, ob SG Markerproteine darin abgelagert werden oder nicht. Bei der Bestimmung von Voraussetzungen für die Rekrutierung von TDP-43 und FUS in SGs konnte ich feststellen, dass eine zytosolische Umverteilung notwendig ist, damit TDP-43 und FUS in SGs sequestriert werden können. Des Weiteren konnte ich zeigen, dass beide Proteine ihre Haupt-RNA-bindende Domäne, sowie die Glycin-reiche Domäne für die Lokalisierung in SGs benötigen. Eine detaillierte Analyse der Proteinzusammensetzung von FUS Ablagerungen in ALS und FTD hat aufgedeckt, dass FUS Ablagerungen in FTD-Patienten nicht nur FUS, sondern alle FET (FUS, Ewing sarcoma protein (EWS), TATA binding protein-associated factor 15 (TAF15)) Familienproteine beinhalten. Ich konnte zeigen, dass diese cytosolische Ablagerung von FET Proteinen in Zellkultur durch eine Hemmung des Transportin-vermittelten Kerntransports nachgestellt werden kann, da dies zur zytosolischen Anhäufung aller FET Proteine und deren Rekrutierung in SGs führt. Im Gegensatz zu FTD Fällen enthalten FUS Ablagerungen in ALS nur FUS, nicht aber EWS und TAF15. In Zellkultur-Experimenten konnte ich zeigen, dass ALS-assoziierte FUS Mutationen zur zytosolischen Umverteilung von FUS führen, welches dann durch nachfolgenden zellulären Stress in SGs rekrutiert wird. Diese SGs enthalten FUS, jedoch nicht EWS oder TAF15, was beweist, dass mutiertes FUS nicht wildtypisches EWS oder TAF15 sequestrieren kann. Darüber hinaus habe ich an zwei Publikationen mitgearbeitet, in denen gezeigt wurde, dass Defekte im Kernimport an der Pathogenese von ALS und FTD beteiligt sind. ALS-assoziierte FUS Mutationen sind häufig im Prolin-Tyrosin Kernlokalisierungs-Signal (PY-NLS) lokalisiert und zerstören so den Transportin-vermittelten Kernimport und führen zur zytosolischen Misslokalisierung von FUS. EWS und TAF15 enthalten ebenfalls ein PY-NLS und werden daher über Transportin in den Kern importiert. Die Interaktion zwischen Transportin und den FET Proteinen kann durch Arginin-Methylierung moduliert werden, welche die Transportin-Bindung reduziert. In FTD Patienten mit FUS Ablagerungen scheint diese post-translationale Modifikation gestört zu sein, da FUS Ablagerungen in diesen Fällen hypomethyliertes FUS enthalten. Diese Daten liefern Beweise dafür, dass Defekte im Kernimport und die Sequestrierung von FUS und TDP-43 in SGs aufeinanderfolgende Schritte in der Pathogenese von ALS und verschiedenen Varianten von FTD sind

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid–liquid phase separation

Liquid–liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease

FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations

Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing's sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing's sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing's sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing's sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma patholog

Electron Transfer Function versus Oxygen Delivery: A Comparative Study for Several Hexacoordinated Globins Across the Animal Kingdom

Caenorhabditis elegans globin GLB-26 (expressed from gene T22C1.2) has been studied in comparison with human neuroglobin (Ngb) and cytoglobin (Cygb) for its electron transfer properties. GLB-26 exhibits no reversible binding for O2 and a relatively low CO affinity compared to myoglobin-like globins. These differences arise from its mechanism of gaseous ligand binding since the heme iron of GLB-26 is strongly hexacoordinated in the absence of external ligands; the replacement of this internal ligand, probably the E7 distal histidine, is required before binding of CO or O2 as for Ngb and Cygb. Interestingly the ferrous bis-histidyl GLB-26 and Ngb, another strongly hexacoordinated globin, can transfer an electron to cytochrome c (Cyt-c) at a high bimolecular rate, comparable to those of inter-protein electron transfer in mitochondria. In addition, GLB-26 displays an unexpectedly rapid oxidation of the ferrous His-Fe-His complex without O2 actually binding to the iron atom, since the heme is oxidized by O2 faster than the time for distal histidine dissociation. These efficient mechanisms for electron transfer could indicate a family of hexacoordinated globin which are functionally different from that of pentacoordinated globins

Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein

ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import

The majority of familial ALS (fALS)-associated mutations occurs within the nuclear localization signal (NLS) and impairs nuclear import. Nuclear import of FUS depends on Transportin and interference with this pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules

Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS

Fused in sarcoma (FUS) is a nuclear protein that carries a proline-tyrosine nuclear localization signal (PY-NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY-NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN-binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN-mediated nuclear import of ALS-associated FUS mutants. The unmethylated arginine-glycine-glycine domain preceding the PY-NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS-FUS patients contain methylated FUS, while inclusions in FTLD-FUS patients are not methylated. Together with recent findings that FUS co-aggregates with two related proteins of the FET family and TRN in FTLD-FUS but not in ALS-FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis

FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations

Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing's sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing's sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing's sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing's sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology

Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexp