HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells

The makings of anisosomes Phase separation of proteins within the cell can produce a liquid-inside-a-liquid phase resembling oil droplets in water. Yu et al. now report that an RNA-binding protein called TDP-43, in which mutation and aggregation are linked to amyotrophic lateral sclerosis and frontotemporal dementia, phase separates into complex droplets, which they named anisosomes. This process occurred when TDP-43 lost its ability to bind RNA through disease-causing mutation or posttranslational acetylation. Anisosomes have spherical shells of TDP-43 (with properties of a liquid crystal) surrounding centers of the protein chaperone HSP70. Chaperone activity was required to maintain liquidity. Anisosomes formed in neurons in vivo when proteasome activity was inhibited and were converted into aggregates when adenosine triphosphate (ATP) levels fell. Science, this issue p. eabb4309 Neurodegeneration-linked TDP-43 protein phase-separates into liquid crystals inside living cells. INTRODUCTION Aggregation of the RNA binding protein TDP-43 (TAR DNA-binding protein 43) is a common pathological hallmark shared by several age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The predominantly nuclear TDP-43 normally undergoes liquid-liquid phase separation (LLPS), in which a homogeneous solution separates into two compartments resembling oil droplets in vinegar. RATIONALE Intranuclear TDP-43 can phase-separate under physiological conditions. Cellular stress can induce cytoplasmic TDP-43 liquid droplets, which can transition to a solid state, suggesting that TDP-43 aggregation observed in neurodegeneration could be initiated by LLPS. The mechanisms that drive phase separation and aggregation remain unclear. RESULTS We identified key regulatory mechanisms of TDP-43 phase separation in cultured cells and in neurons of the rodent nervous system. RNA binding–deficient TDP-43, produced by ALS- or FTD-causing mutations or posttranslational acetylation in its RNA recognition motifs, phase-separated into anisosomes (i.e., droplets with symmetrical liquid spherical shells and liquid cores). RNA-free TDP-43 was found to be enriched in anisosomal shells at concentrations 50 times those of the surrounding nucleoplasm. Acetylation promoted anisosomal formation by abolishing RNA interaction with TDP-43. Anisosomal shells exhibited birefringence (i.e., evidence of a liquid crystal compartment formed from proteins within living cells). Shells were densely packed, as determined with cryo–electron tomography, producing a membraneless, selective barrier to some nuclear proteins and RNAs. Our mathematical modeling predicted that anisosomes were driven by a core component that self-interacted, weakly bound TDP-43, and did not bind RNA. Guided by these concepts, we used proximity labeling and quantitative proteomics to identify HSP70 chaperones as the primary anisosomal core components. HSP70 chaperones selectively bound to and stabilized RNA-unbound TDP-43. Inhibiting adenosine triphosphate (ATP)–dependent chaperone activity of the HSP70 family or reducing cellular ATP levels induced rapid conversion of TDP-43 anisosomes into uniform gels. Transient proteasome inhibition, mimicking the known reduction in proteasome activity during aging, provoked TDP-43 demixing into anisosomes in neurons in rodents. Postmortem ATP reduction was sufficient to convert anisosomes into aggregates similar to those found in neurodegenerative disease. CONCLUSION We identified how phase separation of the RNA-binding protein TDP-43 can be regulated through RNA binding, disease-causing mutation, posttranslational modification, or chaperone activity inside cells. RNA binding–deficient TDP-43 demixed into anisosomes with cores that could be characterized as a “liquid inside a liquid inside a liquid. ” Chaperone activity of the HSP70 family was required to maintain liquidity of anisosomal shells and cores. When ATP levels fell, anisosomes converted into protein aggregates, consistent with being precursors of the pathological aggregates found in patient brain tissues. These findings suggest an essential partnership between TDP-43 and HSP70 chaperones in driving RNA-unbound TDP-43 phase separation into anisosomes and preventing TDP-43 aggregation. TDP-43 phase transition is regulated by its RNA affinity and HSP70 activity. RNA binding protein TDP-43 forms aggregates in degenerating neurons, a pathological feature associated with aging, genetic, and/or environmental factors. Although naturally demixed, RNA binding–proficient TDP-43 is largely soluble in the nucleus, with a small proportion demixed (A and B). Its RNA affinity is eliminated by acetylation, which drives most TDP-43 into anisosomes—intranuclear membraneless compartments with symmetrically aligned shells and cores in which RNA-free TDP-43 is enriched in the shells. HSP70 stabilizes RNA-free TDP-43 and is enriched in the anisosomal core (C). When ATP-dependent chaperone activity of HSP70 is reduced by ATP depletion, TDP-43 anisosomes collapse into gels (D), which may be precursors of intranuclear and cytoplasmic aggregates observed in degenerating neurons (E). The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding–deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named “anisosomes”, have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)–dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43–containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.