Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of 'slow Wallerian degeneration' (Wld (S)), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld(S). Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld(S)-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld(S) expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld(S) or NMNAT2.

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.

The phosphoinositide family of phospholipids, defined here as PtdIns, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, play pivotal roles in organising the location and activity of many different proteins acting on biological membranes, including those involved in vesicle and protein trafficking through the endolysosomal system and receptor signal transduction at the plasma membrane. Accurate measurement of the cellular levels of these lipids, particularly the more highly phosphorylated species, is hampered by their high polarity and low cellular concentrations. Recently, much progress has been made in using mass spectrometry to measure many different lipid classes in parallel, an approach generally referred to as 'lipidomics'. Unfortunately, the acidic nature of highly phosphorylated phosphoinositides makes them difficult to measure using these methods, because they yield low levels of useful ions; this is particularly the case with PtdIns(3,4,5)P3. We have solved some of these problems by methylating the phosphate groups of these lipids with TMS-diazomethane and describe a simple, integrated approach to measuring PtdIns, PtdInsP, PtdInsP2 and PtdInsP3 classes of lipids, in parallel with other phospholipid species, in cell and tissue extracts. This methodology is sensitive, accurate and robust, and also yields fatty-acyl compositions, suggesting it can be used to further our understanding of both the normal and pathophysiological roles of these important lipids.

The spindle assembly checkpoint (SAC) is essential in mammalian mitosis to ensure the equal segregation of sister chromatids. The SAC generates a mitotic checkpoint complex (MCC) to prevent the anaphase-promoting complex/cyclosome (APC/C) from targeting key mitotic regulators for destruction until all of the chromosomes have attached to the mitotic apparatus. A single unattached kinetochore can delay anaphase for several hours, but how it is able to block the APC/C throughout the cell is not understood. Present concepts of the SAC posit that either it exhibits an all-or-nothing response or there is a minimum threshold sufficient to block the APC/C (ref. 7). Here, we have used gene targeting to measure SAC activity, and find that it does not have an all-or-nothing response. Instead, the strength of the SAC depends on the amount of MAD2 recruited to kinetochores and on the amount of MCC formed. Furthermore, we show that different drugs activate the SAC to different extents, which may be relevant to their efficacy in chemotherapy.

Naive CD8+ T cells rely upon oxidation of fatty acids as a primary source of energy. After antigen encounter, T cells shift to a glycolytic metabolism to sustain effector function. It is unclear, however, whether changes in glucose metabolism ultimately influence the ability of activated T cells to become long-lived memory cells. We used a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8+ T cells. We found that cells exhibiting limited glucose incorporation had a molecular profile characteristic of memory precursor cells and an increased capacity to enter the memory pool compared with cells taking up high amounts of glucose. Accordingly, enforcing glycolytic metabolism by overexpressing the glycolytic enzyme phosphoglycerate mutase-1 severely impaired the ability of CD8+ T cells to form long-term memory. Conversely, activation of CD8+ T cells in the presence of an inhibitor of glycolysis, 2-deoxyglucose, enhanced the generation of memory cells and antitumor functionality. Our data indicate that augmenting glycolytic flux drives CD8+ T cells toward a terminally differentiated state, while its inhibition preserves the formation of long-lived memory CD8+ T cells. These results have important implications for improving the efficacy of T cell-based therapies against chronic infectious diseases and cancer.

Fundamental cellular processes such as cell movement, division or food uptake critically depend on cells being able to change shape. Fast acquisition of three-dimensional image time series has now become possible, but we lack efficient tools for analysing shape deformations in order to understand the real three-dimensional nature of shape changes.

In order to manage the rise in life expectancy and the concomitant increased occurrence of age-related diseases, research into ageing has become a strategic priority. Mouse models are commonly utilised as they share high homology with humans and show many similar signs and diseases of ageing. However, the time and cost needed to rear aged cohorts can limit research opportunities. Sharing of resources can provide an ethically and economically superior framework to overcome some of these issues but requires dedicated infrastructure. Shared Ageing Research Models (ShARM) ( www.ShARMUK.org ) is a new, not-for-profit organisation funded by Wellcome Trust, open to all investigators. It collects, stores and distributes flash frozen tissues from aged murine models through its biorepository and provides a database of live ageing mouse colonies available in the UK and abroad. It also has an online environment (MICEspace) for collation and analysis of data from communal models and discussion boards on subjects such as the welfare of ageing animals and common endpoints for intervention studies. Since launching in July 2012, thanks to the generosity of researchers in UK and Europe, ShARM has collected more than 2,500 tissues and has in excess of 2,000 mice registered in live ageing colonies. By providing the appropriate support, ShARM has been able to bring together the knowledge and experience of investigators in the UK and Europe to maximise research outputs with little additional cost and minimising animal use in order to facilitate progress in ageing research.

