Placental DNA methylation marks are associated with maternal depressive symptoms during early pregnancy.

Maternal depressive symptoms during pregnancy are a significant risk factor for adverse developmental and health outcomes of the offspring. The molecular mechanisms mediating the long-term effects of this exposure are not well understood. Previous studies have found association between prenatal exposure to maternal psychological distress and placental DNA methylation of candidate genes, which can influence placental barrier function and development of the fetus. Our objective in this study was to determine epigenome wide association of maternal depressive symptoms in early pregnancy with the placental DNA methylation. For this purpose we examined DNA methylomes of 92 placental samples by using reduced representation bisulfite sequencing. The placental samples were collected after deliveries of 39 girls and 59 boys, whose mothers had Edinburgh Postnatal Depression Score ranging from 0 to 19 at gestational week 14. According to our results maternal depressive symptoms are associated with DNA methylation of 2833 CpG sites, which are particularly over-represented in genic enhancers. The genes overlapping or nearest to these sites are functionally enriched for development of neurons and show expression enrichment in several regions of developing brain. The genomic regions harboring the DNA methylation marks are enriched for single nucleotide polymorphisms associated with mental disease trait class. Potential cellular signaling cascades mediating the effects include inflammatory and hormonal pathways. As a conclusion our results suggest that maternal depressive symptoms during early pregnancy are associated with DNA methylation marks in placenta in genes, which are important for the development and long-term health of the brain. Whether similar marks can be detected in exposed children remains to be elucidated in further studies.

Mechanisms of Transthyretin Inhibition of IAPP Amyloid Formation.

Amyloid-formation by the islet amyloid polypeptide (IAPP), produced by the ß-cells in the human pancreas, has been associated with the development of type II diabetes mellitus (T2DM). The human plasma-protein transthyretin (TTR), a well-known amyloid-inhibiting protein, is interestingly also expressed within the IAPP producing ß-cells. In the present study, we have characterized the ability of TTR to interfere with IAPP amyloid-formation, both in terms of its intrinsic stability as well as with regard to the effect of TTR-stabilizing drugs. The results show that TTR can prolong the lag-phase as well as impair elongation in the course of IAPP-amyloid formation. We also show that the interfering ability correlates inversely with the thermodynamic stability of TTR, while no such correlation was observed as a function of kinetic stability. Furthermore, we demonstrate that the ability of TTR to interfere is maintained also at the low pH environment within the IAPP-containing granules of the pancreatic ß-cells. However, at both neutral and low pH, the addition of TTR-stabilizing drugs partly impaired its efficacy. Taken together, these results expose mechanisms of TTR-mediated inhibition of IAPP amyloid-formation and highlights a potential therapeutic target to prevent the onset of T2DM.

Longitudinal Imaging Using PET/CT with Collagen-I PET-Tracer and MRI for Assessment of Fibrotic and Inflammatory Lesions in a Rat Lung Injury Model.

Non-invasive imaging biomarkers (IBs) are warranted to enable improved diagnostics and follow-up monitoring of interstitial lung disease (ILD) including drug-induced ILD (DIILD). Of special interest are IB, which can characterize and differentiate acute inflammation from fibrosis. The aim of the present study was to evaluate a PET-tracer specific for Collagen-I, combined with multi-echo MRI, in a rat model of DIILD. Rats were challenged intratracheally with bleomycin, and subsequently followed by MRI and PET/CT for four weeks. PET imaging demonstrated a significantly increased uptake of the collagen tracer in the lungs of challenged rats compared to controls. This was confirmed by MRI characterization of the lesions as edema or fibrotic tissue. The uptake of tracer did not show complete spatial overlap with the lesions identified by MRI. Instead, the tracer signal appeared at the borderline between lesion and healthy tissue. Histological tissue staining, fibrosis scoring, lysyl oxidase activity measurements, and gene expression markers all confirmed establishing fibrosis over time. In conclusion, the novel PET tracer for Collagen-I combined with multi-echo MRI, were successfully able to monitor fibrotic changes in bleomycin-induced lung injury. The translational approach of using non-invasive imaging techniques show potential also from a clinical perspective.

Increased chronic pain in patients with ulcerative colitis is mostly associated to increased disease activity. A cross-sectional case-control study.

