David J. Kozlowski

Phosphatidylinositol transfer protein-|[alpha]| in netrin-1-induced PLC signalling and neurite outgrowth

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-α (PITPα) in neurite extension in response to netrin-1, an extracellular guidance cue. PITPα interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PITPα binding to DCC and to phosphatidylinositol (5) phosphate [PI(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PIP2). In addition, the stimulated PIP2 hydrolysis requires PITPα. Furthermore, cortical explants of PITPα mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PITPα mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PITPα expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PITPα in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PITPα regulation.

DNA damage response and Ku80 function in the vertebrate embryo

Cellular responses to DNA damage reflect the dynamic integration of cell cycle control, cell–cell interactions and tissue-specific patterns of gene regulation that occurs in vivo but is not recapitulated in cell culture models. Here we describe use of the zebrafish embryo as a model system to identify determinants of the in vivo response to ionizing radiation-induced DNA damage. To demonstrate the utility of the model we cloned and characterized the embryonic function of the XRCC5 gene, which encodes Ku80, an essential component of the nonhomologous end joining pathway of DNA repair. After the onset of zygotic transcription, Ku80 mRNA accumulates in a tissue-specific pattern, which includes proliferative zones of the retina and central nervous system. In the absence of genotoxic stress, zebrafish embryos with reduced Ku80 function develop normally. However, low dose irradiation of these embryos during gastrulation leads to marked apoptosis throughout the developing central nervous system. Apoptosis is p53 dependent, indicating that it is a downstream consequence of unrepaired DNA damage. Results suggest that nonhomologous end joining components mediate DNA repair to promote survival of irradiated cells during embryogenesis.

Knockdown of zebrafish Lgi1a results in abnormal development, brain defects and a seizure-like behavioral phenotype

Epilepsy is a common disorder, typified by recurrent seizures with underlying neurological disorders or disease. Approximately one-third of patients are unresponsive to currently available therapies. Thus, a deeper understanding of the genetics and etiology of epilepsy is needed to advance the development of new therapies. Previously, treatment of zebrafish with epilepsy-inducing pharmacological agents was shown to result in a seizure-like phenotype, suggesting that fish provide a tractable model to understand the function of epilepsy-predisposing genes. Here, we report the first model of genetically linked epilepsy in zebrafish and provide an initial characterization of the behavioral and neurological phenotypes associated with morpholino (MO) knockdown of leucine-rich, glioma-inactivated 1a (lgi1a) expression. Mutations in the LGI1 gene in humans have been shown to predispose to a subtype of autosomal dominant epilepsy. Low-dose Lgi1a MO knockdown fish (morphants) appear morphologically normal but are sensitized to epilepsy-inducing drugs. High-dose Lgi1a morphants have morphological defects which persist into adult stages that are typified by smaller brains and eyes and abnormalities in tail shape, and display hyperactive swimming behaviors. Increased apoptosis was observed throughout the central nervous system of high-dose morphant fish, accounting for the size reduction of neural tissues. These observations demonstrate that zebrafish can be exploited to dissect the embryonic function(s) of genes known to predispose to seizure-like behavior in humans, and offer potential insight into the relationship between developmental neurobiological abnormalities and seizure.

Theoretical Investigation of the Formation of a New Series of Antioxidant Depsides from the Radiolysis of Flavonoid Compounds

