JP2003504007A - Different gene expression in specific regions of the brain in neurodegenerative diseases - Google Patents

Abstract

  (57) [Summary] The invention provides: a nucleic acid molecule (labeled and may be provided in a kit) that can function as a PCR primer for the detection of Contactin mRNA; a method of detecting the presence of a neurodegenerative disease (expressed in a sample) Measuring the amount of contactin protein or contactin mRNA), a method for identifying which patients are responsive to treatment for a neurodegenerative disease; whether they have increased compared to control cells. Or a composition comprising isolated cells or cells in culture that expresses a reduced amount of contactin; reducing the expression of a composition or compound, including contactin, and a pharmaceutical composition identified by this method A method for screening compounds for or enhancing activity; a method for treating a neurodegenerative disease; Method of identifying a pharmaceutical targets for compounds that modulate the expression or activity and target Contactin identified by a method such as.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates generally to the fields of molecular biology, neurology, neurodegenerative diseases, and their diagnosis and treatment.

BACKGROUND OF THE INVENTION Neurodegenerative diseases afflict humans with various weaknesses, such as memory loss, skeletal muscle loss, and tight motor control, or coma. Several neurodegenerative diseases (eg Alzheimer's disease (AD), Parkinson's disease (PD), chronic Lewy body disease (DLB), chronic vascular dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis) Disease (A
LS), lactic acidosis and seizures of epileptic clonic myoparalysis (MELAS)
, And the uneven red fiber syndrome (MERRF) of epileptic clonic muscular paralysis
May be or be associated with mitochondrial deficiency.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder associated with altered mitochondrial function and substantia nigr.
a) Deletion and / or atrophy of neurons containing dopamine in the compacts of pars compacta. Like Alzheimer's disease (AD), PD also afflicts the elderly. It is characterized by bradykinesia (slow behavior), rigidity, and tremors at rest. The L-Dopa treatment alleviates tremors in most patients for some time, but eventually the tremors become increasingly uncontrollable, thereby allowing the patient to ingest himself or his own basic hygiene. Meeting the above needs becomes difficult or impossible.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinsonism in animals and humans, at least in part, through its effects on mitochondria. It is shown. MPTP is converted to its active metabolite, MPP +, in dopamine neurons. It is then concentrated in mitochondria. MPP + then
It selectively inhibits the mitochondrial enzyme NADH: ubiquinone oxidoreductase (“complex I”). This increases the production of free radicals,
It leads to diminished production of adenosine triphosphate and ultimately death of affected dopamine neurons.

Mitochondrial complex I is composed of 40-50 subunits; most are encoded by the nuclear genome and seven by the mitochondrial genome. Since Parkinson's syndrome can be induced by exposure to mitochondrial toxins that affect the activity of complex I, this is probably due to a deficiency in the complex I protein but a similar biochemical deficiency in the activity of complex I. Can affect the etiology of PD. Indeed, deficiencies in the activity of mitochondrial complex I have been reported in the blood and brain of PD patients (Parke).
r et al., Am. J. Neurol. 26: 719-723, 1989).

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. It is characterized by neuronal loss and / or atrophy in discrete areas of the brain,
And this is achieved by extracellular accumulation of β-amyloid and intracellular accumulation of nerve fiber tangles. It is a unique human disease that affects more than 13 million people worldwide. This is also a tragic disease, which is also endemic. Many individuals who are productive lives that survive normally and productively
AD slowly attacks them as they age, and the disease progressively robs their memory and other memory functions. Eventually, they become unaware of their families and love humans, and they often need lasting care until they eventually die.

There is evidence that a defect in oxidative phosphorylation within mitochondria is at least partially responsible for sporadic AD. The enzyme cytochrome c oxidase (COX), which makes part of the mitochondrial electron transport chain (ETC), is present in normal amounts in Ad patients; however, in AD patients and at necropsy. The catalytic activity of this enzyme in the brain of AD patients at time has been found to be abnormally low. This suggests that COX is deficient in AD patients, leading in some ways to reduced catalytic activity that causes or contributes to the symptoms that are characteristic of AD.

Local defects in metabolism of energy in mitochondria, accompanied by achieving an increase in oxidative stress, may be associated with AD. It is well established that energy metabolism is impaired in AD brains (Palmer et al. Br.
ain Res. 645: 338-42, 1994; Pappolla et al., Am.
． J. Pathol. 140: 621-28, 1992; Jeandel et al., G.
erontol. 35: 275, 1989; Balazs et al., Neuroche.
m. Res. 19: 1131-37, 1994; Mecocci et al., Ann. N
eurol. 36: 747-751, 1994; Gsell et al. Neuro
chem. 64: 1216-23, 1995). For example, a localized specific defect in the metabolism of energy in the AD brain has been reported in numerous positron emission tomography studies (Kuhl et al., J. Cereb. Blood F.
low Metab. 7: S406, 1987; Grady et al., J. Am. Clin.
Exp. Neuropsychol. 10: 576-96, 1988; Haxb.
y et al., Arch. Neurol. 47: 753-60, 1990; Azari et al., J. Am. Cereb. Blood Flow Metab. 13: 438-47,
1993). Metabolic defects in the neocortex of the temporal parietal muscle of AD patients appear to be predictive of cognitive decline over the years. Cutaneous fibroblasts derived from AD patients exhibit reduced glucose utilization and increased glucose oxidation. This leads to the formation of the end product of glycosylation (Yan et al. Proc. Natl.
Acad. Sci. USA 91: 7787-91, 1994). Cortical tissue derived from post-mortem AD brain has reduced activity of the mitochondrial enzyme pyruvate dehydrogenase (Sheu et al., Ann. Neurol. 17: 444-49, 1).
985) and decreased activity of α-ketoglutarate dehydrogenase (M).
Astrogiacomo et al. Neurochem. 6: 2007-14,
1994). Both of these are key enzymes in the metabolism of the enzyme. Functional magnetic resonance spectroscopy studies have shown increased levels of inorganic phosphate as compared to phosphocreatine in AD brains. This suggests an accumulation of precursors that results from mitochondrial production of reduced ATP (Petteg).
rew et al., Neurobiol. of Agging 15: 117-32, 19
94; Pettigrew et al., Neurobiol. of Aging 16:
973-75, 1995). Furthermore, levels of pyruvate, but not glucose or lactate, have been reported to be increased in the cerebrospinal fluid of AD patients, which indicates cerebral mitochondrial electron transport chain (ETC) activity. Consistent with the deletion (Parnetti et al., Neurosci. Lett. 199: 231-3.
3, 1995).

Signs of oxidative damage are hallmarks of the etiology of AD, and, as mentioned above, reactive oxygen species (ROS) are important mediators of neuronal degeneration. Indeed, autopsy studies indicate that protein marker, DNA, and lipid peroxidation are increased in AD brains (Palmer et al. Brain Res. 645: 3).
38-42, 1994; Pappolla et al., Ann. J. Pathol. 14
0: 621-28, 1992; Jeandel et al., Gerontol. 35: 2
75-82, 1989; Balazs et al., Arch. Neurol. 4: 864
, 1994; Mecocci et al., Am. Neurol. 36: 747-51, 1
994; Smith et al., Proc. Natl. Acad. Sci. USA 88
: 10540-43, 1991). In hippocampal tissue derived from AD but not from control, carbonyl formation, an indicator of protein oxidation, is increased in neuronal cytoplasm, as well as in neuronal and glial nuclei (Smith et al., Nature 382: 120-). 21, 1996). Tangles of nerve fibers also appear to be prominent sites of protein oxidation (Sc
hweers et al., Proc. Natl. Acad. Sci. USA 92:84.
63, 1995: Brass et al., Arch. Neurol. 4: 864,199
0). Incubation of cortical tissue from AD brains taken at necropsy under stressed and unstressed conditions demonstrated increased free radical production compared to non-AD controls. did. further,
The activities of key antioxidant enzymes, especially catalase, are reduced in AD (G
sell et al. Neurochem. 64: 1216-23, 1995). This suggests that the AD brain is vulnerable to increased ROS production. Thus, oxidative stress may contribute significantly to the etiology of mitochondrial-related diseases such as AD. In AD, there may be mitochondrial deficiency and / or elevated ROS.

One hallmark of AD that is the etiology is the death of a population of selected neurons within discrete areas of the brain. Cell death in AD is assumed to be apoptotic. Because there are signs of programmed cell death (PCD) and no indication of active gliosis and necrosis (Smale et al. Exp.
Neurolog. 133: 225-230, 1995; Cotman et al., Mo.
lec. Neurobiol. 10: 19-45, 1995). Neuronal and synaptic loss as a result of cell death in AD are closely associated with clinical diagnosis of AD and highly correlated with the degree of dementia in AD (DeKosky et al.,
Ann. Neurology 27: 457-464, 1990).

Mitochondrial deficiency is believed to be important in a cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB).
J. 9: 1277-87, 1995). And this may be responsible for the apoptotic cell death in AD brain neurons. Among others, altered mitochondrial physiology may be an early event in PCD (Zamzami et al., J. Am.
Exp. Med. 182: 367-77, 1995: Zamzami et al. E
xp. Med. 181: 1661-72, 1995), and elevated elevated reactive oxygen species (ROS) caused by such altered mitochondrial function may initiate an apoptotic cascade (Ausserer et al., Mol.
． Cell. Biol. 14: 5032-42, 1994). In some cell types (including neurons), the mitochondrial membrane potential (ΔΨm
A) precedes the disruption of nuclear DNA that achieves apoptosis. In cell-free systems, a fraction rich in mitochondria, but not in the nucleus, can induce nuclear apoptosis (Newmeyer et al., Cell 70: 353-64,199.
4). Disruption of mitochondrial respiratory activity leads to altered cellular metabolic states, such as elevated intracellular ROS, which can lead to mitochondrial-related diseases and, in addition, pathogenic events through apoptotic mechanisms. Can be further induced.

Oxidatively stressed mitochondria can release preformed soluble factors that can induce chromosomal enrichment, an event that precedes apoptosis (
Marchetti et al., Cancer Res. 56: 2033-38, 199.
6). In addition, members of the Bcl-2 family of anti-apoptotic gene products are located within the mitochondrial outer membrane (Monaghan et al., J. Histoc).
hem. Cytochem. 40: 1819-25, 1992). And these proteins appear to protect the membrane from oxidative stress (Korsm).
eyer et al., Biochim. Biophys. Act. 1271: 63, 19
95). Localization of Bcl-2 to this membrane appears to be essential for the regulation of apoptosis (Nguyen et al., J. Biol. Chem. 269: 1).
6521-24, 1994). Thus, alterations in mitochondrial pathology may be important mediators of apoptosis. To the extent that apoptotic cell death is a hallmark of neuronal loss in AD, mitochondrial failure can be important for the progression of this disease and also contribute to other mitochondrial-related diseases. Can also be a factor.

[0013] As to whether the underlying defects of the disease associated with altered mitochondrial function can be of mitochondrial origin or non-mitochondrial origin, and the underlying defects of altered mitochondrial function have been identified. Whether or not this is the case, the present invention provides reagents suitable for determining the risk of, or the presence of, a disease associated with such altered mitochondrial function, and for treating such a disease. To provide a method that is useful for identifying

Chronic lewy body disease (DLB), or Lewy body dementia, is a degenerative disorder of the central nervous system (CNS). It is typically present in elderly patients, initially as psychosis or progressive deteriorating dementia. These may be preceded by tremors, bradykinesia, or other manifestations of rigidity Parkinson's syndrome. DLB pathology reveals a chronic distribution of encapsulation of neurons in the cytoplasm known as Lewy bodies, particularly the brainstem nucleus, basal forebrain, and hypothalamic neurons. D
LB can also be achieved by one or more muscle stiffness, dysphagia, involuntary movements of skeletal muscle.

Vascular dementia, or “multiple infarction”, refers to a variety of disorders characterized by progressively worsening cognitive ability caused by multiple infarct events in cerebral vessels. Impaired memory and intellectual capacity are typically achieved by local neurological signs of vascular dementia.

[0016] Multiple sclerosis is a human chronic disease associated with demyelination of the central nervous system, which, as a succession of phrases characterized by remission tendencies and relapse symptoms (“relapse / sedation” disease), Or often as either a consistently progressive disease leading to paralysis. The anatomical pathological features of the disease include well delimited patches of demyelination in the white matter of the brain and spinal cord. It is believed that inflammation, genetic, environmental, and etiological factors contribute to the pathogenesis of multiple sclerosis.

Amyotrophic lateral sclerosis (ALS) is commonly diagnosed as a progressive motor neuron disease. The disease is characterized by degeneration of motor neurons in the cortex, brain stem, and spinal cord. The onset of this disease is a cascade of about 3 to 6 and is a heterogeneous fate. The cause of ALS is unknown and symptoms (
For example, it is diagnosed when an imbalanced weakness of the extremities, localized fasciculations of the extremities, or spasticity of the legs) is noticed.

Lactic acidosis and seizures of epileptic clonic myoparalysis (MELAS) are diseases characterized by seizure-like episodes and lactic acidosis. Stroke-like episodes can be facilitated by metabolic stress. The disease results in neuropathology, including multiple infarct-like lesions with varying degrees of generalized cerebral and cerebellar atrophy. They are not associated with areas of blood vessels in the central nervous system.

The bumpy red fiber syndrome (MERRF) of epileptic clonic muscle paralysis is characterized by impaired sensory and motor abilities, lactic acidosis, encephalopathy, seizure-like episodes, seizures, and muscle weakness. To be The disease results in microscopic degeneration of tissues of the central nervous system, accompanied by neuronal loss.

Human contactin protein and its homologue from mouse (F3 protein) and its homologue from chicken (F11 protein) are cell surface adhesion proteins involved in the adhesion of cells to substrates. Contactin contains an Ig-like domain and multiple fibronectin III-like domains (Brummendorf et al., J. Neurochem.
Istry 61: 1207-1219 (1993)). Unlike many cell adhesion molecules, contactin, although not a transmembrane protein, is instead bound to the cell surface through linkage to glycosylphosphatidylinositol (GPI) in the outer leaflet (Id.) Of plasma. . In human tissue, relatively high levels of major contactin mRNA (6.5 kb) resulted in three minor transcripts (9.7 kB).
b, 4.4 kb, and 3.4 kb)), but nevertheless low levels of expression of multiple forms of contactin mRNA result in adult lung, Found in the pancreas, kidney, and skeletal muscle (6.8 kb, and 6.0 kb) (Reid et al., Molecular Brain Resea).
rch 21: 1-8 (1994)). High levels of expression of multiple forms of contactin mRNA are found in neuroblastoma and retinoblastoma cell lines (6
． 8 kb, 6.0 kb, and 4.2 kb) (Id.). Expression of contactin in developing neural tissue is complex, transient, and temporally regulated. Contactin is probably a cell recognition molecule N in neurite outgrowth.
It is believed to have a role by binding to g-CAM and / or by interacting with the extracellular matrix glycoprotein restrictin (Faivre-Sarrailh et al., J. Neurosci. 12: 2).
57-267 (1992), Brummendorf et al., Neuron 10:
711-7272 (1993)). Adult neuronal stem cells can arise from hematopoietic cells, including cells of the spinal cord and lymphoid lineages (Bjornson et al., S
science 283: 534-537 (1999)); thus, contactin mRNA, contactin DNA, or contactin protein can be detected in blood.

The identification of the underlying causes of neurodegenerative diseases is often difficult to ascertain, and reliable diagnosis, prognosis, and treatment of such diagnoses are being developed. There is a clear need to provide improved methods and compositions for treating these neurodegenerative diseases. The present invention, such as those described above,
These needs are met by providing an initial recognition of a role for contactin in neurodegenerative diseases, and further provide other related advantages.

SUMMARY OF THE INVENTION The present invention provides for between altered expression levels (eg, increase or decrease in a statistically significant manner) of specific nucleic acid molecules in cells for neurodegenerative diseases. Part of the explanation of the correlation. Here, nucleic acid molecules exhibiting altered expression levels encode specific products including: contactin FREAC-2, APCL,
LAP, COX7C, PAF, 6PTS1, VDAC1, UNK-Br40, U
NK-Br42, a novel brain-expressed EFHD homolog, and various additional novel products, as well as members of known products as described herein. Accordingly, the disclosure of the present invention relates, in a related part, to a plurality of such nucleic acid molecules having altered expression levels in one or more cell types or tissues in at least one neurodegenerative disease. . But here
Nucleic acid molecules encoding contactin include one representative example of nucleic acid molecules having altered expression levels associated with neurodegenerative diseases. Thus, while abundant reference is made to altered expression levels of contactin-encoding nucleic acid molecules, such as a representative example, the present invention is directed to products having novel sequences and known sequences. It will be appreciated that it is well related to some of the additional nucleic acid molecules described herein that encode other products, including those that have.

Thus, in more detail, according to the invention, a correlation between the cellular expression level of the cell adhesion molecule contactin and the presence or risk of a neurodegenerative disease is provided, which results , Quantitatively altering or modulating (ie, increasing or decreasing) cellular expression of contactin has been implicated in neurodegenerative diseases as compared to the absence of such diseases.

[0024] In one aspect, the present invention provides an oligonucleotide primer capable of specifically amplifying DNA or RNA encoding contactin, or a nucleic acid sequence complementary thereto. These primers are SEQ ID NO: 1 or part thereof, SEQ ID NO: 2 or part thereof, SEQ ID NO: 3 or part thereof, SEQ ID NO: 4 or part thereof, SEQ ID NO: 5 or part thereof, SEQ ID NO: 6 or part thereof. part,
Alternatively it contains an isolated nucleic acid molecule which is identical or substantially identical to the nucleotide sequence which is SEQ ID NO: 7 or a part thereof. In a particular embodiment, the isolated nucleic acid molecule comprises a detectable label. The invention also provides a kit containing an oligonucleotide primer as is apparent herein.

Another aspect of the invention is to provide a method for detecting the risk of having a neurodegenerative disease or the presence of that disease in a subject. This method relates to the amount of contactin protein in a sample from a control subject who is known not to be at risk of having a neurodegenerative disease or the disease is absent. Comparing the amount of contactin protein in a test sample from the subject. The invention also provides, in certain embodiments, a method of detecting a risk of having a neurodegenerative disease or the presence of that disease in a subject. This method compares the amount of contactin mRNA in a sample from a control subject who is not at risk of having a neurodegenerative disease or is known not to have that disease to the test. Comparing the amount of contactin mRNA in a test sample from the body. In certain embodiments, the test and control samples are from the central nervous system. In certain other embodiments, contactin mRNA is measured by the polymerase chain reaction method. In certain further embodiments, the polymerase chain reaction method comprises amplification with a forward primer containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. In certain other embodiments, the polymerase chain reaction method comprises amplification with a reverse primer containing the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7.

