DSCAM promotes axon fasciculation and growth in the developing optic pathway

Acknowledgments We thank Drs. Robert Burgess, Carol Mason, and Eloisa Herrera for helpful discussions; Dr. Thomas Theil for his invaluable advice on the slice culture methods; Francesca Lamb and Emma Smith for technical assistance; and the Institute of Medical Sciences Microscopy and Imaging Facility for assistance with confocal microscopy. This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) doctoral training award studentship and a BBSRC project grant (BB/J00815X/1). Freely available online through the PNAS open access option.Peer reviewedPublisher PD

Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction.

Nicotinamide adenine dinucleotide (NAD) is a REDOX cofactor and metabolite essential for neuronal survival. Glaucoma is a common neurodegenerative disease in which neuronal levels of NAD decline. We assess the effects of nicotinamide (a precursor to NAD) on retinal ganglion cells (the affected neuron in glaucoma) in normal physiological conditions and across a range of glaucoma relevant insults including mitochondrial stress and axon degenerative insults. We demonstrate retinal ganglion cell somal, axonal, and dendritic neuroprotection by nicotinamide in rodent models which represent isolated ocular hypertensive, axon degenerative, and mitochondrial degenerative insults. We performed metabolomics enriched for small molecular weight metabolites for the retina, optic nerve, and superior colliculus which demonstrates that ocular hypertension induces widespread metabolic disruption, including consistent changes to α-ketoglutaric acid, creatine/creatinine, homocysteine, and glycerophosphocholine. This metabolic disruption is prevented by nicotinamide. Nicotinamide provides further neuroprotective effects by increasing oxidative phosphorylation, buffering and preventing metabolic stress, and increasing mitochondrial size and motility whilst simultaneously dampening action potential firing frequency. These data support continued determination of the utility of long-term nicotinamide treatment as a neuroprotective therapy for human glaucoma

Adhesion molecules in establishing retinal circuitry

The formation of neural circuits requires molecular mechanisms to confer cell identity, to establish appropriate dendritic arbors, and to space cell bodies within groups of homotypic neurons. Recent work in a variety of organisms has implicated cell adhesion molecules in these processes. The DSCAMs in particular have functions including cell identity and self-avoidance through repulsion in Drosophila , differential adhesion and synaptic pairing in chick retina, and the masking of adhesion within specific cell types in the mouse retina. These differences in molecular function between different organisms, and potentially different cell types within a single tissue, emphasize how seemingly subtle distinctions may be important for deciphering this molecular adhesion code

Distinct expression patterns of mitochondrially localized YFP in neuronal subsets in the retina of three transgenic mouse lines

BACKGROUND: Transgenic labels that allow the visualization of specific populations of neurons have proven to be powerful tools for research. Further developing such resources to label additional cell types and specific organelles within these cell will provide additional experimental opportunities. FINDINGS: The retinal expression profile of a mitochondria-localized yellow fluorescent protein (YFP) in each of three transgenic mouse lines was determined. Each line, Mito-R, Mito-Y and Mito-Z, expresses YFP in distinct and reproducible populations of retinal neurons. In the Mito-R line, YFP is expressed in most or all retinal ganglion cells (RGCs) and photoreceptors making this line useful for studying axonal transport in diseases such as glaucoma and photoreceptor degeneration related to transport of mitochondria into the inner segments. In the Mito-Y line, YFP is expressed in many cell types in the dorsal retina and in a rough mosaic population of RGCs in the rest of the retina, making this line useful for study of how retinal mosaics are organized. In the Mito-Z line, YFP is expressed in a subset of RGCs, amacrine cells, bipolar cells and photoreceptors. The Mito-Z line is inserted on the X-Chromosome, resulting in X-inactivation mosaicism in female mice carrying a single copy of the transgene. In the female hemizygous retina, expression is present in distinct clonal columns, making this transgenic line useful for analysis of clonal proliferation and lateral migration of retinal neurons. CONCLUSION: The retinal expression profiles of three transgenic mouse lines that express a mitochondrially localized YFP were characterized in this study. These lines will allow researchers to isolate and identify cell types within the retina and to study retinal mitochondrial trafficking and disease

Defects in eye development in transgenic mice overexpressing the heparan sulfate proteoglycan agrin

The importance of heparan sulfate proteoglycans (HSPGs) in neurodevelopment is becoming increasingly clear. However, studies on HSPGs are hampered by pleiotropic effects when synthesis or modification of heparan sulfate itself is targeted, and by redundancy when the core proteins are altered. Gain-of-function experiments can sometimes circumvent these issues. Here we establish that transgenic mice overexpressing the HSPG agrin have severe ocular dysgenesis. The defects occur through a gain-of-function mechanism and penetrance is dependent on agrin dosage. The agrin-induced developmental defects are highly variable, and include anophthalmia, persistence of vitreous vessels, and fusion of anterior chamber structures. A frequently observed defect is an optic stalk coloboma leading to the misdifferentiation of the optic stalk as retina, which becomes continuous with the forebrain. The defects in optic-stalk differentiation correlate with reduced sonic hedgehog immunoreactivity and overexpansion of the PAX6 domain from the retina into the optic stalk. The ocular phenotypes associated with agrin overexpression are dependent on genetic background, occurring with high penetrance in inbred C57BL/6J mice. Distinct loci sensitizing C57BL/6J mice to agrin-induced dysgenesis were identified. These results indicate that agrin overexpression will provide a tool to explore the molecular interactions of the extracellular matrix and cell surface in eye development, and provide a means for identifying modifier loci that sensitize mice to developmental eye defects

