Myelin basic protein mRNA levels affect myelin sheath dimensions, architecture, plasticity, and density of resident glial cells.

Myelin basic protein (Mbp) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both Mbp protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role Mbp plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk.

Self-labelled encoder-decoder (SLED) for multi-echo gradient echo-based myelin water imaging.

PURPOSE: Reconstruction of high quality myelin water imaging (MWI) maps is challenging, particularly for data acquired using multi-echo gradient echo (mGRE) sequences. A non-linear least squares fitting (NLLS) approach has often been applied for MWI. However, this approach may produce maps with limited detail and, in some cases, sub-optimal signal to noise ratio (SNR), due to the nature of the voxel-wise fitting. In this study, we developed a novel, unsupervised learning method called self-labelled encoder-decoder (SLED) to improve gradient echo-based MWI data fitting. METHODS: Ultra-high resolution, MWI data was collected from five mouse brains with variable levels of myelination, using a mGRE sequence. Imaging data was acquired using a 7T preclinical MRI system. A self-labelled, encoder-decoder network was implemented in TensorFlow for calculation of myelin water fraction (MWF) based on the mGRE signal decay. A simulated MWI phantom was also created to evaluate the performance of MWF estimation. RESULTS: Compared to NLLS, SLED demonstrated improved MWF estimation, in terms of both stability and accuracy in phantom tests. In addition, SLED produced less noisy MWF maps from high resolution MR microscopy images of mouse brain tissue. It specifically resulted in lower noise amplification for all mouse genotypes that were imaged and yielded mean MWF values in white matter ROIs that were highly correlated with those derived from standard NLLS fitting. Lastly, SLED also exhibited higher tolerance to low SNR data. CONCLUSION: Due to its unsupervised and self-labeling nature, SLED offers a unique alternative to analyze gradient echo-based MWI data, providing accurate and stable MWF estimations.

Transcriptional regulators of the Golli/myelin basic protein locus integrate additive and stealth activities.

Myelin is composed of plasma membrane spirally wrapped around axons and compacted into dense sheaths by myelin-associated proteins. Myelin is elaborated by neuroepithelial derived oligodendrocytes in the central nervous system (CNS) and by neural crest derived Schwann cells in the peripheral nervous system (PNS). While some myelin proteins accumulate in only one lineage, myelin basic protein (Mbp) is expressed in both. Overlapping the Mbp gene is Golli, a transcriptional unit that is expressed widely both within and beyond the nervous system. A super-enhancer domain within the Golli/Mbp locus contains multiple enhancers shown previously to drive reporter construct expression specifically in oligodendrocytes or Schwann cells. In order to determine the contribution of each enhancer to the Golli/Mbp expression program, and to reveal if functional interactions occur among them, we derived mouse lines in which they were deleted, either singly or in different combinations, and relative mRNA accumulation was measured at key stages of early development and at maturity. Although super-enhancers have been shown previously to facilitate interaction among their component enhancers, the enhancers investigated here demonstrated largely additive relationships. However, enhancers demonstrating autonomous activity strictly in one lineage, when missing, were found to significantly reduce output in the other, thus revealing cryptic "stealth" activity. Further, in the absence of a key oligodendrocyte enhancer, Golli accumulation was markedly and uniformly attenuated in all cell types investigated. Our observations suggest a model in which enhancer-mediated DNA-looping and potential super-enhancer properties underlie Golli/Mbp regulatory organization.

TIE: A Method to Electroporate Long DNA Templates into Preimplantation Embryos for CRISPR-Cas9 Gene Editing.

Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR components, including long plasmid-sized DNA templates, into the sub-zona pellucida space. There they are retained, supporting subsequent electroporation. We show that this simple and well-tolerated method achieves intracellular reagent concentrations sufficient to effect precise gene edits.