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Biomedical subjects

I R Griffiths

Publications and source records attributed to I R Griffiths.

At least 19 recordsLinked to original sources
PubMed1999

Distinct phenotypes associated with increasing dosage of the PLP gene: implications for CMT1A due to PMP22 gene duplication.

Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.

Animals↗
PubMed1999

The "CMT rat": peripheral neuropathy and dysmyelination caused by transgenic overexpression of PMP22.

We have generated a transgenic rat model of Charcot-Marie-Tooth disease type 1A (CMT1A) providing formal proof that this neuropathy can be caused by increased expression of peripheral myelin protein-22 (PMP22). Heterozygous PMP22-transgenic rats develop muscle weakness and gait abnormalities as well as reduced nerve conduction velocities and EMG abnormalities, which closely resemble recordings in patients with CMT1A. Dys- and demyelination, Schwann cell hypertrophy, and "onion bulb" formation are also similar to findings in humans. When bred to homozygosity, transgenic rats completely fail to elaborate myelin, but all myelin-forming Schwann cells segregate with axons in the normal one-to-one ratio. Although arrested at this "promyelin" stage, differentiation proceeds in homozygous rats at the molecular level, as demonstrated by high-level expression of myelin structural genes. Intracellular trafficking of the wild-type protein is not visibly impaired, even when strongly overexpressed, suggesting that PMP22 blocks myelin assembly in a late Golgi/cell membrane compartment of the affected Schwann cell.

Animals↗
PubMed1999

Splicing pattern, transcript start distribution, and DNA sequence of the mouse gene (Mobp) encoding myelin-associated oligodendrocytic basic protein.

We have cloned the mouse gene Mobp, encoding the family of myelin-associated oligodendrocytic basic proteins (MOBP), to facilitate elucidation of its genomic organization and regulation. We report near complete sequence analysis of the Mobp gene (>11 kb), including complete sequence of all exons and their associated splice junctions. The Mobp gene comprises eight discrete exons and encompasses a genomic region in excess of 15 kb. We provide a definitive analysis of the alternative splicing events and exon usage required in the generation of the reported splice variants of Mobp transcripts. We identify sequences corresponding to the coding regions of all reported protein isoforms. Consequently, we demonstrate that sequence regions, predicted to encode unique portions of two putative protein isoforms in the rat (MOBP 71 and MOBP 99), are not fully conserved between the rat and the mouse: we predict that the mouse equivalents are two distinct polypeptides of 73 amino acids, MOBP73A and MOBP73B, respectively. We have analyzed sequence from 63 oligo-capped, cloned cDNA fragments and identify six transcription start points associated with the Mobp gene at postnatal day 26. This study provides the platform for a more detailed analysis of the function of the Mobp gene product and subsequent evaluation of its possible involvement in known neuropathies.

5' Untranslated Regions↗
PubMed1999

The early phenotype associated with the jimpy mutation of the proteolipid protein gene.

The jimpy mutation of the X-linked proteolipid protein (Plp) gene causes dysmyelination and premature death of the mice. The established phenotype is characterised by severe hypomyelination, increased numbers of dead oligodendrocytes and astrocytosis. The purpose of this study was to define the earliest cellular abnormalities in the cervical spinal cord. We find that on the first and third postnatal days the amount of myelin in jimpy spinal cord is approximately 20% of wild-type. However, the total glial cell density, the number of dead glial cells and the number and distribution of Plp-positive cells, as assessed by in situ hybridization, are similar to wild-type during the first week of life. Immunostaining of cryosections has identified that jimpy spinal cords express on schedule, a variety of antigens associated with mature oligodendrocytes. Dissociated oligodendrocytes, cultured for 18 hours to reflect their in vivo differentiation, express MBP and surface myelin-associated glycoprotein at the same frequency as wild-type. By comparison, the proportion of jimpy oligodendrocytes expressing surface myelin/oligodendrocyte glycoprotein is reduced by approximately 34%. In vivo, however, only a small minority of axons is surrounded by a collar of myelin-associated glycoprotein, suggesting that the majority of jimpy oligodendrocytes fail to make appropriate ensheathment of axons. Although the DM20 isoform is expressed in the embryonic CNS prior to myelin formation, the cellular abnormalities appear to correspond to the time at which the Plp isoform becomes predominant. The results suggest that the primary abnormality in jimpy is the inability of oligodendrocytes to properly associate with, and then ensheath, axons and that oligodendrocyte death compounds, rather than initiates, the established phenotype.

