Hsp70 and Hsp40 functionally interact with soluble mutant huntingtin oligomers in a classic ATP-dependent reaction cycle

Abstract

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.

FIGURE 1.

The conformation-dependent anti-oligomer antibody A11 detects mutant htt oligomers but not monomers or fibrils. A, schematic of the GST-mutant htt exon 1 fusion protein with 53Q (HD53Q) showing a PreScission protease site between GST and the mutant htt fragment (not drawn to scale) and the locations of epitopes for MW8, MW1, and c-Myc antibodies. B, morphology of HD20Q and HD53Q aggregation reactions over time, analyzed by AFM. Representative AFM images of 6 μm aggregation reactions at 0, 1, and 5 h are shown. Zoomed in panels are 1 μm by 1 μm. Controls are denatured HD20Q and HD53Q in reaction buffer with 6 m guanidinium HCl. Analysis of oligomers and fibrils per unit area at 0, 1, and 5 h is presented. C, dot-blots of HD53Q and HD20Q after incubation with protease for various times, probed with antibodies as labeled. D, percentage of A11 reactivity relative to Myc reactivity (100%) in dot-blots of three independent experiments, quantified by densitometry using Image J. *, p < 0.05 versus HD20Q. ns, not significant at 1 h by t test. The values are the means ± S.E. E, filter trap assays of HD53Q and HD20Q after incubation with protease for various times. Insoluble HD53Q oligomers and fibrils were trapped and detected by MW8 but not by A11. F, quantification of filter trap assay of three independent experiments, quantified by densitometry using Image J. The values are the means ± S.E.

FIGURE 2.

A subset of soluble mutant htt oligomers are detected by the A11 antibody. Supernatant obtained after aggregation of HD53Q and HD20Q (6 μm) for the indicated times was analyzed by SEC on a Superdex 200 column. A, nondenatured fractions were vacuumed onto a nitrocellulose membrane using a slot-blot manifold and probed with anti-htt (MW1) and A11 antibodies. Arrows indicate positions of molecular mass markers (kDa). The black box indicates A11 detection of HD53Q aggregates at 3 h. B, antibody detection, quantified by densitometry with Image J. Soluble htt species were quantified with MW1 and compared with htt oligomers detected by A11.

FIGURE 3.

A11 detects soluble mutant htt oligomers with a globular structure. A, AFM images of SEC fraction 16 from HD20Q and HD53Q aggregation reactions (3 h) (see Fig. 2). Scale bars, 400 nm. B, subunit composition of A11-reactive HD53Q oligomers. Based on corrected volume measurement and the molecular mass of HD53Q, the numbers of molecules/oligomers in A11-reactive fractions were calculated for each fraction from AFM images. Darker shades represent a greater abundance of oligomers composed of that number of molecules. Arrows indicate the size range where ∼200–500-kDa A11 reactive oligomers would be observed. C, fraction 16 was applied to nitrocellulose (100-nm pore size) through a slot-blot manifold and analyzed with A11 and MW1 antibodies. D, number of aggregates/μm² in AFM images. The values are the means ± S.E. E, height, volume, and diameter histograms of oligomers from fraction 16 observed by AFM.

FIGURE 4.

Hsp70 and Hsp40 associate specifically with soluble mutant htt oligomers and attenuate their formation in an ATP-dependent manner. A, analysis of the Hsp70/Hsp40 interaction with soluble HD53Q oligomers. Supernatant of HD53Q aggregation reaction (3 h) alone or a mixture of HD53Q with Hsp70, Hsp40, or both, was loaded onto a Superdex 200 column, and the fractions were analyzed by SDS-PAGE/Western blot with the indicated antibodies. Molecular chaperones were added after 3 h of HD53Q aggregation (when oligomers predominate) and incubated for 45 min before fractionation by SEC. Arrows indicate positions of molecular mass standards (kDa). B, effect of Hsp70/Hsp40 on formation of A11-reactive oligomers. Supernatants from 3 h HD53Q aggregation reactions in the absence or presence of Hsp70 and Hsp40 (chaperones added at 0 h) were loaded onto a Superdex 200 column. Fractions were placed on a nitrocellulose membrane (100-nm pore size) and probed with A11 or anti-htt (Myc) antibodies.

FIGURE 5.

Mutant htt oligomers inhibit Hsp70/Hsp40-dependent luciferase refolding. Denatured luciferase (100 nm) refolded by Hsp70/Hsp40 after 30 min in the presence and absence of HD53Q oligomers from a 3-h aggregation reaction or control protein. The molar ratios (chaperone:oligomer) were estimated based on predicted subunit composition of A11 oligomers after 3 h of aggregation (∼10 molecules/oligomer) in Fig. 3B. The values are the means ± S.E. of three independent experiments. ***, p < 0.001; ns, not significant (one-way analysis of variance).

FIGURE 6.

Hsp70 and Hsp40 suppress formation of A11-reactive mutant htt oligomers and toxicity in PC12 cells. A, analysis of HD103Q-EGFP inclusion bodies by fluorescence microscopy. The addition of hormone (5 μm ponasterone A) to cell medium induced HD103Q-EGFP expression and caused the formation of GFP-positive inclusion bodies. B, the soluble supernatant of a homogenate from HD103Q-expressing PC12 cells (60 μg) in the absence and presence of Hsp70/Hsp40 overexpression was fractionated by SEC on a Superdex 200 column. Fractions were analyzed by slot-blot manifold with GFP and A11 antibodies. The boxed area indicates A11-reactive fractions. C, Western blot analysis of PC12 cell lysate (20 μg) with anti-GFP, anti-Hsp70 (inducible form), and anti-Hsp40 antibodies before and after HD103Q-EGFP expression. Overexpression of Hsp70 and Hsp40 increased the level of these chaperones and slightly reduced insoluble HD103Q-EGFP aggregates that remain in the well (anti-GFP). D, A11 reactivity in fractions 14–20, quantified by densitometry. The values are the means ± S.D. of three experiments. **, p < 0.01; ***, p < 0.001 (Student's t test). E, expression of a mutant htt fragment in PC12 cells causes an increase in caspase 3 activity that is suppressed when Hsp70/Hsp40 is overexpressed. Caspase 3 activity is shown as the ratio of values from induced relative to noninduced cells.

FIGURE 7.

Proposed model for Hsp70/Hsp40 function in mutant htt misfolding in HD. Expansion of the polyQ repeat of mutant htt causes misfolding and formation of discrete subsets of misfolded species (toxic and benign monomers and oligomers), with only toxic conformations recognized specifically by Hsp40 (DNAJB). A, Hsp40 transfers mutant htt substrates to ATP-bound Hsp70 (open conformation). B, in the open state, Hsp70, with Hsp40, forms a trimeric complex with misfolded mutant htt species, and Hsp40 stimulates the ATPase activity of Hsp70. Hydrolysis of ATP leads to ADP-Hsp70 bound state (closed state) and release of Hsp40 and Pi. C, in the ADP-bound state, Hsp70 shows a high affinity for misfolded mutant htt species. Nucleotide exchange factors (NEF) likely mediate the exchange of ADP for ATP, resulting in an open state of Hsp70 and conversion of toxic to benign conformers. D, the benign conformers are transferred to another chaperone system or are competent for degradation by the proteasome or by autophagy.