The present invention is in the field of clinical and diagnostic testing, and relates generally to a method of detecting axonal damage and associated disease states.
Axonal degeneration is a primary feature of brain injury in humans (Hayes et al., 1995). The present invention describes several methods of assessing axon degeneration in humans by measuring proteins that are localized in axons. These axonal proteins are released following head injury into the extracellular space and are transported to cerebrospinal fluid (CSF). Methods are disclosed in the present invention for measuring these axonal proteins in the CSF and blood of patients.
Tau protein is a major microtubule associated structural protein localized primarily in axons (Binder et al. 1985; Kosik and Finch 1987). The localization of tau in axons is thought to result from the preferential sequestration of tau mRNA in the proximal portion of axons (Litman et al., 1993) and the selective stabilization of tau in axons (Kanai and Hirakawa 1995). Human tau proteins are encoded by a single gene and at least six alternately spliced isoforms have been identified that demonstrate an apparent molecular weight of 48 kilodalton (kDa) to 68 kDa (Goedert et al. 1989 and FIG. 5). Under normal conditions, little or no tau is released extracellularly. This disclosure teaches that tau is released under clinical conditions associated with axon damage.
Neurofilament proteins, similar to tau, are structural neuronal proteins found in central nervous system axons (Shaw, 1986). Neurofilaments that are the subject of the present invention consist of four separate protein elements: 1) a light neurofilament subunit (neurofilament-L) with an apparent molecular weight of 68 kDa, 2) a medium-sized neurofilament subunit (neurofilament-M) with an apparent molecular weight of 160 kDa, 3) a heavy neurofilament subunit (neurofilament-H) with an apparent molecular weight of 200 kDa, and 4) neurofilament66/xcex1-intemexin (neurofilament66) with an apparent molecular weight of 66 kDa (Lee and Cleveland, 1996). These neurofilaments are each encoded on a separate gene (Julien et al., 1987; Myers et al., 1987; Lees et al., 1988; Chan and Chiu, 1995). Following head injury neurofilaments are depleted from degenerating axons and gain access to the CSF (Hayes et al., 1995).
One clinical condition associated with axonal degeneration is head trauma. Axonal injury, clinically referred to as diffuse axonal injury, accounts for about half of the primary lesions observed in closed head trauma and is one of the most frequent causes of poor clinical outcome. MRI is the procedure of choice for detecting diffuse axonal injury, however, MRI routinely underestimates the true extent of the damage.
As our understanding of the nervous system and its related disorders increases, a wider range of therapeutic and diagnostic agents will become available. Once these agents have been identified, it will be necessary to detect such diagnostic markers in the central nervous system. Unfortunately, the existence of the blood-brain barrier limits the free passage of many types of molecules from the blood to cells of the central nervous system making it necessary for diagnostic tests to be performed on CSF and blood, serum or plasma.
The physiological basis for the blood-brain barrier is the brain capillaries, which are made of endothelial cells (Goldstein, et al., Scientific American, 255: 74-83 (1986); Pardridge, W. M., Endocrin. Rev. 7:314-330 (1986)). These endothelial cells are different from those found in other tissues of the body. In particular, they form tight junctions between themselves. The actual blood-brain barrier is formed by these high-resistance tight intercellular junctions that form a continuous wall against the passive movement of molecules from the blood to the brain. These cells are also different in that they have few pinocytotic vesicles, which in other tissues allow somewhat unselective transport across the capillary wall. In addition, continuous gaps or channels running through the cells, which would allow unrestrained passage, are absent.
One function of the blood-brain barrier is to protect the brain from fluctuations in blood chemistry. However, this isolation of the brain from the bloodstream is not complete. There does exist an exchange of nutrients and waste products. The presence of specific transport systems within the capillary endothelial cells assures that the brain receives, in a controlled manner, all of the compounds required for normal growth and function. The obstacle presented by the blood-brain barrier is that, in the process of protecting the brain, it excludes many potentially useful therapeutic and diagnostic agents.
There are several techniques that either physically break through the blood-brain barrier or circumvent it to deliver therapeutic or diagnostic agents. Among these are intrathecal injections, surgical implants, and osmotic techniques.
Intrathecal injection administers agents directly into the brain ventricles and spinal fluid by puncturing the membranes surrounding the brain. Sustained dosages of agents directly into the spinal fluid can be attained by the use of infusion pumps that are implanted surgically. These spinal fluid delivery techniques are used to treat brain cancers, infections, inflammation and pain, but only penetrate into a minute fraction of the brain due to diffusion gradients and the density of neural tissues.
Clinicians prefer to avoid intrathecal injections because they frequently are ineffective and can be dangerous. Substances injected intrathecally are distributed unevenly, slowly and incompletely in the brain. Since the volume of the spinal fluid is small, increases in intracerebral pressure can occur with repeated injections. Furthermore, improper needle or catheter placement can result in seizure, bleeding, encephalitis and a variety of other severe side effects.
