Methods for assaying for antidepressant therapy markers

Abstract

The present invention relates generally to methods for assaying for agents that modify the association of G sα with components of the plasma membrane or cytoskeleton of cells. The present invention also relates generally to methods of assaying for agents having antidepressant activity via analysis of the association of G sα with components of the plasma membrane or cytoskeleton of cells using a fluorescent analog of G sα and fluorescence resonance energy transfer (FRET).

1. A method for assaying for an agent or agents having an activity that modifies association of G sα  with components of the plasma membrane or cytoskeleton of cells comprising the step of:
 (a) contacting said agent or agents with cultured cells; 
 (b)(a) determining whether there has been a modification ofthe association of G sα  with components of the plasma membrane or cytoskeleton of the cells in step (a)treated with the agent or agents via comparison to a control cell culture lacking cells not treated with said agent or agents; and  
 (c)(b) identifying an agent or agents that modify the association of G sα  with components of the plasma membrane or cytoskeleton of cells from a difference in the modification of the association of G sα  with components of the plasma membrane or cytoskeleton of the treated compared to untreated cells in step (a). 
 
     
     
       2. A method for assaying for an agent or agents having antidepressant activity comprising the step of:
 (a) contacting said agent or agents with cultured cells; 
 (b)(a) determining whether there has been a modification of association of a fluorescent G sα  analog with components of the plasma membrane or cytoskeleton of the cells in step (a) treated with the agent or agents via comparison to a control cell culture lacking cells not treated with said agent or agents; and  
 (c)(b) identifying an agent or agents having antidepressant activity from a difference in the modification of the association of the fluorescent G sα  analog with components of the plasma membrane or cytoskeleton of the treated compared to untreated cells in step (a), wherein an agent or agents having antidepressant activity increases the modification of decreases the association of fluorescent G sα  analog with components of the plasma membrane or cytoskeleton of the cells in step (a). 
 
     
     
       3. The method of  claim 2  wherein the modification is further defined as enhanced coupling between the fluorescent G sα  analog and adenylyl cyclase. 
     
     
       4. The method of  claim 3  wherein the adenylyl cyclase is fluorescently labeled. 
     
     
       5. The method of claim  4  3, wherein the interaction coupling between the fluorescent G sα  analog and the fluorescently labeled adenylyl cyclase is monitored using fluorescence resonance energy transfer (FRET) wherein an increase in FRET is observed when said agent or agents possess as a measure of antidepressant activity. 
     
     
       6. A method to determine whether an individual is clinically depressed, the method comprising:
 (a) measuring the percentage of G sα  within and outside caveolin-enriched cell membranes or cytoskeletons from tissue samples of the individual; and   (b) determining that the individual is clinically depressed if the percentage of G sα  in the caveolin-enriched membranes or cytoskeletons is closer to the percentage in individuals known to be depressed compared to the percentage in control individuals.    
     
     
       7. The method of claim 6 wherein the percentage of G sα  is determined by extraction from membranes and cytoskeletons by specific detergent solubility of G sα  in cell membranes.  
     
     
       8. The method of claim 6 wherein the tissue samples are blood cells.  
     
     
       9. The method of claim 7 wherein the specific detergent comprises octoxynol-9; t-octylphenoxypolyethoxyethanol (Triton™ X-100) and tert-octylphenoxypoly (ethoxyethanol) (Triton™ X-114).  
     
     
       10. A method of determining whether there is a modification of an association of G sα  with components of plasma membranes or cytoskeletons of cells in tissues, the method comprising:
 (a) measuring the percentage of G sα  in relatively insoluble domains and in more soluble domains using specific detergents; and   (b) determining there is a modification if there is a shift of G sα  from caveolin-enriched membrane regions which are relatively insoluble to caveolin-poor regions which are relatively soluble.    
     
     
       11. The method of claim 10 wherein the modification results in enhanced coupling between G sα  and adenylyl cyclase.  
     
     
       12. The method of claim 10, wherein the specific detergents are octoxynol-9; t-octylphenoxypolyethoxyethanol (Triton™ X-100) and tert-octylphenoxypoly (ethoxyethanol) (Triton™ X-114), and the ratio of G sα  in octoxynol-9; t-octylphenoxypolyethoxyethanol (Triton™ X-100) and tert-octylphenoxypoly (ethoxyethanol) (Triton™ X-114) extracts is used to determine the modification.  
     
     
       13. A method to determine the effectiveness of antidepressant therapy in a treated individual, the method comprising:
 (a) comparing the location of G sα  within and outside cholesterol-rich domains in cell membranes or cytoskeletons of tissues from the individual after treatment, to the location of G sα  in tissues of the individual prior to treatment;   (b) determining that the antidepressant treatment is effective if G sα  has translocated from cholesterol-rich to cholesterol-poor domains, where it can more easily bind to adenylyl cyclase.    
     
     
       14. A biological marker for depression and related mood disorders, the biological marker comprising the percentage of G sα  protein associated with caveolin-enriched membrane regions relative to the percentage associated with caveolin-poor membrane regions.  
     
     
       15. A method to detect a biological marker associated with response of cells to antidepressants, the method comprising:
 (a) measuring G sα  that is in strongly hydrophobic regions of membranes of cells;   (b) measuring G sα  that is in less hydrophobic regions of membranes of cells; and   (c) detecting a biological marker if there is a difference between the amount of G sα  in (a) compared to (b).    
     
     
       16. The method to detect the marker of claim 15, wherein the difference is more G sα  in (a) than in (b).  
     
     
       17. The method to detect the biological marker of claim 15 further defined as measured by the ratio of G sα  protein soluble in octoxynol-9; t-octylphenoxypolyethoxyethanol (Triton™ X-100) and tert-octylphenoxypoly (ethoxyethanol) (Triton™ X-114), wherein octylphenoxypolyethoxyethanol (Triton™ X-100) extracts include more soluble G sα  from the less hydrophobic, less caveolin-enriched domains, than do the tert-octylphenoxypoly (ethoxyethanol) Triton™ X-114 extracts.  
     
     
       18. The method to detect the biological marker of claim 17 wherein the ratio becomes greater than 1 in response to antidepressants.