1. Field of the Invention
The present invention relates generally to medicine. More specifically, the invention is directed to methods relating to treating or preventing dementia.
2. Background Information
Dementia is a neurological disease that results in loss of intellectual capacity and is associated with widespread reduction in the number of nerve cells and brain tissue shrinkage. Memory is the mental capacity most often affected. The memory loss may first manifest itself in simple absentmindedness, a tendency to forget or misplace things, or to repeat oneself in conversation. As the dementia progresses, the loss of memory broadens in scope until the patient can no longer remember basic social and survival skills and function independently. Dementia can also result in a decline in the patient""s language skills, spatial or temporal orientation, judgment, or other cognitive capacities. Dementia tends to run an insidious and progressive course.
Dementia results from a wide variety of distinctive pathological processes. The most common pathological process to cause dementia is Alzheimer""s disease, which results in Alzheimer""s-type dementia (AD). The second most common cause is multi-infarct, or vascular dementia, which results from hypertension or other vascular conditions. Dementia can also result from infectious disease, such as in Creutzfeldt-Jakob disease. Dementia occurs in Huntington""s disease, which is caused by an autosomal dominant gene mutation, and in Parkinson""s disease, which is associated with a motor disorder. Dementia also occurs from head injury and tumors.
Rare before age 50, AD affects nearly half of all people past the age of 85, which is the most rapidly growing portion of the United States population. As such, the current 4 million AD patients in the United States are expected to increase to about 14 million by the middle of the next century.
No method of preventing AD is known and treatment is primarily supportive, such as that provided by a family member in attendance. Stimulated memory exercises on a regular basis have been shown to slow, but not stop, memory loss. A few drugs, such as tacrine, result in a modest temporary improvement of cognition but do not stop the progression of dementia.
A hallmark of AD is the accumulation in brain of extracellular insoluble deposits called amyloid plaques, and abnormal lesions within neuronal cells called neurofibrillary tangles. The presence of amyloid plaques, together with neurofibrillary tangles, are the basis for definitive pathological diagnosis of AD. Increased plaque formation is associated with increased risk of AD.
The major components of amyloid plaques are the amyloid xcex2-peptides, also called Axcex2 peptides, which consist of three proteins having 40, 42 or 43 amino acids, designated as the Axcex21-40, Axcex21-42, and Axcex21-43 peptides. The amino acid sequences of the Axcex2 peptides are known and the sequence of the Axcex21-42 is identical to that of the Axcex21-40 peptide, except that the Axcex21-42 peptide contains two additional amino acids at its carboxyl (COOH) terminus. Similarly, the amino acid sequence of the Axcex21-43 peptide is identical to that of the Axcex21-42 peptide except that the Axcex21-43 peptide contains one additional amino acid at its carboxyl terminus. The Axcex2 peptides are thought to cause the nerve cell destruction in AD, in part, because they are toxic to neurons in vitro and in vivo.
The Axcex2 peptides are derived from larger amyloid precursor proteins (APP proteins), which consist of four proteins, designated as the APP695, APP714, APP751, and APP771 proteins, which contain 695, 714, 751 or 771 amino acids, respectively. The different APP proteins result from alternative ribonucleic acid splicing of a single APP gene product. The amino acid sequences of the APP proteins are also known and each APP protein contains the amino acid sequences of the Axcex2 peptides.
Proteases are believed to produce the Axcex2 peptides by recognizing and cleaving specific amino acid sequences within the APP proteins at or near the ends of the Axcex2 peptides. Such sequence specific proteases are thought to exist because they are necessary to produce from the APP proteins the Axcex21-40, Axcex21-42, and Axcex21-43 peptides consistently found in plaques.