In a particular embodiment, the invention provides a method of detecting a risk of having a neurodegenerative disease or the presence of that disease in a subject. This method relates to the amount of RNA in a sample from a control subject who is known not to be at risk of having a neurodegenerative disease or the absence of that disease, relative to the subject. And comparing the amount of contactin mRNA in the test sample from. Here, the RNA of the control sample does not encode contactin. In certain embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis, multiple sclerosis, MELAS, or MERRF, and in certain preferred embodiments, the neurodegenerative disease is multiple sclerosis. Illness.

The invention also provides, in another aspect, a method of screening reagents for use in treating a patient having a neurodegenerative disease. The method comprises determining an initial level of expression of contactin in a first sample derived from at least a human patient, followed by contacting the patient with a candidate reagent (
Where the determining step determines the amount of contactin protein or the amount of contactin RNA); and a second sample from the patient after contacting the patient with the candidate reagent. Comparing the first level of contactin expression to the second level of contactin expression determined in (wherein the change in the level of contactin expression indicates that the reagent is associated with a neurodegenerative disease). Demonstrating its suitability for use in treating a patient who has).

Another aspect of the present invention is the genomic and mitochondrial DN of different biological origin.
It is to provide a cybrid cell line containing immortal cells having A and cells capable of differentiating. Here, the cells express contactin. In certain embodiments, the cells are neural cells, and in certain other embodiments, the cells are human cells. In certain other embodiments, the cells are human central nervous system cells.

In yet another aspect, the invention provides a method of identifying a factor that can alter the expression of contactin in a cell. The method comprises comparing the level of contactin expression in the at least one cell before and after contacting the at least one cell with the candidate agent, and identifying in the array reagents that may alter contactin expression. The step of performing is included. In a particular embodiment, the candidate agent is
Includes test compounds that are small molecules, nucleic acid molecules, antisense nucleic acid molecules, or ribozymes. In another embodiment, at least one cell comprises a cybrid cell. In another embodiment, the invention provides factors capable of altering contactin, as identified by the methods described herein. In a particular embodiment, the agent is a small molecule, protein, polypeptide, antibody, nucleic acid molecule, antisense molecule, or ribozyme. In another embodiment, the present invention provides a pharmaceutical composition containing the agent in a pharmaceutically acceptable carrier. A further aspect of the invention is to provide a method of treating a patient having a neurodegenerative disease comprising the step of administering to said patient an effective amount of such a pharmaceutical composition.

The invention also provides, in another aspect, treating a patient having a neurodegenerative disease, comprising administering to the patient an effective amount of a pharmaceutical composition capable of altering the expression of contactin. Provide a way to do. In a particular embodiment, the present invention provides a pharmaceutical target containing a cellular component that binds to a factor capable of altering the expression of contactin, as described above. In certain other embodiments, the invention comprises contacting a biological sample known to exhibit contactin expression or activity with a compound that modulates contactin expression or activity. A method of identifying a target is provided.

In another embodiment, the invention provides for Alzheimer's disease or another in a first subject having or suspected of having such a neurodegenerative disease. Methods are provided for determining risk or the presence of a neurodegenerative disease. The method comprises at least one differentially expressed nucleic acid molecule associated with Alzheimer's disease or another neurodegenerative disease in each of the first and second biological samples containing mitochondrial DNA. Determining the presence or absence of, and thereby determining the risk of Alzheimer's disease or another neurodegenerative disease or its presence. Here, the first biological sample described above is obtained from a first subject, and the second sample described above is at risk of disease associated with altered mitochondrial function or Obtained from a second subject known to be absent. Here, the presence of at least one differentially expressed nucleic acid molecule associated with Alzheimer's disease or another neurodegenerative disease in a first biological sample and the corresponding in a second biological sample The absence of the corresponding differentially expressed nucleic acid molecule having a nucleotide sequence that represents an increased risk of Alzheimer's disease. In certain further embodiments, the determining step comprises the step of subjecting each of the first biological sample and the second biological sample described above to a population of isolated nucleic acid molecules immobilized on a solid support. Contacting with an array of the containing nucleic acids under conditions and for a time sufficient to allow hybridization of the DNA from the sample to the isolated nucleic acid molecule (here Where the isolated nucleic acid molecule comprises at least one differentially expressed nucleic acid molecule associated with Alzheimer's disease or another neurodegenerative disease), as well as: (i
A.) The amount of hybridization of the nucleic acid molecule differentially expressed and associated with Alzheimer's disease or another neurodegenerative disease in the first sample to the array of nucleic acids, (ii) differentially expressed and the first Comparing the amount of nucleic acid hybridization of a second sample corresponding to a nucleic acid molecule associated with Alzheimer's disease or another neurodegenerative disease in the sample of: At least one associated with degenerative disease
Determining the presence or absence of three differentially expressed nucleic acid molecules.

These and other aspects of the invention will be apparent with reference to the following detailed description. Further, various references are set forth herein and describe more specific specific aspects of the invention and are therefore hereby incorporated by reference in their entireties.

DETAILED DESCRIPTION OF THE INVENTION The present invention comprises, in part, expression levels of certain novel molecules described herein, as described in greater detail below, and 1 Unexpected findings of expression levels of certain known molecules associated with one or more neurodegenerative diseases include:
Related. As noted above, abundant reference is made herein to altered expression levels of contactin-encoding nucleic acid molecules, such as in representative examples, but the invention is not limited to other It is understood that it also relates to some of the additional nucleic acid molecules described herein that encode the product. These products include products having novel sequences as well as products having known sequences.
Thus, the present invention relates in part to the surprising observation that contactin, a recognition molecule associated with GPI-linked neuronal cells (as a representative example), is associated with neurodegenerative diseases. In certain embodiments, a neurodegenerative disease can be correlated with altered (eg, increased or decreased) levels of contactin in a statistically significant manner. For example, as provided herein, an altered level of contactin expression can be observed as an increase or decrease in the amount of contactin protein in the sample compared to the control sample. As another example, altered levels of contactin can be detected as altered levels of contactin mRNA, as also described in further detail below.

Thus, the present invention provides for a contactin protein or mRNA in a control sample from a second subject known to be at risk of not having or having a neurodegenerative disease. The level of contactin expression, such as the amount of contactin protein or contactin mRNA in the test sample, to the risk of having a neurodegenerative disease or its presence in a subject. Partly related to the method. The present invention also relates to methods of correlating contactant expression with the suitability of agents for treating neurodegenerative diseases in at least one subject, thereby appearing to respond to a particular treatment. Methods are provided for identifying these patients with neurodegenerative diseases.

The present invention also relates in part to oligonucleotide primers capable of specifically amplifying DNA or RNA encoding contactin, or a nucleic acid sequence complementary thereto. Such a primer can be used to
It can be a nucleic acid molecule that can act as a PCR primer for the detection of RNA or contactin DNA. Such nucleic acid molecules can also be labeled and provided in a kit.

With respect to the surprising relationship between contactin and neurodegenerative diseases, the present invention further provides compositions and methods for drug screening assays. Drug screening assays, for example, of cybrid cells to identify agents that may be useful in the treatment of neurodegenerative diseases when formulated in pharmaceutical compositions as provided herein. Including use. Similarly, the present disclosure provides methods for identifying molecular targets for pharmaceutical agents that alter contactin expression levels, as well as related therapeutic methods.

Neurodegenerative diseases to which the present invention is applied include, but are not limited to, Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD), chronic Lewy body disease (DLB). , Vascular dementia, and other neurodegenerative diseases.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Have. Generally, the nomenclature used herein, cell culture, chemistry, microbiology, molecular biology, laboratory procedures in cell chemistry, and cell cultures as described below are Well known and commonly used in the art. Conventional methods such as those provided in the art and various general references are used for these procedures (Sambrook.
Et al., Molecular Cloning: A Laboratory Man.
ual, Second Edition, Cold Spring Harbor Press, Col
d Spring Harbor, N.D. Y. (1989)). When terms are provided in the singular, we also intend the plural of these terms. The nomenclature used herein and the laboratory procedures described below are well known and commonly used in the art. As used throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: "Membrane-permeable derivative" increases the membrane-permeability of a compound. A chemical derivative of the compound. These derivatives become better able to cross cell membranes because hydrophilic groups are masked to provide more hydrophobic derivatives. Also, the masking group can be designed to be cleaved from the compound within the cell, making the compound more hydrophilic once inside the cell. Since the substrate is more hydrophilic than the transmembrane derivative, it is preferentially localized intracellularly (Tsien et al., US Pat. No. 5,741,657, issued Apr. 21, 1998). .

“Isolated polypeptide” refers to a genomic polynucleotide, cDNA, or of synthetic origin, or a combination thereof. By these origins,
An isolated polynucleotide is operably linked to a polynucleotide that (1) is not associated with cells in which the isolated polynucleotide is naturally found, or (2) is not naturally linked. ing. The isolated polynucleotide can be operably linked to a promoter, enhancer, or other regulatory sequence, if desired.

“Isolated protein” refers to cDNA, recombinant RNA, or of synthetic origin, or a combination thereof. By its origin, the isolated protein is (1) not associated with proteins normally found in nature,
Or (2) isolated from normally existing cells, or (3) isolated from other proteins (eg, free of cellular proteins) from the same cell source. , Or (4) expressed by cells from different species, or (5) not naturally occurring.

“Polypeptide” is used herein as a genetic term such as an analog of a naturally occurring protein, fragment, or polypeptide sequence.

“Active fragment” refers to a fragment of a parent molecule such as an organic molecule, nucleic acid molecule, or protein or polypeptide, or combination thereof.
They retain at least one activity of the parent molecule.

“Naturally occurring” refers to the fact that a subject can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and that has not been intentionally modified by humans in the laboratory is a naturally occurring polypeptide or polynucleotide. There is.

“Operably linked” refers to the juxtaposition in which the components so described are in a relationship that enables them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

“Regulatory sequences” refers to polynucleotide sequences that effect expression of coding and non-coding sequences to which they are linked. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences will generally be
It includes a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and transcription termination sequence. It is contemplated that the term regulatory sequence may include components whose presence may affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. .

A “polynucleotide” is at least 10 modified from either ribonucleotides or deoxynucleotides, or any type of nucleotide.
Refers to a multimolecular form of nucleotides of base length. This term refers to DNA or R
A includes single-stranded and double-stranded forms.

[0047]   "Genomic polynucleotide" refers to a portion of the genome.

An “active genomic polynucleotide” or “active portion of the genome” is upregulated, downregulated, either directly or indirectly by a biological process, Alternatively, it refers to a region of the genome that can be both of them.

In the context of biological process (es), “direct” is by the contact or binding of one molecule to another (the same type of molecule or a different type of molecule). It usually refers to causing the process directly without the need for the intermediate steps involved. For example, molecule A contacts molecule B, which causes molecule B to exert effect X, which is part of the biological process.

“In the context of biological process (es),“ indirectly ”means 2
Indirectly requiring intermediate steps that are usually caused by one or more direct steps. For example, molecule A contacts molecule B to exert effect X and then effect Y.

“Sequence homology” refers to the percentage of base matches between two nucleic acid sequences, or the percentage of amino acid matches between two amino acid sequences. Sequence homology is given, for example, as a percentage of 50%. This percentage indicates the percentage length match of the sequence derived from the desired sequence compared to some other sequences. Gaps (either of the two sequences) are allowed to maximize the match; gap lengths of 15 bases or less are commonly used, preferably 6 bases or less, more preferably 2 bases or less. . When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally less than 17 of the 20 possible oligonucleotide base pair matches. Target base match (85%)
Preferably less than 9 of the 10 possible base pair matches (
90%), and most preferably one of the 20 possible base pair matches.
A match of less than 9 (95%).

“Selectively hybridize” refers to being detectable and specifically binding. Polynucleotides, oligonucleotides, and fragments thereof, selectively bind to strands of the target nucleic acid under hybridization and wash conditions that minimize a significant amount of detectable binding to non-specific nucleic acids. Hybridize. Conditions of high stringency can be used to achieve selective hybridization conditions known in the art. In general,
The nucleic acid sequence homology between the polynucleotide, oligonucleotide, and fragments thereof and the nucleic acid sequence of interest is at least 30%, and more typically and preferably, at least 40%, 50%, 60 %, 70%, 80%
, Or 90%.

Hybridization and wash conditions are typically performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For example, if the full length polynucleotide sequence is known in the art, the full length polynucleotide sequence can be labeled,
It is then used as a hybridization probe to isolate genomic clones from the appropriate target library. Representative hybridization conditions and methods for screening plaque lifts and for other purposes are known in the art (Benton and Davis, Science).
196: 180 (1978); Sambrook et al., Supra (1989)).

Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for the best match. Gaps (in either of the two sequences that match) are allowed to maximize the match; gap lengths of 5 or less are preferred and 2 or less are more preferred. Alternatively, and preferably, the sequences of the two proteins (or polypeptide sequences derived from those of at least 30 amino acids in length) are such that when the term is used herein, they have a mutation data matrix. Are homologous if they have an alignment score of at least 5 (standard deviation units) and a gap penalty of 6 or more using the program ALIGN with (Dayhoff, Atlas of Protein Sequence.
and Structure, National Biomedical Re
search Foundation, Volume 5, pp. 101-110 (1972).
, And Supplement 2, 1-10). Two sequences or parts thereof are more preferably homologous if their amino acids are 30% or more identical when optimally aligned using the ALIGN program.

“Corresponding” means that the polynucleotide sequence is homologous (eg, evolutionarily identical in all respects) to the reference polynucleotide, or that the polypeptide sequence is It is identical to all or part of the nucleotide sequence of the reference sequence. In contrast, the term "complementary" is used herein to mean a sequence in which the complementary sequence is homologous to all or part of a reference polynucleotide sequence. For example, the nucleotide sequence TATA
C corresponds to the reference sequence TATAC and is complementary to the reference sequence GTATA.

The following terms are used to describe the sequence relationships between two or more polynucleotides: “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”. , And "substantial identity". A reference sequence is a defined sequence used as a basis for sequence comparisons; the reference sequence may be a subset of a large sequence (eg, as a full-length cDNA given in the sequence listing or a segment of a gene sequence). Alternatively, it may contain the complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides in length, and often at least 50 nucleotides in length. Each of the two polynucleotides may include (1) a sequence that is similar between the two polynucleotides (eg, a portion of the complete polynucleotide sequence), and (2) further differing sequences between the two polynucleotides. As such, sequence comparisons between two (or more) polynucleotides typically include two “comparison windows” over the “comparison window” to identify and compare local regions of sequence similarity. This is done by comparing the sequences of the polynucleotides. Comparison window, as used herein, refers to a conceptual segment of at least 20 consecutive nucleotide positions. Here, a polynucleotide sequence can be compared to a reference sequence of at least 20 contiguous nucleotides, and a portion of the polynucleotide sequence in the comparison window can be compared for optimal alignment of the two sequences (this May contain up to 20% additions and deletions (eg gaps) when compared to (without additions or deletions). Optimal alignment of sequences for aligning comparison windows is accomplished by a local homology algorithm (Smith and Waterman,
Adv. Appl. Math. , 2: 482 (1981)) by the homology alignment algorithm (Needleman and Wunsch, J. Mo.
l. Biol. , 48: 443 (1970)) by a search method for similarity (Pearson and Lipman, Proc. Natl. Acad. S).
ci. U. S. A. 85: 2444 (1988)), GAP, BESTFIT,
By computerized implementation of these algorithms, such as FASTA, and TFASTA (Wisconsin Genetics Software
are Page Release 7.0, Genetics Comput
er Group, Madison, WI), or by visual inspection. Preferably the best alignment produced by various methods
(Eg, the result with the highest percentage of homology over the comparison window) is
To be selected.

“Sequence identity” means that two polynucleotide sequences are identical (eg, on a nucleotide-by-nucleotide basis) over the window of comparison.

“Percentage of sequence identity” is the comparison of two optimally aligned sequences over a window of comparison, determining the number of positions in both sequences that result in the same nucleobase. Getting the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (eg window size),
It is then calculated by multiplying the result by 100 to obtain the percent sequence identity.

“Substantial identity,” as used herein, refers to a characteristic of a polynucleotide sequence. Here, the polynucleotide is often at least 25 to 50 over a comparison window of positions of at least 20 nucleotides.
Have a sequence identity of at least 30% when compared to a reference sequence over a comparison window of nucleotides, preferably at least 50 to 60% sequence identity, more usually at least 60%. Includes sequences with% sequence identity. Here, the percentage of sequence identity is calculated by comparing the reference sequence to a polynucleotide sequence which may contain deletions or additions of less than 20% of the total of the reference sequence over the comparison window.

“Substantial identity,” when applied to a polypeptide herein, for example, uses the default gap weight to program GAP or BEST.
When optimally aligned by FIT, the two peptide sequences share at least 30% sequence identity, preferably at least 40% sequence identity, and more preferably at least 50% sequence identity, And most preferably it means to share at least 60% sequence identity. Preferably,
The positions of non-identical residues differ by conservative amino acid substitutions.

“Conservative amino acid substitution” refers to the interchangeability of residues having chains of similar size. For example, the group of amino acids having an aliphatic side chain is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having an aliphatic-hydroxyl side chain is serine and threonine. Yes, the group of amino acids having side chains containing amides is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; The group of amino acids with a basic side chain is lysine, arginine, and histidine; and the group of amino acids with a side chain containing sulfur is cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamine-asparagine; and aspartic acid-glutamic acid.

“Modulation” refers to the ability to either enhance or inhibit a functional property of a biological activity or process (eg, enzymatic activity or receptor binding). Such enhancement or inhibition may be incidental to the appearance of specific events (eg activation of signaling pathways) and / or may be prominent only in certain cell types.

A “modulator” is a chemical agent (naturally or non-naturally occurring) such as a biological fluid macromolecule (eg, nucleic acid, protein, non-peptide, or organic molecule). ), Or biological material (eg, prokaryotes, bacteria,
Eukaryote, plant, fungus, multicellular organism, or animal, invertebrate, vertebrate,
Mammals and humans). These include whole organisms or parts thereof, cells, organs, tissues, body fluids, whole cultures or parts of cultures, or extracts of environmental samples or parts thereof, where appropriate. . Modulators are typically inhibitors (directly or indirectly) of a biological process (es) or activators (eg, agonists, partial antagonists, partial agonists, antagonists, new Transformant of an organism or an inhibitor of antineoplastic cytotoxicity of cell growth, cell growth promoting factor, antiviral agent, antibacterial agent, antibacterial agent, antibiotic agent, etc.) It is evaluated by inclusion in the assay described in. The activity of the modulator may be known, unknown, or partially known.