BACKGROUNDS/AIMS: Musculoskeletal symptoms are common in patients with ulcerative colitis (UC), but no study has compared the prevalence of chronic pain to controls from a general population. METHODS: Patients with UC (n = 1164) and controls (n = 3867) were sent questionnaires comprising demography, history of pain, pain localization and UC patients' Patient-Simple Clinical Colitis Activity Index. Chronic regional pain (ChRP) and chronic widespread pain (ChWP) were defined as having pain for at least 3 months. RESULTS: The response rate for the patients with UC was 49.0% and for the control persons 61.7% (p < .001). The reported prevalence of ChRP and ChWP was higher in patients with UC versus controls (33.1% vs. 24.2%; p < .001 and 19.8% vs. 12.5%; p < .001). The patients with UC reported significantly more pain in the regions 'lower back', 'hip/upper leg' and 'lower leg/foot' compared to controls. The patients with P-SCCAI ≥ 5 (n = 121) reported more ChWP than patients with P-SCCAI <5 (n = 426) (46.3% vs. 12.7%; p < .001) and controls (n = 2425) (46.3 vs. 12.5%; p < .001) in all body regions. No significant difference in ChWP was found between patients with P-SCCAI <5 and controls (12.7% vs. 12.5%; p = .917). CONCLUSIONS: Patients with UC reported more chronic pain than controls from the general population, especially from the lower back and hip region. Higher UC disease activity was associated with more pain in all body regions.

Apolipoprotein E Interferes with IAPP Aggregation and Protects Pericytes from IAPP-Induced Toxicity.

Apolipoprotein E (ApoE) has become a primary focus of research after the discovery of its strong linkage to Alzheimer's disease (AD), where the ApoE4 variant is the highest genetic risk factor for this disease. ApoE is commonly found in amyloid deposits of different origins, and its interaction with amyloid-ß peptide (Aß), the hallmark of AD, is well known. However, studies on the interaction of ApoEs with other amyloid-forming proteins are limited. Islet amyloid polypeptide (IAPP) is an amyloid-forming peptide linked to the development of type-2 diabetes and has also been shown to be involved in AD pathology and vascular dementia. Here we studied the impact of ApoE on IAPP aggregation and IAPP-induced toxicity on blood vessel pericytes. Using both in vitro and cell-based assays, we show that ApoE efficiently inhibits the amyloid formation of IAPP at highly substoichiometric ratios and that it interferes with both nucleation and elongation. We also show that ApoE protects the pericytes against IAPP-induced toxicity, however, the ApoE4 variant displays the weakest protective potential. Taken together, our results suggest that ApoE has a generic amyloid-interfering property and can be protective against amyloid-induced cytotoxicity, but there is a loss of function for the ApoE4 variant.

Apolipoprotein E impairs amyloid-ß fibril elongation and maturation.

Alzheimer's disease (AD) is strongly linked to amyloid depositions of the Aß peptide (Aß). The lipid-binding protein apolipoprotein E (ApoE) has been found to interfere with Aß amyloid formation and to exert a strong clinical impact to the pathology of AD. The APOE gene exists in three allelic isoforms represented by APOE ε2, APOE ε3, and APOE ε4. Carriers of the APOE ε4 variant display a gene dose-dependent increased risk of developing the disease. Aß amyloids are formed via a nucleation-dependent mechanism where free monomers are added onto a nucleus in a template-dependent manner. Using a combination of surface plasmon resonance and thioflavin-T assays, we here show that ApoE can target the process of fibril elongation and that its interference effectively prevents amyloid maturation. We expose a complex equilibrium where the concentration of ApoE, Aß monomers, and the amount of already formed Aß fibrils will affect the relative proportion and formation rate of mature amyloids versus alternative assemblies. The result illustrates a mechanism which may affect both the clearance rate of Aß assemblies in vivo and the population of cytotoxic Aß assemblies.

p120ctn-Mediated Organ Patterning Precedes and Determines Pancreatic Progenitor Fate.

The mechanism of how organ shape emerges and specifies cell fate is not understood. Pancreatic duct and endocrine lineages arise in a spatially distinct domain from the acinar lineage. Whether these lineages are pre-determined or settle once these niches have been established remains unknown. Here, we reconcile these two apparently opposing models, demonstrating that pancreatic progenitors re-localize to establish the niche that will determine their ultimate fate. We identify a p120ctn-regulated mechanism for coordination of organ architecture and cellular fate mediated by differential E-cadherin based cell sorting. Reduced p120ctn expression is necessary and sufficient to re-localize a subset of progenitors to the peripheral tip domain, where they acquire an acinar fate. The same mechanism is used re-iteratively during endocrine specification, where it balances the choice between the alpha and beta cell fates. In conclusion, organ patterning is regulated by p120ctn-mediated cellular positioning, which precedes and determines pancreatic progenitor fate.

The immunomodulatory quinoline-3-carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis.

