SOX2 overexpression affects neural differentiation of human pluripotent NT2/D1 cells.

SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics.

Non-assisted versus neuro-navigated and XperCT-guided external ventricular catheter placement: a comparative cadaver study.

BACKGROUND AND PURPOSE: Accurate placement of an external ventricular drain (EVD) for the treatment of hydrocephalus is of paramount importance for its functionality and in order to minimize morbidity and complications. The aim of this study was to compare two different drain insertion assistance tools with the traditional free-hand anatomical landmark method, and to measure efficacy, safety and precision. METHODS: Ten cadaver heads were prepared by opening large bone windows centered on Kocher's points on both sides. Nineteen physicians, divided in two groups (trainees and board certified neurosurgeons) performed EVD insertions. The target for the ventricular drain tip was the ipsilateral foramen of Monro. Each participant inserted the external ventricular catheter in three different ways: 1) free-hand by anatomical landmarks, 2) neuronavigation-assisted (NN), and 3) XperCT-guided (XCT). The number of ventricular hits and dangerous trajectories; time to proceed; radiation exposure of patients and physicians; distance of the catheter tip to target and size of deviations projected in the orthogonal plans were measured and compared. RESULTS: Insertion using XCT increased the probability of ventricular puncture from 69.2 to 90.2 % (p = 0.02). Non-assisted placements were significantly less precise (catheter tip to target distance 14.3 ± 7.4 mm versus 9.6 ± 7.2 mm, p = 0.0003). The insertion time to proceed increased from 3.04 ± 2.06 min. to 7.3 ± 3.6 min. (p < 0.001). The X-ray exposure for XCT was 32.23 mSv, but could be reduced to 13.9 mSv if patients were initially imaged in the hybrid-operating suite. No supplementary radiation exposure is needed for NN if patients are imaged according to a navigation protocol initially. CONCLUSION: This ex vivo study demonstrates a significantly improved accuracy and safety using either NN or XCT-assisted methods. Therefore, efforts should be undertaken to implement these new technologies into daily clinical practice. However, the accuracy versus urgency of an EVD placement has to be balanced, as the image-guided insertion technique will implicate a longer preparation time due to a specific image acquisition and trajectory planning.

Active personal dosemeters in interventional radiology: tests in laboratory conditions and in hospitals.

The work package 3 of the ORAMED project, Collaborative Project (2008-11) supported by the European Commission within its seventh Framework Programme, is focused on the optimisation of the use of active personal dosemeters (APDs) in interventional radiology and cardiology (IR/IC). Indeed, a lack of appropriate APD devices is identified for these specific fields. Few devices can detect low-energy X rays (20-100 keV), and none of them are specifically designed for working in pulsed radiation fields. The work presented in this paper consists in studying the behaviour of some selected APDs deemed suitable for application in IR/IC. For this purpose, measurements under laboratory conditions, both with continuous and pulsed X-ray beams, and tests in real conditions on site in different European hospitals were performed. This study highlights the limitations of APDs for this application and the need of improving the APD technology so as to fulfil all needs in the IR/IC field.

Identification of Golli and myelin basic proteins in human brain during early development.

The myelin basic protein gene (Mbp) encodes for the major myelin structural proteins and it is included in the Golli-Mbp gene complex. Previously, we observed MBP-like proteins in the human central nervous system (CNS) at developmental stages preceding myelination. In an effort to distinguish between Golli (HOG5 and HOG7) and MBP mRNAs and to determine their spatiotemporal distribution, we performed in situ hybridization using two human Golli specific probes: one corresponding to exon 5a absent from all MBP transcripts, and the other corresponding to exon 5c specific for HOG5. HOG7 transcript was observed first, in 5 gestational week-old embryos, whereas both Golli transcripts were detected at 6-7 weeks gestation in the proliferative zones of the entire CNS. Golli proteins immunoreactivity was observed in microglia and early neurons of the developing telencephalon. During midgestation (17-22 weeks gestation), at the onset of myelination, MBP and Golli mRNAs were observed in the telencephalic subventricular zone and occasionally in the future cerebral cortex. Developmental expression of the human Golli-Mbp indicates that the two Golli proteins have different onset of expression, distribution and possibly function. These results support the hypothesis that at least one of them, HOG7, may be involved in the regulation of early neurogenesis, while both may have additional, still undefined function at the onset of myelination.

