METHODS AND COMPOSITIONS FOR TREATING NEURODEGENERATIVE DISORDERS AND ALZHEIMER'S DISEASE AND IMPROVING NORMAL MEMORY

Abstract

The disclosure relates generally to neurodegenerative disorders and more specifically to a group of presenilin/G-protein/c-src binding polypeptides and methods of use for modulating signaling and progression of Alzheimer's disease.

Description

DESCRIPTION OF DRAWINGS

FIG. 1 shows a representative study to determine if PS-1 is a GPCR. Extracts of different cell cultures were analyzed in order to determine whether G_o interacts with PS-1, including the necessary controls. In each lane, the particular cell extracts were first immunoprecipitated with a monoclonal Ab (MAb) directed to PS-1; the immunoprecipitate was then dissolved and subjected to SDS-PAGE electrophoresis, and the resulting gel was Western blotted with an antibody directed to G_o (this antibody recognizes both G_oA and G_oB) Lane 1 is a control of an extract of untransfected ES (PS-1^−/−/PS-2^−/−) cells. As expected, this extract showed that no G_oA (or G_oB) was immunoprecipitated with Ab to PS-1. Lane 2 is an extract of ES cells, that had first been transfected with PS-1 only, but not with G_oA. No protein band was observed for G_oA; this was another control experiment. Lane 3 is an extract of the ES cells transfected with both PS-1 and G_oA. In this extract, G_oA is immunoprecipitated along with the PS-1, showing that PS-1 was bound to G_oA, but not G_oB. If PS-1 without its C-terminal “tail” (lane 4), which protrudes from the membrane into the aqueous intracellular compartment), is transfected into ES double null cells along with G_oA (lane 6), little or no G_oA is immunoprecipitated along with the PS-1 tailess, showing that the C-terminal domain of PS-1 is the principal region of G_oA binding to PS-1.

FIG. 2 shows a Western blot of a similar experiment to that of FIG. 1 but with PS-2 instead of PS-1. Lanes 2 and 4 show that tail-less PS-2, unlike tail-less PS-1, still binds G_oA (and G_oB), and therefore that the binding sites for G_oA and G_oB are not confined to the C-terminal domain of PS-2, as is the case for PS-1 (FIG. 1) Lane 1 is untransfected ES (PS-1^−/−/PS-2^−/−). Lane 2 is PS-2+G_oA. Lane 3 is Tail-less PS-2+G_oA. Lane 4 is PS-2+G_oB. Lane 5 is Tail-less PS-2+G_oB.

FIG. 3 involves an independent way of demonstrating G_oA binding to PS-1. [³⁵S]-GTPγS, an analog of GTP, makes a covalent bond to the active site of a G-protein, that is blocked by a prior reaction with Pertussis toxin (PTx). In lane 2, there is shown an 8-fold increase in ³⁵S-incorporation into G_oA that is immunoprecipitated with antibody to PS-1, but not into G_oB (lane 4). Therefore, PS-1 binds to G_oA (that has reacted with [³⁵S]-GTPγS to identify it as a G-protein (lane 2), but also to a lesser extent to GOB than to G_oA (lane 4). The ³⁵S bindings to G_oA and G_oB are blocked by prior treatment with PTx (lanes 3 and 5).

FIG. 4 is a graph depicting ³⁵SGTPγS incorporation in extracts of ES cells transfected with cDNA for PS-2 and G-protein G_oA.

The following two experiments are designed to determine if mouse PS is a GPCR in vivo in the normal mouse brain. FIG. 5 shows the ³⁵S-GTPγS incorporation in extracts of mouse brain that could be immunoprecipitated with monoclonal antibodies to PS-1.

FIG. 6 shows the ³⁵S-GTPγS incorporation in extracts of mouse brain that could be immunoprecipitated with monoclonal antibodies to PS-2. Therefore, endogenous PS-1 and PS-2 in mouse brain are GPCRs.

