Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice.

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.

Brain cell type-specific endocytosis of arylsulfatase A identifies limitations of enzyme-based therapies for metachromatic leukodystrophy.

Enzyme replacement therapies, allogeneic bone marrow transplantation and gene therapies are treatment options for lysosomal storage diseases caused by inherited deficiencies of soluble lysosomal enzymes. Independent from the approach, the enzyme must be delivered to lysosomes of deficient patient cells. Little is known about the dissemination of enzyme within a tissue where cells compete for uptake via different receptor systems, binding affinities and endocytic rates. To evaluate dissemination and lysosomal targeting of a lysosomal enzyme in the CNS, we analysed receptor-mediated endocytosis of arylsulfatase A (ASA) by different types of brain-derived cell lines and primary murine brain cells. For ASA expressed by chinese hamster ovary cells for enzyme replacement therapy of metachromatic leukodystrophy, endocytic rates decline from microglia to neurons and astrocytes and to oligodendrocytes. Only immature oligodendrocytes endocytose significant amounts of enzyme. Uptake by non-microglial cells is due to mannose 6-phosphate receptors, whereas several receptor systems participate in endocytosis by microglial cells. Interestingly, ASA expressed by microglial cells cannot be taken up in a mannose 6-phosphate dependent manner. The resulting failure to correct non-microglial cells corroborates in vivo data and indicates that therapeutic effects of allogeneic bone marrow transplantation and hematopoietic stem cell gene therapy on metachromatic leukodystrophy are independent of metabolic cross-correction of neurons, astrocytes and oligodendrocytes by receptor-mediated endocytosis.

Spin-Selective Electron Transport Through Single Chiral Molecules.

The interplay between chirality and magnetism is a source of fascination among scientists for over a century. In recent years, chirality-induced spin selectivity (CISS) has attracted renewed interest. It is observed that electron transport through layers of homochiral molecules leads to a significant spin polarization of several tens of percent. Despite the abundant experimental evidence gathered through mesoscopic transport measurements, the exact mechanism behind CISS remains elusive. This study reports spin-selective electron transport through single helical aromatic hydrocarbons that are sublimed in vacuo onto ferromagnetic cobalt surfaces and examined with spin-polarized scanning tunneling microscopy (SP-STM) at a temperature of 5 K. Direct comparison of two enantiomers under otherwise identical conditions revealed magnetochiral conductance asymmetries of up to 50% when either the molecular handedness is exchanged or the magnetization direction of the STM tip or Co substrate is reversed. Importantly, the results rule out electron-phonon coupling and ensemble effects as primary mechanisms responsible for CISS.

Osteomodulin Gene Expression Is Associated With Plaque Calcification, Stability, and Fewer Cardiovascular Events in the CPIP Cohort.

BACKGROUND: Stable atherosclerotic plaques are characterized by thick fibrous caps of smooth muscle cells, collagen, and macrocalcifications. Identifying factors of plaque stability is necessary to design drugs to prevent plaque rupture and symptoms. Osteomodulin, originally identified in bones, is expressed by bone synthesizing osteoblasts and involved in mineralization. In the present study, we analyzed osteomodulin expression in human carotid plaques, its link with plaque phenotype, calcification, and future cardiovascular events. METHODS: Osteomodulin gene expression (OMD; n=82) was determined by RNA sequencing and osteomodulin protein levels by immunohistochemistry (n=45) in carotid plaques obtained by endarterectomy from patients with or without cerebrovascular symptoms from the CPIP (Carotid Plaque Imaging Project) cohort, Skåne University Hospital, Sweden. Plaque components were assessed by immunohistochemistry, RNA sequencing, and multiplex analysis. Patients were followed for cardiovascular events or cardiovascular death during a median of 57 or 70 months, respectively, using national registers. RESULTS: OMD levels were increased in plaques from asymptomatic patients compared to symptomatics. High OMD levels were associated with fewer cardiovascular events during follow-up. OMD correlated positively with smooth muscle α-actin (ACTA2; r=0.73, P=10-13) and collagen (COL1A2; r=0.4, P=0.0002), but inversely with CD68 gene expression (r=-0.67, P=10-11), lipids (r=-0.37, P=0.001), intraplaque hemorrhage (r=-0.32, P=0.010), inflammatory cytokine, and matrix metalloproteinase plaque contents. OMD was positively associated with MSX2 (Msh Homeobox 2) (r=0.32, P=0.003), a marker of preosteoblast differentiation, BMP4 (bone morphogenetic protein) (r=0.50, P=0.000002) and BMP6 (r=0.47, P=0.000007), plaque calcification (r=0.35, P=0.016), and was strongly upregulated in osteogenically stimulated smooth muscle cells, which was further increased upon BMP stimulation. Osteomodulin protein was present in calcified regions. Osteomodulin protein levels were associated with plaque calcification (r=0.41, P=0.006) and increased in macrocalcified plaques. CONCLUSIONS: These data show that osteomodulin mRNA and protein levels are associated with plaque calcification in human atherosclerosis. Furthermore, osteomodulin mRNA, but not protein levels, is associated with plaque stability.

Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure.

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P's availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.

The E3 ubiquitin ligase MID1 catalyzes ubiquitination and cleavage of Fu.

SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control.

Efficacy of enzyme replacement therapy in an aggravated mouse model of metachromatic leukodystrophy declines with age.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency of arylsulfatase A (ASA). Previous studies in ASA-knockout mice suggested enzyme replacement therapy (ERT) to be a promising treatment option. The mild phenotype of ASA-knockout mice did, however, not allow to examine therapeutic responses of the severe neurological symptoms that dominate MLD. We, therefore, generated an aggravated MLD mouse model displaying progressive demyelination and reduced nerve conduction velocity (NCV) and treated it by weekly intravenous injections of 20 mg/kg recombinant human ASA for 16 weeks. To analyze the stage-dependent therapeutic effects, ERT was initiated in a presymptomatic, early and progressed disease stage, at age 4, 8 and 12 months, respectively. Brain sulfatide storage, NCV and behavioral alterations were improved only in early, but not in late, treated mice showing a clear age-dependent efficacy of treatment. Hematopoietic stem cell transplantation (HSCT) for late-onset variants is the only therapeutic option for MLD to date. ERT resembles a part of the HSCT rationale, which is based on ASA supply by donor cells. Beyond ERT, our results, therefore, corroborate the clinical observation that HSCT is only effective when performed in early stages of disease.

Spatiotemporal sphingosine-1-phosphate receptor 3 expression within the cerebral vasculature after ischemic stroke.

Sphingosine-1-phosphate receptors (S1PRs) are promising therapeutic targets in cardiovascular disease, including ischemic stroke. However, important spatiotemporal information for alterations of S1PR expression is lacking. Here, we investigated the role of S1PR3 in ischemic stroke in rodent models and patient samples. We show that S1PR3 is acutely upregulated in perilesional reactive astrocytes after stroke, and that stroke volume and behavioral deficits are improved in mice lacking S1PR3. Further, we find that administration of an S1PR3 antagonist at 4-h post-stroke, but not at later timepoints, improves stroke outcome. Lastly, we observed higher plasma S1PR3 concentrations in experimental stroke and in patients with ischemic stroke. Together, our results establish S1PR3 as a potential drug target and biomarker in ischemic stroke.

Enantioselective Adsorption on Magnetic Surfaces.

From the beginning of molecular theory, the interplay of chirality and magnetism has intrigued scientists. There is still the question if enantiospecific adsorption of chiral molecules occurs on magnetic surfaces. Enantiomer discrimination was conjectured to arise from chirality-induced spin separation within the molecules and exchange interaction with the substrate's magnetization. Here, it is shown that single helical aromatic hydrocarbons undergo enantioselective adsorption on ferromagnetic cobalt surfaces. Spin and chirality sensitive scanning tunneling microscopy reveals that molecules of opposite handedness prefer adsorption onto cobalt islands with opposite out-of-plane magnetization. As mobility ceases in the final chemisorbed state, it is concluded that enantioselection must occur in a physisorbed transient precursor state. State-of-the-art spin-resolved ab initio simulations support this scenario by refuting enantio-dependent chemisorption energies. These findings demonstrate that van der Waals interaction should also include spin-fluctuations which are crucial for molecular magnetochiral processes.