Implication of the extracellular disulfide bond on myelin protein zero expression.

Mutations in myelin protein zero (P0) are responsible for several peripheral neuropathies. We studied transport and membrane integration of the truncated P0 mutants using transfected oligodendroglial cell line (Oln93). Starting with rat cDNA, we produced two P0 deletions. The first, called P0-Tyr contains a 66 amino acid deletion in the extracellular domain and a tyrosine at the new position 32. In the second, called P0-Cys, the tyrosine 32 is replaced by a cysteine. This replacement restores a disulfide bond in the extracellular domain. Our results show that P0 proteins, truncated or not, were expressed in the plasma membrane of the transfected cells. Transcription rates of both mutants were normal. However, P0-Tyr was detected in only 3-5% of the cells compared to the P0-Cys and the wild type. Thus, the disulfide bond in the extracellular domain is important for stability and correct addressing of the P0 protein.

The transcriptional regulator Yin Yang 1 activates the myelin PLP gene.

Inauguration of the myelin program in developing oligodendrocytes requires the activation of those genes that encode the myelin proteins and the enzymes responsible for the synthesis and degradation of myelin lipids. An activator of the most abundantly expressed myelin protein, proteolipid protein (PLP), has been identified in a yeast one-hybrid system. The ubiquitously expressed zinc finger protein Yin Yang 1 (YY1) recognizes the myelin PLP promoter in vitro and in vivo. When overexpressed in an oligodendrocyte cell line, YY1 enhances transcription of the PLP promoter. A truncated version of YY1 that includes only the four zinc finger domains has little effect. The binding site for YY1 in the PLP promoter (site 3) fits the YY1 consensus site and DNA-protein complexes containing site 3 can be supershifted with an antibody directed against YY1 protein. Moreover, oligonucleotides with a mutated version of the PLP promoter site 3 are unable to bind to nuclear proteins or to compete for binding in a gel shift system. Finally, mutation of this site greatly reduces the activity of a 1-kb PLP promoter region in transfected glial cells. Our results suggest that PLP is a target gene for the transcriptional regulator YY1. This versatile transcription factor and nuclear matrix protein may boost transcription of the PLP gene to meet the demands of actively myelinating oligodendrocytes.

Analysis of cis-acting sequences from the myelin oligodendrocyte glycoprotein promoter.

Myelin oligodendrocyte glycoprotein (MOG), a minor component of the myelin sheath, appears to be implicated in the late events of CNS myelinogenesis. To investigate the transcriptional regulation of MOG, 657 bp of the 5'-flanking sequence of the murine MOG gene, previously shown to induce the highest level of transcription in an oligodendroglial cell line, was analyzed by in vitro footprinting and electrophoretic mobility shift assays. This region contains at least three sites that contact nuclear proteins in vitro. Each region described in this study binds specific nuclear proteins and enhances transcription in the OLN-93 glial cell line. More specifically, a region located at position -93 to -73 bp, which displays 100% homology in mouse and human MOG promoters, presents distinct binding affinities between brain and liver nuclear proteins. The results obtained by supershift assay and site-directed mutagenesis reveal that this region contains an essential positive element (TGACGTGG) related to the cyclic AMP-responsive element CREB-1 and are additional evidence for the involvement of the cyclic AMP transduction pathway in oligodendrocyte development.

[A case report of pyridoxine-responsive homocystinuria]. / Slucaj piridoksinresponzivne homocistinurije.

