Frontotemporal dementia-like disease progression elicited by seeded aggregation and spread of FUS.

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fused in sarcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic Frontotemporal lobar degeneration (FTLD). Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.

Frontotemporal dementia-like disease progression elicited by seeded aggregation and spread of FUS.

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fu sed in s arcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic FTLD. Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.

Design principles to tailor Hsp104 therapeutics.

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics.

CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

Nuclear-import receptors as gatekeepers of pathological phase transitions in ALS/FTD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the cytoplasmic mislocalization and aberrant phase transitions of prion-like RNA-binding proteins (RBPs). The common accumulation of TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), and other nuclear RBPs in detergent-insoluble aggregates in the cytoplasm of degenerating neurons in ALS/FTD is connected to nuclear pore dysfunction and other defects in the nucleocytoplasmic transport machinery. Recent advances suggest that beyond their canonical role in the nuclear import of protein cargoes, nuclear-import receptors (NIRs) can prevent and reverse aberrant phase transitions of TDP-43, FUS, and related prion-like RBPs and restore their nuclear localization and function. Here, we showcase the NIR family and how they recognize cargo, drive nuclear import, and chaperone prion-like RBPs linked to ALS/FTD. We also discuss the promise of enhancing NIR levels and developing potentiated NIR variants as therapeutic strategies for ALS/FTD and related neurodegenerative proteinopathies.

Mo' m1A, mo' problems.

CAG-repeat expansions underlie fatal neurodegenerative disorders. In a lodestar study published in a recent issue of Nature, Sun et al.1 identify a writer and eraser of N1-methyladenosine (m1A) modifications of CAG-repeat RNA. They establish that m1A modifications in CAG-repeat expanded RNA promote neurodegeneration and aberrant phase transitions of TDP-43. These findings suggest therapeutic strategies for CAG-repeat expansion disorders.

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.

Regulation of Biomolecular Condensates by Poly(ADP-ribose).

Biomolecular condensates are reversible compartments that form through a process called phase separation. Post-translational modifications like ADP-ribosylation can nucleate the formation of these condensates by accelerating the self-association of proteins. Poly(ADP-ribose) (PAR) chains are remarkably transient modifications with turnover rates on the order of minutes, yet they can be required for the formation of granules in response to oxidative stress, DNA damage, and other stimuli. Moreover, accumulation of PAR is linked with adverse phase transitions in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In this review, we provide a primer on how PAR is synthesized and regulated, the diverse structures and chemistries of ADP-ribosylation modifications, and protein-PAR interactions. We review substantial progress in recent efforts to determine the molecular mechanism of PAR-mediated phase separation, and we further delineate how inhibitors of PAR polymerases may be effective treatments for neurodegenerative pathologies. Finally, we highlight the need for rigorous biochemical interrogation of ADP-ribosylation in vivo and in vitro to clarify the exact pathway from PARylation to condensate formation.

Repetitive elements in aging and neurodegeneration.

Repetitive elements (REs), such as transposable elements (TEs) and satellites, comprise much of the genome. Here, we review how TEs and (peri)centromeric satellite DNA may contribute to aging and neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Alterations in RE expression, retrotransposition, and chromatin microenvironment may shorten lifespan, elicit neurodegeneration, and impair memory and movement. REs may cause these phenotypes via DNA damage, protein sequestration, insertional mutagenesis, and inflammation. We discuss several TE families, including gypsy, HERV-K, and HERV-W, and how TEs interact with various factors, including transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) and the siRNA and piwi-interacting (pi)RNA systems. Studies of TEs in neurodegeneration have focused on Drosophila and, thus, further examination in mammals is needed. We suggest that therapeutic silencing of REs could help mitigate neurodegenerative disorders.

C-terminal frameshift variant of TDP-43 with pronounced aggregation-propensity causes rimmed vacuole myopathy but not ALS/FTD.

Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. Using genome-wide linkage analysis and whole exome sequencing in an extended five-generation family with an autosomal dominant rimmed vacuole myopathy, we identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43p.Trp385IlefsTer10) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43Trp385IlefsTer10 does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43p.Trp385IlefsTer10 behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43p.Trp385IlefsTer10 showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43p.Trp385IlefsTer10 is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. Our study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology.

Phase Separation in Biology and Disease; Current Perspectives and Open Questions.

In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ∼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.

A minimal construct of nuclear-import receptor Karyopherin-ß2 defines the regions critical for chaperone and disaggregation activity.

Karyopherin-ß2 (Kapß2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapß2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapß2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapß2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapß2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapß2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapß2 recapitulate all salient features of Kapß2 activity. By contrast, Kapß2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapß2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.

Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy.

BACKGROUND: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-ß1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. METHODS: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. RESULTS: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. CONCLUSION: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.

Advanced Molecular Tweezers with Lipid Anchors against SARS-CoV-2 and Other Respiratory Viruses.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses.

Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.

The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.

Sexually dimorphic RNA helicases DDX3X and DDX3Y differentially regulate RNA metabolism through phase separation.

Sex differences are pervasive in human health and disease. One major key to sex-biased differences lies in the sex chromosomes. Although the functions of the X chromosome proteins are well appreciated, how they compare with their Y chromosome homologs remains elusive. Herein, using ensemble and single-molecule techniques, we report that the sex chromosome-encoded RNA helicases DDX3X and DDX3Y are distinct in their propensities for liquid-liquid phase separation (LLPS), dissolution, and translation repression. We demonstrate that the N-terminal intrinsically disordered region of DDX3Y more strongly promotes LLPS than the corresponding region of DDX3X and that the weaker ATPase activity of DDX3Y, compared with DDX3X, contributes to the slower disassembly dynamics of DDX3Y-positive condensates. Interestingly, DDX3Y-dependent LLPS represses mRNA translation and enhances aggregation of FUS more strongly than DDX3X-dependent LLPS. Our study provides a platform for future comparisons of sex chromosome-encoded protein homologs, providing insights into sex differences in RNA metabolism and human disease.