Alpha-synuclein expression in oxytocin neurons of young and old bovine brains

Abstract

Understanding of central nervous system mechanisms underlying age-related infertility remains limited. Fibril α-synuclein, distinct from its monomeric form, is implicated in age-related diseases. Notably, fibril α-synuclein spreads among neurons, similar to prions, from damaged old neurons in cortex and hippocampus to healthy neurons. However, less is known whether α-synuclein propagates into oxytocin neurons, which play crucial roles in reproduction. We compared α-synuclein expression in the oxytocin neurons in suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), paraventricular hypothalamic nucleus (PVN), and posterior pituitary (PP) gland of healthy heifers and aged cows to determine its role in age-related infertility. We analyzed mRNA and protein expression, along with Congo red histochemistry and fluorescent immunohistochemistry for oxytocin and α-synuclein, followed by confocal microscopy with Congo red staining. Both mRNA and protein expressions of α-synuclein were confirmed in the bovine cortex, hippocampus, SCN, SON, PVN, and PP tissues. Significant differences in α-synuclein mRNA expressions were observed in the cortex and hippocampus between young heifers and old cows. Western blots showed five bands of α-synuclein, probably reflecting monomers, dimers, and oligomers, in the cortex, hippocampus, SCN, SON, PVN, and PP tissues, and there were significant differences in some bands between the young heifers and old cows. Bright-field and polarized light microscopy did not detect obvious amyloid deposition in the aged hypothalami; however, higher-sensitive confocal microscopy unveiled strong positive signals for Congo red and α-synuclein in oxytocin neurons in the aged hypothalami. α-synuclein was expressed in oxytocin neurons, and some differences were observed between young and old hypothalami.

Keywords: Aging; Paraventricular hypothalamic nucleus; Posterior pituitary gland; Suprachiasmatic nucleus; Supraoptic nucleus.

Fig. 1.

Western blot analyses of α-synuclein in brain sections: positive control (+, whole mouse brain), negative control (–, buffer only), cortex (Cort), hippocampus (Hipp), suprachiasmatic nucleus and supraoptic nucleus (S & S), paraventricular hypothalamic nucleus and supraoptic nucleus (P & S), and posterior pituitary (PP) tissues from post-pubertal heifers (Y; n = 6) and older cows (O; n = 6), detected with anti-α-synuclein antibody (A). Representative photos of membrane stained by Revert 700 total protein stain (B). Relative α-synuclein protein levels normalized to the amount of total protein based on all densities in each lane (C, D, E, F, and G). Headings indicate the results of the two-way ANOVA. P-values indicate significant differences by t-test between the young and old specimens. ANOVA, analysis of variance; N.S., non-significant.

Fig. 2.

Relative SNCA mRNA levels, presented as mean ± SEM, in various brain regions: cortex (Cort) (A), hippocampus (Hipp) (B), suprachiasmatic nucleus and supraoptic nucleus (S & S) tissue (C), paraventricular hypothalamic nucleus and supraoptic nucleus (P & S) tissue (D), and posterior pituitary (PP) tissue (E), comparing healthy, post-pubertal, growing, nulliparous heifers (young group; n = 6) to old, multiparous cows (old group; n = 6), as measured by RT-qPCR. Data were normalized to the geometric means of YWHAZ and SDHA levels. The P-values in the upper of each graph represent the results of non-paired t-test. RT-qPCR, quantitative reverse transcription-polymerase chain reaction; SEM, standard error of mean; N.S., non-significant.

Fig. 3.

Fluorescence photomicrographs of oxytocin, α-synuclein, Congo red, and DAPI in the SCN of young and old bovines. Images were captured with laser confocal microscopy for oxytocin (light blue), α-synuclein (green), Congo red (red), and DAPI (blue). In the merged photos, the yellow arrows indicate the cell body. Scale bars, 20 μm. SCN, suprachiasmatic nucleus.

Fig. 4.

Fluorescence photomicrographs of oxytocin, α-synuclein, Congo red, and DAPI in the SON of young and old bovines. Images were captured with laser confocal microscopy for oxytocin (light blue), α-synuclein (green), Congo red (red), and DAPI (blue). In the merged photos, the arrows indicate the cell body. Scale bars, 20 μm. SON, supraoptic nucleus.

Fig. 5.

Fluorescence photomicrographs of oxytocin, α-synuclein, Congo red, and DAPI in the PVN of young and old bovines. Images were captured with laser confocal microscopy for oxytocin (light blue), α-synuclein (green), Congo red (red), and DAPI (blue). In the merged photos, the arrows indicate the cell body. Scale bars, 20 μm. PVN, paraventricular hypothalamic nucleus.

Fig. 6.

Fluorescence photomicrographs of oxytocin, α-synuclein, and Congo red in the PP glands of young and old bovines. Images were captured with laser confocal microscopy for oxytocin (light blue), α-synuclein (green), Congo red (red), and DIC (gray). In the merged photos, the yellow stars indicate the blood vessels. Scale bars, 20 μm. PP, posterior pituitary; DIC, differential interference contrast.