Cereblon Regulates the Proteotoxicity of Tau by Tuning the Chaperone Activity of DNAJA1

Abstract

Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play a neuroprotective role because of their ability to unfold and refold abnormal proteins. The progressive nature of neurotoxic events makes it important to discover endogenous factors that affect pathologic and molecular phenotypes of neurodegeneration in animal models. Here, we identified microtubule-associated protein tau, and chaperones Hsp70 (heat shock protein 70) and DNAJA1 (DJ2) as endogenous substrates of cereblon (CRBN), a substrate-recruiting subunit of cullin4-RING-E3-ligase. This recruitment results in ubiquitin-mediated degradation of tau, Hsp70, and DJ2. Knocking out CRBN enhances the chaperone activity of DJ2, resulting in decreased phosphorylation and aggregation of tau, improved association of tau with microtubules, and reduced accumulation of pathologic tau across brain. Functionally abundant DJ2 could prevent tau aggregation induced by various factors like okadaic acid and heparin. Depletion of CRBN also decreases the activity of tau-kinases including GSK3α/β, ERK, and p38. Intriguingly, we found a high expression of CRBN and low levels of DJ2 in neuronal tissues of 5XFAD and APP knock-in male mouse models of Alzheimer's disease. This implies that CRBN-mediated DJ2/Hsp70 pathway may be compromised in neurodegeneration. Being one of the primary pathogenic events, elevated CRBN can be a contributing factor for tauopathies. Our data provide a functional link between CRBN and DJ2/Hsp70 chaperone machinery in abolishing the cytotoxicity of aggregation-prone tau and suggest that Crbn^-/- mice serve as an animal model of resistance against tauopathies for further exploration of the molecular mechanisms of neurodegeneration.

Keywords: DNAJA1; cereblon; chaperone; neurodegeneration; tau phosphorylation; ubiquitin.

Figure 1.

Hsp70 and DJ2 are endogenous substrates of CRL4^CRBN. A, DJ2 levels are high in the organs of Crbn^−/− mice. Organs of five male mice from each group were homogenized in Tris buffer and immunoblotted (α-CRBN; Abnova). Statistical analysis is shown in the adjacent panel (Student’s t test). B, The mRNA level of DJ2 is similar in the organs of Crbn^−/− mice. The expression of DJ2 in various tissues was analyzed by quantitative RT-PCR. The level of DJ2 mRNA expression in five male mice from each group was normalized to the GAPDH mRNA level. The relative DJ2 mRNA level is shown as a mean of triplicates of quantitative PCR in each sample, and error bars indicate ± SD; p > 0.05, Student’s t test. C, SH-SY5Ycells were lysed in Tris buffer, immunoprecipitated with rabbit IgG control or α-CRBN antibody, and probed with the indicated antibodies (α-CRBN; Sigma-Aldrich). For the CRBN panel, the asterisk shows a nonspecific band, bands at 55 kDa show heavy chains of IgG and CRBN antibody, and the band at 52 kDa shows a band for CRBN (n = 6). D, His-DJ2 (top) and His-Hsp70 (bottom) were subjected to GST pull-down assay with GST-CRBN and blotted with antibodies against DJ2, Hsp70, and CRBN (n = 3). E, CRBN binds to DJ2 but not DJ6. SH-SY5Y cells were transiently cotransfected with Myc-CRBN and FLAG-DJ2 or FLAG-DJ6. Cells were lysed and immunoprecipitated with mouse IgG control or α-Myc antibody (n = 3). F, G, DJ2 and Hsp70 undergo CRBN-mediated ubiquitylation. SH-SY5Y cells were transiently transfected with HA-Ub and lysed in Ubn buffer, and IP was performed with α-DJ2 and α-HSP70 antibodies respectively (α-CRBN=Sigma). Asterisks show nonspecific bands (n = 5). H, SH-SY5Y cells were transiently transfected with FLAG-CRBN and immunoprecipitated with Flag M2 agarose beads. In vitro ubiquitylation of endogenous, coprecipitated DJ2 and Hsp70 was conducted in the presence of E1 + E2 and Ub. Me-Ub was added where indicated (n = 5). I, SH-SY5Y cells were treated with thalidomide (500 nm) for 24 h, then transiently transfected with HA-Ub (in the presence of thalidomide), and ubiquitylation assay was performed. J, K, DJ2 and HSP70 are accumulated in Crbn^−/− cells. WT (Crbn^+/+) and KO (Crbn^−/−) MEF cells were treated with 2.5 µg/ml CHX or 0.5 µg/ml MG132, respectively, and harvested at the indicated time points (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001; Student’s t test was used to calculate the p value for WT and KO cells at the time points mentioned.

