The present invention provides brain cells, such as normal brain cells, apolipoprotein E deficient brain cells, or apoE4 containing brain cells, that are treated with a compound which can modulate integrins and/or integrin receptors to produce increased sequestration of and/or accumulation of and/or uptake of A.beta., and/or changes in cathepsin D content and/or lysosomal dysfunction, and/or microglia activation in the brain cells. The present invention also provides methods for producing such cells and methods for using the cells for screening an agent or substance that modulates the sequestration of and/or accumulation of and/or uptake of A.beta., and/or lysosomal dysfunction, and/or changes in cathepsin D content and/or microglia activation in the brain cells. The method further provides a new therapeutic target, antagonism of glutamate receptors, for the treatment of neurodegenerative diseases which are characterized by inter alia, abnormal amyloid uptake and/or accumulation.

SUMMARY OF THE INVENTION

The present invention provides a model for neurodegenerative diseases, including Alzheimer's disease and other age-related neurodegenerative diseases, wherein the model provides brain cells, or brain tissue containing the same. The invention further provides a method for increasing or decreasing characteristics and changes indicative of neurodegenerative diseases in such cells. These changes especially include increasing sequestration of and/or accumulation of and/or uptake of A.beta., and/or lysosomal dysfunction, and/or microglia activation. The present invention also provides a model wherein brain cells comprise a marked microglia activation and increases in the levels and/or activity of cathepsin D. As described above, many currently available in vivo and in vitro models of neurodegenerative diseases and aged brain lack some or all of these key features.

The present invention is based on, in part, the discovery that integrins and/or integrin receptors can modulate the sequestration and/or accumulation and/or uptake of A.beta. in cultured brain cells. Further, the present invention is also based upon the discovery that glutamate receptors within the brain, for example the NMDA-subtype of glutamate receptors, can modulate the sequestration and/or accumulation and/or uptake of A.beta. in cultured brain cells. Specifically, the treatment of brain cells with agent(s) capable of modulating integrins and/or integrin receptors surprisingly triggered the sequestration and/or accumulation and/or uptake of A.beta.. Further, agent(s) which affect glutamate receptors within the brain, for example the NMDA-subtype of glutamate receptors, blocked the modulation of the sequestration and/or accumulation and/or uptake of A.beta. in brain cells treated with agent(s) capable of modulating integrins and/or integrin receptors. These results can be observed in any suitable brain cells including, e.g., normal brain cells, brain cells derived from transgenic animals, etc.

Among many types of brain cells suitable for embodiments of the invention, hippocampal brain cells treated with agent(s) capable of modulating integrins and/or integrin receptors produced sequestration and/or accumulations and/or uptake of A.beta. at significantly enhanced levels when compared with hippocampal brain cells not treated with agent(s) capable of modulating integrins and/or integrin receptors. The enhanced sequestration and/or accumulations and/or uptake of A.beta. are typically formed within a few days of treatment, and morphologically mimic early stage amyloid sequestration and/or accumulations and/or uptake found in the brains of Alzheimer's patients. Such levels of sequestration and/or accumulations and/or uptake of A.beta. was not achievable in brain cells in vitro even with prolonged treatment with A.beta. without the presence of agent(s) capable of modulating integrins and/or integrin receptors. Therefore, if brain cells with robust sequestration and/or accumulations and/or uptake of A.beta. is desired, the use of agent(s) capable of modulating integrins and/or integrin receptors can be preferably used in embodiments of the invention. Thus, the present invention provides, among other things, brain cells in vitro comprising enhanced levels of sequestration and/or accumulations and/or uptake of A.beta. which can be used as a model for neurodegenerative diseases, including Alzheimer's disease.

Accordingly, in one aspect, the invention provides an in vitro method of increasing sequestration and/or accumulation and/or uptake of A.beta., the method comprising: (a) contacting cultured brain cells with agent(s) capable of modulating integrins and/or integrin receptors; and (b) determining the sequestration of and/or accumulation of and/or uptake of A.beta. in the cell culture.

In another aspect, the invention provides a method comprising, contacting brain cells with a compound that is capable of modulating integrins and/or integrin receptors, thereby producing properties of a brain afflicted with a neurodegenerative disease, wherein the properties include increased sequestration of and/or accumulation of and/or uptake of A.beta..

In yet another aspect, the invention provides brain cells in vitro that have been cultured in a medium capable of modulating integrins and/or integrin receptors in the brain cells, wherein the brain cells comprise an increased amount of sequestration of and/or accumulation of and/or uptake of A.beta. compared to a control.

In yet another aspect, the invention provides brain cells in vitro, wherein the brain cells have been treated with a compound that is capable of modulating integrins and/or integrin receptors, thereby producing properties of a brain afflicted with a neurodegenerative disease, wherein the properties include increased sequestration of and/or accumulation of and/or uptake of A.beta..

