The long-term objectives of this project are to understand the molecular mechanisms regulating cytoskeletal gene expression in mammalian neurons after axonal injury, and to determine the molecular locus at which peripheral and central neurons differ in their injury response. Rat dorsal root ganglion (DRG) neurons exhibit robust increases in their steady-state levels of specific tubulin mRNAs and rapid downregulation of neurofilament (NF) mRNA levels after axotomy. In contrast, CNS neurons such as retinal ganglion neurons (RGN) fail to upregulate tubulin mRNA levels after injury and decrease NF mRNAs more slowly. Currently, it is not known whether such differences are due to disparities in genomic activation at the level of transcription or to differences in post- transcriptional mechanisms affecting mRNA stability.
The specific aims of this proposal are to test the following 2 hypotheses. First: The transcription of cytoskeletal genes is differentially affected by axotomy in peripheral (DRG) and central (RGN) neurons. We will test this hypothesis by using intron-specific DNA probes for in situ hybridization to detect primary, unspliced RNA transcripts (""""""""pre-mRNAs"""""""") of NF-M, betaIII and alpha1-tubulin genes in DRG and RGN nuclei in histological sections at various times after axotomy. We will also directly assess the transcription rate of cytoskeletal mRNAs in isolated nuclei from normal and axotomized DRG neurons using nuclear run-off methods. Second: The stability of specific cytoskeletal mRNAs in peripheral and central neurons is differentially affected by axotomy. We will examine differences in the turnover (half-life) of major cytoskeletal mRNAs in axotomized RGN as well as DRG neurons in vivo using a 3/H-uridine pulse-chase paradigm, and using a transcriptional-blockade paradigm involving microinjection of actinomycin D or DRB(5,6-dichloro-1-beta-ribofuranosylbenzimidazole) and monitoring the subsequent loss of mRNAs with time. Finally, to examine the role of protein synthesis and polysome-association in the turnover of cytoskeletal mRNAs in axotomized neurons, we will microinject different translation inhibitors (cycloheximide or puromycin) into the DRG or eye immediately before, or shortly after, axotomy and determine if we can prevent axotomy-induced changes in the levels of specific cytoskeletal mRNAs. The health-relatedness of this proposal is that the information gained will significantly enhance our understanding of the molecular underpinnings of effective regeneration in mammalian neurons, and may enable the development of molecular strategies to enhance regeneration in neurons which fail to regenerate following injury in the future.
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