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HSF1 physically neutralizes amyloid oligomers to empower overgrowth and bestow neuroprotection

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Science Advances  11 Nov 2020:
Vol. 6, no. 46, eabc6871
DOI: 10.1126/sciadv.abc6871
  • Fig. 1 HSF1 is required for megalencephaly driven by constitutively active PI3K.

    (A) Brains were collected at 15 days postnatal (n = 3 mice). Photo credit: Zijian Tang, NCI. (B) Kaplan-Meier survival curves (n = 8 to 15 mice). (C) Immunoblotting of brain lysates (n = 3 mice). (D) Quantitation of the numbers of nuclei in frozen brain tissues (n = 5 mice). (E) Quantitation of caspase 3 activities in brain lysates (n = 5 mice). (F and G) Enzyme-linked immunosorbent assay (ELISA) quantitation of detergent-insoluble amyloid fibrils (AFs) and detergent-soluble prefibrillar AOs (PAOs) in brain lysates (n = 5 mice). (H) Representative staining images of paraffin brain sections (from three brains). (I) ELISA quantitation of PAOs levels in detergent-insoluble fractions of brain lysates (n = 5 mice). (J) Aβ1–42 (2 μM) peptides were incubated with 100-μg detergent-soluble fractions of brain lysates and the Aβ1–42 fibrillation was monitored by thioflavin T (ThT) binding (n = 5 mice). (K) ELISA quantitation of endogenous mouse Aβ1–42 levels in the soluble fractions of brain lysates (n = 5 mice). (L) Representative immunohistochemistry images of paraffin brain sections (from three brains). Arrowheads denote plaque-like Aβ deposits. Scale bars, 100 μm for main images; 20 μm for insets. Statistical analyses: Log-rank test for (B); two-way analysis of variance (ANOVA) for (J); and one-way ANOVA for the rest. (A) to (D) and (J) were done once; (E) to (G) and (I) were repeated twice; (K) was repeated thrice; (H) and (L) were repeated thrice with different sets of brains. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 2 AOs are causally related to apoptosis.

    (A) Quantitation of caspase 3 activities in p110*-expressing astrocytes treated with and without 10 μM CR for 2 days (n = 3 lines of astrocytes). (B) Primary Hsf1fl/fl astrocytes were first stably transduced with lentiviral short hairpin RNAs. To delete Hsf1, these astrocytes were transiently transduced with adenoviral green fluorescent protein (GFP) or Cre. Both PAOs and AFs were quantitated by ELISA (n = 3 lines of astrocytes). (C) Quantitation of the mitochondrial membrane potentials in Pten-deficient astrocytes with and without Hsf1 deletion by fluorescence-activated cell sorting (FACS) using JC-1 dyes. The red/green (FL2-H/FL1-H) fluorescence ratios were calculated using geometric means (n = 3 lines of astrocytes). (D) Rescue of the mitochondrial membrane potentials in Pten-deficient astrocytes by neutralizing AOs. Following transfection of 100-ng antibodies for 2 days, Pten-deficient astrocytes with Hsf1 deletion were stained with JC-1 dyes (n = 3 lines of astrocytes). (E) Schematic depiction of the roles of HSF1 in supporting the tissue overgrowth. In addition to maintaining the balance between protein quantity and quality, which suppresses the initiation of amyloidogenesis, HSF1 also plays a critical role in antagonizing the amyloid-induced cytotoxicity, once amyloidogensis becomes inevitable. Thereby, HSF1 empowers overgrowth. Statistical analyses: one-way ANOVA. (A) to (D) were repeated thrice with different sets of astrocytes. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 3 HSF1 enables hepatomegaly.

