The effect of Jun dimerization on neurite outgrowth and motif binding.

Axon regeneration is a necessary step toward functional recovery after spinal cord injury. The AP-1 transcription factor c-Jun has long been known to play an important role in directing the transcriptional response of Dorsal Root Ganglion (DRG) neurons to peripheral axotomy that results in successful axon regeneration. Here we performed ChIPseq for Jun in mouse DRG neurons after a sciatic nerve crush or sham surgery in order to measure the changes in Jun's DNA binding in response to peripheral axotomy. We found that the majority of Jun's injury-responsive changes in DNA binding occur at putative enhancer elements, rather than proximal to transcription start sites. We also used a series of single polypeptide chain tandem transcription factors to test the effects of different Jun-containing dimers on neurite outgrowth in DRG, cortical and hippocampal neurons. These experiments demonstrated that dimers composed of Jun and Atf3 promoted neurite outgrowth in rat CNS neurons as well as mouse DRG neurons. Our work provides new insight into the mechanisms underlying Jun's role in axon regeneration.

Injury type-dependent differentiation of NG2 glia into heterogeneous astrocytes.

The glial scar is comprised of a heterogeneous population of reactive astrocytes. NG2 glial cells (also known as oligodendrocyte progenitor cells or polydendrocytes) may contribute to this heterogeneity by differentiating into astrocytes in the injured CNS, but there have been conflicting reports about whether astrocytes comprise a significant portion of the NG2 cell lineage. By using genetic fate mapping after spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) in mice, the goal of this study was to confirm and extend upon previous findings, which have shown that NG2 cell plasticity varies across CNS injuries. We generated mice that express tdTomato in NG2 lineage cells and express GFP under the Aldh1l1 or Glt1 promoter so that NG2 glia-derived astrocytes can be detected by their expression of GFAP and/or GFP. We found that astrocytes comprise approximately 25% of the total NG2 cell lineage in the glial scar by 4 weeks after mid-thoracic contusive SCI, but only 9% by the peak of functional deficit after EAE. Interestingly, a subpopulation of astrocytes expressed only GFP without co-expression of GFAP, uncovering their heterogeneity and the possibility of an underestimation of NG2 glia-derived astrocytes in previous studies. Additionally, we used high performance liquid chromatography to measure the level of tamoxifen and its metabolites in the spinal cord and show that genetic labeling of NG2 glia-derived astrocytes is not an artifact of residual tamoxifen. Overall, our data demonstrate that a heterogeneous population of astrocytes are derived from NG2 glia in an injury type-dependent manner.

Enhanced Transcriptional Activity and Mitochondrial Localization of STAT3 Co-induce Axon Regrowth in the Adult Central Nervous System.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor central to axon regrowth with an enigmatic ability to act in different subcellular regions independently of its transcriptional roles. However, its roles in mature CNS neurons remain unclear. Here, we show that along with nuclear translocation, STAT3 translocates to mitochondria in mature CNS neurons upon cytokine stimulation. Loss- and gain-of-function studies using knockout mice and viral expression of various STAT3 mutants demonstrate that STAT3's transcriptional function is indispensable for CNS axon regrowth, whereas mitochondrial STAT3 enhances bioenergetics and further potentiates regrowth. STAT3's localization, functions, and growth-promoting effects are regulated by mitogen-activated protein kinase kinase (MEK), an effect further enhanced by Pten deletion, leading to extensive axon regrowth in the mouse optic pathway and spinal cord. These results highlight CNS neuronal dependence on STAT3 transcriptional activity, with mitochondrial STAT3 providing ancillary roles, and illustrate a critical contribution for MEK in enhancing diverse STAT3 functions and axon regrowth.

Hyperactivated Stat3 boosts axon regeneration in the CNS.

Axonal regeneration after spinal cord injury (SCI) is intrinsically and extrinsically inhibited by multiple factors. One major factor contributing to intrinsic regeneration failure is the inability of mature neurons in the central nervous system (CNS) to activate regeneration-associated transcription factors (TFs) post-injury. A prior study identified TFs overexpressed in neurons of the peripheral nervous system (PNS) compared to the CNS; some of these could be involved in the ability of PNS neurons to regenerate. Of these, signal transducer and activator of transcription 3 (STAT3), as well its downstream regeneration-associated targets, showed a significant upregulation in PNS neurons relative to CNS neurons, and a constitutively active variant of Stat3 (Stat3CA) promoted neurite growth when expressed in cerebellar neurons (Lerch et al., 2012; Smith et al., 2011). To further enhance STAT3's neurite outgrowth enhancing activity, Stat3CA was fused with a viral activation domain (VP16). VP16 hyperactivates TFs by recruiting transcriptional co-factors to the DNA binding domain (Hirai et al., 2010). Overexpression of this VP16-Stat3CA chimera in primary cortical neurons led to a significant increase of neurite outgrowth as well as Stat3 transcriptional activity in vitro. Furthermore, in vivo transduction of retinal ganglion cells (RGCs) with AAV constructs expressing VP16-Stat3CA resulted in regeneration of optic nerve axons after injury, to a greater degree than for those expressing Stat3CA alone. These findings confirm and extend the concept that overexpression of hyperactivated transcription factors identified as functioning in PNS regeneration can promote axon regeneration in the CNS.

Expression profile of G-protein ßγ subunit gene transcripts in the mouse olfactory sensory epithelia.

Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gßγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gß and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Our reverse transcriptase PCR (RT-PCR) and realtime qPCR analyses of all known Gß (ß1,2,3,4,5) and Gγ (γ1,2,2t,3,4,5,7,8,10,11,12,13) subunits indicate presence of multiple Gß and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH) experiments, which reveal the expression patterns of two Gß subunits and four Gγ subunits in the MOE as well as one Gß and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gßγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs), while Gß1 is present in both mature and immature OSNs. Interestingly, we also found Gß1 to be the dominant Gß subunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3, and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gßγ gene transcripts in three post-natal developmental stages (p0, 7, and 14) and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gß and Gγ in sensory neurons of the olfactory system.