Model for Alzheimer's disease and other neurodegenerative diseases

Abstract

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.

1 - 12 . (Cancelled)  
     
     
         13 . A method of 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 decreases an effective concentration of cholesterol 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 said maintaining; and    (D) determining whether the presence of said substance has an effect on said one or more characteristics,    wherein said characteristics are selected from the group consisting of:    (1) the formation of neurofibrillary tangles,    (2) an increase in the phosphorylation of tau,    (3) an increase in tau proteolytic fragments,    (4) an increased production and/or release of brain-produced cytokines TGF-beta, IL-1b or LPS,    (5) an increased microglia reaction or microglial activation,    (6) increased indications of brain inflammatory reactions,    (7) decrease in the levels of p35,    (8) decreased activity of cyclin dependent protein kinase 5 (cdk5), and    (9) increased levels of mitogen activated protein kinase (MAPK).    
     
     
         14 . The method of  claim 13 , wherein said characteristics comprise an increase in the density of neurofibrillary tangles in said brain cells.  
     
     
         15 . The method of  claim 13 , wherein said characteristics comprise an increase in the amount of phosphorylated tau in said brain cells.  
     
     
         16 . The method of  claim 13 , wherein said characteristics comprise an increase in the amount of tau proteolytic fragments in said brain cells.  
     
     
         17 . The method of  claim 13 , wherein said condition comprises contacting said brain cells with an inhibitor of cholesterol synthesis.  
     
     
         18 . The method of  claim 13 , wherein said condition comprises contacting said brain cells with a member of the family of compounds know as statins.  
     
     
         19 . The method of  claim 17 , wherein said inhibitor is selected from the group consisting of mevastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin, cerivastatin, and mimetics thereof.  
     
     
         20 . The method of  claim 19 , wherein said inhibitor is mevastatin.  
     
     
         21 . The method of  claim 13 , wherein said brain cells are in the form of a brain slice.  
     
     
         22 . The method of  claim 21 , wherein said brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.  
     
     
         23 . The method of  claim 13 , wherein said brain cells are in vivo.  
     
     
         24 . The method of  claim 13 , wherein said brain cells are apolipoprotein E-deficient brain cells.  
     
     
         25 . The method of  claim 13 , wherein said brain cells are apolipoprotein E4-containing brain cells.  
     
     
         26 . The method of  claim 13 , wherein said cells are also contacted with a cathepsin D-increasing compound.  
     
     
         27 - 39 . (Cancelled)  
     
     
         40 . A method of determining the effect of a substance on the inhibition of characteristics of neurodegenerative disease in brain cells, said method comprising: 
 (A) exposing brain cells to a condition that decreases an effective concentration of cholesterol 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 a cysteine protease inhibitor before, during and/or after said exposing or said maintaining;    (D) adding said substance before, during and/or after said exposing or said maintaining; and    (E) determining whether the presence of said inhibitor has an effect on the inhibition of the development of said one or more characteristics,    wherein said characteristics are selected from the group consisting of:    (1) the formation of neurofibrillary tangles,    (2) an increase in the phosphorylation of tau,    (3) an increase in tau proteolytic fragments,    (4) an increased production and/or release of brain-produced cytokines TGF-beta, IL-1b, TNF, or LPS,    (5) an increased microglia reaction or microglial activation,    (6) increased indications of brain inflammatory reactions,    (7) decrease in the levels of p35,    (8) decreased activity of cyclin dependent protein kinase 5 (cdk5), and    (9) increased levels of mitogen activated protein kinase (MAPK).    
     
     
         41 . The method of  claim 40 , wherein said characteristics comprise an increase in the density of neurofibrillary tangles in said brain cells.  
     
     
         42 . The method of  claim 40 , wherein said characteristics comprise an increase in the amount of phosphorylated tau in said brain cells.  
     
     
         43 . The method of  claim 40 , wherein said characteristics comprise an increase in the amount of tau proteolytic fragments in said brain cells.  
     
     
         44 . The method of  claim 40 , wherein said condition comprises contacting said brain cells with an inhibitor of cholesterol synthesis.  
     
     
         45 . The method of  claim 40 , wherein said condition comprises contacting said brain cells with a member of the family of compounds know as statins.  
     
