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

Abstract

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.

Fig. 1.. RNA-binding deficient TDP-43 naturally de-mixes into intranuclear liquid spherical shells.

(A) Schematic of TDP-43 wildtype and mutant proteins. All proteins were tagged at the carboxyl-terminus with the bright GFP variant, clover. (B) U2OS cells induced to express TDP-43 variants develop many more nuclear droplets than do cells expressing wildtype TDP-43. (Dashed boxes) Examples of nuclear droplets and magnified at the lower right corners. (C) Orthogonal projections demonstrate that TDP-43^2KQ-clover droplets have nearly perfectly round spherical shells. (D) Spherical shell formation is dose dependent. (Lower right) TDP-43^2KQ-clover expression level (relative to endogenous TDP-43) in each clone (determined as in Fig. S1C–F) is marked at the lower right of each image. (E-F) Quantified percentages of cells containing (E) TDP-43 droplets and (F) number of nuclear droplets per cell in U2OS cells expressing clover-tagged TDP-43 variants. (G) Liquid-like character of intranuclear TDP-43^2KQ-clover spherical shells, demonstrated by rapid fluorescence recovery after photobleaching (FRAP). (H) Dynamic fusion of TDP-43^2KQ-clover spherical shells visualized by live cell differential interference contrast (DIC) microscopy. Two spherical shells (1–2μm) fuse into one (>2 μm) within 10 seconds. Inhibition of HDAC and proteasome activity synergize to enhance TDP-43 anisosome formation in neuron-like SHSY-5Y cells (I) and in iPSC-derived motor neurons (J).

Fig. 2.. TDP-43 intranuclear liquid spherical shells are anisotropic compartments of anisosomes.

(A) Schematic of complete extinction microscopy. The light path contains two polarizers: the front polarizer can be adjusted from 0° to 90°, and the back polarizer has a fixed angle. Rotating the front polarizer result in a change of polarized light. (B) Only anisotropic materials can be distinguished by complete extinction microscopy because the birefringent property of the anisotropic material allows some light to pass the back polarizer. (C) The same nucleus containing TDP-43 liquid spherical shells is imaged under normal or polarized light sources (images are pseudocolored to display intensity of light). Phase-separated nucleoli (marked by “n”) cannot be detected when the polarized light is perpendicular to the filter. (Dashed box) Magnified view of the dynamic anisotropic sub-domains. (D) Maximum intensity projection of images in (C) (167 frames in 30 seconds of live imaging under complete extinction conditions). Arrow points to membranes, a known anisotropic structure – the lipid bilayer. (E) Quantification of signal intensity of nucleoplasm, nucleoli, and anisosomes.

Fig. 3.. TDP-43 anisosomes are membraneless compartments with densely packed shells visualized by transmission electron microscopy (TEM) and cryo-electron tomography (CryoET).

TEM examples of nuclei from (A) a naïve U2OS cell, (B) a U2OS cell expressing TDP-43^WT-clover, and (C) a U2OS cell expressing TDP-43^2KQ-clover (see additional examples in Fig. S3). (D) Immunogold labeling with a GFP antibody of (left) a TDP-43^WT-clover droplet or (right) a TDP-43^2KQ-clover anisosome. (Dashed boxes) Enlarged views of the distribution of gold particles (see Fig. S4 for additional examples). (E) Schematic of 20-minute temperature shift and determination of the structural changes with TEM. (F) A temperature shift-induced structural change visualized by TEM. (Dashed boxes) Magnified views of each condition. At 42°C, no irregular structure was observed in the nucleoplasm without the induction of anisosomes. At 39 °C, anisosomes become irregular shape, while at 42 °C, irregular shape and less electron dense. (Dashed red circles) Irregular electron dense compartments. (G) Cryo-electron tomography of an anisosome and (H) density plot of the area enclosed in the dashed black rectangle.

Fig. 4.. Proteolytic stress in neurons of the adult nervous system induces TDP-43-containing anisosomes, which convert into aggregates as ATP levels fall.

(A) Schematic of experimental steps for inducing transient proteolytic stress in Sprague Dawley rats by intravenous (i.v.) administration of the proteasome inhibitor bortezomib (BTZ). (B) TEM images of intranuclear spherical shells found in nuclei of DRG sensory neurons from mice treated as in (A). (Dashed red square) area magnified at right. (C) Immunogold labelling for endogenous TDP-43 in bortezomib-induced spherical shells. (D) Schematic of induction of TDP-43-containing spherical shells in mouse DRG neurons by partial proteasome inhibition and then examined after immediate or delayed tissue collection. Images of DRGs collected (E) immediately after perfusion or (F) after a 2-hour postmortem delay during which ATP levels falls sufficiently to initiate rigor mortis. Anisosomes seen in freshly fixed samples convert to amorphous dense intranuclear structures after postmortem delay.

Fig. 5.. TDP-43 anisosomes, intact during mitosis, are selective barriers for RNA and some nuclear proteins.