FIG. 7 shows immunofluorescence microscopic labeling of fixed cells. a) Double immunofluorescence microscopic labeling of untransfected, fixed but not permeabilized, DAMI cells with primary rat Mab #1563 to human PS-1 N-terminal domain (Panel 1) and FITC conjugated anti-rat IgG secondary antibody (green) shows cell-surface immunolabeling of endogenous PS-1 amino terminal domain. Panel 2 shows the same cells do not express appreciable amounts of cell-surface β-APP when labeled with Mab #348 to the β-APP extracellular domain and TRITC-conjugated anti-mouse IgG secondary antibody (red). Panel 3 shows the Nomarski images of cells in panels 1 and 2. b) Double Immunofluorescence microscopic labeling of β-APP-transfected, fixed but not permeabilized, DAMI cells shows cell-surface expressed β-APP when labeled with Mab #348 to the β-APP extracellular domain and TRITC-conjugated secondary antibody (red, Panel 2). Panels 1 and 3, the same cells treated as for FIG. 7a. c) Immunofluorescence microscopic labeling of PS-1-transfected, fixed but not permeabilized, DAMI cells shows high expression of cell-surface PS-1 (Panel 1) but not β-APP (Panel 2) when labeled with the same primary and secondary antibodies described in a. Panel 3 shows the Nomarski image of cells in panels 1 and 2. These experiments show that transfection of the DAMI cells with PS-1 does not call forth cell surface expression of β-APP. d) Immunofluorescence microscopic labeling of β-APP-transfected, fixed but not permeabilized ES cells, double-null for PS-1 and PS-2. Cells show cell-surface expressed β-APP when labeled with Mab #348 to the β-APP extracellular domain and TRITC-conjugated secondary antibody (red; Panel 2). Panel 1 shows the result of labeling with primary rat Mab #1563 to human PS-1 N-terminal domain and FITC conjugated appropriate secondary antibody, indicating the expected absence of PS-1 on the surfaces of ES double-null cells. Panel 3 shows Nomarski image of cells in Panels 1 and 2. e) Immunofluorescence microscopic labeling of untransfected, fixed but not permeabilized ES cells, double-null for PS-1 and PS-2. Cells show cell-surface expressed endogenous mouse β-APP when labeled with Mab #348 to the β-APP extracellular domain and TRITC-conjugated secondary antibody (red; Panel 2). Panels 1 and 3 labeled as in d; no cell surface labeling for PS-1 (Panel 1; green) is observed in these untransfected ES cells. Bar, 20 μm.

FIG. 8 shows that within minutes after mixing β-APP-only expressing transfected ES cells with PS-1 only expressing transfected DAMI cells, a transient protein tyrosine phosphorylation process arises in the mixed cell culture, as detected by ELISA analyses of the cell extracts. This activity peaked at ˜8-10 mins after mixing (a). The same experiment carried out in the presence of 25 μg purified soluble β-APP (b) or 25 μg purified peptide of N-terminal domain of PS-1 fused to FLAG (c) showed none of the increases observed in (a). The addition of 25 μg of purified peptide of the non-specific N-terminal domain of PS-2 fused to FLAG (d), however, resulted in very similar transient increases in protein tyrosine kinase activity to (a).

FIG. 9. Experiments to determine the nature of the tyrosine phosphorylating enzyme activity in FIG. 6. Src family kinase assay with synthetic peptides. a and b: β-APP:PS-1 interaction with separately transfected DAMI cells as a function of time after mixing. Src kinase activity was assayed using the Src family substrate peptide {lys19}cdc2(6-20)-NH₂ (black bars) and control peptides {lys19Phe15}cdc2(6-20)NH2 (white bars) and {lys19ser14val12}cdc2(6-20)NH2 (gray bars) for both the β-APP:PS-1 (a) and control pcDNA3:PS-1 (b) interactions. c and d: β-APP:PS-2 interaction with separately transfected DAMI cells as a function of time after mixing. Src kinase activity was assayed using the Src family substrate peptide {lys19}cdc2(6-20)-NH₂ (black bars) and control peptides {lys19Phe15}cdc2(6-20)NH2 (white bars) and {lys19ser14val12}cdc2(6-20)NH₂ (gray bars) for both the β-APP:PS-2 (c) and control pcDNA3:PS-2 (d) interactions.

FIG. 10. Inhibition of tyrosine kinase activity. ELISAs to demonstrate tyrosine kinase activity of DAMI cells which had been separately transfected with β-APP and PS-1 and mixed in the presence and absence of 10 μg/ml Herbimycin A (a) and 10 nM PP2 (b), as a function of time after mixing.