Age Factors↗
PubMed1999

Subdural injection of contrast medium as a complication of myelography.

In a retrospective evaluation of 654 canine and feline myelograms, 58 were found to have been complicated by injection of the contrast medium into the subdural space. The medium was present predominantly dorsal to the spinal cord, with a sharp dorsal border and an undulating ventral border. Confirmation that this myelographic appearance was due to subdural localisation was achieved using fresh cadavers injected with contrast medium labelled with Indian ink. It was further showed that the dorsal accumulation of contrast medium was due to the denticulate ligaments which restricted the ventral extension of the contrast medium.

Animals↗
PubMed1999

Reduced levels of a specific myelin-associated oligodendrocytic basic protein isoform in shiverer myelin.

Myelin-associated oligodendrocytic basic protein (MOBP) and myelin basic protein (MBP) share many structural similarities. MOBP is synthesised by mature oligodendrocytes and localised at the major dense line (MDL), suggesting a role in the myelin compaction process. The shiverer mouse, a deletion mutant of the myelin basic protein (Mbp) gene, has poorly compacted myelin with essentially no MDL. In this study we compare the developmental expression of the Mobp gene in wild-type and shiverer mice. The significant finding is that one of the two abundant MOBP isoforms, the approximately 20-kD species, is poorly incorporated into shiverer myelin. The absence is specific to shiverer and is not a feature of dysmyelinating mutants with an abnormal intraperiod line. Our data suggest that incorporation of this MOBP isoform into shiverer myelin may be influenced by the presence of MBP or be a consequence of a disrupted MDL.

Aging↗
PubMed1998

Oligodendrocytes are not inherently programmed to myelinate a specific size of axon.

Current studies support the morphological classification of oligodendrocytes proposed by Del Rio Hortega ([1922] Bol. R. Soc. Esp. Hist. Nat. 10:25-29; [1924] C.R. Soc. Biol. 91:818-820), in which cells either myelinate multiple internodes that are associated with small axons, or they myelinate restricted/single internodes of large-diameter axons. The reasons why an oligodendrocyte myelinates a particular calibre of axon are unknown. Because progenitors are generated in restricted, subventricular zones, an intrinsic program would imply that germinal centres contain a mixture of cells, each committed to myelinate axons of a particular size. Conversely, each cell could have the potential ability to myelinate any size axon. We tested this latter hypothesis that oligodendrocyte progenitors are uncommitted in their ability to myelinate a particular axon size. We introduced oligodendrocyte lineage cells from the optic nerve, which normally encounter only small-diameter axons, to a myelin-deficient environment containing a large range of axon sizes. Dissociated, mixed glial cells from the optic nerve were characterised immunocytochemically and were grafted into the spinal cord ventral column of neonatal, myelin-deficient rat mutants. Examination of the patches of myelin produced by these cells at different times after transplantation revealed that optic nerve oligodendrocytes were capable of producing a widespread, nonselective myelination of axons that were destined to have both small or large calibres. Thus, an axonal or local signal, and not an intrinsic program, is probably responsible for the previously described oligodendrocyte diversity.

Animals↗
PubMed1998

Molecular cloning and transfection studies of M6b-2, a novel splice variant of a member of the PLP-DM20/M6 gene family.

The present study documents the nucleic acid and deduced amino acid sequence of M6b-2, a novel splice variant of the M6b gene, which belongs to the PLP-DM20/M6 gene family. M6b-2 differs from the previously published M6b by a novel 40-amino acid insertion which is characterised by a high proline content, two casein kinase, and one tyrosine kinase consensus sequences. M6b-2 mRNA is enriched in perinatal central nervous system (CNS), and although it declines during development, it does persist into adulthood. Transient transfection studies coupled to secondary structure and hydrophobicity analysis suggest that the novel polypeptide in M6b-2 lies at the cytoplasmic face of the plasma membrane. It is therefore possible that the function(s) of M6b-2 may be regulated intracellularly by phosphorylation during CNS development.

Aging↗
PubMed1998

Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene.

