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High expression of cytochrome b 5 reductase isoform 3/cytochrome b 5 system in the cerebellum and pyramidal neurons of adult rat brain

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Abstract

Cytochrome b 5 reductase (Cb 5R) and cytochrome b 5 (Cb 5) form an enzymatic redox system that plays many roles in mammalian cells. In the last 15 years, it has been proposed that this system is involved in the recycling of ascorbate, a vital antioxidant molecule in the brain and that its deregulation can lead to the production of reactive oxygen species that play a major role in oxidative-induced neuronal death. In this work, we have performed a regional and cellular distribution study of the expression of this redox system in adult rat brain by anti-Cb 5R isoform 3 and anti-Cb 5 antibodies. We found high expression levels in cerebellar cortex, labeling heavily granule neurons and Purkinje cells, and in structures such as the fastigial, interposed and dentate cerebellar nuclei. A large part of Cb 5R isoform 3 in the cerebellum cortex was regionalized in close proximity to the lipid raft-like nanodomains, labeled with cholera toxin B, as we have shown by fluorescence resonance energy transfer imaging. In addition, vestibular, reticular and motor nuclei located at the brain stem level and pyramidal neurons of somatomotor areas of the brain cortex and of the hippocampus have been also found to display high expression levels of these proteins. All these results point out the enrichment of Cb 5R isoform 3/Cb 5 system in neuronal cells and structures of the cerebellum and brain stem whose functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia, as well as in brain areas highly prone to undergo oxidative stress-induced neurodegeneration.

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Acknowledgments

AKSA was recipient of a post-doctoral fellowship of the Junta de Extremadura and DMS was recipient of a pre-doctoral fellowship of the Spanish Ministerios de Ciencia e Innovación y Economía y Competitividad. The technical help of Dr. José Antonio Tapia with confocal microscopy images acquisition is gratefully acknowledged. This work has been financed with research project BFU2011-30178 of the Spanish Ministerio de Economía y Competitividad and grants to research groups CCV008/BBB008 and CTS005 of the Plan Regional de I + D+I of the Junta/Gobierno de Extremadura (Spain). These fellowships and grants have been co-financed with the European Fund for Economic and Regional Development (FEDER).

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    Authors and Affiliations

    1. Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06006, Badajoz, Spain

      A. K. Samhan-Arias, D. Marques-da-Silva, R. Lagoa & C. Gutierrez-Merino

    2. Department of Human Anatomy and Embryology, Faculty of Medicine, University of Extremadura, 06006, Badajoz, Spain

      C. López-Sánchez, R. Lagoa & V. García-Martínez

    3. ESTG-Polytechnic Institute of Leiria, Leiria, Portugal

      R. Lagoa

    4. Minimally Invasive Surgery Centre, Cáceres, Spain

      V. Garcia-Lopez

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    1. A. K. Samhan-Arias
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    Correspondence to C. Gutierrez-Merino.

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    A. K. Samhan-Arias and C. López-Sánchez equally contributed to this work and should be considered first authors.

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    429_2015_1036_MOESM1_ESM.ppt

    Figure S1. Western blotting of adult rat brain lysates (2.5 μg of protein per lane) with rabbit anti-C b 5 R isoform 3 (anti-DIA1; Protein Tech Group: 10894-1-AP, dilution 1:100) and with anti-C b 5 (Santa Cruz Biotechnology: sc 33174, dilution 1:100). Details of the experimental protocol are given in the section II.A of Materials and Methods (PPT 151 kb)

    429_2015_1036_MOESM2_ESM.ppt

    Figure S2 (I) Control for Figure 1: The primary antibodies were replaced with rabbit IgG and slices were later treated with the secondary antibody used for the immunohistochemistry images (biotinylated goat anti-rabbit IgG). Raw light micrographs images of coronal sections at cerebellar and brain stem level showed lack of significant staining with biotinylated goat anti-rabbit IgG (A-C) when compared with the staining observed after immunohistochemistry with anti-Cb 5R isoform 3 (A-C images shown in the Figure 1) and anti-Cb 5 (D-F images shown in the Figure 1). Scale bars: I-A (1 mm), I-B (200 μm) and I-C (100 μm). Similar images were obtained using the secondary antibody alone. (II) Control for Figure 3: The primary antibodies were replaced with rabbit IgG and slices were later treated with the secondary antibody used for the immunohistochemistry images (biotinylated goat anti-rabbit IgG). Raw light micrographs images of coronal sections at cerebellar and brain stem level showed lack of significant staining with biotinylated goat anti-rabbit IgG (A,B) when compared with the staining observed after immunohistochemistry with anti-Cb 5R isoform 3 and anti-Cb 5 (images shown in the Figure 3). Scale bars: II-A (1 mm) and II-B (100 μm). Similar images were obtained using the secondary antibody alone. (III) Control for Figures 10 and 11. “Biotinylated goat anti-rabbit IgG” are raw images of controls done with the secondary antibody used for the immunohistochemistry images (biotinylated goat anti-rabbit IgG) alone, and these images showed lack of significant staining with biotinylated goat anti-rabbit IgG when compared with the staining observed after immunohistochemistry with anti-Cb 5R isoform 3 and anti-Cb 5 (images shown in Figures 10 and 11). (A) somatomotor areas of cerebral cortex (Mo) and hippocampal formation (Hip) shown with higher magnification in (B) and (C). Image B shows the somatomotor areas of cerebral cortex with higher magnification. Image C shows the Ammon´s horn of hippocampal formation with higher magnification. Inserted abbreviations in image (C): pyramidal layer (p) of Ammon´s horn of hippocampal formation (CA1, CA2 and CA3: Pyramidal fields). Scale bars: IIIA (0.75 mm), IIIB (100 μm) and IIIC (100 μm) (PPT 651 kb)

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    Samhan-Arias, A.K., López-Sánchez, C., Marques-da-Silva, D. et al. High expression of cytochrome b 5 reductase isoform 3/cytochrome b 5 system in the cerebellum and pyramidal neurons of adult rat brain. Brain Struct Funct 221, 2147–2162 (2016). https://doi.org/10.1007/s00429-015-1036-5

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