Figure 2.

The N-terminal Lon domain of CRBN binds to the C-terminal domain of DJ2. A, C, Schematic diagram of the rCRBN and hDJ2 constructs used in B and D with HA and Myc tags, respectively. L, Linker; CTD, C-terminal domain; Lon-N, N-terminal Lon domain; Lon-C, C-terminal Lon domain. B, D, SH-SY5Y cells were transiently cotransfected with the genetic constructs indicated. After 24 h, cellular extracts were immunoprecipitated with the antibodies mentioned. A band at ∼55 kDa represents IgG heavy chains (HC), and at ∼25 kDa represents IgG light chains (LC). Asterisk (*) indicates a nonspecific band (n = 3). E, G, Schematic diagram of the deletion–mutant constructs of rCRBN and hDJ2 used in F and H with HA and Myc tags, respectively. F, H, SH-SY5Y cells were transiently cotransfected with the genetic constructs indicated. After 24 h, cellular extracts were immunoprecipitated and blotted with the antibodies mentioned (n = 3). I, Structural model of DJ2-CRBN interaction predicted by ClusPro, a fully automated algorithm for protein–protein docking. A number of structural models of DJ2 docked onto the CRBN were analyzed using ClusPro. PDB files of the crystal structure of CRBN (4TZ4) and structural model of DJ2 generated by the I-Tasser server were used. This binding mode was selected after combining the docking data with co-IP data. J, E152 and F153 of CRBN are critical for the binding of CRBN to DJ2. SH-SY5Y cells were transiently cotransfected with Myc-DJ2 and the indicated plasmids. After 24 h, cellular extracts were immunoprecipitated with α-Myc antibody. A band at ∼55 kDa represents IgG HCs and the asterisk indicates a nonspecific band. n = 5. K, Four residues of DJ2 (D281–T284) are critical for the binding of DJ2 to CRBN. SH-SY5Y cells were transiently cotransfected with HA-CRBN and indicated plasmids. IP was performed with the HA antibody after 24 h (n = 5).

Figure 3.

Localization of specific lysine residues responsible for the ubiquitination of DJ2. A, J-domain and C-terminal domain of DJ2 accommodate most of the Ubn sites. The PhosphositePlus and GGbase databases were used to identify the experimentally reported lysines that are ubiquitinated in DJ2. B, K32 and K350 are the major ubiquitination sites in DJ2. SH-SY5Ycells were transfected with EV, WT DJ2, or various lysine mutants. HA-Ub was cotransfected. The Ubn assay was performed as described in Figure 1F (n = 5). C, D, SH-SY5Ycells were transfected with EV, WT DJ2, or lysine mutants. Cells were treated with 2.5 µg/ml CHX and harvested at the indicated time points for immunoblot analysis. Equal amounts of cell lysates were examined by SDS-PAGE and probed with the specified antibodies. The relative ratio of DJ2/GAPDH protein level, normalized to that at zero time, is shown. *p < 0.05, **p < 0.01, and ***p < 0.001. Student’s t test was used to calculate p values for WT and KO cells at the time points mentioned from five independent experiments. E, SH-SY5Ycells were transfected with EV, WT DJ2. or lysine mutants along with HA-Ub. Cells were harvested after 24 h, and Ubn assay was performed.

Figure 4.