“Test compound” refers to a chemical agent, compound, composition, or extract that is tested for being a putative modulator by at least one method of the present invention. A test compound can be a candidate reagent and can be a small molecule (eg, small molecule, drug, protein, or peptide, or active fragment thereof, eg, antibody, nucleic acid molecule (eg, DNA, RNA, or combinations thereof). ), Antisense molecules, ribozymes, or other organic or inorganic molecules (eg, lipids, carbohydrates,
Or any combination thereof)). A test compound containing a nucleic acid molecule is
It can be provided in a vector (eg, a viral vector such as a retroviral vector, an adenovirus, or an adeno-associated viral vector), a liposome, a plasmid, or can be provided with a lipofection reagent. The test compound once identified can be an agonist, an antagonist, a partial agonist, or an inverse agonist of the target. Test compounds are usually not known to bind the target of interest. "Control test compound" refers to a compound known to bind to a target (eg, known agonist, antagonist, partial agonist, or inverse agonist). The test compound typically does not include the compound added to the mixture as a control condition that modifies the function of the target to determine the specificity of the signal in the assay. Such control compounds or conditions include the following chemicals: (1) chemicals that non-specifically or substantially disrupt protein structure (eg, urea or guandium,
Denaturing agents such as sulfhydryl reagents (eg, dithiothritol and β-mercaptoethanol), (2) chemicals that generally inhibit cellular metabolism (eg, mitochondrial uncouple), and (3) protein electricity. Chemicals that nonspecifically disrupt static or hydrophobic interactions (eg, high salt concentrations or surfactants at concentrations sufficient to nonspecifically disrupt hydrophobic or electrical interactions). . The term test compound also typically does not include compounds known to be unsuitable for therapeutic use of a particular indicator due to the toxicity of the subject. Normally, various predetermined concentrations of test compounds are used to determine their activity. If the molecular weight of the test chemical is known, concentrations in the following ranges can be used: between about 0.001 micromolar and about 10 millimolar, preferably between about 0.01 micromolar and about 1 millimolar. , More preferably between about 0.1 micromolar and about 100 millimolar. If the extraction uses a test compound, the concentration of test chemical used can be expressed by weight relative to the volume basis. Under these circumstances, the following concentration ranges may be used: between about 0.001 microgram / ml and about 1 mg / ml, preferably between about 0.01 microgram / ml and about 100 microgram / ml. , And more preferably between about 0.1 microgram / ml and about 10 mg / ml.

“Target” refers to a biochemical substance involved in a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. The therapeutic composition or compound typically binds to the target in order to alter or modulate its function. As used herein, targets include cell surface receptors, G proteins, G protein coupled receptors, kinases, phosphatases, ion channels, lipases, phospholipases, nuclear receptors, intracellular structures, tubules, tubulins and the like. However, the present invention is not limited to these.

“Label” or “labeled” refers to the incorporation of a detectable marker (eg,
Or by the attachment of a labeled compound) or by attachment of a moiety such as biotin to a polypeptide that can be detected by binding of a secretory moiety such as marked avidin. Various methods of labeling polypeptides, nucleic acids, carbohydrates, and other biological or organic molecules are known in the art. Such a sign
It may carry a variety of information such as radioactivity, fluorescence, color, chemiluminescence, or other readouts known in the art or later developed. Readout information includes enzymatic activity (eg, β-galactosidase, β-lactamase, horseradish peroxidase, alkaline phosphatase, luciferase); radioisotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, or ¹³¹ I); It may be based on fluorescent proteins (eg green fluorescent protein); or other fluorescent labels (eg FITC, rhodamine, and lanthanoids). Where appropriate, these labels can be the expression product of the reporter gene, as the term is understood in the art. Examples of reporter genes are β-lactamase (Tsien et al., Issued April 21, 1998, US Pat. No. 5,741,657), and green fluorescent protein (Tsien et al., Issued July 7, 1998). US Patent No. 5,777,
079; Cormack et al., US Pat. No. 5, issued September 8, 1998.
804,387).

“Substantially pure” refers to the species or species of interest for which a superior species or activity exists (eg, on a molecular basis where it is richer than any other individual species or activity in the composition). Refers to activity. And preferably, the substantially purified fraction is the composition in which the species or activity of interest comprises at least about 50% (on a molar, weight or activity basis) of all macromolecules or activity present. Generally, a substantially pure composition is greater than about 80% of all macromolecular species or activity present in the composition, more preferably about 85%, 90%, 95%, and 99%. Including more. Most preferably, the species or activity of interest is purified for essential homogeneity.
Here, the contaminating species or activity cannot be detected by conventional detection methods. Here, the composition consists essentially of a single macromolecular species or activity. The inventors recognize that the activity may be directly or indirectly caused by a single species or multiple species in the composition, especially the extract.

A “pharmaceutical agent or drug” is a chemical, composition that is capable of inducing a desired therapeutic effect when properly administered by the appropriate dose, regimen, route, time and mode of administration. Thing or activity.

“Pharmaceutically effective amount” refers to the appropriate dose of administration, regime, route, time, and mode of delivery, which is associated with delivery of an amount of a compound or composition to produce the desired effect. Such a pharmaceutically effective amount can be obtained using the methods described herein or in the United States Foods and Drug Admi.
determination (USFDA).

“Sample” refers to any biological sample, preferably derived from a test animal such as a mouse, rat, rabbit, or monkey, or a patient such as a human. The sample may be neural tissue, central nervous tissue, internal organs (eg, pancreas, liver, lung, kidney, muscle, skeletal muscle, urine, stool, blood, body fluid from body cavities, or central nervous system).
Can be from any tissue or body fluid, such as, or can be a sample from various body cavities such as the mouth or nose. Samples derived from urine and feces
It includes cells of the immunological, ureteral, or gastrointestinal tract and can be a rich source of samples. Such samples may be prepared using methods known in the art (eg biopsy,
Can be obtained using aspiration, scraping, or simple withdrawal). Samples can be taken from test animals or patients that are either alive or dead.

“Ribozyme” means an enzymatic RNA molecule capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action is sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by intranucleotide (end).
Incleolytic). RNA encoding contactin
Within the scope of the present invention is an engineered hammerhead motif ribozyme molecule capable of specifically and efficiently catalyzing cleavage within the nucleotide. A specific ribozyme cleavage site within any possible RNA target has the sequence GUA, GU
Initially identified by scanning the target RNA target for ribozyme cleavage sites, including U, and GUC. Once identified, a short RNA sequence of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may lead to secondary structural features that may render the oligonucleotide inoperable. Can be evaluated. The suitability of candidate targets can also be assessed by testing their accessibility to hybridization with complementary oligonucleotides using a ribonuclease protection assay.

“Contactin” refers to contactin mRNA, contactin DNA, and contactin protein. By "contactin protein" is meant a protein that exhibits at least one activity of at least one human contactin F3 or F11. A "contactin mRNA" is an mRNA molecule that encodes a contactin protein. Preferably the contactin mRNA is derived from nuclear DNA, which may be derived from mitochondrial DNA. "Contactin DNA
Is a DNA molecule that encodes a contactin protein. Preferably, the contactin DNA is nuclear DNA, but it can be mitochondrial DNA.

Other technical terms used herein are McGraw-Hill Di.
motionary of Chemical Terms and Stedm
They have their usual meaning in the art in which they are used, as described by various technical dictionaries such as an's Medical Diary.

As noted above, the present invention provides for the expression of contactin in cells (typically other novel ones described herein that are within the scope of the invention as described above). Products and the expression of known products) are associated with neurodegenerative diseases. As a non-limiting introduction to the breadth of the present invention, the present invention includes several general and useful aspects including: (i) Contactin mR in a sample.
A nucleic acid molecule that can act as a PCR primer for the detection of NA or contactin DNA; (ii) providing a sample from a patient, and of the contactin protein, contactin DNA, or contactin mRNA expressed in the sample Detecting the presence of a neurodegenerative disease (eg, multiple sclerosis, AD, PD, DLB, or other neurodegenerative disease as provided herein) comprising measuring the amount. (Iii) a method for identifying a patient having a neurodegenerative disease that appears to respond to treatment of a neurodegenerative disease; (iv) normal when compared to control cells Cells and cell lines, including cybrid cell lines expressing different, increased or decreased amounts of contactin; (v) contact And compounds or compounds or compounds (including pharmaceutical compositions identified by these methods that reduce or enhance contactin expression) to alter expression (eg, increase or decrease) of compounds (Vi) a method of treating a neurodegenerative disease using the composition or compound identified by the method of the invention; and (vii) contactin DNA, contactin mRNA, or contactin protein in a cell. A pharmaceutical target for a compound that alters or modulates an amount and a method for identifying a target identified by a method of the invention.

These aspects of the invention, as well as other aspects described herein, are obtained by using the methods, articles of manufacture, and compositions of matter described herein. , Can be achieved. It is further appreciated that various aspects of the invention may be combined to achieve a desired embodiment of the invention, in order to gain a full appreciation of the scope of the invention.

Nucleic Acid Molecules The present invention relates to contactin mRNA or contactin DNA (or other nucleic acid molecule encoding other products as described herein) in a sample.
Nucleic acid molecule that can function as a PCR primer for the detection of Such nucleic acid molecules can be detectably labeled and provided in a kit.

Human contactin protein and its homologue from mouse (F3 protein) and its homologue from chicken (F11 protein) are cell surface proteins, which are associated with the adhesion of cells to substrates. Contactin contains an Ig-like domain and multiple fibronectin III-like domains. Contactin, but not a transmembrane protein, adheres to glycosylphosphatidylinositol (GPI) on the outer membrane of the cell surface. Relatively high levels of contactin mRNA (6.5 kb) resulted in three minor transcripts (9.7 kb,
Although expressed in adult brain with 4.4 kb, and 3.4 kb), low levels of expression of multiple forms of contactin mRNA result in adult lung, pancreas, kidney, and skeletal muscle (6. Seen at 8 kb and 6.0 kb). High levels of expression of multiple forms of contactin mRNA are found in neuroblasts and retinoblasts of the cancerous cells (6.8 kb, 6.0 kb, and 4.2 kb). Expression of contactin in developing neural tissue is complex and transient, with binding to the cell recognition molecule Ng-CAM, and with the extracellular matrix glycoprotein restrictin in neurite outgrowth. It is thought to have an interaction role. Adult neural stem cells can give rise to hematopoietic cells, spinal cord cells, and lymphoid cells (Bjorson et al., Science 283: 534 et al. (1998)).
Thus, contactin mRNA, contactin DNA, or contactin protein can be detected in blood.

Various human contactin mRNA sequences have been reported. Reid and H
emperly is a human contactin mR having 3360 nucleotides
Reported NA (registration number Z21488) (Reid and Hemper
ly, Bran Res. 21: 1-8 (1994)). Berglund and Ranscht report an mRNA (registration number U07819) that encodes the human contactin 1 precursor having 3314 nucleotides (Berglu.
nd and Ranscht, Genomics 21: 571-582 (199).
4)). Berglund and Ranscht have mRNAs (registration number U078) encoding a human contactin 2 precursor having 3335 nucleotides.
20) (Bergrund and Ranscht, Genomics)
21: 571-582 (1994)). Watanabe et al., MRNA encoding bovine F3 / F11 / contactin having 3412 nucleotides.
(Watanabe et al., Gene 160: 245-248 (199).
5)). Hosoya et al. Report an mRNA encoding rat F3 having 3214 nucleotides (Hosoya et al., Neurosci, Lett.
． 186: 2-3 (1995)). Some sequences of Neuro-1, human contactin, mouse F3, and chicken F11 have been compared (Reid et al., Mol. Brain Res. 21: 1-8 (1994)). Also, PCR primers for mouse F3 have been reported (Reid et al., Mol. Br.
ain Res. 21: 1-8 (1994)). Functional domains of chicken F11 have been mapped using deletions of various regions of chicken F11 (
Brummendorf et al., Neuron 10: 711-727 (1993).
). These functional domains have been compared to surface molecules that have similar functions (Brummendorf and Rathjen, J. Neuroche.
m. 61: 1207-1219 (1993)).

We intend that there are various allelic variants of contactin that may have sequences different from those reported in the literature. Allelic variants can have sequences that differ from the reported sequences, such as sequences that naturally occur by deletions, insertions, or substitutions. Allelic variants may have different structures or different functions than the contactins reported in the literature. Such allelic variants are contemplated by the inventors to encode contactin.

In addition to allelic variants, we encode contactins that contain deletions, insertions, or substitutions of different nucleotides, whereby the same, functionally equivalent contactin, or parental An altered nucleic acid sequence that results in a polynucleotide encoding contactin that retains at least one activity of contactin is contemplated. Such altered nucleic acid sequences can be made using art-established methods, such as site-directed mutagenesis or random mutagenesis, as they are known in the art. . The protein encoded by such an altered nucleic acid sequence may exhibit equivalent, similar, or different structure or activity when compared to the molecule of the invention. It is believed by the present inventors that such an altered nucleic acid sequence encodes contactin.

The present invention comprises nucleic acid molecules that are useful as primers for use in a specific PCR amplification procedure for the amplification of at least one contactin mRNA or contactin DNA, particularly in a sample of human origin ( For PCR procedures, see US Pat. No. 4,683,195; US Pat. No. 4,965,188;
And Innis et al., PCR Strategies, Academic Pr.
ess, San Diego (1995)). Such PCR amplification methods are known in the art and include primer extension PCR, real-time PCR, reverse transcriptase PCR (Freeman et al., BipTechnique).
s 26: 112-126 (1999)), inverse PCR (Triglia et al., Nu.
cleic Acids Res. 16: 8186 (1988)), capture PCR
(Lagerstrom et al., PCR Methods Appl. 1:11.
1-119 (1991)), differential primer extension (
Fahy and Ghosh, WO 96/3, published October 3, 1996.
No. 0545) and other PCR amplification methods known in the art or later developed (Innis et al., PCR Strategies, Acade).
mic Press, San Diego (1995)).

In operation, PCR methods generally use primer molecules, which are usually chemically synthesized, but these can be made enzymatically or produced by recombinant steps. PCR primers are commonly used under preferred conditions for the identification of specific genes or conditions, two nucleotide sequences (one in sense direction (5 '→ 3') and the other in antisense). Direction (3 '→ 5')
Is included). Same PCR primers, nested set of oligomers,
Alternatively, a degenerate pool of oligomers can be used under less stringent conditions for detection and / or quantification of closely related DNA or RNA sequences.

In addition, methods that can be used to quantify the expression of particular molecules include radiolabels (Melby et al., J. Immunol Methods 159: 235.
-244 (1993)) or biotinylated (Duplaa et al. Anal. Bi.
ochem. 299-236 (1993)) nucleotides, co-amplification of control nucleic acids, and standard curves to which experimental results are written. Quantitation of multiple samples can be facilitated by performing the assay in an ELISA format in which the nucleic acid molecule of interest is presented in various dilutions, and spectrophotometer or colorimetric response yields rapid quantitation. Colorimetric inte
rcalating dyes can be used in such quantitation methods, as described in the examples and known in the art (Freeman).
Et al., BioTechniques, 26: 112-125 (1999); and Spiess et al., BioTechniques 26: 46-50 (1999).
).

These nucleic acid molecules can also be used individually or in combination as probes to identify contactin mRNA or DNA molecules in a sample. These nucleic acid molecules include: 5'-TCAGTAAGGTCTGGTTCACGCTAT-3 '(SEQ ID NO: 1
) 5'-TCCCGTCACTGTAGATTCATTTGA-3 '(sequence number 2
) 5'-CCCCAAGTCTTCTCGCGCTTA-3 '(sequence number 3) 5'-CAACACATTCAGAGATTCCAAGTAGACA-3' (sequence number 4) 5'-TCCCCAAGTCTTCTCGGCTTA-3 '(sequence number 5) 5'-CCCATCCCAGCTCAGAAGAC-3' (sequence number). And 5'-GCCGCAGAAATTGGAAGG-3 '(SEQ ID NO: 7) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6 are forward primers. SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 7 are reverse primers.

The present invention provides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4,
A primer having substantial identity to the nucleic acid molecule of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a portion thereof or an antisense version thereof, is used to detect the contactin RNA or DNA in the sample. As long as it can be specifically amplified, it is included.

Other primers are those in which they are in the sample, especially in samples of human origin.
Of at least one human contactin mRNA or contactin DNA can be utilized in such PCR procedures.
Such PCR primers are described in Altschul et al., Nucleic Aci.
ds Res. 25: 3389-3402 (1997), and can be selected by identifying at least one stretch of contactin DNA or RNA that is unique to at least one contactin DNA or mRNA. The specificity of amplifying at least one contactin mRNA or contactin DNA of such a primer can be determined using the method of the invention as described by the examples. The present invention has substantial identity to a nucleic acid molecule thus identified or a portion thereof, so long as the primer is capable of specifically amplifying at least one contactin mRNA or contactin DNA in the sample. Includes primers.

The invention also includes antisense versions of these nucleic acid molecules. Such an antisense molecule may be DNA or R encoding contactin in a sample.
It is useful as a probe for detecting NA. Such antisense sequences can be determined by deducing antisense sequences from a given sequence. Such antisense molecule can be either DNA or RNA,
And those of skill in the art will, of course, recognize the different base pairs used in the coding schemes for these different nucleic acid molecules. These antisense molecules can be used to regulate gene expression of targeted sequences. Preferably, such antisense molecules target the transcription start site of a gene to prevent transcription or mRNA to prevent translation or binding of the ribozyme to the mRNA.
Target the molecule. Inhibition of gene transcription or translation also includes the ability to open the double helix sufficiently for the binding of polymerases, transcription factors, or regulatory molecules,
This can be accomplished using the "triple helix" base pair methodology. Recent therapeutic benefits of using triple-helix DNA have been reviewed by Gee et al. (Huber and Carr, Molecular an).
d Immunological Approaches, Futura Publ
Ishing Co. , N .; Y. (1994)).

Nucleic acid molecules of the invention can be made by a variety of methods known in the art. For example, nucleic acid molecules can be made using synthetic procedures or molecular biology techniques known in the art (see Sambrook et al., Supra).

The length of the nucleic acid molecule of the present invention can be easily selected by the person skilled in the art depending on the particular purpose for which the nucleic acid molecule is used. For PCR primers, the length of the nucleic acid molecule is preferably between about 10 nucleotides and about 50 nucleotides,
More preferably, it is between about 12 and about 30 nucleotides, and most preferably, between about 15 and about 20 nucleotides. For probes, the length of the nucleic acid molecule is preferably between about 20 nucleotides and about 1000 nucleotides, more preferably about 100 nucleotides.
It is between nucleotides and about 500 nucleotides in length, and most preferably between about 200 nucleotides and about 400 nucleotides.

