J. Paul Taylor

<h2>Summary</h2> The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

Article22 February 2018Open Access Transparent process Tau protein liquid–liquid phase separation can initiate tau aggregation Susanne Wegmann Corresponding Author Susanne Wegmann [email protected] orcid.org/0000-0002-5388-2479 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Bahareh Eftekharzadeh Bahareh Eftekharzadeh Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Katharina Tepper Katharina Tepper German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Katarzyna M Zoltowska Katarzyna M Zoltowska Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Rachel E Bennett Rachel E Bennett Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Simon Dujardin Simon Dujardin Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Pawel R Laskowski Pawel R Laskowski orcid.org/0000-0002-8118-9030 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Danny MacKenzie Danny MacKenzie Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Tarun Kamath Tarun Kamath Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Caitlin Commins Caitlin Commins Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Charles Vanderburg Charles Vanderburg Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Allyson D Roe Allyson D Roe Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Zhanyun Fan Zhanyun Fan Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Amandine M Molliex Amandine M Molliex Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Amayra Hernandez-Vega Amayra Hernandez-Vega Max-Planck Institute for Molecular Cell Biology & Genetics, Dresden, Germany Search for more papers by this author Daniel Muller Daniel Muller orcid.org/0000-0003-3075-0665 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Anthony A Hyman Anthony A Hyman Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Eckhard Mandelkow Eckhard Mandelkow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck Institute for Metabolism Research, Hamburg Outstation c/o DESY, Hamburg, Germany CAESAR Research Center, Bonn, Germany Search for more papers by this author J Paul Taylor J Paul Taylor Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Bradley T Hyman Corresponding Author Bradley T Hyman [email protected] orcid.org/0000-0002-7959-9401 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Susanne Wegmann Corresponding Author Susanne Wegmann [email protected] orcid.org/0000-0002-5388-2479 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Bahareh Eftekharzadeh Bahareh Eftekharzadeh Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Katharina Tepper Katharina Tepper German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Search for more papers by this author Katarzyna M Zoltowska Katarzyna M Zoltowska Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Rachel E Bennett Rachel E Bennett Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Simon Dujardin Simon Dujardin Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Pawel R Laskowski Pawel R Laskowski orcid.org/0000-0002-8118-9030 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Danny MacKenzie Danny MacKenzie Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Tarun Kamath Tarun Kamath Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Caitlin Commins Caitlin Commins Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Charles Vanderburg Charles Vanderburg Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Allyson D Roe Allyson D Roe Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Zhanyun Fan Zhanyun Fan Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Amandine M Molliex Amandine M Molliex Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Amayra Hernandez-Vega Amayra Hernandez-Vega Max-Planck Institute for Molecular Cell Biology & Genetics, Dresden, Germany Search for more papers by this author Daniel Muller Daniel Muller orcid.org/0000-0003-3075-0665 Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Anthony A Hyman Anthony A Hyman Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Search for more papers by this author Eckhard Mandelkow Eckhard Mandelkow German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Max-Planck Institute for Metabolism Research, Hamburg Outstation c/o DESY, Hamburg, Germany CAESAR Research Center, Bonn, Germany Search for more papers by this author J Paul Taylor J Paul Taylor Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Bradley T Hyman Corresponding Author Bradley T Hyman [email protected] orcid.org/0000-0002-7959-9401 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Author Information Susanne Wegmann *,1,‡, Bahareh Eftekharzadeh1,‡, Katharina Tepper2,‡, Katarzyna M Zoltowska1, Rachel E Bennett1, Simon Dujardin1, Pawel R Laskowski3, Danny MacKenzie1, Tarun Kamath1, Caitlin Commins1, Charles Vanderburg1, Allyson D Roe1, Zhanyun Fan1, Amandine M Molliex4, Amayra Hernandez-Vega5, Daniel Muller3, Anthony A Hyman3, Eckhard Mandelkow2,6,7, J Paul Taylor4,8 and Bradley T Hyman *,1 1Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA 2German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 3Department for Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland 4Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA 5Max-Planck Institute for Molecular Cell Biology & Genetics, Dresden, Germany 6Max-Planck Institute for Metabolism Research, Hamburg Outstation c/o DESY, Hamburg, Germany 7CAESAR Research Center, Bonn, Germany 8Howard Hughes Medical Institute, Chevy Chase, MD, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 617 230 7184; E-mail: [email protected] *Corresponding author. Tel: +1 617 726 3987; E-mail: [email protected] The EMBO Journal (2018)37:e98049https://doi.org/10.15252/embj.201798049 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase-separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho-tau isolated from human Alzheimer brain. Droplet-like tau can also be observed in neurons and other cells. We found that tau droplets become gel-like in minutes, and over days start to spontaneously form thioflavin-S-positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases. Synopsis The microtubule binding protein tau can undergo liquid-liquid phase separation under physiological conditions. Phosphorylated, FTD-mutant, and Alzheimer's disease brain tau is capable of forming droplets that can initiate the formation of aberrant, aggregated tau “seeds”. LLPS may play a role in different tauopathies. Full-length human tau can undergo liquid-liquid phase separation in neurons. In vitro studies show importance of phosphorylation and Frontotemporal Dementia mutations for tau LLPS. AD brain tau is competent of forming droplets. Tau droplets can transition into aggregates. Tau LLPS is a potential mechanism for aggregation initiation in tauopathies. Introduction Tau protein is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD) and of various other forms of intracellular inclusions in frontotemporal dementias (FTDs). Tau is classically described as a soluble neuron-specific microtubule binding (MTB) protein; however, the connection between tau (mis)function and neurodegeneration is uncertain. It is clear, for example, that post-translational modifications (PTMs) and tau mutations predisposing to aggregation are both associated with neurodegeneration. Recently, soluble hyperphosphorylated high molecular weight tau was identified as a bioactive form, which can be released and taken up by neurons and initiate templated misfolding of cytoplasmic tau in neurons (Takeda et al, 2015). This soluble hyperphosphorylated tau is, however, clearly distinct from aggregated fibrillary tau in neurofibrillary tangles, despite both being implicated in tau toxicity. Tau is an exceptionally soluble protein, and the molecular mechanisms that link soluble tau to aggregated tau are unknown. We now report that tau—similar to several other neurodegenerative disease-associated proteins such as the prion-domain harboring RNA binding proteins FUS, TDP43, hnRNPA1 (King et al, 2012)—can undergo liquid–liquid phase separation (LLPS), and we suggest that this observation may provide a biological mechanism for tau aggregation in neurodegenerative diseases. The longest isoform of tau in the human CNS contains a MTB region that contains four pseudo-repeats (R1–R4) plus flanking proline-rich regions (P1, P2, and P3; Gustke et al, 1994), a shorter (≈40 aa) C-terminal tail, and a long (≈250 aa) flexible N-terminal half of tau, which projects from the surface of microtubules in the MT-bound state (Goode et al, 1997), and forms a polyelectrolyte brush around fibrillary aggregates of tau (Sillen et al, 2005; Wegmann et al, 2013). The lack of a fixed tertiary protein structure classifies tau as an intrinsically disordered protein (IDP). Proteins that contain intrinsically disordered regions often have multiple biological functions (Wright & Dyson, 2014), and some of them aggregate in protein aggregation diseases, such as huntingtin protein in Huntington's disease, α-synuclein in Parkinson's disease, TDP43 and FUS in ALS, and tau in Alzheimer's disease and tauopathies (Uversky et al, 2008). Recent studies revealed that the RNA binding and stress granule-associated proteins FUS (Patel et al, 2015), hnRNPA1 (Molliex et al, 2015), and TDP43 (Conicella et al, 2016) have the ability to reversibly form intracellular membrane-less organelles. These reversible droplets represent physiologically active protein or protein-nucleic acid “bioreactors” (Hyman et al, 2014), which form through a process known as LLPS (Brangwynne et al, 2015). Such transient membrane-less organelles have multiple cellular functions, such as p-granule formation to establish intracellular gradients of RNA transcription (Brangwynne et al, 2009), the enrichment of RNA binding proteins in stress granules (Lin et al, 2015; Molliex et al, 2015), concentrating transcription factors in nucleoli (Berry et al, 2015), and the initiation of microtubule spindle formation (Jiang et al, 2015). However, the functional phase separation of nuclear proteins was shown to be disrupted by C9orf72 GR/PR dipeptide repeats (Lee et al, 2016) and is related to protein aggregation in neurodegeneration (Schmidt & Rohatgi, 2016). In most cases, the phase transition of these proteins is driven by so-called low complexity domains (LCDs) in their sequence, a term of somewhat inconclusive nomenclature that is often used to describe protein domains of low amino acid variance leading to inhomogeneous charge distribution or polarity distribution along the peptide backbone (Nott et al, 2015). For example, FUS, TDP-43, and hnRNPA1 contain “prion-like” LCDs that drive their phase separation. However, in the case of tau, no typical low complexity domain (LCD) exists in the protein sequence, but the intrinsic disorder and the inhomogeneous charge distribution of full-length tau (Lee et al, 1988) led us to postulate that tau may undergo a similar phase separation. In fact, recent reports using recombinant tau constructs support the argument that tau, despite having no defined LCDs, can undergo LLPS facilitated by crowding agents or RNA in vitro (Ambadipudi et al, 2017; Hernandez-Vega et al, 2017; Zhang et al, 2017). We now show that full-length human tau protein can efficiently undergo LLPS to form condensed liquid tau droplets in cell physiological conditions. We observed the formation of tau droplets not only ex vivo for post-translationally modified recombinant tau, but also in neurons in vitro and with strong evidence even in vivo, using high molecular weight hyperphosphorylated tau isolated from human Alzheimer brain. Importantly, tau LLPS is enabled and regulated by physiological and pathological-like tau phosphorylation, by disease-associated mutations, and can be induced in in vitro aggregation conditions. Similar to FUS and hnRNPA1 proteins (Molliex et al, 2015; Murakami et al, 2015; Patel et al, 2015), droplets of pathological tau “mature” through a viscous gel phase into protein aggregates; the tau aggregates become thioflavin-S positive, suggesting the emergence of the beta-pleated sheet conformation present in tau aggregates in vivo. It appears that the phase separation of tau at physiological protein concentrations may be catalyzed by an interplay of electrostatic interactions in the unstructured N-terminal half of tau, combined with hydrophobic interactions of the C-terminal MTB domain, which can stabilize tau droplets possibly through β-sheet structures. These findings complement and provide additional biological relevance to the recent reports showing in vitro tau LLPS of repeat domain constructs at rather high concentrations (Ambadipudi et al, 2017) and in the presence of aggregation triggering polyanionic RNA (Zhang et al, 2017). Together, our data strongly support the hypothesis that intracellular phase separation of tau leads to subcellular foci of high local concentration of tau, which may be important for physiological roles of tau, and—in the setting of aberrant phosphorylation or disease-relevant pro-aggregation mutations—lead to tau aggregation. By analogy to hnRNPA1 (Molliex et al, 2015)-, TDP-43 (Conicella et al, 2016)-, FUS (Murakami et al, 2015)-, and C9orf72-derived dipeptide repeats (Lee et al, 2016), tau LLPS could act to initiate tau aggregation in AD and FTD. We suggest that protein LLPS may be a biophysical mechanism underlying multiple protein aggregation and neurodegenerative diseases. Results Intrinsically disordered tau protein can accumulate in droplet-like assemblies in neurons Several disordered proteins with LCDs have been shown to undergo LLPS leading to liquid droplet formation in aqueous solution upon increasing the molecular crowding in the solution (Mitrea et al, 2016). Cells use LLPS to reversibly assemble membrane-less organelles such as stress granules, p-granules, or nucleoli (reviewed in Mitrea et al, 2016; Hyman et al, 2014). In a number of LLPS proteins, including Ddx4 (Nott et al, 2014), domains of low amino acid complexity introduce an unequal charge distribution along the protein backbone resulting in multivalency of these polypeptides with alternating patches of high positive or negative charge densities (Toretsky & Wright, 2014). The phase separation of these proteins is often driven by electrostatic interactions within the LCDs, or between them. Tau is intrinsically disordered (Fig 1A, Appendix Fig S1), and the distribution of charges in the longest human isoform of tau (2N4R, 441 aa) at physiological pH 7.4 shows per se a similar multivalent pattern (Wegmann et al, 2013): The N-terminus (≈aa 1–120) has a negative net charge, the middle domain (≈aa 121–250) has a large excess of positive charges, the repeat domain (TauRD, ≈aa 251–390) has a moderate excess of positive charges, and the C-terminus (≈aa 391–441) is negatively charged (Fig 1B). Figure 1. Droplet-like condensation of intrinsically disordered human tau protein in neurons Protein sequence and disorder prediction (PONDR) of the longest human tau isoform [2N4R, 441 amino acids (aa)]. The highly disordered N-terminal half (projection domain) contains two N-terminal repeats (N1, N2) and two proline-rich regions (P1, P2). The repeat domain (TauRD) consists of four pseudo-repeats (R1–R4), can transiently adopt short stretches of β-strand structure in R2 and R3, facilitates microtubule binding together with P2 + P3, and mediates the aggregation of tau. Single amino acid and average (sliding window of 6 aa) charge distribution along the tau441 sequence (at pH 7.4) reveal the following charged domains in tau441: the negatively charged N-terminal end (≈aa 1–120), a strongly positively charged middle domain (aa 121–250), the positively charged TauRD (aa 251–400), and a negatively charged C-terminal end (aa 401–441). Intracellular droplet-like accumulations of GFP-tau441 (white arrows) in primary cortical mouse neurons. Expression of GFP alone does not lead to droplets. Droplet dynamics in neurons is shown in Movie EV1. FRAP reveals incomplete recovery of droplet-like tau in neuronal cell bodies indicating an immobile fraction of GFP-tau441 molecules of ≈30%. Neurons growing in microfluidic chambers extend their axons in microgrooves (Takeda et al, 2015). Time-lapse imaging (#1–5, time interval ≈3 s) shows the movement of droplet-like GFP-tau441 (white arrows) in axons over time. Immunofluorescent labeling of neurons expressing GFP-tau441 shows that tau accumulations (white arrows) do not co-localize with lysosomes (LAMP1). In murine neuroblastoma cells (N2a) expressing GFP-tau441, droplet-like tau occurs in cells having a critical GFP-tau441 concentration (white star). In cells with low expression level (white square), GFP-tau binds to microtubule bundles; in cells with medium GFP-tau441 (white circle), excess GFP-tau441 fills the cell bodies. The graph shows the GFP fluorescence intensity in cell bodies with (pink) and without (gray) droplets. Cross-sectional profiles of cells with droplets suggest a similar tau concentration of GFP-tau on microtubules (white lines, black traces) and in droplets (pink lines and traces). Data information: In (D), data are presented as mean ± s.e.m., n = 9 droplets; data have been fitted with a one-phase exponential fit, r2 = 0.08215. In (G), average fluorescence intensity in cell bodies is plotted as mean ± s.e.m., n = 45 for “− droplets”, n = 17 for “+ droplets”. Download figure Download PowerPoint Based on this knowledge about the disorder and inhomogeneous charge distribution of tau, we postulated that human tau may also be able to undergo LLPS in neurons. To test this hypothesis, we expressed GFP-tagged full-length tau (GFP-tau441) in primary cortical mouse neurons. The expression of GFP-tau441 led to the formation of mobile intracellular droplet-like tau accumulations in the cytosol (Fig 1C, Movies EV1–EV3), and fluorescence recovery after photobleaching (FRAP) of intraneuronal GFP-tau441 droplets revealed a fast recovery rate with an immobile tau molecule fraction of ≈30% (Figs 1D and EV1A). Some droplet-like tau accumulations in axons of neurons grown in microfluidic chambers moved retro- as well as anterograde (Fig 1E, Movie EV4). Interestingly, GFP-tau441 droplets (Movies EV1–EV3) appeared less mobile compared to droplets formed by the N-terminal projection domain of tau441 (aa 1–256), GFP-tau256 (Movies EV5,EV6, EV7), maybe because of the ability of full-length GFP-tau441 but not GFP-tau256 to bind microtubules via the repeat domain. Click here to expand this figure. Figure EV1. Tau phosphorylated in cells can undergo LLPS Example FRAP images of GFP-tau441 droplet (green circle and arrowhead) in primary neurons. Recovery after photobleaching event appears rapid but incomplete. FRAP of tau aggregates in HEK TauRDP301S-CFP/YFP cells shows no recovery after photobleaching of the aggregates that have been initiated with brain lysate (5 μg/μl total protein) from human tauP301L transgenic mice (rTg4510 line). Insets show example images taken before bleaching and at t = 0 and 300 s. FRAP parameters were the same as in (A, C). Graph represents mean ± s.e.m. of n = 3 aggregates. FRAP of GFP-PolyQ aggregates that formed in primary cortical mouse neurons after overexpression for 24 h. PolyQ aggregates show no recovery after photobleaching. Insets show example images taken before bleaching and at t = 0 and 300 s. FRAP parameters were the same as in (A, B). Graph represents mean ± s.e.m. of n = 4 aggregates. Example FRAP experiment for GFP-tau441 expressed in primary cortical neurons. Cytosolic soluble GFP-tau441 shows rapid and complete recovery, whereas microtubule-bound GFP-tau441 in a neighboring cell recovers slightly slower. FRAP parameters were the same as in (A– C). Expression of AAV8 Dendra2-tau441 in the cortex of wild-type mice allows the visualization of droplet-like tau in cortical neuronal cell bodies (1–3) and processes (4–6) in vivo by two-photon microscopy. GFP expressing control neurons show a homogenous GFP distribution instead. Cell lysates from murine N2a cells and primary cortical mouse neurons (DIV7) expressing GFP-tau256 or GFP-tau441 were analyzed by Western blot for the content of human tau (Tau13) and phospho-tau using antibodies PHF-1 or a mix of p-Tau antibodies. Most abundant phosphorylation sites previously found in p-tau441 and deP-tau441 (*) by mass spectrometry (Mair et al, 2016). Most of these phospho-sites, for which specific antibodies were available, could be verified (red; blue = not detected) in the p-tau441 preparation used in this manuscript. Download figure Download PowerPoint We did not observe the fusion or fission of GFP-tau441 droplets in neurons, which can be seen as a typical behavior of liquid droplets. However, the observed FRAP recovery of the GFP-tau droplets excludes the possibility that the observed spherical droplets resemble large tau aggregates previously reported for mutant tau expressed in neurons (Hoover et al, 2010). Tau aggregates induced in HEK TauRDP301S-CFP/YFP cells, or GFP-polyQ aggregates in primary neurons, did not show recovery after photobleaching (Fig EV1B and C), whereas soluble GFP-tau in the cytosol and bound on microtubules showed fast recovery (Fig EV1D). Moreover, the expression of wild-type tau does not lead to intracellular tau aggregation, neither in vitro (Lim et al, 2014) nor in vivo. Furthermore, GFP-tau441 droplets did not co-localize with membrane-bound organelles like lysosomes (Fig 1F), endosomes (Appendix Fig S2A), or the endoplasmic reticulum (ER, Appendix Fig S2B). Interestingly, when tau441 N-terminally fused to the fluorescent protein Dendra2, Dendra2-tau441, was expressed in the cortex of living wild-type mice upon stereotactical injection of AAV Dendra2-tau441 into the somatosensory cortex, two-photon imaging through a cranial window revealed a heterogeneous distribution of unconverted green Dendra2-tau441 with spherical droplet-shaped accumulations in the cell bodies of neurons in cortex layer 2/3 (Fig EV1E, neuron #1–3). Some of these accumulations also occurred along neuritic projections (Fig EV1E, #4–6). In contrast, the distribution of GFP in neurons of the control AAV GFP-injected hemisphere was homogeneous in cell bodies and projections of transduced neurons. These data indicated that tau LLPS may also occur in the living brain. Notably, in N2a cells, GFP-tau droplets could mostly be observed in cells with sufficiently high GFP-tau expression levels (Fig 1G), whereas in primary neurons, droplets could also occur in neurons with rather low or medium GFP-tau441 expression levels. This indicated that tau LLPS in neurons may be regulated by additional factors. In neurons, tau is phosphorylated at multiple sites (Fig EV1F; Iqbal et al, 2005). Many of tau's physiological and pathological phosphorylation sites are located in the positively charged middle domain of the N-terminal half and in the repeat domain (Johnson & Stoothoff, 2004), where phosphorylation causes a local increase or a change in domain charge from positive to neutral or negative, and hence can change intra- and intermolecular interactions of tau. However, most previous in vitro studies on tau aggregation utilized recombinant non-phosphorylated tau from Escherichia coli, and in these studies, tau LLPS has not been observed. Phosphorylated human full-length tau undergoes LLPS in vitro We decided to describe the conditions for tau LLPS in more detail and produced recombinant full-length human tau (p-tau441, aa 1–441; Fig 2A) in SF9 insect cells, which are able to introduce PTMs including phosphorylation into recombinant tau (Tepper et al, 2014). Previously, the phosphorylation sites found in p-tau441 by mass spectrometry were reported to be similar to the phosphorylation of tau extracted from AD brains (Mair et al, 2016; Fig EV1G), with phosphorylation in the repeat domain (R1–R4), in the proline-rich region (P1 + P2), and some in the N-terminal insets (N1 and N2) of tau441 (Fig 2A). Here, we used Sf9 p-tau441 protein from the same source that was expressed and purified under identical conditions, and verified most of the reported phosphorylation sites in p-tau441 by Western blot analysis (Fig EV1G). Figure 2. Liquid droplet characteristics of p-tau441 Qualitative distribution of phosphorylation sites in p-tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al, 2016)]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p-tau441 in presence of molecular crowding (12.5% w/v Ficoll-400). No phase separation is observed without crowding agent or in the absence of p-tau441 protein. Liquid droplets formed by p-tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl-acetate and visualized by transmission electron microscopy (TEM). p-tau441 droplets are decorated with gold particles after immunogold labeling using anti-tau antibody K9JA. Shortly after formation (15 min), p-tau441 droplets stop to coalesce and often occur as doublets or triplets. With time (60 min), droplets grow in size but remain colloidal. Droplet fusion is shown in Movie EV1. p-tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p-tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p-tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium

<h2>Summary</h2> The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.

