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Histological detection of dynamic glial responses in the dysmyelinating Tabby-jimpy mutant brain

Abstract

Oligodendrocytes (OLs) are glial cells that form myelin sheaths surrounding the axons in the central nervous system (CNS). Jimpy (jp) mutant mice are dysmyelinating disease models that show developmental abnormalities in myelinated OLs in the CNS. The causative gene in jp mice is the proteolipid protein (PLP) located on the X chromosome. Mutations in the jp allele result in exon 5 skipping and expression of abnormal PLP containing a C-terminal frame shift. Many lines of evidence suggest that abnormal PLP in OLs results in endoplasmic reticulum (ER) stress and cell death. To histologically detect glial responses in the jp mutant brain, we performed staining with lineage-specific markers. Using OL markers and OL progenitor cell marker staining, we identified reduced numbers of OL lineage cells in the jp mutant brain. Nuclear staining of the transcription factor Olig1 was observed in the Tabby-jp brain, whereas cytoplasmic Olig1 staining was observed in the wild-type brain at postnatal day 21, suggesting that active myelination was present in the mutant brain. Many microglial cells with activated morphology and intensive staining of CD11b microglia marker were observed in the internal capsule of the mutant brain, a region of white matter containing residual OLs. Activated astrocytes with high glial fibrillary acidic protein-immunoreactivity were also mainly observed in white matter. Finally, we performed in situ hybridization using C/EBP homologous protein (CHOP) antisense probes to detect ER stressed cells. CHOP mRNA was strongly expressed in residual OLs in the Tabby-jp mutant mice at postnatal stages. These data show that microglia and astrocytes exhibit dynamic glial activation in response to cell death of OLs during Tabby-jp pathogenesis, and that CHOP antisense probes may be a good marker for the detection of ER-stressed OLs in jp mutant mice.

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Acknowledgements

This study was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; Grant-in-Aid for Scientific Research (B) (15H04667, H.T.), Grant-in-Aid for Scientific Research on Innovative Areas, “Glial assembly” (25117007, H.T.) and Grant-in-Aid for Exploratory Research (16K15168, H.T.); and a Grant from the Niigata University Kyowakai Society (M.I.). We thank Dr. Tetsushi Kagawa for PLP plasmid; Dr. Yukiko Mori and Ms. Satoko Yamagiwa for technical assistance; Dr. Yoshihide Yamaguchi, Dr. Mari Tada, Dr. Akiyoshi Kakita, Dr. Kazunori Imaizumi, Dr. Takashi Kudo for discussion; all Takebayashi laboratory members, especially Dr. Norihisa Bizen and Dr. Masato Yano, for providing input and advice, and Dr. Sugata Takahashi for continual support.

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Correspondence to Hirohide Takebayashi.

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Movie S1 Ta-jp mutant mouse (2 weeks old). One Ta-jp mutant (Ta-jp/Y) and two control littermates (+/Y) at P14. The Ta-jp mutant has smaller body than wild-type mice and shows a shivering phenotype and ataxic gait (MP4 15735 kb)

Movie S2 Ta-jp mutant mouse (3 weeks old). One Ta-jp mutant (Ta-jp/Y) and one control littermate (+/Y) at P21. The Ta-jp mutant has smaller body than wild-type mice and shows shivering phenotype with extended and spastic hindlimbs (MP4 6848 kb)

Fig. S1a,b Ta-jp mutant mice. a Ta-jp mutant mouse (Ta-jp/Y, right) and control wild-type mouse (X/Y, left) at P14. b Female heterozygous mouse (Ta-jp/X, right) and wild-type mouse (X/X, left) at P21. Female heterozygous mouse showed hair color phenotype, stripes in the back (PPTX 247 kb)

Fig. S2a,b Increased expression of microglia marker CD11b in the white matter. a, b CD11b immunostaining in the coronal sections of wild-type mouse (a) and Ta-jp mutant mouse (b) at P21. Corpus callosum (a′, b′) and internal capsule (a″,b″) were shown in higher magnifications. Lots of CD11b-positive microglia were observed in the internal capsule of Ta-jp mutant (b″). Counterstaining was performed with Mayer’s Hematoxylin. Bars: a, b 1 mm; a′, b′, a′′, b′′ 50 μm (PPTX 1465 kb)