    (A) Kaplan-Meier survival curves (n = 10 to 12 mice). (B) Quantitation of caspase 3 activities in liver lysates (n = 5 mice). (C and D) ELISA quantitation of AFs and soluble PAOs in liver lysates (n = 5 mice). (E) Representative photographs of livers. As one set of livers was harvested at a later time point, the weights of each set of livers are normalized (n = 3 mice). Photo credit: Zijian Tang, NCI. (F) Kaplan-Meier survival curves (n = 8 to 11 mice). (G) Quantitation of caspase 3 activities in liver lysates (n = 5 mice). (H and I) ELISA quantitation of AFs and soluble PAOs in liver lysates (n = 5 mice). (J) Representative staining images of frozen liver sections (from three livers, and the TUNEL staining was performed by two individuals). Scale bars, 20 μm for main images; 10 μm for insets. (K) ELISA quantitation of insoluble PAOs in liver lysates (n = 5 mice). (L) Aβ1–42 peptides (2 μM) were incubated with 100-μg soluble liver lysates, and the Aβ1–42 fibrillation was monitored (n = 5 mice). (M) ELISA quantitation of endogenous Aβ1–42 levels in the soluble fractions of liver lysates (n = 5 mice). Statistical analyses: log-rank test for (A) and (F); two-way ANOVA for (L); and one-way ANOVA for the rest. (A), (E), (F), (L), and (M) were done once; (B) to (D), (G) to (I), and (K) were repeated twice; and (J) was repeated thrice with different sets of livers. H&E, hematoxylin and eosin. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 4 Hsf1 deficiency leads to HSP60 loss and mitochondrial damage.

    (A to D) Quantitation of PAOs and AFs in p110*-expressing astrocytes treated with and without 50 μM 4EGI-1 or 20 μM LY2584702 overnight. All cells were cotreated with 20 μM Q-VD-OPH to block apoptosis (n = 3 lines of astrocytes). (E) Representative images of HSP60 immunofluorescence in two lines of astrocytes. Scale bars, 10 μm. (F) Immunoblotting of HSP60 and TOM20 in detergent-soluble and detergent-insoluble fractions of brain lysates (n = 3 mice). Equal amounts of detergent-insoluble fractions, following re-solubilization by sonication, were loaded, shown by ponceau red staining. (G) Quantitation of Hsp mRNAs in brains by quantitative reverse transcription polymerase chain reaction (n = 5 mice). (H) Detection of HSP60 polyubiquitination in brain lysates by immunoprecipitation (IP) with the EasyBlot reagents (n = 3 mice). (I) Representative images of HSP60 immunofluorescence in three frozen brains. βIII-tubulin served as a neuronal marker. Scale bars, 10 μm. (J) Immunoblotting of the mitochondrial and cytosolic fractions of astrocytes (representative images of two lines of astrocytes). Equal amounts of the same fractions were loaded. TOM20 and β-actin were used as the mitochondrial and cytosolic markers, respectively. Statistical analyses: one-way ANOVA. (F) to (H) were done once; (E) and (J) were repeated twice with different sets of astrocytes; and (A) to (D) and (I) were repeated thrice with different sets of astrocytes or brains. DAPI, 4′,6-diamidino-2-phenylindole. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5 Loss of HSP60 causes mitochondrial damage, mitophagy, and apoptosis.

    (A) Quantitation of mitochondrial mass in astrocytes treated with 10 μM CR or 20 μM CQ for 6 days by FACS (FL1-H, geometric means) using MitoView Green (n = 3 lines of astrocytes). (B) Immunoblotting of astrocytes transduced with lentiviral LacZ or HSP60 (multiplicity of infection, MOI = 10) for 6 days (representative images of two lines of astrocytes). Equal amounts of the same fraction were loaded. (C) JC-1 staining of astrocytes described in (B). The contour plot represents one line of astrocytes. The red/green (FL2-H/FL1-H) fluorescence ratios from three lines of astrocytes are quantitated in fig. S4F. (D) Quantitation of caspase 3 activities in astrocytes described in (B) (n = 3 lines of astrocytes). (E) Detection of molecular changes in hGFAP-Cre+; Hsf1+/+ astrocytes transfected with control or Hsp60-targeting siRNAs for 4 days (representative images of three lines of astrocytes). Equal amounts of the same fraction were loaded. (F) Quantitation of caspase 3 activities in astrocytes described in (E) (n = 3 lines of astrocytes). Statistical analyses: one-way ANOVA. (B) was repeated twice with different sets of astrocytes; (A) and (C) to (F) were repeated thrice with different sets of astrocytes. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 6 HSF1 shields HSP60 from the attack of AOs.

