Phenotypic analysis of rumpshaker mutation on two different genetic backgrounds

rumpshaker (rsh) is a recessive X-linked point mutation (Ile186Thr) which produces dysmyehnation in the murine central nervous system. This study compared the behavioural and pathological aspects of the mutation on the C57BL/6 background (C57 rsh) to those on the C3H background (C3H rsh). C3H rsh is more mildly affected behaviourally and pathologically than C57 rsh. C3H rsh has normal longevity and is also able to reproduce successfully. In contrast, C57 rsh develops seizures at about postnatal day (P25) and dies between 4-5 weeks. Both C3H rsh and C57 rsh fail to myelinate properly and develop dysmyelination in the CNS with the C57 rsh being more severely affected. This feature coincides with stages of active myelinogenesis. Western blot analyses strongly supports this finding as the levels of major myelin proteins are considerably reduced in the C57 rsh compared with C3H rsh. There are significant differences between the mutants and their normal littermates on both genetic backgrounds and between the two mutants themselves. The total glial density is notably increased in the C57 rsh which is coupled with an increase in dead cells in the cervical spinal cord white matter. In vivo BrdU labelling shows that proliferation rates and the number of dividing cells are markedly greater in the C57 rsh. In spite of the differences between the two strains, C3H rsh and C57 rsh both maintain the numbers of oligodendrocytes. Nonetheless, the activated macrophage/microglial cells are more marked in C57 rsh CNS white matter than C3H rsh. Surprisingly, the actual number of astrocytes remained unchanged, however, their activation is represnted by an increase in GFAP, as shown by immunostaining and western immunoblotting. This study suggests that there is no direct causal link between the dysmyelination and the oligodendrocyte number or even the death of oligodendrocye lineages. However, it provides evidence to support the notion that the severity of the disease correlates with the number of apoptotic cells, the reduced amount of myelin and the activation of microglia/macrophages. While the rsh mutation on either genetic background is similarly heritable, the differences in phenotypes are likely attributable to differences in genetic background, thus suggesting the importance of modifying loci in determining the phenotype

The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD

Genetic background determines phenotypic severity of the Plp rumpshaker mutation

The rumpshaker mutation of the proteolipid protein (Plp) gene causes dysmyelination in man and mouse. We show that the phenotype in the mouse depends critically on the genetic background in which the mutation is expressed. On the C3H background there is normal longevity whereas changing to a C57BL/6 strain results in seizures and death at around postnatal day 30. The more severe phenotype is associated with less myelin and reduced levels of major myelin proteins. There are also more apoptotic cells, including oligodendrocytes, increased numbers of proliferating cells, increased numbers of NG2+ oligodendrocyte progenitors and increased microglia compared to the milder phenotype. The number of mature oligodendrocytes is similar to wild-type in both strains of mutant, however, suggesting that increased oligodendrocyte death is matched by increased generation from progenitors. The dichotomy of phenotype probably reflects the influence of modifying loci. The localization of these putative modifying genes and their mode of action remain to be determined. (C) 2003 Wiley-Liss, Inc

Modulation of rumpshaker phenotype with wild-type PLP/DM20 suggests several pathogenic mechanisms

The rumpshaker mutation of the murine myelin proteolipid protein 1 (P1p1) gene generates misfolded PLP/DM20 protein, resulting in dysmyelination, increased oligodendrocyte apoptosis, and death prior to P40 when expressed on the C57 BL/6 background. In this study, we used transgenic complementation to normalize the levels of PLP/DM20 in myelin with wild-type protein to determine whether loss of normal PLP function or gain of toxic function is responsible for dysmyelination in the rumpshaker. Restoring myelin PLP/DM20 levels extended the survival time to at least P60, significantly reduced the density of apoptotic cells, increased myelin volume, and restored normal periodicity of myelin. Biochemical analysis found that several myelin proteins that are reduced in rumpshaker, including MAG, CNP, and SirT2, are markedly elevated at peak myelination (P20) in the rumpshaker transgenic mouse. Myelin. basic protein, however, remained low at peak myelination but was restored at P60 when myelin had matured and entered into a maintenance phase. Markers of the unfolded protein response (UPR), BiP and XBP1, remained activated with the introduction of wild-type PLR These data demonstrate that restoring wild-type PLP/DM20 levels in rumpshaker improves the phenotype and the integrity of myelin, but hypomyelination persists and stress pathways remain activated. This suggests that both gain- and loss-of-function mechanisms are involved in the pathogenesis of the rumpshaker