Quinoline-3-carboxamides (Q substances) are small molecule compounds with anti-inflammatory properties. In this study, we used one of these substances, Paquinimod, to treat a novel model for chronic liver inflammation and liver fibrosis, the NOD-Inflammation Fibrosis (N-IF) mouse. We show that treatment of N-IF mice significantly reduced inflammation and resulted in the regression of fibrosis, even when the treatment was initiated after onset of disease. The reduced disease phenotype was associated with a systemic decrease in the number and reduced activation of disease-promoting transgenic natural killer T (NKT)-II cells and their type 2-cytokine expression profile. Paquinimod treatment also led to a reduction of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages.

Transthyretin Interferes with Aß Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates.

The pathological Aß aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aß peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aß fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aß.

Deficiency in plasmacytoid dendritic cells and type I interferon signalling prevents diet-induced obesity and insulin resistance in mice.

AIMS/HYPOTHESIS: Obesity is associated with glucose intolerance and insulin resistance and is closely linked to the increasing prevalence of type 2 diabetes. In mouse models of diet-induced obesity (DIO) and type 2 diabetes, an increased fat intake results in adipose tissue expansion and the secretion of proinflammatory cytokines. The innate immune system not only plays a crucial role in obesity-associated chronic low-grade inflammation but it is also proposed to play a role in modulating energy metabolism. However, little is known about how the modulation of metabolism by the immune system may promote increased adiposity in the early stages of increased dietary intake. Here we aimed to define the role of type I IFNs in DIO and insulin resistance. METHODS: Mice lacking the receptor for IFN-α (IFNAR-/-) and deficient in plasmacytoid dendritic cells (pDCs) (B6.E2-2 fl/fl .Itgax-cre) were fed a diet with a high fat content or normal chow. The mice were analysed in vivo and in vitro using cellular, biochemical and molecular approaches. RESULTS: We found that the development of obesity was inhibited by an inability to respond to type I IFNs. Furthermore, the development of obesity and insulin resistance in this model was associated with pDC recruitment to the fatty tissues and liver of obese mice (a 4.3-fold and 2.7-fold increase, respectively). Finally, we demonstrated that the depletion of pDCs protects mice from DIO and from developing obesity-associated metabolic complications. CONCLUSIONS/INTERPRETATION: Our results provide genetic evidence that pDCs, via type I IFNs, regulate energy metabolism and promote the development of obesity.

A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis.

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.

E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes.

Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic ß-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD) mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs) to NOD islets around 8-9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse.

Global and 3D spatial assessment of neuroinflammation in rodent models of Multiple Sclerosis.

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.

Imaging dynamics of CD11c⁺ cells and Foxp3⁺ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes. METHODS: We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets. RESULTS: We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour. CONCLUSIONS/INTERPRETATION: Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.

Drosophila cdk5 is needed for locomotive behavior and NMJ elaboration, but seems dispensable for synaptic transmission.

Cyclin-dependent kinase 5 (Cdk5) functions in postmitotic neuronal cells and play roles in cell differentiation, cell migration, axonal guidance, and synaptic function. Here, we demonstrate that Drosophila cdk5 is dispensable for adult viability and fertility, a feature that allows us to study its physiological function in the whole animal model. For the adult, cdk5 is needed for proper locomotion and flight performance. Larvae lacking cdk5 in the presynaptic tissue display abnormal crawling motion, and their neuromuscular junctions (NMJ) are elongated and contain a higher number of boutons that are smaller. As a result of these two counteracting effects, the total synaptic area/NMJ is similar to wild type, leading to normal synaptic transmission, indicating that a compensatory mechanism is capable of correcting the problem caused by the lack of cdk5. futsch, the Drosophila MAP1B homolog, is also involved in NMJ morphogenesis, and analysis of the NMJ phenotype of the double mutant futsch(K68); cdk5(-) indicates that cdk5 is epistatic to futsch in this process.

Production, characterization and crystallization of the Plasmodium falciparum aquaporin.

The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae.

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use.

Aquaporins in yeasts and filamentous fungi.

Recently, genome sequences from different fungi have become available. This information reveals that yeasts and filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budding yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. Whereas Aqy1 is a spore-specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmotic signals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the otherwise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1-like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, plays a central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gating have been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has a long N-terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins, only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi are a fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compare orthologous sequences from a large number of different organisms facilitates functional and structural studies.

Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling.

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Delta gpd2Delta mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Delta gpd2Delta mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Delta1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Delta1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Delta1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Delta gpd2Delta mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Delta gpd2Delta mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.