Patent 6,803,233

The present invention provides a model for studying the development of, and/or pathologies associated with neurodegenerative diseases, and agents that can alter such development and/or pathologies. The model of the invention is especially useful as an Alzheimer's disease model. The model of the invention provides brain cells and a method for increasing neurodegenerative disease characteristics in such cells, especially, induction of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases by selectively increasing the concentration of cathepsin D to an effective level, and/or by lowering the concentration of cholesterol in such cells. The model also provides a method of reversing such effects, by inhibiting cysteine protease and mitogen activated kinase activity, and especially, by inhibiting calpain, and/or MAP kinase.

DETAILED DESCRIPTION OF THE INVENTION

I. Characteristics of the Neurodegenerative Disease Brain Cells of the Invention, or Brain Tissue Containing the Same

The present invention provides a novel method for triggering brain cells, or brain tissue containing the same, to induce the characteristics of a brain cell or tissue from a brain that is afflicted with a neurodegenerative disease. The present invention also provides novel methods for inhibiting or preventing the development of such characteristics of such neurodegenerative disease in the brain cells.

The model of the present invention is based on, in part, the discovery that experimental lysosomal dysfunction, decreases in cholesterol concentration, and especially a combination of both, rapidly induce the formation of one or more brain cell characteristics that are indicative of a decline of neuron functioning and are associated with, and especially in combination, definitive for, neurodegenerative diseases and especially age-related neurodegenerative diseases such as Alzheimer's disease. According to the invention, exposing a brain cell to conditions that increase the concentration of cathepsin D ("cathepsin D-increasing compound"), or exposing a brain cell to conditions that decrease the concentration of cholesterol ("cholesterol decreasing compound"), or both, surprisingly trigger the hyperphosphorylation of the protein tau and tau fragments, the production of neurofibrillary tangles, the production of tau fragments, increases in cytokine activity and levels, microglia activation, and induction of brain inflammatory reactions and other indicia or characteristics of neurodegenerative diseases. Such effects are even more surprisingly enhanced when apoE-deficient or apoE4-containing brain cells, or brain tissue containing the same, are use in the model. Such exposure can come from altering the environmental conditions to which the brain cell is exposed, or, preferably by contacting or treating brain cells, or brain tissue containing the same, with a compound capable of inducing such effective levels of cathepsin D and/or of decreasing the concentration of cholesterol

Brain cells, or tissue containing the same, maintained under conditions in which cathepsin D is selectively induced, and/or cholesterol is selectively decreased, are characterized by the de novo appearance and/or accumulation of, one or more characteristics of neurodegenerative diseases in the cells. The accumulation of such characteristics is relative to the levels present in the cells at the start of the treatment or exposure to the indicated condition, and/or relative to the levels present in similar cells not contained with, or otherwise maintained in the absence of, the cathepsin D-increasing compound and/or compounds capable of selectively decreasing cholesterol. Such characteristics include:

(1) the formation of neurofibrillary tangles,

(2) the hyperphosphorylation of tau,

(3) the fragmentation of tau, that is, tau proteolysis and especially, increased amounts of the 15-35 kDa forms of tau ("tau fragments"),

(4) increased production and/or release of brain-produced pro-inflammatory cytokines especially TGF-beta (tumor growth factor beta or TGF1), TGF-alpha, IL1 (interleukin-1), IL1-alpha (interleukin-1alpha), IL1-beta (interleukin-1beta), IL6 (interleukin-6), IL10 (interleukin-10), TNF (tumor necrosis factor), TNF-alpha (tumor necrosis factor alpha) and LPS (lipopolysaccharide), and most especially TGF-beta, IL-1beta and LPS,

(5) increased microglia reaction and/or activation,

(6) increased indications of brain inflammatory reactions, such as, for example, increased positive staining for HLA-DR (which detects reactive microglia); increased positive staining for glial fibrillary acidic protein (GFAP; to detect reactive astrocytes); the extracellular accumulation of complement proteins, complement inhibitors, acute phase reactants, growth factors, heat shock proteins, proteoglycans, lipoproteins, cathepsins, cystatins, coagulation factors, proteases, protease inhibitors, integrin adhesion molecules, etc., and

(7) increased conversion of p35 to p25

(8) changes in the levels and activities of protein kinases, for example, cyclin dependent protein kinase 5 (cdk5) and mitogen activated protein kinase (MAPK).

Additional characteristics useful as an indicator of a brain afflicted neurodegenerative diseases can include, e.g., an increased amount of lysosomes, the appearance of basophilic granules in the mossy fiber terminal zone, the presence of secondary lysosomes with lipofuscin, amyloid deposition, amyloid plaques, neuritic plaques, synaptic loss, neuritic degeneration, neuronal death, increased glial elements (astrocytes, microglia), fragmentation of the amyloid precursor protein, increases in the levels of Cathepsin D, etc.

The above characteristics, and others describe herein, are indicative of a decline of neuron functioning. Such decline may be the result of a direct effect of the characteristic or an indirect effect. Characteristics that are the result of a direct effect are those found in the neurons, for example, an increase in tau phosphorylation or tau proteolysis and fragmentation. Characteristics that are the result of an indirect effect are those that are found in brain cells other than neurons, for example, induction of glial activation.

The first, and most preferable characteristic of brain cells, or brain tissue containing the same, cultured under conditions that selectively increase cathepsin D, and/or that selectively decreases levels of cholesterol, according to the method of the invention is the formation of neurofibrillary tangles in the cells. The formation of neurofibrillary tangles refers to the appearance and/or accumulation of intraneuronal deposits that are composed mainly of paired helical filaments. Generally, such tangles can be seen with light microscopy. The method of the invention is especially characterized by its ability to induce the appearance of "early tangles" in the brain cells, or brain tissue containing the same. "Early tangles" refer to intraneuronal tangles typically found at an early stage of neurodegenerative disease, such as Alzheimer's disease. Under appropriate conditions as described herein, such "early tangles" are typically formed, or enhanced levels are detectable, within a few days of culture or treatment. Such early tangles may appear in the brain cells in any of day 1, 2, 3, 4, 5 or 6, or even beyond, after initiation of the appropriate condition in culture or after administration of the appropriate agent(s) in vivo. Preferably, such early tangles appear 2-6 days in culture embodiments. However, longer periods are acceptable, especially in the in vivo models, because in vivo models are not constrained by the same viability considerations as in vitro models. Morphologically, such tangles mimic early-stage tangles (i.e., intracellular tangles) found in the brain of Alzheimer's patients.

As described above, it was previously shown that neurofibrillary tangles may contribute to the cognitive decline associated with neurodegenerative diseases and may also trigger neuronal cell death in the brain. However, many currently available in vivo and in vitro models of neurodegenerative diseases lack this or other key features associated with brain cells from patients inflicted with these conditions. Thus, the present invention advantageously provides a model brain cell system, wherein the brain cells contain, or can be induced to contain, among other things, neurofibrillary tangles. The appearance and/or disappearance of such tangles, as a result of the presence or absence of various therapeutic candidates or culture conditions, can be monitored and used to assess the value of therapeutic candidates that might be useful for the treatment of such conditions or diseases.

A further characteristic of brain cells maintained according to the method of the invention so as to induce the formation of indicia of neurodegenerative disease is the accumulation of levels (amounts or concentrations) of phosphorylated tau that are greater than levels found in control cells or in the same cells at the beginning of the culture. Phosphorylated tau, and especially abnormally phosphorylated and/or hyperphosphorylated tau, can be assayed in the cells as a whole, or in subfractions, for example, in soluble and/or insoluble fractions thereof, including paired helical filaments.

A further characteristic of brain cells incubated according to the method of the invention so as to induce the formation of neurodegenerative disease indicia is the production or accumulation of greater amounts of proteolytic fragments of tau, and specifically, tau fragments having a molecular weight of 15-35 kDa fragments (tau fragments), when compared to control cells or to the same cells at the beginning of the culture, especially fragments having an apparent molecular weight of 33 or 29 kDa. The 29 kDa tau fragment results from cleavage at amino acids 200-257. Larger fragments may also be seen. For example, the appearance or accumulation of a fragment with an apparent molecular weight of 40 kDa is an indicia of neurodegenerative disease. Such proteolytic products of tau can be unphosphorylated and/or phosphorylated and can include hyperphosphorylated forms. As above, tau fragments can be measured in the cells as a whole, or in soluble and/or insoluble fractions thereof, including paired helical filaments.

A further characteristic of brain cells incubated according to the method of the invention so as to induce the formation of neurodegenerative disease indicia is the increased production and/or release and/or accumulation of brain-produced cytokines especially, TGFb (tumor growth factor-beta, or TGF-beta), IL-1b (interleukin 1 beta) and LPS (lipopolysaccharide), when compared to control cells or to the same cells at the beginning of the culture. As above, cytokines and LPS can be measured in the cells as a whole, or in soluble and/or insoluble fractions thereof, including the medium.