(A) A U2OS nucleus stably expressing H2B^mCherry, a fluorescent histone marker and TDP-43^5FL-clover. (B,C) Fluorescence imaging of nuclear RNA-binding proteins (B) hnRNPA2B1, hnRNPH1 and hnRNPK (each carboxy-terminally tagged with mRuby2) or (C) EGFP-labeled FUS, iavNP or EGFP alone. (D) Schematic for fluorescent labeling of cellular RNA with “CLICK” chemistry. (E) Imaging of anisosomes of TDP-43^2KQ and nuclear RNA (labeled as in (D)). (F) Quantification of fluorescence intensity of the RNA in (E). (G) Imaging of TDP-43 anisosomes in mitotic and post-mitotic cytoplasm and post-mitotic reassembly within daughter nuclei. (H) An imaging series of TDP-43^2KQ-clover anisosomes during mitosis and early interphase. (Solid lines) The cell plasma membrane; (dashed lines) outline of the nuclear envelope. The numbers 1–7 denote specific cytoplasmic TDP-43^2KQ-clover droplets, each of which becomes smaller within 10 minutes. (m) Anisosomes reforming within micronuclei produced by mitotic chromosome segregation errors.

Fig. 6.. Mathematical modeling predicts that a third component is required for anisosomal formation and maintenance.

(A) Schematic of modeling of anisosome formation by TDP-43^2KQ mutant in a simple mixture of TDP-43, RNA, and a proposed Y molecule that has high affinity for itself, low affinity for TDP-43, and no RNA binding. (B) A snapshot from the model in (A) of de-mixing at 3 and 1000 seconds after starting from a uniform solution. (C) The volume fraction of each species from modeling in (A and B) at a 1000 second time point. The ratio of volume fraction between the shell and the center of the anisosome for TDP-43^2KQ is about 2. Clover fluorescence intensity across TDP-43^2KQ anisosomes. (D) An optical slice of an anisosome with (E) intensity of the dashed line in (D). (F) The quantified ratio between the shell and the center, with a mean of 2.28 ± 0.56 (SD). (G) Measurement of the ratio of gold particle density in immunogold-labeling of TDP-43^2KQ-clover in an anisosome and (H) quantification of the ratio of gold particle density between the shell and the center.

Fig. 7.. The HSP70 family of molecular chaperones are specifically enriched in cores of TDP-43 anisosomes.

(A) Schematic of APEX2-mediated proximity labeling followed by quantitative proteomic analyses (see also Fig. S10). (B) Quantitative proteomic analyses identify HSP70 family proteins to be enriched in the cores of de-mixed TDP-43 anisosomes. Y axis is p-value at log₁₀ scale; X axis is fold enrichment on log₂ scale. The size and color of each spot correspond to the total spectral counts of LC/MS-MS reads on log₂ scale. (C) Maximum intensity projections of immunofluorescence images of U2OS nuclei expressing (green) TDP-43^2KQ-clover anisosomes and (red) HSP70 family proteins tagged by mRuby2. (White boxes) Boxed areas that are magnified at right.

Fig. 8.. The ATP-dependent chaperone activity of the HSP70 family chaperones regulate anisosomal structure and liquidity of both shells and cores.

(A) Imaging of anisosomes formed after transient induction of TDP-43^2KQ-clover in cells stably expressing HSPA6^mRuby2 after (left) addition of an HSP70 ATPase inhibitor (VER155008) and (right) after a subsequent 0.5 hour wash out of the inhibitor. (B) (Top) Schematic of FRAP of anisosomes formed in cells in (A) in the absence of or after addition the HSP70 inhibitor. (Middle) FRAP of HSPA1L^mRuby2 in TDP-43^2KQ-clover anisosomes. (Bottom) FRAP of a portion of a TDP-43^2KQ-clover and HSPA1L^mRuby2-containing intranuclear droplet in cells treated with the HSP70 inhibitor. (C) Kinetics of the FRAP experiments in (B). HSP70^mRuby2 quickly recovers after bleaching (the red curve, τ < 2 seconds), but is much less dynamic after HSP70 inhibitor treatment (the purple curve, τ is between 40 and 42.5 seconds). In the HSP70 inhibitor, only a small proportion of TDP-43^2KQ-clover recovers, which recovers slowly. (D) Live DIC and fluorescence imaging of TDP-43^2KQ-clover in anisosomes after addition and wash out of the HSP70 inhibitor. (E) Schematic of addition and imaging of diffuse cytoplasmic TDP-43^{NLSm/5FL-clover} after addition of the HSP70 inhibitor. (F) Live fluorescence imaging of TDP-43^{NLSm/5FL-clover} assembly into cytoplasmic droplets upon addition of the HSP70 inhibitor. (G) FRAP of cytoplasmic droplets from (F)

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 of TDP-43 into anisosomes, an intranuclear membraneless compartment with symmetrically aligned shell and core in which RNA-free TDP-43 is enriched in the shell. 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).