Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease.

Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD)1,2. Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD1,3-5. Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1jp) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.

Initiation of apoptosis in the developing avian outflow tract myocardium.

Apoptosis occurs within the cardiac outflow tract (OFT) myocardium during normal development of chick hearts. This peak of apoptosis occurs at stage 30-31 and coincides with dramatic remodeling of the OFT, suggesting that apoptosis occurs to allow proper alignment of the great vessels over their respective ventricles. The signals that initiate apoptosis in this setting are unknown. The aim of this study was to characterize the cells undergoing apoptosis in the cardiac OFT myocardium and the cells that may influence this process. Two cell populations that may initiate apoptosis of the cardiomyocytes are the cardiac neural crest (CNC) cells and epicardial cells. We examined stage 30-31 chick embryos that had undergone removal of the CNC cells or had delayed epicardial growth for alterations of apoptosis. Removal of the CNC cells did not reduce the levels or pattern of apoptosis in the OFT myocardium. In contrast, impeding the growth of the epicardium over the OFT resulted in a 57% reduction in apoptotic cells in the OFT myocardium. Analysis of the apoptotic cells within the OFT myocardium showed that as many as 92% of them expressed cardiomyocyte markers. In the quail, the endothelial marker QH1 identified a component from the epicardium, endothelial cells, in regions where apoptosis is elevated in the OFT myocardium. These results suggest that a component from the epicardium, possibly endothelial cells, is required for the initiation of apoptosis in OFT cardiomyocytes.

The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.