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. 2015 May;25(5):701-13.
doi: 10.1101/gr.182444.114.

Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Martin Stroedicke  1 , Yacine Bounab  1 , Nadine Strempel  1 , Konrad Klockmeier  1 , Sargon Yigit  1 , Ralf P Friedrich  1 , Gautam Chaurasia  2 , Shuang Li  1 , Franziska Hesse  1 , Sean-Patrick Riechers  1 , Jenny Russ  1 , Cecilia Nicoletti  3 , Annett Boeddrich  1 , Thomas Wiglenda  1 , Christian Haenig  1 , Sigrid Schnoegl  1 , David Fournier  1 , Rona K Graham  4 , Michael R Hayden  4 , Stephan Sigrist  5 , Gillian P Bates  6 , Josef Priller  3 , Miguel A Andrade-Navarro  1 , Matthias E Futschik  7 , Erich E Wanker  1
Affiliations

Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Martin Stroedicke et al. Genome Res. 2015 May.

Abstract

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.

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Figures

Figure 1.
Figure 1.
Predicting caudate nucleus-specific, dysregulated HTT-associated proteins by interaction network filtering. (A) Data integration strategy for interaction network filtering by differentially expressed genes. By systematic integration (three filtering steps) of protein interaction and gene expression data (PPI1-3 and ΔEG1-3), a caudate nucleus-specific HD network (PPI4) with potentially dysregulated, disease-relevant HTT-associated proteins was created. (B) Potential function of HTT-associated proteins predicted though systematic HTT interaction network filtering. In PPI4, 13 potentially dysregulated proteins are directly or indirectly linked to HTT. The orange ring indicates known HD therapy targets (HDTTs). (C) Effects of the computationally predicted proteins CRMP1, KLF6, HMGA1, and CFLAR on mutant HTT aggregation in cell-based assays. Potential modulator proteins were coproduced as mCherry-tagged fusions with YFP-tagged HTTex1Q79 fusion protein in SH-EP neuroblastoma cells. Formation of YFP-HTTex1Q79 aggregates was quantified after 48 h by high-content fluorescence imaging. Data are represented as mean ± SEM. (***) P ≤ 0.001, two-sided t-test with unequal variance.
Figure 2.
Figure 2.
Expression of CRMP1 in brains of HD transgenic mice and cell models. (A) Endogenous CRMP1 protein levels are reduced in brain tissues of 12-mo-old YAC128 HD transgenic mice compared to controls. Protein extracts prepared from striatal tissues of wild-type and transgenic HD mice were analyzed by SDS-PAGE and immunoblotting using the anti-CRMP1 antibody 504-518. (B) Quantification of ∼65 kDa CRMP1 bands in A. Values represent the means of three independent experiments. (**) P ≤ 0.01, two-sided t-test with unequal variance. (C) Endogenous CRMP1 levels are altered in brains of R6/2 HD transgenic mice compared to controls. Protein extracts were analyzed by SDS-PAGE and immunoblotting using the anti-CRMP1 antibody 504-518. (D) Quantification of CRMP1 protein bands shown in C. Values represent the means of three independent experiments. (*) P < 0.1; (**) P ≤ 0.01, two-sided t-test with unequal variance. (E) Heat stress induces endogenous CRMP1 protein levels in SH-SY5Y neuroblastoma cells. Cell lysates were analyzed by SDS-PAGE and immunoblotting. (F) Quantification of CRMP1 and HSPA1A protein bands shown in E. Values represent the means of three independent experiments. (**) P ≤ 0.01, two-sided t-test with unequal variance. (G) Analysis of CRMP1 and HSPA1A protein expression in SH-SY5Y cells under heat stress conditions by immunofluorescence microscopy. Data in B, D, and F are represented as mean ± SEM.
Figure 3.
Figure 3.
CRMP1 protein production influences polyQ-mediated HTT aggregation and toxicity in PC12 cells. (A,E) SDS-PAGE and immunoblot analysis of PC12 cells overproducing HTTQ103-EGFP fusion protein. Cells treated with si-CRMP1 RNA (A) or overproducing a HA-tagged CRMP1 protein (E) were analyzed. (B,F) Detection of insoluble HTTQ103-EGFP aggregates in si-CRMP1-treated (B) and HA-CRMP1 overproducing (F) PC12 cells. Insoluble aggregates (black dots) were detected by filter retardation assay. (C,G) Quantification of insoluble HTTQ103-EGFP aggregates shown in B and F by image analysis: n = 3; (***) P ≤ 0.001, two-sided t-test. (D,H) Quantification of caspase-3/7 activity in si-CRMP1-treated (D) and HA-CRMP1 overproducing (H) PC12-HTTQ103-EGFP cells: n = 3; (***) P ≤ 0.001, two-sided t-test. Data in C, D, G, and H are represented as mean ± SEM.
Figure 4.
Figure 4.
