Activating Transcription Factor 3 (ATF3) is a Highly Conserved Pro-regenerative Transcription Factor in the Vertebrate Nervous System.

The vertebrate nervous system exhibits dramatic variability in regenerative capacity across species and neuronal populations. For example, while the mammalian central nervous system (CNS) is limited in its regenerative capacity, the CNS of many other vertebrates readily regenerates after injury, as does the peripheral nervous system (PNS) of mammals. Comparing molecular responses across species and tissues can therefore provide valuable insights into both conserved and distinct mechanisms of successful regeneration. One gene that is emerging as a conserved pro-regenerative factor across vertebrates is activating transcription factor 3 (ATF3), which has long been associated with tissue trauma. A growing number of studies indicate that ATF3 may actively promote neuronal axon regrowth and regeneration in species ranging from lampreys to mammals. Here, we review data on the structural and functional conservation of ATF3 protein across species. Comparing RNA expression data across species that exhibit different abilities to regenerate their nervous system following traumatic nerve injury reveals that ATF3 is consistently induced in neurons within the first few days after injury. Genetic deletion or knockdown of ATF3 expression has been shown in mouse and zebrafish, respectively, to reduce axon regeneration, while inducing ATF3 promotes axon sprouting, regrowth, or regeneration. Thus, we propose that ATF3 may be an evolutionarily conserved regulator of neuronal regeneration. Identifying downstream effectors of ATF3 will be a critical next step in understanding the molecular basis of vertebrate CNS regeneration.

Quantitative Assessment of Neurite Outgrowth Over Growth Promoting or Inhibitory Substrates.

The use of sensory neurons and assessment of neurite outgrowth in vitro is an important part of understanding neuronal development and plasticity. Cultures of rat dorsal root ganglion (DRG) neurons provide quantitative results very quickly and, when grown on growth promoting or inhibitory substrates, can be utilized to study axonal growth, neurotrophic dependence, and structure and function of growth cones. Since we are interested in axon regeneration and targeting, we have sought to promote neurite outgrowth by refining the techniques of growing DRG neurons in culture. This chapter describes detailed methods for the dissection and purification of DRG neurons and quantitative assessment of neurite on promoting or inhibitory substrates.

Compartmentalized Neuronal Culture for Viral Transport Research.

Neuron-invading viruses usually enter via the peripheral organs/tissues of their mammalian hosts and are transported to the neurons. Virus trafficking is critical for transport or spread within the nervous system. Primary culture of neurons is a valuable and indispensable method for neurobiological research, allowing researchers to investigate basic mechanisms of diverse neuronal functions as well as retrograde and anterograde virus transport in neuronal axons. Primary ganglion sensory neurons from mice can be cultured in a compartmentalized culture device, which allows spatial fluidic separation of cell bodies and distal axons. These neurons serve as an important model for investigating the transport of viruses between the neuronal soma and distal axons. Alphaherpesviruses are fascinating and important human and animal pathogens, they replicate and establish lifelong latent infection in the peripheral nervous system, the mechanism of the viral transport along the axon is the key to understand the virus spread in the nervous system. In this review, we briefly introduce and evaluate the most frequently used compartmentalization tools in viral transport research, with particular emphasis on alphaherpesviruses.

Chronic morphine regulates TRPM8 channels via MOR-PKCß signaling.

Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCß), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCß-mediated reduction of TRPM8 desensitization. This MOR-PKCß dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCß as important targets for opioid-induced cold hypersensitivity.