    (A and B) Representative images of AO-HSF1 and AO-HSP60 co-IP from three (A) and two (B) brains with the EasyBlot reagents (performed by two individuals). (C) In vitro reconstitution of interactions among Aβ1–42, HSP60, and HSF1 using recombinant proteins at a 1:1:1 molar ratio. After incubation, reconstituted mixtures were centrifuged to collect the supernatants and pellets for immunoblotting (representative images of three experiments). (D) Aβ1–42, HSP60, and A11 or OC antibodies were reconstituted in vitro at a 1:1:1 molar ratio at RT for 4 hours (representative images of three experiments). (E) Immunoblotting of HSF1 and HSP60 in immortalized Rosa26-CreERT2; Hsf1fl/fl MEFs transfected with Aβ42–1 or Aβ1–42 for 2 days (representative images of three experiments). (F) HSF1:Aβ42:HSP60 molar ratios in soluble Hsf1+/+ brain lysates (n = 5 mice). (G) ELISA quantitation of soluble AOs in P*H+ brain lysates before and after IP of Aβ (n = 5 mice, two-tailed paired Student’s t test). Biotinylated A11 and OC Abs were used to detect AOs. (G) was done once; (F) was repeated twice; (B) was repeated twice with different sets of brains; (A) was repeated thrice with different sets of brains; and (C) to (E) were repeated thrice with the same cell line or reagents. n.s., not significant, P > 0.05; *P < 0.05; ***P < 0.001.

  • Fig. 7 HSF1 protects HSP60 independently of transcription.

    (A) The dual HSF1 reporter plasmids were cotransfected with HSF1 mutants into HEK293T cells (n = 3 experiments). (B and C) Quantitation of the mitochondrial membrane potential (B) and caspase activities (C) in p110*-expressing astrocytes transduced with lentiviral LacZ, HSF11–529, HSF11–323, or HSF1324–529 (MOI = 10) for 6 days (n = 3 lines of astrocytes). (D) PLA visualization of PAO-HSP60 interactions in astrocytes described in (B) and (C) (representative images of two lines of astrocytes). Scale bars, 10 μm. (E) Schematic depiction of the model wherein the AO:HSF1 molar ratio determines the fates of both HSP60 and HSF1. Excessive HSF1 fully neutralizes AOs and protects HSP60. However, the AO-HSF1 complexes may either remain soluble or become nonamyloid aggregates, depending on their interaction stoichiometry (AO << HSF1 or AO < HSF1). When AOs rise and become excessive, on the one hand, AOs that are not neutralized by HSF1 start attacking HSP60, leading to HSP60 misfolding and aggregation. On the other hand, the stoichiometry of AO-HSF1 interactions may be reversed (AO > HSF1), which would cause HSF1 misfolding and aggregation. Statistical analyses: one-way ANOVA. (D) was repeated twice with different sets of astrocytes; (B) and (C) were repeated thrice with different sets of astrocytes; and (A) was repeated thrice with the same cell line. n.s., not significant, P > 0.05; *P < 0.05; ***P < 0.001.

  • Fig. 8 HSF1 blocks in vitro amyloidogenesis.

    (A) HiLyte Fluor 488–labeled Aβ1–42 was incubated with Dabcyl-labeled HSF1 or GST at a 1:1 molar ratio (n = 3 experiments). (B) Quantitation of the fibrillation of 0.8 μM Aβ1–42 incubated with recombinant HSF1 proteins in vitro at increased molar ratios (n = 3 experiments). (C to E) ELISA quantitation of amyloids after incubation of Aβ1–42 with GST or HSF1 for 48 hours (n = 3 experiments). Aβ42–1 served as the negative control. (F) Quantitation of the fibrillation of 0.8 μM Aβ1–42 incubated with normal rabbit IgG, A11, or OC Abs in vitro at a 1:1 molar ratio (n = 3 experiments). (G) Quantitation of the nephelometric turbidities of Aβ1–42 described in (B) (n = 3 experiments). (H) Visualization of in vitro fibrillation of Aβ1–42 coincubated with either GST or HSF1 at 37°C for 48 hours by transmission electron microscopy (representative images of three experiments). Scale bars, 600 nm for 9300×; 100 nm for 30,000×. (I) Detection of the aggregation of 0.2 μM Aβ1–42 incubated with HSF1 at a 1:32 molar ratio for 48 hours by filter trap assays. The aggregates were stained with ponceau red (representative images of two experiments). The curves in (B), (F), and (G) are fitted with the Boltzmann sigmoid equation. Statistical analyses: two-way ANOVA for (A), (B), (F), and (G); and one-way ANOVA for (C) to (E). (I) was repeated twice; and (A) to (H) were repeated thrice. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 9 HSF1 contains amyloids formed in vivo.