Intracerebroventricular enzyme infusion corrects central nervous system pathology and dysfunction in a mouse model of metachromatic leukodystrophy.

Arylsulfatase A (ASA) catalyzes the desulfation of sulfatide, a major lipid component of myelin. Inherited functional deficiencies of ASA cause the lysosomal storage disease (LSD) metachromatic leukodystrophy (MLD), which is characterized by intralysosomal accumulation of sulfatide, progressive neurological symptoms and early death. Enzyme replacement therapy (ERT) using intravenous injection of active enzyme is a treatment option for many LSDs as exogenous lysosomal enzymes are delivered to lysosomes of patient's cells via receptor-mediated endocytosis. Efficient treatment of MLD and other LSDs with central nervous system (CNS) involvement is, however, hampered by the blood-brain barrier (BBB), which limits transfer of therapeutic enzymes from the circulation to the brain parenchyma. To bypass the BBB, we infused recombinant human ASA (rhASA) by implanted miniature pumps into the cerebrospinal fluid (CSF) of a conventional and a novel, genetically aggravated ASA knockout mouse model of MLD. rhASA continuously delivered to the lateral ventricle for 4 weeks penetrated the brain parenchyma and was targeted to the lysosomes of brain cells. Histological analysis revealed complete reversal of lysosomal storage in the infused hemisphere. rhASA concentrations and sulfatide clearance declined with increasing distance from the infusion site. Correction of the ataxic gait indicated reversal of central nervous system dysfunctions. The profound histopathological and functional improvements, the requirement of low enzyme doses and the absence of immunological side effects suggest intracerebroventricular ERT to be a promising treatment option for MLD and other LSDs with prevailing CNS disease.

Ivacaftor therapy post myocardial infarction augments systemic inflammation and evokes contrasting effects with respect to tissue inflammation in brain and lung.

Acquired cystic fibrosis transmembrane regulator (CFTR) dysfunctions have been associated with several conditions, including myocardial infarction (MI). Here, CFTR is downregulated in brain, heart, and lung tissue and associates with inflammation and degenerative processes. Therapeutically increasing CFTR expression attenuates these effects. Whether potentiating CFTR function yields similar beneficial effects post-MI is unknown. The CFTR potentiator ivacaftor is currently in clinical trials for treatment of acquired CFTR dysfunction associated with chronic obstructive pulmonary disease and chronic bronchitis. Thus, we tested ivacaftor as therapeutic strategy for MI-associated target tissue inflammation that is characterized by CFTR alterations. MI was induced in male C57Bl/6 mice by ligation of the left anterior descending coronary artery. Mice were treated with ivacaftor starting ten weeks post-MI for two consecutive weeks. Systemic ivacaftor treatment ameliorates hippocampal neuron dendritic atrophy and spine loss and attenuates hippocampus-dependent memory deficits occurring post-MI. Similarly, ivacaftor therapy mitigates MI-associated neuroinflammation (i.e., reduces higher proportions of activated microglia). Systemically, ivacaftor leads to higher frequencies of circulating Ly6C+ and Ly6Chi cells compared to vehicle-treated MI mice. Likewise, an ivacaftor-mediated augmentation of MI-associated pro-inflammatory macrophage phenotype characterized by higher CD80-positivity is observed in the MI lung. In vitro, ivacaftor does not alter LPS-induced CD80 and tumor necrosis factor alpha mRNA increases in BV2 microglial cells, while augmenting mRNA levels of these markers in mouse macrophages and differentiated human THP-1-derived macrophages. Our results suggest that ivacaftor promotes contrasting effects depending on target tissue post-MI, which may be largely dependent on its effects on different myeloid cell types.

Transforming growth factor-ß2 is associated with atherosclerotic plaque stability and lower risk for cardiovascular events.

