MicroRNAs modulate Schwann cell response to nerve injury by reinforcing transcriptional silencing of dedifferentiation-related genes.

In the peripheral nervous system, Schwann cells (SCs) surrounding damaged axons undergo an injury response that is driven by an intricate transcriptional program and is critical for nerve regeneration. To examine whether these injury-induced changes in SCs are also regulated posttranscriptionally by miRNAs, we performed miRNA expression profiling of mouse sciatic nerve distal segment after crush injury. We also characterized the SC injury response in mice containing SCs with disrupted miRNA processing due to loss of Dicer. We identified 87 miRNAs that were expressed in mouse adult peripheral nerve, 48 of which were dynamically regulated after nerve injury. Most of these injury-regulated SC miRNAs were computationally predicted to inhibit drivers of SC dedifferentiation/proliferation and thereby re-enforce the transcriptional program driving SC remyelination. SCs deficient in miRNAs manifested a delay in the transition between the distinct differentiation states required to support peripheral nerve regeneration. Among the miRNAs expressed in adult mouse SCs, miR-34a and miR-140 were identified as functional regulators of SC dedifferentiation/proliferation and remyelination, respectively. We found that miR-34a interacted with positive regulators of dedifferentiation and proliferation such as Notch1 and Ccnd1 to control cell cycle dynamics in SCs. miR-140 targeted the transcription factor Egr2, a master regulator of myelination, and modulated myelination in DRG/SC cocultures. Together, these results demonstrate that SC miRNAs are important modulators of the SC regenerative response after nerve damage.

Congenital hypomyelinating neuropathy with lethal conduction failure in mice carrying the Egr2 I268N mutation.

Mouse models of human disease are helpful for understanding the pathogenesis of the disorder and ultimately for testing potential therapeutic agents. Here, we describe the engineering and characterization of a mouse carrying the I268N mutation in Egr2, observed in patients with recessively inherited Charcot-Marie-Tooth (CMT) disease type 4E, which is predicted to alter the ability of Egr2 to interact with the Nab transcriptional coregulatory proteins. Mice homozygous for Egr2(I268N) develop a congenital hypomyelinating neuropathy similar to their human counterparts. Egr2(I268N) is expressed at normal levels in developing nerve but is unable to interact with Nab proteins or to properly activate transcription of target genes critical for proper peripheral myelin development. Interestingly, Egr2(I268N/I268N) mutant mice maintain normal weight and have only mild tremor until 2 weeks after birth, at which point they rapidly develop worsening weakness and uniformly die within several days. Nerve electrophysiology revealed conduction block, and neuromuscular junctions showed marked terminal sprouting similar to that seen in animals with pharmacologically induced blockade of action potentials or neuromuscular transmission. These studies describe a unique animal model of CMT, whereby weakness is due to conduction block or neuromuscular junction failure rather than secondary axon loss and demonstrate that the Egr2-Nab complex is critical for proper peripheral nerve myelination.

In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.