Jorge David Miranda, Ph.D. - US grants

The objective of this project is to understand the possible role that eph receptor tyrosine kinases (RPTK) may play during neuronal differentiation. The RN33B cell line differentiates in vivo after transplantation into the neonatal and adult rat brain. This cell line shows neurite outgrowth formation and a morphology typical of endogenous neurons when transplanted in the rat cerebral cortex or hippocampus. The high levels of eph RPTK ligand expression in the central nervous system, along with the expression of at least one eph RPTK ligand expression in the central nervous system, along with expression of at least one eph RTK in RN33B cells, has led us to hypothesize that this protein could mediate the morphogenesis observed in vivo. In addition, sever eph RPTKs have been implicated in axonal targeting, suggesting that this family of RPTKs are involved in the process of neural differentiation. To address the question, we will screen and sequence clones derived from RN##B cells using RT-PCR with degenerate oligonucleotides for the conserved regions of the eph RPTK. Once the type(s) of eph RPTK has been determined, developmental studies at the mRNA and protein level will be done with Northern blotting, Western blotting and immunohistochemistry techniques on RN33B cells in vitro. The possible role of eph RPTK in differentiation will be tested by the use of a dominant negative construct, to inactivate the RN33B cell eph RPTK, and analyze its effect on morphogenesis after transplanted into the neonatal rat brain. The analysis will make a significant contribution to the understanding of neuronal differentiation in vitro and in vivo, when a neural cell line is used as a model system to study differentiation in culture during neural grafting.

The goal of this study is to investigate the role of Eph A receptor protein tyrosine kinases (RPTK) and their ligands (ephrins) in producing a non- permissive environment for axonal regeneration after spinal cord injury (SCI). Molecular biology, protein biochemistry, neuroimaging tracing strategies and behavioral assays will be used in conjunction with injured rats at the T10 level to analyze the expression of these proteins. The first aim will focus on the spatial and temporal expression of Eph A and ephrin A, mRNAs and proteins, after spinal after Central Nervous System (CNS) lesion at the nuclei and amino acid level. The expression pattern will moderate spinal cord contusion, In the second aim, we will determine the possible role of Eph A RPTK and ephrins as repulsive molecular cues that restrict axonal regeneration and functional recovery after SCI. For this study, fusion proteins expressing the amino terminus of the Eph A RPTKs or ephrins A will be temporal and spatial expression of individual Eph A RPTKs and ephrins, both on the regenerating fibers and in the local environment, may lead to novel therapeutic strategies to enhanced regeneration and functional recovery after SCI.

Spinal cord injury (SCI) in adult rats initiates a cascade of events producing a non-permissive environment for axonal regeneration. This non-favorable environment could be due to the expression of repulsive factors like the Eph receptors and their ligands (ephrins). The Eph receptors and their respective ephrins play a major in axonal pathfinding and target recognition during ventral nervous system (CNS) development, and their action is mediated by repellent forces between receptor and ligand. The possible role that these ligands play after CNS trauma is unknown. The goal of this pilot study is to investigate the role of Ephrin B ligands in producing a non-permissive environment for axonal regeneration after SCI. Molecular biology, protein biochemistry, and neuroimaging tracing strategies will be used to analyze the expression and function of these proteins in rats injured at the T10 level.. The first aim will focus on the spatial and temporal expression of ephrin B, mRNAs and proteins, after spinal cord trauma. These experiments will provide temporal and spatial information about ephrin B expression after central nervous system (CNS) lesion at the nucleic and amino acid level. The expression pattern will be correlated both with the lack of regeneration of specific supraspinal pathways and the non-favorable regions for axonal outgrowth and circuit reconnection generated at the lesion site after moderate spinal cord contusion. In the second aim, we will determine the possible role of ephrins B as repulsive molecular cues that restrict axonal regeneration after SCI. For this study, specific polyclonal antibodies against the amino terminus of the ephrin B ligands or fusion proteins expressing the amino terminus of the ephrins B will be administered to contused rats to black the endogenous receptor-ligand interaction. Therefore, defining the specific temporal and spatial expression of individual ephrins B, both on the regenerating fibers and in the local microenvironment, may lead to novel therapeutic strategies to enhanced regeneration and functional recovery after SCI.

