An Ansatz for undecidable computation in RNA-world automata

In this Ansatz we consider theoretical constructions of RNA polymers into automata, a form of computational structure. The basis for transitions in our automata are plausible RNA-world enzymes that may perform ligation or cleavage. Limited to these operations, we construct RNA automata of increasing complexity; from the Finite Automaton (RNA-FA) to the Turing Machine equivalent 2-stack PDA (RNA-2PDA) and the universal RNA-UPDA. For each automaton we show how the enzymatic reactions match the logical operations of the RNA automaton, and describe how biological exploration of the corresponding evolutionary space is facilitated by the efficient arrangement of RNA polymers into a computational structure. A critical theme of the Ansatz is the self-reference in RNA automata configurations which exploits the program-data duality but results in undecidable computation. We describe how undecidable computation is exemplified in the self-referential Liar paradox that places a boundary on a logical system, and by construction, any RNA automata. We argue that an expansion of the evolutionary space for RNA-2PDA automata can be interpreted as a hierarchical resolution of the undecidable computation by a meta-system (akin to Turing's oracle), in a continual process analogous to Turing's ordinal logics and Post's extensible recursively generated logics. On this basis, we put forward the hypothesis that the resolution of undecidable configurations in RNA-world automata represents a mechanism for novelty generation in the evolutionary space, and propose avenues for future investigation of biological automata

Emergent properties of microglia

More than 80 years ago, Pio Del Rio-Hortega recognized that one of the "main controversial points in regard to the microglia" is "whether it belongs to the reticulo-endothelial system [i.e. monocytes and macrophages] and possesses the ordinary characteristics of this system or has a more specialized function." The notion of microglia having functions that are different from those of other macrophages has gained significant support in recent years. The brain represents a unique environment and shows species, developmental and regional specialization. Thus, any consideration of microglial activity has to be thought of in this tissue context. Contexts may be normal (health, physiology) or disease conditions showing either primary or secondary microglial involvement. Subclinical, reversible "soft pathologies" (Kreutzberg) such as pain that involves microglia also exist. Here, we examine a multilayered approach to understanding microglia that illustrates the emergent character of the microglial (population) phenotype. Accordingly, terms such as microglial "activation" and microgliosis, which are of increasing importance for our understanding of neurological disorders, need to be filled with refined meaning. It is suggested that the pathophysiological context guides nomenclatorial considerations; for example, development, trauma or pain-associated microglia is preferred over the traditional but less distinctive "microglial activation." This should also help to tease out the different functional subtypes currently hidden under the umbrella term "neuroinflammation," which is being applied so widely that it has become effectively useless in practice and even inhibits research progress because both true and pseudo-inflammation are covered by this term.6 page(s

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons

Transactivating DNA-binding protein-43 (TDP-43) deposits represent a typical finding in almost all ALS patients, more than half of FTLD patients and patients with several other neurodegenerative disorders. It appears that perturbation of nucleo-cytoplasmic transport is an important event in these conditions but the mechanistic role and the fate of TDP-43 during neuronal degeneration remain elusive. We have developed an experimental system for visualising the perturbed nucleocytoplasmic transport of neuronal TDP-43 at the single-cell level in vivo using zebrafish spinal cord. This approach enabled us to image TDP-43-expressing motor neurons before and after experimental initiation of cell death. We report the formation of mobile TDP-43 deposits within degenerating motor neurons, which are normally phagocytosed by microglia. However, when microglial cells were depleted, injury-induced motor neuron degeneration follows a characteristic process that includes TDP-43 redistribution into the cytoplasm, axon and extracellular space. This is the first demonstration of perturbed TDP-43 nucleocytoplasmic transport in vivo, and suggests that impairment in microglial phagocytosis of dying neurons may contribute towards the formation of pathological TDP-43 presentations in ALS and FTLD

Motor neuron-expressed microRNAs 218 and their enhancers are nested within introns of Slit2/3 genes

miR218-1 and miR218-2 are embedded in introns of SLIT2 and SLIT3, respectively, an arrangement conserved throughout vertebrate genomes. Both miR218 genes are predicted to be transcribed in the same orientation as their host genes and were assumed to be spliced from Slit2/3 primary transcripts. In zebrafish miR218 is active in cranial nerve motor nuclei and spinal cord motor neurons, while slit2 and slit3 are expressed predominantly in the midline. This differential expression pattern suggested independent regulation of miR218 genes by distinct enhancers. We tested conserved noncoding elements for regulatory activity by reporter gene transgenesis in zebrafish. Two human enhancers, 76 kb and 130 kb distant from miR218-2, were identified that drove GFP expression in zebrafish in an almost complete miR218 expression pattern. In the zebrafish slit3 locus, two enhancers with identical activity were discovered. In human SLIT2 one enhancer 52 kb upstream of miR218-1 drove an expression pattern very similar to the enhancers of miR218-2. This establishes that miR218-1/-2 regulatory units are nested within SLIT2/3 and that they are duplicates of an ancestral single locus. Due to the strong activity of the enhancers, unique transgenic lines were created that facilitate morphological and gene functional genetic experiments in motor neurons

