Linda L. Phillips, PhD - US grants

The experiments of this proposal are designed to identify the proteins produced within the neuropil of the dentate gyrus during periods of neuronal sprouting and synapse growth. The long term objective of this approach is to define some of the molecular processes that may be involved in synaptogenesis during normal development and in response to lesion. Proposed experiments are based on four discoveries about the model system (rat's dentate gyrus): 1) 3H-leucine incorporation into protein increases over the denervated dentate neuropil concurrent with synapse formation, 2) most of the polyribosomes in the dentate neuropil are associated with dendritic spines, 3) the number of spine associated polyribosomes is high during developmental synaptogenesis, decreasing as the synapse matures and 4) the incidence of spine associated polyribosomes increases during reactive synaptogenesis. The working hypotheses are: A) local synthesis of proteins within the neuropil plays a key role in regulation of synapse growth, and B) part of the local synthesis is carried out by the polyribosomes that are selectively positioned beneath synapses. We will begin to address these hypotheses using in situ hybridization to determine the distribution of selected mRNAs within the dentate gyrus during synapse formation and reinnervation. As a second index of local synthesis we will determine tissue levels of the same proteins within dissected neuropil and cell body layers. Finally, using the hippocampal slice preparation we will radiolabel polypeptides made in the dentate neuropil and then apply standard biochemical methods (i.e., 1 and 2 dimensional gel electrophoresis and immunoblotting) to characterize the newly synthesized proteins. This approach will tell us: 1) whether mRNAs for major cytoskeletal and synaptic proteins vary as a function of reactive synaptogenesis, 2) if these are regional differences in the level of these proteins within the granule neurons and 3) what profile of proteins are made locally within the dentate neuropil and how these compare to those synthesized in the cell body.

[unreadable] DESCRIPTION (provided by applicant): The extent of long-term functional recovery following traumatic brain injury (TBI) is clearly associated with the capacity for adaptive synaptic plasticity within injured circuitry. Recent evidence supports a role for extracellular matrix proteins (ECMs) and their regulatory metalloproteinases (MMPs) in neurite growth and synaptic reorganization after CNS trauma. Given that these molecules are found within brain regions vulnerable to TBI, we have begun to examine members of the gelatinase and stromelysin MMP families during injury-induced synaptic plasticity. We hypothesize that the interaction of MMPs and their ECM substrates during synaptic reorganization determines the success of long-term recovery following TBI. Specifically, we posit that MMPs control ECM dissociation during removal of degenerating terminals, and subsequently regulate distribution of ECM associated molecules involved with synaptogenesis. Our approach will first identify the spatio-temporal pattern of MMP expression and functional activity after unilateral entorhinal lesion (UEC), an insult which induces the well-defined process of reactive synaptogenesis within the hippocampus. The neuronal plasticity induced in this model results in adaptive restoration of synaptic structure and function. With the UEC pattern as a baseline for comparison, we will profile MMP expression and function after brain trauma using the rat TBI model which combines excessive neuroexcitation of percussive injury with targeted hippocampal deafferentation of entorhinal lesion (TBI+BEC insult). We have shown that this model reliably produces a persistent, maladaptive synaptic plasticity and severe long-term cognitive deficits. Initially, we will examine both protein (LM/EM immunohistochemistry, Western blots) and mRNA (RT-PCR, Northern blot and/or in situ hybridization) expression for select MMPs (gelatinases A and B; stromelysin) and their associated ECM substrates (collagenase IV, chondroitin sulfated proteoglycan, enascin) after injury. Additional experiments will determine how effects on protein and mRNA are correlated with MMP enzyme activity (gel zymography and chromogenic enzyme assay). Next, we will establish whether these injury-induced changes in MMPs/ECMs are associated with alterations in electrophysiological measures of synaptic plasticity (LTP, paired-pulse facilitation, current-source-density analysis) and changes in cognitive outcome (Morris Water Maze performance). Finally we will test the association between MMPs and synaptic reorganization following TBI by: 1) applying specific MMP inhibitors and assessing the extent of synaptic plasticity generated, and 2) enhancing injury-induced plasticity with compounds targeting NMDA and dopamine receptors and then assessing MMP expression and functional activity. Together, these studies will establish whether or not MMPs play a role in regenerative processes evoked by TBI and potentially identify novel therapies for brain trauma victims.