Fig. S3a–f Characterization of neurons and astrocytes in the brain of Ta-jp mutant. a, b NF-H immunostaining in the coronal sections of wild-type brain (a) and Ta-jp mutant brain (b) at P21. Cerebral cortex (a′, b′) and internal capsule (a″, b″) are shown at higher magnification. Strong NF-H signals were observed in the axons of cortex and internal capsule of Ta-jp mutant. c, d NF-M immunostaining in the coronal sections of wild-type brain (c) and Ta-jp mutant brain (d) at P21. Cerebral cortex (c′, d′) and internal capsule (c″, d″) are shown at higher magnification. Strong NF-M signals were observed in the axons of internal capsule of Ta-jp mutant. (e, f) GFAP immunostaining in the coronal sections of wild-type brain (e) and Ta-jp mutant brain (f) at P21. Activated astrocytes with strong GFAP imuunoreactivity were observed in the white matter of Ta-jp mutant brain (f). Pictures of corpus callosum (e′, f′) and internal capsule (e″, f″) are shown at higher magnification. Insets High magnification picture of dot-lined rectangle area. Counterstaining was performed with Mayer’s hematoxylin. Bars af 1 mm; a′f′a″f″ 50 μm; insets of a′d′, e″, f″ 20 µm; insets of a″d″ 10 µm (PPTX 5167 kb)

Fig. S4a–d Double staining of CHOP ISH and IHC of glial markers. a Double staining experiment using CHOP probe (purple) and Olig2 antibody (brown) in the coronal sections of Ta-jp mutant brain at P21. b Double staining experiment using CHOP probe (purple) and CC-1 antibody (brown) in the coronal sections of Ta-jp mutant brain at P21. c Double staining experiment using CHOP probe (purple) and GFAP antibody (brown) in the coronal sections of Ta-jp mutant brain at P21. d Double staining experiment using CHOP probe (purple) and Iba1 antibody (brown) in the coronal sections of Ta-jp mutant brain at P21. Note that CHOP signal co-localizes with OL makers, Olig2 (a″) and CC-1 (b″) in internal capsule. Bars ad 500 μm, a′d′ 100 μm, a″d″ 40 μm, insets 20 μm (PPTX 3776 kb)

Fig. S5a–c Apoptotic marker staining. a, b Immunostaining using anti-cleaved caspase-3 antibody in the coronal sections of wild-type brain (a) and Ta-jp mutant brain (b) at P21. Positive cells are indicated by arrowheads. The insets show cleaved caspase-3 positive cells noted by small rectangles. c Quantification of anti-cleaved caspase-3 positive cells in the cerebral cortex, corpus callosum and internal capsule at P21 (n = 3). More number of cleaved caspase-3 positive cells were observed in the white matters of Ta-jp mutant brain (arrowheads). Bars a, b 1 mm; a′, b′, a″, b″ 50 μm; insets 10 μm (PPTX 1541 kb)

Movie S1 Ta-jp mutant mouse (2 weeks old). One Ta-jp mutant (Ta-jp/Y) and two control littermates (+/Y) at P14. The Ta-jp mutant has smaller body than wild-type mice and shows a shivering phenotype and ataxic gait (MP4 15735 kb)

Movie S2 Ta-jp mutant mouse (3 weeks old). One Ta-jp mutant (Ta-jp/Y) and one control littermate (+/Y) at P21. The Ta-jp mutant has smaller body than wild-type mice and shows shivering phenotype with extended and spastic hindlimbs (MP4 6848 kb)

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Ikeda, M., Hossain, M.I., Zhou, L. et al. Histological detection of dynamic glial responses in the dysmyelinating Tabby-jimpy mutant brain. Anat Sci Int 93, 119–127 (2018). https://doi.org/10.1007/s12565-016-0383-5

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Keywords

  • Oligodendrocyte
  • Dysmyelination
  • Proteolipid protein
  • ER stress
  • CHOP