Nervous system-specific transmembrane proteasome complex that modulates neuronal signaling through extracellular signaling via brain activity peptides
The inventors surprisingly found that neural stimulation caused the synthesis and degradation of proteins into peptides which were then secreted into the cell media within minutes of stimulation by a novel neural-specific and membrane bound proteasome (neuronal membrane proteasome or NMP) that is transmembrane in nature. These secreted, activity-induced, proteasomal peptides (SNAPPs) range in size from about 500 Daltons to about 3000 Daltons. Surprisingly none of the peptides appear to be those previously known to have any neuronal function. Moreover, these SNAPPs have stimulatory activity and are heretofore a new class of signaling molecules. Moreover, the NMP appears to play a highly significant role in aspects of neuronal signaling known to be critical for neuronal function. The inventors have gone on to develop all tools to study this novel mechanisms including protocols and practice for generation and purification of SNAPPs as well as a new and specific inhibitor of the NMP allowing for selective control of this process in the nervous system. The present invention provides methods of making and using these SNAPPs for both laboratory and clinical purposes, the screening for molecules which modulate NMP function in vivo and in vitro, and methods for diagnosis of NMP related diseases.
1. A method for screening for compounds which inhibit Neuronal Membrane Proteasome (NMP) mediated production of secreted neuronal-activity induced proteasomal peptides (SNAPPs) comprising the steps of:
a) providing a plurality of in vitro cultures comprising a plurality of neurons in a growth medium;
b) providing one or more test cultures by contacting the neurons of at least a first culture with a test compound and with a known neuronal proteasomal inhibitor for a period of time sufficient to allow NMP mediated production of SNAPPs into the growth medium;
c) providing a negative control by contacting the neurons of at least a second culture for a period of time sufficient with a carrier or vehicle which will not inhibit NMP mediated production of SNAPPs into the growth medium;
d) providing a positive control by stimulating the neurons of a third culture for a period of time sufficient with a known neuronal proteasomal stimulator to allow NMP mediated production of SNAPPs into the growth medium;
e) removing at least a portion of the growth medium of the cultures of b) to d) and performing an isolation step to purify the SNAPPs from the cultures of b) to d);
f) quantifying the amount of SNAPPs isolated in e) from the cultures of b) to d); and
g) determining that the test compound is an inhibitor of NMP mediated production of SNAPP secretion when the quantity of SNAPPs isolated from b) are significantly reduced when compared with the amount of SNAPPs in c) and/or d).
2. The method of claim 1 , wherein the quantification of SNAPPs in the sample is performed using HPLC (LC-MALDI) or fractionation of an HPLC column directly into an electrospray mass spectrometer (LC/MS-ESI).