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P J Brophy

Publications and source records attributed to P J Brophy.

At least 37 records · Page 2Linked to original sources
PubMed1997

The secretory epithelial cells of the choroid plexus employ a novel kinesin-related protein.

The proteins of the kinesin superfamily (KIFs) are microtubule-based molecular motors whose functions include the transport of membrane-bound organelles. We have isolated the cDNA encoding a novel kinesin by reverse transcription and polymerase chain reaction using degenerate primers that flank the highly conserved motor domain. The deduced amino acid sequence of this protein shows considerable similarity to both KIF1A and KIF1B thus defining it as a new member of the monomeric KIF1/unc104 family. The C-terminal domain of KIF1D is the most divergent by comparison with the other members of the family, which supports the view that the tail region is responsible for conferring specificity on the interactions of these kinesins with their cargoes. In the adult rat brain KIF1D mRNA is expressed in neurons in the hippocampus and in the Purkinje cells of the cerebellum. However, the levels of KIF1D are particularly high in the choroid plexus which is a polarised epithelium that lines the lateral, third and fourth ventricles. The major function of the epithelial cells in the choroid plexus is to produce cerebrospinal fluid, which suggests that KIF1D plays an important role in their secretory function.

Amino Acid Sequence↗
PubMed1997

A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system.

Two isoforms of the Ca2+-sensitive, actin-binding protein gelsolin have been identified thus far; one is an intracellular protein, cytoplasmic gelsolin, and the other is a secretory protein called plasma gelsolin. Gelsolin expression in the mammalian CNS appears to be localized mainly to oligodendrocytes where it is presumed that the cytoplasmic isoform predominates. Here, we show that oligodendrocytes not only contain cytoplasmic gelsolin, but they also express a novel gelsolin isoform that we have named gelsolin-3. Cytoplasmic gelsolin, plasma gelsolin, and gelsolin-3 arise by alternative splicing from the same gene. The N-terminal amino acid sequence unique to gelsolin-3 is shown to be encoded by a single exon in a region previously thought to be an intron in the human gelsolin gene. In situ hybridization analysis confirmed that gelsolin-3 mRNA is localized primarily to oligodendrocytes in rat brain. In other tissues, gelsolin-3 shows a more restricted pattern of expression than cytoplasmic gelsolin. These data support the view that the gelsolin isoforms have differential roles in the regulation of the actin cytoskeleton.

Amino Acid Sequence↗
PubMed1995

Microtubule-associated proteins in developing oligodendrocytes: transient expression of a MAP2c isoform in oligodendrocyte precursors.

The morphological differentiation of oligodendrocytes is characterized by the formation of multiple, microtubule-rich processes which endow these cells with the ability to myelinate many axons simultaneously. Since microtubule-associated proteins (MAPs) strongly influence the structure and function of microtubules, we have investigated their expression in cultured differentiating oligodendrocytes in order to gain insights into MAP function during process formation and stabilization. MAP1B has been compared with two other structural MAPs: MAP4, which is an ubiquitously expressed protein, and MAP2, which hitherto was thought to be confined to neurons and reactive astrocytes. Immunofluorescence microscopy showed that the colocalization of MAP4 with microtubules in oligodendrocyte processes is not as extensive as found previously for MAP1B (Vouyiouklis and Brophy: J Neurosci Res 35:257-267, 1993). Nevertheless, like MAP1B, the expression of MAP4 increases during oligodendrocyte differentiation. In contrast, the expression of MAP2 is transiently elevated in preoligodendrocytes but declines precipitously at the onset of terminal differentiation. Cells of the oligodendrocyte lineage exclusively express a novel isoform of MAP2c which is primarily localized in the cell bodies of preoligodendrocytes. This suggests that MAP2c assists in the initiation of process extension rather than in the stabilization of microtubules in the cytoplasm-filled membranous extensions of mature cells. MAP-tau was not expressed at any developmental stage by oligodendrocytes. The distinct subcellular localizations and patterns of developmental expression of MAP1B, MAP4, and MAP2c suggest that these MAPs have different roles in the regulation of the microtubule network during the differentiation of myelin-forming oligodendrocytes.

Amino Acid Sequence↗
PubMed1995

Novel E-cadherin-mediated adhesion in peripheral nerve: Schwann cell architecture is stabilized by autotypic adherens junctions.