    (A and B) Quantitation of the changes in nephelometric turbidities (A) and soluble PAOs (B) of 100-μg detergent-soluble brain lysates incubated at 37°C with shaking for 48 hours (n = 5 mice). (C and D) Quantitation of the changes in nephelometric turbidities (C) and soluble PAOs (D) of 100-μg detergent-soluble fractions of Hsf1-deficient brain lysates supplemented with 200-ng GST or HSF1 proteins (n = 5 mice). (E) Schematic depiction of the multilayered regulations of amyloidogenesis by HSF1. When HSF1 is slightly or moderately excessive (AOs < HSF1), the low interaction stoichiometry fully blocks the self-propagation of both PAOs and FAOs and, importantly, stops their attack on HSP60. This low stoichiometry, however, is inadequate to block the assembly of FAOs into mature AFs, only achieving partial impairments. Under this scenario, FAOs either continue to mature into fibrils (very low stoichiometry) or are transformed into amorphous aggregates (intermediately low stoichiometry), owing to the increased antagonizing force of HSF1. When HSF1 is considerably excessive (AOs << HSF1), the high interaction stoichiometry stabilizes FAOs at the soluble, nontoxic state, preventing the formation of both AFs and amorphous aggregates. The curves are fitted with the Boltzmann sigmoid equation. Statistical analyses: two-way ANOVA for (A) and (C); two-tailed paired Student’s t test for (B) and (D). (A) to (D) were done once. n.s., not significant, P > 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 10 HSF1 protects human neurons against Aβ.

    (A and B) PLA visualization of HSP60–Aβ1–42 interactions (A, representative images of three experiments performed by two individuals) or HSF1-Aβ1–42 interactions (B, a single experiment) in cultured primary human neurons. Following transduction with lentiviral LacZ or HSF1 mutants (MOI = 20) for 4 days, primary human neurons were transfected with 1 μM biotinylated Aβ42–1 or Aβ1–42 overnight. βIII-tubulin (green) was costained as a neuronal marker. Scale bars, 10 μm. (C) Measurement of the viabilities of primary human neurons transfected with 10 μM Aβ42–1 or Aβ1–42 for 24 hours (n = 3 experiments, one-way ANOVA). The neurons were transduced with lentivirus as described in (A) before transfection with Aβ42–1 or Aβ1–42. (D) Visualization of HSP60-PAOs interactions in the brains of patients with AD by bright-field PLA using a mouse anti-HSP60 Ab (LK1) and the rabbit anti-PAOs (A11) Ab (representative images of five experiments performed by two individuals). The brain sections on tissue arrays are from three patients with late-onset AD and three aged normal controls. Scale bars, 20 μm for low magnification; 10 μm for high magnification. (B) was done once; (A) and (C) were repeated thrice; and (D) was repeated five times. n.s., not significant, P > 0.05; ***P < 0.001.

  • Fig. 11 Implications of HSF1 in human AD.

    (A and B) Detection of AO-HSP60 and AO-HSF1 interactions by co-IP with the EasyBlot reagents in AD whole brain or hippocampus lysates (500 μg) with addition of 200 ng of recombinant GST or HSF1. The lysates are from two patients with late-onset AD and two aged normal controls, distinct from the ones on the tissue arrays. N, normal controls. (C) Detection of HSP60 and HSF1 polyubiquitination in AD brain lysates by IP with the EasyBlot reagents. (D) Immunoblotting of AKT/mTOCR1 signaling in AD brain lysates. (E) Schematic depiction of the model wherein HSF1 safeguards the mitochondria against amyloids. With sufficient HSF1, the attack on HSP60 by AOs is blocked. Accordingly, HSP60 preserves the stability of the mitochondrial proteome. Continuous rise of AOs overwhelms and destabilizes HSF1, causing its deficiency. Thus, those nonneutralized AOs start attacking HSP60, a decisive event devastating the entire mitochondrial proteome. The ensuing mitochondrial damage instigates both apoptosis and mitophagy. (F) Schematic depiction of the dual roles of HSF1 in antagonizing amyloidogenesis. Through transcriptional up-regulation of HSPs, HSF1 ensures appropriate protein quality to sustain proteomic stability, thereby preventing the emergence of amyloids, which represents an indirect mechanism. Nonetheless, once amyloidogenesis becomes inevitable, HSF1, by physically sequestering AOs, both alleviates the buildup of amyloids and safeguards the mitochondria to avert toxicity, which represents a direct mechanism. (A) to (D) were done once.

Supplementary Materials

  • Supplementary Materials

    HSF1 physically neutralizes amyloid oligomers to empower overgrowth and bestow neuroprotection

    Zijian Tang, Kuo-Hui Su, Meng Xu, Chengkai Dai

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