A further characteristic of brain cells incubated according to the method of the invention so as to induce the formation of neurodegenerative disease indicia is increased microglia reaction and/or activation. Microglial reaction and/or activation refers to the fact that when injury or disease affect nerve cells, microglia in the central nervous system become "active," causing inflammation in the brain, similar to the manner in which white blood cells act in the rest of the body. Microglia act like the monocyte phagocytic system. Microglia can be activated by numerous materials including complement proteins and beta amyloid protein. Activated microglia generate large quantities of superoxied anions, with hydroxyl radicals, singlet oxygen species and hydrogen peroxide being a downstream product, any of which can be assayed in the preparations utilized in such methods of the invention. Such microglial activation may be used with other indicia in the model of the invention in the embodiments in which brain architecture is retained to some degree, for example, when a brain slice is employed, or when brain cells are in vivo.

Reactive microglia and astrocytes are characterized by their cell bodies becoming larger, their processes becoming thicker, by an increase in the GFAP and ED-1 staining, by a proliferation and clustering of microglia and astrocytes, by infiltration of peripheral inflammatory cells, for example, white blood cells, and by formation of gliosis, etc., as compared to that found in the non-reactive state.

A further characteristic of brain cells incubated according to the method of the invention so as to induce the formation of neurodegenerative disease indicia is the appearance of increased indications of brain inflammatory reactions. Increased indications of brain inflammatory reactions can include indications such as, for example, increased positive staining for HLA-DR (which detects reactive microglia); increased positive staining for glial fibrillary acidic protein (GFAP; to detect reactive astrocytes); the extracellular accumulation of complement proteins, complement inhibitors, acute phase reactants, growth factors, heat shock proteins, proteoglycans, lipoproteins, cathepsins, cystatins, coagulation factors, proteases, protease inhibitors, integrin adhesion molecules, etc. Such indicia of brain inflammatory reactions may be used with other indicia in the model of the invention in the embodiments in which brain architecture is retained to some degree, for example, when a brain slice is employed, or when brain cells are in vivo.

A further characteristic of brain cells incubated according to the method of the invention so as to induce the formation of neurodegenerative disease indicia is a change in the levels and activities of protein kinases, for example, cyclin dependent protein kinase 5 (cdk5) and mitogen activated protein kinase (MAPK).

To be useful in the methods of the invention, it is not necessary that all of the brain cells in a sample or preparation exhibit at least one of the above characteristics. Rather, such preparations are useful even if only some of the brain cells contained therein exhibit such characteristics. Preparations of brain cells in accordance with embodiments of the invention preferably contain at least some cells that contain neurofibrillary tangles, but not all the cells in the preparation need to exhibit such tangles. In a preferred embodiment, such changes are found in the neurons that are in the brain cell preparations. In another preferred embodiment such changes are found in brain cells in vivo.

Not all the characteristics need to be induced by the same agent or at the same time or to the same degree in preparations intended to induce brain cells to exhibit the characteristics of neurodegenerative diseases. Preferably, upon treatment with the agent that induces lysosomal dysfunction so as to increase cathepsin D, or with the agent that decreases an effective concentration of cholesterol, the brain cell's biochemistry, physiology or morphology is changed to include at least one or more of:

(1) the formation of neurofibrillary tangles,

(2) the hyperphosphorylation of tau, and/or

(3) the fragmentation of tau, that is, tau proteolysis and especially, increased amounts of the 15-35 kDa forms of tau.

The rest of the characteristics:

(4) increased production and/or release of brain-produced pro-inflammatory cytokines especially TGF-beta, TGF-alpha, IL1, IL1-alpha, IL1-beta, IL6, IL10, TNF, TNF-alpha and LPS and most especially TGF-beta, IL-1beta and LPS,

(5) increased microglia reaction and/or activation,

(6) increased indications of brain inflammatory reactions,

(7) increased conversion of p35 to p25

(8) changes in the levels and activities of protein kinases, for example, cyclin dependent protein kinase 5 (cdk5) or mitogen activated protein kinase (MAPK), are preferably not relied on solely but, if assayed, are assayed along with any of

the formation of neurofibrillary tangles,

the hyperphosphorylation of tau, and/or

the fragmentation of tau, that is, tau proteolysis and especially, increased amounts of the 15-35 kDa forms of tau, as indicators of the appearance or disappearance of characteristics of neurodegenerative disease in the methods and cultures of the invention.

Pro-inflammatory cytokines including IL1-alpha, IL1-beta, IL6, and IL10, TNF, TFN-alpha and TGF (alpha or beta), and especially TGFbeta1 (also referred to as TGF1), and TNF-alpha, are useful as indicators of glial activation. Levels of these cytokines, including levels of their mRNAs, can be quantitated, for example, by RT-PCR, to assay for glial activation and lysosomal dysfunction. Assays for such factors are known in the art.

Activation of the MAP kinase pathways can be monitored as an indication of glial activation or as an indication of an increased brain inflammatory condition. The pathways can be summarized as follows. There are two IL1/TNF-activated kinase cascades, one of which involves the p38 homologues of MAP kinase and the other of which involves the p54 homologues of MAP kinase. IL1, TNF, TGF1beta, etc. activate both pathways. The activation of kinases or phosphatases of either or of both of these cascades can be assayed as an indicator of glial activation and/or the induction of a brain inflammatory reaction.

The activity of any of a variety of kinases can be assayed as indicators of glial activation or brain inflammatory reactions. Such kinases include, for example, GCK1 (Germinal center kinase), PAK kinase (for example, as identified in Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S. and Lim, L. (1994) A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 367,40-46), MLK3 (mixed lineage kinase-3, also known as SPRK or PTK1), MLK1, MLK2, MLK4, DLK (also known as Muk), SEK1, SEK2, SAPK (stress activates protein kinases alpha/beta/gamma), MKK3, MKK6, p38 MAPK (alpha/beta/gamma/delta), MAPKAPK2, elk1, c-jun, ATF-2 and hsp27 (heat shock protein -25, also known as hsp25 and hsp28). Increased activity of the end targets of such cascades, for example, an increased activity of transcription factors CHOP/GADD 153 and MEF2C, can also be monitored as an indication of an induced inflammatory state of the brain cells. Conversely, decreased phosphorylation, or a decreased activity, is indicative of a lesser inflamed, or non-inflamed state of the brain cells. In a preferred embodiment, the activation or inactivation of cyclin dependent protein kinase 5 (cdk5) and mitogen activated protein kinase (MAPK) are assayed as an indication of an inflammatory or non-inflammatory state of the brain cells.

Thus, using the instant invention, agents that enhance or retard the formation of one or more of the characteristics of neurodegenerative disorders can be identified, including: neurofibrillary tangles, tau proteolytic fragments (and especially the formation of the 15-35 kDa forms of tau), or agents that enhance or retard the formation of tau hyperphosphorylation, and increased production and/or release of brain-produced cytokines especially TGF-beta, IL1-beta, TNF-alpha and LPS, an increased microglia reaction and/or activation, increased indications of brain inflammatory reactions, and changes, especially, increases in the levels and activities of protein kinases, for example, cyclin dependent protein kinase 5 (cdk5) and mitogen activated protein kinase (MAPK).

The above characteristics are also often seen in "brain aging" and are also useful as models thereof. Age-related neurodegenerative diseases and neurodegenerative diseases are characterized by many of the same properties including, e.g., an increased amount of neurofibrillary tangles and/or lysosomes, the appearance of basophilic granules in the mossy fiber terminal zone, the presence of secondary lysosomes with lipofuscin, amyloid deposition, amyloid plaques, neuritic plaques, synaptic loss, neuritic degeneration, neuronal death, increased glial elements (astrocytes, microglia), the fragmentation of the amyloid precursor protein, increased levels of Cathepsin D, inflammation, etc. Brain aging refers to a condition in which brain cells mimic the biochemistry or physiology of brain cells taken from a mature or elderly animal, especially human. Brain aging is manifested by significant changes in lysosomal functions and chemistry, e.g., the proliferation of secondary lysosomes filled with lipofuscin, decreases in cathepsin L activities and increases in the levels of cathepsin D. Additional characteristic features of human brain aging, as is found in neurodegenerative diseases, and especially Alzheimer's disease, include depletion of synaptic proteins, meganeurite formation, induction of early-stage tangles, and accompanying tau proteolysis.

II. Sources of Brain Cells

Any suitable source of brain cells can be used in embodiments of the invention. Typically, brain cells are derived from a mammal, such as a mouse, rat, guinea pig, rabbit, etc. Primary cell cultures, including human primary cell cultures, can also be used in the methods of the invention. Brain cells can be derived from a normal animal (e.g., comprising a wild-type apoE gene in the chromosome) or other suitable animals. For example, apoE-deficient brain cells or apoE4-containing brain cells can be used in embodiments of the invention. A preferred embodiment includes in vivo brain cells.

Among many types of brain cells suitable for embodiments of the invention, brain cells cultured from apoE-deficient brain cells or from apoE4-containing brain cells are especially preferred because they produce neurofibrillary tangles at significantly enhanced levels when compared to brain cells from control (normal) brains. The relatively high density with which such tangles form in apoE-deficient brain cells, or in apoE4-containing brain cells was not achievable by treatment with only a cathepsin D-increasing compound in normal brain cells even with a prolonged treatment with the same cathepsin D-increasing compound. However, a high density in normal brain cells was achievable by treating the cells under conditions in which cholesterol synthesis was inhibited or cholesterol levels were lowered.