Abstract Kozlowski, D., Marsal, P., Steel, M., Mokrini, R., Duroux, J-L., Lazzaroni, R. and Trouillas, P. Theoretical Investigation of the Formation of a New Series of Antioxidant Depsides from the Radiolysis of Flavonoid Compounds. Radiat. Res. 168, 243–252 (2007). This paper deals with the formation of a series of antioxidant depsides obtained from flavonoid solutions irradiated with γ rays. These reactions take place in radiolyzed alcohol solutions, a medium that is very rich in many different highly reactive species and that hosts specific reactions. We focus on the first step of those reactions, i.e., reactivity of the solute (flavonoid) with the alkoxy radicals CH3O· and CH3CH2O· formed in methanol and ethanol, respectively, and their carbon-centered isomers: the 1-hydroxy-methyl (·CH2OH) and the 1-hydroxy-ethyl (CH3·CHOH) radicals. Among the different flavonoid groups of molecules, only flavonols are transformed. To establish the structure–reactivity relationship that explains why the radiolytic transformation occurs only for those compounds, the process is rationalized theoretically, with Density Functional Theory calculations, taking into account the solvent effects by a Polarizable Continuum Model and a microhydrated environment (one or two water molecules surrounding the active center). The first redox reaction, occurring between the flavonol and the reactive species formed upon irradiation of the solvent, is studied in terms of (1) the O-H bond dissociation enthalpy of each OH group of the flavonoids and (2) electron abstraction from the molecule. We conclude that the reaction, initiated preferentially by the alkoxy radicals, first occurs at the 3-OH group of the flavonol. It is then followed by the formation of a peroxyl radical (after molecular oxygen or superoxide addition). The different cascades of reactions, which lead to the formation of depsides via C-ring opening, are discussed on the basis of the corresponding calculated energetic schemes.

Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo

Abstract Bladen, C. L., Flowers, M., Miyake, K., Podolsky, R. H., Barrett, J., Kozlowski, D. J. and Dynan, W. S. Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo. Radiat. Res. 168, 149–157 (2007). Quantitative studies of radiation cytotoxicity have been performed mostly in cells in culture. For a variety of reasons, however, the response of cells in culture may not reflect the response for cells in situ in a whole organism. We describe here an approach for quantification of radiation-induced cell death in vivo using the transparent embryo of the zebrafish, Danio rerio, as a model vertebrate system. Using this system, we show that the number of TUNEL-positive cells within a defined region increases approximately linearly with radiation dose up to 1 Gy. The results are consistent with predictions of a linear-quadratic model. The use of alternative models, accommodating a response threshold or low-dose hypersensitivity, did not significantly improve the fit to the observed data. Attenuation of the expression of the 80-kDa subunit of Ku, an essential protein for the nonhomologous end-joining pathway of repair, led to a dose reduction of 30- to 34-fold, possibly approaching the limit where each double-strand break causes a lethal hit. In both the Ku80-attenuated and the control embryos, apoptotic cells were distributed uniformly, consistent with a cell-autonomous mechanism of cell death. Together, these results illustrate the potential of the zebrafish for quantitative studies of radiation-induced cell death during embryogenesis and in vivo.

Developmental regulation and expression of the zebrafish connexin43 gene

We cloned and sequenced the zebrafish (Danio rerio) connexin43 (Cx43α1) gene. The predicted protein sequence shows a high degree of sequence conservation. Transcript analyses revealed multiple transcription start sites and a potential alternative transcript encoding a N‐terminally truncated Cx43α1 protein. Maternal Cx43α1 transcripts were detected, with zygotic expression initiated before gastrulation. In situ hybridization revealed many Cx43α1 expression domains, including the notochord and brain, heart and vasculature, many resembling patterns seen in mammalian embryos. Of interest, a reporter construct under control of the mouse Cx43α1 promoter was observed to drive green fluorescent protein expression in zebrafish embryos in domains mimicking the native Cx43α1 expression pattern in fish and mice. Sequence comparison between the mouse and zebrafish Cx43α1 promoter sequences showed the conservation of several transcription factor motifs, which otherwise shared little overall sequence homology. The conservation of protein sequence and developmental gene regulation would suggest that Cx43α1 gap junctions are likely to have conserved roles in vertebrate embryonic development. Developmental Dynamics 233:890–906, 2005.

Loss of Zebrafish lgi1b Leads to Hydrocephalus and Sensitization to Pentylenetetrazol Induced Seizure-Like Behavior

Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.