CRMP1 overproduction mitigates mutant HTT-induced photoreceptor degeneration and motor impairment in HD transgenic flies. (A–F) Investigation of eye sections. Analysis of eye sections in control flies (A) and transgenic flies overproducing CRMP1 (B) or HTT336Q128 (C) alone. Coproduction of the control protein GFP does not influence HTT336Q128-induced retina degeneration (D), whereas coproduction of human CRMP1 (E) or Drosophila CRMP (F) mitigates HTT336Q128-induced retinal degeneration. Arrows indicate retinal degeneration. (G) Quantification of retinal degeneration in control and HD transgenic flies (A–F): n ≥ 9 flies per genotype; (***) P ≤ 0.001, two-sided t-test with unequal variance. (H) CRMP1 significantly improves climbing abilities of HTT336Q128 overproducing HD transgenic flies. Data were analyzed using Graphpad Prism 5: n ≥ 16 flies per genotype; P = 0.0017; linear regression followed by F-test. (I–J) Immunofluorescence analysis of eye imaginal discs of HD transgenic flies. (I) MYC-CRMP1 colocalizes with HTT336Q128 in multiple inclusion bodies with a diameter of 0.2–0.4 μm. (J) In contrast to the control protein GFP, coproduction of the proteins CRMP1 and CRMP reduced both the number as well as the average size of HTT336Q128 inclusion bodies. (K) Quantification of the number of HTT336Q128 inclusion bodies in HD flies coproducing modulator proteins. An area of 35.71 μm2 in the middle of the posterior third of eye imaginal discs was systematically analyzed: n ≥ 5; (***) P ≤ 0.001, two-sided t-test with unequal variance. Data in G, H, and K are represented as mean ± SEM.
Figure 5.
Figure 5.
A mutant variant of CRMP1 (D408V) shows impaired activity in cell-based assays and HD transgenic flies. (A) Multiple sequence alignment of CRMP1 protein sequences. The aspartic acid residue 408 in human CRMP1 is conserved in a large number of related proteins (yellow box). (B) Effects of mCherry-tagged CRMP1 and CRMP1-D408V fusion proteins on aggregation of YFP-tagged HTTex1Q79 in cell-based assays. Formation of insoluble YFP-HTTex1Q79 aggregates in SH-EP cells was quantified by high-content fluorescence imaging. Aggregates were detected by YFP fluorescence of HTT aggregates or by immunostaining using the anti-HTT antibody MW8. MW8 preferentially detects insoluble HTT aggregates. Data are represented as mean ± SEM. (***) P ≤ 0.001, two-sided t-test with unequal variance. (C) Retinal degeneration in control flies carrying the GMR-GAL4 driver (GMR) alone and in flies coexpressing HTT336Q128 with CRMP1-D408V or the GFP control protein under control of the GMR driver. Analysis of eye sections reveals that in contrast to wild-type CRMP1, neither GFP nor CRMP1-D408V affects HTT336Q128-induced retinal degeneration compared to the GMR flies. (D–E) Effects of mCherry-tagged CRMP1 or CRMP1-D408V on aggregation of YFP-tagged ATXN1Q82 (D) or TARDBP (E) in cell-based assays. (D) YFP-ATXN1Q82 or (E) YFP-TARDBP aggregates were detected in SH-EP cells by YFP fluorescence and quantified by high-content fluorescence imaging. Data are represented as mean ± SEM. (***) P ≤ 0.001, two-sided t-test with unequal variance.
Figure 6.
Figure 6.
A mutant variant of CRMP1 (D408V) shows impaired activity in cell-free HTT aggregation assays. (A) The proteins His-CRMP1 and His-CRMP1-D408V were produced in E. coli and purified to >90% homogeneity by affinity chromatography. Protein expression and purity was confirmed by Coomassie staining of SDS gels or immunoblotting (IB). (B) Analysis of spontaneous HTTQ49 aggregation in the presence of modulator proteins in cell-free assays by filter retardation assay. SDS-insoluble HTTQ49 protein aggregates were detected on filter membranes using the anti-HTT antibody CAG53b. Representative results from three independent experiments are shown. (C) Analysis of spontaneous HTTQ49 aggregation in the presence of modulator proteins by dot-blot assays. The conversion of soluble HTTQ49 into insoluble protein aggregates was monitored using the antibody 3B5H10. Representative results from three independent experiments are shown.
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References

    1. Abraham VC, Taylor DL, Haskins JR. 2004. High content screening applied to large-scale cell biology. Trends Biotechnol 22: 15–22. - PubMed
    1. Apostol BL, Illes K, Pallos J, Bodai L, Wu J, Strand A, Schweitzer ES, Olson JM, Kazantsev A, Marsh JL, et al.2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum Mol Genet 15: 273–285. - PubMed
    1. Aylward EH. 2007. Change in MRI striatal volumes as a biomarker in preclinical Huntington's disease. Brain Res Bull 72: 152–158. - PubMed
    1. Behrends C, Langer CA, Boteva R, Böttcher UM, Stemp MJ, Schaffar G, Rao BV, Giese A, Kretzschmar H, Siegers K, et al.2006. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers. Mol Cell 23: 887–897. - PubMed
    1. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Methodol 57: 289–300.

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