Chaperone activity of DJ2 prevents template-assisted aggregation of tau. A, Full-length hTau-40 (5 μm) was incubated at 37°C with the indicated combinations of proteins (5 μm) for 90 h, all without heparin (control experiment) except the one mentioned (Tau+heparin, maroon line). Samples were collected at the time points mentioned and kept at −80°C until the collection of all samples. ThT 5 μm was added after 90 h to observe the degree of aggregation and fluorescence emission that was measured at 480 nm, with excitation at 440 nm. Student’s t test and one-way ANOVA was used for the statistical analysis of data. Experiments were repeated three times with three samples (n = 9) for all data points. Labels and colors shown in the inset of D are also applicable for all other panels (A–C). p Values shown here are calculated for “tau + heparin” with respect to zero time point. Statistical analysis of different combinations with respect to “tau without heparin” and “tau with heparin” is shown in Extended Data Figure 4-1. B, Experiment detailed in A was repeated with heparin added to all the samples, except the negative control. Heparin was included in all the reactions except the negative control (tau without heparin, orange line), at a concentration of 2.5 μm. Student’s t test and one-way ANOVA were used for the statistical analysis of data (Extended Data Figure 4-1). The experiment was repeated five times with three samples (n = 15) for all data points in every experiment. C, Using hTau-K18 mutant, experiment detailed in A was repeated with heparin added to all the samples, except the negative control. hTau-K18 (5 μm) was incubated at 37°C for 4 h. One-way ANOVA was used for the statistical analysis of data. The experiment was repeated five times with three samples (n = 15) for all data points in every experiment. D, To monitor the effects of chaperones on Alzheimer-like PHF formation, experiment detailed in A was repeated with full-length hTau-40 (5 μm). Samples were incubated at 37°C with the indicated combinations of proteins (5 μm) for 9 d, with heparin added to all the samples, except the negative control. One-way ANOVA was used for the statistical analysis of data. Experiment was repeated five times with three samples (n = 15) for all data points in every experiment. Complete statistical analysis of A–D is shown in Extended Data Figure 4-1. E, SH-SY5Y cells were treated with monomeric and aggregated forms of hTau-K18 (top panel) and full-length hTau-40 (bottom panel) with or without DJ2 and CRBN as shown. F, MTT assay was performed after 48 h to assess cell viability. Error bars represent the SEM. Student’s t test was used for quantification of data with a 95% significance level in Excel (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5.

Knocking out CRBN improves the association of tau with microtubules by decreasing tau phosphorylation. A, Schematic representation of the BiFC assay. hTau-40 is attached to nonfluorescent N- or C-terminal fragment of Venus fluorescence protein (VN173 or VC155). The Venus fluorescence turns on only when the phosphorylated tau assembles. B, CRBN^+/+ and CRBN^−/− SH-SY5Y cells were transfected with siRNA targeting DJ2 to find out the optimal conditions for knockdown of DJ2. siRNA targeting GAPDH was used as positive control (n = 4). C, Effects of CRBN on phosphorylation-mediated tau dimerization. CRBN^+/+ and CRBN^−/− SH-SY5Y cells were transfected withVN173 and VC155 empty vectors (BiFC-EV) or VN173–tau and VC155–tau (BiFC–tau) constructs and treated with OA (30 nm). CRBN^−/− SH-SY5Y cells transfected with siRN-DJ2 were also used (bottom panel). Hoechst dye was used as a counterstain. Cells were imaged under fluorescence microscope (n = 5). D, Cells from C were subjected to Western blot analysis. ImageJ was used to quantify the fluorescence Intensity and Western blots, and is represented as the mean ± SD (n = 5). Scr-si, Scramble siRNA; DJ2-si, DJ2 siRNA. Student’s t test was used to calculate statistical significance. E, CRBN^−/− (KO) and CRBN^+/+ (WT) HEK293 T cells were treated with OA for 16 h and stained with tubulin tracker green, tau antibody (secondary antibody Alexa Fluor-594), and Hoechst dye. The cells were then imaged with an FV1000 confocal laser-scanning microscope (n = 5).

Figure 6.

CRBN tunes the chaperone activity of DJ 2 to prevent tau phosphorylation and tau pathology. A, HEK293T cells were subjected to CRISPR/Cas9-mediated KO of CRBN or nontargeting gRNA sequences [negative control (NC) vector]. B, Myc-tau was transiently transfected to both cell lines, and Western blot was performed against the pathologic phospho-tau sites (tau panel) and chaperones (chaperone panel). Heat map shows the statistical analysis of five independent experiments, whereas the intensity of each box shows the mean value (n = 5). C, SH-SY5Y cells were transiently cotransfected with Myc-tau and FLAG-DJ2, and immunoprecipitated with mouse IgG or α-FLAG antibody, SE (short exposure), and LE (long exposure). D, SH-SY5Y cells were transiently cotransfected Myc-tau and FLAG-CRBN; and immunoprecipitated with α-FLAG antibody (n = 3).