In yet another aspect, the invention provides a screening method comprising: (a) contacting brain cells in vitro, with a compound that is capable of modulating integrins and/or integrin receptors in the brain cells, wherein the compound is capable of increasing the sequestration of and/or accumulation of and/or uptake of A.beta.; (b) contacting the brain cells with an agent; and (c) determining whether the agent modulates the amount of sequestration of and/or accumulation of and/or uptake of A.beta. in the brain cells treated with the agent compared to the brain cells that are not treated with the agent.

In yet another aspect, the invention provides a method of increasing the sequestration of and/or accumulation of and/or uptake of A.beta. in any suitable brain cells, the method comprising: (a) contacting the brain cells in a medium which is capable of modulating integrins and/or integrin receptors; and (b) determining the sequestration of and/or accumulation of and/or uptake of A.beta. in the brain cells.

In another aspect, the invention provides a method comprising: (a) culturing brain cells; and (b) contacting the brain cells with a compound which is capable of modulating integrins and/or integrin receptors, thereby producing properties of a brain afflicted with a neurodegenerative disease, wherein the properties include increased sequestration of and/or accumulation of and/or uptake of A.beta..

In yet another aspect, the invention provides brain cells in vitro that have been cultured in a medium which modulates integrins and/or integrin receptors in the brain cells, wherein the brain cells comprise an increased sequestration of and/or accumulation of and/or uptake of A.beta. compared to a control.

In yet another aspect, the invention provides brain cells in vitro, wherein the brain cells have been treated with a compound that modulates integrins and/or integrin receptors in the brain cells, thereby producing properties of a brain afflicted with a neurodegenerative disease, wherein the properties include increased sequestration of and/or accumulation of and/or uptake of A.beta..

In yet another aspect, the invention provides a screening method comprising: (a) contacting brain cells in vitro, with a compound that modulates integrins and/or integrin receptors in the brain cells, wherein the modulation of integrins and/or integrin receptors is capable of increasing the sequestration of and/or accumulation of and/or uptake of A.beta. in the brain cells; (b) contacting the brain cells with an agent; and (c) determining whether the agent modulates the amount of sequestration of and/or accumulation of and/or uptake of A.beta. in the brain cells treated with the agent compared to the brain cells that are not treated with the agent.

In yet another aspect, the invention provides a method for determining the effect of a substance on characteristics of neurodegenerative disease in brain cells, said method comprising: (A) exposing brain cells to a condition that modulates integrins or integrin receptors in said cells, (B) maintaining said cells for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells, (C) adding said substance before, during and/or after said exposing or maintaining; and (D) determining whether the presence of said substance has an effect on one or more of said characteristics.

Another aspect of the invention provides method of obtaining brain cells having characteristics of neurodegenerative disease comprising (A) culturing brain cells, (B) exposing said brain cells to a condition that modulates integrins or integrin receptors in said cells, and (C) maintaining said cells or brain tissue for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells.

Another aspect of the invention is directed to an in vitro method for increasing at least one or more characteristics of neurodegenerative disease in brain cells, wherein said characteristics are selected from the group consisting sequestration of A.beta., accumulation of A.beta., uptake of A.beta., lysosomal dysfunction and microglia activation, said in vitro method comprising:

(A)exposing brain cells in culture to a condition that modulates integrins or integrin receptors in said cells wherein said modulation results in increase in characteristics of neurodegenerative disease in said cells, and (B) maintaining said cells in culture for a time sufficient to increase one or more characteristics of a neurodegenerative disease in said cells.

Another aspect of the invention is directed to a method for determining the effect of a substance on inhibition of characteristics of neurodegenerative disease in brain cells, said method comprising: (A) exposing brain cells to a condition that modulates integrins or integrin receptors in said cells, (B) maintaining said cells for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells, (C) adding said substance before, during and/or after said exposing or maintaining; and (D) determining whether the presence of said substance inhibits one or more of said characteristics.

The invention is also directed to a method for determining the effect of a substance on inhibition of characteristics of neurodegenerative disease in brain cells, said method comprising: (A) exposing brain cells to a condition that modulates integrins or integrin receptors in said cells, (B) maintaining said cells for a time sufficient to induce one or more characteristics of a neurodegenerative disease in said cells, (C) adding said substance before, during and/or after said exposing or maintaining; and (D) determining whether the presence of said substance inhibits one or more of said characteristics.

Aspects of the invention include methods drawn to the effect of the substance on characteristics selected from the group consisting of sequestration of A.beta., accumulation of A.beta., uptake of A.beta. and lysosomal dysfunction, changes in cathepsin D content and microglia activation. Additional embodiments look at increases or decreases of these characteristics, such as where the changes are at least about 10% compared to a control.

