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. 2017 Sep 12;114(37):E7803-E7811.
doi: 10.1073/pnas.1710549114. Epub 2017 Aug 28.

Differential HspBP1 Expression Accounts for the Greater Vulnerability of Neurons Than Astrocytes to Misfolded Proteins

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Free PMC article

Differential HspBP1 Expression Accounts for the Greater Vulnerability of Neurons Than Astrocytes to Misfolded Proteins

Ting Zhao et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Although it is well known that astrocytes are less vulnerable than neurons in neurodegenerative diseases, the mechanism behind this differential vulnerability is unclear. Here we report that neurons and astrocytes show markedly different activities in C terminus of Hsp70-interacting protein (CHIP), a cochaperone of Hsp70. In astrocytes, CHIP is more actively monoubiquitinated and binds to mutant huntingtin (mHtt), the Huntington's disease protein, more avidly, facilitating its K48-linked polyubiquitination and degradation. Astrocytes also show the higher level and heat-shock induction of Hsp70 and faster CHIP-mediated degradation of various misfolded proteins than neurons. In contrast to astrocytes, neurons express abundant HspBP1, a CHIP inhibitory protein, resulting in the low activity of CHIP. Silencing HspBP1 expression via CRISPR-Cas9 in neurons ameliorated mHtt aggregation and neuropathology in HD knockin mouse brains. Our findings indicate a critical role of HspBP1 in differential CHIP/Hsp70 activities in neuronal and glial cells and the greater neuronal vulnerability to misfolded proteins in neurodegenerative diseases.

Keywords: Huntington; chaperone; misfolding; neurodegeneration; polyglutamine.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CHIP is more actively involved in mHtt degradation in astrocytes. (A) More abundant monoubiquitinated CHIP (Ub1-CHIP) is present in cultured astrocytes than in neurons. (B) CHIP knockdown decreased Ub1-CHIP in astrocytes (n = 3 independent experiments). (C) Transfected CHIP-myc in cultured astrocytes was immunoprecipitated by anti-myc. Immunoblotting of the immunoprecipitates with anti-ubiqutitin indicated the presence of monoubiquitinated CHIP in astrocytes. (D) Expression of transfected mHtt (Htt-130Q) selectively increases the levels of CHIP and Ub1-CHIP in astrocytes but not in neurons [n = 3 independent experiments (astrocytes) and n = 5 independent experiments (neurons)]. (E) Immunofluorescent staining shows the colocalization of transfected mHtt (Htt-130Q) with endogenous CHIP in cultured astrocytes. (Scale bar, 5 μm.) (F) CHIP knockdown increases both soluble and aggregated mHtt (Htt-130Q) in astrocytes (n = 3 independent experiments). (G) Colocalization of CHIP with mHtt aggregates was detected in glial cells in the corpus callosum, but not in neuronal cells in the striatum and cortex, in HD140Q KI mice at the age of 12 mo. Arrowheads indicate nuclear mHtt aggregates; arrows indicate cytoplasmic aggregates in glial cells or neuropil mHtt aggregates in neurons. (Scale bar, 10 μm.) (H) Association of mHtt with CHIP and Ub1-CHIP was detected in HD140Q KI astrocytes but not in KI neurons. Quantitative data beneath the blots are represented as mean ± SEM (error bar). *P < 0.05, **P < 0.01 (unpaired two-tailed Student’s t test); ns, not significant.
Fig. S1.
Fig. S1.
Expression of huntingtin in primary cultures and the brain of HD knockin mouse. (A) Wild-type (23Q) or mutant (130Q) huntingtin was transfected in astrocytes and neurons, and their expression was confirmed by Western blotting. (B) mHtt aggregates are formed in both NeuN-positive neurons and GFAP-positive astrocytes of 12-mo-old HD 140Q KI mice. However, CHIP puncta, which are indicative of localization of CHIP in mHtt aggregates, are found in astrocytes of the corpus callosum, but not in neurons of the striatum. (Scale bar, 10 μm.)