The nucleic acid molecule of the present invention may be linked to a detectable label so as to form a labeled nucleic acid molecule. Such labeled nucleic acid molecules can be made using methods known in the art. Such labeled nucleic acid molecules can be used to detect contactin DN in a sample using established nucleic acid hybridization methods (eg, solid phase hybridization or in situ hybridization).
It is useful alone or in combination as a probe to detect A or contactin mRNA. Such labeled probes can be used in PCR procedures that utilize labeled primers, including multiple labeled primers such as those known in the art (eg, fluorescence resonance energy transfer ( FR
ET) based amplification procedure).

Labeled or unlabeled nucleic acids of the invention can be provided separately or in combination in a kit to carry out at least one method of the invention.
The nucleic acid molecule can be provided in one or a separate container along with other reagents, buffers, or substances used in performing at least one method of the invention. The kit may be provided in a container, such as a package container, which may optionally include instructions for carrying out at least one method of the invention. The instructions may be provided in any language or format, particularly a language and format that is targeted to the end user so that the end user may perform at least one method of the present invention.

Methods for Detecting the Presence of Neurodegenerative Diseases The present invention also includes methods for detecting the presence of neurodegenerative diseases such as multiple sclerosis. The method comprises providing a sample from a patient and measuring the amount of expressed contactin protein, contactin DNA, or contactin mRNA in the sample.

Samples for use in this method can be taken from any tissue, organ, or body fluid from the patient. Such samples can be prepared using methods known in the art (eg,
Biopsy, aspiration, or scraping). Preferred samples include samples derived from the nervous system, including the central nervous system, pancreas, lung, kidney, blood, mouth, nasal cavity, urine, stool, and skeletal muscle. Preferably, the sample is derived at least in part from the patient's central nervous system. The samples can be used when they are taken from the patient, or can be processed, for example, by thin section preparation, homogenization, or culturing cells in the sample using established methods. Can be used for. Once the sample is prepared for a particular detection method, contactin mRNA, contactin DNA in the sample
, Or the amount of contactin protein can be determined.

Contactin mRNA in a sample can be measured using a variety of established methods, such as the PCR method known in the art. Such PCR methods utilize suitable nucleic acid molecules of the invention, as they are discussed in previous sections and examples. Contactin mRNA can also be measured using hybridization methods such as blot analysis (eg Northern blot analysis or slot / dot blot analysis) and in situ hybridization such as methods known in the art.

Contactin DNA can be measured using established methods, such as hybridization methods known in the art. Such hybridization methods utilize suitable nucleic acid molecules of the invention, as they are discussed in the previous section. Contactin DNA can also be hybridized by methods such as blot analysis such as Southern blot analysis or slot / dot blot analysis.
Alternatively, it can be measured using in situ hybridization, such as methods known in the art. Contactin DNA may also be detected by fluorescent in situ hybridization (FI) using the nucleic acid molecules of the invention, as is known in the art.
SH) method can be used to determine in chromosomes or cell preparations (Verma et al., Human Chromosomes: A Manual of Basic).
Techniques, Pergamon Press, N.M. Y. (1988
)).

Contactin protein in a sample can be determined using various methods. For example, immunological methods such as ELISA, Western blot analysis, or immunocytochemical analysis can be utilized. Generally, these methods use a primary antibody specific for contactin. Such antibodies are known in the art or can be made using established methods known in the art (Faivre-Sarrailh et al., J. Nurosci., 12: 257-.
267 (1992); Brummendorf et al., Neuron 10: 711.
-727 (1993); and Pesheva et al., Neuron 10: 69-.
82 (1993)). The primary antibody can be attached to a detectable label so that binding of the primary antibody to contactin in the sample can be detected. Alternatively, the primary antibody is not attached to a detectable label. In this example, specifically binds to the primary antibody (preferably at the Fc region of the primary antibody)
Secondary antibody is used. The secondary antibody is attached to a detectable label so that binding of the primary antibody to contactin is detected.

Then, preferably, contactin mRNA, contactin D in the sample
The amount of NA, or contactin protein, is compared to the amount of contactin mRNA, contactin DNA, or contactin protein in the control sample. Appropriate control samples are readily selected by one of ordinary skill in the art.
For example, suitable control samples include samples taken from normal patients who do not have a particular disease state, or who are known to express normal amounts of contactin. Such controls can be derived from the same tissue, organ, or body fluid from which the test sample was derived, so that valid comparisons can be made. The amount of contactin mRNA, contactin DNA, or contactin protein in the control sample can be provided in the form of charts or other literature or databases so that the control sample does not need to be processed for every assay. Contactin mRNA in control samples
Differences between the amount of contactin mRNA, DNA, or protein in a sample that is statistically different from the amount of protein, DNA, or protein may be associated with neurodegenerative diseases (especially amyotrophic lateral sclerosis, multiple (A group consisting of sclerosis, MELAS, and MERRF).

The method of the present invention also includes control mRNA, control DN in a sample.
A, or measuring the amount of control protein. Here, the control mRNA or control DNA does not encode contactin,
And the control protein is not contactin. Preferably, the control mRNA, control DNA, or control protein is associated with a constitutively, consistently or highly expressed protein such as actin, ribosomal RNA or GAPDH. In this example, the control is an internal control, resulting in contactin mRNA, contactin DNA.
, Or the amount of contactin protein and the amount of control mRNA, control DNA, or control protein is detected in the same sample. Control mRNA, control DNA, or control protein can be detected using the methods described herein for detecting contactin mRNA, contactin DNA, or contactin protein.
The amount of control mRNA, control DNA, or control protein is preferably contactin mRNA, contactin DNA,
Or compared to the amount of contactin protein. Preferably, the contactin / control mRNA, DNA, or protein ratio is determined and compared to normal values obtained from samples derived from normal samples. Differences in the ratio to control mRNA, DNA, or protein that are statistically different from the normal value consist of neurodegenerative diseases (especially amyotrophic lateral sclerosis, multiple sclerosis, MELAS, and MERRF). By group).

In certain other preferred embodiments, the nucleic acid molecules having specific nucleotide sequences are different populations of DNA, or different populations of oligonucleotide primers provided herein, which are , Immobilized as an array of nucleic acids on a solid support), can be efficiently detected, screened, and / or quantified by high throughput hybridization methodologies directed to independently probe . Typically, the solid support can be silica, quartz, or glass, or allows for suitable hybridization, washing, and detection steps known in the art and provided herein. Can be any other material that can immobilize nucleic acids to a solid support in the manner described. In a preferred embodiment, solid phase nucleic acid arrays are described, for example, in US Pat.
Precisely spaced as described in U.S. Pat. No. 00,992 (eg, WO 95/21944; Schena et al., 1995, Science).
270: 467-470, 1995; Pease et al., 1994, Proc. Na
tl. Acad. Sci. USA 91: 5022; Lipshutz et al., 19
95 Biotechniques 19: 442-447).

Detection of hybridized (eg, double-stranded) nucleic acids on an array of nucleic acids is accomplished by any known procedure (eg, spectrophotometer or potentiometer (eg, MALDI).
-By MS)). In certain preferred embodiments,
The array comprises less than 5500 nt long oligonucleotides, in other preferred embodiments less than 500 nt long oligonucleotides, in other preferred embodiments less than 100 nt long oligonucleotides, and other preferred embodiments. In embodiments, it comprises an oligonucleotide less than 50 nt in length.
For high throughput screening of arrays of nucleic acids, the format is preferably
Responsive to automation. For example, it is preferred that the automated device for use in accordance with the high throughput screening embodiments of the present invention be under the control of a computer or other programmable controller. The controller can continuously monitor the results of each step of nucleic acid accumulation, washing, hybridization, detection, and related processes, and can automatically alter the test palladium in response to these results. .

Methods for Identifying Which Patient Having a Neurodegenerative Disease Responds to Treatment of a Neurodegenerative Disease The present invention relates to which patient having a neurodegenerative disease For responding to the treatment of neurodegenerative diseases. Such a method comprises the steps of: providing a sample from a group of patients having the above-described neurodegenerative disease; a contactin protein, contactin mRNA, or contactin present in the sample. Measuring the amount of DNA; providing said treatment to said patient; measuring the extent, frequency, rate, or range of said patient's response to said treatment; and in said sample Determining whether there is a correlation between the amount of contactin protein, contactin DNA, or contactin mRNA of, and the extent, frequency, rate, or range of the response. Here: 1) When the above correlation is a positive correlation, the presence of the above positive correlation indicates that the sample has an increased amount of contactin protein, contactin DNA, or contactin mRNA. Is shown to respond to the above treatments; and 2) if the above correlation is a negative correlation, the presence of a negative correlation indicates the presence of contactin. Patients providing a sample with a reduced amount of protein, contactin DNA, or contactin mRNA are likely to respond to the above treatments.

In practicing this method, the sample is from a patient diagnosed with a neurodegenerative disease (eg, multiple sclerosis, amyotrophic lateral sclerosis, MELAS, or MERRF). Offered by the group. The sample can be any tissue, organ,
Alternatively, it may be in the form of body fluid, but is preferably derived from at least a portion of neurological tissue, preferably central nervous tissue. The group of patients is preferably one or more, more preferably at least 4 or more, and most preferably at least 9 or more. The amount of contactin mRNA, contactin DNA, contactin protein is measured in these samples using methods known in the art or described herein.

The patient is then provided with a treatment that can regress, ameliorate, reduce the severity, slow the progression, or cure the neurodegenerative disease.
Treatment can be any treatment, including administering the compound or composition or not administering them. For example, treatment can include the use of a companion animal or human, massage, humor, or other treatment that does not involve the targeted administration of the compound or composition to a patient by any route of administration. Treatment also includes the step of administering the compound or composition to the patient. This includes traditional or non-traditional drugs or treatments, such as herbal or aroma remedies. Patient response is monitored using criteria and endpoints associated with or established for neurodegenerative diseases. For example, the extent, frequency, rate, or extent of patient response to treatment can be measured using established methods and endpoints for neurodegenerative diseases. The patient should die during the procedure, a sample can be taken from the body, and death is recorded as the disease progresses.

Samples are taken from the patient at least once during the course of treatment. Preferably, the sample is taken from the same tissue, organ, or body fluid from which the original sample was taken. The amount of contactin mRNA, contactin DNA, or contactin protein is measured in these samples.

Patient response correlates with changes in the amount of contactin mRNA, contactin DNA, or contactin protein in a sample derived from the patient. A positive correlation if the presence of the correlation indicates that the patient providing the sample with increased amounts of contactin mRNA, contactin DNA, or contactin protein appears to respond to treatment. There is a relationship. A negative correlation exists if the presence of the correlation indicates that the patient providing the sample with a reduced amount of contactin mRNA, contactin DNA, or contactin protein responds to the treatment. To do.

For both positive and negative correlations, a response may or may not be desired. The desired response is one that regresses, ameliorates, reduces the severity of, slows progression, or cures a neurodegenerative disease. The unwanted response is any response that is not the desired response.

Cells Showing Increased or Decreased Amounts of Contactin The present invention also includes increased or decreased amounts of contactin (eg, contactin mRNA, contactin DNA, or contactin when compared to control cells. Protein) or a composition of matter comprising cells in culture. A cell can be a part of tissue, an organ, or a body fluid, or a part thereof. The cells can also be cultured in vitro.

The cells of the invention may be derived from a sample taken from a test animal or patient (eg, human patient). Such cells can be part of a primary cell culture or continuous cell line. Preferably, the cells of the invention are a population of clones. Primary cell cultures can be obtained using methods known in the art. A continuous cell line can be created by repeating the passage of a population of cells in culture until a continuous cell line is obtained. Alternatively, primary cells can be made into a continuous cell line by immobilizing the cell line using methods known in the art. For example, primary cells can be fused with polyethylene glycol or cell lines that have been immobilized using electrical charges. Alternatively, primary cells can be infected with transforming viruses such as retroviruses to create immortalized cell lines.

Cells can also be engineered using methods known in the art such that the cells exhibit increased or decreased amounts of contactin mRNA, contactin DNA, or contactin protein. For example, increased amount of contactin mR
Cells displaying NA, contactin DNA, or contactin protein can be made by transfecting a cell line with a nucleic acid molecule encoding the contactin protein. The nucleic acid molecule can be provided in a vector and can be operably linked to regulatory sequences such as CMV promoter or LTR elements. As a result, relatively high levels of contactin are expressed in the cell. Nucleic acid molecules encoding contactin can also be provided in the vector. It is operably linked to a nucleic acid sequence that facilitates homologous recombination to a known region of the genome so that the nucleic acid molecule encoding contactin can be expressed under the control of an endogenous promoter (1994). See Smith et al., WO 94/24301, published October 27). Nucleic acid molecules encoding contactin can also be provided in the vector. It is not operably linked to regulatory sequences or nucleic acid molecules that promote homologous recombination, so that the nucleic acid molecule encoding contactin is randomly integrated into the cell's genome (April 1998). See Whitney, WO 98/13353, published 2).
Increased amounts of contactin mRNA, contactin DNA, or contactin protein in these cells can be identified using the methods of the invention.

Cells exhibiting reduced amounts of contactin can be isolated using methods known in the art.
It can be made by transfecting a cell line with a nucleic acid molecule that encodes an antisense molecule for contactin DNA or contactin mRNA, or a nucleic acid molecule that encodes a ribozyme that can degenerate contactin mRNA. Reduced amount of contactin mRNA, contactin DN, in these cells
A, or contactin protein, can be identified using the methods of the invention.

Cells exhibiting increased or decreased amounts of contactin mRNA, contactin DNA, or contactin protein can be used to make cybrid cells using methods known in the art (October 1995). See Hernstadt et al., WO 95/26973, published 12th). Briefly, cells are rendered essentially devoid of mitochondria by exposure to ethidium bromide. These cells are then fused with platelets derived from a patient, such as a patient with a neurodegenerative disorder involving mitochondria or a normal patient. The fused cell line contains the cellular nuclear elements of platelets and mitochondria, including the mitochondrial DNA of platelets. The amount of contactin mRNA, contactin DNA, or contactin protein in these cybrids can be measured using the method of the invention and compared to cells prior to fusion with platelets.

In addition, increased or decreased amounts of contactin mRNA, contactin D
Cybrids exhibiting NA, or contactin protein, can be made. The cells are exposed to ethidium bromide to create cells that are essentially devoid of mitochondria. Cells that are essentially devoid of mitochondria (eg, the human SH-SY5Y neuroblastoma cell line) are fused with platelets from normal patients or patients with neurodegenerative disorders. The amount of contactin mRNA, contactin DNA, or contactin protein in these cybrids can be measured using the method of the invention, and increased or decreased amounts of contactin mRNA, contactin DNA, or contactin protein To identify the indicated cybrid cell lines, one can compare to the amount of contactin mRNA, contactin DNA, or contactin protein expressed in the parental cells.

Methods for Screening Compounds for Activity to Reduce or Enhance Contactin Expression The invention includes methods of screening test compounds for activity to reduce or enhance contactin expression. The method comprises providing at least one cell; contacting at least one cell as described above with at least one test compound; and a change in contactin mRNA, contactin DNA, or contactin protein in at least one cell. The step of measuring is included. The present invention also includes compositions containing pharmaceutical compositions that include at least one test compound identified by these methods.

In operation of this method, the cells of the invention are contacted with at least one test compound. Then, contactin mRNA and contactin DNA in cells
, Or a change in the amount of contactin protein is measured using at least one method of the invention. Preferably, the amount of contactin mRNA, contactin DNA, or contactin protein exhibited by the cell is known, but need not be, before the cell is contacted with the test compound. A compound that increases or decreases the amount of contactin mRNA, contactin DNA or contactin protein in a cell is a contactin mRNA in the cell.
, A putative therapeutic agent that increases or decreases the amount of contactin DNA or contactin protein.

Test compounds that increase the amount of contactin mRNA, contactin DNA, or contactin protein in cells reduce cell motility and reduce Ng-CA.
It has putative therapeutic activities that enhance the binding of cells to M, increase the interaction of cells with the extracellular matrix glycoprotein restrictin, and promote neurite outgrowth. Test compounds that decrease the amount of contactin mRNA, contactin DNA, or contactin protein in cells increase cell motility, decrease cell binding to Ng-CAM, and reduce cells and extracellular matrix glycoprotein restrictin. Neurodegenerative diseases (eg, multiple sclerosis, lateral sclerosis, MELAS)
, Or MERRF) has a putative therapeutic treatment activity. Identified test compounds can be evaluated using the methods presented herein.

Test Compound Pharmacology and Toxicity The structure of test compounds can be determined or confirmed by methods known in the art such as mass spectroscopy. Structures, activities, and their potency can be confirmed for test compounds that have been stored under various conditions for extended periods of time.

Identified test compounds can be evaluated for specific activity using art-recognized methods and methods disclosed herein. For example, if the identified test compound is found to have anti-cancer cell activity in vitro, the test compound has putative pharmacological properties as a chemotherapeutic agent for treating cancer. Such nexuses are known in the art for several diseases and are expected to be discovered over time. Based on such nexus, appropriate confirmation of in vitro and in vivo models of pharmacological activity and toxicity will be selected and performed. The methods described herein can also be used to assess pharmacological selectivity and specificity and toxicity.

Identified test compounds can be evaluated for toxicological effects using known methods (Lu, Basic Toxicology, Fundamental).
s, Target Organs and Risk Assessment,
Hemisphere Publishing Corp. , Washington
on (1985); Culbreth, US Pat. No. 5,196,313 (19).
(Issued March 23, 1993) and Benet, No. 5,567,952 (issued October 22, 1996)). For example, the toxicology of test compounds can be established by determining toxicity in vitro for cell lines, such as mammalian (eg, human) cell lines. The test compound is, for example,
Treatment with tissue extracts (eg, liver preparations (eg, microsomal preparations)) to determine the increased or decreased toxicological properties of test compounds after being metabolized by the whole organism Can be done. The results of these types of studies are predictive of the toxicological properties of chemicals in animals such as mammals, including humans.