Previous studies (Blank, W. F., M. B. Bunge, and R. P. Bunge. 1974. Brain Res. 67:503-518) showed that Schwann cell paranodal membranes were disrupted in calcium free medium suggesting that cadherin mediated mechanisms may operate to maintain the integrity of the paranodal membrane complex. Using antibodies against the fifth extracellular domain of E-cadherin, we now show by confocal laser and electron immunomicroscopy that E-cadherin is a major adhesive glycoprotein in peripheral nervous system Schwann cells. E-Cadherin is not found, however, in compact myelin bilayers. Rather, it is concentrated at the paranodes, in Schmidt-Lanterman incisures, and at the inner and outer loops. At these loci, E-cadherin is associated with subplasmalemmal electron densities that coordinate in register across several cytoplasmic turns of a single Schwann cell. F-Actin and beta-catenin, two proteins implicated in cellular signaling, also co-localize to E-cadherin positive sites. These complexes are autotypic adherens-type junctions that are confined to the plasma membrane synthesized by a single Schwann cell; E-cadherin was never observed between two Schwann cells, nor between Schwann cells and the axon. Our findings demonstrate that E-cadherin and its associated proteins are essential components in the architecture of the Schwann cell cytoplasmic channel network, and suggest that this network has specialized functions in addition to those required for myelinogenesis.

Amino Acid Sequence↗
PubMed1995

Periaxin expression in myelinating Schwann cells: modulation by axon-glial interactions and polarized localization during development.

Periaxin is a newly described protein that is expressed exclusively by myelinating Schwann cells. In developing nerves, periaxin is first detected as Schwann cells ensheathe axons, prior to the appearance of the proteins that characterize the myelin sheath. Periaxin is initially concentrated in the adaxonal membrane (apposing the axon) but, during development, as myelin sheaths mature, periaxin becomes predominately localized at the abaxonal Schwann cell membrane (apposing the basal lamina). In permanently axotomized adult nerves, periaxin is lost from the abaxonal and adaxonal membranes, becomes associated with degenerating myelin sheaths and is phagocytosed by macrophages. In crushed nerves, in which axons regenerate and are remyelinated, periaxin is first detected in the adoxonal membrane as Schwann cells ensheathe regenerating axons, but again prior to the appearance of other myelin proteins. Periaxin mRNA and protein levels change in parallel with those of other myelin-related genes after permanent axotomy and crush. These data demonstrate that periaxin is expressed by myelinating Schwann cells in a dynamic, developmentally regulated manner. The shift in localization of periaxin in the Schwann cell after completion of the spiralization phase of myelination suggests that periaxin participates in membrane-protein interactions that are required to stabilize the mature myelin sheath.

Animals↗
PubMed1994

Microwave frequency dependence of ESR spectra from spin labels undergoing two-site exchange in myelin proteolipid membranes.

Measurement at two microwave frequencies allows the unambiguous assignment of two-component spin-label ESR spectra such as are observed frequently from biological membranes and reconstituted protein-lipid complexes. Consistent spectral subtractions were obtained with 9 and 34 GHz ESR spectra of spin-labeled lipids from lipid-protein complexes for two related myelin proteins, and the 34 GHz difference spectra further showed restriction of axial lipid rotation at the hydrophobic protein surface. Extension of lineshape simulations with the exchange-coupled Bloch equations to 34 GHz, by allowing for nonaxial g tensors and including linear dispersion distortions, yielded consistent rates of lipid exchange at the protein interface and reflected the different lipid selectivities for the two proteins. The present data at two microwave frequencies leave little doubt that the spin-label ESR spectra from these myelin protein-lipid complexes consist of two components in slow exchange.

Algorithms↗
PubMed1994

Periaxin, a novel protein of myelinating Schwann cells with a possible role in axonal ensheathment.

We report the cloning and subcellular localization of a novel Schwann cell-specific protein of 147 kd that we have named periaxin. Periaxin has a remarkable domain of repetitive pentameric units in the primary sequence. It is expressed in the first uncompacted whorls of membrane that ensheathe the axon, and further synthesis of the protein in the rat sciatic nerve parallels the deposition of myelin. In mature myelin, periaxin colocalizes with the myelin-associated glycoprotein in the cytoplasm-filled periaxonal regions of the sheath but is excluded from compact myelin. We propose that periaxin has a role in axon-glial interactions, possibly by interacting with the cytoplasmic domains of integral membrane proteins such as myelin-associated glycoprotein in the periaxonal regions of the Schwann cell plasma membrane.

Amino Acid Sequence↗
PubMed1993

Interaction of two complementary fragments of the bovine spinal cord myelin basic protein with phosphatidylglycerol bilayers, studied by 2H and 31P NMR spectroscopy.