The cultured brain cells, in particular apoE-deficient brain cells, even without the treatment with a cathepsin D-increasing compound, have some residual amount of neurofibrillary tangles. However, the initial density of neurofibrillary tangles in these untreated brain cells is too low to be regarded as an adequate model for neurodegenerative diseases, such as Alzheimer's disease.

If brain cells with a higher density of neurofibrillary tangles are desired, apoE-deficient brain cells or apoE4-containing brain cells can be preferably used in embodiments of the invention. Alternatively, such brain cells, and even normal brain cells, can be exposed to the cholesterol limiting embodiments of the invention as described below.

Preferably, when apoE-deficient brain cells are used, they are derived from a transgenic animal. For example, apoE-deficient brain cells useful in the method of the invention can be derived from a transgenic animal that carries an altered or ablated and/or expresses an altered endogenous apolipoprotein E gene (one or both alleles) that results in undetectable or significantly less amounts of apolipoprotein E proteins. These transgenic animals are sometimes referred to as apoE "knockout" animals. "Knock-out" transgenics can be transgenic animals having a heterozygous knock-out of the apoE gene or a homozygous knock-out of the apoE gene. For example, for use in the methods of the invention, the function and/or expression of the apoE protein in the apoE "knockout" animal is typically less than about 30%, preferably less than about 10%, more preferably less than about 5%, still more preferably less than about 1%, compared to a normal animal with the wild-type apoE genes. Most preferably, apoE-deficient brain cells are derived from apoE-knockout animals that have no apoE (i.e., null) gene expression.

Typically, apoE4-containing brain cells can be derived from a transgenic animal that comprises an exogenous apoE gene, e.g., a human apoE4 gene, polymorphic variants, interspecies homologs, or other conservatively modified variants thereof. Preferably, in these transgenic animals that comprise an exogenous apoE4 gene, their endogenous apoE gene is completely or partly knocked out.

Transgenic animals comprising apoE-deficient brain cells can be produced by recombinant methods known in the art. For example, the endogenous apoE gene function can be altered or ablated by, e.g., the deletion of all or part of the coding sequence, or insertion of a sequence, or substitution of a stop codon. In another example, the non-coding sequence of the apoE gene in the chromosome can be modified by, e.g., deleting the promoter region, the 3' regulatory sequences, enhancers and/or other regulatory sequences of the apoE gene in the chromosome. In yet another example, apoE-deficient transgenic animals can be produced by introducing an anti-sense construct that blocks the expression of the endogenous apoE gene products. In some cases, it may be desirable to produce conditional "knock-out" transgenic animals, wherein the alteration in the apoE gene can be induced by, e.g., exposure of the animal to a substance that promotes the apoE gene alteration postnatally. Preferably, both alleles of the apoE gene in the chromosome are altered in these transgenic animals.

The methods for producing transgenic animals are well known and described in, e.g., U.S. Pat. Nos. 5,464,764, and 5,627,059, the disclosures of which are incorporated herein by reference. In particular, the following references describe methods for producing apoE-deficient homozygous rodents: Plump et al., Cell 71:343-353 (1992); and Gordon et al., Neuroscience Letters 199:1-4 (1995), the disclosures of which are incorporated herein by reference. Moreover, some apoE-deficient transgenic animals are commercially available. For example, apoE-deficient homozygous mice, such as strain, are available from the Jackson laboratory, Bar Harbor, Me.

Moreover, apoE4-containing brain cells can be derived from a transgenic animal that comprises an exogenous apoE gene. For example, an exogenous apoE gene can be a human apoE4 gene, its interspecies homologs, polymorphic variants, or conservatively modified variants thereof. In human, three isoforms (apoE2, apoE3 and apoE4) express variants of apoE. Among these isoforms, apoE4 is known in the art to encode an apoE protein that is deficient in various functions. For example, compared to apoE3 that stimulates neurite extension, apoE4 was shown to inhibit neurite extension. Nathan et al., Sco. Neurosci. 20(Part 2):1033 (1994). It has also been suggested that, in vitro, tau interacts with apoE3, but not with apoE4. Stritmatter et al., Exp. Neurol. 125:163-171 (1994). Moreover, the human apoE4 isoform has been described as a risk factor of Alzheimer's disease (see, e.g., Peterson et al., JAMA 273:1274-1278 (1995)). Since brain cells comprising an apoE4 gene appear to lack many normal functions that other apoE isoforms possess, like the apoE-deficient brain cells, transgenic animals that comprise an apoE4 gene or its variants may also be used as a source of brain cells in embodiments of the invention.

Such transgenic animals can be produced using various apoE nucleotide sequences known in the art or conservatively modified variants thereof. For example, the human apoE4 gene has the Genbank accession number M10065. The mouse apoE gene has the Genbank accession number D00466. Other homologs or polymorphic variants of apoE genes can also be readily identified. For example, homologs or polymorphic variants of a known apoE gene can be isolated using nucleic acid probes by screening libraries under stringent hybridization conditions. Exemplary stringent hybridization conditions are as follows: a hybridization in a buffer containing 50% formamide, 5xSSC, and 1% SDS, at 42^oC., or 5xSSC, 1% SDS, at 65^oC., with wash in 0.2xSSC, and 0.1% SDS at 65^oC. In some cases, moderately stringent conditions may be used to clone homologs or polymorphic variants of a known apoE gene. An example of a moderately stringent condition includes a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37^oC., and a wash in 1xSSC at 45^oC. The source of homologs can be any species, e.g., rodents, primates, bovines, canines, human, etc.

Preferably, the exogenous apoE gene is operably linked with a mammalian apoE promoter, such as human apoE4 regulatory sequences. This construct can be introduced into an animal using methods known in the art. In these transgenic animals comprising an exogenous apoE gene (e.g., human apoE4 gene), preferably the endogenous apoE gene is partially or completely knocked out so that the endogenous apoE expression or function is insubstantial. Moreover, methods for producing transgenic animals comprising various human apoE isoforms are described in, e.g., U.S. Pat. Nos. 6,046,381 and 5,767,337, the disclosure of which are herein incorporated by reference.

Preferably, apoE4-containing brain cells are also derived from transgenic animals that are genetically modified. As above, for use in the methods of the invention, the function and/or expression of the apoE4 protein in the apoE4 animal is typically less than about 30%, preferably less than about 10%, more preferably less than about 5%, still more preferably less than about 1%, compared to a normal animal with the wild-type apoE genes.

In some embodiments, it may be desirable to use modified or mutated versions of apoE genes. For example, a modified version of a human apoE4 gene, when introduced into a transgenic animal, may be capable of producing a higher density of neurofibrillary tangles compared to the unmodified human apoE4 gene. Techniques for in vitro mutagenesis of cloned genes are well-known in the art and can be readily applied for making a modified or mutated apoE gene. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press (1989). The functional effect of a modified or mutated apoE gene can be further tested in vivo or in vitro. For example, a transgenic animal comprising a modified or mutated apoE gene can be produced using the methods known in the art. The change in the properties in apoE brain cells (e.g., the neurofibrillary tangle or phosphorylated tau fragment production) can be determined using the methods described below.

III Brain Cell Preparations

Brain cells and preparations containing the same can be prepared and processed in any suitable manner. For example, the brain can be processed in the form of tissue sections, such as brain slices. Alternatively, the brain tissues can be processed in the form of dissociated cells. Whether in the form of brain slices, dissociated cells, or other forms, they will be generically referred to as "brain cells" herein, unless otherwise indicated.

In one embodiment, an in vivo model is used. Such in vivo models have an advantage in that they retain the native brain architecture and environment. The effects that are brought about by the methods of the invention are presented against the background of a physiological environment that is more likely to mimic such conditions in humans. In vivo models are also more amenable to long term analysis than are primary cultures, or brain slice cultures. Another advantage is that multiple samples can be taken at the same time from the same animal and from different parts of the brain.

Preferably, the brain is processed in the form of brain slices so that neuronal circuitry or other biological functions are maintained, but environmental (culture) conditions can be closely monitored and normalized. A suitable thickness of the brain slice is readily determinable by those of skill in the art, and may be varied depending on the culture condition or subsequent analysis methods. For example, the brain can be sliced in the thickness of about 200 .mu.m to about 800 .mu.m, preferably about 350 .mu.m to about 400 .mu.m. The entire brain or portions of the brain can be processed into slices. For example, suitable brain slices may include a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice. Since neurofibrillary tangles tend to develop more prominently in the hippocampal region, a hippocampal slice is preferably used.

Alternatively, the brain can be processed into dissociated brain cells. The entire brain or selected regions of the brain (e.g., the hippocampal region) can be dissociated and maintained in a culture. Generally, the brain tissue is dissected, minced and digested in an enzyme (e.g., trypsin) for a suitable period of time. Then cells are centrifuged and plated at a low density in culture plates, and cultured. The methods for dissociating cells are well-known in the art. See, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994), incorporated herein by reference.

