Maternal Choline Availability Alters the Localization of p15Ink4B and p27Kip1 Cyclin-Dependent Kinase Inhibitors in the Developing Fetal Rat Brain Hippocampus

Abstract

Previously we have shown that changes in maternal dietary choline are associated with permanent behavioral changes in offspring. Importantly, in adult male rats, feeding a choline-deficient diet increases the localization of cyclin-dependent kinase inhibitors (CDKIs) in the liver, whereas young adult CDKI knockout mice (p15Ink4B or p27Kip1) exhibit behavioral abnormalities. Thus, maternal dietary choline-CDKI interactions could underlie the changes we observe in fetal hippocampal development and cognitive function in offspring. Here, timed-pregnant rats on embryonic day E12 were fed the AIN-76 diet with varying levels of dietary choline for 6 days, and, on E18, fetal brain sections were collected, and the localization of CDKI proteins was studied using immunohistochemistry and an unbiased image analysis method. In choline-supplemented animals compared to controls, the number of cells with nuclear immunoreactivity for p15Ink4b CDKI protein was decreased 2- to 3-fold in neuroepithelial ventricular zones and adjacent subventricular zones corresponding to the fimbria, primordial dentate gyrus and Ammon’s horn regions in the fetal hippocampus. In contrast, maternal dietary choline deficiency significantly decreased nuclear p15Ink4b immunoreactivity in the neuroepithelial layer of the dentate gyrus. Unlike p15Ink4b, the CDKI protein p27Kip1 was observed almost exclusively in the cytoplasm, though the protein was distributed throughout the proliferating and postmitotic zones in the E18 fetal hippocampus. Maternal dietary choline supplementation decreased the cytoplasmic staining intensity for p27Kip1 throughout the fetal hippocampus compared to control animals. Choline deficiency increased the staining intensity of p27Kip1 throughout the hippocampus in association with increased expression of MAP-1 and vimentin proteins. These results link maternal dietary choline availability to CDKI protein immunoreactivity and commitment to differentiation during fetal hippocampal development.

Maternal dietary choline availability alters the balance of netrin-1 and DCC neuronal migration proteins in fetal mouse brain hippocampus

“…Surprisingly, netrin-1 and DCC showed similar changes in the CD and CS groups. This is in contrast to mitotic inhibition, apoptosis, and cell proliferation in fetal developing hippocampus which do not track in the same direction in the CD and CS groups [2][3][4]7,14], suggesting not only that each of these components of neurogenesis has a different choline dose-response, but also that the consequences of netrin-DCC signaling for precursor cell migration are dependent on additional molecular interactions. For example, recent reports suggest a critical role for cyclic nucleotides in regulating the response to guidance cues.…”

“…We previously showed that, depending on the availability of maternal dietary choline, precursor cells in ventricular zone undergo proliferation and commit to differentiate into neuronal cell types (e.g., calretinin-containing cells) [8] known to regulate memory, or undergo apoptosis. We previously described how choline availability affects pro-apoptotic signal transduction in neuronal-type cells [18,28] and hepatocytes [1,4,6,7,9]. We are beginning to understand how maternal dietary choline availability during pregnancy affects not only the generation, but also the targeted migration of precursor neuronal-type cells in the fetal hippocampus [2,3,14].…”

“…In addition, netrin-1 and DCC immunoreactivity were not detected in adult mouse liver. Analysis of netrin-1 and DCC immunoreactivity was also performed using cortical neuronal precursor cells obtained at day E14 from C57BL/6 mice (QBM Cell Science, Germantown, MD) and maintained in cell culture as described previously [26].Image analysis of fetal brain slices and neural precursor cells was performed on a Nikon FXA microscope equipped with an Optronics DEI 750 low light level integrating CCD camera (Optronics Engineering, Goleta, CA) and the public domain NIH Image program version 1.61 as described previously [4]. Digital fluorescence images of netrin-1 and DCC-labeled brain sections or neuronal precursor cells were acquired using narrow band-pass emission filters, gray scale images were prepared and Scion image software were then used to measure the integrated optical density of netrin-1 and DCC immunoreactivity.…”

“…Image analysis of fetal brain slices and neural precursor cells was performed on a Nikon FXA microscope equipped with an Optronics DEI 750 low light level integrating CCD camera (Optronics Engineering, Goleta, CA) and the public domain NIH Image program version 1.61 as described previously [4]. Digital fluorescence images of netrin-1 and DCC-labeled brain sections or neuronal precursor cells were acquired using narrow band-pass emission filters, gray scale images were prepared and Scion image software were then used to measure the integrated optical density of netrin-1 and DCC immunoreactivity.…”

Betaine rescue of an animal model with methylenetetrahydrofolate reductase deficiency

“…Intrauterine choline deficiency has been shown to result in structural defects in the hippocampus of rats [36] and mice [37][38][39]. Choline availability altered the timing, genesis, migration and differentiation of progenitor neuronal-type cells in the fetal hippocampus.…”