INTRODUCTION: Homocystinuria is a rare, congenital, autosomal-recessive, metabolic disease biochemically characterized by homocysteinemia and homocystinuria and by multi-system clinical disorders. It is a biochemical abnormality of methionine metabolism caused either by transulfuration pathway disorders or by disorders of homocysteine remethilation into methionine and as such it can be a result of numerous specific and different genetic lesions. CASE REPORT: Homocystinuria is most commonly caused by deficiency of cystationine beta synthetase enzyme which catalyses the synthesis of cystathionine from homocysteine and serine in the methinione pathway. This results in accumulation of homocysteine and methionine in plasma and leads to excretion of excessive urinary homocysteine. Depending on specific property of the mutant enzyme molecule some patients respond to very high doses of pyridoxine with decreases of methionine and homocystine and some not. Pyridoxine responsiveness is based on the presence of small residual activity of cystathionine beta synthetase which is not present in nonresponsive patients. Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is characterized by numerous different clinical abnormalities, but changes in four organ systems are dominant (eye, skeletal, central nervous and vascular system). CASE DESCRIPTION: Six years ago a six year-old boy was admitted to the hospital with vision problems. Ophthalmologic examination revealed a lens dislocation and because of many stigmates the child was sent to endocrinologist. The child had a marfanoid stature, with pectus carinatum and genu valgum, ataxic gait with motoric discoordination, muscle tone which ranged from hypotonia to hypertonia of extrapvramidal type and low intellectual abilities. A simple cyanide-nitroprusside test of patient's urine was positive suggesting homocystinuria. The diagnosis was established after plasma and urine amino acid analysis by HPLC. Concentration of homocystine and methionine were 52 mumol/l and 57 mumol/l in plasma and 249 mumol/du and 55 mumol/du in urine, respectively. After four months of treatment with pyridoxine there were not any significant changes in plasma homocysteine and methionine, but at the same time decrease in urine of these two amino acids were more than 2.5 times higher. This confirms that the patient has a pyridoxine-responsive type of homocystinuria and the dose was increased to 60 mg/day and 600 mg/day. This results in further decline of homocysteine and methionine in plasma and urine which persists up to now (for six years).

Myelin basic protein immunoreactivity in the human embryonic CNS.

Myelin basic protein (MBP) is a major myelin constituent produced by oligodendrocytes in the central nervous system (CNS). Expression of MBP was considered to be a marker for oligodendrocyte differentiation and myelination in the developing CNS. In this study, expression of myelin basic protein (MBP) and its messenger RNA (mRNA) was examined in human embryos and fetuses ranging in age from 5 to 20 gestational weeks (g.w.). We were able to demonstrate that MBP antibody labels cells in both human nervous and non-nervous tissues beginning from early embryonic life (5-6 g.w.). MBP positive (MBP+) cells were rounded, with either no cell processes or only 1-2 short processes, and were located in caudal regions of the CNS. MBP+ cells were also observed in the non-nervous tissue, such as leptomeninges, choroid plexus, and connective tissues. A number of MBP+ cells in nervous and non-nervous tissues were morphologically similar to macrophages and showed a positive reaction to macrophage-microglia markers: lectin (RCA-1) and the monoclonal antibody (EBM-11) to human macrophage antigen CD68, whereas they were negative for neuronal, astroglial, or marker for oligodendrocyte progenitors. At the same embryonic age, 5 g.w. and onward, the MBP mRNA was observed in the CNS by in situ hybridization. The results of this study show that MBP immune reaction is spread in a large area of the CNS prior to myelin appearance. In addition, for the first time it has been demonstrated that the same population of cells could be labelled with both MBP and macrophage markers. These results indicate that MBP, or MBP-related proteins, could represent a link between the immune and nervous system during early development. Thus, besides the well established role in myelination, these proteins might have an additional and still unknown function in development.