Molecular characterization and expression pattern of taurine transporter in zebrafish during embryogenesis.

Taurine and its transporter (TauT) are expressed in preimplantation embryos, but their role in embryogenesis is not known. To investigate the role of TauT during embryonic development, we cloned and functionally characterized the zebrafish TauT. The zebrafish TauT cDNA codes for a protein of 625 amino acids which is highly homologous to mammalian TauT. When expressed in mammalian cells, zebrafish TauT mediates taurine uptake in a Na(+)/Cl(-)-dependent manner with a Na(+):Cl(-):taurine stoichiometry of 2:1:1. In the zebrafish embryo, taurine and TauT mRNA are present during early cleavage stages, indicating that both the transporter and its substrate are maternally derived. During embryogenesis, zygotic expression of TauT mRNA is evident in the retina, brain, heart, kidney, and blood vessels. Knockdown of TauT by antisense morpholino oligonucleotides leads to cell death in the central nervous system and increased mortality. These findings suggest a specific role for TauT during development in vertebrates.

Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio)

There are multiple populations of gonadotropin-releasing hormone (GnRH) neurons that have distinct physiological and behavioral functions. Teleost fish have a population of GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb that is thought to play a neuromodulatory role in multiple physiological systems, including olfactory, visual, and reproductive. We used transgenic zebrafish in which the GnRH3 promoter drives expression of a green fluorescent protein to identify GnRH3 neurons during development in live embryos. Unlike with hypophysiotropic GnRH neurons of zebrafish, TN-GnRH3 neurons are of neural crest origin and are one of the first populations of GnRH neurons to develop in the early embryo. Using a combination of optical imaging and electrophysiology, we showed that during the first 3 days post-fertilization, TN-GnRH3 neurons increase in number, extend neural projections, move in association with tissue expansion, and acquire an adult-pattern of spontaneous action potential firing. Early during development, about half of the neurons were quiescent/non-firing. Later, at 3 days post-fertilization, there was an increase in the proportion of neurons showing action potential firing and an increase in the number of neurons that showed an adult-like tonic or beating pattern of action potential firing with a firing frequency similar to that seen in adult TN-GnRH3 neurons. This study represents the first neurophysiological investigation of developing GnRH neurons in live embryos--an important advancement in understanding their potential non-reproductive roles during embryogenesis.

Phylogenetic analysis of the NEEP21/calcyon/P19 family of endocytic proteins: evidence for functional evolution in the vertebrate CNS.

Endocytosis and vesicle trafficking are required for optimal neural transmission. Yet, little is currently known about the evolution of neuronal proteins regulating these processes. Here, we report the first phylogenetic study of NEEP21, calcyon, and P19, a family of neuronal proteins implicated in synaptic receptor endocytosis and recycling, as well as in membrane protein trafficking in the somatodendritic and axonal compartments of differentiated neurons. Database searches identified orthologs for P19 and NEEP21 in bony fish, but not urochordate or invertebrate phyla. Calcyon orthologs were only retrieved from mammalian databases and distant relatives from teleost fish. In situ localization of the P19 zebrafish ortholog, and extant progenitor of the gene family, revealed a CNS specific expression pattern. Based on non-synonymous nucleotide substitution rates, the calcyon genes appear to be under less intense negative selective pressure. Indeed, a functional group II WW domain binding motif was detected in primate and human calcyon, but not in non-primate orthologs. Sequencing of the calcyon gene from 80 human subjects revealed a non-synonymous single nucleotide polymorphism that abrogated group II WW domain protein binding. Altogether, our data indicate the NEEP21/calcyon/P19 gene family emerged, and underwent two rounds of gene duplication relatively late in metazoan evolution (but early in vertebrate evolution at the latest). As functional studies suggest NEEP21 and calcyon play related, but distinct roles in regulating vesicle trafficking at synapses, and in neurons in general, we propose the family arose in chordates to support a more diverse range of synaptic and behavioral responses.