<h2>Summary</h2> RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-β2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-β1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-β2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. <i>In vivo</i>, Karyopherin-β2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2’s ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2’s biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

<h3>Background</h3> Cerebellar ataxias are the result of diverse disease processes that can be genetic or acquired. Establishing a diagnosis requires a methodical approach with expert clinical evaluation and investigations. We describe the causes of ataxia in 1500 patients with cerebellar ataxia. <h3>Methods</h3> All patients were referred to the Sheffield Ataxia Centre, UK, and underwent extensive investigations, including, where appropriate genetic testing using next-generation sequencing (NGS). Patients were followed up on a 6-monthly basis for reassessment and further investigations if indicated. <h3>Results</h3> A total of 1500 patients were assessed over 20 years. Twenty per cent had a family history, the remaining having sporadic ataxia. The commonest cause of sporadic ataxia was gluten ataxia (25%). A genetic cause was identified in 156 (13%) of sporadic cases with other causes being alcohol excess (12%) and cerebellar variant of multiple system atrophy (11%). Using NGS, positive results were obtained in 32% of 146 patients tested. The commonest ataxia identified was EA2. A genetic diagnosis was achieved in 57% of all familial ataxias. The commonest genetic ataxias were Friedreich9s ataxia (22%), SCA6 (14%), EA2 (13%), SPG7 (10%) and mitochondrial disease (10%). The diagnostic yield following attendance at the Sheffield Ataxia Centre was 63%. <h3>Conclusions</h3> Immune-mediated ataxias are common. Advances in genetic testing have significantly improved the diagnostic yield of patients suspected of having a genetic ataxia. Making a diagnosis of the cause of ataxia is essential due to potential therapeutic interventions for immune and some genetic ataxias.

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin1,2. However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn−/− microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn−/− microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration. In the absence of progranulin, microglia enter a disease-specific state that causes endolysosomal dysfunction and neurodegeneration, and these microglia promote TDP-43 granule formation, nuclear pore defects and cell death specifically in excitatory neurons via the complement activation pathway.

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

To expand the clinical phenotype of autosomal dominant congenital spinal muscular atrophy with lower extremity predominance (SMA-LED) due to mutations in the dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) gene.Patients with a phenotype suggestive of a motor, non-length-dependent neuronopathy predominantly affecting the lower limbs were identified at participating neuromuscular centers and referred for targeted sequencing of DYNC1H1.We report a cohort of 30 cases of SMA-LED from 16 families, carrying mutations in the tail and motor domains of DYNC1H1, including 10 novel mutations. These patients are characterized by congenital or childhood-onset lower limb wasting and weakness frequently associated with cognitive impairment. The clinical severity is variable, ranging from generalized arthrogryposis and inability to ambulate to exclusive and mild lower limb weakness. In many individuals with cognitive impairment (9/30 had cognitive impairment) who underwent brain MRI, there was an underlying structural malformation resulting in polymicrogyric appearance. The lower limb muscle MRI shows a distinctive pattern suggestive of denervation characterized by sparing and relative hypertrophy of the adductor longus and semitendinosus muscles at the thigh level, and diffuse involvement with relative sparing of the anterior-medial muscles at the calf level. Proximal muscle histopathology did not always show classic neurogenic features.Our report expands the clinical spectrum of DYNC1H1-related SMA-LED to include generalized arthrogryposis. In addition, we report that the neurogenic peripheral pathology and the CNS neuronal migration defects are often associated, reinforcing the importance of DYNC1H1 in both central and peripheral neuronal functions.

To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.

Following years of rapid progress identifying the genetic underpinnings of amyotrophic lateral sclerosis (ALS) and related diseases such as frontotemporal dementia (FTD), remarkable consistencies have emerged pointing to perturbed biology of heterogeneous nuclear ribonucleoproteins (hnRNPs) as a central driver of pathobiology. To varying extents these RNA-binding proteins are deposited in pathological inclusions in affected tissues in ALS and FTD. Moreover, mutations in hnRNPs account for a significant number of familial cases of ALS and FTD. Here we review the normal function and potential pathogenic contribution of TDP-43, FUS, hnRNP A1, hnRNP A2B1, MATR3, and TIA1 to disease. We highlight recent evidence linking the low complexity sequence domains (LCDs) of these hnRNPs to the formation of membraneless organelles and discuss how alterations in the dynamics of these organelles could contribute to disease. In particular, we discuss the various roles of disease-associated hnRNPs in stress granule assembly and disassembly, and examine the emerging hypothesis that disease-causing mutations in these proteins lead to accumulation of persistent stress granules.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.

Prion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorder.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Mutations in the stress granule protein T-cell restricted intracellular antigen 1 (TIA1) were recently shown to cause amyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD). Here, we provide detailed clinical and neuropathological descriptions of nine cases with TIA1 mutations, together with comparisons to sporadic ALS (sALS) and ALS due to repeat expansions in C9orf72 (C9orf72+). All nine patients with confirmed mutations in TIA1 were female. The clinical phenotype was heterogeneous with a range in the age at onset from late twenties to the eighth decade (mean = 60 years) and disease duration from one to 6 years (mean = 3 years). Initial presentation was either focal weakness or language impairment. All affected individuals received a final diagnosis of ALS with or without FTD. No psychosis or parkinsonism was described. Neuropathological examination on five patients found typical features of ALS and frontotemporal lobar degeneration (FTLD-TDP, type B) with anatomically widespread TDP-43 proteinopathy. In contrast to C9orf72+ cases, caudate atrophy and hippocampal sclerosis were not prominent. Detailed evaluation of the pyramidal motor system found a similar degree of neurodegeneration and TDP-43 pathology as in sALS and C9orf72+ cases; however, cases with TIA1 mutations had increased numbers of lower motor neurons containing round eosinophilic and Lewy body-like inclusions on HE stain and round compact cytoplasmic inclusions with TDP-43 immunohistochemistry. Immunohistochemistry and immunofluorescence failed to demonstrate any labeling of inclusions with antibodies against TIA1. In summary, our TIA1 mutation carriers developed ALS with or without FTD, with a wide range in age at onset, but without other neurological or psychiatric features. The neuropathology was characterized by widespread TDP-43 pathology, but a more restricted pattern of neurodegeneration than C9orf72+ cases. Increased numbers of round eosinophilic and Lewy-body like inclusions in lower motor neurons may be a distinctive feature of ALS caused by TIA1 mutations.

Approximately 1% of human proteins harbor a prion-like domain (PrLD) of similar low complexity sequence and amino acid composition to domains that drive prionogenesis of yeast proteins like Sup35. PrLDs are over-represented in human RNA-binding proteins and mediate phase transitions underpinning RNP granule assembly. This modality renders PrLDs prone to misfold into conformers that accrue in pathological inclusions that characterize various fatal neurodegenerative diseases. For example, TDP-43 and FUS form cytoplasmic inclusions in amyotrophic lateral sclerosis (ALS) and mutations in TDP-43 and FUS can cause ALS. Here, we review our recent discovery of discrete missense mutations that alter a conserved gatekeeper aspartate residue in the PrLDs of hnRNPA2/B1 and hnRNPA1 and cause multisystem proteinopathy and ALS. The missense mutations generate potent steric zippers in the PrLDs, which enhance a natural propensity to form self-templating fibrils, promote recruitment to stress granules and drive cytoplasmic inclusion formation. PrLDs occur in ~250 human proteins and could contribute directly to the etiology of various degenerative disorders.

A preclinical therapy to treat neurodegeneration is developed that selectively targets the AF-2 domain of the androgen receptor while sparing other functions of this receptor. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.

Several rare inherited disorders have been described that show phenotypic overlap with Paget's disease of bone (PDB) and in which PDB is a component of a multisystem disorder affecting muscle and the central nervous system. These conditions are the subject of this review article. Insertion mutations within exon 1 of the TNFRSF11A gene, encoding the receptor activator of nuclear factor kappa B (RANK), cause severe PDB-like disorders including familial expansile osteolysis, early-onset familial PDB and expansile skeletal hyperphosphatasia. The mutations interfere with normal processing of RANK and cause osteoclast activation through activation of nuclear factor kappa B (NFκB) independent of RANK ligand stimulation. Recessive, loss-of-function mutations in the TNFRSF11B gene, which encodes osteoprotegerin, cause juvenile PDB and here the bone disease is due to unopposed activation of RANK by RANKL. Multisystem proteinopathy is a disorder characterised by myopathy and neurodegeneration in which PDB is often an integral component. It may be caused by mutations in several genes including VCP, HNRNPA1, HNRNPA2B1, SQSTM1, MATR3, and TIA1, some of which are involved in classical PDB. The mechanisms of osteoclast activation in these conditions are less clear but may involve NFκB activation through sequestration of IκB. The evidence base for management of these disorders is somewhat limited due to the fact they are extremely rare. Bisphosphonates have been successfully used to gain control of elevated bone remodelling but as yet, no effective treatment exists for the treatment of the muscle and neurological manifestations of MSP syndromes.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.

The genetics of human disease serves as a robust and unbiased source of insight into human biology, both revealing fundamental cellular processes and exposing the vulnerabilities associated with their dysfunction. Over the last decade, the genetics of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have epitomized this concept, as studies of ALS-FTD-causing mutations have yielded fundamental discoveries regarding the role of biomolecular condensation in organizing cellular contents while implicating disturbances in condensate dynamics as central drivers of neurodegeneration. Here we review this genetic evidence, highlight its intersection with patient pathology, and discuss how studies in model systems have revealed a role for aberrant condensation in neuronal dysfunction and death. We detail how multiple, distinct types of disease-causing mutations promote pathological phase transitions that disturb the dynamics and function of ribonucleoprotein (RNP) granules. Dysfunction of RNP granules causes pleiotropic defects in RNA metabolism and can drive the evolution of these structures to end-stage pathological inclusions characteristic of ALS-FTD. We propose that aberrant phase transitions of these complex condensates in cells provide a parsimonious explanation for the widespread cellular abnormalities observed in ALS as well as certain histopathological features that characterize late-stage disease.