Alternatively, or in addition to these in vitro studies, the toxicological properties of test compounds in animal models (eg, mouse, rat, rabbit, dog, or monkey) have been established. Can be determined using (Lu, supra (1985); and Creasey, Drug Disposition in Humans.
, The Basis of Clinical Pharmacology,
Oxford University Press, Oxford (1979)
checking). Depending on the toxicity of the test compound, the target organ, tissue, locus, and putative mechanism, one of ordinary skill in the art would know the appropriate dose, LD ₅₀ value, appropriate to determine the toxicological properties of the test compound, It will not be difficult to determine the route of administration and the regimen. In addition to animal models, human clinical trials have shown that United States
It may be performed according to established procedures such as those set forth by Food and Drug Administration (USFDA) or other equivalent government agencies. These toxicity studies provide the criteria for determining the efficacy of test compounds in vivo.

Efficacy of Test Compounds Efficacy of test compounds can be established using a number of art-recognized methods, such as in vitro methods, animal models, or human clinical trials. (See Creamey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, HIV
The ability of test compounds to prolong the in vitro survival time of cells infected with L.
Recognized as an acceptable model for identifying chemicals that are predicted to be effective for treating V infection or AIDS (Daluge et al., Antimi).
cro. Agents Chemother. 41: 1082-1093 (19)
95)). In addition, cyclosporin A (CsA)
The ability to prevent cell proliferation has been established as an acceptable model for identifying chemicals that are predicted to be effective as immunosuppressants (Suthanthi.
See ran et al., supra (1996)). In vitro acceptable or animal models are available for almost all classes of therapeutic agents, diseases, or conditions. Those skilled in the art are assisted with a wide variety of such models, which are available in the literature or by the USFDA or National Institutes of Health (NIH). In addition, these in vitro methods may use tissue extracts such as liver preparations (eg, microsomal preparations) to provide a reliable indication of metabolic effects for test compounds.

Similarly, acceptable animal models can be used to establish the efficacy of test compounds for treating various diseases or conditions. For example, rabbit knees are an acceptable model for test factors for efficacy in treating arthritis (Shaw).
And Lacy, J. et al. Bone Joint Surg. (Br.) 55:19.
7-205 (1973)). Hydrocortisone, which is approved for use in humans to treat arthritis, is effective in this model. This confirms the validity of this model (McDonough
, Phys. Ther. 62: 835-839 (1982)). When selecting an appropriate model for determining the efficiency of a test compound, one of ordinary skill in the art will be familiar with the appropriate model, dose and route of administration, regimen, and endpoint (endpoint) according to the description in the field, USFDA, or NIH. ), And therefore not be an overburden. In addition to animal models, human clinical trials can be used to determine the efficiency of test compounds. The USFDA or equivalent government agency has established procedures for such research.

Selectivity of Test Compounds The in vitro and in vivo methods described above also establish the selectivity of candidate modulators. It will be appreciated that chemical agents may be selective or even selective in a wide variety of biological processes. Panels of cells can be used to determine the specificity of a test compound if they are known in the art (4 1998).
See Whitney et al., WO 98/13353, published March 2). Selectivity is, for example, in the field of chromatography, where selectivity of a chemical that is toxic to cancerous cells but not to non-cancerous cells is clearly desired, it is obvious. Selective modulators are preferred. Because
This is because selective modulators have few side effects in the clinical setting.
Selectivity of test compounds can be established in vitro by testing toxicity,
And the effect of the test compound can be established in vitro by testing the toxicity and effect of the test compound on a population of cell lines. This demonstrates various cellular pathways and sensitivities. The data obtained from these in vitro toxicity studies can be extended to studies in animal models, including human clinical trials to determine the toxicity, efficacy, and selectivity of test compounds. .

The selectivity, specificity, and toxicity of the test compound, as well as the general pharmacology, make additional test compounds based on the structure / property relationships of the test compound originally identified as having activity. Can often be improved. Test compounds can be modified to improve various properties such as affinity, longevity in blood, toxicity, specificity, and membrane permeability. Such purified test compounds can be subjected to further assays as they are known in the art or as described herein. Methods for making and analyzing such compounds or compositions are known in the art (eg, Agrafiotis et al., US Pat. No. 5,574,656).

Pharmaceutical Compositions The present invention also includes a test compound in a pharmaceutical composition containing a pharmaceutically acceptable carrier prepared for storage and, preferably, subsequent administration. It has a pharmaceutically effective amount of test compound in a pharmaceutically acceptable carrier or diluent.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sci.
incomes. Mack Publishing Co. , (AR Genna
Ro edited (1985)). Preservatives, stabilisers, dyes and even flavoring agents may still be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents may be used.

The test compounds of the invention may be formulated as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions, injectable drugs, and the like, And can be used. Injectables may be prepared in either convenient forms, either liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like. In addition, if desired, the injectable pharmaceutical composition can contain minor amounts of nontoxic auxiliary substances such as wetting agents, pH buffering agents and the like. If desired, adsorption-enhancing preparations such as liposomes can be used.

The pharmaceutically effective amount of test compound required for administration will depend on the route of administration, the type of animal or patient being treated, and the particular physical characteristics of the animal considered. The dosage may be adjusted to achieve the desired effect, but will depend on such factors as weight, diet, parallel pharmaceutical agents, and other factors that those skilled in the medical arts will recognize. In practicing the methods of this invention, the pharmaceutical compositions may be used alone or in combination with each other or in combination with other therapeutic or diagnostic agents. These products are expressed in vivo, preferably in a mammalian patient (
Preferably in humans) or in vitro. For their use in vivo, the pharmaceutical compositions may be administered in various dosage forms in various ways, including parenteral, intravenous, subcutaneous, intramuscular, colon, rectal, intranasal, or intraperitoneal. Can be administered to a patient. Such methods can also be used in testing the activity of test compounds in vivo.

As will be apparent to those of skill in the art, useful in vivo administered dosages and particular modes of administration will be the age, weight and type of patient to be treated, the particular pharmaceutical composition used. , And depending on the particular application for which the pharmaceutical composition is used. Determination of the effective dose level, which is the dose level required to achieve the desired result, can be accomplished by one of skill in the art using routine methods as discussed above, and USFDA Or it may be guided by an agency such as NIH. Typically, human clinical application of the product is initiated at lower dosage levels, with dosage levels being increased until the desired effect is achieved. Alternatively, applicable in vitro studies can be used to establish useful doses and routes of administration of test compounds.

In non-human animal studies, application of the pharmaceutical composition is initiated at a high dose level and the dosage is adjusted until the desired effect is no longer achieved or the adverse side effects have disappeared. Be reduced. The dosage of test compounds of the present invention can fall within a wide range depending on the desired effect, therapeutic index, route of administration, and purity and activity of the test compound. Typically, the dose will be about 1 ng / kg to about 10 n
g / kg, preferably between about 10 ng / kg and about 1 mg / kg, more preferably between about 100 ng / kg and about 100 microgram / kg, and most preferably about 1 microgram / kg. To about 10 micrograms / kg.

Extraction formulations, routes of administration, and dosages can be selected by the individual physician in view of the patient's condition (Fingle et al., The Pharmacologica.
l Basis of Therapeutics (1975)).
. It should be noted that the attending physician knows how and when to end, discontinue, or adjust dosing due to toxicity, organ failure, or other adverse effects. Conversely, the attending physician also knows to adjust treatment to higher levels when the clinical response is inadequate. The magnitude of the administered dose in the management of the disorder of interest will vary with the severity of the condition being treated and the route of administration. The severity of the condition can be assessed in part by, for example, standard prognostic assessment methods. In addition, dosages and perhaps dose frequencies, including for veterinary applications, will also vary according to the age, weight, and response of the individual patient.

Depending on the specific condition being treated, such pharmaceutical compositions may be formulated,
And it can be administered systemically or locally. Techniques for prescribing and administering are described in R
emington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. , Easton, PA (19
90). Suitable routes of administration include oral, rectal, transdermal, ear,
Ocular, vaginal, transmucosal, or enteral administration; parenteral delivery (intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection) Can be mentioned).

For injection, the pharmaceutical composition of the present invention is an aqueous solution (preferably Han).
ks solution, Ringer's solution, or in a physiologically compatible buffer such as saline buffer). For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The use of a pharmaceutically acceptable carrier for formulating the pharmaceutical compositions disclosed herein for practicing the invention at a dose suitable for systemic administration is within the scope of the invention. is there. With proper choice of carrier and proper manufacturing practice, the compositions of the present invention, particularly those formulated as solutions, may be administered parenterally (eg, by intravenous injection). . The pharmaceutical composition can be formulated readily using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers are formulated for oral ingestion by the patient to be treated with the test compounds of the invention as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like. To enable that.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents can be encapsulated in liposomes and then administered as described above. Substantially all of the molecules present in the aqueous solution at the time of the liposome formulation are entrapped in or in the liposomes thus formed. The liposome contents are all protected from the external microenvironment,
Further, the liposome dissolves the cell membrane, so that the liposome is efficiently delivered into the cytoplasm of the cell. Moreover, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amount of a pharmaceutical composition is well within the capability of those skilled in the art, especially with reference to the detailed disclosure provided herein. In addition to the active ingredient, these pharmaceutical compositions contain excipients and additives which facilitate the processing of the active chemical agents into pharmaceutically usable preparations. Any pharmaceutically acceptable carrier may be included. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules or solutions. The pharmaceutical compositions of the invention are in a manner known per se (eg by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes). Can be manufactured. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active chemical in water-soluble form.

Additionally, suspensions of the active chemicals can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the chemicals to allow for the preparation of highly concentrated solutions.

A pharmaceutical composition for oral use comprises mixing a solid excipient with an active chemical agent, optionally granulating the resulting mixture, and mixing the granules. And then, if desired, adding suitable additives,
It can be obtained by obtaining the core of a tablet or dragee. Suitable excipients include, among others, bulking agents such as sugars (including lactose, sucrose, mannitol, or sorbitol); cellulosic preparations (such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, Methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone), if desired, a disintegrant (eg, crosslinked polyvinylpyrrolidone, agar, alginic acid, or salts thereof (eg, alginate)). Dragee cores may be provided with suitable coatings.Dyes or pigments may be added to tablets or dragee coatings to identify or characterize different combinations of active doses. Profit .

The test compounds and pharmaceutical compositions of this invention containing such test compounds are useful for treating various diseases in patients, including humans. As shown in the examples, the test compounds of the present invention have antibacterial, bactericidal, antiviral, anticancer cell, antitumor and cytotoxic activity. Patients in need of such treatment are treated with a test compound of the invention, preferably to reduce the number or growth rate of bacteria, microorganisms, cancer cells, or tumor cells in the patient as described above, or as described above. It may be provided in a pharmaceutical composition in an amount effective to reduce the infectivity of the virus in a patient. Amount, dose, route of administration, regimen, and endpoint are all
Using the procedures described herein, or in an appropriate government agency (eg,
United States Food and Drug Admin
ratio).

Methods of Treating Neurodegenerative Diseases The invention also relates to methods of treating neurodegenerative diseases using the compositions or compounds identified by the methods of the invention. As shown herein, compounds that increase or decrease the amount of contactin mRNA, contactin DNA, or contactin protein in cells have putative therapeutic activity. These therapeutic activities can be confirmed using the methods of the invention. Preferably, this aspect of the invention is therapeutic, therapeutic, transient, reversible, prophylactic in nature using appropriate endpoints for such treatment. , Administering a pharmaceutical composition of the invention by a route of administration sufficient to provide treatment that is protective or prophylactic.

Suitable endpoints or parameters for the treatment of multiple sclerosis include: cognitive impairment, ocular neritis, diplopia, dizziness, weakness, tremor, spasticity, hystagmus, ataxia. Symptom, visual loss, fatigue, sphincter deficiency, progression of disorders such as gait dysfunction or the amount of immunoglobulin in spinal fluid, or a decrease in the frequency or appearance of symptom features of such disorders. Suitable endpoints or parameters for the treatment of lateral sclerosis include the following: progression of disorders such as loss of function of upper or lower neurons resulting in progressive skeletal muscle wasting or weakness, or A decrease in the frequency or appearance of characteristic symptoms of such disorders. Suitable endpoints or parameters for the treatment of MELAS include: local or generalized epileptic seizures, dementia, headache, vomiting, hemiblindness, cortical blindness, hearing loss, elevated serum lactate. , Or the progression of disorders such as encephalopathy, or a decrease in the frequency or appearance of characteristic symptoms of such disorders. Suitable endpoints or parameters for the treatment of MERRF include the following: eg epileptic seizures, ataxia, lactic acidosis, dysarthia, atrophy of the eyes, hearing impairment, dementia, ocular concussion, spasticity. , Muscle weakness, or progression of disorders such as increased serum pyruvate, or a decrease in the frequency or appearance of characteristic symptoms of such disorders.

Another aspect of the invention is a method of treating a neurodegenerative disease. The method comprises the steps of: providing a sample from a patient having the neurodegenerative disease described above; contactin protein in the sample, contactin DN
A, or measuring the amount of contactin mRNA; and administering to the patient an effective amount of the composition of the invention. Here, there is a positive correlation between the amount of contactin protein or contactin mRNA present in the patient-derived sample and the efficiency of the compound. This aspect of the invention is directed to obtaining a sample from a patient having a neurodegenerative disease and in a sample.
The methods described herein are used to measure the amount of NA, contactin DNA, or contactin protein. Sample is contactin mR
A suitable composition (e.g., a pharmaceutical composition of the invention) is said to have an amount of contactin mRNA, contactin DNA, or contactin protein if it exhibits increased or decreased amount of NA, contactin DNA, or contactin protein. Is administered to the patient to adjust the level to normal levels. The pharmaceutical composition may be administered in an effective amount by a suitable route using a suitable regime. The efficacy of such treatment can be measured using the appropriate endpoint or parameter for the neurodegenerative disease being treated. Preferably, such treatment is therapeutic in nature, therapeutic, palliative, revertive, preventative, protective, or prophylactic.

Methods for Identifying Pharmaceutical Targets, and Pharmaceutical Targets Identified by Such Methods The present invention provides methods for identifying pharmaceutical targets for compounds that modulate contactin expression or activity. , And targets identified by the methods of the invention. The present invention also includes pharmaceutical targets identified by such methods.

A method for identifying a pharmaceutical target comprises the following steps: a compound that regulates the expression or activity of contactin, such as the amount of contactin mRNA, contactin DNA, or contactin protein in a cell. Providing; and identifying cellular components that bind these compounds. In practice, this method involves increasing or decreasing the amount of normal or normal amount of contactin mRNA,
Providing a cell, such as a cell of the invention, that expresses contactin DNA or contactin protein. Alternatively, an extract of cells can be provided. The cells or extracts thereof are then treated with the contactin mRNA in the sample.
, A contactin DNA, or a compound that regulates the amount of contactin protein. The binding of the compound to the cellular component is then detected. The cellular component that binds the compound can be multiple sclerosis, lateral sclerosis, MELAS, or M
It is a putative therapeutic target for the treatment of neurodegenerative diseases such as ERRF.

The binding of compounds to cellular components can be detected using various methods.
For example, the compound can be attached to a detectable label so that the location of the compound can be monitored in the cell. The compound-cellular component can then be isolated using affinity chromatography or immunoprecipitation methods using antibodies specific for the compound or label. Preferably, a gel shift assay can be used to detect compound-cellular component binding. For example, a compound attached to a detectable label will react in the gel in response to an electric current due to various parameters, including the molecular weight of the compound attached to the detectable label. It has a specific mobility. Binding of a compound to a cellular component modifies the mobility of the compound-cellular component complex through the gel due to the increased molecular weight of the complex as compared to the cellular component and compound alone. Let The area of the gel containing the compound-cellular component may be recovered, and the cellular component may have reactivity with the antibody, molecular weight, localization of the cellular component in the cell, and cellularity. Is identified using methods known in the art such as the activity of the components of

Furthermore, the ability of a compound to modulate a signal transduction pathway can be determined. The ability of a compound to modulate an identified signaling pathway identifies such signaling pathway as a therapeutic target. Various cells containing reporter genes that report increased or decreased activity of signaling pathways in response to compounds are known in the art. Such cells can also be made using methods known in the art (Whitney, WO 98/133, published Apr. 2, 1999).
53; Evans et al., US Pat. No. 5,2, issued Mar. 29, 1994.
98,429; and Skarnes et al., Genes and Develo.
pment 6: 903-918 (1992)). The compounds of the invention can be contacted with such cells, and expression of the reporter gene monitored to identify signal transduction pathways regulated by the compound. The signaling pathway thus identified is itself a pharmaceutical target, as are the individual components of the identified signaling pathway.

The following examples are provided by way of illustration and not by way of limitation.

EXAMPLES Example 1 Amplification of Contactin mRNA This example demonstrates that the method used to detect contactin mRNA is specific for contactin mRNA.

A cDNA preparation containing the nucleic acid encoding contactin was cloned into Clonte.
ch (Palo Alto, CA: Catalog Number: 64020-1) was prepared from total RNA derived from human brain tissue. cDNA preparation was oligod
T-primer was used to make using Superscript II kit by GIBCO.

Contactin nucleic acids in the samples were measured by the SYBR® Green detection system using the Real-Time PCR amplification method and primers: 5′-TCAGTAAGGTCTGGTTCACGCTAT-3 ′ (SEQ ID NO: 1), 5′-TCCCGTCACTGTAGATTCATTTTGA. -3 '(SEQ ID NO: 2) 5'-CCCCAAGTCTTCTCGCGCTTA-3' (SEQ ID NO: 3) 5'-CAACACATTCAGAATTTCAAGTAGACACA-3 '(SEQ ID NO: 4) 5'-TCCCCAAGTCTTCTCGGGCTTA-3' (SEQ ID NO: 5) 5'-CCCATCCCAAGCTCAGAAC '(SEQ ID NO: 6), and 5'-GCCGCAGAAATTTGGAAGG-3' (SEQ ID NO: 7). SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6 are forward primers. SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 7 are reverse primers. Preferred primer pairs were SEQ ID NO: 6 and SEQ ID NO: 7; SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 5 and SEQ ID NO: 4; and SEQ ID NO: 3 and SEQ ID NO: 4.

PCR amplification products were separated using a 4% agarose gel containing the appropriate molecular weight markers. The gel was stained using ethidium bromide and showed a single band of exactly the size expected for the PCR product, all derived from contactin. Therefore, these results demonstrate that these primer pairs specifically amplify contactin mRNA.

Example 2 Quantitative Real-Time PCR Optimization The use of real-time PCR to quantify the level of specific nucleic acids has been described in the art (Freeman et al., BioTechniques 26). : 112
-125 (1999), and for a recent summary, Heid et al., Geno.
me Research 6: 986-994 (1996); Gibson et al.,
Genome Research 6: 995-1001 (1996), U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,683,195; U.S. Pat.
965,188; US Pat. No. 5,035,996; SYBR® G
reen PCR and RT-PCR Reagents, Protoco
1, Applied Biosystems, 1998; and Spiess.
Et al., BioTechniques 26: 46-50 (1990), all of which are incorporated herein by reference). For ease of understanding, a brief description of quantitative real-time PCR (Q-RTPCR) is given below.