AIMS: Transforming growth factor-beta (TGF-ß) exists in three isoforms TGF-ß1, -ß2, and -ß3. TGF-ß1 has been suggested to be important for maintaining plaque stability, yet the role of TGF-ß2 and -ß3 in atherosclerosis remains to be investigated.This study explores the association of the three isoforms of TGF-ß with plaque stability in the human atherosclerotic disease. METHODS AND RESULTS: TGF-ß1, -ß2, and -ß3 proteins were quantified in 223 human carotid plaques by immunoassays. Indications for the endarterectomy were: symptomatic carotid plaque with stenosis >70% or without symptoms and >80% stenosis. Plaque mRNA levels were assessed by RNA sequencing. Plaque components and extracellular matrix were measured histologically and biochemically. Matrix metalloproteinases and monocyte chemoattractant protein-1 (MCP-1) was measured with immunoassays. The effect of TGF-ß2 on inflammation and protease activity was investigated in vitro using THP-1 and RAW264.7 macrophages. Patients were followed longitudinally for cardiovascular (CV) events.TGF-ß2 was the most abundant isoform and was increased at both protein and mRNA levels in asymptomatic plaques. TGF-ß2 was the main determinant separating asymptomatic plaques in an Orthogonal Projections to Latent Structures Discriminant Analysis. TGF-ß2 correlated positively to features of plaque stability and inversely to markers of plaque vulnerability. TGF-ß2 was the only isoform inversely correlated to the matrix-degrading matrix metalloproteinase-9 and inflammation in the plaque tissue. In vitro, TGF-ß2 pre-treatment reduced MCP-1 gene and protein levels as well as matrix metalloproteinase-9 gene levels and activity. Patients with plaques with high TGF-ß2 levels had a lower risk to suffer from future CV events. CONCLUSIONS: TGF-ß2 is the most abundant TGF-ß isoform in human plaques and may maintain plaque stability by decreasing inflammation and matrix degradation.

Transport of arylsulfatase A across the blood-brain barrier in vitro.

Enzyme replacement therapy is an option to treat lysosomal storage diseases caused by functional deficiencies of lysosomal hydrolases as intravenous injection of therapeutic enzymes can correct the catabolic defect within many organ systems. However, beneficial effects on central nervous system manifestations are very limited because the blood-brain barrier (BBB) prevents the transfer of enzyme from the circulation to the brain parenchyma. Preclinical studies in mouse models of metachromatic leukodystrophy, however, showed that arylsulfatase A (ASA) is able to cross the BBB to some extent, thus reducing lysosomal storage in brain microglial cells. The present study aims to investigate the routing of ASA across the BBB and to improve the transfer in vitro using a well established cell culture model consisting of primary porcine brain capillary endothelial cells cultured on Transwell filter inserts. Passive apical-to-basolateral ASA transfer was observed, which was not saturable up to high ASA concentrations. No active transport could be determined. The passive transendothelial transfer was, however, charge-dependent as reduced concentrations of negatively charged monosaccharides in the N-glycans of ASA or the addition of polycations increased basolateral ASA levels. Adsorptive transcytosis is therefore considered to be the major transport pathway. Partial inhibition of the transcellular ASA transfer by mannose 6-phosphate indicated a second route depending on the insulin-like growth factor II/mannose 6-phosphate receptor, MPR300. We conclude that cationization of ASA and an increase of the mannose 6-phosphate content of the enzyme may promote blood-to-brain transfer of ASA, thus leading to an improved therapeutic efficacy of enzyme replacement therapy behind the BBB.

Deposition of Chiral Heptahelicene Molecules on Ferromagnetic Co and Fe Thin-Film Substrates.

The discovery of chirality-induced spin selectivity (CISS), resulting from an interaction between the electron spin and handedness of chiral molecules, has sparked interest in surface-adsorbed chiral molecules due to potential applications in spintronics, enantioseparation, and enantioselective chemical or biological processes. We study the deposition of chiral heptahelicene by sublimation under ultra-high vacuum onto bare Cu(111), Co bilayer nanoislands on Cu(111), and Fe bilayers on W(110) by low-temperature spin-polarized scanning tunneling microscopy/spectroscopy (STM/STS). In all cases, the molecules remain intact and adsorb with the proximal phenanthrene group aligned parallel to the surface. Three degenerate in-plane orientations on Cu(111) and Co(111), reflecting substrate symmetry, and only two on Fe(110), i.e., fewer than symmetry permits, indicate a specific adsorption site for each substrate. Heptahelicene physisorbs on Cu(111) but chemisorbs on Co(111) and Fe(110) bilayers, which nevertheless remain for the sub-monolayer coverage ferromagnetic and magnetized out-of-plane. We are able to determine the handedness of individual molecules chemisorbed on Fe(110) and Co(111), as previously reported for less reactive Cu(111). The demonstrated deposition control and STM/STS imaging capabilities for heptahelicene on Co/Cu(111) and Fe/W(110) substrate systems lay the foundation for studying CISS in ultra-high vacuum and on the microscopic level of single molecules in controlled atomic configurations.