Myelin basic protein immunoreactivity in the human embryonic CNS

Myelin basic protein (MBP) is a major myelin constituent produced by oligodendrocytes in the central nervous system (CNS). Expression of MBP was considered to be a marker for oligodendrocyte differentiation and myelination in the developing CNS. In this study, expression of myelin basic protein (MBP) and its messenger RNA (mRNA) was examined in human embryos and fetuses ranging in age from 5 to 20 gestational weeks (g.w.). We were able to demonstrate that MBP antibody labels cells in both human nervous and non-nervous tissues beginning from early embryonic life (5-6 g.w.). MBP positive (MBP+) cells were rounded, with either no cell processes or only 1-2 short processes, and were located in caudal regions of the CNS. MBP+ cells were also observed in the non-nervous tissue, such as leptomeninges, choroid plexus, and connective tissues. A number of MBP+ cells in nervous and non-nervous tissues were morphologically similar to macrophages and showed a positive reaction to macrophage-microglia markers: lectin (RCA-1) and the monoclonal antibody (EBM-11) to human macrophage antigen CD68, whereas they were negative for neuronal, astroglial, or marker for oligodendrocyte progenitors. At the same embryonic age, 5 g.w. and onward, the MBP mRNA was observed in the CNS by in situ hybridization. The results of this study show that MBP immune reaction is spread in a large area of the CNS prior to myelin appearance. In addition, for the first time it has been demonstrated that the same population of cells could be labelled with both MBP and macrophage markers. These results indicate that MBP, or MBP-related proteins, could represent a link between the immune and nervous system during early development. Thus, besides the well established role in myelination, these proteins might have an additional and still unknown function in development.

[Homocystinuria--biochemical, clinical and genetic aspects]. / Homocistinurija--biohemijski, klinicki i genetski aspekti.

Homocystinuria is a rare autosomal recessive disease characterized by homocystinuria and multisystemic clinical disorders. The term denotes a biochemical abnormality of methionine metabolism caused both by transsulfuration pathway disorders and remethylation of homocysteine into methionine, and as such it can be a result of numerous specific and different genetic lesions. Homocystinuria is most commonly caused by deficiency of cystathionine beta-synthase (CBS) activity (EC 4.2.1.22). In this lesion, depending on specific characteristics of mutant enzyme molecules, in regard to existence of residual activity, responsive and nonresponsive homocystinuria can be differed regarding clinical response to high doses of pyridoxine. Although there are numerous different clinical abnormalities, changes on four organ systems are dominant. The most common symptoms of homocystinuria include lens dislocation, vascular disorders, skeletal abnormalities and mental retardation. Laboratory findings are the first diagnostic procedure, while determination of enzymatic activity is a direct parameter for making diagnosis. Prenatal diagnosis and early detection are extremely important for the course and prognosis of the disease as they enable application of currently available therapy as soon as possible. The presently available therapy can, only in such cases, prevent occurrence of serious clinical symptoms, prevent their advancement to some extent or improve reversible clinical manifestations.

Intracellular transport of the DM-20 bearing shaking pup (shp) mutation and its possible phenotypic consequences.

Paralytic tremor (pt) in rabbits and shaking pup (shp) in dogs are allelic dysmyelinated mutants of the proteolipid protein (Plp) gene. Both mutations affect the same amino acid, histidine36, which is replaced by glutamine in pt and by proline in shp. Phenotypic expression of these two mutations is very different. Paralytic tremor presents a much milder form of dysmyelination than shaking pup. The number of oligodendrocytes in the mutant rabbit is normal, while in the dog, the oligodendrocyte number is reduced due to early death or incomplete maturation. We have previously reported an abnormal intracellular transport of the PLPpt, whereas DM-20pt was normally transported to the cell membrane. In the present study, we show that the transport of the two isoforms containing the shp mutation is impaired in transfected Cos-7 cells. Cotransfecting cells with different ratios and combinations of mutated PLP and DM-20 cDNAs, we demonstrated that DM-20pt, but not DM-20shp, facilitates intracellular trafficking and integration into the plasma membrane of either of the two mutated PLPs. The phenotypic difference between these two allelic mutations can result from differences in DM-20 protein trafficking and sorting. These results show that the loss of function of PLP is not position-dependent but depends on the nature of the mutation.