Spinal cord injury (SCI) generates a cascade of events that lead to inhibition of axonal regeneration. These molecular and biochemical changes represents the presence of repulsive factors that may restrict or block neurite outgrowth after CNS trauma. Members of the Eph subfamily of receptor tyrosine kinases (RTKs)have been associated with axonal pathfinding, target recognition and synapse formati on during development. It has been shown that these roles are accomplished by repulsive interactions caused after ligands binding. However, the role of EphAs in inhibiting axonal outgrowth in adult injured spinal cord is unknown. The expression of some of these receptors after injury was examined with standardized semi-quantitative RT-PCRanalysis. Results showed that several EphA's RTKs were induced after the injury and this enhanced expression persisted for several days. The expression of the EphA's after SCI were localized by immunocytochemistry and the results indicated that at the lesion epicenter, the immunoreactivity was focused in the lateral and ventral region of the white matter. These results suggest that these EphA's may be involved in the establishment of the non-permissive environment for axonal regeneration after CNS trauma. It is the objective of this proposal to block EphA's gene expression with antisense technology, reducing the nonpermissive environment generated after contusion to the spinal cord. Behavioral (BBB, grid walking, narrow-beam crossing, righting reflex and climbing test), physiological (transcranial magnetic motor evoked potentials) and anatomical tracing studies will be performed to monitor axonal regeneration and functional recovery after blockade of these repulsive proteins in SCI. In addition, neurotrophic factors will be used together with the antisense oligonucleotides to enhance the axonal outgrowth across the injury site. Establishing the EphA recept or roles, both on the regenerating fibers and in the local microenvironment, may lead to novel therapeutic strategies to enhance regeneration and functional recovery after SCI.

Excessive alcohol drinking among human adolescents is a major social and biomedical problem in the United States and Puerto Rico. Moreover, early initiation of alcohol use or misuse leads to greater risk of lifetime alcohol use disorders. Recent human brain imaging studies clearly show that the prefrontal cortex (PFC), which underlies various executive cognitive functions, undergoes extensive structural and functional re-organization from adolescence to adulthood. This is consistent with the notion that heightened synaptic plasticity is a cardinal feature of adolescent brain development. Although usually adaptive and beneficial, heightened plasticity may lead to greater vulnerability to substance abuse. Indeed, the mechanism underlying synaptic plasticity are similar to the mechanisms mediating ethanol dependence. Research performed in animal models is needed because studies involving the administration of alcohol to human adolescents are illegal. We have recently developed an adolescent C57BL/6J (B6) mouse model that shows greater propensity for ethanol drinking behavior. The use of the B6 strain may be especially valuable given its wealth of available genetic information (i.e., Mouse Genome Project). Studies proposed in this application are to combine our 86 adolescent drinking model with in-vivo neurochemical and pharmacological approached that have never been employed during the adolescent period. Our primary objective is to determine the role of extracellular glutamate homeostasis in the PFC and its projections to the nucleus accumbens (NAC). Our working hypothesis is that elevated glutamatergic transmission in the PFC-NAC circuit leads to greater propensity for alcohol drinking during adolescence. We also propose to study the effects of adolescent drinking on dendritic spines in the PFC, which are the major postsynaptic components of glutamatergic synapses. It is anticipated that prefrontal spine plasticity will be severely altered following adolescent alcohol drinking experience. Collectively, these studies will generate new and novel information regarding the role of synaptic glutamate transmission in the PFC-NAC circuitry in mediating adolescent alcohol drinking. This will provide valuable insight into this crucial clinical and social issue, as well as facilitate development of new glutamate- and neuroplasticity-based pharmacotherapies that reduce harmful consequences of alcohol abuse.