The methods of the invention are also drawn to obtaining brain cells and the use of brains cells wherein the brain cells may be in vivo or in vitro such as in the form of a brain slice. The brain slice may include a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice. Brain cells may also be obtained from a non-human transgenic animal. Such animals may comprise a human apolipoprotein E4 gene or and animal where an endogenous apolipoprotein E gene of the non-human transgenic animal are ablated. Apolipoprotein E deficient brain cells or apolipoprotein E4 containing brain cells cultured in a medium which selectively increases are also included in the invention.

The methods of the invention are also drawn to culturing brain slices or the cells therein in a medium that comprises an antagonist or modulator of an integrin. The modulator or antagonist may be selected from the group consisting of neutralizing and/or function blocking antibodies for integrin subunits alphal, alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, alpha8, beta1, beta2, beta3, beta4, beta5, beta6, beta7 and beta8. The methods of the invention are further drawn to a peptide selected from the group of peptides consisting of RGD, RGDS (SEQ. ID NO.1), GRGDS (SEQ. ID NO.2), GRGDSP (SEQ. ID NO.3), GRGDTP (SEQ. ID NO.4), mimetics thereof and disintegrins such as echistatin found in snake venom.

The methods of the invention are also directed to determining visually the amount of: sequestration of A.beta., accumulation of A.beta., uptake of A.beta., lysosomal dysfunction or microglia activation is determined visually. The determinations may be done visually and also using a capture reagent. The capture reagents may include is an antibody that binds to A.beta., lysosomes, cathepsin D or a microglia element.

The methods of the invention are also directed to either contacting the brain cells simultaneously with the compound that modulates integrins and/or integrin receptors or contacting the brains cells with the compound that modulates integrins and/or integrin receptors prior to contact with the substance whose effect is being determined.

Another aspect of the invention is directed to a method for alleviating the symptoms of disease states having at least one of the following characteristics selected from the group consisting of intracellular uptake of amyloid protein, amyloid accumulation and/or plaque formation, said method comprising: (A) administering to a patient in need thereof a composition comprising an effective amount of an NMDA receptor antagonist, and (B) determining the effectiveness of treatment with said composition, (C) increasing or decreasing the composition based on the determinative testing, and (D) alleviating symptoms of the disease. Determinative testing may include methods of brain imaging such as MRI or PET. Additional, determinative testing may include electroencephalogram analysis as well as cognitive testing.

The invention is also directed to a pharmaceutical composition comprising a compound capable of sufficiently inhibiting the activity of the NMDA receptors in an amount effective to alleviate one or more symptoms of disease states associated with at least one characteristic selected from the group consisting of abnormal accumulation, abnormal molecular organization of amyloid protein and/or amyloid plaques and said composition also includes a suitable carrier or pharmaceutical excipient. Embodiments of the invention may include a composition comprising at least one of the compounds selected from a group consisting of magnesium, ketamine, dextromethorphan, amantadine, dexanabinol, AP3, AP5, AP6, AP7, 4C3HPG, 4CPG, CGS 19755, chlorophenylglutamic acid, CPP, MK-801, PCP, ibogaine, noribogaine, ifenprodil, fiupirtine, selfotel, D-CPP-ene, procyclidine, trihexyphenidyl, CP-101606, CP-98113, GVI150526, AR-R15896AR, NPS 1506, NPC 12626, LY274614, LY 2835959, SDZ 220-040, SDZ 220-040, SDZ 220-581, SDZ 221-653 and memantine.

Another aspect of the invention is directed to a method for inhibiting the intracellular accumulation of amyloid comprising: (A) contacting brain cells with a glutamate receptor antagonist and (B) determining whether the intracellular accumulation of amyloid is inhibited.
 

Claim 1 of 26 Claims

1. A method for determining the effect of a substance on sequestration, uptake or accumulation of amyloid in brain cells, said method comprising: (A) exposing brain cells in vitro to an integrin antagonist, wherein said antagonist is selected from the group consisting of function blocking anti-.beta.5 subunit integrin antibody, function blocking anti-.beta.1 subunit integrin antibody, an RGD peptide capable of modifying integrin adhesion, RGDS peptide, GRGDS peptide, GRGDSP peptide, GRGDTP peptide and echistatin, (B) maintaining said cells for a time sufficient to induce sequestration, uptake or accumulation of amyloid in said cells as a result of said antagonist, (C) adding said substance before, during and/or after said exposing or maintaining; and (D) determining whether the presence of said substance has an effect on said antagonist induced sequestration, uptake or accumulation of amyloid.

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