One embodiment of the present invention involves the development and use of an alternative procedure for quantifying axon damage in patients with CNS injury. Another embodiment of the present invention involves a method for quantifying axonal degeneration which is the central feature of neurodegenerative disorders including Alzheimer""s disease. Alzheimer""s disease is a progressive, degenerative disease that attacks the brain and results in impaired memory, thinking and behavior. Alzheimer""s disease is the most common form of dementia, which is the loss of intellectual function so severe it interferes with daily life. Since being first described by Dr. Alois Alzheimer in 1906, it has become the fourth leading cause of death among adults in the United States between the ages of 75 and 84. The clinical mortality and morbidity seen in Alzheimer""s patients directly results from neuronal death in the brain. In neurodegenerative disorders including Alzheimer""s disease, neuronal death is always accompanied by axonal degeneration.
Patents that discuss tau protein in serum include U.S. Pat. Nos. 5,492,812, 5,861,257, 5,733,734 and 5,601,985.
To determine the existence vel non of disease or trauma states associated with axonal damage in a patient, it is desirable to be able to ascertain whether a patient has such axonal damage and to quantify such damage.
In view of the present disclosure or through practice of the present invention, other advantages or problem solutions may become apparent.
The present invention includes a method of determining the extent of axonal damage in the human CNS, novel cleaved forms of tau proteins and neurofilament proteins associated with axonal damage, and monoclonal antibodies (MAbs) raised against novel cleaved forms of tau protein useful in such a method.
In general terms, the method of the present invention is a method of determining axonal damage in the human CNS, the method comprising the steps: (a) obtaining a sample of CSF or blood from the human central nervous system of a patient; (b) treating the sample of CSF or blood with monoclonal antibody(ies) binding to novel cleaved form(s) of tau proteins or neurofilament proteins described in the present application; and (c) detecting the presence and/or level of the cleaved form(s) of the tau proteins or neurofilament proteins bound to the monoclonal antibody(ies).
The method of the present invention may also include the step of comparing the amount of the cleaved form(s) of the tau proteins or neurofilament proteins bound to the monoclonal antibody(ies) in step (c) to control samples selected from the group consisting of those representing a normal undamaged axon state and those representing an axonal damage state.
The method of the present invention may be used to detect cleaved form(s) of tau proteins or neurofilament proteins. The cleaved tau protein(s) of particular interest in the present method are those having an apparent molecular weight less than 50 kDa and being in a phosphorylated or non-phosphorylated state, particularly those fragments in the range of about 30 kDa to 50 kDa. The present method also has been forced to characterize the multiple protein bands comprising these 30 kDa to 50 kDa tau protein fragments. The cleaved neurofilament proteins of particular interest in the present method occur in human CSF or blood where they demonstrate an apparent molecular weight: 1) for neurofilament-L less than 68 kDa, 2) for neurofilament-M less than 160 kDa, 3) for neurofilament-H less than 200 kDa, and for neurofilament66 less than 66 kDa.
The method of the present invention may be used to determine whether axonal damage has occurred and to what extent, and thus can be used to determine the existence or likelihood of any disease state associated with axonal damage, such as CNS injuries, including primary neuronal injuries (e.g., cortical contusion, diffuse axonal injury, subcortical gray matter injury and primary brain stem injury), primary hemorrhages (e.g., subdural hematoma, epidural hematomas, intracerebral hematoma and diffuse hemorrhages), primary vascular injuries (e.g., arterial psuedoaneurysm, arterial dissection/occlusion), dural sinus laceration/occlusion, traumatic pia-arachnoid injuries, cranial nerve injuries and secondary traumatic lesions (e.g., infarction, hypoxic injury, diffuse brain swelling/edema, secondary hemorrhage), central nervous system tumor, neurodegenerative diseases of the central nervous system including Alzheimer""s disease, spinal cord injury, acute cerebral vascular accident or axonal damage following ingestion of drug(s) or poison(s).
The present invention also includes cleaved forms of tau proteins having an apparent molecular weight less than 50 kDa particularly those fragments in the range of about 30 kDa to 50 kDa, and being in a substantially isolated and/or purified form. The present invention also includes cleaved forms of neurofilament proteins in a substantially isolated form occuring in human CSF where they demonstrate an apparent molecular weight: 1) for neurofilamnent-L less than 68 kDa, 2) for neurofilament-M less than 160 kDa, 3) for neurofilament-H less than 200 kDa, and for neurofilament66 less than 66 kDa. These substances may be used as standards or controls in tests using the present invention.
Substantially isolated and/or purified MAbs raised against at least one tau protein and/or its cleaved forms, and having been screened against human CSF are also included in the present invention. As used herein, a substantially isolated or substantially purified monoclonal antibody consists of a single hybridoma clone recognizing a single protein epitope essentially free of contaminating or interfering molecules. It is preferred that such MAbs are those raised against tau protein fragments, and having been screened against human CSF, said monoclonal antibody being in a substantially isolated form, and said tau protein fragment comprising the peptide sequence including the amino acids from serine199 to serine396 of tau protein, and lacking the native N-terminal and C-terminal amino acids. These substances are useful in the methods of the present invention.
The present invention can also be used to ascertain or predict the severity of neurologic trauma, such as intercranial lesions, or the neurologic disease states giving rise to tau or neurofilament proteins in CSF or blood, such as those disease states discussed above and/or to ascertain or predict clinical outcome following such trauma.
USPTO Backgrounds
USPTO Backgrounds