Brain cells in the form of slices or dissociated cells can be maintained in a culture. Suitable culture conditions for brain cells are well-known in the art. For example, brain cells can be placed onto culture plates, preferably on a support, such as a matrix or membrane, which allows cells to attach. Any suitable medium can be used in maintaining the culture of brain cells. Typically, the culture of brain cells is maintained in a medium that has all the essential nutrients. The culture medium generally has a neutral pH, e.g., between about pH 7.2 to about 7.8, and is maintained at a temperature between about 4^oC. to about 40^oC., typically at about 37^oC. The culture of brain cells is typically maintained in an atmosphere that contains CO₂, preferably at 5% CO₂. In general, the culture can be maintained for at least about 60 days with a periodic replacement of culture medium.

IV. Assays for Neurofibrillary Tangles, Phosphorylated Tau and Tau Fragments

Determination of the neurofibrillary tangles, phosphorylated tau and/or tau fragments production can be qualitative or quantitative. In some applications, it may be sufficient to visually inspect the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments. For example, it may be useful to visually observe the timing and the pattern of neurofibrillary tangle development at different regions of the brain. In other applications, it may be desirable to quantitate the neurofibrillary tangles, phosphorylated tau and/or tau fragments production. Quantitation would be particularly useful in a screening assay for agents that modulate the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments.

Any suitable methods known in the art can be used to determine the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments. For example, brain cells, in the form of brain slices, brain sections, dissociated cells, or other suitable forms, can be stained using conventional staining methods. For example, the brain cells can be fixed and stained with a silver stain (Bielschowsky) stain or toluidine blue. Then the stained neurofibrillary tangles can be visualized by microscopy.

To assess the appearance and density of tangles, light microscopic evaluation of tangles and tangle formation can be performed by scanning at 40xobjective magnification. Immunoreactive elements can be plotted and images digitized and stored for any desired area. Especially, the following areas are preferentially examined: (1) the entorhinal cortical layers II/III; (2) CA1 str. pyramidale; (3) CA1 str. oriens; (4) subiculum str. oriens. Typically, five to seven serial sections are collected per brain slice. The bottom section (i.e., that on the Millipore filter side of the explant in the examples below) is generally neuron-poor and therefore not evaluated. Analysis preferably focuses on the dense paired helical filament (PHF) tau-immunoreactive elements that are greater than or equal to 2 .mu.m in diameter. Comparisons from aligned serial sections can be used to identify structures that are present in more than one section so that individual elements are not double-counted. With this correction, the densely stained structures can be quantified and the result expressed per unit area for each field of analysis. In addition, the types of paired helical filament tau-positive structures can be catalogued as shown in FIG. 8. Differences in treatment regimens that affect the qualities as well as the number of tangles can thus be determined.

An absolute value of the density of tangles in the models of the invention is not required. Rather, a relative increase in the density of tangles, as compared to the density found in similar preparations but from wild-type or in controls, is indicative of the appearance of neurodegenerative disease changes in the cells of the method of the invention. In a preferred embodiment, the number of tangles per unit of space is 20-30% higher in the cells of the invention that have been exposed to a cathepsin D-increasing compound and/or a compound that decreases an effective concentration of cholesterol than it is in normal or control cells. In a preferred embodiment, such density is greater than 30% and may be even 100% or more higher than the wild-type or control cells (as such wild-type or control cells may lack tangles completely).

Morphologically, cells that contain an increased cathepsin D generally have lysosomes that are round in shape and that are distributed homogenously in the cell body. Changes in the shape, size and numbers of lysosomes and changes in the localization of enzyme activity from lysosomal localization to cytoplasmic localization can also be used as indexes by which to assess the degree of neurodegenerative disease characteristics that have been induced in the cells according to the invention.

In another example, the brain cells can be stained by immunostaining, and the neurofibrillary tangles, phosphorylated tau and/or tau fragments production can be visualized. In immunostaining, suitable capture reagents, such as antibodies that specifically bind to neurofibrillary tangles, phosphorylated tau and/or tau fragments, can be used. Preferably, antibodies preferentially bind to neurofibrillary tangles, phosphorylated tau and/or tau fragments and do not significantly cross-react with other proteins in the brain cells. For example, the antibodies that specifically bind to phosphorylated tau proteins and/or tau protein fragments have less than 50%, preferably less than 30%, more preferably less than 10% crossreactivity with native tau proteins that are not phosphorylated.

Examples of mouse monoclonal antibodies that preferentially bind phosphorylated tau and/or tau protein fragments over the tau found in normal adult brain include antibodies 8D8, RT97, 121.5, BF10 (Miller et al., EMBO J. 5:269-276 (1986)); AT8 (Bierat et al., EMBO J. 11:1593-1597 (1992)); SMI31, SMI34, SMI310 (Sternberger et al., Proc. Natl. Acad. Sci. USA 82:4274-4276 (1985); Sternberger & Sternberger, Proc. Natl. Acad. Sci. USA 80:6129-6130 (1983)); and ALZ-50 (Wolozin et al., Science 232:648-650 (1986)). Preferably, AT8 is used in embodiments of the invention to bind phosphorylated tau protein and/or tau protein fragments.

In immunostaining, an antibody against neurofibrillary tangles, phosphorylated tau and/or tau fragments is added to brain cells, and the brain cells are incubated for a sufficient time to allow binding between the antibody and neurofibrillary tangles, phosphorylated tau and/or tau fragments. The antibody may be labeled with a variety of labels that are detectable. Useful labels include magnetic beads (e.g., DYNABEADS), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). Alternatively, the antibody may be unlabeled, and a label may be coupled indirectly. For example, an unlabeled primary antibody can be added to the culture to bind neurofibrillary tangles, phosphorylated tau and/or tau fragments, and then a labeled secondary antibody can be used to amplify the signal for detection.

Means of detecting labels are well known to those of skill in the art. For example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels may be detected simply by observing the color associated with the label.

Alternatively, the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments can be determined using cell lysate in an immunoassay. An immunoassay can be performed in any of several formats. These formats include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay. For a review of the general immunoassays, see, e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991). A general overview of applicable technology can also be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988). See, also, U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.

In one embodiment, immunoblotting can be used to quantify the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments produced in brain cells treated with or without a cathepsin D-increasing compound and/or brain cells treated with or without (i.e., in the presence or absence of) a cysteine protease inhibitor. Generally, brain cells are disrupted in an electrophoresis sample buffer and are treated to obtain a fraction that contains proteins. The proteins are separated by gel electrophoresis and transferred to a membrane that binds the proteins nonspecifically. The location of neurofibrillary tangles, phosphorylated tau and/or tau fragments on the membrane is determined using, e.g., a labeled primary antibody or an unlabeled primary antibody, followed by a labeled secondary antibody. A detectable label may be, e.g., a radio-label or a fluorescent label or, an enzyme label. Then the membrane comprising a detectable label can be scanned, and digitized images can be quantitatively analyzed by densitometry.

In another embodiment, a sandwich assay can be performed by preparing a brain cell lysate sample, and placing the sample in contact with a solid support on which is immobilized a plurality of antibodies that bind neurofibrillary tangles, phosphorylated tau and/or tau fragments. The solid support is then contacted with detection reagents for neurofibrillary tangles, phosphorylated tau and/or tau fragments. After incubation of the detection reagents for a sufficient time to bind a substantial portion of the immobilized neurofibrillary tangles, phosphorylated tau and/or tau fragments, any unbound labeled reagents are removed. The detectable label associated with the detection reagents is then detected. For example, in the case of an enzyme used as a detectable label, a substrate for the enzyme that turns a visible color upon action of the enzyme is placed in contact with the bound detection reagent. A visible color will then be observed in proportion to the amount of the neurofibrillary tangles, phosphorylated tau and/or tau fragments in the sample.

According to the invention, the production of tau proteolytic fragments of a size from 15-35 kDa is increased. The size of tau proteolytic fragments can be determined using techniques known in the art, for example, gel electrophoresis, and especially SDS gel electrophoresis, or 2D gel electrophoresis.

The above described detection methods are merely exemplary, and other suitable detection methods will be apparent and can be readily substituted by one of skill in the art.

V. Treatment of Brain Cells with Cathepsin D-Increasing Compounds to Induce or Enhance the Characteristics of Neurodegenerative Diseases

In a preferred embodiment, brain cells (e.g., normal brain cells, apoE-deficient brain cells, or apoE4-containing brain cells) are cultured in a medium that provides an effective concentration of cathepsin D as a result of an agent or compound that selectively increases the concentration or amount of cathepsin D in the brain cells. An effective concentration of cathepsin D can be induced, or increased, in a brain cell by either increasing the amount or concentration of cathepsin D or by stimulating the catalytic activity of cathepsin D. The "effective concentration" of cathepsin D is the concentration that will achieve the indicated result.

According to the invention, increasing the concentration of cathepsin D in the brain cells to an effective level results in the increased production of neurofibrillary tangles, the major component of which are abnormally phosphorylated tau proteins and tau protein fragments. Tau protein fragments are generally also hyperphosphorylated and are composed mainly of fragments containing the microtubule binding domains and flanks, and are generally of 27-35 kDa in size. In a preferred embodiment, the proteolysis of tau to fragments of a size of 15-35 kDa is examined. The inventors have discovered a phosphorylated tau fragment of 33 kDa that is thought to become a component of the tangles. Therefore, in an especially preferred embodiment, the amount or levels of the 33 kDa tau fragment are detected.