miR-124 Contributes to the functional maturity of microglia

During early development of the central nervous system (CNS), a subset of yolk-sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self-renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue-specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR-124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR-124 in the mpeg1 expressing yolk-sac-derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR-124 was used to assess microglial development in a miR-124 loss-of-function environment. Following the induction of miR-124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR-124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR-124 overexpression in differentiated neurons. Conversely, expression of the miR-124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR-124 overexpression. This study provides in vivo evidence that miR-124 activity has a key role in the development of functionally mature microglia

Development of ramified microglia from early macrophages in the zebrafish optic tectum

Microglia, the resident macrophage precursors of the brain, are necessary for the maintenance of tissue homeostasis and activated by a wide range of pathological stimuli. They have a key role in immune and inflammatory responses. Early microglia stem from primitive macrophages, however the transition from early motile forms to the ramified mature resident microglia has not been assayed in real time. In order to provide such an assay, we used zebrafish transgenic lines in which fluorescent reporter expression is driven by the promoter of macrophage expressed gene 1 (mpeg1; Ellet et al. [2011]: Blood 117(4): e49-e56,). This enabled the investigation of the development of these cells in live, intact larvae. We show that microglia develop from highly motile amoeboid cells that are engaged in phagocytosis of apoptotic cell bodies into a microglial cell type that rapidly morphs back and forth between amoeboid and ramified morphologies. These morphing microglia eventually settle into a typical mature ramified morphology. Developing microglia frequently come into contact with blood capillaries in the brain, and also frequently contact each other. Up to 10 days postfertilization, microglia were observed to undergo symmetric division. In the adult optic tectum, the microglia are highly branched, resembling mammalian microglia. In addition, the mpeg1 transgene also labeled highly branched cells in the skin overlying the optic tectum from 8-9 days postfertilization, which likely represent Langerhans cells. Thus, the development of zebrafish microglia and their cellular interactions was studied in the intact developing brain in real time and at cellular resolution.12 page(s

Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Keywords: Zebrafish, Optogenetics, Oxidative stress, Motor neurons, Microscop

Neuronal cell culture from transgenic zebrafish models of neurodegenerative disease

We describe a protocol for culturing neurons from transgenic zebrafish embryos to investigate the subcellular distribution and protein aggregation status of neurodegenerative disease-causing proteins. The utility of the protocol was demonstrated on cell cultures from zebrafish that transgenically express disease-causing variants of human fused in sarcoma (FUS) and ataxin-3 proteins, in order to study amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-3 (SCA3), respectively. A mixture of neuronal subtypes, including motor neurons, exhibited differentiation and neurite outgrowth in the cultures. As reported previously, mutant human FUS was found to be mislocalized from nuclei to the cytosol, mimicking the pathology seen in human ALS and the zebrafish FUS model. In contrast, neurons cultured from zebrafish expressing human ataxin-3 with disease-associated expanded polyQ repeats did not accumulate within nuclei in a manner often reported to occur in SCA3. Despite this, the subcellular localization of the human ataxin-3 protein seen in cell cultures was similar to that found in the SCA3 zebrafish themselves. The finding of similar protein localization and aggregation status in the neuronal cultures and corresponding transgenic zebrafish models confirms that this cell culture model is a useful tool for investigating the cell biology and proteinopathy signatures of mutant proteins for the study of neurodegenerative disease

Induction of Gliotoxin Secretion in Aspergillus fumigatus by Bacteria-Associated Molecules

Aspergillus fumigatus is the most common causative agent of mold diseases in humans, giving rise to life-threatening infections in immunocompromised individuals. One of its secreted metabolites is gliotoxin, a toxic antimicrobial agent. The aim of this study was to determine whether the presence of pathogen-associated molecular patterns in broth cultures of A. fumigatus could induce gliotoxin production. Gliotoxin levels were analyzed by ultra-performance liquid chromatography and mass spectrometry. The presence of a bacteria-derived lipopolysaccharide, peptidoglycan, or lipoteichoic acid in the growth media at a concentration of 5 mu g/ml increased the gliotoxin concentration in the media by 37%, 65%, and 35%, respectively. The findings reveal a correlation between the concentrations of pathogen-associated molecular patterns and gliotoxin secretion. This shows that there is a yet uncharacterized detection system for such compounds within fungi. Inducing secondary metabolite production by such means in fungi is potentially relevant for drug discovery research. Our results also give a possible explanation for the increased virulence of A. fumigatus during bacterial co-infection, one that is important for the transition from colonization to invasiveness in this pulmonary disease