5-Lipoxygenase in mouse cerebellar Purkinje cells

Abstract

It has been suggested that the enzymatic pathway of 5-lipoxygenase (5-LOX) influences brain functioning and pathobiology. The mRNAs for both the enzyme 5-LOX and its activating protein FLAP have been found in the cerebellum. In this work, we investigated the cellular expression of 5-LOX in the adult mouse cerebellar cortex. We used the in situ mRNA hybridization assay, immunocytochemistry, laser capture microdissection, and our previously developed method for assaying the DNA methylation status of a putative mouse 5-LOX promoter. Since both 5-LOX mRNA in situ hybridization signal and FLAP immunoreactivity co-localize with calbindin 28 kD immunoreactivity (a Purkinje cell marker) but not with S-100β immunoreactivity (a Bergmann glia marker), the suggestion is that the 5-LOX pathway is expressed in cerebellar Purkinje cells. We found that methylation in the sites targeted by methylation-sensitive restriction endonucleases AciI and HinP1I but not BstUI and HpaII was greater in DNA samples obtained from a high-5-LOX-expressing cerebellar region (Purkinje cells) versus a low-5-LOX-expressing region (the molecular cell layer), suggesting a possible epigenetic contribution to the cell-specific 5-LOX expression in the cerebellum. We propose that Purkinje cell-localized 5-LOX and FLAP expression may be involved in the cerebellar synthesis of leukotrienes and/or could influence the Dicer-mediated microRNA formation and processes of neuroplasticity.

Fig. 1

Double-labeling of 5-LOX mRNA and CaBP-28 (Purkinje cell marker) in mouse cerebellum. Cellular localization of 5-LOX mRNA was characterized by in situ hybridization using antisense (A, D) and sense (B, negative control) RNA probes. Note a strong green 5-LOX mRNA signal in the cell bodies in A and its absence in B. The red immunofluorescence of the Purkinje cell marker CaBP-28 is shown in C. Merging the 5-LOX/CaBP-28 immunofluorescences demonstrated the colocalization of the 5-LOX mRNA-specific signal and the Purkinje cell-specific signal (yellow, D). Images were obtained with a ×20 objective lens.

Fig. 2

Double-labeling of 5-LOX mRNA and S-100β (marker of Bergmann glia) in mouse cerebellum. Panels A and D show the 5-LOX mRNA signal obtained with an antisense probe; panel B shows a negative control obtained with a sense probe. Panel C shows the red immunofluorescence of the Bergmann glia marker S-100β. Merging the 5-LOX/S-100β immunofluorescences demonstrated that the two signals do not overlap (D). Images were obtained with a ×20 objective lens.

Fig. 3

5-LOX mRNA content of the Purkinje cell layer (PC) and the molecular layer (ML). The content of 5-LOX mRNA in the LCM cerebellar samples was measured by quantitative PCR (see text). Data are expressed as mean ± SEM (n = 5); *p<0.05 vs. PC.

Fig. 4

5-LOX protein content in the Purkinje cell layer (PC) and the molecular layer (ML). The 5-LOX protein content in the LCM cerebellar samples was measured by quantitative Western blot using β-actin content as a control (an example is shown in panel A). The intensity of the 5-LOX signals was normalized by corresponding β-actin signals (B). Data are expressed as mean ± SEM (n = 5), *p<0.05 vs. PC.

Fig. 5

5-LOX DNA methylation levels in the Purkinje cell layer and the molecular layer. The assay of DNA methylation was performed in the LCM cerebellar samples by methylation-sensitive endonuclease assay using four endonucleases: AciI, BstUI, HinP1I, and HpaII. The results are reported in units (CV multiplied by a factor of 1000, see text for details) and are expressed as mean ± SEM (n = 5), *p<0.001 vs. corresponding Purkinje cells.

Fig. 6

FLAP immunoreactivity in the cerebellum; colocalization with the immunoreactivities of CaBP-28 and S-100β. Panels A-C show double-labeling of FLAP (A, green) and the Purkinje cell marker CaBP-28 (B, red); merging of both immunofluorescences demonstrated the FLAP/CaBP-28 colocalization (C, yellow). Panels D-F show double-labeling of FLAP (D, green) and the Bergmann glia marker S-100β (E, red); merging of both immunofluorescences demonstrated that the two signals do not overlap (F). Images were obtained with a ×20 objective lens.