Preferably, such increase in the concentration of cathepsin D activity or levels is brought about or induced by contacting the brain cells with a cathepsin D-increasing compound throughout the entire period of culture during which it is desired to maintain such selectively increased concentrations or amounts of cathepsin D. Alternatively, the brain cells can be exposed in intermittent fashion of desired intervals, or, alternatively, only once at a desired point in the culture period.

A selective inhibitor of cathepsin B and L (i.e., ZPAD) can be used to selectively increase cathepsin D activity or levels relative to such activity or levels of cathepsin B and L. By changing the ratio of cathepsin B and/or L to cathepsin D, the cathepsin D concentration is increased to a concentration effective to induce the appearance or increase in the desired indicia of neurodegenerative disease. Selective inhibitors of cathepsin B and L have been reported to induce abnormally phosphorylated tau fragments in cultured hippocampal slices of normal rodents. Abnormally phosphorylated tau fragments assemble into structures having the appearance, size and epitopes of early-stage neurofibrillary tangles in human brain. See Bi et al., Exp. Neurol. 158:312-317 (1999). However, the density of neurofibrillary tangles produced in the normal rodent hippocampal slices was very sparse compared to the density of neurofibrillary tangles seen in the brain of Alzheimer's patients.

Surprisingly, when apoE-deficient or apoE4-containing brain cells are treated with a cathepsin D-increasing compound, levels of neurofibrillary tangles and phosphorylated tau proteins and fragments were elevated and greatly induced. In particular, when apoE-deficient brain cells were used, a dramatically increased production of neurofibrillary tangles, phosphorylated tau protein, and phosphorylated tau fragments was observed. Typically, the amount of neurofibrillary tangles or phosphorylated tau fragments seen in these treated apoE-deficient brain cells is at least twice, sometimes at least ten times greater than the amount of these materials seen in normal brain cells treated with the same compound. Also, the amount of neurofibrillary tangles or phosphorylated tau fragments seen in these treated apoE-deficient brain cells is at least ten times greater than the amount of these materials seen in apoE-deficient brain cells that are not treated with the compound. The density of neurofibrillary tangles in these apoE-deficient brain cells treated with a cathepsin D-increasing compound is sufficiently high that it mimics the density of neurofibrillary tangles typically found in the brain of Alzheimer's disease patients. Since apoE4-containing brain cells lack many normal function of apoE, like the apoE-deficient brain cells, apoE4-containing brain cells can also be used in embodiments of the invention.

Preferably, a cathepsin D-increasing compound increases the effective concentration of cathepsin D in brain cells by at least about 30%, preferably at least about 50%, more preferably at least about 80%, most preferably at least about 100%, compared to a control (e.g., brain cells untreated with the compound). As described previously, cathepsin D exists in three forms in the brain-the inactive proenzyme, the active single chain and the active heavy chain. Any compound that increases one or more of these cathepsin D forms can be used in embodiments of the invention.

Any suitable cathepsin D-increasing compound can be used in embodiments of the invention. Some of these compounds include inhibitors of cathepsin B and/or cathepsin L. Examples of these inhibitors include chloroquine, N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone, N-CBZ-L-phenylalanyl-L-phenylalanine-diazomethylketone, beta-amyloid (amyloid beta protein), and mimetics thereof.

Other suitable cathepsin D-increasing compounds, and/or agents which mimic the activity of Cathepsin D (e.g., an inhibitor of cathepsin B and/or L or a modulator or an agonist of cathepsin D) are readily determinable by those skilled in the art. For example, a test compound can be contacted with brain cells. Then the activity or the amount of cathepsin D in brain cells can be measured using, e.g., an immunoassay using antibodies against cathepsin D. For example, antibodies such as Cathepsin D (Ab-2), Calbiochem can be used.

The activity or the amount of cathepsin D is then compared with a control amount (e.g., the amount of cathepsin D in brain cells that are not treated with the test compound). A test compound is referred to as a "cathepsin D-increasing compound" if it increases the activity or the amount of any one or more of cathepsin D forms (i.e., the inactive proenzyme, the active single chain or the active heavy chain) by, e.g., at least about 30%, preferably at least about 50%, more preferably at least about 80%, most preferably at least about 100%, compared to a control.

Brain cells can be contacted with a cathepsin D-increasing compound at any suitable time. For example, brain cells can be contacted with a cathepsin D-increasing compound when the culture is first established, or at a later time after maintaining the culture for a few days. Preferably, brain cells are contacted with a cathepsin D-increasing compound for a period of 1-18 days, preferably for a period of 4-8 days. To induce neurofibrillary tangles and phosphorylated tau fragments, a cathepsin D-increasing compound is typically added at a concentration of 0.1 .mu.M to about 500 .mu.M, more typically at a concentration of about 1 nM to about 100 .mu.M.

Other modulatory compounds, in addition to a cathepsin D-increasing compound(s), can be, for example, added in the culture medium, or at least placed in contact with the brain cells or tissue containing such cells, to further facilitate the production of neurofibrillary tangles or other neurodegenerative characteristics or features in brain cells. Examples of modulatory compounds include oxidative free radicals (Fe³⁺, H₂ O₂, etc.), or inflammatory factors (TGFb, IL-1b, TNFalpha, LPS, etc.).

Typically, brain cells in a culture are treated with a cathepsin D-increasing compound under a condition such that the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments is increased by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 80%, or at least about 100%, or at least about 150%, or at least about 200%, compared to a control (e.g., brain cells that are cultured in substantially the same condition but without the cathepsin D-increasing compound). Also, brain cells that are treated with a cathepsin D-increasing compound generally produce neurofibrillary tangles, phosphorylated tau and/or tau fragments at a significantly higher level, typically at least two times, sometimes ten times, more than normal brain cells treated with the same compound. Preferably, the treatment conditions (e.g., concentration of a cathepsin D-increasing compound, a period of incubation, etc.) are selected so that the density of neurofibrillary tangles, phosphorylated tau and/or tau fragments produced in apoE-deficient brain cells or in apoE4-containing brain cells is similar to the density of these materials in aging brain or the brain of patients with Alzheimer's disease or other neurodegenerative diseases.

Brain cells produced in accordance with the present invention, under conditions in which cathepsin D levels or activity is increased, have a variety of applications. For example, the brain cells can be used as an in vitro assay system to screen libraries or identify agents that modulate the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain, especially agents that decrease or prevent the accumulation of such characteristics. These agents can be further tested in other systems and/or in vivo to confirm their efficacy in modulating the production of neurofibrillary tangles in brain cells and possibly other conditions and/or pathologies associated with neurodegenerative diseases, such as the cognitive decline seen in persons afflicted with such disorders. In another example, the brain cells can be used to study the morphological pattern of neurofibrillary tangle formation in the brain. In another example, the brain cells can be used to study the effect of neurofibrillary tangle formation in normal aging. Such morphological studies would provide additional information regarding the pathological process of neurodegenerative diseases.

VI. Treatment of Brain Cells with a Cholesterol Decreasing Compound to Induce or Enhance the Characteristics of Neurodegenerative Diseases

According to another embodiment of the invention, decreasing intracellular cholesterol levels in brain cells, for example, by inhibiting cholesterol synthesis, can be used to induce the characteristics of neurodegenerative diseases, and especially Alzheimer's disease, in that brain cell--even in cells from normal animals. In a preferred embodiment, the brain cell in which cholesterol is decreased is a neuron and the characteristics that are monitored are the formation of tangles and tau fragmentation. In another embodiment, the brain cells in which cholesterol is decreased are glia cells and the characteristics that are monitored are glia activations, glia reactions, and/or cytokine production and/or release.

Exposing the brain cell to such cholesterol-lowering agents or conditions mimics results found when using apoE-deficient brain cells, or brain cells that contain the apoE4 isotype. The advantage of the cholesterol-limiting treatment (i.e., inhibition of cholesterol synthesis and/or lowering of cholesterol levels) is that relatively high tangle densities can be obtained in normal cells by such treatment, densities that are otherwise only obtainable in cells in apoE-deficient brain cells, or in apoE4-containing brain cells. Accordingly, for the first time, high densities of neurofibrillary tangles and the appearance of other characteristics of neurodegenerative diseases can be induced in brain cells from normal animals in a relatively short period of time, and thus be useful as a model for studying such diseases and for identifying agents useful to treat or prevent the same. A combined inhibition of cholesterol synthesis and lysosomal dysfunction can be used to further dramatically enhance the neurodegenerative effects brought about by either manipulation alone.

Therefore, in another embodiment, the characteristics of neurodegenerative diseases, such as, for example

(1) neurofibrillary tangles,

(2) the hyperphosphorylation of tau,

(3) the fragmentation of tau, that is, tau proteolysis and especially, increased amounts of the 15-35 kDa forms of tau,

(5) increased microglia reaction and/or activation,

(6) increased indications of brain inflammatory reactions

(7) increased conversion of p35 to p25

(8) changes in the levels and activities of protein kinases, for example, cyclin dependent protein kinase 5 (cdk5) and mitogen activated protein kinase (MAPK),

are induced by exposing brain cells to a condition that, or by contacting brain cells with a compound that, inhibits cholesterol synthesis or otherwise decreases the levels of cholesterol.