Until recently, the traditional means of measuring the products of specific PCR reactions was the "end-point" analytical method. Here, the reaction products are measured and identified after the amplification reaction is complete. In contrast, "real-time" PCR monitors the amplification reaction in a thermal cycler as they progress. Q-RTPCR provides improved quantitation. Because quantitation is most accurately achieved during the linear range of amplification, and more information about the amplification reaction is obtained for each cycle.

For example, a standardized (ie, relative to a passive reference dye that does not bind DNA) fluorescence intensity (“ΔR _n ”), which measures the magnitude of the signal generated by a given set of PCR conditions. Shown) can be measured during each cycle. From such data, the cycle in which a statistically significant increase in ΔR _n is first detected can be determined. A "marginal cycle" or "C _T value" is determined on one logarithm of the originally detected signal and provides a quantitative measure of the amount of input nucleic acid template of interest present in the original sample. .

In order to optimize the PCR reaction for use in the SYBR® Green detection system, which does not discriminate between different amplification products, different concentrations of different primers were used. Used to normalize the rate of amplification. The results are shown in Table 1.

Preferred primer pairs and concentrations are such that the average C _T is relatively low (ie
Sensitivity), and the average ΔR _n is relatively high (ie, large range of signal magnitudes), reflecting these examples. Based on these criteria, preferred primer pairs and concentrations include: SEQ ID NO: 3 (900 nM) and SEQ ID NO: 4 (900 nM), SEQ ID NO: 1 (300 nM) and SEQ ID NO: 2 (300 nM), SEQ ID NO: 1 (900 nM) and SEQ ID NO: 2 (900 nM), and SEQ ID NO: 6 (300 nM) and SEQ ID NO: 7 (900 nM).

To normalize sample to sample variation, an internal RNA normalizer is used in Q-RTPCR. The RNA normalization group includes endogenous RNA species (eg, constitutively expressed protein-like actin or glyceraldehyde-3-phosphate dehydrogenase (GADPH).
), Or mRNA encoding ribosomal RNA (eg, 18S or 28S rRNA); RNA molecules produced in vitro can also be used as a normalization group. Therefore, the results of Q-RTPCR analysis can often be presented as relative amounts.

For example, if the normalization group is actin and the nucleic acid being quantified is the nucleic acid encoding contactin, the normalization group is according to a standard curve generated for both gene sequences for each RNA sample. The relative amount of contactin RNA is determined relative to that of actin. A standard curve was typically prepared using different amounts of input RNA between about 3 and 4 in triplicate reactions. The standard curve is plotted as logarithmic input concentration (x-axis) versus _Ct (y-axis, also on a logarithmic scale). For each standard curve, the slope (m) and y-intercept (b) are
Calculate using standard analytical software.

The logarithmic input quantity (nN) for the normalized group is calculated for a given C _T (C _T ⁰ ). For example, if C _T ⁰ = 20: nN = (20−b _N ) / _mn For a specific target (T) sequence of interest, reach a log input quantity equal to C _T ^T (nN). C _T ), which is required for C _T ^{T, is} determined from the following formula: C _T ^T = (m _T × nN) + b _T Normalized target C _T (normalized C _T ^T ) Calculate: Normalized C _T ^T = C _T ^T- C _T ^{0 In} this example, the efficiency of these PCR reactions is compared to the efficiency for a control reaction using a pair of primers for actin. did. A standard curve to determine the change in Ct for these reactions was used to determine the concentration of primer used versus Taqman SYBR® for contactin.
It was determined in comparison with the results obtained for actin by plotting the signal by the Green detection system (Table 2). The slope of the contactin plot was subtracted from the results for the actin plot. An absolute difference of less than about 0.1 is considered a preferred assay. The contactin primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) preferably uses an actin primer as a control. A preferred actin primer pair is 5'-CTGGAACGGTGAA.
GGTGACA-3 ′ (SEQ ID NO: 8) (forward primer) and 5′-CGGCC
It was ACATGTGAACTTTG-3 '(SEQ ID NO: 9) (reverse primer). These conditions gave an absolute difference of 0.063.

[0157]

[Table 1]

[0158]

[Table 2] Example 3 Contactin mRNA Expression in Neurological Samples Human brain samples were obtained as soon as possible postmortem from the cerebral occipital pole after mortem. Samples were obtained from patients diagnosed with multiple sclerosis (MS) and control patients. Freeze the biological sample and store at -80 ° C,
It was then shipped on dry ice to facilitate analysis.

Total RNA was purified using the TRIzol® Reagent supplied by Life Technologies®, one version of the manufacturer's instructions (one version of TRIzol® Reagent is US Pat.
, 346,994), as described in Example 2,
Isolated from biological sample. Briefly, tissue samples were treated with TRIzol® Reagent (1 mL of TRI per 50 mg to 100 mg tissue).
Homogenized in zol® Reagent). The homogenized sample was incubated for about 5 minutes at about 15 ° C to 30 ° C. Chloroform (1 ml of TRIzol® used for the first homogenization)
0.2 ml) and the mixture is shaken vigorously for 15 seconds, incubated for 2 or 3 minutes at about 15 ° C to 30 ° C, and about 12,000x.
Centrifuge for 15 minutes at about 2 ° C to 8 ° C. The aqueous phase was transferred to a new container, and the RNA therein was treated with 1 ml of TRIz used for the first homogenization.
Precipitated by mixing with 0.5 ml of isopropyl alcohol per ol® Reagent. The mixture was incubated at about 15 ° C to 30 ° C for about 10 minutes and centrifuged at 12,000g for 10 minutes at about 2 ° C to 8 ° C. The supernatant was removed and the resulting pellet was washed once with 75% ethanol and collected by centrifugation at about 12,000 xg for 5 minutes at 2-8 ° C. The resulting pellet was collected and dried.

Contactin mRNA and actin mRNA in the pellet were amplified and quantified as described in Examples 1 and 2. The percent expression of contactin mRNA was calculated as [(contactin mRNA / actin mRNA) x 100]. For the control sample, the percent expression of contactin mRNA was 22.84. For multiple sclerosis samples, the percent expression of contactin mRNA was 95.22. From these results, M
A sample of neurological tissue derived from a patient diagnosed with S showed contactin mR.
It is proven to have an elevated level of NA.

Example 4 Assay for Contactin Protein and mRNA in Biological Samples Contains materials from central nervous system tissue, lung tissue, kidney tissue, skeletal muscle, epithelium, blood, or amniotic fluid. The biological samples that are being analyzed by MS, ALS, MELAS, ME
Prepared according to known methods from patients diagnosed with or suspected of having RRF, and / or other neurological disorders. Control samples from individuals, preferably age-matched and gender-matched, or generally related individuals, who do not clearly have such neurological disorders Collect. The biological sample can be from a living individual or a dead individual. Biological samples are evaluated for levels of contactin protein and / or mRNA encoding contactin.

In order to detect the contactin protein and to quantify its level,
Biological samples are stained using immunological methods with antibodies that are specific for contactin, or evaluated by immunoassay. As such an antibody, for example, the monoclonal antibody Neuro-1 (Reid et al.,
Molecular Brain Research 21: 1-8 (1994)
)) And the antibodies described by Brummendorf et al. (Neuro)
n 10: 711-727 (1993)). The detailed description of the invention and the methods described herein in Examples 1 to 3 are used to detect and quantify the level of mRNA encoding contactin.

Consistent with the results obtained in Example 3, MS, ALS, MELAS, ME
The amount of contactin in samples from patients with RRF, and / or other neurological disorders is altered compared to the amount of contactin in control samples.

Example 5 Screening of Test Compounds to Increase or Decrease Expression of Contactin mRNA or Contactin Protein Express normal, increased or decreased amount of contactin mRNA or contactin protein Cells are provided. Such cells can contain, for example, an expression construct containing a nucleic acid encoding a contactin protein (Reid et al., Molecular Brain Research 21: 1-8 (199).
4), Ogawa et al., Neurosci. Lett. 218: 173-176 (
1996)). It is expected that an increase in the amount of contactin mRNA and protein will occur if the nucleic acid encoding contactin is transcribed in the "sense" orientation. In contrast, a reduction in the amount of contactin mRNA and protein would be expected if the nucleic acid encoding contactin were transcribed in the "antisense" orientation.

These cells are contacted with individual members of a library of test compounds known to have pharmaceutical activity. The amount of contactin mRNA and contactin protein is measured in such cells before and after being contacted with a test compound using the method of the invention. A test compound that increases or decreases the amount of contactin mRNA or contactin protein has been identified,
And these are putative therapeutic agents that increase or decrease the amount of contactin mRNA or contactin protein in a patient.

Example 6 Differential Genetic Expression in Brain Brain Samples of AD Individuals diagnosed with Alzheimer's disease (AD sample), individuals diagnosed with MS, or control individuals (control or “ Human brain tissue from the cerebral occipital poles of the "C" sample) was obtained immediately after mortem and stored frozen at -80 ° C. All poly A + RNA was isolated from frozen brain tissue after thawing and extracting the tissue according to standard protocols. Poly (A) + RN
A was prepared from 3 different areas of the brain by autopsy of 5 normal (control) humans and 7 Alzheimer's disease patients. A preparation of radiolabeled cDNA derived from poly (A) + RNA was used to generate essentially a set of labeled probes using the reaction of the Superscript II kit and oligo-dT as a primer. Manufacturer (Life Techno
Logged) protocol was prepared. Label the probe with Gen
ome Systems. Inc. (St. Louis, MO) GDA 1.3
(Gene Discovery Array) and hybridized.

(A selection of assay systems for gene discovery is described, for example, in Genome.
Systems, Inc. (GDA), Research Genetics,
Inc. Or Clonetech Laboratories, Inc. (P
alo Alto, CA), or high density microarrays (eg, Affym)
etrix GeneChip and Genome Systems / In
It can be modified to include a filter system by Cyte / Synteni (GEM)). For example, Affymetrix Inc. By 40,000
40,000 oligonucleotides representing 3 human genes (35,000
GeneChi containing 0 ESTs and about 6,000 full-length cDNAs)
p or Genome Systems Inc. , GEM chips containing 7,000 cDNAs displaying 7,000 ESTs with about 4,000 known human genes according to G., can optionally be screened as a high throughput microarray. 3.) The probed membrane was washed and exposed to a Phosphoimager screen to produce a series of digital image files showing the intensity of hybridization of the probe to individual locations on the membrane. Manufacturer (Genome Systems. Inc., St. Louis, MO)
According to, each individual position on the filter has a DNA sequence derived from a specific known gene. Although the manufacturer's identification of genes whose DNA sequences are in place is generally accurate, some variation was found in some of the examples described herein.

Image files containing 18,000 different brain gene expression profiles of normal and Alzheimer's patients were first processed for background normalization and mean values (Genome Systems, Inc.
． so). These results reduce the genetic sequences present in higher amounts in AD patients compared to control samples (ie, "genes that are upregulated" in AD), and in AD patients compared to control samples It was used to rank the degree of hybridization of the labeled probe at each position with respect to both genes present in abundant amounts (ie, genes "downregulated" in AD).

The ranked data were further analyzed by an algorithm to analyze clusters of genes that are altered in the diseased brain, particularly with respect to altered gene expression profiles in some affected areas of the brain. Used and analyzed as described herein. Such an algorithm is disclosed in US patent application Ser. No. 09 / 397,380 filed Sep. 15, 1999. Its contents are incorporated herein by reference. The results are shown in Table 3,
It is summarized in Table 4A. In Alzheimer's disease (AD) brains, 28 genes were down-regulated by more than 5 fold and 38 genes were up by more than 5 fold as compared to normal control (C) brains. It was regulated.

[0170]

[Table 4A]

[0171]

[Table 4B]

[0172]

[Table 5]

[0173]

[Table 3] Example 7 Quantitative Real-Time PCR Analysis of Differentially Expressed Genes Quantitative real-time PCR (Q-RTPCR) was performed on the differentially expressed genes of AD described in the previous example. It was used to measure some expression. The Q-RTPCR techniques, as they are applied to the contactin sequence, are described in Example 2.
In detail. In this example, the same basic technique was performed, essentially as in the previous example, as described in detail below, with the following exclusions.

Each pair of oligonucleotide primers (one in the forward direction and one in the reverse direction) was not derived from contactin, but differentially expressed genes in each AD for Q-RTPCR experiments. Had a sequence derived from Oligonucleotide probes with sequences that specifically hybridize to each of the differentially expressed genes in AD were also prepared (see "Table B"). Specific nucleic acids are detected and Q-RTPCR, as well as primers and SYBR (
ABI PRISM 7700 using registered trademark Green PCR reagents
It was measured by the Sequence Detection System.

The internal RNA normalization group was used in Q-RTPCR to normalize sample-to-sample variation. The RNA normalization group was treated with the endogenous RN
Species A (eg, constitutively expressed proteinaceous actin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or ribosomal RN)
MRNA encoding A (eg 18S or 28S RNA). RNA molecules produced in vitro can also be used as a normalization group.
Therefore, the results of Q-RTPCR analysis can often be expressed as relative amounts.
The putative amount of RNA differentially expressed in AD was calculated as “Use Bulletin
Manufacturers # 2 (version December 11, 1997) (
PE Corp. , PE Applied Biosystems Divs
Ion, Foster City, CA), ABI PRISM 7
700 Sequence Detection System, “Compe
rative C _T Method "and was determined by comparison with normalized group essentially according to the protocol and formula have not been described on pages 11-15 of the section which is entitled; The above examples some of these formulas 2 To provide.

Human brain samples were tested for various areas of the brain (eg, lower temporal cortex (ITC)).
, Medial frontal cortex (MFC), occipital pole (OP), temporal parietal region, and cerebellum). RNA was isolated from the biological sample essentially according to the method described in Example 3. Briefly, total RNA was isolated from biological samples using TRIzol® Reagent supplied by Life Technologies®, essentially according to the manufacturer's instructions (TRIzol. (One version of Reagent® is described in US Pat. No. 5,346,994). Briefly, tissue samples were homogenized in TRIzol® Reagent (2 mL TRIzol® Reagent per 50 mg to 100 mg tissue). The homogenized sample was incubated for about 5 minutes at about 15 ° C to 30 ° C. Chloroform (0.2 ml per 1 ml TRIzol used for the first homogenization) was added and the mixture was shaken vigorously for 15 seconds, incubated for 2 or 3 minutes at about 15 ° C to 30 ° C and about 1
Centrifuged at 2,000 xg for 15 minutes at about 2-8 ° C. The aqueous phase was transferred to a new container and the RNA in it was used for the first homogenization of 1 m.
Precipitated by mixing with 0.5 ml isopropyl alcohol per 1 TRIzol® Reagent. Mix the mixture at about 15 ° C to 30 ° C
Incubated at 15 ° C. for 15 minutes and centrifuged at about 12,000 × g for 15 minutes at about 2 ° -8 ° C. The supernatant was removed, and the resulting pellet was washed once with 75% ethanol and recollected by centrifugation at approximately 7,500 xg for 5 minutes at 2-8 ° C. The resulting pellet was collected, dried and resuspended in buffer.

Data showing the expression of genes differentially expressed in 10 AD as determined by Q-RTPCR are shown in Table 5. The data in Table 5 are presented as the ratio of expression levels in AD samples compared to the expression levels in control (C) brain.
Ratios greater than 1.0 indicate gene sequences that are up-regulated in AD brains compared to control brains, while ratios less than 1.0 indicate AD compared to control brains. 3 shows a gene sequence that is down-regulated in the human brain.

Example 8 Features and Functions of Genes Differentially Expressed in Neurodegenerative Diseases Without wishing to be bound by any theory, the above differentially expressed in neurodegenerative diseases A gene (and the protein encoded by it) has the characteristics and functions reported in the scientific literature. A summary of studies to date on each of the differentially expressed genes and their gene products (if known) is provided below.

Contactin Human contactin protein, which is upregulated in MS brain as described in the previous example, and its homologue from mouse (F3 protein) and chicken. Its homolog (F11 protein) is a cell surface adhesion protein that is involved in cell adhesion to substrates. Contactin contains an Ig-like domain and multiple fibronectin III-like domains (Brummendorf et al., J. Neurochemistry).
61: 1207-1219 (1993)). Unlike many cell adhesion molecules,
Contactin, although not a transmembrane protein, is instead bound to the cell surface through linkage to glycosylphosphatidylinositol (GPI) in the outer leaflet (Id.) Of the plasma membrane. In human tissues, relatively high levels of major contactin mRNA (6.5 kb) resulted in three minor transcripts (9.7 kb, 4.4).
kb, and 3.4 kb)) in the adult brain, but nevertheless low levels of multiple forms of contactin mRNA are expressed in the adult lung,
Found in pancreas, kidney, and skeletal muscle (6.8 kb, and 6.0 kb) (
Reid et al., Molecular Brain Research 21: 1-.
8 (1994)). High levels of expression of multiple forms of contactin mRNA are found in neuroblastoma and retinoblastoma cell lines (6.8 kb, 6.0 k).
b, and 4.2 kb) (Id.). Expression of contactin in developing neural tissue is complex, transient, and temporally regulated. Contactin is believed to have a role in neurite outgrowth, presumably by binding to the cell recognition molecule Ng-CAM and / or by interacting with the extracellular matrix glycoprotein restrictin (Faivr).
e-Sarrailh et al. Neurosci. 12: 257-267 (19
92), Brummendorf et al., Neuron 10: 711-11272 (
1993)). Adult neural stem cells can give rise to hematopoietic cells, including cells of the spinal cord and lymphoid lineages (Bjornson et al., Science 283: 534).
-537 (1999)); thus, contactin mRNA, contactin D
NA, or contactin protein, can be detected in blood.

COX7c COX7c (down-regulated in AD brain as described in previous examples) corresponds to the VIIc subunit of complex IV (cytochrome c oxidase) of the ETC chain. In mammals, COX7c is a mitochondrial protein, which is encoded by the nuclear rather than mitochondrial genome. A number of the above assays for the activity and properties of COX7c and / or ETC subunit IV are useful screening assays to identify chemical and biological treatments for AD and other neurodegenerative disorders. Can act as.