The interaction of two complementary fragments of myelin basic protein from bovine spinal cord with bilayers of dimyristoylphosphatidylglycerol has been studied by broad line 2H and 31P NMR. The fragments, produced by cleavage at the single tryptophan, consist of an N-terminal portion of molecular mass 12.6 kDa and a C-terminal portion of molecular mass 5.8 kDa. The phosphatidylglycerol lipid was deuterated at all three segments of the glycerol headgroup. The approximately linear dependence of the 2H quadrupole splittings and 31P chemical shift anisotropy on protein/lipid ratio in the complexes indicates that the lipids interacting with the protein fragments were in fast exchange on the NMR time scale (approximately 10(-4)-10(-5) s). The relative gradients of the dependence on protein/lipid ratio of both these parameters decrease with the size of the protein fragment and correlate reasonably well with both the net charge on the protein and the lipid binding stoichiometries in the absence of salt. The results are therefore consistent with a model in which the perturbation of the quadrupole splittings either is determined by the net surface potential or is constant for the different protein fragments. Either possibility is consistent with the reduced activity of the fragments relative to the whole protein.

Animals↗
PubMed1993

Microtubule-associated protein MAP1B expression precedes the morphological differentiation of oligodendrocytes.

The microtubule-associated protein MAP1B is believed to play an important role in the outgrowth of neurites from neurons (Tucker and Matus, Dev Biol 130: 423-434, 1988). We have investigated the possibility that MAP1B might participate in the formation of processes in cultured oligodendrocytes by an analysis of the expression of MAP1B during oligodendrocyte progenitor development. The appearance of the antigens recognized by the monoclonal antibodies A2B5, O4, and O1 which define distinct stages in the maturation of progenitors, was compared with the developmental expression of MAP1B. MAP1B is first detectable in O4+ preoligodendrocytes prior to the acquisition of galactocerebroside and immediately before they develop the complex process-bearing morphology characteristic of terminally differentiated myelin-forming oligodendrocytes in the CNS. In contrast, astrocytes have negligible amounts of MAP1B. These results demonstrate that the expression of MAP1B precedes the development of the mature oligodendrocyte phenotype and suggest that interactions between microtubules and MAP1B might have a role in the formation and stabilization of myelin-forming processes.

Animals↗
PubMed1993

Spin label saturation transfer EPR determinations of the stoichiometry and selectivity of lipid-protein interactions in the gel phase.

Lipid-protein interactions with the myelin proteolipid protein incorporated in the gel phase of dimyristoylphosphatidylcholine bilayers have been studied by saturation transfer EPR spectroscopy of spin-labelled phospholipids. The integrated intensities of the saturation transfer EPR spectra from spin-labelled phosphatidylcholine are linearly dependent on the protein/lipid ratio, and correspond to a fixed stoichiometry of approximately 11 lipids per monomer associated with the protein in the gel phase. The normalized saturation transfer intensities of spin-labelled phosphatidic acid, on the other hand, display a non-linear dependence on the protein/lipid ratio that can be described well by a selectivity for interaction with the protein in the gel phase with an average association constant relative to phosphatidylcholine of approx. 5.2. These values for the stoichiometry and selectivity of lipid-protein interaction in the lipid gel phase obtained from saturation transfer EPR spectroscopy are comparable to those found previously in fluid phase lipids by conventional EPR spectroscopy.

Dimyristoylphosphatidylcholine↗
PubMed1993

Oligodendrocyte development and differentiation in the rumpshaker mutation.

The jimpy rumpshaker (jprsh) mutation is an amino acid substitution in exon 4 (Ile186-->Thr) of the proteolipid protein (PLP) gene on the X chromosome. Affected mice show moderate hypomyelination of the central nervous system (CNS) with increased numbers of oligodendrocytes in the white matter of the spinal cord, a feature distinguishing them from other PLP mutations such as jp, in which premature cell death occurs with reduced numbers of oligodendrocytes. Myelin sheaths of jprsh immunostain for myelin basic protein (MBP) and DM-20, but very few contain PLP. This study examines the differentiation of oligodendrocytes cultured from the spinal cords of young mutant and wild type mice using various surface and cytoplasmic antigenic markers to define the stage of development. The majority of oligodendrocytes from mutant mice progress normally to express MBP; approximately 30%, relative to wild type, contain DM-20 at the in vivo age of 16 days, but very few immunostain for PLP or the O10 and O11 markers. The morphology of mutant cells in respect to membrane sheets and processes appears similar to normal. The jprsh oligodendrocyte is, therefore, characterized by a failure to express the markers indicative of the most mature cell; however, it is probably able to achieve a normal period of survival. These data, taken in conjunction with previous results, suggest that the PLP gene has at least two functions; one, probably involving PLP, is concerned with a structural role in normal myelin compaction; the other, perhaps related to DM-20 (or another lower molecular weight proteolipid), is essential for cell survival. The mutation in jprsh at residue 186 suggests that this region, which is common to PLP and DM-20, is not critical for this latter function.