According to this embodiment, to increase the production of such characteristics, and especially neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases, brain cells are contacted with a compound capable of decreasing levels of cholesterol or inhibiting cholesterol synthesis or otherwise capable of decreasing the concentration of cholesterol ("cholesterol-lowering compound"). This compound can preferably decrease either the concentration of cholesterol or the synthesis of cholesterol in cells and thus decrease the availability of cholesterol within the cells.

In one aspect, the invention provides cultured brain cells, and methods for producing the brain cells, wherein the brain cells have been treated with a compound that increases cathepsin D to an effective concentration and with a compound that decreases cholesterol levels or inhibits cholesterol synthesis to an sufficient low concentration to result in or to produce increased amounts of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases compared to such indicia in a control (e.g., brain cells that are untreated with said compound(s)).

Embodiments of the invention include methods comprising: (a) culturing brain cells; and (b) contacting the brain cells with a compound that increases an effective concentration of cathepsin D and with a compound that decreases an effective concentration of cholesterol, thereby producing properties of a brain afflicted with neurodegenerative disease, wherein the properties include increased neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases and/or related biochemical changes.

In some embodiments, a method for increasing neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in brain cells comprises: (a) culturing the brain cells in a medium which selectively increases an effective concentration of cathepsin D and that decreases the concentration of cholesterol in the medium and cells; and (b) optionally, determining the production of and/or levels of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases.

Preferably, a cholesterol-lowering compound decreases the effective concentration of cholesterol in brain cells by at least about 30%, preferably at least about 50%, more preferably at least about 80%, most preferably at least about 100%, compared to a control (e.g., brain cells untreated with the compound). Any compound that lowers cholesterol levels (for example, by inhibiting cholesterol synthesis or stimulating cholesterol degradation or lowering the availability of cholesterol) can be used in embodiments of the invention. Examples which can be used in embodiments of the invention include compounds which decrease either the concentration of cholesterol, or the synthesis of cholesterol, or decreases the availability of cholesterol in cells.

Any suitable cholesterol-lowering compound can be used in embodiments of the invention. Some of these compounds include inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA Reductase) inhibitors. Examples of these inhibitors include members of the statin class of compounds, such as, for example, mevastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin, cerivastatin, and mimetics thereof.

A further class of compounds includes agents which decrease the availability of cholesterol within cells. Examples of this class include agents which bind, immobilize, and/or otherwise separate cholesterol from other elements found within cells.

Other suitable cholesterol-lowering compounds, and/or agents which modulate the activity of cholesterol are readily determinable by those skilled in the art. For example, a test compound can be contacted with cells. Then the activity the amount of cholesterol in cells can be measured using, e.g., an immunoassay using antibodies against cholesterol. Alternatively, a test compound can be contacted with cells and the activity or amount of HMG-CoA reductase (an enzyme involved in cholesterol synthesis in cells) and/or other entities involved in cholesterol synthesis, degradation, storage, and/or transport can be measured using assays known to one skilled in the art.

The activity or the amount of cholesterol and/or the activity or amount of HMG-CoA reductase and/or other entities involved in cholesterol synthesis, degradation, storage, and/or transport is then compared with a control amount (e.g., the amount of cholesterol and/or the activity or amount of HMG-CoA reductase and/or other entities involved in cholesterol synthesis, degradation, storage, and/or transport in cells that are not treated with the test compound). A test compound is referred to as a "cholesterol-lowering compound" if it decreases the activity or the amount of cholesterol and/or the activity or amount of HMG-CoA reductase and/or modulates other entities involved in cholesterol synthesis, degradation, storage, and/or transport by, e.g., at least about 30%, preferably at least about 50%, more preferably at least about 80%, most preferably at least about 100%, compared to a control.

Brain cells can be contacted with a cholesterol-lowering compound at any suitable time. For example, brain cells can be contacted with a cholesterol-lowering compound when the culture is first established, or at a later time after maintaining the culture for a few days. Preferably, brain cells are contacted with a cholesterol-lowering compound for a period of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 90, 120, 150, or 240 days, or preferably, for in vitro experiments, for a period of 4, 5, 6, 7, 8 or 9 days, while in vivo experiments preferably have a duration of 30-120 days, or any appropriate period of time to achieve the desired effect. To induce the formation of neurofibrillary tangles, and/or phosphorylated tau, and/or tau fragments, and/or microglial reactions, and/or cytokine reactions, or any other of the indicia of neurodegenerative brain disease discussed above. For in vitro experiments a cholesterol-lowering compound is typically added at a concentration of 0.1 .mu.M to about 500 .mu.M, more typically at a concentration of about 1 nM, 10 nM or 100 nM to about 100 .mu.M, and especially 20 .mu.M, or any appropriate amount that achieves the desired effect. For in vivo experiments a cholesterol-lowering compound is typically added at a dose of 0.5 to about 50 mgs/kg body weight of the animal, more typically about 5-40 mgs/kg, and especially 10-20 mgs/kg, or any appropriate amount that achieves the desired effect. More than one cholesterol-lowering compound can be administered at the same time, or sequentially at different times, to the brain cell preparation or animal.

Other modulatory compounds, in addition to such cholesterol-lowering compound(s), can be added in the culture medium to further facilitate the production of neurofibrillary tangles or any of the other neurodegenerative features in brain cells, especially tau fragmentation. Examples of useful modulatory compounds in this regard include agents capable of modulating those kinases and/or phosphatases that are involved in cholesterol metabolism or that interact with cholesterol to affect cell function, amyloid beta peptide, oxidative free radicals (Fe³⁺, H₂ O₂, etc.), or inflammatory factors (TGF-beta, IL-1b, TNFalpha, LPS, etc.).

Typically, brain cells in a culture are treated with a cholesterol-lowering compound under a condition such that the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases is increased by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 80%, or at least about 100%, or at least about 150%, or at least about 200%, compared to a control (e.g., brain cells that are cultured in substantially the same condition but without the cholesterol-lowering compound). Also, brain cells that are treated with a cholesterol-lowering compound generally produce neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases at a significantly higher level, typically at least two times, sometimes ten times more than normal brain cells treated with the same compound. Preferably, the treatment conditions (e.g., concentration of a cholesterol-lowering compound, the period of contact with the brain cells, etc.) are selected so that the density of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases produced is similar to the density of these materials and/or reactions in aging brain or the brain of patients with Alzheimer's disease or other neurodegenerative diseases.

VII. Treatment of Brain Cells with a Cysteine Protease Inhibitor to Prevent or Reverse the Characteristics of Neurodegenerative Diseases

According to a further model of the invention, the above indicia of neurodegenerative disease can be prevented or reversed by exposing brain cells to a cysteine protease inhibitor, and preferable a calpain inhibitor. Specifically such protease inhibitor, and especially calpain inhibitor, reverses the effects of the lysosomal dysfunction and/or cholesterol-lowering, and decreases, or prevents the formation of:

(1) neurofibrillary tangles,

(2) the hyperphosphorylation of tau,

(3) the fragmentation of tau, that is, tau proteolysis and especially, increased amounts of the 15-35 kDa forms of tau,

(5) increased microglia reaction and/or activation,

(6) increased indications of brain inflammatory reactions

(7) increased conversion of p35 to p25

(8) changes in the levels and activities of protein kinases, for example, cyclin dependent protein kinase 5 (cdk5) and mitogen activated protein kinase (MAPK).

Cysteine protease inhibitors, and specifically calpain inhibitors, are therefore useful to identify agents or compounds that might modulate the effects of the cysteine protease inhibitor, for example, induce or enhance the effects, or interfere with the same.

The term "calpain inhibitor" refers to a compound that inhibits the proteolytic action of calpain-I or calpain-II, or both, but preferably calpain-I. The term calpain inhibitors as used herein include those compounds having calpain inhibitory activity in addition to or independent of their other biological activities. A wide variety of compounds have been demonstrated to have activity in inhibiting the proteolytic action of calpains. Examples of calpain inhibitors that are useful in the practice of the invention include N-acetyl-leucyl-leucylmethional (ALLM or calpain inhibitor II), N-acetyl-leucyl-leucyl-norleucinal (ALLN or calpain inhibitor 1), calpain inhibitor III (carbobenzoxy-valyl-phenylalanal; Z-Val-Phe-CHO), calpain inhibitor IV (Z-LLY-FMK; Z-LLY-CH₂ F where Z=benzyloxycarbonyl), calpain inhibitor V (Mu-Val-HPh-FMK where Mu is morphlinoureidyl and Hph is homophenylalanyl), calpeptin (benzyloxycarbonyldipeptidyl aldehyde; Z-Leu-Nle-CHO), calpain inhibitor peptide (Sigma No. C9181), calpastatin, acetyl-calpastatin (acetyl calpain inhibitor fragment, 184-210), leupeptin, mimetics thereof and combinations there, AK275, MDL28170 and E64. Additional calpain inhibitors are described in the following U.S. patents, incorporated herein by reference, U.S. Pat. Nos. 5,716,980; 5,714,471; 5,693,617; 5,691,368; 5,679,680; 5,663,294, 5,661,150; 5,658,906; 5,654,146; 5,639,783; 5,635,178; 5,629,165; 5,622,981; 5,622,967; 5,621,101; 5,554,767; 5,550,108; 5,541,290; 5,506,243; 5,498,728; 5,498,616; 5,461,146; 5,444,042; 5,424,325; 5,422,359; 5,416,117; 5,395,958; 5,340,922; 5,336,783; 5,328,909; 5,135,916.