(FREAC-2) Forkhead-related activator-2 (Forkhead Related)
Activator-2) (FREAC-2), which is up-regulated in the brain of AD as described in previous examples, was described by Pierrou et al. (EM).
BO J. 13: 5002, 1994) first cloned from a human cDNA library. FREAC-2 is a "winged helix (win
“Ged helix” or “forkhead” transcription factor family members (for a review, see Kaufman et al., Me.
(chanism of Development 57: 3-20, 1996).
. FREAC-2 binds to DNA as a monomer and is responsible for embryogenesis, tumorigenesis,
And play a regulatory role in the maintenance of differentiated cells. Published reports suggest that FREAC-2 expression is restricted to the lung and placenta; thus, elevated expression of FREEAC-2 in the AD brain results in the development and / or maintenance of AD. Can be an abnormal process that plays a role in. There are various activities of FREAC-2, including but not limited to its binding to DNA (Hell
qvist et al. Biol. Chem. 271: 4482-4490, 199
6).

(APCL) APCL (“Adenomatous Polypopsis Coli L
The "ike" (adenomatous polyposis coli-like) gene, which is upregulated in AD brains as described in previous examples, is specifically expressed in the brain. It encodes a protein of 2023 amino acid residues. APCL has various in vitro and in vivo activities and functions. Each of these is A
It has been tested for a significant degree that may be associated with D and its role in other neurodegenerative diseases.

For example, APCL binds β-catenin in vitro, and overexpression of APCL in SW480 cells results in a reduced pool of intracellular β-catenin (
Nakagawa et al., Cancer Research 58: 5176-51.
81, 1988). Upregulation of APCL as seen in AD is therefore expected to reduce intracellular levels of β-catenin. In the brains of AD patients who also have a mutation in presenilin-1, a protein that complexes with β-catenin and stabilizes β-catenin, the levels of β-catenin are reduced and / or Alternatively, the β-catenin protein is inappropriately targeted intracellularly (Zhang et al., Nature 395: 698-702,
1998; Nishimura et al., Nature Medicine 5:16.
4-169, 1999). Furthermore, decreased β-catenin signaling increases neuronal vulnerability to apoptosis and acidic stress induced by amyloid-β protein (Zhang et al., Nature 39).
5: 698-702, 1998).

Members of the Tcf family of transcription factors that associate with β-catenin mediate transactivation of downstream genes involved in neural development (Cho et al. Mec.
hanisms of Development 77: 9-18, 1998).
. Thus, according to a non-limiting theory, β-catenin may be associated with the onset or progression of AD and other neurodegenerative disorders.

(LAP) Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression that is upregulated in the brain of AD. Neither the physiological substrates nor the functional significance of LAP are known. Mice with a LAP deficiency created by targeted disruption of the LAP gene are fertile and usually develop, and microscopic examination of various peripheral organs reveals that locally conserved material Podocyte with the appearance of different superstructures
And reveals progenitor lysosomal storage in tubular epithelial cells of the kidney. In the central nervous system of such mice, lysosomal storage is associated with the progression of progressive astrocyte formation and activation of astrocytes, in the presence of cellular microglial cells, ventricles. It was detected locally in ependymal cells and astrocytes to varying degrees. Behavioral and neuromotor analysis allowed the distinction between control and deficient mice, but approximately 7% of deficient animals developed the commonly referred Keiren seizure (Saftig et al., J. Biol. Che.
m. 272: 18628-18635, 1997).

(PAF) Peroxisome Assembly Factor-1 (peroxisome assembly factor-1) (PAF), which is downregulated in AD, is responsible for the normal assembly of peroxisomes in the liver and other organs. Needed. Defects in the assembly of peroxisomes have many detrimental effects, including the widespread distribution of iron in all organs found in Zellweger syndrome and other disorders. Zellweger's syndrome is typically fetal up to the age of 6 months, but some first attempts at assessing gene therapy potential for defective peroxisomal assembly hold promise. Young, SP and Aisen P .: T
he Liver and Iron. The Liver Biology
and Pathology (IM Arias, JL Boyer)
, N .; Fausto, W.F. B. Jakoby & D. Schachter)
609, Raven Press, New York, 1994).

(6PTS-1) 6-pyruvoyl-tetrahydropterin synthase (6PTS-1, aka
PTPS), which is down-regulated in AD, is the second enzyme in the biosynthetic pathway from GTP to tetrahedrobiopterin (BH4). Second, BH4 is an essential cofactor for NO synthesis and aromatic amino acid hydroxylases. The latter is, among other things, responsive to the biosynthesis of monoamine neurotransmitters (Turri et al., Biol. Chem. 379: 1441-1447, 19).
98). Thus, downregulation of 6PTS-1 can lead to diminished synthesis of certain neurotransmitters in AD and other neurodegenerative disorders.

VDAC-1 VDAC-1 (which is downregulated in AD brain) is one of several voltage-gated anion channels (VDAC, also known as mitochondrial porin). Is one. VDAC is a small pore-forming protein in the outer membrane of mitochondria found in all eukaryotes, and is the binding site for several cytoplasmic enzymes, including isoforms of hexokinase and glycerol kinase. . VDAC also processes ATP when in the open state, allowing bound kinases to preferentially access mitochondrial ATP, and a possible mechanism for regulation of adenine nucleotide flux. (For a review, see Sampson et al., J. Biol. C).
hem. 272: 18966-18973, 1997; Mannella, J .;
Bioenerg. Biomembr. 29: 6525-6531, 1997).
.

Several VDAC isoforms have been found in mammals (Blachly).
-Dyson et al., Genomics 20: 162-167, 1994). In vitro, VDAC processes a variety of small metabolites, and in vivo they act as binding sites for several cytoplasmic kinases involved in intermediate metabolism, yet The specific physiological role of the isoform is unknown. Mouse embryonic stem cells lacking each isoform are viable but show a 30% reduction in oxygen consumption. VDAC-1 and VDAC-2
Deficient cells show reduced cytochrome c oxidase activity, but nevertheless VDAC-3 deficient cells have normal activity (Wu et al., Biochim.
． Biophys. Acta 1452: 168-178, 1999).

It has been suggested that the mitochondrial permeability transition (MPT) pore may contain VDAC molecules (Szaboe et al., FEBS Lett. 330: 206-10).
, 1993). In support of this proposal, in vitro experiments promoted the pre-apoptotic proteins Bax and Bak to open VDAC, but nevertheless the anti-apoptotic protein Bcl-x (L
) Demonstrates closing the VDAC by binding directly to it.
Bax and Bak allow cytochrome c to pass outside the VDAC of liposomes, but such passage is blocked by Bcl-x (L). These results indicate that the Bcl-2 family of proteins binds to VDAC to regulate mitochondrial membrane potential and regulate cytochrome c release during apoptosis (Shimizu et al. Nature 39).
9: 483-487, 1999).

Novel EFHD-Like Proteins Expressed in Brain A large number of EF-hand (EFHD) proteins have been identified, recognizing many different subfamilies, such as over 40 (Kawasaki et al., Biometal 11: 277-295, 1998). The EF-hand motif is composed of two α-helices “E” linked by a Ca (2 +)-binding loop.
] And “F”. EF-hand has been identified in many Ca (2 +)-binding proteins by amino acid sequence similarity and has been confirmed in several crystal structures. Functional EF-hands are prone to pairing. To date, the family of EF-hand homologs has
It contains over 160 various Ca (2 +)-regulated proteins, which have a wide range of functions. Among these, troponin C, myosin regulatory light chain, parvalbumin, S-100 protein, and calbindin (cal).
bindins) 9 and 28 kDa are calmodulins.

The most prominent feature of the EF-hand family members is their ability to bind calcium (Ca2 +) with dissociation constants in the micromolar range, and the ability to regulate the activity of many enzymes. The activity of the EHFD protein is generally believed to be regulated by a stimulus-induced increase in cytosolic free Ca2 + (Weinman, J. Biol. Buccale 1: 90-98).
, 1991). With the exception of Troponin-C, all subfamilies and unique EF-hand homologs presented in vertebrates can be found in the CNS (P
ersecini et al., Trends Neurosci. 11: 462-46
7, 1989). The novel brain-expressed EFHD-like proteins described herein are up-regulated in AD, as described in previous examples. Assays for various activities and properties of novel EFHD-like proteins expressed in the brain include, but are not limited to, the intracellular calcium assay. As a non-limiting example, one set of intracellular calcium assays that may be used in the present application is described, for example, in co-pending US patent application Ser. No. 60/176, which is incorporated herein by reference. No. 384.

Example 9 Quantitative Real-Time PCR Analysis of Expression of Genes Differentially Expressed in AD in Other Neurodegenerative Diseases The present invention provides a consistent manner in a variety of neurodegenerative diseases. Identify differentially expressed genes (“shared genes”), as well as genes that are differentially expressed in one particular neurodegenerative disease but not in another (“disease-specific gene”) Used to The expression patterns of several AD up-regulated and AD down-regulated genes in neurodegenerative diseases other than AD according to the previous examples were further tested as follows.

Quantitative real-time PCR (Q-RTPCR) was used to measure the expression of these genes in samples from other diseases. Human brain samples from various regions (eg, for the inferior temporal cortex (ITC), medial frontal cortex (MFC), cerebral posterior pole (OP), temporal parietal and cerebellum). , Obtained as soon as possible after death. Samples were obtained from patients diagnosed with Alzheimer's disease (AD), vascular dementia (VaD), chronic Lewy Lewy body disease (DLB), Parkinson's disease (PD), and control (C) patients. Biological samples were frozen and stored at -80 ° C and shipped on dry ice.

Q-RTPCT, VDAC-1, FREAC-2, COX7c, and AP
Primers and probes specific for CL were used to perform on brain samples from a dead person with one or more neurodegenerative disorders. The results from these experiments were grouped according to the grade and degree of AD in each death. That is, "sAD" indicates severe AD and "mAD"
Indicates mild AD, and "No AD" indicates no AD features. However, it should be noted that deaths in each of these classes of AD may nevertheless have one or more other neurodegenerative disorders, such as PD.

The results of these experiments for VDAC-1, FREAC-2, COX7c, and APCL are shown in Tables 6, 7, 8 and 9, respectively. The Q-RTPCR primers and probe were used in the following reactions. Each probe was designed to be complementary to a portion of the mRNA of interest (ie, having an antisense sequence when compared to the sense strand present in the mRNA). The part to which the probe hybridizes was placed between the two primers used for amplification, ie in the amplification product. The target mRNA was labeled with a fluorescent dye (“
Quencher "). This acted to quench the signal from the second fluorescent dye attached to the oligonucleotide probe. TA
MRA is typically used as a quencher, and various dyes can be used for labeling the probe (eg 6-FAM, TET, JOE, etc.).

Q-RTPCR was performed according to the instructions provided by the manufacturer of the instrument (http://www.pebiodocs.com/pebiodocs/
04303859. pdf), which is hereby incorporated by reference, in particular, pp. 11-15 therein, prior to initiating the polymerase chain reaction, the probe in the mRNA of interest. It hybridized specifically to the complementary sequence. The fluorescent signal by the probe was suppressed due to the close proximity of the probe dye to the quencher dye present in the mRNA. Therefore, at time = 0, little or no fluorescence was detected due to the dye linked to the probe. But P
During CR, the labeled probe is a mRNA that hybridizes to the helicase and / or exonuclease activity of the polymerase due to it.
Separated from. In either event, the dye initially present in the probe becomes physically separated from the quencher molecule when PCR is continued,
And as a result, the fluorescence signal corresponding to the probe molecule which changed the position increased with time. This signal is monitored over time, and this reflects the amount of mRNA of interest present in the sample (ie, higher amounts of mRNA molecule are required for additional PCR cycles and / or at varying concentrations). Primers and probes are required to separate the quencher dye molecule from the reporter dye molecule). These parameters and measurements were used to determine the amount of mRNA of interest in the sample.

[0198]

[Table 6]

[0199]

[Table 7]

[0200]

[Table 8]

[0201]

[Table 9] Table 7, like the other Tables 6, 8 and 9, evaluates the data from the above experiments in various ways. In order to understand the various results from data analysis, it must first be recognized that lower "Δ" Ct values correspond to higher levels of mRNA in question. “Δ” due to two areas of the brain that are thought to have AD
Ct values are compared in several ways. These analyzes of the data use some of the formulas shown in the previous examples.

[0202] To begin, the ratio "2 ^- .DELTA..delta ^Ct" is when compared with the expression in the cerebellum, showing a difference in expression of FREAC-2 in TPC patient (e.g., lane 2
6, second from the rightmost column). The most right column of the table, the ratio by three subgroups of patients ^- the mean of the "2 .DELTA..delta ^Ct" (and standard deviation, SD) shows a. These values are for patients with severe AD (sAD), patients with moderate AD (m
AD) and patients without AD (control), respectively,
3.5, 1.8, and 1.6.

Since the results described above are ratios, these can be achieved in two different ways. A ratio of 3.5 for the relative levels of FREEC-2 mRNA in TPC when compared to cerebellum was that "additional" FREEC-2 mRNA was TPC.
Indicates that it exists inside. This can be understood in two ways: FREAC.
-2 can be downregulated in the cerebellum of AD and / or FRE
AC-2 can be upregulated in TPC. Comparison of the other values in Table D2 to another helps distinguish these causes for the differential expression of FREAC-2 in the AD brain.

For example, determine the mean “Δ” C _t for TPC FREAC-2 in each patient subgroup: the values for sAD, mAD, and no AD are 9.9, 11.2, respectively. , And 11.6 (see lanes 7, 15, and 24 of Table D2). 9. For sAD, because the lower “Δ” Ct TPC corresponds to higher levels of FREEAC-2 mRNA.
A value of 9 indicates that FREEC-2 mRNA is more abundant in AD TPC when compared to 11.2 and 11.6 for mAD and no AD, that is, FREEAC-2 is TPC of AD. It is shown to be up-regulated in.

Similarly, the mean “Δ” Ct is determined for cerebellar FREAC-2 in each patient subgroup: values for sAD, mAD, and no AD are 11.5, 11. 9 and 12.1 (see lanes 8, 16 and 25 of Table D2). 11 for sAD as the lower “Δ” Ct TPC corresponds to higher levels of FREEAC-2 mRNA.
． A value of 5 indicates that FREAC-2 mRNA is more abundant in the cerebellum of patients with sAD when compared to 11.9 and 12.1 for mAD and no AD, ie It is shown to be upregulated in the cerebellum. However, the difference between the mean “Δ” Ct's for cerebellar FREAC-2 was less pronounced than the difference for TPC, indicating that the differences in the amount of FREEAC-2 in the various cerebellums were small. Suggest that. Moreover, increased levels of FREEAC-2 in the cerebellum are associated with "additional" FREEAC-2.
To achieve the ratio indicating that is present in the TPC.
It requires a level higher than the normal level of 2. Putting all the data together,
This result suggests that FREAC-2 is upregulated in TPC of sAD patients.

All publications, including patent and academic literature, referenced in this application are to the same extent as if each were individually incorporated by reference, in their entirety for all purposes. Incorporated herein by reference.

From the above, specific embodiments of the present invention are described herein for purposes of illustration, but various modifications can be made without departing from the spirit and scope of the invention. It is clear that it can be broken. Accordingly, the invention is not limited except as limited by the appended claims.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61P 25/28 4C086 48/00 31/04 4C087 A61P 25/00 31/12 25/28 35 / 00 31/04 C12Q 1/02 31/12 1/68 A 35/00 G01N 33/15 Z C12N 5/10 33/50 Z C12Q 1/02 33/53 D 1/68 M G01N 33/15 33 / 566 33/50 C12N 15/00 ZNAA 33/53 5/00 B A61K 37/02 33/566 37/24 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, K E, LS, MW , MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ , BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX , MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Davis, Robert E. United States California 92130, San Diego, Glencliff Way 13272 F term (reference) 2G045 AA40 DA12 DA13 DA14 DA36 FB02 FB03 4B024 AA01 AA11 CA04 DA03 EA04 FA02 FA18 GA11 HA12 HA17 4B063 QA08 QA19 QQ43 QR32 QR62 QS25 AQAQBQAQBQAQAQBQAQBQAQBQAQBQAQAQBQAQBQAQAQBQAQBQAQAQAQAQAQAQAQAQA CA44 CA46 4C084 AA02 AA13 AA17 BA01 CA53 ZA01 ZA02 ZA16 ZB26 ZB33 ZB35 4C086 AA01 EA16 MA01 MA04 NA14 ZA01 ZA02 ZA16 ZB26 ZB33 ZB35 4C087 AA01 BC83 CA12 NA14 ZA01 ZA02 B33 ZB33 ZB33 ZB33 ZB33

Claims (53)