Amyotrophic lateral sclerosis (ALS) is a multi-system disease characterized primarily by progressive muscle weakness. Cognitive dysfunction is commonly observed in patients; however, factors influencing risk for cognitive dysfunction remain elusive. Using sparse canonical correlation analysis (sCCA), an unsupervised machine-learning technique, we observed that single nucleotide polymorphisms collectively associate with baseline cognitive performance in a large ALS patient cohort (N = 327) from the multicenter Clinical Research in ALS and Related Disorders for Therapeutic Development (CReATe) Consortium. We demonstrate that a polygenic risk score derived using sCCA relates to longitudinal cognitive decline in the same cohort and also to in vivo cortical thinning in the orbital frontal cortex, anterior cingulate cortex, lateral temporal cortex, premotor cortex, and hippocampus (N = 90) as well as post-mortem motor cortical neuronal loss (N = 87) in independent ALS cohorts from the University of Pennsylvania Integrated Neurodegenerative Disease Biobank. Our findings suggest that common genetic polymorphisms may exert a polygenic contribution to the risk of cortical disease vulnerability and cognitive dysfunction in ALS.

Homozygous loss-of-function mutations in optineurin (OPTN) are a rare cause of amyotrophic lateral sclerosis (ALS), whereas heterozygous loss-of-function mutations have been suggested to increase ALS disease risk. We report a patient with ALS and frontotemporal dementia (FTD) from the Clinical Research in ALS and Related Disorders for Therapeutic Development (CReATe) Consortium carrying compound heterozygous loss-of-function variants in OPTN. Quantitative real-time mRNA expression analyses revealed a 75–80% reduction in OPTN expression in blood in the OPTN carrier as compared to controls, suggesting at least partial nonsense-mediated decay of the mutant transcripts. This case report illustrates the diverse inheritance patterns and variable clinical presentations associated with OPTN mutations, and underscores the importance of complete OPTN gene screening in patients with ALS and related disorders, especially in the context of clinical genetic testing.

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules, uncovering a role for stress granules in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID introduces a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

Amyotrophic lateral sclerosis (ALS) is a degenerative condition of the brain and spinal cord in which protein-coding variants in known ALS disease genes explain a minority of sporadic cases. There is a growing interest in the role of noncoding structural variants (SVs) as ALS risk variants or genetic modifiers of ALS phenotype. In small European samples, specific short SV alleles in noncoding regulatory regions of SCAF4, SQSTM1, and STMN2 have been reported to be associated with ALS, and several groups have investigated the possible role of SMN1/SMN2 gene copy numbers in ALS susceptibility and clinical severity.Using short-read whole genome sequencing (WGS) data, we investigated putative ALS-susceptibility SCAF4 (3'UTR poly-T repeat), SQSTM1 (intron 5 AAAC insertion), and STMN2 (intron 3 CA repeat) alleles in African ancestry patients with ALS and described the architecture of the SMN1/SMN2 gene region. South African cases with ALS (n = 114) were compared with ancestry-matched controls (n = 150), 1000 Genomes Project samples (n = 2,336), and H3Africa Genotyping Chip Project samples (n = 347).There was no association with previously reported SCAF4 poly-T repeat, SQSTM1 AAAC insertion, and long STMN2 CA alleles with ALS risk in South Africans (p > 0.2). Similarly, SMN1 and SMN2 gene copy numbers did not differ between South Africans with ALS and matched population controls (p > 0.9). Notably, 20% of the African samples in this study had no SMN2 gene copies, which is a higher frequency than that reported in Europeans (approximately 7%).We did not replicate the reported association of SCAF4, SQSTM1, and STMN2 short SVs with ALS in a small South African sample. In addition, we found no link between SMN1 and SMN2 copy numbers and susceptibility to ALS in this South African sample, which is similar to the conclusion of a recent meta-analysis of European studies. However, the SMN gene region findings in Africans replicate previous results from East and West Africa and highlight the importance of including diverse population groups in disease gene discovery efforts. The clinically relevant differences in the SMN gene architecture between African and non-African populations may affect the effectiveness of targeted SMN2 gene therapy for related diseases such as spinal muscular atrophy.

