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The expression of PLP/DM-20 mRNA is restricted to the oligodendrocyte-lineage cells in the adult rat spinal cord

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Abstract

Proteolipid protein (PLP) is the major component of myelin; its gene encodes two major splicing variants: PLP and DM-20. Compared with PLP, DM-20 lacks the amino acids encoded by exon IIIb. The expression of PLP/DM-20 in cells outside the oligodendrocyte-lineage is unclear. To address this issue, we analyzed the detailed expression pattern of PLP/DM-20 mRNA in the adult rat spinal cord by in situ hybridization (ISH) with a cRNA probe complementary to DM-20 mRNA, which has been used to detect both PLP and DM-20 both mRNA. ISH did not label the cells expressing NeuN nor glial fibrillary acidic protein but detected those expressing Olig2, indicating that PLP/DM-20 mRNA are expressed only in oligodendrocyte-lineage cells. This cell population was expected to contain NG2-expressing oligodendrocyte precursor cells (OPCs), because some exhibited the expression of glutathione S-transferase pi isoform in the nucleus. A recent publication showed that OPCs express PLP but not DM-20 mRNA. However, no OPCs were detected. We performed ISH with a cRNA probe that specifically recognizes PLP mRNA to successfully detect some OPCs. Additionally, OPCs were detected by ISH with a cRNA probe complementary to DM-20 mRNA that was digested via alkaline hydrolysis prior to ISH. These findings collectively demonstrate that PLP and DM-20 mRNA expression is restricted to oligodendrocyte-lineage cells, and imply that the undigested cRNA probe complementary to the full-length DM-20 mRNA sequence only recognizes DM-20 mRNA and not the PLP counterpart when applied to ISH without denaturation/digestion methods.

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Abbreviations

AH:

Anterior horn

ALP:

Alkaline phosphatase

BSA:

Bovine serum albumin

CF:

Cuneate funiculus

CNS:

Central nervous system

cRNA:

Complementary RNA

CST:

Corticospinal tract

DAPI:

4′,6-Diamidino-2-phenylindole

DEPC:

Diethylpyrocarbonate

DIG:

Digoxigenin

GFAP:

Glial fibrillary acidic protein

GM:

Gray matter

GST-pi:

Glutathione S-transferase pi isoform

OPC:

Oligodendrocyte precursor cell

PB:

Phosphate buffer

PBS:

Phosphate buffered saline

PCR:

Polymerase chain reaction

PK:

Proteinase K

PLP:

Proteolipid protein

SD:

Standard deviation

WM:

White matter

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Acknowledgments

The authors thank Dr. Bernard Zalc, Research Center of the Brain and Spinal Cord Institute, INSERM U975 (Paris, France) for generously providing the pBS-DM-20 plasmid and Dr. John Alberta, Department of Cancer Biology, Dana-Farber Cancer Institute (Boston, MA) for kindly providing the Olig2 antibody. The authors are also grateful to Ms. Nice Mamiya, Ms. Yukari Hirohara, and Dr. Yukie Kyakumoto for their excellent technical assistance. This study was supported by a Grant-in-Aid for Young Scientists (B) (22790181) and a Grant-in-Aid for Scientific Research on Innovative Areas (23124501, 25124701) from the Japan Society for the Promotion of Science (JSPS).

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  1. Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan

    Kazuya Takeda, Mari Dezawa & Masaaki Kitada

  2. Faculty of Medicine, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan

    Kazuya Takeda

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  1. Kazuya Takeda
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Takeda, K., Dezawa, M. & Kitada, M. The expression of PLP/DM-20 mRNA is restricted to the oligodendrocyte-lineage cells in the adult rat spinal cord. Histochem Cell Biol 145, 147–161 (2016). https://doi.org/10.1007/s00418-015-1384-5

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