Immunology of Pregnancy and Systemic Consequences.

Pregnancy is an immunological paradox, with renowned Nobel Prize winning transplantation biologist Sir Peter Brian Medawar being the first to introduce this concept back in 1953. This concept considers how the maternal immune system can tolerate the developing fetus, which is 50% antigenically foreign to the uterus. There have been significant advances in our understanding of the immune system in regulating fertility, pregnancy and in complications of these, and what was once considered a paradox can be seen as a highly evolved system. Indeed, the complexity of the maternal-fetal interface along with our ever-advancing knowledge of immune cells and mediators means that we have a better understanding of these interactions, with gaps still present.  This chapter will summarise the key aspects of the role of the immune system at each stage of pregnancy and highlight the recent advances in our knowledge.

α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation.

The factors regulating cellular identity are critical for understanding the transition from health to disease and responses to therapies. Recent literature suggests that autophagy compromise may cause opposite effects in different contexts by either activating or inhibiting YAP/TAZ co-transcriptional regulators of the Hippo pathway via unrelated mechanisms. Here, we confirm that autophagy perturbation in different cell types can cause opposite responses in growth-promoting oncogenic YAP/TAZ transcriptional signalling. These apparently contradictory responses can be resolved by a feedback loop where autophagy negatively regulates the levels of α-catenins, LC3-interacting proteins that inhibit YAP/TAZ, which, in turn, positively regulate autophagy. High basal levels of α-catenins enable autophagy induction to positively regulate YAP/TAZ, while low α-catenins cause YAP/TAZ activation upon autophagy inhibition. These data reveal how feedback loops enable post-transcriptional determination of cell identity and how levels of a single intermediary protein can dictate the direction of response to external or internal perturbations.

Active immunotherapy and alternative therapeutic modalities for Alzheimer's disease.

As knowledge of Alzheimer's disease (AD) progression improves, the field has recognized the need to diversify the pipeline, broaden strategies and approaches to therapies, as well as delivery mechanisms. A better understanding of the earliest biological processes of AD/dementia would help inform drug target selection. Currently there are a number of programs exploring these alternate avenues. This meeting will allow experts in the field (academia, industry, government) to provide perspectives and experiences that can help elucidate what the pipeline looks like today and what avenues hold promise in developing new therapies across the stages of AD. The focus here is on Active Immunotherapies and Alternative Therapeutic Modalities. This topic includes active vaccines, antisense oligomers, and cell-based therapy among others, and highlights new clinical developments that utilize these modalities.

Leucine regulates autophagy via acetylation of the mTORC1 component raptor.

Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.

A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.

Autophagy involves engulfment of cytoplasmic contents by double-membraned autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This "scission" step is, thus, critical for autophagosome formation, is defective in a human disease, and influences the way we consider how autophagosomes are formed.

A Comprehensive Profile of Chemokine Gene Expression in the Tissues of the Female Reproductive Tract in Mice.

Homeostatic leukocyte trafficking into and within the female reproductive tract (FRT) contributes to fertility and reproductive health. It is unclear how this process is regulated in the anatomically distinct reproductive tissues, or whether the genes involved are affected by cyclical changes in reproductive hormones. In tissues such as skin and intestine, mouse studies have defined evolutionarily conserved molecular mechanisms for tissue-specific homing, interstitial positioning, and leukocyte egress. Chemokine family members are invariably involved, with the chemokine expression profile of a tissue regulating leukocyte content. Reproductive tissues (ovary, vagina, cervix, uterine horn) of 8 week old virgin female C57BL/6 mice (n = 20) were collected, and expression of mRNA for leukocyte markers and chemokines conducted by qPCR. Lymphocytic and myeloid cell populations within the uterus, cervix, bone marrow and PALN from virgin C57BL/6 mice were determined by flow cytometric analysis. Variation in leukocyte content between reproductive tissues is evident, with the uterus and cervix containing complex mixtures of lymphocytes and myeloid cells. Twenty-six chemokine genes are expressed in the FRT, many by several component tissues, some preferentially by one. Most striking are Xcl1 and Ccl28, which are restricted to the uterus. Ccl20 and genes encoding CXCR2 ligands are primarily transcribed in cervix and vagina. Ovary shows the lowest expression of most chemokine genes, with the notable exception of Ccl21 and Ccl27. We also identify eight chemokines in the vagina whose expression fluctuates substantially across the oestrous cycle. These data reveal complex chemokine networks within the FRT, and provide a framework for future studies of homeostatic leukocyte trafficking into and within these tissues.Abbreviations: BM: bone marrow; DC: dendritic cell; DN: double negative; FRT: female reproductive tract; FSC: forward scatter; NK: natural killer; PALN: para-aortic lymph node; SSC: side scatter; Tregs: regulatory T cells.

Targeting the Synapse in Alzheimer's Disease.