Mutations of the proteolipid protein (Plp) gene cause a generalized central nervous system (CNS) myelin deficit in Pelizaeus-Merzbacher disease of man and various tremor syndromes in animal models. X-linked spastic paraplegia is also due to Plp gene mutations but has a different clinical profile and more restricted pathology involving specific tracts and regions. We have shown previously that PLP overexpression in mice homozygous for a Plp transgene results in premature arrest of CNS myelination and premature death. Here, we demonstrate that a low-level increase in Plp gene expression in transgenic mice causes significant axonal degeneration and demyelination with predilection for specific tracts. Following normal motor development, aged mice develop progressive myelin loss, axonal swellings with resultant Wallerian degeneration, and marked vacuolation of the neuropil associated with ataxia, tremor, and seizures. The age of onset and severity of the phenotype is a function of Plp gene dosage. The corticospinal tracts, optic nerve, fasciculus gracilis cerebellum, and brainstem are particularly involved. Although oligodendrocyte cell bodies show little abnormality, their inner adaxonal tongue is often abnormal, suggesting a perturbation of the axon/glial interface that may underlie the axonal changes. We conclude that abnormal expression of an oligodendrocyte-specific gene can cause axonal damage, a finding that is relevant to the pathogenesis of PLP-associated disorders and probably to other myelin-related diseases.

Age of Onset↗
PubMed1998

Mouse models of myelin diseases.

Dys- and demyelination are the common endpoints of several inherited diseases of glial cells, which elaborate myelin and which maintain the myelin sheath very much like an "external" cellular organelle. Whereas some of the genes that are affected by mutations appear to be glial-specific, other genes are expressed in many cell types but their defect is restricted to oligodendrocytes or Schwann cells. Many of the disease genes and their encoded proteins have been studied with the help of mouse models, and a number of different molecular pathomechanisms have emerged which have been summarized in Figure 8. Some of the new concepts in the field, which have been addressed in this review, have only emerged because similar pathomechanisms were discovered for different myelin proteins. Mouse models have clearly helped to address both, the molecular pathology of myelin diseases and the normal function of myelin genes, but as discussed in this review, these questions turned out to be very different. Despite the progress in understanding the role of the abundant myelin proteins, there also remain a number of open questions that concern, among other things, the initial axon-glia recognition, the assembly process of the myelin sheath, and the long-term interaction of axons with their myelinating glia. Finally, animal models of human neurological diseases should not be restricted to the study of pathology, but they should also contribute to the development of experimental treatments. It is encouraging that a few attempts have been made.

Animals↗
PubMed1997

Modification of Schwann cell phenotype with Plp transgenes: evidence that the PLP and DM20 isoproteins are targeted to different cellular domains.

The X-linked proteolipid protein (Plp) gene encodes PLP, the major protein of central nervous system myelin, and its alternative RNA splice product, termed DM20. Schwann cells also express the Plp gene but, in contrast to oligodendrocytes, neither protein is incorporated into peripheral myelin. In the present study, we use different transgenes encoding PLP and DM20 to modify the expression of these proteins in myelin-forming Schwann cells of wild-type and jimpy mice. Increasing the level of PLP, either singly or in combination with DM20, leads to the incorporation of PLP into the compacted myelin sheath; however, DM20 always remains restricted to cytoplasmic regions of the Schwann cell. The insertion of PLP into the membrane does not appear to depend on a cooperativity of the two isoproteins. The presence of PLP does not visibly alter the ultrastructure and periodicity of peripheral nervous system (PNS) myelin. The results indicate that the absence of PLP in the peripheral myelin of normal animals most probably reflects the very low amounts of this isoprotein synthesised by Schwann cells. The preferential incorporation of PLP, as opposed to DM20, in peripheral myelin may indicate that a myelin targeting signal is present in the PLP-specific region of the molecule.

Animals↗
PubMed1997

Developmental expression of the murine Mobp gene.

