Calbindin D28k: Distribution and Regulation in AD

Calcium is an essential component of all cells, including nervous system cells (i.e.: neurons) . Although calcium is critical for the normal maintenance of neurons, at high concentrations it can be deadly. Therefore, cells have elaborate mechanisms for maintaining the concentration of calcium within the cell (intracellular calcium). One such mechanism exploits the ability of specific cellular proteins to bind calcium (calcium binding proteins) which in turn will decrease the amount of free/unbound calcium. This “buffering” of calcium, between free and bound forms, helps to establish a physiological concentration of calcium within the cell. When the concentration of calcium goes off balance the neuron will no longer function normally and will die. It has been proposed that imbalances in calcium regulation occur in certain neurodegenerative diseases such as Alzheimer’s disease. We are interested in studying whether these imbalances in calcium buffering are related to the expression of one of the calcium binding proteins; human calbindin D28k. We are also interested in better understanding how the expression of the gene coding for calbindin D28k is regulated in normal as well as in diseased brains.

In the first part of this grant, we propose to examine the localization of calbindin in the human brain and to determine if there are any changes in calbindin expression in brain tissue from Alzheimer’s disease patients and similarly aged patients. We will use an assay called jn situ hybridization to determine the levels of the messenger RNA (mRNA) which codes for calbindin. This technique is based on the observation that synthetically produced mRNA (antisense RNA) which has a sequence complementary to the neurons endogenous mRNA (sense RNA) will under the proper incubation conditions bind to this sense mRNA forming RNA:RNA hybrids. These hybrids are easily detectable since the synthetically produced antisense mRNA is radiolabelled. An additional advantage of this technique is that it can be used on thin sections of frozen postmortem human brain. Data provided in this proposal indicate that we have already identified mRNA for calbindin in specific regions of human brain obtained at autopsy.

n the second part of this grant, we propose to study the regulation of calbindin D28k gene expression . We will be able to accomplish these studies by utilizing a combination of cell culture techniques and extracts from nuclei obtained from postmortem human brain. Our first goal is to determine whether this gene is regulated by changes in the levels of intracellular calcium. This is a very important question since it would provide a mechanism for the cell to recover from insults which either increase or decrease this essential ion. The second important goal of this research is to identify what other factors may influence the amount of calbindin D28k mRNA being expressed. One such molecule that we are interested in studying is the glucocorticoid receptor. Steroid regulation has been implicated in many aspects of aging and neurodegenerative diseases. The cell culture system which we will use for these studies will allow us to manipulate the cellular environment so we can address these questions more directly. Lastly, we want to compare the activity and/or level or the factors which regulate calbindin D28k transcript ion between normal human brain and Alzheimer’s disease brain. This can be accomplished by preparing nuclear extracts from postmortem human brain and testing these extracts for various activities corresponding to these factors. It is interesting to hypothesize that changes in Alzheimer’s disease brains may be due in part to a disruption in the regulation of calcium via the calcium binding protein, calbindin D28k .

Furthermore, the levels of calbindin D28k may be different in Alzheimer’s disease brains when compared to normal brains because certain regulatory factors are missing or no longer active. We provide data in this proposal that postmortem human brain tissue can be used as a source of transcriptionally active extract and for purification of regulatory factors. We feel that the studies we have proposed will provide new insights into one of the mechanisms of calcium regulation in the neurodegenerative disease, Alzheimer’s disease.