[Claims] 1. SEQ ID NO: 1 or part thereof, SEQ ID NO: 2 or part thereof, SEQ ID NO: 3 or part thereof, SEQ ID NO: 4 or part thereof, SEQ ID NO: 5 or part thereof, SEQ ID NO: 6 or part thereof A contactin, comprising an isolated nucleic acid molecule that is identical or substantially identical to a nucleotide sequence selected from the group consisting of a portion and SEQ ID NO: 7 or a portion thereof. DNA or R
An oligonucleotide primer capable of specifically amplifying NA or a nucleic acid sequence complementary thereto. 2. The isolated nucleic acid molecule comprises a detectable label.
The composition according to. A kit comprising: SEQ ID NO: 1 or part thereof, SEQ ID NO: 2 or part thereof, SEQ ID NO: 3 or part thereof, SEQ ID NO: 4 or part thereof, SEQ ID NO: 5 or An isolated nucleic acid molecule that is identical or substantially identical to a nucleotide sequence selected from the group consisting of a portion thereof, SEQ ID NO: 6 or a portion thereof, and SEQ ID NO: 7 or a portion thereof. At least one oligonucleotide primer capable of specifically amplifying the contained DNA or RNA encoding contactin, or a nucleic acid sequence complementary thereto. 4. The kit of claim 3, wherein the at least one nucleic acid molecule comprises a detectable label. 5. A method for detecting the risk of having a neurodegenerative disease or the presence of that disease in a subject comprising the steps of: not being at risk of having a neurodegenerative disease. Or comparing the amount of contactin protein in a test sample derived from the subject to the amount of contactin protein in a sample derived from a control subject known to be absent of said disease Process. 6. A method of detecting the risk of having a neurodegenerative disease or the presence of that disease in a subject, comprising the steps of: not having or at risk of having a neurodegenerative disease. Of contactin mRNA in a test sample derived from a subject relative to the amount of contactin mRNA in a sample derived from a control subject known to be absent
Comparing the amount of NA. 7. The method according to claim 5, wherein the test sample and the control sample are from the central nervous system. 8. The method of claim 6, wherein the contactin mRNA is measured by the polymerase chain reaction method. 9. The polymerase chain reaction method comprises SEQ ID NO: 1 and SEQ ID NO: 3.
9. The method of claim 8, comprising amplification with a forward primer containing a nucleotide sequence selected from the group consisting of :, SEQ ID NO: 5, and SEQ ID NO: 6. 10. The method of polymerase chain reaction comprises amplification using a reverse primer containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 7. The method described in. 11. A method of detecting the risk of having a neurodegenerative disease or the presence of that disease in a subject comprising the steps of: not having or at risk of having a neurodegenerative disease Comparing the amount of contactin mRNA in a test sample derived from a subject to the amount of RNA in a sample derived from a control subject known to be free of disease, wherein: And the RNA of the control sample does not encode contactin. 12. The method according to claim 5, wherein the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, multiple sclerosis, MELAS, and MERRF. 13. The method according to claim 5, wherein the neurodegenerative disease is multiple sclerosis. 14. A method of screening reagents for use in treating a patient having a neurodegenerative disease, comprising the steps of: in a first sample derived from at least one patient. Determining an initial level of contactin expression, followed by contacting the patient with a candidate reagent, wherein the determining step comprises determining the amount of contactin protein, and contactant RNA A second level of contactin expression determined in a second sample from said patient after contacting said patient with said candidate reagent; To the first level of expression of contactin, wherein the change in the level of expression of contactin is determined by the reagent Indicating that it is suitable for use in treating a patient having a degenerative disease, process. 15. A cybrid cell line comprising immortalized cells having genomic and mitochondrial DNA of different biological origin and cells capable of differentiating, said cells being contactin. Expressing a cell line. 16. The cybrid cell line of claim 15, wherein the cells are nerve cells. 17. The cybrid cell line of claim 15, wherein the cells are human cells. 18. The cybrid cell line of claim 15, wherein the cells are human central nervous system cells. 19. A method of identifying a reagent capable of altering the expression of contactin in a cell, comprising the steps of: in at least one cell before and after contacting said at least one cell with a candidate reagent. Comparing the levels of contactin expression in E. coli, as well as identifying reagents by which the expression of contactin can be altered. 20. The candidate reagent comprises a test compound selected from the group consisting of small molecules, nucleic acid molecules, antisense nucleic acid molecules, and ribozymes.
9. The method according to 9. 21. The method of claim 19, wherein the at least one cell comprises a cybrid cell. 22. A reagent capable of altering the expression of contactin identified by the method of claim 19. 23. The reagent according to claim 22, which is selected from the group consisting of small molecules, proteins, polypeptides, antibodies, nucleic acid molecules, antisense molecules, and ribozymes. 24. A pharmaceutical composition comprising the reagent of claim 22 in a pharmaceutically acceptable carrier. 25. A method of treating a patient having a neurodegenerative disease comprising the step of administering to the patient an effective amount of the pharmaceutical composition of claim 24. 26. A method of treating a patient having a neurodegenerative disease, comprising the steps of: administering to said patient an effective amount of a pharmaceutical composition capable of altering the expression of contactin. 27. A pharmaceutical target containing a cellular component that binds to the reagent of claim 22. 28. A method of identifying a pharmaceutical target comprising contacting a compound that regulates contactin expression or activity with a biological sample known to exhibit contactin expression or activity. 29. A risk for Alzheimer's disease in a first subject having or suspected of being at risk of having such a disease, comprising the steps of: Method for determining its presence: The presence of at least one differentially expressed nucleic acid molecule associated with Alzheimer's disease in each of the first and second biological samples containing mitochondrial DNA Or a step of determining the absence, wherein the first biological sample is obtained from a first subject and the second sample is at risk of disease associated with altered mitochondrial function. A step obtained from a second subject that is known to be absent or absent, wherein the step involving Alzheimer's disease in the first biological sample is The presence of at least one differentially expressed nucleic acid molecule in series and the absence of the corresponding differentially expressed nucleic acid molecule having the corresponding nucleotide sequence in the second biological sample is , Showing an increased risk of Alzheimer's disease and thereby determining the risk of Alzheimer's disease or its presence. 30. The method of claim 29, wherein the determining step comprises: each of the first biological sample and the second biological sample immobilized on a solid support. An array of nucleic acids containing a population of isolated nucleic acid molecules, and under conditions sufficient and sufficient to allow hybridization of the DNA from the sample to the isolated nucleic acid molecules. Contacting for a period of time, wherein the isolated nucleic acid molecule comprises at least one differentially expressed nucleic acid molecule associated with Alzheimer's disease; and: ) Differentially expressed, and the amount of hybridization of the nucleic acid molecule associated with Alzheimer's disease in the first sample to an array of nucleic acids, (ii) differentially expressed, And comparing the amount of hybridization of the nucleic acid molecule of the second sample corresponding to the nucleic acid molecule associated with Alzheimer's disease in the first sample, and thereby at least 1 associated with Alzheimer's disease. Determining the presence or absence of three differentially expressed nucleic acid molecules. 31. The method of claim 29, wherein said differentially expressed nucleic acid molecule is selected from the group consisting of: (i) a sequence as set forth in any one of SEQ ID NOs: 1-15, And SEQ ID NO: 1
A nucleic acid molecule having a nucleotide sequence selected from the group consisting of the sequences shown in 6 and capable of hybridizing to the isolated polynucleotide; (ii) one of SEQ ID NOs: 17 to 50 (Iii) SEQ ID NO: 52, which has a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID NO: 51 and the sequence shown in SEQ ID NO: 51; To 67, and a nucleic acid having a nucleotide sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 68 and hybridizable to an isolated polynucleotide Molecule; (iv) having a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID NO: 69 and the sequence shown in SEQ ID NO: 70 A nucleic acid molecule capable of hybridizing to the isolated polynucleotide; and a nucleotide sequence that is the reverse complement of (v) (i), (ii), (iii), or (iv) nucleic acid molecule. A nucleic acid molecule having: 32. A method for identifying a molecular target for therapeutic intervention to treat Alzheimer's disease comprising the steps of: (a) (i) versus (ii) below. Steps of comparing (i) At least one expression level of at least one gene product in a first sample from a subject known or suspected of having Alzheimer's disease (Ii) at least one expression level of said gene product in a second sample from a subject who is not at risk or has no Alzheimer's disease; and (b) said second Detecting the altered expression level of at least one said gene product in said first sample as compared to the expression level of the gene product in said sample, wherein said The gene product is selected from the group consisting of: (i) a nucleotide selected from the group consisting of the sequence shown in any one of SEQ ID NOS: 1 to 15 and the sequence shown in SEQ ID NO: 16 Has an array,
A nucleic acid molecule capable of hybridizing to the isolated polynucleotide; (ii) selected from the group consisting of the sequence shown in any one of SEQ ID NOS: 17 to 50 and the sequence shown in SEQ ID NO: 51 A nucleic acid molecule that has a nucleotide sequence and is capable of hybridizing to an isolated polynucleotide; (iii) a sequence set forth in any one of SEQ ID NOs: 52-67, and set forth in SEQ ID NO: 68. A nucleic acid molecule that has a nucleotide sequence selected from the group consisting of sequences and is capable of hybridizing to an isolated polynucleotide; (iv) the sequence set forth in SEQ ID NO: 69, and set forth in SEQ ID NO: 70. A nucleic acid component having a nucleotide sequence selected from the group consisting of sequences and capable of hybridizing to an isolated polynucleotide. And (v) (i), (ii), (iii), or (iv) a nucleic acid molecule having a nucleotide sequence that is the reverse complement of the nucleic acid molecule, and (vi) (i), ( ii), (iii), (iv), or (v) a polypeptide encoded by a nucleic acid molecule, wherein the gene product exhibiting a detectably altered expression level is for treating Alzheimer's disease. Subscriptive or code for the therapeutic intervention. 33. A method for identifying a molecular target for therapeutic intervention to treat Alzheimer's disease comprising the steps of: (a) (i) versus (ii) below. Steps of comparing (i) At least one expression level of at least one gene product in a first sample from a subject known or suspected of having Alzheimer's disease (Ii) at least one expression level of said gene product in a second sample from a subject who is not at risk or has no Alzheimer's disease; and (b) said second Detecting the altered expression level of at least one said gene product in said first sample as compared to the expression level of the gene product in said sample, wherein said The further expression level is a reduced expression level and the gene product is selected from the group consisting of: (i) a sequence as set forth in any one of SEQ ID NOs: 1-15, and a sequence Having a nucleotide sequence selected from the group consisting of the sequences set forth in No. 16,
A nucleic acid molecule capable of hybridizing to the isolated polynucleotide; (ii) a nucleic acid molecule having a nucleotide sequence that is the reverse complement of the nucleic acid molecule of (i), and (iii) (i) or ( ii) a polypeptide encoded by a nucleic acid molecule, wherein said gene product exhibiting detectably altered expression levels is or is a molecular target for therapeutic intervention to treat Alzheimer's disease. Code 34. A method for identifying a molecular target for therapeutic intervention to treat Alzheimer's disease comprising the steps of: (a) (i) versus (ii) below. Steps of comparing (i) At least one expression level of at least one gene product in a first sample from a subject known or suspected of having Alzheimer's disease (Ii) at least one expression level of said gene product in a second sample from a subject who is not at risk or has no Alzheimer's disease; and (b) said second Detecting the altered expression level of at least one said gene product in said first sample as compared to the expression level of the gene product in said sample, wherein said The additional expression level is an increased expression level and the gene product is selected from the group consisting of: (i) a sequence as set forth in any one of SEQ ID NOs: 17-50, and a sequence A nucleic acid molecule having a nucleotide sequence selected from the group consisting of the sequences set forth in No. 51 and capable of hybridizing to an isolated polynucleotide; (ii) the reverse complement of the nucleic acid molecule of (i) And a polypeptide encoded by the nucleic acid molecule of (iii) (i) or (ii), wherein the gene product exhibiting a detectably altered expression level is: , Is a molecular target for or encodes a therapeutic intervention for treating Alzheimer's disease. 35. The first sample comprises mitochondria derived from a subject known to have or suspected of being at risk of having Alzheimer's disease. The method according to any one of claims 32 to 34, which comprises cybrid cells containing the same. 36. A subject to which the first sample is known to have, or is suspected of being at risk of having, a disease associated with altered mitochondrial function. Derived mitochondria (mitochondr)
Ion) -containing cells. 37. The second sample contains mitochondria derived from a subject who is not at risk of a disease associated with altered mitochondrial function or is absent thereof. 35. The method of any one of claims 32-34, wherein the method comprises resident cells. 38. The first sample comprises mitochondria derived from a subject known to have or suspected of having a risk of having Alzheimer's disease. Containing a cybrid cell, wherein the second sample comprises a cybrid cell containing a mitochondria derived from a subject who is not at risk of Alzheimer's disease or is free of Alzheimer's disease. 35. The method of any one of claims 32-34. 39. The at least one gene product having an altered expression level in the first sample has an increased expression level as compared to the expression level of the gene product in the second sample. 33. The method of claim 32. 40. The at least one gene product having an altered expression level in the first sample has a reduced expression level as compared to the expression level of the gene product in the second sample. 33. The method of claim 32. 41. A method of identifying a reagent for treating Alzheimer's disease, which comprises the steps of: (a) knowing or having the risk of having Alzheimer's disease. Contacting a first sample from a subject suspected of having with a candidate reagent under conditions and for a time sufficient to express at least one gene product, Wherein the gene product is selected from the group consisting of: (i) a group consisting of the sequence shown in any one of SEQ ID NOS: 1-15 and the sequence shown in SEQ ID NO: 16 Having a nucleotide sequence selected from
A nucleic acid molecule capable of hybridizing to the isolated polynucleotide; (ii) selected from the group consisting of the sequence shown in any one of SEQ ID NOS: 17 to 50 and the sequence shown in SEQ ID NO: 51 A nucleic acid molecule that has a nucleotide sequence and is capable of hybridizing to an isolated polynucleotide; (iii) a sequence set forth in any one of SEQ ID NOs: 52-67, and set forth in SEQ ID NO: 68. A nucleic acid molecule that has a nucleotide sequence selected from the group consisting of sequences and is capable of hybridizing to an isolated polynucleotide; (iv) the sequence set forth in SEQ ID NO: 69, and set forth in SEQ ID NO: 70. A nucleic acid component having a nucleotide sequence selected from the group consisting of sequences and capable of hybridizing to an isolated polynucleotide. And (v) (i), (ii), (iii), or (iv) a nucleic acid molecule having a nucleotide sequence that is the reverse complement of the nucleic acid molecule, and (vi) (i), ( ii), (iii), (iv), or (v) the polypeptide encoded by the nucleic acid molecule; (b) the expression level of the gene product in the first sample in the absence of a candidate reagent. By comparison with at least one expression level of at least one gene product in the first sample in the presence of the candidate reagent; and (c) is not at risk for Alzheimer's disease? Or determining the expression level of at least one of the gene products in a second sample from a subject in the absence of Alzheimer's disease, wherein in the absence of the candidate reagent, The gene product exhibits an altered expression level in the first sample as compared to the expression level of the gene product in the second sample, and: (i) non-existence of the candidate reagent If, in the presence, the gene product exhibits increased expression levels in the first sample as compared to the second sample, the reagent and the first
Contacting the sample with a sample reduces the expression level of the gene product in the first sample, and (ii) compares the gene product with the second sample in the absence of the candidate reagent. And then exhibiting a reduced expression level in the first sample, the step of contacting the reagent with the first sample increases the expression level of the gene product in the first sample, And thereby identifying reagents for treating Alzheimer's disease. 42. The first sample comprises mitochondria derived from a subject known to have or suspected of being at risk of having Alzheimer's disease. 42. The method of claim 41, which comprises cybrid cells containing. 43. A subject to which the first sample is known to have, or is suspected of being at risk of having, a disease associated with altered mitochondrial function. Derived mitochondria (mitochondr)
42. The method of claim 41, wherein the method comprises cells containing (ion). 44. The mitochondria derived from a subject, wherein the second sample is not at risk for a disease associated with altered mitochondrial function or is free of such disease. 42. The method of claim 41, which comprises cells containing. 45. The first sample comprises mitochondria derived from a subject known to have, or suspected of being at risk of having, Alzheimer's disease. Containing a cybrid cell, and the second sample contains mitochondria derived from a subject who is not at risk of Alzheimer's disease or is free of such disease. 42. The method of claim 41, which comprises cybrid cells that are 46. The at least one gene product having an altered expression level in the first sample has an increased expression level as compared to the expression level of the gene product in the second sample. Item 41. The method according to Item 41. 47. The at least one gene product having an altered expression level in the first sample has a decreased expression level as compared to the expression level of the gene product in the second sample. Item 41. The method according to Item 41. 48. Administering to a subject in need thereof a therapeutically effective amount of a composition containing a reagent identified according to the method of any one of claims 41-47. A method of treating Alzheimer's disease. 49. A method of treating a patient known to have or suspected of being at risk of having Alzheimer's disease, including: (a) Comparing (i) to (ii) (i) at least in a first sample from a subject known or suspected of having Alzheimer's disease At least one expression level of one gene product, (ii) at least one of the gene product in a second sample from a subject who is not at risk or has no Alzheimer's disease Expression level; and (b) an altered expression level of at least one of said gene products in said first sample as compared to the expression level of said gene product in said second sample. Wherein the altered expression level is selected from the group consisting of increased expression level and decreased expression level, and wherein the gene product is selected from the group consisting of: Step (i) having a nucleotide sequence selected from the group consisting of the sequence shown in any one of SEQ ID NOS: 1 to 15 and the sequence shown in SEQ ID NO: 16,
A nucleic acid molecule capable of hybridizing to the isolated polynucleotide; (ii) selected from the group consisting of the sequence shown in any one of SEQ ID NOS: 17 to 50 and the sequence shown in SEQ ID NO: 51 A nucleic acid molecule that has a nucleotide sequence and is capable of hybridizing to an isolated polynucleotide; (iii) a sequence set forth in any one of SEQ ID NOs: 52-67, and set forth in SEQ ID NO: 68. A nucleic acid molecule that has a nucleotide sequence selected from the group consisting of sequences and is capable of hybridizing to an isolated polynucleotide; (iv) the sequence set forth in SEQ ID NO: 69, and set forth in SEQ ID NO: 70. A nucleic acid component having a nucleotide sequence selected from the group consisting of sequences and capable of hybridizing to an isolated polynucleotide. And (v) (i), (ii), (iii), or (iv) a nucleic acid molecule having a nucleotide sequence that is the reverse complement of the nucleic acid molecule, and (vi) (i), ( ii), (iii), (iv), or a polypeptide encoded by the nucleic acid molecule of (v), (c) having a therapeutically effective amount of a composition selected from the group consisting of: having Alzheimer's disease Administering to a patient known to be or suspected of being at risk of having Alzheimer's disease: (i) decreasing the expression level of a gene product exhibiting an increased expression level in said first sample (Iii) a composition containing a reagent that increases the expression level of a gene product exhibiting a reduced expression level in the first sample, (iii) the composition One A composition containing a reagent that is a modulator capable of inhibiting a biological activity or a biological process associated with a gene product exhibiting increased expression levels in a sample, and (iv) associated with the gene product Comprising a reagent that is a modulator capable of enhancing or activating a biological activity or a biological process, wherein the gene product is reduced in the first sample. Selected from the group consisting of a gene product exhibiting expression levels, and a gene product exhibiting increased expression levels in the first sample. 50. The altered expression level detected in step (b) is an increased expression level of at least one gene product, and the composition administered in step (c) comprises the gene product. 50. The method of claim 49, comprising a reagent that reduces the expression level of A., wherein said reagent is selected from the group consisting of antisense nucleic acids and ribozymes. 51. The altered expression level detected in step (b) is a reduced expression level of at least one gene product, and the composition administered in step (c) comprises the gene product. A nucleic acid molecule encoding the gene product, a nucleic acid expression vector containing the nucleic acid molecule encoding the gene product, and a nucleic acid molecule encoding the gene product. 50. The method of claim 49, selected from the group consisting of encoded proteins. 52. The altered expression level detected in step (b) is an increased expression level of at least one gene product, and the composition administered in step (c) comprises said first Comprising a reagent that is a modulator capable of enhancing or activating a biological activity or process associated with a gene product exhibiting a reduced expression level in a sample of: Can be bound to,
The method of claim 49. 53. The altered expression level detected in step (b) is a reduced expression level of at least one gene product, and the composition administered in step (c) comprises the first A reagent that is a modulator capable of inhibiting a biological activity or process associated with a gene product exhibiting an increased expression level in a sample of, wherein the modulator binds to the gene product. 50. The method of claim 49, which can be.