In this report we describe the developmental expression of the murine (Mobp) gene encoding myelin-associated oligodendrocytic basic protein. We have characterized three Mobp cDNA clones which have been used as probes. Murine Mobp splice variant-1 (mmsv-1), a portion of 3' untranslated region (UTR), is homologous to 3' UTR sequences found in the rat Mobp splice variants rOP1, Mobp81-A and Mobp-99. The mmsv-2 sequence, encoding 81 amino acids, closely resembles the rat Mobp81-A splice variant. The mmsv-3 cDNA, encoding 170 amino acids corresponding closely to the rat rOPRP1 splice variant, detects a single mRNA species present in low levels from E12 onward, suggesting this MOBP may have a function alternative or additional to involvement in myelin formation. The mmsv-1 probe detects an mRNA species abundantly expressed in the postnatal central nervous system (CNS) but barely detectable at E18. This mRNA is located initially in the cell bodies of oligodendrocytes, moving distally into their processes as myelination proceeds. The most abundant mmsv(s) in the adult CNS are present at detectable levels after expression of the myelin basic protein (Mbp) gene and marginally after or coincident with the proteolipid protein (Plp) gene. The level of the abundant, late-expressed mRNA correlates closely with the capacity to form myelin and the maturity of oligodendrocytes, as shown in two hypomyelinated mutants, rumpshaker and jimpy, which represent mildly and severely affected phenotypes, respectively.

Animals↗
PubMed1997

Phenotypic severity of murine Plp mutants reflects in vivo and in vitro variations in transport of PLP isoproteins.

Mutations of the major myelin gene, proteolipid protein (Plp), cause Pelizaeus-Merzbacher disease and some forms of spastic paraplegia in man and dysmyelinating phenotypes in animals. The clinical severity is markedly heterogeneous, ranging from relatively mild to severe and fatal. Point mutations, or frame shifts, which are predicted to result in translation of structurally altered proteins account for many of these cases, including 3 of the allelic murine conditions. Plp(jp-rsh), Plp(jp-msd), and Plp(jp) represent an increasing severity of clinical and pathological phenotypes, respectively. In this study we determined whether there was any correlation between the severity of phenotype and the transport of the predicted abnormal protein. We examined the ability of the two products of the Plp gene, PLP and DM20, to insert into the plasma membrane of transfected BHK or COS-7 cells, and into the myelin sheath of oligodendrocytes. With these complementary in vitro and in vivo approaches we find that proteins of Plp(jp-rsh), associated with the mildest phenotype, have a far greater ability to insert into the cell membrane or myelin than those associated with the severe phenotypes. Additionally, altered DM20 is more readily transported to the cell surface and to myelin than the PLP isoprotein. Interestingly, the two clonal cell lines chosen for transient transfection differ in their ability to fold DM20 from Plp(jp-rsh) and Plp(jp-msd) mice correctly, as inferred by staining for the conformation-sensitive O10 epitope. In the case of Plp(jp), which is associated with the most severe phenotype, no PLP or O10 staining is present at the cell surface or in myelin. The perturbation in trafficking observed for altered Plp(jp) PLP and DM20 in oligodendrocytes does not extend to other myelin membrane proteins, such as MAG and MOG, nor to wild type PLP co-expressed in the same cell, all of which are correctly inserted into myelin. As Plp-knockout mice do not have a dysmyelinating phenotype it seems unlikely that absence of PLP and/or DM20 in the membrane is responsible for the pathology. It remains to be determined whether the perturbation in protein trafficking is associated with the dysmyelination, or if the altered product of the mutant alleles acquire a novel function which is deleterious to myelin production by oligodendrocytes.

Animals↗
PubMed1997

Assembly of CNS myelin in the absence of proteolipid protein.

Two proteolipid proteins, PLP and DM20, are the major membrane components of central nervous system (CNS) myelin. Mutations of the X-linked PLP/DM20 gene cause dysmyelination in mouse and man and result in significant mortality. Here we show that mutant mice that lack expression of a targeted PLP gene fail to exhibit the known dysmyelinated phenotype. Unable to encode PLP/DM20 or PLP-related polypeptides, oligodendrocytes are still competent to myelinate CNS axons of all calibers and to assemble compacted myelin sheaths. Ultrastructurally, however, the electron-dense 'intraperiod' lines in myelin remain condensed, correlating with its reduced physical stability. This suggests that after myelin compaction, PLP forms a stabilizing membrane junction, similar to a "zipper." Dysmyelination and oligodendrocyte death emerge as an epiphenomenon of other PLP mutations and have been uncoupled in the PLP null allele from the risk of premature myelin breakdown.