Animals↗
PubMed1993

The distribution of myelin basic protein mRNAs within myelinating oligodendrocytes.

The nervous system contains oligodendrocytes with processes that are greatly extended in space. It is now clear that there are numerous complex, poorly understood mechanisms by which polypeptides are synthesized and delivered to their sites of function in these cells. One mechanism is by the active positioning of mRNAs encoding certain proteins to restricted intracellular subdomains. Perhaps the best studied example of this in the vertebrate CNS is the translocation of myelin basic protein mRNAs to the forming myelin sheath, where the newly synthesized polypeptides, which avidly associate with membranes, can be directly incorporated into the myelin membrane. Evidence for this conclusion is presented here in the context of related work on the general phenomenon of mRNA translocation that is under analysis in other systems.

Animals↗
PubMed1993

Exchange rates at the lipid-protein interface of the myelin proteolipid protein determined by saturation transfer electron spin resonance and continuous wave saturation studies.

The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.

Animals↗
PubMed1992

Rumpshaker: an X-linked mutation causing hypomyelination: developmental differences in myelination and glial cells between the optic nerve and spinal cord.

The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.

Animals↗
PubMed1992

Developmental expression of major myelin protein genes in the CNS of X-linked hypomyelinating mutant rumpshaker.

Rumpshaker (rsh) is an X-linked mutation causing hypomyelination of the CNS of mice and has recently been identified as an allele of jimpy (jp). The mutation (known as jprsh) differs in several respects from other X-linked myelin mutants, including jp, in that mice have normal longevity, oligodendrocyte numbers are not decreased, and cell death is not a feature. Myelin sheaths are deficient in immunostainable PLP protein. The present study examines the developmental expression of the major myelin protein genes and translatability of PLP and MBP mRNA. Differences between the spinal cord and brain of mutants are evident in that mRNA levels are more markedly decreased in the brain. Protein levels are severely reduced in both locations and to a proportionately greater extent than the mRNA, particularly in the spinal cord where PLP RNA and protein are approximately 80% and 10-20%, respectively, of age-matched wild type mice. DM-20 protein, the other major product of the PLP gene, is disproportionately expressed in rumpshaker as is a 10 kDa proteolipid. In vitro translation studies indicate a marked decrease in PLP translation products from mutant RNA. There is no deficiency in the number of PLP mRNA-expressing oligodendrocytes although the abundance per cell is reduced. The data suggest that the phenotypic effects of the mutation may be associated with reduced translation of major myelin proteins, in particular PLP and its incorporation into compact myelin. However, the mutation is compatible with survival of oligodendrocytes and their differentiation to the stage of expressing PLP/DM-20 mRNA.

Aging↗
PubMed1992

Schwann cells of the myelin-forming phenotype express neurofilament protein NF-M.

Immature Schwann cells of the rat sciatic nerve can differentiate into myelin-forming or non-myelin-forming cells. The factors that influence this divergent development are unknown but certain markers such as galactocerebroside distinguish the two cell populations at an early stage of Schwann cell differentiation. Because myelination requires extensive changes in cell morphology, we have investigated the composition and structure of the Schwann cell cytoskeleton at a time when these cells become committed to myelination. Here we show that Schwann cells express a cytoskeletal protein of M(r) 145 before diverging into the myelin-forming path, i.e., before they acquire cell-surface galactocerobroside. The p145 protein has the characteristics of an intermediate filament (IF) protein and immunoelectron microscopy shows that it colocalizes with vimentin, which suggests that these two proteins can coassemble into IFs. Elevated intracellular cAMP levels, which can mimic some of the early effects of axons on Schwann cell differentiation, induced p145 synthesis, therefore, we conclude that myelin-forming Schwann cells express this protein at a very early stage in their development. Immunological comparisons with other IF proteins revealed a close similarity between p145 and the neurofilament protein NF-M; the identification of p145 as NF-M was confirmed by isolating and sequencing a full-length clone from a Schwann cell cDNA library. These data demonstrate that Schwann cells remodel their IFs by expressing NF-M before acquiring the myelin-forming phenotype and that IF proteins of the neurofilament-type are not restricted to neurons in the vertebrate nervous system.

Amino Acid Sequence↗