Protein Profiles Associated With Context Fear Conditioning and Their Modulation by Memantine

Abstract

Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/ protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein responses and interactions following normal learning. Molecular & Cellular

Protein Dynamics Associated with Failed and Rescued Learning in the Ts65Dn Mouse Model of Down Syndrome

“…Male mice only (age 3–4 months) were used. The wild type littermates have been reported previously [ 39 ].…”

“…Context fear conditioning (CFC) was performed as described [ 16 , 39 , 42 ]. Briefly, mice were placed in a novel cage (Med Associates, St. Albans, VT, Modular Mouse Test Chamber), allowed to explore for three minutes and then given an electric shock (2 s, 0.7 mA, constant electric current).…”

“…Mice were sacrificed by cervical dislocation, without anesthetic to minimize additional alterations to the protein profiles. Brain dissections for hippocampus and cortex, and preparation of protein lysates from subcellular fractions (nuclear-enriched, cytosolic and crude membrane) have been described [ 39 ]. Protein concentrations, determined using the 660 nM Protein Assay kit (Pierce), were within the range of 3–4 mg/ml for all samples.…”

“…Protein concentrations, determined using the 660 nM Protein Assay kit (Pierce), were within the range of 3–4 mg/ml for all samples. Information for each mouse, including for the control mice reported previously [ 39 ], on age, littermates, and weights of individual brain regions is provided in S1 Table .…”

“…Here, we describe the effects of memantine on protein expression in brain regions of Ts65Dn mice exposed to CFC. We have previously described in control mice the responses in 85 proteins/protein modifications to both normal learning in CFC and to memantine [ 39 ]. Here we show that Ts65Dn that have not been trained in CFC already have elevated levels of approximately half the proteins that respond when control mice successfully learn.…”