Abstract Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA–protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2) 58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2) 58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter). https://doi.org/10.7554/eLife.39578.001 Introduction Genetic, pathologic, biophysical, and cell biological evidence has implicated disturbances in stress granules as a primary driver of several common neurodegenerative diseases, including ALS, FTD, and inclusion body myopathy (IBM) (Molliex et al., 2015; Mackenzie et al., 2017; Taylor et al., 2016; Lee et al., 2016; Ramaswami et al., 2013; Buchan et al., 2013; Patel et al., 2015; Hackman et al., 2013). These diseases show substantial clinical and genetic overlap and share the hallmark histopathological feature of cytoplasmic inclusions composed of RNA-binding proteins and other constituents of ribonucleoprotein (RNP) granules in affected neurons and muscle cells. A prominent feature of this end-stage cytoplasmic pathology is ubiquitinated and phosphorylated forms of TDP-43, although a host of other proteins co-localize with these pathological inclusions, including related RNA-binding proteins and ubiquitin-binding proteins such as SQSTM1, UBQLN2, OPTN, and VCP (Neumann et al., 2006; Mackenzie et al., 2007; Mackenzie and Neumann, 2016; Williams et al., 2012; Deng et al., 2011). Many mutations that cause ALS-FTD and/or IBM impact RNA-binding proteins that are building blocks of stress granules (e.g., TDP-43, hnRNPA1, hnRNPA2B1, hnRNPDL, TIA1, matrin 3, and FUS). Furthermore, these mutations largely cluster in low-complexity, intrinsically disordered regions (IDRs) and in many cases have been shown to change the dynamic properties of stress granules (Mackenzie et al., 2017; Hackman et al., 2013; Kim et al., 2013; Liu-Yesucevitz et al., 2010). Another set of disease-causing mutations impact ubiquitin-binding proteins (e.g., UBQLN2, VCP, p62/SQSTM1, and OPTN) whose functions intersect with disassembly and/or clearance of stress granules (Buchan et al., 2013; Dao et al., 2018; Chitiprolu et al., 2018). Furthermore, pathological poly-dipeptides arising from repeat-expanded C9orf72, the most common genetic cause of ALS-FTD, insinuate into stress granules and other membrane-less organelles, perturbing their dynamics and/or functions (Lee et al., 2016; Boeynaems et al., 2017). Several ALS-, FTD-, and IBM-causing mutations cause aberrant phase separation and change the biophysical and material properties of stress granules, generally resulting in poorly dynamic membrane-less organelles that, it has been suggested, may evolve into the cytoplasmic pathology found in end-stage disease (Mackenzie et al., 2017; Buchan et al., 2013; Kim et al., 2013). However, no direct evidence has demonstrated that perturbation of phase separation is sufficient to drive neurotoxicity or that ALS-FTD-associated inclusions represent the endpoint of a formerly dynamic stress granule. Moreover, capitalizing on mechanistic links between stress granules and disease to identify therapeutic targets has been limited by models employing exogenous stressors (e.g., heat shock, arsenite) to initiate stress granule assembly, with numerous nonspecific and pleiotropic effects. Stress granules are comparatively large (~50 nm to ~3 μm) biomolecular condensates that rapidly form in the cytoplasm in response to a wide variety of stressors (Protter and Parker, 2016; Panas et al., 2016). Like other RNP granules, stress granules are believed to arise at least in part through liquid-liquid phase separation (LLPS), a biophysical phenomenon in which RNA-protein complexes separate from the surrounding aqueous cytoplasm to create a functional cellular compartment with liquid properties (Molliex et al., 2015; Protter and Parker, 2016). Stress granule assembly is a complex process that involves a cascade of events, including the dismantling of polysomes and reorganization of mRNPs into discrete cytoplasmic foci that contain >400 different protein constituents (Jain et al., 2016; Markmiller et al., 2018; Youn et al., 2018) and >1800 different RNAs (Khong et al., 2017). The assembly of RNP granules, including stress granules, is driven in part by the collective behavior of many macromolecular interactions, including RNA-RNA interactions, protein-RNA interactions, conventional interactions between folded protein domains, as well as weak, transient interactions mediated by low complexity IDRs of proteins – particularly those present in RNA-binding proteins (Banani et al., 2017). While there is consensus about the major underlying forces that drive RNP granule assembly, the precise mechanisms that orchestrate the assembly of distinct types of RNP granules are largely unknown, although general principles have been suggested by in vitro studies (Banani et al., 2016). In this conceptual framework, RNP granules and other biomolecular condensates are established and maintained by a small number of essential constituents defined as scaffolds, whereas the remaining constituents are considered clients (Banani et al., 2016). Although at least six proteins have been suggested to be ‘essential’ elements of stress granules (Markmiller et al., 2018; Youn et al., 2018; Kedersha et al., 2016; Gilks et al., 2004; Kwon et al., 2007), until recently it was unknown which of these proteins (if any) are true scaffolds for stress granules. In related work that informs the study presented here, we performed a whole-genome screen that identified G3BP as a uniquely essential scaffold in stress granule assembly (Yang, Mathieu et al., unpublished). Moreover, we found that an oligomerization domain within G3BP that is essential to stress granule assembly could be functionally replaced by heterologous oligomerization domains, which suggested the possibility of engineering temporal and spatial control of stress granule assembly without the confounding influences of stress (Yang, Mathieu et al., unpublished). We built upon a previously described system, termed ‘OptoDroplets,’ which uses optogenetic oligomerization of proteins as a means to control intracellular LLPS (Shin et al., 2017). In this system, light-sensitive chimeric proteins are assembled from the IDRs of various RNP granule proteins combined with the light-sensitive oligomerization domain of Arabidopsis thaliana cryptochrome 2 (CRY2) photolyase homology region (PHR) to generate fusion proteins that undergo LLPS in living cells upon blue light activation. Whereas enforced aggregation of IDRs drives LLPS and thereby leads to OptoDroplet formation, it is not anticipated that droplets formed by the IDRs of any given RNP granule protein will initiate the full cascade of bona fide RNP granule assembly. However, we reasoned that adapting this OptoDroplet system might provide a means of testing the hypothesis that enforced LLPS of key stress granule constituents could distinguish between stress granule scaffolds and clients, in which LLPS of a scaffold protein would initiate a process that faithfully reconstitutes the assembly of a stress granule, whereas LLPS of a client protein would not. Moreover, if we succeeded in optical induction of stress granules, it would provide the first opportunity to examine the consequences of protracted stress granule assembly without the confounding variable of exogenous stress. Herein we report that light-based activation of Opto-G3BP1, a chimeric protein assembled from the IDR and RNA-binding domain of G3BP1 combined with CRY2PHR, initiated the rapid assembly of dynamic, cytoplasmic, liquid granules that were composed of canonical stress granule components, including PABP, TDP-43, TIA1, TIAR, eIF4G, eIF3η, ataxin 2, GLE1, FUS, and polyadenylated RNA, thereby establishing the identity of G3BP1 as a scaffold protein for stress granules. To differentiate these complex assemblies formed by LLPS of the scaffold protein G3BP1 from the relatively homogenous clusters formed by LLPS of client proteins, we termed these structures OptoGranules. Importantly, we found that persistent or repetitive assembly of OptoGranules is cytotoxic and is accompanied by the evolution of these granules to neuronal cytoplasmic inclusions characteristic of ALS-FTD. Results To test whether optogenetically induced LLPS of a stress granule scaffold protein could faithfully reconstitute the assembly of a bona fide stress granule, we first investigated G3BP1 as a potential scaffold protein. G3BP1 (and its close paralog G3BP2) has been suggested to be an essential nucleator of stress granule assembly (Kedersha et al., 2016), and a genome-wide screen recently identified G3BP1/2 as a uniquely essential protein for stress granule assembly (Yang, Mathieu et al., unpublished). G3BP1 has an N-terminal 142-amino acid dimerization domain, termed the NTF2L domain, that is essential for nucleation of stress granule assembly. Remarkably, the NTF2L domain can be replaced by generic dimerization domains, and the resulting chimeric proteins are able to fully nucleate stress granule assembly in living cells (Yang, Mathieu et al., unpublished). Thus, the domain architecture of G3BP1 is ideal for engineering light-inducible stress granule assembly by replacing the NTF2L domain of G3BP1 with the blue light-dependent dimerization domain CRY2PHR in frame with the fluorescent proteins mCherry or mRuby. We named this construct ‘Opto-G3BP1’ and also created an ‘Opto-Control’ construct referring to CRY2PHR-mCherry (or mRuby) alone (Figure 1a). Figure 1 with 4 supplements see all Download asset Open asset OptoGranules are light-inducible dynamic stress granules. (a) Design of Opto-G3BP1 and Opto-Control constructs. (b) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 MW/cm2) in a defined ROI. Representative images are shown from n = 3 independent experiments. (c) Quantification of data in cells treated as in (b). Five cells with similar expression levels were counted. Granule numbers are shown relative to the granule number at the peak of OptoGranule assembly. Error bars represent s.e.m. (d-f) U2OS cells were stably transfected with Opto-Control or Opto-G3BP1, or stable Opto-G3BP1 cells were transiently transfected with G3BP1-GFP, and stimulated with a blue-light laser (power density ~4.5 W/cm2) for 3 mins. Regions marked with yellow circles were photobleached and monitored for fluorescence recovery. Data are shown as representative images (d), relative fluorescence intensity of photobleached region over time (e), and relative mobile fraction derived from (e) (f). For (e, f) n = 15 cells for Opto-Control; n = 12 for Opto-G3BP1; n = 14 for G3BP1-GFP. Data are representative of n = 3 independent experiments. Data shown as mean + s.d. ns, not significant by one-way ANOVA with Dunnett’s test. (g) U2OS cells transiently transfected with Opto-G3BP1 and the stress granule marker GFP-TIA1 were stimulated with a blue-light laser (power density ~2.5 MW/cm2) for 5 msec. Cells were sequentially imaged by 561 nm and 488 nm channels; we note that the 488 nm channel used for imaging also activates Opto-G3BP1 (power density 2.2 W/cm2). Representative images are shown from n = 3 independent experiments. (h-j) U2OS cells stably expressing Opto-Control or Opto-G3BP1 constructs were stimulated for 6 hr without or with continuous blue light (~2 mW/cm2) using custom-made LED arrays for global activation. Cells were immunostained with PABP antibody (h), TDP-43 antibody (i), or RNA fluorescence in situ hybridization using FAM-labelled oligo (dT)20 as a probe (j). Scale bars, 10 µm in all micrographs. https://doi.org/10.7554/eLife.39578.002 We next generated U2OS cell lines stably expressing Opto-Control or Opto-G3BP1 constructs with comparable expression levels of Opto-G3BP1 and endogenous G3BP1 (Figure 1—figure supplement 1a). Within seconds of blue light activation, Opto-G3BP1 in U2OS cells assembled into numerous, spherical cytoplasmic granules that exhibited liquid behaviors (Figure 1b and Videos 1 and 2). A 5-millisecond pulse of blue light using a 488 nm vector laser (~2.5 MW/cm2) was sufficient to initiate robust induction of these cytoplasmic granules, and these granules spontaneously disassembled over a period of approximately 5 min (Figure 1b,c). These granules were highly dynamic, exhibiting liquid behaviors such as fusion to form larger granules and relaxation to a spherical shape (Video 2). In contrast, under the same conditions, Opto-Control expression remained diffuse, with a modest amount of nuclear and cytoplasmic clusters (Figure 1b and Video 1). To confirm the dynamic nature of the optically induced granules, we performed fluorescence recovery after photobleaching (FRAP) to monitor recovery rates and mobile fractions of individual granules (Figure 1d–f), finding that these properties were very similar between Opto-G3BP1 and the conventional stress granule marker G3BP1-GFP. Furthermore, Opto-G3BP1 localized to spontaneous stress granules induced by expression of ALS mutant proteins (FUS R521C, TDP-43 ΔNLS, TIA1 A381T) even in the absence of blue light activation, demonstrating that the Opto-G3BP1 protein behaves similarly to endogenous G3BP1 (Figure 1—figure supplement 1b). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Opto-Control fails to assemble light-dependent cytoplasmic clusters. U2OS cells stably expressing Opto-Control were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 MW/cm2) in a defined ROI. See Video 2 for corresponding Opto-G3BP1 condition. https://doi.org/10.7554/eLife.39578.