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The Alopecia Areata Phenotype Is Induced by the Water Avoidance Stress Test in Cchcr1-Deficient Mice

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The Alopecia Areata Phenotype Is Induced by the Water Avoidance Stress Test in Cchcr1-Deficient Mice bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. The alopecia areata phenotype is induced by the water avoidance stress test in cchcr1- deficient mice. Qiao-Feng Zhao1, Nagisa Yoshihara1, Atsushi Takagi1, Etsuko Komiyama1, Akira Oka3, Shigaku Ikeda*1, 2 1Department of Dermatology and Allergology, and 2Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan 3The Institute of Medical Sciences, Tokai University, Kanagawa, Japan *Corresponding author: Shigaku Ikeda, MD, PhD, Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo- ku, Tokyo 113-8421, Japan Tel: 81-3-5802-1089, Fax: 81-3-3813-2205, E-mail: [email protected] Running title: AA phenotype induced in cchcr1-deficient mice COI: The authors have no conflicts of interest to declare. p. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. ABSTRACT Background: We recently discovered a nonsynonymous variant in the coiled-coil alpha- helical rod protein 1 (CCHCR1) gene within the alopecia areata (AA) risk haplotype; mice engineered to carry the risk allele displayed a hair loss phenotype. Objective: To further investigate the involvement of the CCHCR1 gene in AA pathogenesis. Methods: We developed an AA model using C57BL/6N cchcr1 gene knockout mice. Mice (6-8 weeks) were divided into two groups: cchcr1 -/- mice and wild-type (WT) mice. Both groups were subjected to a water avoidance stress (WAS) test. Results: Eight weeks after the WAS test, 25% of cchcr1 mice exhibited noninflammatory foci of alopecia on the dorsal skin. The foci resembled human AA in terms of gross morphology, trichoscopic findings and histological findings. Conclusions: Our results strongly suggest that CCHCR1 is associated with AA pathogenesis and that cchcr1 -/- mice are a good model for investigating AA. Author summary Alopecia areata is thought to affect 1-2% of the population. In severe alopecia areata, changes in appearance significantly reduce the patient's quality of life, but there is no p. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. established treatment. Its pathogenic mechanism is thought to cause an autoimmune disease of the hair bulb of growing hair. Many genes and factors associated with AA onset form a background that easily causes such an immune reaction, and the causative genes related to alopecia areata are being elucidated worldwide. We were able to identify the CCHCR1 gene as one of the causative genes by genome analysis. In this study, we created CCHCR1-deficient mice using Cre/loxP technology and confirmed that 25% of CCHCR1-deficient mice that underwent the WAS test for psychological stimulation developed hair loss similar to that observed in human alopecia areata. This suggests that the CCHCR1 gene is a disease susceptibility gene for alopecia areata. Introduction Alopecia areata (AA) is a complex genetic and tissue-specific autoimmune disease characterized by nonscarring hair loss that may begin as patches that can coalesce and progress to cover the entire scalp and/or whole body [1]. Most authors tend to accept the hypothesis that AA is caused by a T-cell mediated autoimmune response targeting an unknown antigen in anagen-stage hair follicles [2,3]. Previous genome-wide association studies (GWAS) have implicated a number of p. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. immune and nonimmune loci in the etiology of AA, although none have yet been demonstrated to be causative for the disease, and none have been functionally confirmed to be involved in AA pathogenesis [4,5]. Alleles of the human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) on chromosome 6p21.3 have so far shown the strongest associations with AA across different ethnic groups [4,5]. The largest reported genome-wide meta-analysis of AA identified HLA-DRβ1 as a key etiologic driver [4]. However, no convincing susceptibility gene has yet been pinpointed in the MHC. We recently discovered a nonsynonymous variant (rs142986308, p. Arg587Trp) in the MHC associated with AA susceptibility in CCHCR1 (coiled-coil alpha-helical rod protein 1), which encodes a novel component of hair shafts. In addition, our results demonstrate that mice carrying this amino acid substitution display a patchy hair loss phenotype. We further identified keratin abnormalities in the hair shaft and comparative differential expression of hair-related keratin genes not only in alopecic mice but also in hair follicles from AA patients with the risk variant. Thus, our study identified a novel AA susceptibility variant that was validated by functional analysis [6]. p. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. The purpose of this study was to investigate the effect of CCHCR1 gene deficiency on AA pathogenesis. First, we generated C57BL/6N mice with deletion of the cchcr1 gene using Cre/loxP technology. Because cchcr1−/− mice develop normally and exhibit no obvious behavioral or physical phenotypic defects and because psychological stress is known to be a triggering factor in AA patients [7], the water avoidance stress (WAS) test, which is well known to stimulate psychological stress in mice [8,9], was performed on cchcr1 −/− and WT mice. Results First, we generated mice with deletion of cchcr1 using Cre/loxP technology. The final recombined floxed allele is presented in Fig. 1a. Cchcr1−/− mice were obtained by intercrossing heterozygous (cchcr1 +/−) mice. After identifying Cre-mediated deletion and the inactivation of cchcr1, the expression of cchcr1 was assessed by PCR (Fig. 1b). We also analyzed the protein and RNA levels of cchcr1 in the skin (Fig. 1c, 1d). The cchcr1 −/− mice were born at the expected ratios and had normal weights. The mice developed normally, were fertile, and exhibited no obvious behavioral or physical phenotypic defects. Next, a WAS test was performed on cchcr1-/- mice and WT mice (n=9) (both 6-8 weeks p. 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. after birth). The WAS test was performed for two hours a day, five times per week, for two weeks. Eight weeks after the WAS test, in contrast to WT mice (Fig. 2c), approximately 25% of cchcr1 -/- mice spontaneously exhibited noninflammatory foci of alopecia in the dorsal area (Fig. 2a, Table 1). Hair loss may also develop within a sharply demarcated, localized area or diffusely on the dorsal side. The skin of the hair loss area has a normal appearance. The hair-pull test showed an increase in dystrophic anagen hairs. Characteristic phenotypes of AA (short stubs of hair and exclamation mark hairs) were observed in the hair loss area by dermoscopy. We also identified white dots and tapering hairs as signs of AA (Fig. 2a). Eighteen weeks after the WAS test, the localized hair loss spontaneously recovered (Fig. 2b). Regrowth began one to three months after hair loss and was followed by the recurrence of hair loss in the same or other areas. In addition, AA showed a variable prognosis in each mouse and could recur. In aging AA mice, intralesional hair regrowth was a highly characteristic feature of AA; the newly sprouted hairs were white and became thinner as they gradually approached the dorsum, and the majority of the hair follicles were catagen and telogen follicles. p. 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423031; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. The AA-like skin lesions in cchcr1 -/- mice induced by WAS were tested by HE staining, immunohistochemistry and scanning and transmission electron microscopy (SEM and TEM). The histopathologic appearance of AA mice included a variety of features. The follicles showed little inflammatory lymphocytic infiltration in the peribulbar region (Fig. 3a). We also observed the infiltration of plasma cells and an increase in the number of hair follicles as well as miniaturized anagen follicles.
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