Dynamic gain and loss of synapses is fundamental to healthy brain function. While Alzheimer's Disease (AD) treatment strategies have largely focussed on beta-amyloid and tau protein pathologies, the synapse itself may also be a critical endpoint to consider regarding disease modification. Disruption of mechanisms of neuronal plasticity, eventually resulting in a net loss of synapses, is implicated as an early pathological event in AD. Synaptic dysfunction therefore may be a final common biological mechanism linking protein pathologies to disease symptoms. This review summarizes evidence supporting the idea of early neuroplastic deficits being prevalent in AD. Changes in synaptic density can occur before overt neurodegeneration and should not be considered to uniformly decrease over the course of the disease. Instead, synaptic levels are influenced by an interplay between processes of degeneration and atrophy, and those of maintenance and compensation at regional and network levels. How these neuroplastic changes are driven by amyloid and tau pathology are varied. A mixture of direct effects of amyloid and tau on synaptic integrity, as well as indirect effects on processes such as inflammation and neuronal energetics are likely to be at play here. Focussing on the synapse and mechanisms of neuroplasticity as therapeutic opportunities in AD raises some important conceptual and strategic issues regarding translational research, and how preclinical research can inform clinical studies. Nevertheless, substrates of neuroplasticity represent an emerging complementary class of drug target that would aim to normalize synapse dynamics and restore cognitive function in the AD brain and in other neurodegenerative diseases.

Author Correction: Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration.

Leucine Signals to mTORC1 via Its Metabolite Acetyl-Coenzyme A.

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of cell growth and metabolism. Leucine (Leu) activates mTORC1 and many have tried to identify the mechanisms whereby cells sense Leu in this context. Here we describe that the Leu metabolite acetyl-coenzyme A (AcCoA) positively regulates mTORC1 activity by EP300-mediated acetylation of the mTORC1 regulator, Raptor, at K1097. Leu metabolism and consequent mTORC1 activity are regulated by intermediary enzymes. As AcCoA is a Leu metabolite, this process directly correlates with Leu abundance, and does not require Leu sensing via intermediary proteins, as has been described previously. Importantly, we describe that this pathway regulates mTORC1 in a cell-type-specific manner. Finally, we observed decreased acetylated Raptor, and inhibited mTORC1 and EP300 activity in fasted mice tissues. These results provide a direct mechanism for mTORC1 regulation by Leu metabolism.

Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis.

Contact inhibition enables noncancerous cells to cease proliferation and growth when they contact each other. This characteristic is lost when cells undergo malignant transformation, leading to uncontrolled proliferation and solid tumor formation. Here we report that autophagy is compromised in contact-inhibited cells in 2D or 3D-soft extracellular matrix cultures. In such cells, YAP/TAZ fail to co-transcriptionally regulate the expression of myosin-II genes, resulting in the loss of F-actin stress fibers, which impairs autophagosome formation. The decreased proliferation resulting from contact inhibition is partly autophagy-dependent, as is their increased sensitivity to hypoxia and glucose starvation. These findings define how mechanically repressed YAP/TAZ activity impacts autophagy to contribute to core phenotypes resulting from high cell confluence that are lost in various cancers.

The RAB11A-Positive Compartment Is a Primary Platform for Autophagosome Assembly Mediated by WIPI2 Recognition of PI3P-RAB11A.

Autophagy is a critical pathway that degrades intracytoplasmic contents by engulfing them in double-membraned autophagosomes that are conjugated with LC3 family members. These membranes are specified by phosphatidylinositol 3-phosphate (PI3P), which recruits WIPI2, which, in turn, recruits ATG16L1 to specify the sites of LC3-conjugation. Conventionally, phosphatidylinositides act in concert with other proteins in targeting effectors to specific membranes. Here we describe that WIPI2 localizes to autophagic precursor membranes by binding RAB11A, a protein that specifies recycling endosomes, and that PI3P is formed on RAB11A-positive membranes upon starvation. Loss of RAB11A impairs the recruitment and assembly of the autophagic machinery. RAB11A-positive membranes are a primary direct platform for canonical autophagosome formation that enables autophagy of the transferrin receptor and damaged mitochondria. While this compartment may receive membrane inputs from other sources to enable autophagosome biogenesis, RAB11A-positive membranes appear to be a compartment from which autophagosomes evolve.

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.

Polyglutamine tracts regulate autophagy.

Expansions of polyglutamine (polyQ) tracts in different proteins cause 9 neurodegenerative conditions, such as Huntington disease and various ataxias. However, many normal mammalian proteins contain shorter polyQ tracts. As these are frequently conserved in multiple species, it is likely that some of these polyQ tracts have important but unknown biological functions. Here we review our recent study showing that the polyQ domain of the deubiquitinase ATXN3/ataxin-3 enables its interaction with BECN1/beclin 1, a key macroautophagy/autophagy initiator. ATXN3 regulates autophagy by deubiquitinating BECN1 and protecting it from proteasomal degradation. Interestingly, expanded polyQ tracts in other polyglutamine disease proteins compete with the shorter ATXN3 polyQ stretch and interfere with the ATXN3-BECN1 interaction. This competition results in decreased BECN1 levels and impaired starvation-induced autophagy, which phenocopies the loss of autophagic function mediated by ATXN3. Our findings describe a new autophagy-protective mechanism that may be altered in multiple neurodegenerative diseases.