FIG. 11 shows β-APP:PS-1 intercellular interaction: C-Src activity in extracts of mixed cells. a. Western Immunoblot. β-APP:PS-1 interactions with mixtures of separately transfected DAMI cells. Western immunoblot with primary anti-PTyr polyclonal antibodies (Panel 1) and anti-pp60c-src monoclonal antibodies (Panel 2) from the same experiment in which β-APP-transfected DAMI cells were mixed with PS-1-transfected DAMI cells for 0-12 mins. Panel 3: Antibody labeling of control pp60c-src protein with the pp60c-src antibodies. Panel 4: Western immunoblots with primary anti-PTyr antibodies, as in Panel 1, from experiments in which β-APP-transfected ES double-null cells were interacted with PS-1-transfected DAMI cells. b. Autoradiograph of in-vitro phosphorylated proteins. Extracts of separately transfected β-APP and PS-1 DAMI cell mixtures at 0-12 mins after mixing were first immunoprecipitated with antibodies to c-Src and then phosphorylated in vitro with γ³²P-ATP. Autophosphorylation reactions were subjected to SDS-PAGE followed by autoradiography.

FIG. 12 shows β-APP:PS-2 intercellular interaction: C-Src activity in extracts of mixed cells. a. Western Immunoblot. β-APP:PS-2 interaction in extracts of separately transfected and mixed DAMI cells as a function of time after mixing. Panels 1 and 2: Same as FIG. 9a except that PS-2-transfected DAMI cells replaced PS-1-transfected cells in the intercellular interaction with β-APP and cells were mixed from 1-20 mins. b. Autoradiograph of in-vitro phosphorylated proteins. Same extracts as in part a. Same as 5b except that PS-2-transfected DAMI cells replaced PS-1-transfected DAMI cells in the intercellular interaction with β-APP.

FIG. 13 shows β-APP:PS-2 intercellular interaction: Activity of Lyn and Fyn in extracts of mixed cells. a and b. Western Immunoblots: β-APP:PS-2 interaction. Western immunoblot with primary anti-Lyn polyclonal antibodies (a, Panel 1) and anti-Fyn polyclonal antibodies (b, Panel 1) from the same experiment in which β-APP-transfected DAMI cells were mixed with PS-2-transfected DAMI cells for 0-20 mins and extracts made. No change with time in concentration of either Lyn or Fyn protein was observed. Panel 2: Antibody labeling of control Lyn (a) and Fyn (b) protein with their respective antibodies. c and d. Autoradiograph of in-vitro phosphorylated proteins: β-APP:PS-2 interaction. Extracts of mixtures of β-APP and PS-2 mixed transfected cells at 0-20 mins after mixing were first immunoprecipitated with antibodies to Lyn (c) or Fyn (d) and then phosphorylated in vitro with γ³²P-ATP. Autophosphorylation reaction products were subjected to SDS-PAGE followed by autoradiography.

FIG. 14 illustrates intracellular domains of PS.

FIG. 15 shows the effect of intercellular β-APP:PS interactions on Aβ production.

Claims

1. A method of identifying an agent that modulates presenilin G-protein coupled receptor (GPCR) activity, the method comprising: a) contacting presenilin, or fragment thereof, with a G-protein under conditions that would permit binding of the G-protein to presenilin;b) prior to, simultaneously with, or subsequent to a), contacting presenilin, or fragment thereof, with an agent;c) monitoring presenilin-mediated binding to the G-protein; andd) determining whether the agent modulates presenilin binding to the G-protein thereby identifying an agent that modulates presenilin G-protein coupled receptor (GPCR) activity.

2. The method of claim 1 wherein the modulating is by inhibition of presenilin binding to the G-protein.

3. The method of claim 1 wherein the modulating is by activating presenilin binding to the G-protein.

4. The method of claim 1 wherein the presenilin is presenilin-1 (PS-1).

5. The method of claim 4 wherein the PS-1 comprises amino acid residues 1 to about 430.

6. The method of claim 1 wherein the presenilin is presenilin-2 (PS-2).

7. The method of claim 1 wherein the G-protein is selected from the group consisting of Go, Gs, Gi, Gz and Gq.

8. The method of claim 7 wherein the G-protein is Go comprising a Ga subunit.

9. The method of claim 8 wherein the Gα subunit is GoA.

10. The method of claim 8 wherein the Gα subunit is GoB.

11. The method of claim 1, wherein the agent is selected from the group consisting of a naturally occurring or synthetic polypeptide or oligopeptide, a peptidomimetic, a small organic molecule, a polysaccharide, a lipid, a fatty acid, a polynucleotide, an RNAi or siRNA, an asRNA, and an oligonucleotide.