Controlling integration specificity of a yeast retrotransposon

Retrotransposons and retroviruses integrate nonrandomly into eukaryotic genomes. For the yeast retrotransposon Ty5, integration preferentially occurs within domains of heterochromatin. Targeting to these locations is determined by interactions between an amino acid sequence motif at the C terminus of Ty5 integrase (IN) called the targeting domain, and the heterochromatin protein Sir4p. Here we show that new Ty5 integration hot spots are created when Sir4p is tethered to ectopic DNA sites. Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either IN or Sir4p that prevent their interaction. Ty5 target specificity can be altered by replacing the IN-targeting domain with other peptide motifs that interact with known protein partners. Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners are tethered. These findings define a mechanism by which retrotransposons shape their host genomes and suggest ways in which retroviral integration can be controlled

Cell autonomy of DSCAM function in retinal development

Cell adhesion molecules (CAMs) provide identifying cues by which neural architecture is sculpted. The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for many neurodevelopmental processes in different species and also has several potential mechanisms of activity, including homophilic adhesion, homophilic repulsion and heterophilic interactions. In the mouse retina, Dscam is expressed in many, but not all neuronal subtypes. Mutations in Dscam cause the fasciculation of dendrites of neighboring homotypic neurons, indicating a role in self-avoidance among cells of a given type, a disruption of the non-random patterning of their cell bodies, and a decrease in developmental cell death in affected cell populations. In order to address how DSCAM facilitates retinal pattering, we developed a conditional allele of Dscam to use alongside existing Dscam mutant mouse strains. Conditional deletion of Dscam reproduces cell spacing, cell number and dendrite arborization defects. Inducible deletion of Dscam and retinal ganglion cell depletion in Brn3b mutant retinas both indicate that these DSCAM-mediated phenotypes can occur independently. In chimeric retinas, in which wild type and Dscam mutant cells are comingled, Dscam mutant cells entangle adjacent wild type cells of the same type, as if both cells were lacking Dscam, consistent with DSCAM-dependent cell spacing and neurite arborization being mediated through homophilic binding cell-to-cell. Deletion of Dscam in specific cell types causes cell-type-autonomous cell body spacing defects, indicating that DSCAM mediates arborization and spacing by acting within given cell types. We also examine the cell autonomy of DSCAM in laminar stratification and find that laminar disorganization can be caused in a non-cell autonomous fashion. Finally, we find Dscam dosage-dependent defects in developmental cell death and amacrine cell spacing, relevant to the increased cell death and other disorders observed in Down syndrome mouse models and human patients, in which Dscam is present in three copies. ► Conditional allele developed to test DSCAM cell autonomy. ► DSCAM‐mediated processes were genetically separated and are distinct. ► Dscam regulates developmental cell death and spacing in a dose dependent manner. ► DSCAM deficient cells induce mutant phenotype on adjacent homotypic wild type cells. ► DSCAM mediates arborization and spacing by acting within cell types

Finding Down Syndrome Cell Adhesion Molecule (DSCAM) Protein Interactions in Pathways of Neuronal Development

Down Syndrome cell adhesion molecule (DSCAM) is a transmembrane protein that has a significant role in proper neurodevelopment by promoting self-avoidance in neurons. This project aims to identify proteins that interact with DSCAM in the mouse brain by using a library of plasmids. The plasmid library was tested for proteins that interact with the C-terminus end of DSCAM in Brewer’s yeast (S. cerevisiae). By using the bait and prey method from a yeast two-hybrid system, mouse brain proteins were tested for true interaction with DSCAM. Proteins that interact with DSCAM allow the yeast to produce their own leucine, which aids in the growth of the yeast on selective media. Proteins that have been identified as possible protein interactors include COX5B, NEFL, ELAV1, and SOD1, among others. By identifying interactions between unknown proteins and DSCAM, we will be better able to map out the protein interactions that lead to the proper development of the brain

Retrotransposon Target Site Selection by Imitation of a Cellular Protein

Mobile elements rely on cellular processes to replicate, and therefore, mobile element proteins frequently interact with a variety of cellular factors. The integrase (IN) encoded by the retrotransposon Ty5 interacts with the heterochromatin protein Sir4, and this interaction determines Ty5's preference to integrate into heterochromatin. We explored the hypothesis that Ty5's targeting mechanism arose by mimicking an interaction between Sir4 and another cellular protein(s). Mutational analyses defined the requirements for the IN-Sir4 interaction, providing criteria to screen for cellular analogues. Esc1, a protein associated with the inner nuclear membrane, interacted with the same domain of Sir4 as IN, and 75% of mutations that disrupted IN-Sir4 interactions also abrogated Esc1-Sir4 interactions. A small motif critical for recognizing Sir4 was identified in Esc1. The functional equivalency of this motif and the Sir4-interacting domain of IN was demonstrated by swapping these motifs and showing that the chimeric IN and Esc1 proteins effectively target integration and partition DNA, respectively. We conclude that Ty5 targets integration by imitating the Esc1-Sir4 interaction and suggest molecular mimicry as a general mechanism that enables mobile elements to interface with cellular processes