Animals↗
PubMed1997

Hindshaker, a novel myelin mutant showing hypomyelination preferentially affecting the spinal cord.

Animals with spontaneous mutations affecting myelin formation have provided useful information about the genetic and cellular mechanisms regulating normal and abnormal myelination. In this paper we describe a novel murine mutation termed hindshaker (hsh), which is inherited in an autosomal recessive manner. Affected mice are characterised by a variable tremor of the hind end which commences at about 2 weeks of age and largely disappears in animals older than 6 weeks. There is hypomyelination affecting predominantly the spinal cord, although the optic nerves and brain are involved to a much lesser degree. The defect of thinly myelinated and naked axons is maximal at 20 days of age and largely resolves with time so that in the adult most axons are myelinated. The myelin structure appears normal and immunostains for the major proteins. Although the distribution of oligodendrocytes in the spinal cord is similar to normal during the period of hypomyelination, there are fewer mature cells. The hsh mutation appears to delay the maturation of oligodendrocytes, particularly in the spinal cord. Additionally, there is a considerable variation in phenotypic expression and in penetrance when the mutation is expressed on different genetic backgrounds, suggesting the hsh locus is subject to the influence of modifying gene(s). Identification of the hsh gene should identify a factor important in the development of oligodendrocytes, particularly those in the spinal cord.

Animals↗
PubMed1997

Expression of the dm-20 isoform of the plp gene in olfactory nerve ensheathing cells: evidence from developmental studies.

The olfactory bulb is a specialized area of the CNS with well-defined areas containing both myelinated, and non-myelinated axons. The presence of plp gene transcripts has been demonstrated previously in the olfactory bulb, but no detailed description of plp gene activity in this complex area of the CNS is available. In this study we describe the developmental expression of the two plp gene isoforms, plp and dm-20, and their products in the mouse olfactory bulb. plp gene activity was present in the non-myelinated olfactory nerve layer of the bulb from E14 through to adult ages. Expression in the deeper layers of the bulb, associated with myelination of the second order axons, was apparent from P10 onwards. dm-20 was the predominant isoform expressed at the transcript level in the olfactory nerve layer and was the only isoprotein demonstrable by immunostaining. This expression was associated with the resident glial cell of the non-myelinated olfactory nerve layer, the olfactory nerve ensheathing cell. Selective expression of the DM-20 isoprotein in the non-myelin forming olfactory nerve ensheathing cells implies a role for DM-20 other than as a structural myelin protein. Further study of this specialized glial cell may prove useful in elucidating the specific functions(s) of the DM-20 isoprotein.

Animals↗
PubMed1996

Evidence that some oligodendrocyte progenitors in the developing optic pathway express the plp gene.

DM-20, a product of the proteolipid protein (plp) gene, has been demonstrated in the spinal cord of the mouse embryo as early as embryonic day 12 (E12) in certain cells, some of which are identifiable as oligodendrocyte progenitors. The present work uses optic pathways of rat and mouse as well-characterized systems for the study of gliogenesis. plp gene expression was monitored with a combination of reverse transcriptase polymerase chain reaction, in situ hybridization, and immunostaining with antibodies to different PLP peptide sequences, combined with O-2A lineage markers. In tissue sections, hybridizing cells were detected initially in the proximal optic tracts between E18 and birth and thereafter progressively in the chiasm and optic nerves. Small unbranched cells expressing DM-20 but not myelin basic protein (MBP) and probably representing progenitors were detectable by immunostaining in similar locations. With increasing postnatal ages, cells representing maturing oligodendrocytes which co-label for PLP and MBP are present in the optic pathways. In vitro analysis of freshly dissociated cells from premyelinated optic nerve demonstrated that the plp gene is expressed in some O-2A progenitor cells as well as mature oligodendrocytes. We also present evidence that increase in expression of the plp gene along the O-2A lineage differentiation is not progressive but that downregulation at the proligodendroblast (O4+/O1-) stage probably occurs. We suggest that progenitors express the dm-20 isoform while oligodendrocytes express predominantly the plp isoform. Not all progenitors express the plp gene at the times studied, indicating that the presence of DM-20 is either transitory in individual cells or that only a sub-population is involved. The function of DM-20 at this early stage of the oligodendrocyte lineage has yet to be determined.

Animals↗