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Opto-G3BP1 assembles light-dependent cytoplasmic clusters. U2OS cells stably expressing Opto-G3BP1 were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 MW/cm2) in a defined ROI. Opto-G3BP1 assembles highly dynamic, liquid-like cytoplasmic granules, and these granules spontaneously disassemble over a period of approximately 5 min. See Video 1 for corresponding Opto-Control condition. https://doi.org/10.7554/eLife.39578.008 To further define the relationship between light-induced Opto-G3BP1 granules and stress-induced stress granules, we next examined their composition. Employing live cell imaging, we documented the dynamic recruitment of the stress granule marker GFP-TIA1 into optically induced granules following light-induced assembly (Figure 1g and Video 3). In contrast, clusters of Opto-Control (olig), a modified form of the Opto-Control construct designed to produce abundant aggregates, did not recruit GFP-TIA1 (Figure 1—figure supplement 1c), nor did they show dynamic behavior by FRAP (Figure 1—figure supplement 1d–f). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Dynamic recruitment of the stress granule marker GFP-TIA1 into light-induced Opto-G3BP1 granules. U2OS cells transiently transfected with Opto-G3BP1 and the stress granule marker GFP-TIA1 were stimulated with a blue-light laser (power density ~2.5 MW/cm2) for 5 msec. Cells were sequentially imaged by 561 nm and 488 nm channels; we note that the 488 nm channel used for imaging (power density 2.2 W/cm2) also activates Opto-G3BP1. https://doi.org/10.7554/eLife.39578.009 We further examined the composition of optically induced Opto-G3BP1 granules by staining activated cells for additional stress granule components. In these experiments, we employed a blue-light LED array that permitted global activation of a larger number of cells. This LED array has a much lower energy density (~2 mW/cm2) than the laser used for dynamic imaging, drives less oligomerization of CRY2PHR, and therefore offers slower kinetics to facilitate monitoring of the recruitment of granule components over time. In cells expressing Opto-G3BP1, but not Opto-Control, all stress granule components that we examined, including PABP, TDP-43, TIA1, TIAR, eIF4G, eIF3η, ataxin 2, GLE1, and FUS, were recruited to optically induced granules (Figure 1h–i and Figure 1—figure supplement 2a–g). Since stress granules represent assemblies of mRNA as well as protein (Panas et al., 2016; Kedersha et al., 1999), we used fluorescent in situ hybridization (FISH) with fluorescently conjugated oligo(dT) probes to examine whether polyadenylated mRNAs were present in these optically induced granules as in canonical stress granules. We found that polyadenylated mRNAs were recruited into optically induced granules that assembled after blue light stimulation but showed no relocalization in cells expressing Opto-Control (Figure 1j). These findings indicate that optically induced Opto-G3BP1 granules are stress granules composed of mRNAs and RNA-binding proteins, including ALS-associated proteins such as TDP-43, ataxin 2, GLE1, FUS, and TIA1. Consistent with prior reports, knockout of endogenous G3BP1 and G3BP2 in U2OS cells abolished stress granule assembly in response to arsenite (Kedersha et al., 2016) (Figure 1—figure supplement 3a). When introduced into these G3BP1/G3BP2 double knockout cells, Opto-G3BP1 (or the analogous chimeric protein Opto-G3BP2) substantially restored stress granule assembly in response to blue light activation, demonstrating that the scaffolding activity of G3BP1 in the chimeric protein is functionally intact (Figure 1—figure supplement 3b,c). The initiation of stress granule assembly in response to enforced LLPS of G3BP1 differs from prior observations made regarding OptoDroplets, which do not typically represent assembly of complex, physiologically assembled membrane-less organelles (Shin et al., 2017). To examine this in more detail, we generated a series of optically inducible chimeric proteins by generating constructs in which CRY2PHR-mCherry was fused with stress granule constituent proteins, including full-length or truncated versions of FUS, TDP-43, and TIA1. Opto-FUS [CRY2PHR-mCherry-FUS(IDR)] and Opto-TDP-43 [CRY2PHR-mCherry-TDP-43(IDR)] did assemble into droplets with blue light activation, as previously reported (Shin et al., 2017), but these OptoDroplets did not recruit stress granule constituents commonly used as markers, including G3BP1 and PABP (Figure 1—figure supplement 4a–c), and were also negative for stress granule constituents VCP, SQSTM1, and the related protein OPTN (Figure 1—figure supplement 4d,e). Similarly, constructs containing the IDR and RNA recognition motifs of FUS or TDP-43 [CRY2PHR-mCherry-FUS (1–371 aa); CRY2PHR-mCherry-TDP-43 (106–414 aa)] assembled into droplets upon blue light activation, but these droplets were also negative for stress granule markers (Figure 1—figure supplement 4f,g). Expression of Opto-constructs using full-length FUS or TDP-43 [CRY2PHR-mCherry-FUS(FL); CRY2PHR-mCherry-TDP-43(FL)] did not produce stress granules with blue light activation (Figure 1—figure supplement 4f,g). Finally, Opto-TIA1, which represents fusion of CRY2 with TIA1 (CRY2PHR-mCherry-TIA1), also assembled into droplets with blue light activation, but did not drive the assembly of stress granules, as illustrated by lack of colocalization with stress granule markers (Figure 1—figure supplement 4h–j). These data indicate that RNP granule assembly cannot be driven by enforced LLPS of any random constituent, but depends upon specific constituents. This conclusion is consistent with the proposition that LLPS initiated by scaffold proteins (e.g., G3BP1) has the capacity to initiate a membrane-less organelle, whereas client proteins (e.g., FUS, TDP-43, TIA1), even when forced to undergo LLPS, cannot reconstitute such a complex assembly (Banani et al., 2017). Thus, we termed Opto-G3BP1-induced stress granules ‘OptoGranules’ to distinguish them from OptoDroplets. Phase transitions are highly dependent on protein concentration, and we therefore hypothesized that the induction of OptoGranule assembly would be dependent on the local concentration of activated G3BP1, similar to the concentration-dependent formation of light-activated OptoDroplets (Shin et al., 2017). To test this prediction, we controlled the local G3BP1 molecular concentration by modulating either the intensity of the activating blue light or the expression level of the Opto-G3BP1 construct. As predicted, we observed a strong positive correlation between blue light intensity and induction of OptoGranules (Figure 2a,b) and, independently, a strong positive correlation between Opto-G3BP1 expression level and induction of OptoGranules (Figure 2c,d). Thus, the OptoGranule system is highly tunable, a useful feature for a variety of studies. Figure 2 with 1 supplement see all Download asset Open asset OptoGranule formation is dependent on the local concentration of activated G3BP1 and dependent on polysome disassembly, but independent of eIF2α phosphorylation. (a) U2OS cells stably expressing Opto-G3BP1 were intermittently exposed to a blue-light laser (488 nm) for activation followed by image acquisition with a 561 nm channel. Blue light intensity was sequentially increased from top to bottom (488 nm power density measurement from top to bottom: 1%, 0.02 W/cm2; 5%, 0.04 W/cm2; 25%, 0.95 W/cm2; 75%, 5.5 W/cm2). Representative images are shown from n = 3 independent experiments. (b) Quantification of data in cells treated as in (a). Error bars represent s.d. (c) U2OS cells with different expression levels of Opto-G3BP1 were intermittently exposed to a 488 nm blue-light laser (90% laser power, power density 6.3 W/cm2) followed by image acquisition with a 561 nm channel. Relative expression levels from top to bottom: 0.19, 0.32, 0.78 and 1 a.u. Representative images are shown from n = 3 independent experiments. (d) Quantification of data in cells treated as in (c). (e) U2OS cells stably expressing Opto-G3BP1 were pre-treated with cycloheximide (CHX) or ISRIB for 30 min and then exposed to 45 min of sodium arsenite (0.5 mM NaAsO2) or 6 hr of continuous blue light (~2 mW/cm2) using custom-made LED arrays for global activation, and immunostained with PABP antibody. (f) Quantification of granule-positive cells from (e). Data are shown as mean ± s.e.m. from n = 3 independent experiments. ****p<0.0001; ns, not significant by one-way ANOVA with Tukey’s post-test. (g) Immunoblot showing phosphorylated eIF2α (P-eIF2α), eIF2α, and actin levels in cells treated with sodium arsenite (0.5 mM NaAsO2) for 45 min, exposed to 42°C heat shock for 1 hr, or activated with blue light for 6 hr. See also Figure 2—figure supplement 1 for sequential probe images. Scale bars, 10 μm in all micrographs. https://doi.org/10.7554/eLife.39578.010 We next examined the role of upstream events in OptoGranule formation and compared these to the cellular triggers associated with conventional stress granule assembly. Given that conventional stress granule formation is typically linked to the disassembly of translating polysomes (Panas et al., 2016), we tested whether polysome disassembly is required for OptoGranule formation. We determined that treatment with cycloheximide, which traps translating mRNAs within polysomes, strongly mitigated the formation of arsenite-induced stress granules and the formation of light-induced OptoGranules (Figure 2e,f), indicating that OptoGranule formation is dependent on polysome disassembly and further illustrating commonality with conventional stress granules. We next tested the role of eIF2α phosphorylation, which integrates stress granule formation downstream of a variety of stressors, such as arsenite and heat shock (Panas et al., 2016). We used the small molecule ISRIB, which binds eIF2B and interrupts eIF2α-mediated translational control (Sidrauski et al., 2015). We found that formation of arsenite-induced stress granules was blocked by ISRIB, as previously documented (Sidrauski et al., 2015), whereas the formation of light-induced OptoGranules was unaffected by ISRIB treatment (Figure 2e,f). Consistent with this finding, Western blotting also showed minimal phosphorylated eIF2α accompanying OptoGranule assembly (Figure 2g, Figure 2—figure supplement 1). Thus, OptoGranule formation depends upon the recruitment of mRNPs from polysomes, but this assembly occurs downstream and independent of the evolutionarily conserved integrated stress response regulated by eIF2α. This observation is consistent with the notion that OptoGranule formation is not driven by the classic signaling pathway for stress granule formation, which increases the concentration of free, uncoated RNA in the cytoplasm, but rather by oligomerization of G3BP1, which increases the valency for RNA binding. Given the accumulating evidence that disturbance of membrane-less organelles such as stress granules may contribute to the initiation or progression of disease, we hypothesized that discrete disturbance in the dynamics or material properties of stress granules should be sufficient to cause cytotoxicity and recapitulate the pathognomonic features of specific diseases. To test this prediction, we examined the consequences of chronic OptoGranule assembly. First, we examined the consequences of continuous blue light activation in cells expressing Opto-G3BP1 or Opto-Control. We found that continuous induction of OptoGranules using a blue-light LED array resulted in progressive loss of cell viability reflected by progressive loss of crystal violet staining and depletion of ATP levels (Figure 3a,b). However, we also noted that chronic exposure to blue light resulted in a moderate amount of cytotoxicity in cells expressing Opto-Control or parental U2OS cells (Figure 3b). Although cells expressing Opto-G3BP1 exhibited significantly greater loss of viability upon exposure to blue light than cells expressing Opto-Control or parental U2OS cells, we sought to eliminate this potentially confounding background toxicity. Figure 3 with 1 supplement see all Download asset Open asset Persistent OptoGranules are cytotoxic and evolve to pathological inclusions. (a,b) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were stimulated with continuous blue light (~2 mW/cm2) for indicated times using custom-made LED arrays and viability was assessed by crystal violet staining (a) or CellTiter-Glo 2.0 luminescence (b). Whiskers represent minimum to maximum from n = 9 biological replicates. ****p<0.0001.; ns, not significant by two-way ANOVA with Tukey’s post-test. (c,d) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were exposed to chronic persistent (c) or chronic intermittent (d) blue light (445 nm) stimulation with live-cell imaging (power density ~0.12 W/cm2) as illustrated in the schematic (left) and assessed for cell survival by counting living cells (right). Blue boxes in schematic indicate the timing of light induction; red line is an idealized graph of the cellular response. Chronic persistent paradigm: n = 26 for Opto-Control and n = 28 for Opto-G3BP1. Chronic intermittent paradigm: n = 7 for Opto-Control and n = 10 for Opto-G3BP1. Data are shown from n = 3 independent experiments. ****p<0.0001 by log-rank (Mantel-Cox) test. (e) Timeline of protein accumulation in OptoGranules in U2OS cells. (f-h) U2OS cells stably expressing Opto-G3BP1 were stimulated with continuous blue light (~2 mW/cm2) for indicated times using custom-made LED arrays and co-immunostained with p-TDP-43 and A11 antibodies (f), SQSTM1 and ubiquitin antibodies (g), or VCP and TDP-43 antibodies (h). (i) quantification of data from (f-h). Error bars represent s.e.m. Images in f-h are representative of n = 3 independent experiments. ***p=0.0002 (2 hr), ***p=0.0001 (3 hr) for TDP-43, **p=0.0048 (2 hr), ***p=0.0002 (3 hr) for A11, **p=0.0051 (5 hr) for ubiquitin, ****p<0.0001 for SQSTM1, ***p=0.0003 for pTDP-43, and ****p<0.0001 for V

