Laser capture microdissection of the ganglion cell layer for analyzing transcriptional responses induced by demyelination
Demyelination and loss of oligodendrocytes deprives neurons of crucial metabolic and trophic support and contributes to neurodegeneration. However, the precise mechanisms that trigger neurodegeneration during demyelination remain unclear. Here, we examine transcriptional differences in neurons that have been demyelinated relative to myelinated neurons. Demyelination is induced by the knockout of Myrf, a transcription factor essential for myelin gene expression and oligodendrocyte identity, from oligodendrocyte lineage cells using Myrffl/fl Sox10 CreERT mice. We performed laser capture microdissection of the ganglion cell layer, which contains the majority of the retinal ganglion cells (RGC), a nearly ubiquitously myelinated population of neurons. We find that dissection of the ganglion cell layer greatly enriches for RGC transcripts relative to controls.  Demyelinated Myrffl/fl Sox10 CreERT mice have upregulation of Hrk and Ecel1 in the ganglion cell layer, which are known to be upregulated during axonal injury and driven by dual leucine zipper kinase (DLK) signaling. These data suggest that demyelination may activate a DLK-mediated signaling cascade that culminates in transcriptional changes that are often associated with apoptosis of neurons in the central nervous system. Overall design: To understand how demyelination affects neuronal transcription, Myrffl/fl Sox10 CreERT mice were injected with tamoxifen at 8 weeks of age to induce CNS-wide demyelination, including of the visual system. Myrffl/fl littermates were used as controls. Transcriptional analysis was performed on laser-microdissected ganglion cell layer or whole retina at 18 weeks of age. The ganglion cell layer of n=6 Myrffl/fl Sox10 CreERT mice and n=6 Myrffl/fl controls, as well as n=4 whole retinae were sequenced. Comparative gene expression was performed between whole retina and the ganglion cell layer, and between the ganglion cell layer of Myrffl/fl Sox10 CreERT and Myrffl/fl mice.