DESCRIPTION (provided by applicant): Millions of Americans are victims of traumatic brain injury (TBI), making it a serious health challenge. TBI often involves significant axonal injury with attendant neuronal deafferentation and synaptic loss. While the brain has an inherent capacity for synaptic reorganization, this plasticity often fails after TBI, resulting in serious functional deficits. We have examined the role of extracellular matrix (ECM) proteins during reactive synaptogenesis induced by TBI. Certain ECM proteins and their regulatory matrix metalloproteinases (MMPs) appear to influence the extent of recovery achieved after TBI. Our initial studies contrasted ECM/MMP response in a recovering adaptive injury (unilateral entorhinal cortical lesion or UEC) and a non-recovering maladaptive insult (fluid percussion TBI + bilateral entorhinal cortical lesion or TBI+BEC). Two gelatinases (MMPs 2,9), stromelysin-1 (MMP 3) and the membrane bound MT-5 MMP all showed change in expression and function which correlated with different phases of reactive synaptogenesis. ECM shifts in agrin, tenascin, RPTP-2 and N-cadherin occurred during postinjury recovery as well. We also found that aberrant MMP activation was linked with failed plasticity after TBI+BEC. When MMP inhibitors were applied, plasticity was altered either positively or negatively depending upon dosing and complexity of injury. Given these results, we now posit that specific matrix enzyme/substrate pairs mediate different phases of pre and postsynaptic recovery, as well as the subsequent synapse stabilization. By contrasting profiles of these matrix proteins after adaptive UEC and maladaptive TBI+BEC, we will determine if individual protein dysfunction at specific phases of synaptogenesis can account for the extent of recovery achieved. We will test the facilitative role of MMP3/agrin during the axonal sprouting phase of synaptic recovery, tenascin/RPTP-2/2-catenin during the synaptogenic period, and MT-5MMP/N-cadherin at synapse maturation. For each molecule we will document protein/mRNA expression, synaptic distribution and, when applicable, binding interactions. Finally, we will manipulate MMP3, RPTP-2 and MT-5MMP by either pharmacological inhibition or siRNA knockdown and examine recovery using structural and physiological indices of synaptic plasticity. These studies will better define matrix role in TBI-induced synaptic plasticity and provide new treatment strategies. PUBLIC HEALTH RELEVANCE: Common feature of head injury is poor recovery of damaged brain connections. The proposed studies will determine the role of extracellular matrix proteins during this recovery and identify matrix manipulations which would improve recovery.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) results in long term debilitation for millions of Americans. A consistent feature of this pathology is traumatic axonal injury (TAI), diffuse in profile and challenging to study. To date, TAI etiology is best documented in large caliber, myelinated axons, which consistently exhibit plasmalemmal, cytoskeletal and mitochondria! pathology. From diagnostic imaging it is clear that the corpus callosum and other subcortical white matter tracts are vulnerable to TAI. These pathways contain both large myelinated and small unmyelinated axons, where fiber type is systematically associated with sensorimotor and associative cortices. Given that TBI induces long-term cognitive deficits, and that TAI in subcortical white matter is poorly understood, our recently published studies have explored the physiological and morphological response of corpus callosum axons to diffuse TBI. Those results show rapid evolution of TAI and greater vulnerability of unmyelinated fibers. In parallel, pilot studies we also find upregulation of extracellular matrix (ECM) proteins and their regulatory metalloproteinases (MMPs) with callosal TAI. Such observations are consistent with the fact that these proteins mediate axonal growth, myelination and fasciculation. Based upon this information, we propose to explore the role of MMP/ECM pathways during degenerative and recovery phases of TAI. We will test the hypothesis that MMP/ECM proteins influence the progression of white matter TAI following TBI and that their activation is regulated by fibrinolytic proteins which permeate the neuropil through breaches in the blood brain barrier. Using the callosal TAI model, we will document protein/mRNA expression of tenascin and phosphacan, ECM proteins known to be present in white matter, and MMPs 2, 3 and 9, known to modify ECM after injury. Temporal profile of MMP activity will be correlated with enzyme expression and screening for adhesion molecule binding partners of these proteins will be performed. Next, we will similarly assess the response of fibrinolytic pathway proteins tPA and plasminogen to callosal TAI. Finally, we will pharmacologically manipulate either fibrinolytic/MMP enzyme activity or degree of axonal protection after injury and test for cause/effect relationships between fibrinolytic/MMP molecules and axonal integrity using functional and morphological endpoints. These studies will establish the role of MMP/ECM proteins in TAI and provide new therapeutic targets forTBI.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a significant U.S. health concern, with over 1.7 million new cases each year. Loss of brain circuitry underlies persistent cognitive deficits suffered by its victims, with axonal damage as a major contributor. Using the injured corpus callosum (CC) as a model, we have shown that postinjury change in matrix metalloproteinases (MMPs) is correlated with distinct myelinated and unmyelinated fiber pathology. MMPs are critical modulators of brain extracellular matrix (ECM) which affect axonal integrity. We found that CC gelatinases MMP2 and 9 peak in activity at different postinjury intervals which are marked by reactive glial response. We also observed that this MMP activity was temporally correlated with unmyelinated axon pathology. Treatment with neuroprotective compounds FK-506 and minocycline resulted in selective, time- dependent reduction of this gelatinase activity and reduced deficits in CC compound action potentials(CAPs). Pilot microarray studies revealed that osteopontin (OPN), a cytokine secreted into the ECM and reciprocally linked to MMP function, was significantly upregulated in injured CC. From these data we hypothesize that gelatinase response to traumatic axonal injury is mediated through acute activation of OPN within reactive glia. We also posit that the time course and glial role in this pathway will differ between unmyelinated and myelinated fibers. To test these hypotheses, the following aims will be explored in the fluid percussion model of TBI : 1) to document OPN/MMP2,9 during axonal injury within fiber environments enriched in unmyelinated (ON, olfactory nerve) and myelinated fibers (IC, internal capsule), and determine if OPN KO and MMP9KO alters these changes, 2) to dissect cell specific OPN/MMP2,9 interaction in vitro using primary CC and ON glial cultures, and 3) to test whether OPNKO or MMPKO alters efficacy of axonal neuroprotective drugs FK-506 and minocycline, then determine if combining optimal drug and OPN/MMP9 manipulation alters functional or structural outcome in the rat model of FPTBI. These studies are likely to identify novel options for regional and fiber targeted therapy in patients suffering from axonal damage after TBI.