Increased proteolytic cleavage of osteoglycin is associated with a stable plaque phenotype and lower risk of cardiovascular events.

BACKGROUND AND AIMS: Extracellular matrix (ECM) remodeling is one of the key components in the formation of vulnerable atherosclerotic plaques and cardiovascular events. We recently showed that the full-length ECM-proteoglycan osteoglycin was associated with plaque vulnerability and future cardiovascular events. In the present study, we aimed to investigate the association of cleaved osteoglycin with plaque phenotype. METHODS: Two-hundred human carotid plaques were analyzed by immunohistochemistry. Cleaved osteoglycin and active caspase-3 were assessed by ELISA. ECM components (collagen, elastin and glycosaminoglycans) were assessed by colorimetric assays in plaque tissue homogenates. Matrix metalloproteinases (MMPs) were assessed using Milliplex. MMP-cleavage of osteoglycin and its effect on apoptosis were studied in vitro. Cardiovascular events were recorded during follow-up using national registries. RESULTS: Plaque levels of cleaved osteoglycin were significantly higher in asymptomatic plaques and correlated to α-actin plaque area, collagen, elastin and inversely to lipids, active. caspase-3 and a histological vulnerability index. Cleaved osteoglycin correlated to several MMPs, especially MMP-12, which was also shown to cleave osteoglycin in vitro. In vitro cleavage of osteoglycin was also associated with less smooth muscle cell apoptosis. Patients with high plaque levels of cleaved osteoglycin had a significantly lower risk to suffer from future cardiovascular events. CONCLUSIONS: The current study shows that cleaved osteoglycin is associated with a stable plaque phenotype and lower risk for future cardiovascular events. Potentially due to reduced cell apoptosis and ability to retain LDL. These results indicate that targeting the cleavage of osteoglycin may be a potential therapeutic strategy to stabilize plaques.

Cyclophane with eclipsed pyrene units enables construction of spin interfaces with chemical accuracy.

Advanced functionality in molecular electronics and spintronics is orchestrated by exact molecular arrangements at metal surfaces, but the strategies for constructing such arrangements remain limited. Here, we report the synthesis and surface hybridization of a cyclophane that comprises two pyrene groups fastened together by two ferrocene pillars. Crystallographic structure analysis revealed pyrene planes separated by ∼352 pm and stacked in an eclipsed geometry that approximates the rare configuration of AA-stacked bilayer graphene. We deposited this cyclophane onto surfaces of Cu(111) and Co(111) at submonolayer coverage and studied the resulting hybrid entities with scanning tunnelling microscopy (STM). We found distinct characteristics of this cyclophane on each metal surface: on non-magnetic Cu(111), physisorption occurred and the two pyrene groups remained electronically coupled to each other; on ferromagnetic Co(111) nanoislands, chemisorption occurred and the two pyrene groups became electronically decoupled. Spin-polarized STM measurements revealed that the ferrocene groups had spin polarization opposite to that of the surrounding Co metal, while the pyrene stack had no spin polarization. Comparisons to the non-stacked analogue comprising only one pyrene group bolster our interpretation of the cyclophane's STM features. The design strategy presented herein can be extended to realize versatile, three-dimensional platforms in single-molecule electronics and spintronics.

Simvastatin therapy attenuates memory deficits that associate with brain monocyte infiltration in chronic hypercholesterolemia.

