Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Quality and quantity of genetic relatedness data affect the analysis of social structure

Kinship plays a fundamental role in the evolution of social systems and is considered a key driver of group living. To understand the role of kinship in the formation and maintenance of social bonds, accurate measures of genetic relatedness are critical. Genotype‐by‐sequencing technologies are rapidly advancing the accuracy and precision of genetic relatedness estimates for wild populations. The ability to assign kinship from genetic data varies depending on a species’ or population's mating system and pattern of dispersal, and empirical data from longitudinal studies are crucial to validate these methods. We use data from a long‐term behavioural study of a polygynandrous, bisexually philopatric marine mammal to measure accuracy and precision of parentage and genetic relatedness estimation against a known partial pedigree. We show that with moderate but obtainable sample sizes of approximately 4,235 SNPs and 272 individuals, highly accurate parentage assignments and genetic relatedness coefficients can be obtained. Additionally, we subsample our data to quantify how data availability affects relatedness estimation and kinship assignment. Lastly, we conduct a social network analysis to investigate the extent to which accuracy and precision of relatedness estimation improve statistical power to detect an effect of relatedness on social structure. Our results provide practical guidance for minimum sample sizes and sequencing depth for future studies, as well as thresholds for post hoc interpretation of previous analyses.     

Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals

Numerous studies have reported correlations between the heterozygosity of genetic markers and fitness. These heterozygosity–fitness correlations (HFCs) play a central role in evolutionary and conservation biology, yet their mechanistic basis remains open to debate. For example, fitness associations have been widely reported at both neutral and functional loci, yet few studies have directly compared the two, making it difficult to gauge the relative contributions of genome‐wide inbreeding and specific functional genes to fitness. Here, we compared the effects of neutral and immune gene heterozygosity on death from bacterial infection in Antarctic fur seal (Arctocephalus gazella) pups. We specifically developed a panel of 13 microsatellites from expressed immune genes and genotyped these together with 48 neutral loci in 234 individuals, comprising 39 pups that were classified at necropsy as having most likely died of bacterial infection together with a five times larger matched sample of healthy surviving pups. Identity disequilibrium quantified from the neutral markers was positive and significant, indicative of variance in inbreeding within the study population. However, multilocus heterozygosity did not differ significantly between healthy and infected pups at either class of marker, and little evidence was found for fitness associations at individual loci. These results support a previous study of Antarctic fur seals that found no effects of heterozygosity at nine neutral microsatellites on neonatal survival and thereby help to refine our understanding of how HFCs vary across the life cycle. Given that nonsignificant HFCs are underreported in the literature, we also hope that our study will contribute toward a more balanced understanding of the wider importance of this phenomenon.     

Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking

Background

Age-related macular degeneration (AMD) is the major cause of blindness in the elderly. Those with the neovascular end-stage of disease have irreversible loss of central vision. AMD is a complex disorder in which genetic and environmental factors play a role. Polymorphisms in the complement factor H (CFH) gene, LOC387715, and the HTRA1 promoter are strongly associated with AMD. Smoking also contributes to the etiology. We aimed to provide a model of disease risk based on these factors.

Methods and Findings

We genotyped polymorphisms in CFH and LOC387715/HTRA1 in a case–control study of 401 patients with neovascular AMD and 266 controls without signs of disease, and used the data to produce genetic risk scores for the European-descent population based on haplotypes at these loci and smoking history. CFH and LOC387715/HTRA1 haplotypes and smoking status exerted large effects on AMD susceptibility, enabling risk scores to be generated with appropriate weighting of these three factors. Five common haplotypes of CFH conferred a range of odds ratios (ORs) per copy from 1 to 4.17. Most of the effect of LOC387715/HTRA1 was mediated through one detrimental haplotype (carriage of one copy: OR 2.83; 95% confidence interval [CI] 1.91–4.20), with homozygotes being at particularly high risk (OR 32.83; 95% CI 12.53–86.07). Patients with neovascular macular degeneration had considerably higher scores than those without disease, and risk of blinding AMD rose to 15.5% in the tenth of the population with highest predicted risk.

Conclusions

An individual''s risk of developing AMD in old age can be predicted by combining haplotype data with smoking status. Until there is effective treatment for AMD, encouragement to avoid smoking in those at high genetic risk may be the best option. We estimate that total absence of smoking would have reduced the prevalence of severe AMD by 33%. Unless smoking habits change or preventative treatment becomes available, the prevalence of AMD will rise as a consequence of the increasing longevity of the population.     

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

The interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis.     

Condensed complexes and the calorimetry of cholesterol-phospholipid bilayers

A recent thermodynamic model describes a reversible reaction between cholesterol (C) and phospholipid (P) to form a condensed complex C(nq)P(np). Here q and p are relatively prime integers used to define the stoichiometric composition, and n is a measure of cooperativity. The present study applies this model to the scanning calorimetry of binary mixtures of cholesterol and saturated phosphatidylcholines, especially work by McElhaney and collaborators. These mixtures generally show two heat capacity peaks, a sharp peak and a broad peak. The sharp heat absorption is largely due to the chain melting transition of pure phospholipid. In the present work the broad heat absorption is attributed to the thermal dissociation of complexes. The best fits of the model to the data require the complex formation to be highly cooperative, with cooperativity n = 12. Detailed comparisons are made between model calculations and calorimetric data. A number of unusual features of the data arise naturally in the model. The principal discrepancy between the calculations and experimental results is a spurious calculated heat absorption peak. This discrepancy is related to the reported relative magnitudes of the integrated broad and sharp heat absorption curves.     

MHC class I genes of the channel catfish: sequence analysis and expression

 Four cDNAs encoding the major histocompatibility complex (MHC) class I α chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I α chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I α and β₂-microglobulin (β₂m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 M _r glycoprotein (presumably class I α) was associated with β₂m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of β₂m with the MHC class I α chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts. Received: 25 March 1998 / Revised: 28 July 1998     

Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons

Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. We present evidence that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, we identified a serine within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity.     

Mouse development and cell proliferation in the absence of D-cyclins

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.