12. The method of claim 1 wherein the contacting is in vitro.

13. The method of claim 1 wherein the contacting is in vivo.

14. The method of claim 1 further comprising contacting the presenilin with β-APP prior to, simultaneously with, or subsequent to contacting the presenilin with the G-protein.

15. A method of identifying an agent that modulates presenilin-mediated Src protein kinase activity, the method comprising: a) contacting presenilin, or fragment thereof, with β-APP under conditions that would permit binding of β-APP to presenilin;b) prior to, simultaneously with, or subsequent to a), contacting presenilin, or fragment thereof, with an agent;c) monitoring presenilin-mediated Src protein kinase activity; andd) determining whether the agent modulates presenilin-mediated Src protein kinase activity.

16. The method of claim 15 wherein the modulating is by inhibition of presenilin-mediated Src protein kinase activity.

17. The method of claim 15 wherein the modulating is by activating presenilin-mediated Src protein kinase activity.

18. The method of claim 15 wherein the presenilin is presenilin-1 (PS-1).

19. The method of claim 15 wherein the presenilin is presenilin-2 (PS-2).

20. The method of claim 1, wherein the agent is selected from the group consisting of a naturally occurring or synthetic polypeptide or oligopeptide, a peptidomimetic, a small organic molecule, a polysaccharide, a lipid, a fatty acid, a polynucleotide, an RNAi or siRNA, an asRNA, and an oligonucleotide.

21. The method of claim 15 wherein the contacting is in vitro.

22. The method of claim 15 wherein the contacting is in vivo.

23. A method of inhibiting the production of Aβ by interfering with the intercellular binding of βAPP and presenilin-1 (PS-1) and/or presenilin-2 (PS-2) comprising: inhibiting the binding of the N-terminal extracellular domain of βAPP with the N-terminal region of PS-1 and PS-2, or PS-1 or PS-2 where an interfering agent is a peptide, a small molecule, a peptidomimetic or an antibody.

24. The method of claim 24, wherein the peptide is a soluble N-terminal domain of PS-1 or -2.

25. A method of inhibiting the production of Aβby contacting a cell expressing PS-1 and/or PS-2 with an agent that inhibits the interaction of PS-1 and/or PS-2 with GoA and GoB.

26. The method of claim 25, wherein the agent interacts with the C-terminal tail and/or other cytoplasmic domain of PS-1 and/or 2 with a GoA and/or GoB.

27. A method of inhibiting the production of Aβ by contacting a cell expressing a PS-1 and/or PS-2 with an agent that interferes with the downstream results of PS-1 and/or PS-2 binding to Go such as Go activation with phospholipase C.

28. A method of inhibiting the production of Aβ by inhibiting the activity of members of the Src family of tyrosine kinases in cells expressing PS-1 and/or PS-2.

29. A method of assaying for inhibitors of Alhzeimer's Disease progression comprising contacting a cell system comprising a first cell expressing βAPP and a second cell expressing PS-1 and/or PS-2 with an agent and measuring the activity of (a) Src family of tyrosine kinases, (b) GoA and/or GoB interaction with PS-1 and/or PS-2, and/or (c) the interaction of N-terminal domain of βAPP with the N-terminal domain of PS-1 and/or PS-2.

30. A method for improving memory, comprising administering to a subject an agent that inhibits the interaction of PS-1 and/or PS-2 with a G-protein.

31. The method of claim 30 wherein the G-protein is selected from the group consisting of Go, Gs, Gi, Gz and Gq.

32. The method of claim 30, wherein the agent interacts with the C-terminal tail and/or other cytoplasmic domains of PS-1 and/or 2 with GoA and/or GoB.

33. The method of claim 30, wherein the agent inhibits the activity of members of the Src family of tyrosine kinases in cells expressing PS-1 and/or PS-2.