Homeobox gene expression in adult dorsal root ganglia: Is regeneration a recapitulation of development?

Neurons of the peripheral nervous system are able to regenerate their peripheral axons after injury, leading to complete recovery of sensory and motor function. The sciatic nerve crush model is frequently used to study peripheral nerve regeneration. Sensory neurons in the dorsal root ganglia (DRGs) corresponding to the sciatic nerve undergo alterations in their gene expression in response to nerve injury, enabling the cells to downregulate their functional mode and to induce their growing mode. Many of the proteins that are reinduced participate in the development of the peripheral nervous system. Therefore, it has been hypothesized that developmental processes are recapitulated during regeneration. The regulation of transcription factor expression in adult DRG neurons is likely to underlie the molecular mechanisms of regeneration-associated gene expression alterations in injured neurons. Homeobox genes were likely candidates because of their functions in neural development. Indeed, it is proven that Schwann cells surrounding the distal nerve fibers repeat their developmental program after nerve injury by reexpressing developmentally expressed homeobox genes. In this thesis, we aimed to use homeobox gene expression as a tool to address the question whether the molecular mechanisms of DRG neuron regeneration recapitulate developmental mechanisms. The studies were initiated with a PCR-based screen for homeobox gene expression in the adult rat DRGs in order to make an inventory of the homeobox gene repertoire used by this tissue. Twenty-two homeobox genes were identified. Selected homeobox genes were quantified in DRGs after sciatic nerve crush in the C57BL/6J mouse strain, which we first characterized both at the functional and at the molecular level. We found that regenerating DRG neurons do not recapitulate their developmental expression of four homeobox genes: DRG11, Isl1, Lmx1b and Pax3. Two of these, DRG11 and Pax3, have known functions in DRG neuronal differentiation, outgrowth and survival during embryonic development. Isl1 is likely to be involved in developmental processes like outgrowth, pathfinding and neuroendocrine phenotype. From the lack of a recapitulation of developmental homeobox gene expression it follows that the transcriptional mechanisms for DRG neuronal differentiation, outgrowth and survival are different between regenerating and developing DRG neurons. Our data are supported by the recent finding that adult DRG neurons utilize neuroactive cytokines rather than neurotrophins for regenerative outgrowth and survival. Transcription factors other than homeobox genes that are induced during regeneration also reflect a difference between regenerating and developing DRGs. We conclude that the molecular mechanisms of regeneration reflect adult gene regulation rather than developmentally controlled gene expression. Differences in the neuronal environment implicate different signaling molecules and different signal transduction, leading to a different gene expression program in regenerating as opposed to developing DRG neurons. In future studies, further unraveling of the signal transduction pathways in regenerating DRG neurons should provide more insight into those mechanisms that are regeneration-specific. This may result in novel strategies to promote the regeneration process

Homeobox gene expression in adult dorsal root ganglia during sciatic nerve regeneration: is regeneration a recapitulation of development?

After damage of the sciatic nerve, a regeneration process is initiated. Neurons in the dorsal root ganglion regrow their axons and functional connections. The molecular mechanisms of this neuronal regenerative process have remained elusive, but a relationship with developmental processes has been conceived. This chapter discusses the applicability of the developmental hypothesis of regeneration to the dorsal root ganglion; this hypothesis states that regeneration of dorsal root ganglion neurons is a recapitulation of development. We present data on changes in gene expression upon sciatic nerve damage, and the expression and function of homeobox genes. This class of transcription factors plays a role in neuronal development. Based on these data, it is concluded that the hypothesis does not hold for dorsal root ganglion neurons, and that regeneration-specific mechanisms exist. Cytokines and the associated Jak/STAT (janus kinase/signal transducer and activator of transcription) signal transduction pathway emerge as constituents of a regeneration-specific mechanism. This mechanism may be the basis of pharmacological strategies to stimulate regeneration

High glucose enhances thrombin responses via protease-activated receptor-4 in human vascular smooth muscle cells

Diabetes is associated with vascular remodeling and increased thrombin generation. Thrombin promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3, and PAR-4. We investigated the effect of high glucose on expression and function of vascular thrombin receptors.In human vascular SMCs, high glucose (25 versus 5.5 mmol/L) induced a rapid and sustained increase in PAR-4 mRNA, protein, and cell surface expression. PAR-1 and PAR-3 expression were not changed. High glucose pretreatment (48 hours) enhanced thrombin or PAR-4-activating peptide but not PAR-1-activating peptide evoked intracellular calcium mobilization, migration, and tumor necrosis factor α gene expression. This enhancement of thrombin-stimulated migration and gene expression by high glucose was abolished by endogenous PAR-4 knockdown. PAR-4 regulation was prevented by inhibition of protein kinase (PK)C-β and -δ isoforms or nuclear factor (NF)κB. Nuclear translocation of NFκB in high glucose-stimulated SMCs led to PKC-dependent NFκB binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Furthermore, in situ hybridization and immunohistochemistry confirmed high abundance of PAR-4 in human diabetic vessels as compared with nondiabetic vessels.High glucose enhances SMC responsiveness to thrombin through transcriptional upregulation of PAR-4, mediated via PKC-β, -δ, and NFκB. This may play an important role in the vascular complications of diabetes