Emily Brinton Stuart and Dr. Edwin D. Lephart, Neuroscience

Introduction

Calbindin-D28K (CALB), a calcium binding protein, is one of the most abundant proteins found in neurons of certain brain areas. It is potentially important in neuron formation and may play a protective role against programmed cell death [1]. CALB helps regulate the amount of calcium in neurons. An over-abundance of intracellular calcium leads to cell degeneration and eventually cell death. Brain sites that are exposed to toxic amounts of calcium experience less neurodegeneration where calcium-binding proteins are present than in areas where the protein is less abundant [4]. It is hypothesized that decreased levels of CALB lead to patterns of cell death seen in neurodegenerative diseases such as Alzheimer’s, Huntington’s, and Parkinson’s disease.

Overview of the Study

To determine the protective role calbindin plays in regulating neuronal cell death, the following were objectives of this project: 1) quantify CALB mRNA species in the medial basal hypothalamic-preoptic area (MBH-POA) in male vs. female rats during different stages of perinatal (around the time of birth) development, 2) examine if the levels of CALB mRNA differ between male and female rats, and 3) examine if CALB mRNA levels vary over developmental intervals in order to compare this mRNA profile to that of the dimorphic MBH-POA CALB expression previously reported [3].

In our study, we collected MBH-POA tissue in male and female rats at gestational day (GD) 18, newborn, and postnatal days (PND) 2 and 5. We then isolated total RNA from the tissue by centrifugation through a cesium chloride step-gradient. Next, we blot hybridized the total RNA and quantified CALB mRNA in each sample using Northern blot analysis and radiolabeled PCR generated probe derived from the coding region of a rat CALB cDNA. The blots were exposed to x-ray films followed by optical density analysis of the films with NIH Imaging version 1.6. The quantified results were expressed as a percentage of the lowest intensity band.

Results

Our results show MBH-POA CALB mRNA levels independent of sex, were lowest at GD18, increased to approximately four-fold in newborns and remained high during postnatal development at PND 2 and PND 5. Male levels were approximately two-fold higher than female values at GD 18 and in newborns. However, at PND 2 MBH-POA CALB mRNA levels were approximately equivalent in males vs. females. At PND 5, female levels were about 1 1’2 times higher than male levels. These mRNA data confirm previous dimorphic protein expressions of MBH-POA CALB during prenatal development and in neonates where males express significantly higher levels than females [3].

Significance

This is a novel finding since the MBH-POA represents an important brain region where physiologic, neuroendocrine functions, and reproductive behaviors are regulated by dimorphic neural structures [2]. These sexually dimorphic neural structures within the MBH-POA may be mediated by the greater abundance of calcium-binding proteins.

While it is known that glucocorticoids regulate MBH-POA CALB during development, the regulatory agents involved in the male vs. female expression of MBH-POA CALB during this interval is not known. However, androgens stimulate CALB expression and may participate in the dimorphic perinatal pattern. (This idea is further supported in that fetal androgen levels peak around GD 18, when CALB mRNA levels in males are two-fold the levels in females.)

The implications of dimorphic expression (i.e. mRNA and protein levels) suggest that neuronal cell loss may be greater in females vs. males due to less abundant CALB levels in females compared to notably higher CALB levels in males during perinatal development [3].

The results presented here are abbreviated. Complete results, including figures and discussion of adult distribution of calbindin-D28k mRNA over 12 brain regions in Sprague-Dawley rats, are contained in a paper accepted for publication in Molecular Brain Research, 1999 (in press).

References

1. Baimbringe, K.G., Celio, M.R., and Rogers, J.H., Calcium-binding proteins in the nervous system, Trends Neurosci., 15(1992)303-308).
2. Lephart, E.D., A review of brain aromatase cytochrome P450, Brain Res. Rev., 22(1996)1- 26.
3. Lephart, E.D., Dimorphic expression of calbindin-D28K in the medial basal hypothalamus from perinatal male and female rats, Dev. Brain Res., 96(1996)231-284.
4. Iacopino, A.M., Quintero, E.M., and Miller, E.K., Calbindin-D28K potential neuroprotective protein, Neurodegeneration, 3(1994)20.

We thank N.A. Jacobson for technical support and A. McMahon and D.C. German (University of Texas Southwestern Medical Center; Dallas, TX) for the rat calbindin cDNA.This work was supported by grants from the National Science Foundation under Grant No. IBN-9507972 (to EDL), The Neuroscience Center at BYU under Grant No. 19-223566 (to EDL), and ORCA grant (to EBS).