Loss of nuclear TDP-43 has been linked to amyotrophic lateral sclerosis (ALS), which also features increased genome damage in affected neurons. Although TDP-43 binds to DNA, its role in the DNA damage response (DDR) has not been investigated. Here, we report that nuclear TDP-43 is a critical component of the DNA double-strand break (DSB) repair machinery in motor neurons. TDP-43 is rapidly recruited at DSB sites where it stably interact with non-homologous end joining (NHEJ)- mediated DSB repair and DDR factors in spinal motor neurons derived from human neural stem cells. Total or nucleus-specific loss of TDP-43 in motor neurons and C. elegans markedly increased genomic DSBs by impairing NHEJ and sensitized cells to DSB stress. TDP-43 pathology strongly correlated with DSB accumulation and DDR activation in the spinal cord of postmortem ALS patients. Our findings link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases, hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth Type 2 (CMT2). In contrast, ALS associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss of function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

SUMMARY To discover novel genetic risk factors underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry matched controls. We observed a significant excess of ultra-rare and rare protein-truncating variants (PTV) among ALS cases, which was primarily concentrated in constrained genes; however, a significant enrichment in PTVs does persist in the remaining exome. Through gene level analyses, known ALS genes, SOD1, NEK1 , and FUS , were the most strongly associated with disease status. We also observed suggestive statistical evidence for multiple novel genes including DNAJC7 , which is a highly constrained gene and a member of the heat shock protein family (HSP40). HSP40 proteins, along with HSP70 proteins, facilitate protein homeostasis, such as folding of newly synthesized polypeptides, and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of degraded proteins can occur leading to aberrant protein aggregation, one of the pathological hallmarks of neurodegeneration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Abstract Liquid-liquid phase separation (LLPS) is an important mechanism of intracellular organization that underlies the assembly of a variety of distinct RNP granules. Fundamental biophysical principles governing LLPS during RNP granule assembly have been revealed by simple in vitro systems consisting of several components, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in live cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells, and allows for a wide variety of studies of membraneless organelles.

