R. Wayne Davies

Myelin-Associated Oligodendrocytic Basic Protein: A Family of Abundant CNS Myelin Proteins in Search of a Function

The myelin-associated oligodendrocytic basic protein (MOBP) family constitutes the third most abundant protein in CNS myelin. The mouse Mobp gene comprises eight exons. Mobp pre-mRNA processing gives rise to at least seven Mobp splice variants which are expressed solely in the oligodendrocyte. The predicted proteins all, with one exception, share a 68 residue amino terminus, encoded by exon 3. The carboxyl termini differ in length, giving rise to the diverse array of the protein isoforms. Like myelin basic protein, MOBP is present in the major dense line of CNS myelin suggesting a role in the compaction or stabilization of myelin. However, Mobp homozygous null mice display no overt clinical phenotype and no defect in the process of myelination. MOBP can induce experimental allergic encephalomyelitis in mice and has been proposed to have a role in the pathogenesis of multiple sclerosis. Despite 10 years of rigorous study, the normal physiological function of MOBP remains unknown.

A candidate gene for human neurodegenerative disorders: a rat PKCγ mutation causes a Parkinsonian syndrome

Rats harboring the agu mutation have altered behavior and brain pathology resembling human Parkinsonian syndromes; notably, they have a movement disorder and age-progressive dysfunction and death of neurons in the midbrain (substantia nigra pars compacta) that use dopamine as a neurotransmitter. We present evidence that this phenotype is due to a mutation in the rat protein kinase Cγ (in rat, Prkcg; in mouse, Prkcc; in human, PRKCG) gene, which generates a premature stop codon, drastically reducing the level of synthesis of the catalytic domain of the brain-specific protein kinase Cγ protein.

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. J. Neurosci Res. 49:133–143, 1997. © 1997 Wiley‐Liss, Inc.

Isolation, characterization, and differentiation of thy1.1-sorted pancreatic adult progenitor cell populations.

We have isolated a novel progenitor cell population from adult rat pancreatic ducts, termed pancreatic-derived progenitor cells (PDPCs). Here, we report the in vitro culture, selection, and characterization of Thy1.1-positive and Thy1.1-negative PDPC subpopulations. These cells exhibit bipotentiality for differentiation into both pancreatic and hepatic cell types. Significantly, they express Pdx-1. Using a serum-free FGF-4-containing differentiation protocol, we have observed a time course of both morphological and gene expression changes indicative of hepatic lineage differentiation for the Thy1.1-positive subpopulation. These cells express albumin and store glycogen, typical features of mature hepatocytes. The Thy1.1-positive subpopulation could also readily be induced to differentiate into a pancreatic lineage with characteristic morphological changes resulting in three-dimensional islet-like structures and the transcriptional expression of insulin and glucagon in addition to Pdx-1. No morphological evidence of islet-like clusters was observed using the Thy1.1-negative population. However, Thy1.1-negative cells grown in pancreatic differentiation medium did show insulin gene transcription. Glucagon was not expressed in the undifferentiated Thy1.1-negative cells, nor was it induced in vitro after differentiation. The detection of Pdx-1 transcriptional expression in both populations indicates their potential as a novel source of non-beta-cell-derived insulin.

Pancreatic-derived pathfinder cells enable regeneration of critically damaged adult pancreatic tissue and completely reverse streptozotocin-induced diabetes.

We demonstrate that intravenous delivery of human, or rat, pancreas-derived pathfinder (PDP) cells can totally regenerate critically damaged adult tissue and restore normal function across a species barrier. We have used a mouse model of streptozotocin (STZ)-induced diabetes to demonstrate this. Normoglycemia was restored and maintained for up to 89 days following the induction of diabetes and subsequent intravenous delivery of PDP cells. Normal pancreatic histology also appeared to be restored, and treated diabetic animals gained body weight. Regenerated tissue was primarily of host origin, with few rat or human cells detectable by fluorescent in situ hybridization (FISH). Crucially, the insulin produced by these animals was overwhelmingly murine in origin and was both types I and II, indicative of a process of developmental recapitulation. These results demonstrate the feasibility of using intravenous administration of adult cells to regenerate damaged tissue. Critically, they enhance our understanding of the mechanisms relating to such repair and suggest a means for novel therapeutic intervention in loss of tissue and organ function with age.