Evidence associates cardiovascular risk factors with unfavorable systemic and neuro-inflammation and cognitive decline in the elderly. Cardiovascular therapeutics (e.g., statins and anti-hypertensives) possess immune-modulatory functions in parallel to their cholesterol- or blood pressure (BP)-lowering properties. How their ability to modify immune responses affects cognitive function is unknown. Here, we examined the effect of chronic hypercholesterolemia on inflammation and memory function in Apolipoprotein E (ApoE) knockout mice and normocholesterolemic wild-type mice. Chronic hypercholesterolemia that was accompanied by moderate blood pressure elevations associated with apparent immune system activation characterized by increases in circulating pro-inflammatory Ly6Chi monocytes in ApoE-/- mice. The persistent low-grade immune activation that is associated with chronic hypercholesterolemia facilitates the infiltration of pro-inflammatory Ly6Chi monocytes into the brain of aged ApoE-/- but not wild-type mice, and links to memory dysfunction. Therapeutic cholesterol-lowering through simvastatin reduced systemic and neuro-inflammation, and the occurrence of memory deficits in aged ApoE-/- mice with chronic hypercholesterolemia. BP-lowering therapy alone (i.e., hydralazine) attenuated some neuro-inflammatory signatures but not the occurrence of memory deficits. Our study suggests a link between chronic hypercholesterolemia, myeloid cell activation and neuro-inflammation with memory impairment and encourages cholesterol-lowering therapy as safe strategy to control hypercholesterolemia-associated memory decline during ageing.

Plasma S1P (Sphingosine-1-Phosphate) Links to Hypertension and Biomarkers of Inflammation and Cardiovascular Disease: Findings From a Translational Investigation.

[Figure: see text].

Site-specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme arylsulfatase A (ASA). Enzyme replacement therapy (ERT) is a therapeutic option for MLD and other lysosomal disorders. This therapy depends on N-linked oligosaccharide-mediated delivery of intravenously injected recombinant enzyme to the lysosomes of patient cells. Because of the importance of N-linked oligosaccharide side chains in ERT, we examined the composition of the three N-linked glycans of four different recombinant ASAs in a site-specific manner. Depending on the culture conditions and the cell line expressing the enzyme, we detected a high variability of the high-mannose-type N-glycans which prevail at all glycosylation sites. Our data show that the composition of the glycans is largely determined by substantial trimming in the medium. The susceptibility for trimming is different for the glycans at the three N-glycosylation sites. Interestingly, which of the glycans is most susceptible to trimming also depends on production conditions. CHO cells cultured under bioreactor conditions yielded recombinant ASA with the most preserved N-glycan structures, the highest mannose-6-phosphate content and the highest similarity to non-recombinant enzyme. Notably, roughly one-third of the N-glycans released from the three glycosylation sites were fucosylated. In the last years, numerous recombinant lysosomal enzymes were used for preclinical ERT trials. Our data show that the oligosaccharide structures were very different in these trials making it difficult to draw common conclusions from the various investigations.

Inhibition of transcription and DNA replication by the papillomavirus E8-E2C protein is mediated by interaction with corepressor molecules.

Papillomavirus genomes replicate as nuclear plasmids at a low copy number in undifferentiated keratinocytes. Papillomaviruses encode the E1 and E2 proteins that bind to the origin of replication and are required for the activation of replication. In addition to E2, several papillomaviruses express an E8-E2C protein, which is generated by alternative splicing and functions as a transcriptional repressor and inhibitor of the E1/E2-dependent replication of the viral origin. Previous analyses suggested that the E8 domain functions as a transferable repression domain. In this report we present evidence that the E8 domain is responsible for the interaction with cellular corepressor molecules such as histone deacetylases, the histone methyltransferase SETDB1, and the TRIM28/KAP-1/TIF1beta/KRIP-1 protein. Whereas the interaction with histone deacetylases is involved only in transcriptional repression, the interaction with TRIM28/KAP-1/TIF1beta/KRIP-1 contributes to the inhibition of E1/E2-dependent replication. The corepressor TRIM28/KAP-1/TIF1beta/KRIP-1 has been described to be part of multicomponent complexes involved in transcriptional regulation and functions as a scaffold protein. Since neither histone deacetylases nor the histone methyltransferase SETDB1 appears to be involved in the inhibition of E1/E2-dependent replication, most likely the modification of non-histone proteins contributes to the replication repression activity of E8-E2C.