     
         46 . The method of  claim 44 , wherein said inhibitor is selected from the group consisting of mevastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin, cerivastatin, and mimetics thereof.  
     
     
         47 . The method of  claim 46 , wherein said inhibitor is mevastatin.  
     
     
         48 . The method of  claim 40 , wherein said brain cells are in the form of a brain slice.  
     
     
         49 . The method of  claim 48 , wherein said brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.  
     
     
         50 . The method of  claim 40 , wherein said brain cells are in vivo.  
     
     
         51 . The method of  claim 40 , wherein said brain cells are apolipoprotein E-deficient brain cells.  
     
     
         52 . The method of  claim 40 , wherein said brain cells are apolipoprotein E4-containing brain cells.  
     
     
         53 . The method of  claim 40 , wherein said cells are also contacted with a compound that increases cathepsin D.  
     
     
         54 - 71 . (Cancelled)  
     
     
         72 . A method of determining the effect of a substance on the inhibition of characteristics of neurodegenerative disease in brain cells, said method comprising: 
 (A) exposing brain cells to a condition that decreases an effective concentration of cholesterol 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 a mitogen activated kinase inhibitor before, during and/or after said exposing or said maintaining;    (D) adding said substance before, during and/or after said exposing or said maintaining; and    (E) determining whether the presence of said inhibitor has an effect on the inhibition of the development of said one or more characteristics,    wherein said characteristics are selected from the group consisting of:    (1) the formation of neurofibrillary tangles,    (2) an increase in the phosphorylation of tau,    (3) an increase in tau proteolytic fragments,    (4) an increased production and/or release of brain-produced cytokines TGF-beta, IL-1b, TNF, or LPS,    (5) an increased microglia reaction or microglial activation,    (6) increased indications of brain inflammatory reactions,    (7) decrease in the levels of p35,    (8) decreased activity of cyclin dependent protein kinase 5 (cdk5), and    (9) increased levels of mitogen activated protein kinase (MAPK).    
     
     
         73 . The method of  claim 72 , wherein said characteristics comprise an increase in the density of neurofibrillary tangles in said brain cells.  
     
     
         74 . The method of  claim 72 , wherein said characteristics comprise an increase in the amount of phosphorylated tau in said brain cells.  
     
     
         75 . The method of  claim 72 , wherein said characteristics comprise an increase in the amount of tau proteolytic fragments in said brain cells.  
     
     
         76 . The method of  claim 72 , wherein said condition comprises contacting said brain cells with an inhibitor of cholesterol synthesis.  
     
     
         77 . The method of  claim 72 , wherein said condition comprises contacting said brain cells with a member of the family of compounds know as statins.  
     
     
         78 . The method of  claim 76 , wherein said inhibitor of cholesterol synthesis is selected from the group consisting of mevastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin, cerivastatin, and mimetics thereof.  
     
     
         79 . The method of  claim 78 , wherein said inhibitor is mevastatin.  
     
     
         80 . The method of  claim 72 , wherein said brain cells are in the form of a brain slice.  
     
     
         81 . The method of  claim 80 , wherein said brain slice is a hippocampal slice, an entorhinal cortex slice, an entorhinohippocampal slice, a neocortex slice, a hypothalamic slice, or a cortex slice.  
     
     
         82 . The method of  claim 72 , wherein said brain cells are in vivo.  
     
     
         83 . The method of  claim 72 , wherein said brain cells are apolipoprotein E-deficient brain cells.  
     
     
         84 . The method of  claim 72 , wherein said brain cells are apolipoprotein E4-containing brain cells.  
     
     
         85 . The method of  claim 72 , wherein said cells are also contacted with a compound that increases cathepsin D.  
     
     
         86 . The method of  claim 72 , wherein said mitogen activated kinase inhibitor is a MAP kinase inhibitor.  
     
     
         87 . The method of  claim 72 , wherein said mitogen activated kinase inhibitor is selected from the group consisting of PD98059, SB203580 and U0126.  
     
     
         88 . The method of  claim 86 , wherein said mitogen activated kinase inhibitor is PD 98059.  
     
     
         89 - 94 . (Cancelled)