The long-term objective of this proposal is to study the role of HuD, a neuronal-specific RNA-binding protein that selectively interacts with GAP-43, during regeneration of axotomized rubrospinal neurons in the mouse. These studies will provide the base knowledge necessary to study molecular events occurring in other motorneuron tracts of the central nervous system after spinal cord injury. The first specific aim is to examine the spatial and temporal expression patterns of HuD protein and GAP-43 mRNA in the red nucleus before and after rubrospinal tractotomy. To accomplish this immunocytochemistry and in situ hybridization will be used on tissue sections of mice subjected to a C3 hemisection of the dorsolateral funiculus compared to sham-operated controls. The second specific aim is to use gene-activating matrices (GAM) to overexpress genes and determine the subsequent response both in the red nucleus and in axotomized axons. To achieve this aim a gene-activating matrix containing HuD cDNA will be implanted into the injury site to transduce cells and examine whether this will promote GAP-43 expression and sprouting of axons across the wound. Concurrently, it will determine if exogenous HuD can undergo retrograde transport to the cell body of an injured neuron and become constitutively expressed, thereby altering endogenous GAP-43 expression. In addition, a GAM containing brain-derived neurotrophic factor (BDNF) cDNA in combination with HuD will be used to amplify and prolong the effects of overexpressing HuD alone. Tract-tracing will be used to determine if regenerating axons sprout into or around the wound site after injury.
The third aim will involve the use of ribonomics technology to identify genes target by HuD and BDNF during regeneration of the red nucleus. These studies have the potential of directly impacting SCI in humans as HuD is endogenously expressed in humans and may be a useful candidate in gene therapy to manipulate the temporal expression of GAP-43 during regeneration.