Cellular “bodies”, i.e. organelles in cells that are not enclosed by membranes, have been characterized as large protein assemblies with liquid-like properties, though the biophysical basis for their formation is currently unclear. Recent work demonstrated that weak, multivalent protein interactions can result in the formation of large higher-order complexes, can undergo liquid demixing phase separation in vitro and may enable the formation of cellular bodies. The inherent size heterogeneity of higher-order complexes renders them difficult to characterize biophysically and structurally. As a result, their size distributions remain largely unquantified, limiting molecular insight into their biological functions. We report novel mechanisms governing the formation of nuclear SPOP bodies and stress granules in which the self-association of folded domains into large homo-oligomers and of long disordered regions play key roles. We have used biophysical, biochemical and cell biological approaches to explore the self-association of proteins in vitro, to quantify the size distribution of the resulting higher-order oligomers, and to relate these to the function of cellular bodies in cells. We further explore how changes in self-association properties of constituent proteins, i.e. the destabilization or rigidification of cellular bodies, can lead to cancer and neurodegenerative diseases. We propose that dynamic, higher-order protein self-association is a general mechanism underlying the formation of cellular bodies. These may serve as hotspots of enzymatic or signaling activity, which can be dynamically turned on or off through the regulated assembly and disassembly of the organelle bodies, respectively.

Cellular “bodies”, i.e. organelles that are not enclosed by membranes, are large protein assemblies with liquid‐like properties, but the biophysical basis for their formation is largely unclear. Recent work demonstrated that weak, multivalent protein interactions, resulting in the formation of large higher‐order complexes, can undergo phase separation in vitro and may enable the formation of cellular bodies. The inherent size heterogeneity of higher‐order complexes renders them difficult to characterize biophysically, limiting molecular insight into their biological functions. We report novel mechanisms governing the formation of nuclear SPOP bodies and stress granules in which the self‐association of folded domains into large homo‐oligomers and of long disordered regions play key roles. We have used biophysical, biochemical and cell biological approaches to characterize protein self‐association in vitro, to quantify the size distribution of the resulting higher‐order oligomers, and to relate these to the function of organelles in cells. We explore how the destabilization or rigidification of cellular bodies can lead to cancer and neurodegenerative diseases. We propose that dynamic protein self‐association is a general mechanism underlying the formation of cellular bodies. These may serve as signaling hotspots that can be dynamically turned on or off through their regulated assembly and disassembly.

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996). The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect. Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Abstract Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 ( CHCHD10 ) are a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia (ALS-FTD). To elucidate how mutations in CHCHD10 induce disease, we generated a Drosophila melanogaster model of CHCHD10 -mediated ALS-FTD. Expression of CHCHD10 S59L in Drosophila caused gain-of-function toxicity in eyes, motor neurons, and muscles, in addition to mitochondrial defects in flies and HeLa cells. TDP-43 and PINK1 formed two axes, driving the mutant-dependent phenotypes. CHCHD10 S59L expression increased TDP-43 insolubility and mitochondrial translocation. Blocking mitochondrial translocation with a peptide inhibitor reduced CHCHD10 S59L -mediated toxicity. PINK1 knockdown rescued CHCHD10 S59L -mediated phenotypes in Drosophila and HeLa cells. The two PINK1 substrates mitofusin and mitofilin were genetic modifiers of this phenotype. Mitofusin agonists reversed the CHCHD10 S59L -induced phenotypes in Drosophila and HeLa cells and increased ATP production in Drosophila expressing C9orf72 with expanded GGGGCC repeats. Two peptides inhibitors of PINK1 mitigated the mitochondrial defects introduced by CHCHD10 S59L expression. These findings indicate that TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways by CHCHD10 S59L generate dominant toxicity. Therefore, inhibiting PINK1 activity may provide a therapeutic strategy for CHCHD10 -associated disease. One Sentence Summary Inhibition of TDP-43 mitochondrial translocation or PINK1 kinase activity mitigates CHCHD10 S59L -mediated mitochondrial toxicity.

Around 180 genes have been found in Arabidopsis that control lowering time based on analysis of transgenic plants [1]. Most of these genes take part in the six main pathways controlling flowering; the photoperiod, vernalization and the ambient temperature pathways that respond to environmental conditions, while the age-dependent, autonomous, and gibberellin pathways involve a response to endogenous signals [2]. Flowering Locus T (FT) is a major gene involved in flowering in plants because FT encodes mobile florigen for floral induction. Mutation of the FT gene can result in delayed flowering. Using a transient CRISPR-Cas9 expression system mediated by virus expression, it is possible to do gene-editing in plants without incorporation of exogenous DNA. We aim to develop these approaches to manipulate flowering time in plants.

OBJECTIVE. To find a treatment for Spinal and bulbar muscular atrophy (SBMA). BACKGROUND. SBMA is an adult-onset neuromuscular disorder caused by the expansion of a CAG repeat in the androgen receptor (AR) gene, encoding a polyglutamine tract in the AR protein. Androgen binding to the polyglutamine-expanded AR promotes heat shock protein dissociation and nuclear translocation of the mutant protein, and these are important steps in the disease pathogenesis. In SBMA, as in other polyglutamine disorders, toxicity is primarily mediated by a toxic gain of function by the mutant protein and the consequent disruption of critical downstream pathways, including transcription, axonal transport, and mitochondrial function. No disease-specific treatment for SBMA is currently available. DESIGN. Here we tested the effects of a small molecule, the curcumin derivative ASC-JM17 in in vitro and in vivo models of SBMA. The compound was selected as candidate for further pharmacological development based on its efficacy in downregulating the levels of AR and its oral bioavailability. RESULTS. We found that ASC-JM17 effectively promotes degradation of polyglutamine-expanded AR protein, induces the HSF1-dependent heat shock response and enhances proteasomal activity through the Nrf1/NFE2L1 pathway. We also showed that ASC-JM17 is a strong inducer of the Nrf2/NFE2L2 antioxidant response pathway, which is a primary cellular defense against oxidative stress. Interestingly, the protective effect of ASC-JM17 in a Drosophila model of the disease was independent of HSF1 but required the Nrf1/2 ortholog CncC suggesting a central role for the proteasome and/or oxidative stress response. Additionally, treatment of a cohort of SBMA transgenic mice by oral administration also resulted in a significant amelioration of the phenotype and of the histological and biochemical alterations associated with the disease. CONCLUSIONS. Taken together, our results establish ASC-JM17 as a candidate for pharmacological intervention in SBMA and other neurodegenerative disorders. Disclosure: Dr. Rinaldi has nothing to disclose. Dr. Bott has nothing to disclose. Dr. Badders has nothing to disclose. Dr. Bautista has nothing to disclose. Dr. Harmison has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Taylor has nothing to disclose. Dr. Dantuma has nothing to disclose. Dr. Fischbeck has nothing to disclose.

Inclusion body myopathy (IBM) associated with paget’s disease of the bone, fronto-temporal dementia and amyotrophic lateral sclerosis (ALS) is a multisystem degenerative proteinopathy (MSP) unified pathologically by ubiquitinated inclusions and TDP-43 accumulation. TDP-43 is an RNA binding protein with a Q/N rich prion-like domain (PrLD). PrLDs in RNA binding proteins mediate the assembly of RNA processing granules. Dominantly inherited mutations in valosin containing protein (VCP) have been previously reported to cause this syndrome. We identified two families with an MSP-like phenotype that did not harbor VCP mutations. Exome sequencing identified identical aspartate to valine mutations in two homologous RNA binding proteins hnRNPA2/B1 and hnRNPA1. These mutations introduce a potent “steric zipper” motif into their PrLD, which accelerates formation of self-seeding fibrils that can cross-seed polymerization of wild-type hnRNP. Disease causing mutations also promote incorporation of hnRNPA2 and A1 into stress granules and drive the formation of cytoplasmic inclusions in animal models. Consistent with this, MSP patient muscle, including VCP-associated MSP, accumulates hnRNPA2/B1, A1 and TDP-43 as sarcoplasmic aggregates that redistribute from their normal nuclear localization. Sporadic inclusion body myositis (sIBM) patient tissue also accumulates hnRNPA2/B1 and A1, as well as TDP-43 aggregates. Other distinctive features in MSP patient muscle biopsies include large regions of myopathic grouping, eosinophilic inclusions and rimmed vacuoles. Dysregulated polymerization of RNA binding proteins with PrLDs is an emerging pathogenic mechanism associated with degenerative phenotypes in IBM, ALS and dementia.

Abstract Ataxia is a common finding in patients seen in neurological practice and can be the result of a wide variety of causes. Although cerebellar degeneration can be chronic and slowly progressive, acute cerebellar swelling due to infarction, oedema or haemorrhage can have rapid and catastrophic effects and is a true neurological emergency. Here we set out to briefly describe the clinical/anatomical correlates of cerebellar disease and to provide a broad differential diagnosis for patients who present with cerebellar ataxia. This article specifically focuses on acquired causes of cerebellar ataxia. A separate complementary article discusses common hereditary causes of cerebellar ataxia. Key Concepts Clinical syndromes associated with cerebellar dysfunction may be divided into syndromes that predominantly affect the midline cerebellar structures (gait and ocular dysfunction predominant) or the cerebellar hemisphere(s) (ipsilateral appendicular ataxia, dysarthria). Acute onset of cerebellar ataxia is most commonly associated with vascular disorders (stroke or haemorrhage), toxins or infectious aetiologies. Subacute onset cerebellar ataxia can result from a variety of causes, including infectious, neoplastic, autoimmune and metabolic abnormalities. Many of these causes of ataxia are treatable. Most slowly progressive cerebellar ataxias result from neurodegenerative aetiologies, either inherited or sporadic.

Ependymomas are classified into nine molecular groups by DNA methylation profiling. PFA tumors are most prevalent, present mainly in infants and have poor outcomes. Genomic studies have not identified a recurrent driver mutation in PFA ependymomas. However, PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3K27-me3) and overexpression of CXorf67, a novel gene of unknown function. A review of PF ependymoma sequencing data revealed recurrent mutations in CXorf67. Subsequent analysis of a large series of ependymomas (n=263) showed that CXorf67 mutations occur in PFA ependymomas at a frequency of 9.4%, but not in non-PFA ependymomas. Targeted sequencing also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. H3 and CXorf67 mutations were mutually exclusive. We used immunoprecipitation (IP) / mass spectrometry (MS) to study proteins bound to CXorf67 in the Daoy cell line, which overexpresses CXorf67. EZH2, SUZ12, and EED, core components of the PRC2 complex were identified among enriched peptides and validated through complementary SUZ12 IP/MS. We also showed that modulation of CXorf67 influences H3K27-me3 levels downstream of PRC2, suggesting a link between overexpression of CXorf67 and the altered global epigenetic state in PFA ependymomas through its interaction with PRC2. Enforced reduction of CXorf67 in Daoy cells restored H3K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3K27-me3 levels. Our results suggest that CXorf67 overexpression may have an oncogenic role in PFA ependymomas, but the selective advantage of CXorf67 mutations is yet to be explained.

