Modeling the natural history of Pelizaeus-Merzbacher disease.

Major gaps in our understanding of the leukodystrophies result from their rarity and the lack of tissue for the interdisciplinary studies required to extend our knowledge of the pathophysiology of the diseases. This study details the natural evolution of changes in the CNS of the shaking pup (shp), a model of the classical form of the X-linked disorder Pelizaeus-Merzbacher disease, in particular in glia, myelin, and axons, which is likely representative of what occurs over time in the human disease. The mutation in the proteolipid protein gene, PLP1, leads to a delay in differentiation, increased cell death, and a marked distension of the rough endoplasmic reticulum in oligodendrocytes. However, over time, more oligodendrocytes differentiate and survive in the spinal cord leading to an almost total recovery of myelination, In contrast, the brain remains persistently hypomyelinated. These data suggest that shp oligodendrocytes may be more functional than previously realized and that their early recruitment could have therapeutic value.

Autophagy promotes oligodendrocyte survival and function following dysmyelination in a long-lived myelin mutant.

The Long-Evans shaker (les) rat has a mutation in myelin basic protein that results in severe CNS dysmyelination and subsequent demyelination during development. During this time, les oligodendrocytes accumulate cytoplasmic vesicles, including lysosomes and membrane-bound organelles. However, the mechanism and functional relevance behind these oligodendrocyte abnormalities in les have not been investigated. Using high-magnification electron microscopy, we identified the accumulations in les oligodendrocytes as early and late autophagosomes. Additionally, immunohistochemistry and Western blots showed an increase in autophagy markers in les. However, autophagy did not precede the death of les oligodendrocytes. Instead, upregulating autophagy promoted membrane extensions in les oligodendrocytes in vitro. Furthermore, upregulating autophagy in les rats via intermittent fasting increased the proportion of myelinated axons as well as myelin sheath thickness in les and control rats. Overall, this study provides insight into the abnormalities described in les as well as identifying a novel mechanism that promotes the survival and function of oligodendrocytes.

Spontaneous optic nerve compression in the osteopetrotic (op/op) mouse: a novel model of myelination failure.

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.

A mutation in the canine gene encoding folliculin-interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination.

Novel mutations in myelin and myelin-associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin-interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract-specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome-wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes.

Characterization of a PLP-overexpressing transgenic rat, a model for the connatal form of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) most frequently results from duplication of the Plp1 gene with a correlation between disease severity and increasing copy number of the gene. Animal models of PMD, in particular those overexpressing the Plp1 gene, have been sought in attempts to provide systems in which potential therapies can be tested. Here we describe a rat model of the severe connatal form of PMD and provide a detailed characterization of its pathology and molecular biology, prior to testing therapeutic approaches. We determined the exact copy number of Plp1, and the resulting effects on RNA and protein expression. Distinct differences in myelin and disparate distributions of myelin protein markers in comparison to wild-type controls were observed. Altered expression of Plp1 also caused an increase in the apoptotic cell death of oligodendrocytes. These results provide the platform from which to test the effectiveness of in vivo therapies.

Assembly properties of the Bacillus subtilis actin, MreB.

The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, it does not require a bound nucleotide for assembly and polymerizes indistinguishably regardless of the nucleotide species bound, with a critical concentration of approximately 900 nM. A number of the presently reported properties of B. subtilis MreB differ significantly from those of T. maritima MreB1 (Bean and Amann [2008]: Biochemistry 47: 826-835), including the nucleotide requirements and temperature and ionic effects on polymerization state. These observations collectively suggest that additional factors interact with MreB to account for its complex dynamic behavior in cells.

Myelin and oligodendrocyte development in the canine spinal cord.

We studied the developmental pattern of oligodendrocyte differentiation and myelin formation in the fetal canine spinal cord from E40 to P0. The pattern of development matches what has been described in the spinal cord of humans, rodents, and many other species. Oligodendrocytes were first found at E40, close to the central canal, with their spread in a tangential manner to the ventral and then lateral columns. Myelin development followed the same pattern but was not seen until E46. A clear subpial zone lacking glial cells and myelin was seen in the lateral column in early development, suggesting that there may also be a radial component of migration of oligodendrocyte progenitor cells (OPCs) from a ventral site. This spatial-temporal developmental pattern seen in wild type matches a delay in myelination of the superficial tracts of the spinal cord seen in a canine myelin mutant, suggesting that the mutation prevents the distribution and differentiation of OPCs at an early, but narrow, window of time during fetal development.

Modeling the Chronic Loss of Optic Nerve Axons and the Effects on the Retinal Nerve Fiber Layer Structure in Primary Disorder of Myelin.

PURPOSE: We determined whether the chronic lack of optic nerve myelination and subsequent axon loss is associated with optical coherence tomography (OCT) changes in the retinal nerve fiber layer (RNFL), and whether this models what occurs in multiple sclerosis (MS) and confers its use as a surrogate marker for axon degeneration. METHODS: Using an animal model of Pelizaeus-Merzbacher disease (shp) bilateral longitudinal measurements of the peripapillary RNFL (spectral-domain OCT), electroretinograms (ERG), and visual evoked potentials (VEP) were performed in affected and control animals from 5 months to 2 years and in individual animals at single time points. Light and electron microscopy of the optic nerve and retina and histomorphometric measurements of the RNFL were compared to OCT data. RESULTS: Of the shp animals, 17% had an average reduction of OCT RNFL thickness on the superior retinal quadrant compared to controls (P < 0.05). Electroretinograms showed normal photopic A- and B-waves but flash VEPs were disorganized in shp animals. Morphologically, the shp retinas and optic nerves revealed significant RNFL thinning (P < 0.001) without retinal ganglion cell (RGC) loss, decrease total and relative retinal axonal area, and loss of optic nerve axons. There was strong positive correlation between OCT and morphometric RNFL thickness measurements (r = 0.878, P = 0.004). CONCLUSION: The loss of optic nerve axons demonstrated in the shp model resulted in moderate thinning of the RNFL confirmed by OCT and histology. These results indicate that OCT-derived RNFL measurement can be a useful surrogate biomarker of optic nerve axon loss and potentially disease progression in demyelinating diseases.