Fig. 2.
Fig. 2.
CHIP enhances K48-linked polyubiquitination on mHtt in astrocytes. (A and B) There are more K48-polyubiquitinated mHtt in KI cortical astrocytes than KI neurons. Ratios of K48-polyubiquitinated mHtt to total immunoprecipitated mHtt are from three independent experiments. (C and D) CHIP knockdown suppressed K48-linked polyubiquitination on endogenous mHtt in HD140Q KI cortical astrocytes. Ratios of K48-polyubiquitinated mHtt to total immunoprecipitated mHtt are from three independent experiments. (E) Expression of mHtt in Htt-130Q transfected astrocytes and HD140Q KI astrocytes did not affect proteasomal function compared with Htt-23Q transfected or wild-type (WT) astrocytes (n = 3 independent experiments). In AD, cultured cells were treated with lactacystin (10 μM for 6 h) to inhibit proteasomal degradation. ***P < 0.001, ****P < 0.0001. ns, not significant. Data are represented as mean ± SEM. Error bars indicate SEM.
Fig. S2.
Fig. S2.
Hsp70-dependent association of CHIP with mHtt. (A) PU-H71 induction promoted the association of transfected Htt-73Q with CHIP (Upper) and also increased the expression of Hsp70 (Lower). The ratios of immunoprecipitated CHIP to Htt-73Q in transfected HEK293 cells untreated (control) or treated with PU-H71 from three independent experiments are also shown. (B) Knocking down Hsp70 via siRNA abolished the coimmunoprecipitation of Htt-73Q with CHIP (Upper) and diminished the level of Hsp70 in transfected HEK293 cells (Lower). Right shows the ratios of CHIP to Htt-73Q in coimmunoprecipitation from three independent experiments. *P < 0.05, ****P < 0.0001 (unpaired two-tailed Student’s t test). Data are represented as mean ± SEM. Error bars indicate SEM.
Fig. S3.
Fig. S3.
More abundant Hsp70 in astrocytes than in neurons. (A) Hsp70 is expressed at a higher level in wild-type cultured cortical astrocytes than neurons (n = 4 independent experiments). (B) The higher level of Hsp70 in the corpus callosum (CC) than in the cortex (CTX) of 3-mo-old wild-type mice (n = 4 independent experiments). (C) Levels of Hsp70 are higher in astrocytes than in neurons in the mouse brain (n = 3 mice) (Scale bar, 20 μm.) *P < 0.05, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed Student’s t test); ns, not significant. Data are represented as mean ± SEM (error bar).
Fig. 3.
Fig. 3.
Differential CHIP activity causes distinct heat-shock responses in astrocytes and neurons. (A) Typical heat shock (42 °C for 1 h) induced Hsp70, Hsp90, CHIP, and Ub1-CHIP specifically in wild-type cortical astrocytes around 30 days in vitro (DIV 30) (Left), but not in wild-type cortical neurons at DIV 10–14 (Right) (at least three independent experiments). (B) Heat-shock stress increased Hsp70 in the corpus callosum, but not in the cortex, of brain slices from wild-type (WT) and HD140Q KI mice at the age of 3 mo (n = 3 independent experiments). CC, corpus callosum; CTX, cortex; HS, heat shock. (C) Induction of Hsp70 by heat shock (42 °C, 1 h) was completely inhibited by CHIP knockdown, and no Hsp90 was induced in wild-type astrocytes at DIV 45–60. (D) CHIP knockdown decreased the expression of Hsp70/Hsp90, but did not affect levels of Hsc70 in DIV 45–60 wild-type astrocytes. (E) Ratios of indicated proteins to GAPDH are from three independent experiments (Upper for C and Lower for D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed Student’s t test). ns, not significant. Data are represented as mean ± SEM (error bar).
Fig. S4.
Fig. S4.
