SLOW AXONAL TRANSPORT IN CNS DEVELOPMENT

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A major problem in contemporary medicine is the failure of regrowth of injured CNS axons. Cytoskeletal proteins have a central role in axonal growth both during developmental and after injury. The process of delivery of cytoskeletal elements is vectorial; transcription, translation and assembly occur largely in the cell body and the products are exported to the axon where important posttranslational modifications occur. The cytoskeleton then continually moves by slow axonal transport to the terminal. Following injury, this vectorial process must apply cytoskeletal elements to the growing regions in order for a new axon to form. One of the possible explanations for mammalian CNS regenerative failure is that some aspect of this vectorial process is suboptimal. We propose to continue studies which examine this hypothesis by conducting both longitudinal studies of a CNS system which undergoes a critical period of development in which regeneration fails and by comparative studies that examine injured peripheral neurons. The hamster corticospinal system provides the CNS model since these neurons elaborate axons entirely postnatally and maintain the ability to regenerate after injury for the first 2 postnatal weeks. After that critical period, injury results in regenerative failure and permanent functional loss. We will first examine changes in the mRNA levels of the low and high molecular weight neurofliament proteins, two different beta tubulins and actin during normal development of corticospinal neuronals using quantitative in situ hybridization with cDNA probes. This will provide information on the initial appearance of transcriptional products, clues on the extent to which major cytoskeletal genes are transcriptionally coregulated, and target changes which occur during the critical period for regrowth of this system. Immunochemical studies of developing corticospinal neurons with specific monoclonal antibodies will complement the studies of mRNA changes by examining both the expression and modification of major cytoskeletal proteins. Second, we will axotomize corticospinal neurons at different developmental stages and determine how cytoskeletal gene expression changes using quantitative in situ hybridization with cDNA probes. Immunochemical studies will provide information about the protein products and changes in their posttranslational modifications (such as NF phosphorylation) that result after injury. Third, we will conduct comparative studies of the injury response of the dorsal root ganglion (DRG) cell to determine the molecular changes mounted