Abstract Our study aims to establish whether CXorf67 has a role in the pathogenesis of posterior fossa (PF) ependymoma. Among molecular groups of ependymoma defined by DNA methylation profiling, PF type-A (PFA) is the commonest. PFA ependymomas present mainly in infants and are difficult to treat, having a 10-year overall survival of approximately 40%. Published genomic studies have not identified a recurrent driver mutation in PFA ependymomas, but they do show widespread epigenetic alterations, including global loss of histone H3 K27-trimethlyation (H3K27-me3). Another childhood PF tumor, the diffuse pontine glioma (DPG), also shows loss of H3K27-me3. In DPGs, loss of H3K27-me3 is associated with H3 K27M mutation, but the mechanism in PFA ependymomas is not yet established. We reexamined published PF ependymoma sequencing data and discovered recurrent mutations in a novel gene, CXorf67. Targeted sequencing in a series of PFA ependymomas revealed CXorf67 mutations in 22/234 (9.4%). CXorf67 is a single-exon gene of unknown function. Its protein product is predicted to be “disordered,” apart from one region towards the N terminus. Mutations in PFA ependymomas are missense, and there is a mutation hotspot in the “ordered” region. CXorf67 mutations are not present in other molecular groups of ependymoma and are rare in other cancers. Analyzing the DNA methylation profiles of 675 PFA ependymomas, we have discovered two subgroups, PFA-1 and PFA-2, and nine subtypes among these two subgroups. All PFA subtypes harbor CXorf67 mutations with the exception of PFA-1f and PFA-2c. Targeted sequencing in our tumor series also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. Two thirds of mutations in HIST1H3B, HIST1H3C, and H3F3A were found in PFA-1f ependymomas, among which H3 mutations were present at a frequency of 35%. Mutations in H3 genes and CXorf67 were mutually exclusive across our series of PFA ependymomas. We used Affymetrix u133v2 arrays to establish that CXorf67 is expressed at high levels in PFA ependymomas, in contrast to low levels in other ependymomas from the PF and supratentorial compartments. A mechanism for CXorf67 overexpression was revealed in a similar comparative analysis of CpG island methylation profiles, which showed that the promoter region of CXorf67 is hypomethylated in PFA tumors, but not in other ependymomas. Using immunohistochemical preparations, we detected expression of CXorf67 at the protein level in the nuclei of PFA ependymomas; PFB and supratentorial tumors were immunonegative. CXorf67 overexpression is found in all PFA molecular subtypes, except PFA-1f, which shows similar levels to those in supratentorial and PFB ependymomas. CXorf67 expression is unrelated to mutation status. Elevated CXorf67 expression is found in the Daoy and U2-OS cancer cell lines. We used immunoprecipitation (IP)/mass spectrometry (MS) to study proteins bound to CXorf67 in Daoy and U2-OS. Analysis of enriched peptides following immunoprecipitation of CXorf67 indicated that it binds EZH2, SUZ12, and EED, three core components of the PRC2 complex. Complementary immunoprecipitation of SUZ12 detected CXorf67. Detecting CXorf67 mutations in almost 10% of PFA ependymomas led to the discovery of overexpression related to promoter region hypomethylation in these tumors. Overexpression of CXorf67 was detected in all PFA ependymoma subtypes except PFA-1f, 35% of which harbor an H3 K27M mutation. The protein product of CXorf67 is found in tumor cell nuclei, where our IP/MS data suggest it is bound to PRC2. Our findings suggest that global loss of H3K27-me3 in PFA ependymomas could be related to overexpression of CXorf67 and its interaction with PRC2, except in tumors where alterations in histone H3 genes are responsible. However, the selective advantage of CXorf67 mutation is yet to be explained. Citation Format: Ji Wen, Jens-Martin Hübner, Wilda Orisme, Gang Wu, Bo Tang, Sujuan Jia, John Easton, Kelly Haupfear, Brian D. Freibaum, Hong Joo Kim, Anthony High, Junmin Peng, Ruth G. Tatevossian, J. Paul Taylor, Stefan M. Pfister, Jinghui Zhang, Kristian W. Pajtler, Marcel Kool, David W. Ellison. Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR08.

Abstract Cerebellar ataxia is a frequent finding among patients seen in neurological practice and may result in a wide variety of aetiologies, both acquired and genetic. Inherited ataxia is a large and important subgroup of the ataxic disorders, and includes metabolic ataxias, autosomal recessive degenerative ataxias, autosomal dominant spinocerebellar ataxias (SCAs), X‐linked and maternally inherited ataxias. Hereditary ataxia is reviewed in this article and acquired ataxias are covered in the accompanying article. Hereditary conditions account for the majority of ataxic syndromes in children and one‐third to one‐half of patients with adult‐onset ataxic syndromes. Classification by mode of inheritance is a useful way of organizing the hereditary ataxias for both clinical and research purposes, and this approach will be used in this article. Key Concepts Understand that there are a wide variety of different inherited causes of ataxia. Some patients without a family history of ataxia still have the disorder on the basis of a genetic mutation. Nucleotide expansion disorders are a common cause of dominantly inherited cerebellar ataxia. The majority of these diseases result from a toxic gain‐of‐function mechanism and they demonstrate genetic anticipation, wherein intergenerational expansion leads to disease with earlier onset and often more severe symptom. Friedreich ataxia is the most common inherited cause of ataxia. It results from decreased expression of the Frataxin protein due to a nucleotide repeat expansion in a noncoding region. A variety of mitochondrial disorders are associated with ataxia as one of the number of neurologic and systemic symptoms. These diseases result from mutations in either nuclear DNA in genes involved in mitochondrial function or in mitochondrial DNA itself. Wilson disease is a disorder of copper metabolism that can present with dystonia, ataxia or neuropsychiatric symptoms and is also associated with liver failure. Early treatment can be curative. Defects in DNA repair underlie many childhood onset forms of inherited ataxia.

Amyotrophic lateral sclerosis (ALS) is a lethal and incurable neurodegenerative disorder commonly associated with repeat expansion in the C9orf72 gene; termed C9-ALS. A major pathological feature of this is the accumulation of arginine-rich (R-rich) dipeptide repeat (DPR) polypeptides in neurons. R-rich DPRs interact with low complexity domains in proteins, accumulate in membraneless organelles (MLOs), alter the material properties of MLOs, and induce cell death. The molecular mechanisms underlying pathogenesis, though, remain unknown. R-rich DPRs infiltrate nucleoli, co-localize with nucleophosmin (NPM1), and alter NPM1 phase separation in vitro. NPM1 is crucial to the maintenance of nucleolar liquid-like properties through its ability to phase separate with proteins and nucleic acids. Elucidating the effects of DPR interactions with NPM1 on the liquid-like properties and overall architecture of nucleoli that ultimately lead to nucleolar dysfunction and cell death is critical to understanding C9-ALS. Here we employ poly(PR) as an archetypal R-rich DPR to (1) identify the interactions mediating phase separation with NPM1; (2) elucidate the mechanisms causing the DPR-dependent dissolution of in vitro NPM1/DPR droplets; and (3) confirm hypotheses stimulated by our biophysical results regarding the mechanisms of DPR-mediated nucleolar disruption in cells. Results from multiple, complementary biochemical and biophysical techniques show that (1) NPM1/DPR interactions are mediated by acidic tracts within the intrinsically disorder region of NPM1, (2) R-rich DPRs dissolve NPM1-containing droplets in vitro by sequestering NPM1 into large saturated complexes, and (3) exogenous poly(PR) induces NPM1 release from nucleoli, disrupting nucleolar organization and function. These results support the hypothesis that R-rich DPRs, mediate their toxic effects in part through saturation/sequestration of NPM1, perturbing NPM1 mediated phase separation in nucleoli, disrupting nucleolar function, and inducing cell death.

SUMMARY Stress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog confers hypersensitivity of the male germline to heat stress in vivo . Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress through fine-tuning phase separation and could aid in the development of male fertility therapies.

ABSTRACT Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form ribonucleoproteins. hnRNPs are a major subclass of evolutionarily conserved RBPs that are primarily concentrated in the nucleus and are heavily involved in pre-mRNA splicing, mRNA stability and transcriptional/translational regulation. During times of stress, standard translational programming is interrupted, and hnRNPs, mRNA, and other RBPs condense in the cytoplasm, forming liquid-liquid phase separated (LLPS) membraneless organelles termed stress granules (SGs). SGs are central to the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). hnRNPs and other RBPs are critical components of SGs. Indeed, the link between SGs, hnRNPs, and neurodegenerative diseases has been established by the identification of additional mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, each of which can directly lead to ALS, IBM and other related neurodegenerative diseases. Here, we report and characterize four novel HNRNPA1 mutations and two known HNRNPA1 mutations, previously reported as being causal for ALS, in a broad spectrum of patients with hereditary motor neuropathy (HMN), ALS, and myopathy. Our results show the different effects of mutations on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics, indicating the possibility of different underlying pathomechanisms for HNRNPA1 mutations with a possible link to the clinical phenotypes.

Phase separation aided percolation transitions of multivalent protein and RNA molecules drive the formation of distinct membraneless ribonucleoprotein condensates such as stress granules and RNA transport granules. A key function of RNP condensates is to ensure that the concentrations of specific types of protein and RNA molecules are maintained below threshold levels. Can a single, multicomponent condensate, irrespective of its material properties, enable an equilibrium buffering of concentrations of different macromolecules, while being permissive about the concentrations of other components? Here, we answer this question by developing a rigorous thermodynamic framework built around analyses of the slopes of tie lines within phase diagrams for systems comprising complex mixtures of linear multivalent macromolecules. The computed phase diagrams generated using the LASSI engine show that desired outcomes are achievable via a predictable interplay between homotypic and heterotypic interactions across a network of macromolecules in a complex mixture. This interplay directly determines the slopes of tie lines in two-component systems and the shapes of tie planes in multicomponent systems. We find that buffering cannot be achieved if the condensate forms purely via heterotypic interactions. However, tuning the interplay between homotypic and heterotypic interactions affords component-specific levels of control over macromolecular concentrations in the dilute phase. Our findings formalize the rules that give rise to heterotypic buffering via multicomponent condensates. They indicate that condensates can enable digital control over the concentrations of some components and analog control over some others. This duality is predictable and tunable thus highlighting the versatility and generalization of the concept of buffering to complex systems where the conventional expectations regarding saturation concentrations are no longer applicable.

The original version of this article unfortunately contained a mistake in Funding section. The correct Funding section is given below.