Alzheimer’s disease is a neurodegenerative disease characterized by changes in normal neuronal function. In most cases, the risk of these changes increases with age and the disease itself is progressive. Currently, neurodegenerative diseases are considered incurable.

Alzheimer’s disease is a brain disorder that primarily affects older persons. Alzheimer’s disease can begin with

forgetfulness and usually progress into more advanced symptoms, including confusion, language disturbances, personality and behaviour changes, impaired judgment and profound dementia. As the disease advances, most patients will eventually need extensive, skilled nursing care, and in the absence of other illnesses, the progressive loss of brain function itself will cause death.

Neuropathic diagnosis of Alzheimer’s disease is typically based upon examination of the brain at autopsy and is determined by the number of plaques and tangles in the neurocortex (frontal, temporal, and parietal lobes), hippocampus and amygdala. A diagnosis of Alzheimer’s disease based upon neuropathologic criteria alone, however, is often difficult because there are a significant number of plaques and tangles in the neurocortex, hippocampus and amygdala of normal elderly people. In addition, the density of neocortical plaques and tangles has only a rough correlation with the degree of dementia. Perhaps the most alarming fact about diagnosis of Alzheimer’s disease is the fact that the only biochemically certain diagnosis depends on examination of a patient’s brain at autopsy. Thus, diagnosis of Alzheimer’s disease in living patients depends on behavioral and more subjective medical criteria

Alzheimer’s disease has been difficult to diagnose and to treat. The discovery that levels of acetylcholinesterase are markedly reduced in the cortex and hippocampus of patients with Alzheimer’s disease has resulted in the development of a number of pharmaceutical compounds which restore or replace cholinergic function. Examples of such compounds include tacrine (“THA”), physostigmine (an irreversible inhibitor of acetylcholinesterase) and physostigmine derivatives. In general, however, those compounds have met with only limited success.
Given the increasing number of individuals with Alzheimer’s disease, it is critical that new methods for diagnosing, treating and monitoring the disease be developed.

Dr. Wilfred Jefferies and his research team (the “Inventors”) have found that p97 is a protein that is attached to the cell membrane by a glycosyl-phosphatidylinositol (“GPI”) anchor. The GPI-anchored protein may be treated with an enzyme that cleaves at the GPI-anchor to provide a cleaved p97 protein that is liberated from the cell in a soluble form. The cleaved p97 can be prepared using a novel, semi-continuous process. Other cleaved GPI-anchored proteins can also be prepared using the same method.
The Inventors have also found a natural soluble form of p97. This form is hydrophilic and is present exclusively in the aqueous phase after Triton-X 114 detergent phase separation. The molecule does not contain ethanolamine and exhibits a slower rate of transport than GPI-anchored p97. The soluble form of p97 may be present in biological fluids such as cerebrospinal fluid (“CSF”), blood, or urine. The Inventors have also shown that p97 is involved in iron transport since the GPI-anchored form expressed on the cell surface binds iron and bound iron is released after phospholipase treatment. The Inventors have thus demonstrated a transferrin independent p97-mediated iron uptake pathway. The presence of p97 was found to double the amount of iron uptake into mammalian Chinese hamster ovary cells (CHO) cells.

A substance which is a stimulant, agonist or antagonist of p97 may be identified by determining the effect of the substance of the binding activity of p97 and iron, or p97 and the transferrin receptor, or the effect of the substance on the expression of p97 in cells capable of expressing p97 including cells genetically engineered to express p97 on their surface.

The invention therefore in one aspect relates to a method of identifying stimulants, agonists or antagonists of p97 comprising reacting a substance suspected of being a stimulant, agonist or antagonist of p97 with p97 and transferrin receptor under conditions such that p97 is capable of binding to the transferrin receptor; measuring the amount of p97 bound to the transferrin receptor; and determining the effect of the substance by comparing the amount of p97 bound to transferrin receptor with an amount determined for a control.

The invention also relates to a method of identifying stimulants, agonists or antagonists of p97 comprising reacting a substance suspected of being a stimulant, agonist or antagonist of p97 with a cell which expresses p97, measuring the amount of p97 expressed by the cell, and determining the effect of the substance by comparing the amount of expression of p97 with an amount determined for a control.

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