Nemours Biomedical Research
Axons damage to the central nervous system (CNS) including brain and spinal cord do not spontaneously regenerate and there are currently no effective treatments, resulting in irreversible and permanent neurophysiological dysfunction. Each year, it is estimated that more than 2 million people in the U.S. alone sustain traumatic brain injuries and approximately 12,000 new cases are annually added to an estimated 400,000 patients with spinal cord injuries. By contrast, axonal injury to the peripheral nervous system (PNS) normally shows a robust regenerative response. These spontaneous regenerative responses of peripheral nerves can be experimentally further enhanced by a pre-conditioning injury to the nerve, for which the peripheral nerve has been given an injury prior to being subjected to another injury. De novo protein synthesis is a key component of this enhanced regeneration.
Although most proteins are synthesized in the cell body, our previous study has shown that many mRNAs are differentially localized into axons of neurons to accomplish local and autonomous de novo protein synthesis. Translation of mRNAs directly in the axonal compartment is now known to contribute to axonal growth and regeneration. Studies on axonal mRNAs will provide invaluable insight into the molecular changes that occur in the regenerating nerve. The long-term goal of our laboratory is to uncover the molecular mechanisms that regulate de novo protein synthesis needed to regenerate axons. Determination of mechanisms that lead to regeneration of peripheral nerves will enable development of new strategies to promote axonal regrowth within the CNS.
Neurons are large polarized cells that provide the long-range communication in the nervous system underlying nearly every function of the brain and spinal cord. mRNA localization into and localized translation within the long processes of neurons provide a means for rapid and autonomous responses to stimuli at distances 10–1,000 fold more than the cell body diameter.
For example, protein synthesis in the post-synaptic compartment (i.e., dendritic spines) leads to persistent changes in synaptic connectivity and efficacy, functions that are thought to underlie learning and memory. Protein synthesis in axons of developing neurons is needed for response to guidance cues (i.e., pathfinding) that determines how the developing nervous system is wired.
In contrast, mature vertebrate axons appear to have limited capacity for local protein synthesis. However, axonal injury to adult nerves triggers localized synthesis of new proteins and these new proteins are needed for the retrograde signaling that underlies the cell body's response to injury. Thus, despite that mature axons seem to have limited translational machinery, axonal injury is able to recruit this machinery to synthesize new proteins that are needed for regeneration, including proteins needed for growth cone formation.
Our previous studies have also shown that this axonal protein synthesis is quite robust during the process of regeneration and is needed to maintain the structure of the distal axon. The pith of the concurrent research is to know the exact cellular/molecular mechanisms that result in different responses to injury between the CNS and PNS neurons to cure currently incurable neurological diseases. This information will allow us to manipulate the properties and eventually to prevent or, at least, lessen loss of function resulting from injury to the central nervous system, e.g., paralysis after spinal cord injury, and finally to promote the recovery.
Successful axon regeneration is likely to be accomplished through a sophisticated coordination of gene expression at both transcriptional and post-transcriptional levels, i.e., newly synthesized proteins through changes of gene expression in the cell body and local translation at the injury site, respectively. However, to date, there is no study that has rigorously investigated how such gene-specific regulation in subcellular domains occurs.
Understanding of molecular and cellular mechanisms underlying coordinated regulation of local protein synthesis during regeneration will be required for appreciating the pathophysiological complexity and for developing future therapeutic agents in nerve regeneration of CNS/PNS neurons.
Small non-coding RNAs, including microRNAs (miRNA), have recently been recognized as a prominent player in post-transcriptional regulation of local protein synthesis. What makes miRNAs very interesting, as gene-specific regulators in nervous system, is their enormous diversities in heterogeneity and function.
Since the first discovery of the existence of miRNA lin-4 miRNA and its regulatory role in cell fate determination of the C. elegans larvae in 1993, 2042 miRNAs have been identified in humans and deposited in database (miRBase, Release of 19) and the number is continuously growing. Of those, almost 50 percent are differentially distributed in distinct subareas of the brain as well as in subcellular domains of neurons and are predicted to control ~ 30 percent of all protein coding genes.
Enormously different miRNAs expressed in neurons provide means to simultaneously control many different gene- specific translations. miRNAs also enable many different synapsing axons and dendritic spines on the same neuron to behave differentially in response to extracellular stimuli, suggesting a fine-tuning of gene expression, rather than a simple on-off expression switch.
In addition, a specific miRNA can have hundreds of target mRNAs, but several different miRNAs can also share the same target transcript. These suggest that miRNAs are capable to simultaneously regulate multiple genes in response to one or multiple extracellular cues. The extreme complexity in miRNA-dependent regulation of axonal gene expression could provide an effective way to ensure the tight and precise control of neuronal gene expression required for regenerative program that is reactivated upon injury to axon.
Although it is now certain that the miRNAs localize into axons and play a role in the coordinated regulation of local protein synthesis in regenerating axons, how these non-coding RNAs translocate into distal process of neurons is completely unknown to date. Interestingly, several recent studies show the presence of Dicer and components of the miRNA-induced silencing complex (miRISC) that are required for processing precursor miRNAs (pre-miRNAs) to mature functional miRNAs both in dendrites and axons.
Processing of a pre-miRNA to mature miRNA locally in neuronal processes could confer a unique advantage for coordinately altering the population of proteins generated in growth cones by targeting mRNA cohorts. Therefore, we hypothesize that specificity of axonally transported pre-miRNAs confers spatiotemporal regulation of local protein synthesis in regenerating axons.
Understanding molecular mechanisms of miRNA localization as well as local miRNA biogenesis in neurons will provides the basis for future research of gene expression regulation to promote functional recovery following CNS injury.
Nemours/Alfred I. duPont Hospital for Children
1600 Rockland Road
Wilmington, DE 19803
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