Development and prevention of neural tube defects in the mouse embryo

The morphogenetic process of cranial neural tube closure was studied in normal and mutant splotch mouse embryos to identify factors contributing to the development of the neural tube defect (NTD), exencephaly. The study noted sex differences in the timing of the neurulation process and strain differences in the initiation of cranial neural tube closure, at the site termed closure 2. Splotch embryos exhibit closure 2 at a rostral level, within the forebrain region, and exencephaly results from failure of elevation of the midbrain neural folds in a proportion of homozygotes. Backcrossing the splotch mutation onto the DBA/2 background caused a caudal shift in the position of closure 2, to the midbrain region, and a reduction in the incidence of exencephaly, suggesting that altering the position of closure 2 may aid midbrain fold elevation and prevent the development of exencephaly. Whole-mount in situ hybridisation revealed that expression patterns of Pax2, Pax5 and Fgf8 remained constant within the forebrain- midbrain region, regardless of variation in the position of closure 2 in different strains. NTD in humans can be prevented by periconceptional supplementation with folic acid, although the mechanism of action of folate is unknown. The deoxyuridine (dU) suppression test was adapted for use in whole embryo culture and several genetic models of NTD were screened for defects of folate metabolism. The splotch mutant showed an abnormal dU suppression test, with excessive incorporation of 3H-thymidine. Administration of thymidine or folic acid prevented the NTD in splotch homozygotes, both in vitro and in vivo. Folic acid also ameliorated the excessive incorporation of 3H-thymidine and prevented the neural crest defects in homozygous embryos. The splotch mutant therefore appears to provide a model for folate-preventable NTD in humans

Autophagy in Neuronal Development and Plasticity.

Autophagy is a highly conserved intracellular clearance pathway in which cytoplasmic contents are trafficked to the lysosome for degradation. Within neurons, it helps to remove damaged organelles and misfolded or aggregated proteins and has therefore been the subject of intense research in relation to neurodegenerative disease. However, far less is understood about the role of autophagy in other aspects of neuronal physiology. Here we review the literature on the role of autophagy in maintaining neuronal stem cells and in neuronal plasticity in adult life and we discuss how these contribute to structural and functional deficits observed in a range of human disorders

Seeing is believing: methods to monitor vertebrate autophagy in vivo.

Autophagy is an intracellular clearance pathway that delivers cytoplasmic contents to the lysosome for degradation. It plays a critical role in maintaining protein homeostasis and providing nutrients under conditions where the cell is starved. It also helps to remove damaged organelles and misfolded or aggregated proteins. Thus, it is not surprising that defects in this pathway are associated with a variety of pathological conditions, such as neurodegeneration, cancer and infection. Pharmacological upregulation of autophagy is considered a promising therapeutic strategy for the treatment of neurodegenerative and infectious diseases. Studies in knockout mice have demonstrated that autophagy is essential for nervous system function, and data from invertebrate and vertebrate models suggest that the efficiency of autophagic processes generally declines with age. However, much of our understanding of the intracellular regulation of autophagy comes from in vitro studies, and there is a paucity of knowledge about how this process is regulated within different tissues and during the processes of ageing and disease. Here, we review the available tools to probe these questions in vivo within vertebrate model systems. We discuss how these tools have been used to date and consider future avenues of research

Functional characterisation of the maturation of the blood-brain barrier in larval zebrafish.

Zebrafish are becoming increasingly popular as an organism in which to model human disease and to study the effects of small molecules on complex physiological and pathological processes. Since larvae are no more than a few millimetres in length, and can live in volumes as small as 100 microliters, they are particularly amenable to high-throughput and high content compound screening in 96 well plate format. There is a growing literature providing evidence that many compounds show similar pharmacological effects in zebrafish as they do in mammals, and in particular humans. However, a major question regarding their utility for small molecule screening for neurological conditions is whether a molecule will reach its target site within the central nervous system. Studies have shown that Claudin-5 and ZO-1, tight-junction proteins which are essential for blood-brain barrier (BBB) integrity in mammals, can be detected in some cerebral vessels in zebrafish from 3 days post-fertilisation (d.p.f.) onwards and this timing coincides with the retention of dyes, immunoreactive tracers and fluorescent markers within some but not all cerebral vessels. Whilst these findings demonstrate that features of a BBB are first present at 3 d.p.f., it is not clear how quickly the zebrafish BBB matures or how closely the barrier resembles that of mammals. Here, we have combined anatomical analysis by transmission electron microscopy, functional investigation using fluorescent markers and compound uptake using liquid chromatography/tandem mass spectrometry to demonstrate that maturation of the zebrafish BBB occurs between 3 d.p.f. and 10 d.p.f. and that this barrier shares both structural and functional similarities with that of mammals

Zebrafish as a model to understand autophagy and its role in neurological disease.

In the past decade, the zebrafish (Danio rerio) has become a popular model system for the study of vertebrate development, since the embryos and larvae of this species are small, transparent and undergo rapid development ex utero, allowing in vivo analysis of embryogenesis and organogenesis. These characteristics can also be exploited by researchers interested in signaling pathways and disease processes and, accordingly, there is a growing literature on the use of zebrafish to model human disease. This model holds great potential for exploring how autophagy, an evolutionarily conserved mechanism for protein degradation, influences the pathogeneses of a range of different human diseases and for the evaluation of this pathway as a potential therapeutic strategy. Here we summarize what is known about the regulation of autophagy in eukaryotic cells and its role in neurodegenerative disease and highlight how research using zebrafish has helped further our understanding of these processes

Building the backbone: the development and evolution of vertebral patterning.

The segmented vertebral column comprises a repeat series of vertebrae, each consisting of two key components: the vertebral body (or centrum) and the vertebral arches. Despite being a defining feature of the vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral component structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, we assemble evidence from ichthyologists, palaeontologists and developmental biologists to consider these issues. Vertebral arch elements were present in early stem vertebrates, whereas centra arose later. We argue that centra are homologous among jawed vertebrates, and review evidence in teleosts that the notochord plays an instructive role in segmental patterning, alongside the somites, and contributes to mineralization. By clarifying the evolutionary relationship between centra and arches, and their varying modes of skeletal mineralization, we can better appreciate the detailed mechanisms that regulate and diversify vertebral patterning

Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome.

Protein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the accumulation of α-synuclein, the main component of Lewy bodies found in Parkinson's disease. Aggresomes are closely-related, cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organise the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to functional deficits and neuronal cell death in Parkinson's disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals

cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases

The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils.

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci.Abbreviations: ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification

Expression and regulation of drug transporters in vertebrate neutrophils.

There remains a need to identify novel pro-resolution drugs for treatment of inflammatory disease. To date, there are no neutrophil-specific anti-inflammatory treatments in clinical use, perhaps due to our lack of understanding of how drugs access this complex cell type. Here we present the first comprehensive description and expression of both major classes of drug transporters, SLC and ABC, in resting human blood neutrophils. Moreover, we have studied the expression of these carriers in the tractable model system, the zebrafish (Danio rerio), additionally examining the evolutionary relationship between drug transporters in zebrafish and humans. We anticipate that this will be a valuable resource to the field of inflammation biology and will be an important asset in future anti-inflammatory drug design