Preferably the concentration of such inhibitor in the fluid, culture medium, milieu, or other environment contacting the brain cells of the invention is a concentration of 1 nM to 1 mM, and preferably 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100 .mu.M and especially 20 .mu.M, or any appropriate amount that achieves the desired effect.

The cysteine protease inhibitor, and especially, the calpain inhibitor, can be added at the beginning of the culture of the brain cells, or intermittently during the culture, as desired. The inhibitor can be one that is active metabolically intracellularly, or that acts by binding to the outer membrane and inducing a cascade that ultimately results in an inhibition and/or reversal of the desired characteristic of neurodegenerative disease that is being measured in the culture. Two or more inhibitors can be simultaneously added, or sequentially added.

Accordingly, a target class of compounds for a screening method can be identified according to the invention by a method comprising: (a) contacting brain cells with a cathepsin D-increasing compound that increases cathepsin D to an effective concentration in the brain cells, and/or contacting brain cells with a cholesterol-lowering compound wherein the increased concentration of cathepsin D and/or the decreased concentration of cholesterol is effective to increase the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells; (b) contacting the brain cells with a cysteine protease inhibitor; and (c) determining whether the cysteine protease inhibitor modulates the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells treated with the cysteine protease inhibitor compared to the brain cells that are not treated with the cysteine protease inhibitor.

The present invention thus provides a novel target--inhibition of tau proteolysis by a cysteine protease inhibitor, and especially by a calpain inhibitor--for intervention and treatment of Alzheimer's disease, neurodegenerative diseases, and related disorders, such as senile dementias, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementias, Parkinsonism, Pick's disease, etc., and for diminishing the occurrence of neurofibrillary tangles and/or tau fragmentation events capable of resulting in the formation of neurofibrillary tangles and/or tau-related pathologies.

That cysteine protease inhibition, and especially calpain inhibition can reverse the characteristics of neurodegenerative diseases, and especially tangle formation, is especially surprising because the art has taught that hyperphosphorylated tau in the paired helical filaments is resistant to degradation by calpain. As much as five times the levels of calpain are needed to completely degrade paired helical filament tau as compared to "normal" tau (Mercken, M. et al., FEBS Lett 38:10-14 (1995); Yang, L.-S. and Ksiezak-Reding, H., Eur. J. Biochem. 233:9-17 (1995))

In a preferred embodiment of this aspect of the invention, the present invention provides a cysteine protease inhibitor, and particularly an inhibitor of the class of cysteine proteases known as calpains, that affects the central nervous system in a manner that alleviates the symptomologies of Alzheimer's disease, senile dementia, and related disorders, such as Pick's disease. Further, the present invention provides the advantage of alleviating the symptomologies of Alzheimer's disease by inhibiting the formation of neurofibrillary tangles and related tau fragmentation events characteristic of such diseases, of which there is a need in the art.

In a preferred embodiment, the present invention provides novel methods for ameliorating certain conditions associated with neurodegenerative disease and/or neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, senile dementia, etc. In accordance with embodiments of the invention, a host afflicted with a neurodegenerative disease, such as Alzheimer's disease, Pick's disease, etc., is treated with a cysteine protease inhibitor, e.g., by administering a pharmaceutically effective amount of an agent capable of inhibiting the activity of a member of the calpain class of proteases.

In another embodiment of the invention brain cells (e.g., normal brain cells, apoE-deficient brain cells, apoE4-containing brain cells, and/or other transgenically altered brain cells) are treated with a cysteine protease inhibitor, e.g., by contacting the brain cells with an agent capable of inhibiting the activity of a member of the calpain class of proteases. The administration of the agent to the host, and/or the contacting of the agent with the brain cells, then results in a decreased amount of tau fragmentation events which can lead to the formation of neurofibrillary tangles, and the decreased formation of neurofibrillary tangles, or the degradation of tangles that have already been formed.

While some features of neurodegenerative disease or neurodegenerative diseases have been partially remedied by other classes of therapeutics, a key feature such as the reduction of tau fragmentation and/or a reduction in the density of neurofibrillary tangles in the brain was missing in these treatment modalities. The present invention advantageously provides a therapeutic treatment for a host and/or a treatment for brain cells, wherein the host and/or the brain cells comprise, among other things, reduced levels of tau fragmentation and reduced levels of neurofibrillary tangles.

In the present invention, any suitable host or brain cells can be treated. Preferably, hosts are human, and are believed to be afflicted with a neurodegenerative disease, such as Alzheimer's disease, Pick's disease, or a related disorder such as senile dementia, etc. Preferably, brain cells are from a mammal, such as rat, mouse, guinea pig, rabbit, etc. In some embodiments, apoE-deficient brain cells or apoE4-containing brain cells, or other brain cells from a transgenic animal, can be treated.

In one aspect, the invention provides compounds, and methods for using such compounds to decrease the formation of neurofibrillary tangles and/or tau fragments compared to a control (e.g., a host not given said compound(s) and/or brain cells that are untreated with said compound(s)). Embodiments of the invention include methods comprising:

(a) identifying a host thought to be afflicted with a disorder or disease believed to comprise abnormal tau fragmentation events and/or increased levels of neurofibrillary tangles; and

(b) administering to such host a compound that inhibits a member of the calpain class of cysteine proteases, wherein, as a result of the administration of the compound, the characteristics of neurodegenerative disease are lessened or decreased, and preferably, there are decreased levels of neurofibrillary tangles, and/or there are decreased levels of tau fragments and/or decreases in related tau-mediated pathologies.

In other embodiments, a method is provided for decreasing neurofibrillary tangles and/or tau fragmentation in brain cells, the method comprising contacting brain cells with a medium under conditions which, or in the presence of sufficient amounts of a compound that, inhibit one or more members of the calpain class of cysteine proteases, and preferably calpain I.

In another embodiment, the invention provides a target class of compounds for a screening method comprising:

(a) contacting brain cells with a cathepsin D-increasing compound and/or a cholesterol-decreasing compound that increases cathepsin D and/or decreases cholesterol in the brain cells to levels effective to increase the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells;

(b) contacting the brain cells with a cysteine protease inhibitor, and preferably an inhibitor of calpain; and

(c) determining whether the cysteine protease inhibitor modulates the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells treated with the cysteine protease inhibitor compared to the brain cells that are not treated with the cysteine protease inhibitor.

VIII. Screening Assays

Screening assays can be performed in vitro or in vivo. To produce brain cells comprising neurofibrillary tangles, phosphorylated tau and/or tau fragments, etc., the methods described above can be used.

The advantage of using brain cells in the form of slices or in vivo animal testing for the screening assays is that since the neuronal circuitry and other biological functions are more intact in brain slices and in vivo, compared to dissociated brain cells, the experimental conditions better mimic the physiological condition of the brain.

Preferably, the concentration of cathepsin D, and/or the synthesis (and/or levels) of cholesterol, and other culture conditions are adjusted so that the density of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells (prior to contacting with an agent) is similar to the density of these materials found in neurodegenerative diseases, such as Alzheimer's disease. ApoE-deficient brain cells or apoE4-containing brain cells can be used.

To screen agents that modulate the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments, brain cells are contacted with a test agent. An "agent" refers to any molecule, including, e.g., a chemical compound (organic or inorganic), or a biological entity, such as a protein, sugar, nucleic acid or lipid, that modulates the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in brain cells. Generally, a test agent is added to the culture medium in the range from 0.1 nM to 10 mM, and/or an animal is administered a dose of 0.5 to 50 mgs/kg.

Agents can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be tested. Known pharmacological agents may be subjected to directed or random chemical modifications, e.g., alkylation, esterification, amidification, etc. to produce a library of structural analogs. Alternatively, a library of randomly or directed synthesized organic compounds or biomolecules (e.g., oligonucleotides and oligopeptides) can be used as a source of agents. Preparation and screening of combinatorial libraries are well known to those of skill in the art. See, e.g., U.S. Pat. No. 5,010,175, PCT Publication No. WO 93/20242, PCT Publication No. WO 92/00091, Chen et al., J. Amer. Chem. Soc. 116:2661 (1994), U.S. Pat. No. 5,539,083.

Since the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments is correlated with the increased concentration of cathepsin D in brain cells, an inhibitor of cathepsin D may be effective in reducing the production of neurofibrillary tangles or other neuropathological lesions. Accordingly, a library of putative cathepsin D inhibitors can be used as a source of agents in a screening assay. Methods for producing a library of potential cathepsin D inhibitors are known. For example, a combinatorial library of agents against the active site of cathepsin D was previously synthesized by others based on the crystal structure of cathepsin D. See Kick et al., Chem. Biol. 4:297-307 (1997). The library of these agents can be screened by methods in accordance with embodiments of the invention.