Figure 7.

Knocking out Crbn attenuates pathologic phosphorylation of tau. A, WBL of WT (Crbn^+/+) and KO (Crbn^−/−) mice were subjected to immunoblot against the selected p-tau epitopes. Adjacent panels show statistics. Phospho/total ratio was calculated and represented as the mean ± SD (n = 3; 9 mice/group). Student’s t test was used. B, Brain tissues of WT (Crbn^+/+) and KO (Crbn^−/−) were cryosectioned and immunostained with AT8 (pTau) and DJ2 antibodies for 16 h, incubated with Alexa Fluor-conjugated secondary antibodies for 1 h, and imaged under confocal microscope equipped with 40× lens. Adjacent panels shows statistics, Student’s t test was used to calculate p value for n = 6. C, WBL of WT (Crbn^+/+) and KO (Crbn^−/−) mice were subjected to immunoblot against the selected tau-kinases. Adjacent panels show statistics. Phospho/total ratio was calculated and represented as the mean ± SD (n = 3; 9 mice/group). Student’s t test was used. D, SH-SY5Y cells were transiently cotransfected with Myc-tau and FLAG-DJ2, and immunoblotted with the indicated antibodies. Student’s t test was used to calculate p value for n = 3.

Figure 8.

Progressive accumulation of pathologic tau is prevented in Crbn knock-out mice. A, B, Brains were removed from 1- and 15-month-old male WT (Crbn^+/+) and KO (Crbn^−/−) mice, and pTau level was measured using Western blot and DAB staining using AT8 antibody. Data represent the mean ± SD (n = 5) using Student’s t test. C, Schematic representation of dissection. Mouse brains were dissected into certain anatomic parts, anterior contralateral (AC), anterior ipsilateral (AI), posterior contralateral (PC), posterior ipsilateral (PI), and cerebellum (Cb), for analysis with Western blotting. The PI region contains the lateral amygdala injection site. D, OA was stereotaxically injected into the brains of WT and KO mice. After 24 h, dissected parts were lysed in RIPA buffer, and blotted with antibodies of the p-tau array as shown. E, The same brain samples used in D were analyzed for three tau kinases (phospho and total forms), as shown in the panels. F, OA was stereotaxically injected into the brains of WT and KO mice. After 7 d, dissected parts were lysed in RIPA buffer and blotted with antibodies of the p-tau array, as shown.

Figure 9.

Progressive accumulation of pathologic tau is prevented in Crbn knock-out mice. A–C, Densitometric analysis of panels D–F, respectively, in Figure 8, with n = 6 in each group of KO and WT mice is shown. Student’s t test was performed. Experiment was performed three times. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 10.

CRBN is a novel contributor for tauopathies. A–C, Brains were removed from 8-month-old male APP knock-in and 5XFAD mice; half of the brain was used for cryosectioning (A, B), and another half for Western blot analysis (C). Hippocampi were immunostained with APP, CRBN (Abnova), and DJ2 antibodies for 16 h, and imaged under a confocal microscope equipped with 100× (A) and 10× (B) lenses. Images were quantified by ImageJ and represented as the mean ± SD (n = 5). Student’s t test was used. D, E, SH-SY5Y cells were transiently cotransfected with Myc-DJ2 and FLAG-CRBN. Cellular extracts were treated with scrambled (D) or DJ2 peptide (E) for 24 h. Cellular extracts were immunoprecipitated with α-FLAG antibody from rabbit (Rb) and immunoblotted with Myc and FLAG antibodies from mouse (Ms) to get rid of the band of IgG heavy chain. F, Sequence and 3D structure of scrambled peptide and DJ2 peptide inhibitor. Models were generated for a span of 51 aa of DJ2 (20 aa on both sides of the peptide) using Swiss model. G, Statistical analysis of D and E; data were represented as the mean ± SD (n = 5). Student’s t test was used.

Figure 11.

Proposed model for the role of CRBN in tau pathology by regulating the availability of chaperones. Atypical disengagement of tau from the microtubules results in a concomitant increase in the cytosolic fraction of p-tau, which is likely to be the key event leading to tauopathies. DJ2-mediated refolding/clearance of cytosolic p-tau may constrain its aggregation and stimulate reassociation with microtubules. CRBN mitigates the DJ2-mediated inhibition of pathologic tau species.