The mammalian central nervous system does not regenerate after injury. An important contributing factor is the presence in myelin of inhibitors of axonal regeneration, including the potent inhibitor myelin-associated glycoprotein (MAG). Recently, we made the novel observation that if neurons are exposed to neurotrophins (primed) before they encounter MAP or myelin, inhibition of axonal growth is COMPLETELY blocked. This finding is supported by results in vivo of our collaborator. Dr. Bregman, who showed that primed neurons extend axons into white matter. We have shown that neurotrophins (NTs) that block MAG/myelin's inhibition increase neuronal cAMP and activate protein kinase A (PKA). This important observation is the first to suggest a general mechanism-elevate [cAMPi]- to overcome all myelin inhibitors and thus establish conditions for CNS regeneration. Now, in collaboration with Dr. Bregman we will systematically test this hypothesis in vivo. In her protocol, rat spinal cord is transected, and embryonic tissue is implanted at the lesion site with a pump to deliver NTs.
In Aim #1 we extend the model to a) test if the cAMP analogue, dibutryl cAMP, leads to even better regeneration; b) test in vivo the specific NTs we have shown to be most effective in vitro; and c) begin to dissect the downstream signals involved in the cAMP-dependent block of MAG/myelin inhibition. For the CREB mutants and 2) by expressing constitutively active CREB in neurons.
In Aims #2 &3, an adenovirus delivery will be developed to introduce cDNAs to express either CREB- related proteins or NTs in primary neurons to test their effects on inhibition by MAP/myelin.
In Aim #4, we will use recombinant adenoviruses to express NTs or CREB-related proteins in vivo. The methodological reasoning is that adenovirus permits injection into either the transplanted embryonic tissue or directly to the proximal end of the transfected spinal cord. Growing axons will thereby be exposed to NTs secreted by infected cells before encountering myelin inhibitors in the host. Adenovirus delivery of CREB-related cDNAs can also infect severed axons for retrograde transport to the neuronal cell body. In summary, these studies will allow us, first to extend our in vitro observations on inhibition of axonal regeneration to an in vivo model. Second, we will bring the important experimental approaches of animal model of spinal cord regeneration as well as viral vector technology to Hunter College to significantly strengthen the Neuroscience Research Program.
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