Impairing committed cholesterol biosynthesis in white matter astrocytes, but not grey matter astrocytes, enhances in vitro myelination

Remyelination is a regenerative process that is essential to recover saltatory conduction and to prevent neurodegeneration upon demyelination. The formation of new myelin involves the differentiation of oligodendrocyte progenitor cells (OPCs) toward oligodendrocytes and requires high amounts of cholesterol. Astrocytes (ASTRs) modulate remyelination by supplying lipids to oligodendrocytes. Remarkably, remyelination is more efficient in grey matter (GM) than in white matter (WM), which may relate to regional differences in ASTR subtype. Here, we show that a feeding layer of gmASTRs was more supportive to in vitro myelination than a feeding layer of wmASTRs. While conditioned medium from both gmASTRs and wmASTRs accelerated gmOPC differentiation, wmOPC differentiation is enhanced by secreted factors from gmASTRs, but not wmASTRs. In vitro analyses revealed that gmASTRs secreted more cholesterol than wmASTRs. Cholesterol efflux from both ASTR types was reduced upon exposure to pro-inflammatory cytokines, which was mediated via cholesterol transporter ABCA1, but not ABCG1, and correlated with a minor reduction of myelin membrane formation by oligodendrocytes. Surprisingly, a wmASTR knockdown of Fdft1 encoding for squalene synthase (SQS), an enzyme essential for the first committed step in cholesterol biosynthesis, enhanced in vitro myelination. Reduced secretion of interleukin-1β likely by enhanced isoprenylation, and increased unsaturated fatty acid synthesis, both pathways upstream of SQS, likely masked the effect of reduced levels of ASTR-derived cholesterol. Hence, our findings indicate that gmASTRs export more cholesterol and are more supportive to myelination than wmASTRs, but specific inhibition of cholesterol biosynthesis in ASTRs is beneficial for wmASTR-mediated modulation of myelination. (Figure presented.)

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC),and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS),while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

AbstractThe only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage

The Neurospora mitochondrial genome:the region coding for the polycistronic cytochrome oxidase subunit I transcript is preceded by a transfer RNA gene

AbstractWe have sequenced a 682 bp fragment of Neurospora crassa mitochondrial DNA to complete the sequence between the gene for cytochrome b and the unassigned reading frame, URF U. The sequence contains a gene for a cysteine tRNA. The 5' end of the 6 kb polycistronic transcript of cytochrome c oxidase subunit 1 is immediately downstream from this tRNA. This shows that also in fungal mitochondria tRNAs can be used as processing signals, whereas palindromic sequences containing double Pst I sites, also present in this region, are not used for processing

Transcriptional Expression of Myelin Basic Protein in Oligodendrocytes Depends on Functional Syntaxin 4:a Potential Correlation with Autocrine Signaling

Myelination of axons by oligodendrocytes is essential for saltatory nerve conduction. To form myelin membranes, a coordinated synthesis and subsequent polarized transport of myelin components are necessary. Here, we show that as part of the mechanism to establish membrane polarity, oligodendrocytes exploit a polarized distribution of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery components syntaxins 3 and 4, localizing to the cell body and the myelin membrane, respectively. Our data further reveal that the expression of myelin basic protein (MBP), a myelin-specific protein that is synthesized "on site" after transport of its mRNA, depends on the correct functioning of the SNARE machinery, which is not required for mRNA granule assembly and transport per se. Thus, downregulation and overexpression of syntaxin 4 but not syntaxin 3 in oligodendrocyte progenitor cells but not immature oligodendrocytes impeded MBP mRNA transcription, thereby preventing MBP protein synthesis. The expression and localization of another myelin-specific protein, proteolipid protein (PLP), was unaltered. Strikingly, conditioned medium obtained from developing oligodendrocytes was able to rescue the block of MBP mRNA transcription in syntaxin 4-downregulated cells. These findings indicate that the initiation of the biosynthesis of MBP mRNA relies on a syntaxin 4-dependent mechanism, which likely involves activation of an autocrine signaling pathway

Addition of PTK787/ZK 222584 can lower the dosage of amsacrine to achieve equal amounts of acute myeloid leukemia cell death

Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express the vascular endothelial growth factors, VEGFA and VEGFC, as well as kinase insert domain-containing receptor (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. This study investigates the role of the VEGFR inhibitor PTK787/ZK 222584 in leukemic cell death, and the possibility of an additional effect on cell death by a chemotherapeutic drug, amsacrine. In three AML cell lines and 33 pediatric AML patient samples, we performed total cell-kill assays to determine the percentages of cell death achieved by PTK787/ZK 222584 and/or amsacrine. Both drugs induced AML cell death. Using a response surface analysis, we could show that, in cell lines as well as in primary AML blasts, an equal magnitude of leukemic cell death could be obtained when lower doses of the more toxic amsacrine were combined with low dosages of the less toxic VEGFR inhibitor. This study shows that PTK787/ ZK 222584 might have more clinical potential in AML when combined with a chemotherapeutic drug such as amsacrine. In future, it will be interesting to study whether the complications and the long-term effects of chemotherapy can be reduced by lowering the dosages of amsacrine, and by replacing it with other drugs with lower toxicity profiles, such as PTK787/ZK 222584

MMP7 cleaves remyelination-impairing fibronectin aggregates and its expression is reduced in chronic multiple sclerosis lesions

Upon demyelination, transient expression of fibronectin precedes successful remyelination. However, in chronic demyelination observed in multiple sclerosis (MS), aggregates of fibronectin persist and contribute to remyelination failure. Accordingly, removing fibronectin (aggregates) would constitute an effective strategy for promoting remyelination. Matrix metalloproteinases (MMPs) are enzymes known to remodel extracellular matrix components, including fibronectin. Here, we examined the ability of MMPs to degrade fibronectin aggregates. Our findings reveal that MMP7 cleaved fibronectin aggregates resulting into a prominent 13kDa EIIIA (16kDa EDA)-containing fragment. MMP7 was upregulated during lysolecithin-induced demyelination, indicating its potential for endogenous fibronectin clearance. In contrast, the expression of proMMP7 was substantially decreased in chronic active and inactive MS lesions compared with control white matter and remyelinated MS lesions. Microglia and macrophages were major cellular sources of proMMP7 and IL-4-activated, but not IFN+LPS-activated, microglia and macrophages secreted significant levels of proMMP7. Also, conditioned medium of IL-4-activated macrophages most efficiently cleaved fibronectin aggregates upon MMP-activating conditions. Yet, coatings of MMP7-cleaved fibronectin aggregate fragments inhibited oligodendrocyte maturation, indicating that further degradation and/or clearance by phagocytosis is essential. These findings suggest that MMP7 cleaves fibronectin aggregates, while reduced (pro)MMP7 levels in MS lesions contribute to their persistent presence. Therefore, upregulating MMP7 levels may be key to remove remyelination-impairing fibronectin aggregates in MS lesions

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A:Implications for Multiple Sclerosis

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro, and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factorcAMPresponse element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination

Testing the effectiveness of a mindfulness-based intervention to reduce emotional distress in outpatients with diabetes (DiaMind): design of a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Approximately 20-40% of outpatients with diabetes experience elevated levels of emotional distress, varying from disease-specific distress to general symptoms of anxiety and depression. The patient's emotional well-being is related to other unfavorable outcomes, like reduced quality of life, sub-optimal self-care, impaired glycemic control, higher risk of complications, and increased mortality rates. The purpose of this study is to test the effectiveness of a new diabetes-specific, mindfulness-based psychological intervention. First, with regard to reducing emotional distress; second, with respect to improving quality of life, dispositional mindfulness, and self-esteem of patients with diabetes; third, with regard to self-care and clinical outcomes; finally, a potential effect modification by clinical and personality characteristics will be explored.</p> <p>Methods/Design</p> <p>The Diabetes and Mindfulness study (DiaMind) is a randomized controlled trial. Patients with diabetes with low levels of emotional well-being will be recruited from outpatient diabetes clinics. Eligible patients will be randomized to an intervention group or a wait-list control group. The intervention group will receive the mindfulness program immediately, while the control group will receive the program eight months later. The primary outcome is emotional distress (anxiety, stress, depressive symptoms), for which data will be collected at baseline, four weeks, post intervention, and after six months follow-up. In addition, self-report data will be collected on quality of life, dispositional mindfulness, self-esteem, self-care, and personality, while complications and glycemic control will be assessed from medical files and blood pressure will be measured. Group differences will be analyzed with repeated measures analysis of covariance.</p> <p>The study is supported by grants from the Dutch Diabetes Research Foundation and Tilburg University and has been approved by a medical ethics committee.</p> <p>Discussion</p> <p>It is hypothesized that emotional well-being, quality of life, dispositional mindfulness, self-esteem, self-care, and blood pressure will improve significantly more in the mindfulness group compared to the control group. Results of this study can contribute to a better care for patients with diabetes with lowered levels of emotional well-being. It is expected that the first results will become available in 2012.</p> <p>Trial registration</p> <p>Dutch Trial Register <a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2145">NTR2145</a>.</p