BRNI Research
Molecular Pathways of Memory
Institute scientists have for many years been investigating the sequence of molecular events responsible for learning, memory consolidation, and permanent memory storage in the brain. Using a multidisciplinary approach to define the synaptic connections between neurons in brain structures involved in learning, BRNI scientists have uncovered molecular pathways that control these synaptic connections. BRNI research has demonstrated that different forms of the enzyme Protein Kinase C (PKC) activate multiple targets during memory acquisition and storage. " In past research they demonstrated that a number of models of learning and memory such as Pavlovian condition of the snaill Hermissenda (Fig. 1), rabbit nictating membrane conditioning, rat spatial maze learning and rat olfactory discrimination learning, all activate PKC by causing it to move from the cell body to the membrane walls of neurons critical for the memory task involved (Fig. 2). Synaptic signals within specific brain areas were found to be amplified by associative learning paradigms that engage these PKC pathways. Some of these synaptic changes involved in memory could then be explained by changes of specific ion channels within the relevant connections between neurons in these specific areas.
Many of the molecular events in these PKC-mediated memory pathways were also shown to be activated only when learning and memory storage occurred. A number of different techniques including genomic microarray analysis, RT-PCR, in situ hybridization (Fig. 3, 4), and immunohistochemical measures demonstrated memory-specific changes of specific proteins and their substrates.
More recently, molecular steps in the PKC pathway implicated at BRNI involve the mRNA stabilizing proteins (Hud, HuC, HuR), the insulin receptor and insulin growth factor, fibroblast growth factor 18, the ryanodine receptor, and, indirectly the cbl-b protein regulator of proteasome degradation. The mRNA stabilizing proteins (also called the ELAV protein class) are a direct target of PKC and control a group of key protein factors that control synaptic development and remodeling. This ELAV-target group includes GAP-43 (growth associated protein), fos, jun, the insulin receptor, and the amyloid precursor protein (or APP).
BRNI scientists also investigate the structural basis of memory acquisition and storage in brain regions critical for mammalian memory such as the hippocampus. Using confocal microscopic visualization of individual hippocampal pyramidal cells and their sites of synaptic contacts, they are measuring memory specific changes of synaptic structure (Fig. 5). These changes were found to be enhanced by the PKC activating drug bryostatin (See Target Indentification and Drug Discovery) and impaired in aged animals with decreased learning ability. Other structurally-specific changes that occur with PKC activation involve movement of the PKC substrate mRNA stabilizing proteins (or ELAV proteins) from the nucleus into the surrounding cell body and into the dendritic tree.
Application to Alzheimer's Disease and Other Neurological Disorders
The molecular pathways of memory described above have been shown by BRNI scientists to converge with molecular pathways important for processing of proteins critical for Alzheimer’s disease pathophysiology. The toxic protein known as beta amyloid or Abeta (Aβ), for example, is generated by enzymatic breakdown by beta-secretase of the Amyloid Precursor Protein (APP). PKC isozymes (α, ε), on the other hand, activate another secretase known as the alpha-secretase to generate the normal breakdown product known as the soluble APP or sAPP. These same PKC isozymes also activate MAP Kinase Erk1/2 that is within the pathways that activate alpha-secretase. Furthermore, PKC α and ε inhibit the enzyme known as glycogen synthase 3-β kinase which is important for phosphorylating the tau protein that contributes to the pathologic Alzheimer’s changes known as “neurofibrillary tangles”. The role of PKC-isozyme pathway dysfunction in the initial causes of Alzheimer’s disease received important support when BRNI scientists demonstrated that the key toxic Alzheimer’s protein Aβ directly inhibits PKC functions. This finding was further confirmed by another laboratory’s demonstration of the structural location on Aβ that directly combines with PKC isozymes.
The direct relevance of these Aβ inhibitory effects on PKC was suggested by BRNI scientists when they first discovered some time ago that Aβ blocked specific potassium channels that were found to be defective in Alzheimer's disease and that were regulated by PKC activators such as bryostatin (See Target Identification and Drug Discovery).
In another related approach, molecular analogues of soluble forms of Aβ activate glutamate receptors in the absence of conventional activators (agonists and co-agonists). These analogues reduced glutamate evoked receptor responses. Furthermore, these effects of soluble oligomer analogues were not observed for other forms such as Aβ monomers or fibrils. Still other recent BRNI studies showed that Dopamine D1 ligands, agonists, and antagonists, directly block NMDA receptors as channel pore blockers. These ligands appear to act like memantine, a NMDA receptor channel blocker, drug currently used clinically for the treatment of Alzheimer's disease.
Finally, BRNI researchers are also exploring molecular mechanisms of pathology seen in other neurodegenerative diseases such as Parkinson’s disease (PD). A team of researchers identified novel, intramolecularly crosslinked species of the α-synuclein monomer, which is present early in PD pathology and represents the major monomeric α-synuclein species in the substantia nigra an area directly affected by PD. The crosslinking is catalyzed by the enzyme tissue transglutaminase (tTG). BRNI scientists also identified tTG-induced crosslinking of collagen in Motor Neuron Disease Inclusion Dementia (MNDID). Other laboratories have reported similar findings with tTG-induced intramolecular crosslinking of the protein tau as a part of early Alzheimer’s disease (AD) pathology. Taken together, these findings suggest that tTG-induced crosslinking might represent a common causal link in multiple neurodegenerative diseases marked by abnormal protein aggregation.
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Zhao W, Cavallaro S, Gusev P, Alkon DL: Nonreceptor tyrosine protein kinase pp60c-src in spatial learning: synapse-specific changes in its gene expression, tyrosine phosphorylation, and protein-protein interactions. Proc Natl Acad Sci USA 97: 8098-8103, 2000.
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Zhao WQ, Chen GH, Chen H, Pascale A, Ravindranath L, Quon MJ, Alkon DL: Secretion of annexin II via activation of insulin receptor and insulin-like growth factor receptor. J Biol Chem. 278:4205-15, 2003.