But the proteases have not been isolated. Nonetheless, they have been named xe2x80x9csecretasesxe2x80x9d because the Axcex2 peptides which they produce are secreted by cells into the extracellular environment. Moreover, the secretases have been named according to the cleavages that must occur to produce the Axcex2 peptides. The secretase that cleaves the amino terminal end of the Axcex2 peptides is called the xcex2-secretase and that which cleaves the carboxyl terminal end of the Axcex2 peptides is called the xcex3-secretase. The xcex3-secretase determines whether the Axcex21-40, Axcex21-42, or Axcex21-43 peptide is produced (see FIG. 1). But since the secretases have not been isolated, the terms xcex2-secretase and xcex3-secretase each could relate to one or several protease species.
In addition to the Axcex2 peptides, proteolytic cleavage of another specific amino acid sequence within the APP proteins is known to occur and to produce xcex1-APP and 10 kilodalton (kDa) fragments. That amino acid sequence lies within the Axcex2 peptide amino acid sequence of the APP proteins. Like the xcex2-secretase and the xcex3-secretase, the protease responsible for that cleavage has also not been isolated but has been named the xcex1-secretase (see FIG. 1). Significantly, the products produced by the xcex1-secretase cleavage, the a-APP and the 10 kilodalton (kDa) fragments, do not form senile plaques.
Proteases can be isolated from tissue homogenates or lysed cell samples, but those samples can contain the proteases from cell organelles in which the product is not produced, but which may be able to cleave in vitro the precursor protein to produce the product. Thus, a problem in using such samples to isolate the secretases has been that proteases which produce the Axcex2 peptide in vitro, but not in vivo, may be erroneously isolated.
The problem can be avoided by isolating the secretase from cell organelles in which the APP proteins are processed in vivo. A cell organelle thought to be a site in which such processing occurs is the secretory vesicles of brain neuronal cells. But methods have not been developed to obtain sufficient amounts of pure secretory vesicles from neuronal cells to assay for secretase activity in those vesicles.
Large amounts of pure secretory vesicles can be obtained from chromaffin cells, neuroendocrine cells of the adrenal medulla, and have been used to obtain proteases. For example, carboxypeptidase H (CPH), prohormone thiol protease (PTP), and the prohormone convertases (PC1 and PC2), which process precursor proteins into peptides having opiate activity have been obtained from such vesicles. But chromaffin cells have not been shown to produce the Axcex2 peptides or have secretase activity.
The xcex2-secretase, xcex3-secretase, and xcex1-secretase must be isolated to understand how the neurotoxic Axcex2 peptides are produced so that AD can be prevented or treated. To isolate the secretase, new methods are needed for assaying for the proteolytic activity of secretases in substantially purified preparations of the cell organelles in which the APP protein is processed in vivo. Moreover, new screening methods for selecting agents that affect the proteolytic activity of the secretases are needed to develop new pharmaceuticals for treating or preventing AD.
The invention satisfies these needs by providing new methods of determining the proteolytic activity of secretases and isolating secretases having that activity. The invention also provides new screening methods for selecting agents that affect the activity of such secretases.
The invention is directed to a method of determining the proteolytic activity of a secretase by obtaining substantially pure vesicles, permeablizing the vesicles and incubating the permeablized vesicles with an APP substrate. The activity of the secretase is determined by detecting the cleavage of the APP substrate which is proportional to that activity. The invention is also directed to a method of isolating the secretase having that activity and the secretase obtained by that isolation method.
The invention is further directed to methods of selecting an agent that alters the cleavage of an APP substrate by a secretase. In one method, substantially pure vesicles are obtained, permeablized, and incubated with an APP substrate with and without the agent. The cleavage of the APP substrate with and without the agent is compared and the agent that alters the cleavage of the APP substrate selected. In another method, an isolated secretase and an APP substrate are incubated with and without the agent, the cleavage of the APP substrate compared and the agent selected that alters the cleavage of the substrate. In yet another method, a cell is selected by determining whether the cells contains vesicles having the proteolytic activity of a secretase and those cells used to select an agent. The production of an APP protein or an APP derived product produced by those cells with and without the agent is compared and the agent is selected that alters the production of the product. Another aspect of the invention is the agent selected by such methods.