The goals of the proposed research are to 1) compare the effects on signal transduction of specific neurotensin (NT) analogs, NT69L and NT79, to those of typical and atypical antipsychotics, and 2) to identify the NT receptors (NTRs) by which these effects are mediated. The long term objective of the proposed research is to better understand the mechanism(s) of action of NT analogs to further their development as potential novel antipsychotic drugs. With this knowledge, NT analogs can be modified to directly target proteins thought to be directly involved in the treatment of neuropsychiatric disease while avoiding those thought to mediate adverse side effects. Neurotensin (NT) is an endogenous thirteen amino acid neuropeptide that acts as a neurotransmitter in the brain. It has been implicated in the pathophysiology of neuropsychiatric diseases including schizophrenia, as well as in the mechanism of action of antipsychotic drugs. NT can elicit effects similar to those seen using antipsychotic drugs, even when administered acutely. However, in animal models NT must be administered directly into the brain to observe its effects. To circumvent this problem, the Richelson laboratory has developed several NT analogs that act as agonists to one or more of the three known NT receptors, NTS1, NTS2, and NTS3. The NT analogs have similar effects as NT, but their mechanisms of action are not fully understood at this time. These analogs, which can be administered peripherally, include NT69L, thought to bind both NTS1 and NTS2, and NT79, thought to preferentially bind NTS2. Comparing the effects of these compounds on protein phosphorylation in regions of the brain implicated in schizophrenia to the effects of established antipsychotic drugs will offer insight into the mechanism(s) of action mediating antipsychotic-like effects. This will further the development of NT analogs as potential antipsychotic drugs by identifying possible molecular targets which mediate the antipsychotic-like properties of the analogs as well as identifying potential adverse side-effects. Three focused research aims are proposed to elucidate the mechanism(s) of action of these analogs: (1) Identify proteins that are differentially phosphorylated in wild-type C57BL mice treated acutely with vehicle, NT69L, and NT79, and compare them to those treated acutely with haloperidol and clozapine, (2) Identify proteins that are differentially phosphorylated between NTS1 knock-out (KO) mice treated acutely with vehicle, NT69L and NT79, and (3) Identify proteins that are differentially phosphorylated between NTS2 KO mice treated acutely with vehicle, NT69L and NT79. Methods include animal injection, brain dissection, extraction of synaptoneurosomal-enriched preparations, and protein separation via two-dimensional gel electrophoresis, phosphoprotein detection and identification of differentially phosphorylated proteins via mass spectrometry.
NT69L and NT79 are compounds that may be therapeutic in the treatment of schizophrenia. Determining the effects of these compounds on proteins in the brain, and comparing them to those caused by established antipsychotic drugs (used to treat schizophrenia), can offer insight to what role each compound may play in treating disease. With this knowledge, these compounds may be designed to more specifically target those proteins involved in disease, increasing their effectiveness as antipsychotics and decreasing potential side effects.