Deficits in the mid-brain raphe nuclei and striatum of the AS/AGU rat, a protein kinase C-γ mutant

The AS/AGU rat carries a recessive mutation (agu) in the gene coding for the gamma isoform of protein kinase C. The rat is characterized by disordered locomotion and progressive dysfunction of the nigrostriatal dopaminergic (DA) system. This dysfunction begins with a failure to release DA within the striatum and culminates in cell loss within the substantia nigra pars compacta. The present study examines another midbrain aminergic system with input to the basal ganglia, the serotonergic (5‐HT) raphe–striatal system originating in the dorsal raphe nucleus. By 3 months after birth, there is a very substantial reduction in the extracellular levels of 5‐HT in the dorsal caudate‐putamen of the mutants compared with controls (c. 70%). This is accompanied by a proportional increase in the levels of the 5‐HT metabolite 5‐hydroxyindole acetic acid (5‐HIAA). At a later age, there are reductions in whole tissue 5‐HT (and increases in 5‐HIAA) in both the striatum and the region containing the dorsal raphe nucleus, as well as numbers of 5‐HT‐immunoreactive cells in the dorsal raphe nucleus. The median raphe appears to be unaffected. The results are seen in terms of an initial dysfunction in transmitter release leading to cell death, perhaps through the formation of free radicals or neurotoxins.

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 ∼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.

Microvesicles as Vehicles for Tissue Regeneration: Changing of the Guards

Purpose of ReviewMicrovesicles (MVs) have been recognised as mediators of stem cell function, enabling and guiding their regenerative effects.Recent FindingsMVs constitute one unique size class of extracellular vesicles (EVs) directly shed from the cell plasma membrane. They facilitate cell-to-cell communication via intercellular transfer of proteins, mRNA and microRNA (miRNA). MVs derived from stem cells, or stem cell regulatory cell types, have proven roles in tissue regeneration and repair processes. Their role in the maintenance of healthy tissue function throughout the life course and thus in age related health span remains to be elucidated.SummaryUnderstanding the biogenesis and mechanisms of action of MVs may enable the development of cell-free therapeutics capable of assisting in tissue maintenance and repair for a variety of age-related degenerative diseases. This review critically evaluates recent work published in this area and highlights important new findings demonstrating the use of MVs in tissue regeneration.

Characterization of the murine splice variant Mobp155: Developmental CNS expression pattern and subcellular localization of epitope-tagged protein

Members of the myelin‐associated oligodendrocytic basic protein (MOBP) family constitute the third most abundant protein in CNS myelin. Although MOBP localizes to the major dense line (MDL) of CNS myelin, the function of the individual isoforms is unknown. Alternative splicing of pre‐Mobp mRNA gives rise to six characterized splice variants in both the mouse and the rat. These splice variants share a common N‐terminal encoded in Mobp exon 3 comprising 68 amino acids. The predicted protein isoforms differ in their C‐termini. Sequence analysis of intron 3 revealed the presence of a putative initiation codon followed by an open reading frame (ORF) encoding 53 amino acids that extends in frame into Mobp exon 4 yielding a predicted MOBP isoform comprising 155 amino acids, designated MOBP155. This newly characterized isoform possessing a novel N‐terminus shares a common C‐terminus with MOBP170. Mobp170 message is detectable at low abundance throughout myelinogenesis. In contrast, the novel splice variant encoding MOBP155 is expressed at modest levels late in CNS development, coincident with the expression of the abundant splice variant, Mobp81A. Immunostaining of Cos7 cells transiently expressing an epitope‐tagged MOBP155 suggested that most of the product was translocated to mitochondria. Although Mobp155 and Mobp170 encode a common predicted C‐terminus they have different expression profiles and their products are targeted to mitochondria and the nucleus, respectively, in transiently transfected Cos7 cells.

DISCOVERING GENES WITH LOCALISED EXPRESSION IN THE MOUSE BRAIN : CDNAS SPECIFIC TO THE SUBSTANTIA NIGRA

Many important phenomena of normal brain physiology and disease are likely to be related to the function of genes expressed in localised regions of the brain. We show that subtracted libraries enriched in clones corresponding to rare mRNAs, which must include genes with very localised and neuron-specific expression, can easily be produced from single-stranded directional cDNA libraries after hybridization to excess photobiotinylated opposite-stranded cDNA (or RNA) from another brain region, followed by the removal of biotinylated molecules. We also demonstrate the use of heterologous probes from anatomically precise small regions of bovine brain to identify cDNA clones that putatively represent mRNAs present at significantly higher levels in a substantia nigra mRNA population enriched for pars compacta mRNA than in the total ventral midbrain or cerebellar mRNA population. Some of these cDNAs may identify genes that play important roles in the specific molecular biology of dopaminergic neurons, including susceptibility to Parkinsons disease.