Polyglutamine tracts regulate beclin 1-dependent autophagy.

Nine neurodegenerative diseases are caused by expanded polyglutamine (polyQ) tracts in different proteins, such as huntingtin in Huntington's disease and ataxin 3 in spinocerebellar ataxia type 3 (SCA3). Age at onset of disease decreases with increasing polyglutamine length in these proteins and the normal length also varies. PolyQ expansions drive pathogenesis in these diseases, as isolated polyQ tracts are toxic, and an N-terminal huntingtin fragment comprising exon 1, which occurs in vivo as a result of alternative splicing, causes toxicity. Although such mutant proteins are prone to aggregation, toxicity is also associated with soluble forms of the proteins. The function of the polyQ tracts in many normal cytoplasmic proteins is unclear. One such protein is the deubiquitinating enzyme ataxin 3 (refs 7, 8), which is widely expressed in the brain. Here we show that the polyQ domain enables wild-type ataxin 3 to interact with beclin 1, a key initiator of autophagy. This interaction allows the deubiquitinase activity of ataxin 3 to protect beclin 1 from proteasome-mediated degradation and thereby enables autophagy. Starvation-induced autophagy, which is regulated by beclin 1, was particularly inhibited in ataxin-3-depleted human cell lines and mouse primary neurons, and in vivo in mice. This activity of ataxin 3 and its polyQ-mediated interaction with beclin 1 was competed for by other soluble proteins with polyQ tracts in a length-dependent fashion. This competition resulted in impairment of starvation-induced autophagy in cells expressing mutant huntingtin exon 1, and this impairment was recapitulated in the brains of a mouse model of Huntington's disease and in cells from patients. A similar phenomenon was also seen with other polyQ disease proteins, including mutant ataxin 3 itself. Our data thus describe a specific function for a wild-type polyQ tract that is abrogated by a competing longer polyQ mutation in a disease protein, and identify a deleterious function of such mutations distinct from their propensity to aggregate.

Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities.

Autophagy is a conserved pathway that delivers cytoplasmic contents to the lysosome for degradation. Here we consider its roles in neuronal health and disease. We review evidence from mouse knockout studies demonstrating the normal functions of autophagy as a protective factor against neurodegeneration associated with intracytoplasmic aggregate-prone protein accumulation as well as other roles, including in neuronal stem cell differentiation. We then describe how autophagy may be affected in a range of neurodegenerative diseases. Finally, we describe how autophagy upregulation may be a therapeutic strategy in a wide range of neurodegenerative conditions and consider possible pathways and druggable targets that may be suitable for this objective.

CCT complex restricts neuropathogenic protein aggregation via autophagy.

Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.

The role of chemokines and their receptors during protist parasite infections.

Protists are a diverse collection of eukaryotic organisms that account for a significant global infection burden. Often, the immune responses mounted against these parasites cause excessive inflammation and therefore pathology in the host. Elucidating the mechanisms of both protective and harmful immune responses is complex, and often relies of the use of animal models. In any immune response, leucocyte trafficking to the site of infection, or inflammation, is paramount, and this involves the production of chemokines, small chemotactic cytokines of approximately 8-10 kDa in size, which bind to specific chemokine receptors to induce leucocyte movement. Herein, the scientific literature investigating the role of chemokines in the propagation of immune responses against key protist infections will be reviewed, focussing on Plasmodium species, Toxoplasma gondii, Leishmania species and Cryptosporidium species. Interestingly, many studies find that chemokines can in fact, promote parasite survival in the host, by drawing in leucocytes for spread and further replication. Recent developments in drug targeting against chemokine receptors highlights the need for further understanding of the role played by these proteins and their receptors in many different diseases.

Autophagy regulates Notch degradation and modulates stem cell development and neurogenesis.

Autophagy is a conserved, intracellular, lysosomal degradation pathway. While mechanistic aspects of this pathway are increasingly well defined, it remains unclear how autophagy modulation impacts normal physiology. It is, however, becoming clear that autophagy may play a key role in regulating developmental pathways. Here we describe for the first time how autophagy impacts stem cell differentiation by degrading Notch1. We define a novel route whereby this plasma membrane-resident receptor is degraded by autophagy, via uptake into ATG16L1-positive autophagosome-precursor vesicles. We extend our findings using a physiologically relevant mouse model with a hypomorphic mutation in Atg16L1, a crucial autophagy gene, which shows developmental retention of early-stage cells in various tissues where the differentiation of stem cells is retarded and thus reveal how modest changes in autophagy can impact stem cell fate. This may have relevance for diverse disease conditions, like Alzheimer's Disease or Crohn's Disease, associated with altered autophagy.

Mammalian Autophagy: How Does It Work?

Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.