An antisense transgenic strategy to inhibit the myelin oligodendrocyte glycoprotein synthesis.

To understand the function of the myelin oligodendrocyte glycoprotein (MOG), a myelin specific protein of the central nervous system, transgenic mice were produced. The transgene is a fusion gene containing 1.9 kb of murine myelin basic protein promoter, 430 bp of rat MOG cDNA in the reverse orientation and 4.5 kb of human proteolipid protein gene. In spite of high expression of antisense MOG mRNA in the oligodendrocytes, MOG synthesis was not inhibited in transgenic mice. This lack of inhibition of MOG underlines the difficulties encountered with antisense transgenic strategies.

Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells.

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport.

Paralytic tremor (pt): a new allele of the proteolipid protein gene in rabbits.

Paralytic tremor (pt) is a sex-linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents approximately 30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP- and DM-20-specific messenger RNAs. Our results showed that both PLP and DM-20 are affected and that the ratio DM-20/PLP was higher in pt rabbits than in age-matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T-->A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.

Myelin proteolipid protein mutation in the rabbit: a new model of Pelizaeus-Merzbacher disease.

Proteolipid protein (PLP) is a major myelin protein of the central nervous system. Mutations of the Plp gene are responsible for a number of sex-linked disorders in humans (Pelizaeus-Merzbacher disease) and in animals. We have identified a novel mutation of the Plp gene which gives rise to the paralytic tremor (pt) phenotype in rabbit. Pt rabbits are hypomyelinated and present very low levels of PLP protein and its mRNA. Sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. Histidine36 is positioned at the boundary of the first transmembrane domain. Therefore, its position can be crucial for the efficient interaction of PLP with other proteins and lipids, and for correct incorporation into the membrane.

Paralytic tremor (pt) rabbit: a sex-linked mutation affecting proteolipid protein-gene expression.

Paralytic tremor (pt) is a neurological sex-linked recessive mutation in rabbits which is characterized by a coarse body tremor and limb paresis. Morphological studies showed that this mutation affects CNS myelination. Although the number of oligodendrocytes is not reduced, myelination is slower, irregular and defective. We have made a biochemical and molecular analysis of 4-wk-old mutant and normal rabbits. The amount of myelin in the mutant represents only approximately 25% of the normal level. Radioimmunoassay for myelin basic protein showed a reduction to approximately 40% in pt whole-brain homogenate but the difference was not significant in purified myelin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of brain homogenates followed by immunoblotting showed that all major myelin proteins are affected by the pt mutation, although to different degrees. While most of the myelin proteins are reduced to approximately 60-80% of the normal level, an important reduction to approximately 30%, was measured for the proteolipid protein (PLP). In purified myelin, the difference in PLP concentration was significant while the other specific proteins were less affected. A similar reduction in myelin-protein gene expression was detected at the mRNA level. Sex-linked transmission, low concentrations of PLP and its specific mRNA in the CNS indicate that the pt mutation primarily affects the expression of the Plp gene.

Developmental expression of major myelin proteins in hypomyelinated pt mutant rabbit.

A term "paralytic tremor" (pt) is attributed to a neurological mutation of Chinchilla rabbits, affecting the development of the central nervous system (CNS). A quantification of myelin protein content indicates the strong CNS hypomyelination during the development (1-120 postnatal days). SDS-PAGE electrophoresis of total brain homogenates, followed by immunoblotting, shows a reduced concentration of major myelin-connected proteins. MBP deficiency corresponds approximately to the level of the hypomyelination, whereas PLP expression is drastically reduced.

Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.

If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin-associated glycoprotein (Mag), proteolipid protein (Plp), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half-lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein-specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.