Autophagy is a conserved survival pathway, which cells and tissues will activate during times of stress. It is characterized by the formation of double-membrane vesicles called autophagosomes inside the cytoplasm. The molecular mechanisms and the signalling components involved require specific control to ensure correct activation. The present chapter describes the formation of autophagosomes from within omegasomes, newly identified membrane compartments enriched in PI3P (phosphatidylinositol 3-phosphate) that serve as platforms for the formation of at least some autophagosomes. We discuss the signalling events required to nucleate the formation of omegasomes as well as the protein complexes involved.

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

NOD2 is an intracellular sensor that contributes to immune defense and inflammation. Here we investigated whether NOD2 mediates its effects through control of microRNAs (miRNAs). miR-29 expression was upregulated in human dendritic cells (DCs) in response to NOD2 signals, and miR-29 regulated the expression of multiple immune mediators. In particular, miR-29 downregulated interleukin-23 (IL-23) by targeting IL-12p40 directly and IL-23p19 indirectly, likely via reduction of ATF2. DSS-induced colitis was worse in miR-29-deficient mice and was associated with elevated IL-23 and T helper 17 signature cytokines in the intestinal mucosa. Crohn's disease (CD) patient DCs expressing NOD2 polymorphisms failed to induce miR-29 upon pattern recognition receptor stimulation and showed enhanced release of IL-12p40 on exposure to adherent invasive E. coli. Therefore, we suggest that loss of miR-29-mediated immunoregulation in CD DCs might contribute to elevated IL-23 in this disease.

The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient's life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn's disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFβ1 reduces production of proinflammatory cytokines, including TNFα, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses.

Recent studies highlight an important role of the aryl hydrocarbon receptor (AhR) at mucosal barriers. Surprisingly, activation of the AhR, required for the maintenance of lymphocytes as well as lymphoid architecture, can be achieved via cues derived from the external environment. This environment contains both beneficial and harmful microorganisms as well as a diverse array of compounds, and the epithelia must offer very sophisticated levels of defence. This is achieved via multifaceted immune recognition diversity and cellular complexity. Mucosal associated tissues, particularly in the gastrointestinal tract, constitute a complex immune organ for local lymphocytes and contain highly organised lymphoid structures. We will discuss the recent observations concerning the AhR in relation to the function and maintenance of innate T cells, with focus on γδ T cells found enriched at epithelial barriers.

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. Disease pathophysiology is complex and not yet fully understood, but is proposed to include the accumulation of misfolded proteins, as aggregates are present in spinal cords from ALS patients and in ALS model organisms. Increasing autophagy is hypothesized to be protective in ALS as it removes these aggregates. Rapamycin is frequently used to increase autophagy, but is also a potent immune suppressor. To properly assess the role of rapamycin-induced autophagy, the immune suppressive role of rapamycin should be negated.

A Hoxd11/lacZ reporter, expressed with a Hoxd11-like axial expression pattern in transgenic mouse embryos, is stimulated in tailbud fragments when cultured in presence of Gdf11, a TGF-β growth/differentiation factor. The same construct is also stimulated by Gdf11 when transiently transfected into cultures of HepG2 cells. Stimulation of the reporter in HepG2 cells is enhanced where it contains only the 332 bp Hoxd11 enhancer region VIII upstream or downstream of a luciferase or lacZ reporter. This enhancer contains three elements conserved from fish to mice, one of which has the sequence of a Smad3/4 binding element. Mutation of this motif inhibits the ability of Gdf11 to enhance reporter activity in the HepG2 cell assay. Chromatin immunoprecipitation experiments show direct evidence of Smad2/3 protein binding to the Hoxd11 region VIII enhancer. The action of Gdf11 upon Hoxd11 in HepG2 cells is inhibited, at least in part, by SIS3, a specific inhibitor of Smad3. SIS3 also produces partial inhibition of Hoxd11/lacZ expression in cultured transgenic tailbuds, indicating that Smad3 may play a similar role in the embryonic expression of Hoxd11. Transgenic mouse experiments show that the Smad binding motif is essential for the axial expression of Hoxd11/lacZ reporter in the embryo tailbud, posterior mesoderm and neurectoderm.

Macrophages play an important role in immunity and homeostasis. Upon pathogen recognition via specific receptors, they rapidly induce inflammatory responses. This process is tightly controlled at the transcriptional level. The DNA binding zinc-finger protein CCCTC-binding factor (Ctcf) is a crucial regulator of long-range chromatin interactions and coordinates specific communication between transcription factors and gene expression processes. In this study, the Ctcf gene was specifically deleted in myeloid cells by making use of the transgenic Cre-LoxP system. Conditional deletion of the Ctcf gene in myeloid cells induced a mild phenotype in vivo. Ctcf-deficient mice exhibited significantly reduced expression of major histocompatibility complex (MHC) class II in the liver. Ctcf-deficient macrophages demonstrated a normal surface phenotype and phagocytosis capacity. Upon Toll-like receptor (TLR) stimulation, they produced normal levels of the pro-inflammatory cytokines IL-12 and IL-6, but manifested a strongly impaired capacity to produce tumor-necrosis factor (TNF) and IL-10, as well as to express the IL-10 family members IL-19, IL-20 and IL-24. Taken together, our data demonstrate a role of Ctcf that involves fine-tuning of macrophage function.

Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTβR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice.

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.

A phosphatidylinositol-phosphate affinity chromatographic approach combined with mass spectrometry was used in order to identify novel PI(3)P and PI(3,5)P2 binding proteins from Arabidopsis thaliana suspension cell extracts. Most of the phosphatidylinositol-phosphate interacting candidates identified from this differential screening are characterized by lysine/arginine rich patches. Direct phosphoinositide binding was identified for important membrane trafficking regulators as well as protein quality control proteins such as the ATG18p orthologue involved in autophagosome formation and the lipid Sec14p like transfer protein. A pentatricopeptide repeat (PPR) containing protein was shown to directly bind to PI(3,5)P2 but not to PI(3)P. PIP chromatography performed using extracts obtained from high salt (0.4M and 1M NaCl) pretreated suspensions showed that the association of an S5-1 40S ribosomal protein with both PI(3)P and PI(3,5)P2 was abolished under salt stress whereas salinity stress induced an increase in the phosphoinositide association of the DUF538 domain containing protein SVB, associated with trichome size. Additional interacting candidates were co-purified with the phosphoinositide bound proteins. Binding of the COP9 signalosome, the heat shock proteins, and the identified 26S proteasomal subunits, is suggested as an indirect effect of their interaction with other proteins directly bound to the PI(3)P and the PI(3,5)P2 phosphoinositides.

Dendritic cells (DCs) comprise distinct populations with specialized immune-regulatory functions. However, the environmental factors that determine the differentiation of these subsets remain poorly defined. Here, we report that retinoic acid (RA), a vitamin A derivative, controls the homeostasis of pre-DC (precursor of DC)-derived splenic CD11b(+)CD8α(-)Esam(high) DCs and the developmentally related CD11b(+)CD103(+) subset within the gut. Whereas mice deprived of RA signaling significantly lost both of these populations, neither pre-DC-derived CD11b(-)CD8α(+) and CD11b(-)CD103(+) nor monocyte-derived CD11b(+)CD8α(-)Esam(low) or CD11b(+)CD103(-) DC populations were deficient. In fate-tracking experiments, transfer of pre-DCs into RA-supplemented hosts resulted in near complete conversion of these cells into the CD11b(+)CD8α(-) subset, whereas transfer into vitamin A-deficient (VAD) hosts caused diversion to the CD11b(-)CD8α(+) lineage. As vitamin A is an essential nutrient, we evaluated retinoid levels in mice and humans after radiation-induced mucosal injury and found this conditioning led to an acute VAD state. Consequently, radiation led to a selective loss of both RA-dependent DC subsets and impaired class II-restricted auto and antitumor immunity that could be rescued by supplemental RA. These findings establish a critical role for RA in regulating the homeostasis of pre-DC-derived DC subsets and have implications for the management of patients with immune deficiencies resulting from malnutrition and irradiation.

The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem cells (ESCs) that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g., insertion of human-specific genes or reporter genes), gene disruption, point mutations, and short- and long-range deletions, inversions. Site-specific modification into the genome of ESCs can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ESCs.

The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (1) delete or insert mutations into genes of interest to determine their function, (2) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g., mice susceptible to infections by human-specific pathogens of interest, (3) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (4) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse's lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice.

The NAD-synthesising enzyme Nmnat2 is a critical survival factor for axons in vitro and in vivo. We recently reported that loss of axonal transport vesicle association through mutations in its isoform-specific targeting and interaction domain (ISTID) reduces Nmnat2 ubiquitination, prolongs its half-life and boosts its axon protective capacity in primary culture neurons. Here, we report evidence for a role of ISTID sequences in tuning Nmnat2 localisation, stability and protective capacity in vivo. Deletion of central ISTID sequences abolishes vesicle association and increases protein stability of fluorescently tagged, transgenic Nmnat2 in mouse peripheral axons in vivo. Overexpression of fluorescently tagged Nmnat2 significantly delays Wallerian degeneration in these mice. Furthermore, while mammalian Nmnat2 is unable to protect transected Drosophila olfactory receptor neuron axons in vivo, mutant Nmnat2s lacking ISTID regions substantially delay Wallerian degeneration. Together, our results establish Nmnat2 localisation and turnover as a valuable target for modulating axon degeneration in vivo.