An agent can be contacted with brain cells at any suitable time. For example, an agent can be contacted with brain cells prior to contacting the brain cells with a cathepsin D-increasing compound, and/or a compound that lowers the cholesterol to an effective concentration in the cells. In another example, the brain cells can be contacted with the agent after the brain cells are contacted with a cathepsin D-increasing compound and/or a compound that lowers cholesterol to an effective concentration in the cells. Preferably, the brain cells can be contacted simultaneously with the agent and the cathepsin D-increasing compound and/or a compound that lowers cholesterol to an effective concentration in the cells. Generally, brain cells are contacted with an agent for a period of time sufficient to allow the agent to penetrate the cells and to take an effect. Typically, the brain cells and an agent are contacted for a period of between about 1 minute to about 30 days, preferably between about 30 minutes to about 6 days. Typically, during this time, the culture of brain cells is maintained at a temperature between about 4^oC. to about 40^oC., preferably at 37^oC., at atmosphere containing about 0 to 10% CO₂. Other suitable experimental conditions are readily determinable by those skilled in the art.

A number of assays known in the art can be used to determine the effect of candidate agents on the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments in brain cells. For example, various staining or immunoassays described above can be used, and the details of these assay techniques will not be repeated in this section. Other suitable assays will be readily determinable by those of skill in the art, and can be applied in detecting the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments.

In determining whether an agent modulates the cathepsin D and/or cholesterol-induced production of neurofibrillary tangles, phosphorylated tau and/or tau fragments in brain cells, experiments are typically carried out with a control. A control can be, e.g., adding no agent or adding a different amount or type of agent and extrapolating and determining the zero amount. A statistically significant difference in a test amount (e.g., brain cells treated with a test agent) and a control amount (e.g., brain cells untreated with a test agent) of neurofibrillary tangles, phosphorylated tau and/or tau fragments indicates that the test agent modulates the production of neurofibrillary tangles of phosphorylated tau fragments. For example, inhibition of neurofibrillary tangles, phosphorylated tau and/or tau fragment production is achieved when the test amount of neurofibrillary tangles or phosphorylated tau or tau fragments relative to the control amount is about 90% (e.g., 10% less than the control), optionally 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 25-0%.

Brain cells in accordance with embodiments of the invention provide a model for the development of the biochemical characteristics of neurodegenerative diseases, such as Alzheimer's disease. In regular rats, mevastatin produces similar types and amounts of pathologies as observed in ApoE-knockout mice, and mevastatin plus ZPAD in regular rats produces results similar to those found in apoE-knockout mice treated with ZPAD. However, just as useful are normal rats treated with an agent that can lower the concentration of cholesterol since neurofibrillary tangles and phosphorylated tau protein/tau fragments are induced at a higher density, mimicking early-stage tangles found in Alzheimer's disease and other neurodegenerative diseases.

ApoE-deficient brain cells and apoE4-containing brain cells provide a cost and time efficient in vitro model to study such diseases. For example, apoE-deficient brain cells or apoE4-containing brain cells produced in accordance with embodiments of the invention can be used to screen agents that may modulate the production of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells. Efficacious agents that are identified by in vitro screening methods described herein can be further tested to determine their efficacy in vivo. Some of these agents can potentially be useful as therapeutic compounds for neurodegenerative diseases, including Alzheimer's disease.

In another aspect, the invention provides screening assays to identify cysteine protease inhibitors that modulate the amount of tau fragments. Additionally, such inhibitors may be assayed for their ability to inhibit the formation of tau fragments in the aforementioned assay system. For example, such screening methods would comprise:

(a) contacting brain cells with an agent capable of modulating the activity or levels of a cysteine protease;

(b) determining whether the agent modulates the amount of neurofibrillary tangles, tau fragmentation and/or the production of phosphorylated tau in the brain cells treated with the agent compared to the brain cells that are not treated with the agent.

Thus, the inhibition of tau proteolysis can be used as an assay for a new calpain inhibitor, especially a calpain inhibitor that has therapeutic utility in the treatment or prevention of neurological disorders, and, especially, Alzheimer's disease and/or Pick's disease.

In another aspect, the invention provides screening assays that identify MAP kinase inhibitors that modulate the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases. Additionally, such inhibitors may be assayed for their ability to inhibit the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the aforementioned assay system. For example, such screening methods can include:

(A) contacting brain cells with an agent that modulates the activity or levels of a MAP kinase; and

(B) determining whether the agent modulates the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the brain cells treated with the agent as compared to the brain cells that are not treated with the agent, and

(C) identifying those agents that decrease or that increase one or more of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the brain cells treated with the agent as compared to the brain cells that are not treated with the agent.

In a further embodiment, such agents are used in brain cells treated with such agent to increase or decrease, respectively, one or more of such neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases that such agent increased or decreased in the screening assay of the invention.

MAP kinase inhibitors that would be useful in this regard are known in the art. PD98059 (2-2(Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) is a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK). SB209580 (4-[5-(4-Fluorophenyl)-2-[4-(methylsulphonyl)phenyl]-1H-imidzaol-4yl]pyrid ine) is a highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK) and also inhibits cycoloxygenase-1 and -2, and thromboxane synthase. PD98059 and SB203580 are especially useful at concentrations of 5-100 .mu.M range. U0126 (1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene; Promega) is a selective inhibitor of MAP kinase kinase. U0126 is more potent and inhibits MEK-1 and MEK-2 with an IC₅₀ value of 0.07 and 0.06 .mu.M, respectively. Preferred concentrations of U0126 are 5-20 .mu.M.

The compounds can be employed in a free base form or in a salt form (e.g., as pharmaceutically acceptable salts). Examples of suitable pharmaceutically acceptable salts include inorganic acid addition salts such as hydrochloride, hydrobronide, sulfate, phosphate, and nitrate; organic acid addition salts such as acetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, salicylate, p-toluenesulfonate, and ascorbate; salts with acidic amino acids such as aspartate and glutamate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic basic salts such as trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, and N,N-dibenzylethylenediamine salt; and salts with basic amino acids such as the lysine salt and arginine salts. The salts may be in some cases be hydrates or ethanol solvates.

The manner in which the compounds are administered in vivo can vary. The compounds can be administered by inhalation (e.g., in the form of an aerosol either nasally or using delivery articles of the type set forth in U.S. Pat. No. 4,922,901 to Brooks et al., the disclosure of which is incorporated herein by reference in its entirety); topically (e.g., in lotion form); orally (e.g., in liquid form within a solvent such as an aqueous or non-aqueous liquid, or within a solid carrier); intravenously (e.g., within a dextrose or saline solution); as an infusion or injection (e.g., as a suspension or as an emulsion in a pharmaceutically acceptable liquid or mixture of liquids); intrathecally; intracerebro ventricularly; or transdermally (e.g., using a transdermal patch). Although it is possible to administer the compounds in the form of a bulk active chemical, it is preferred to present each compound in the form of a pharmaceutical composition or formulation for efficient and effective administration. Exemplary methods for administering such compounds will be apparent to the skilled artisan. For example, the compounds can be administered in the form of a tablet, a hard gelatin capsule or as a time release capsule. As another example, the compounds can be delivered transdermally using the types of patch technologies available from Novartis and Alza Corporation. The administration of the pharmaceutical compositions of the present invention can be intermittent, or at a gradual, continuous, constant or controlled rate to a warm-blooded animal, (e.g., a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, monkey or human). In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered can vary. Administration preferably is such that the active ingredients of the pharmaceutical formulation interact with receptor sites within the body of the subject that effect the functioning of the central nervous system. More specifically, in treating a neurodegenerative disease, administration preferably is such so as to optimize the effect upon those relevant protease and/or kinase subtypes (e.g., those which have an effect upon the functioning of the central nervous system), while minimizing the effects upon protease and/or kinase subtypes in muscle and ganglia. Other suitable methods for administering the compounds of the present invention are described in U.S. Pat. No. 5,604,231 to Smith et al., the disclosure of which is incorporated herein by reference in its entirety

Claim 1 of 218 Claims

What is claimed is:

1. An in vitro method of determining the effect of a substance on characteristics that are indicative of Alzheimer's Disease in rodent brain cells, said method comprising:

(A) exposing said brain cells to a cathepsin D-increasing agent or compound under conditions that increase the concentration or amount of cathepsin D in said cells to an effective concentration,

(B) maintaining said cells for a time that is sufficient to induce, relative to the levels present in the absence of said substance, one or more characteristics indicative of Alzheinier's Disease in said cells as a result of said increase in said cathepsin D,

(D) determining whether the presence of said substance has an effect on the induction of said one or more characteristics, wherein said characteristics are selected from the group consisting of:

(1) the formation of neurofibrillary tangles,

(2) the hyperphosphorylation of tau,

(3) the fragmentation of tau,

(4) the production and/or release of brain-produced cytokines TGF-beta, IL-1b, or TNF,

(5) a microglia reaction or microglial activation,

(6) indications of brain inflammatory reactions,

(7) conversion of p35 to p25,

(8) changes in the level and/or activity of cyclin dependent protein kinase 5 (cdk5), and

(9) changes in the level and/or activity of mitogen activated protein kinases (MAPK), wherein said effect on said induction of any or all of said characteristics in D(1)-D(9) is indicative of the appearance or disappearance, respectively, of said characteristics of Alzheimer's Disease.

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