Increased CHIP, monoubiquitinated CHIP, and Hsp70 after heat shock specifically in HD140Q KI astrocytes. (A) Hsp70 and Hsp90 were not inducible in cultured KI neurons at DIV 10–14 by 30 min of heat-shock stress, and CHIP decreased after heat shock (n = 4 independent experiments). (B) Heat shock for 30 min was able to induce Hsp70, CHIP, and Ub1-CHIP, but not Hsp90, in cultured KI astrocytes at DIV 21–30 (n = 4 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student’s t test); ns, not significant. Data are represented as mean ± SEM. Error bars indicate SEM.
Fig. 4.
Fig. 4.
CHIP mediates faster degradation of misfolded proteins in astrocytes. (AC) Mutant TDP-43 (M337V), TBP-105Q, and α-synuclein (A53T)-cyan fluorescent protein (CFP) were degraded faster by astrocytes than neurons, which was eliminated by CHIP knockdown in astrocytes. *P < 0.05, **P < 0.01 (unpaired two-tailed Student’s t test). ns, not significant. Data are represented as mean ± SEM (error bar).
Fig. 5.
Fig. 5.
High activity of CHIP contributes to astrocytic resistance to hyperthermia. (A) Apparent demise of cultured cortical neurons at DIV 10–12 after heat-shock stress (45 °C, 30 min). The same heat-shock stress (45 °C, 30 min) did not affect viability of cultured cortical astrocytes at DIV 40–45. Neurons and astrocytes were fixed and immunostained at 24 h after heat shock. (B) CHIP knockdown caused death of a portion of CHIP shRNA-transfected astrocytes, which was characterized by shrunken nucleus, at 24 h after heat shock (45 °C, 30 min). Arrows indicate corpse of astrocytes. (C) Quantitative results of Fig. 5 A and B. At least 10 visual fields were randomly picked up for each group, and the live cells in each visual field were quantified. (D and E) TUNEL assay showing increased susceptibility of CHIP knockdown astrocytes to heat-shock stress (45 °C, 30 min). Arrows indicate apoptotic astrocytes (TUNEL-positive cells indicated by green fluorescence). ***P < 0.001, ****P < 0.0001 (unpaired two-tailed Student’s t test); ns, not significant. Data are represented as mean ± SEM. Error bars indicate SEM. (Scale bar, 10 μm.)
Fig. 6.
Fig. 6.
Deficient expression of HspBP1 in astrocytes. (A) Higher levels of HspBP1 in cultured cortical neurons than astrocytes were shown by Western blotting (n = 3 independent experiments). (B) Representative images of immunostaining showed differential expression levels of HspBP1 between cultured neurons and astrocytes. (Scale bar, 5 μm.) (C) Fluorescent immunostaining revealing greater abundance of HspBP1 in neurons than astrocytes in mouse brain. (Scale bar, 20 μm.) (D) Immunostaining demonstrated more HspBP1 expression in neurons in the gray matter of human basal ganglia than that in astrocytes in the white matter of basal ganglia. (Scale bar, 20 μm.) (E) Statistical results for C and D [mice n = 3, human n = 2 (57 y)]. ****P < 0.0001 (unpaired two-tailed Student’s t test). Data are represented as mean ± SEM (error bar).
Fig. S5.
Fig. S5.
Expression of Bag2 in neurons and astrocytes. The levels of Bag2 do not show difference between cultured neurons and astrocytes. Data are represented as mean ± SEM (error bar). ns, not signficant.
Fig. 7.
Fig. 7.
Deficient expression of HspBP1 in astrocytes contributes to high activity of CHIP in astrocytes. (A) Expression of HspBP1 in cultured astrocytes reduced the level of Ub1-CHIP, and major form of CHIP increased correspondingly (n = 3 independent experiments). (B) qRT-PCR showed that overexpression of HspBP1 did not influence transcription of CHIP in cultured astrocytes (n = 3 independent experiments). (C) HspBP1 expression in cultured astrocytes stabilized aggregated and soluble mHtt and suppressed induction of CHIP and Ub1-CHIP by mHtt, which was seen in Fig. 1D (n = 3 independent experiments). (D) Induction of Hsp70, CHIP, and Ub1-CHIP by heat-shock stress at 42 °C for 1 h was significantly inhibited by HspBP1 expression in DIV 30–50 astrocytes (n = 4 independent experiments). (E) Expression of HspBP1 decreased basal levels of Hsp70 in astrocytes (n = 3 independent experiments). *P < 0.05, **P < 0.01, ****P < 0.0001 (unpaired two-tailed Student’s t test). Data are represented as mean ± SEM (error bar).
Fig. 8.
Fig. 8.
Knocking down HspBP1 is neuroprotective in HD 140Q KI mice. (A) HspBP1 knockdown (KD) in cultured neurons induced Ub1-CHIP expression. (B) Hsp70/90 were induced 6 h after heat shock (42 °C for 1 h) in HspBP1 knockdown neurons (n = 3 independent experiment). (CH) HspBP1 KD decreased mHtt aggregates and reactive astrocytes, rescued the loss of synaptophysin in the striatum of HD140Q KI mice at the age of 13–14 mo, compared with the injection with HspBP1 sgRNA alone [control (Con)]. *P < 0.05, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed Student’s t test). (Scale bar, 20 μm.) Data are represented as mean ± SEM (error bar). (I) A proposed model for the differential accumulation and toxicity of misfolded proteins in neurons and astrocytes: CHIP is more active and monoubiquitinated in astrocytes due to deficient HspBP1 expression. Activated CHIP preferentially binds to and polyubiquitinates misfolded proteins and confers higher Hsp70, resulting in the prompt proteasomal degradation of misfolded proteins in astrocytes.
Fig. S6.
Fig. S6.
Genome-editing activity of HspBP1 sgRNA. (A) For increasing specificity and decreasing off-target binding, we used Target Finder, which is generated by Feng Zhang’s laboratory, Massachusetts Institute of Technology, Cambridge, MA to design gRNA. The chosen target sites of sgRNA are shown in bold font, and the PAM sequence is highlighted in red. (B) Genome-editing activity of HspBP1 sgRNA was tested using T7E1 assay. N2a cells were cotransfected with HspBP1 sgRNA and Cas9 plasmids or transfected only with HspBP1 sgRNA plasmid as control. The T7E1 assay was carried out using PCR products of genome DNA from transfected N2a cells. The arrow indicates cleaved PCR products by T7E1 enzyme. MT, mutant; WT, wild type. (C) Western blotting assay showed knockdown of HspBP1 at the protein level in N2a cells that were cotransfected with HspBP1 sgRNA and Cas9 plasmids. (D) Mutations in target HspBP1 gene were identified by TA cloning and subsequent sequencing.
Fig. S7.
Fig. S7.
Knocking down HspBP1 in the mouse striatum using CRISPR-Cas9. (A) Schematic representation of DNA constructs of HspBP1 sgRNA and Cas9. RFP is expressed under the cytomegalovirus (CMV) promoter in the same AAV vector for expressing HspBP1 sgRNA. Viral injection of HspBP1 sgRNA and Cas9 into one striatum of HD 140Q KI mice was performed to knock down endogenous HspBP1, and injection of HspBP1 sgRNA alone into the striatum of the other hemisphere served as a control. (B) In the striatum of HD140Q KI mice, HspBP1 knockdown by CRISPR-Cas9 was validated by Western blotting. (C) Knocking down HspBP1 enhances the level of Hsp70 in the striatum of HD 140Q KI mice (n = 3 mice). RFP labeling indicates cells expressing HspBP1 sgRNA. (Scale bar, 20 μm.) ***P < 0.001 (unpaired two-tailed Student’s t test). Data are represented as mean ± SEM (error bar).

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