Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

The University of Melbourne

Despite its initial identification in neurons exposed to the seizure-inducing drug pentylentetrazole (PTZ), the function of the seizure-related gene SEZ-6 remains obscure. Expression analysis indicates specific expression in the adult brain and testis, and the structure of the predicted protein suggests putative roles in cell-cell recognition and signalling. We report here that type I SEZ-6 mRNA is strongly expressed in highly specific regions in the developing forebrain. Specifically, the pattern of SEZ-6 expression is closely tied with the emergence of the neocortical layers and hippocampus, and implies a forebrain-specific role for this gene during development. In the adult hippocampus, SEZ-6 appears to be a CA1-specific regional marker.

Localized expression of the seizure-related gene SEZ-6 in developing and adult forebrains.

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Serie de an&#xE1;lisis de expresi&#xF3;n g&#xE9;nica utilizando peque&#xF1;as cantidades de material de partida (microSAGE) a&#xFA;n no se ha demostrado de forma concluyente que sea representativo, reproducible o exacta.

MicroSAGE es altamente representativo y reproducible, pero muestra grandes diferencias en la expresi&#xF3;n gen&#xE9;tica entre las muestras obtenidas de tejidos similares

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Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate.

MicroSAGE is highly representative and reproducible but reveals major differences in gene expression among samples obtained from similar tissues.

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MicroSAGE est tr&#xE8;s repr&#xE9;sentatif et reproductible, mais constate de grandes diff&#xE9;rences dans l&#x26;#39;expression g&#xE9;nique chez les &#xE9;chantillons obtenus &#xE0; partir des tissus similaires

MicroSAGE ist sehr repr&#xE4;sentative und reproduzierbare aber offenbart gro&#xDF;e Unterschiede in der Genexpression zwischen den Proben aus &#xE4;hnlichen Gewebe erhalten.

Serial analysis of gene expression utilizzando piccole quantit&agrave; di materiale (microSAGE) di partenza non ha ancora definitivamente dimostrato essere rappresentativo, riproducibile o accurata.

MicroSAGE &egrave; altamente rappresentativi e riproducibili, ma rivela notevoli differenze nell&#39;espressione genica tra i campioni ottenuti da tessuti simili.

Following their generation in the germinal zones, young neurons of the neocortex, hippocampus and cerebellum undergo long-distance migration to reach their final destinations. This locomotive activity depends on active deployment of cytoskeletal elements including the microtubule apparatus. In this study, we report the identification and expression of radial spoke protein 3 (RSP3), a member of a protein cluster responsible for anchoring and modifying dynein motor activity known to be crucial to microtubule sliding. The mouse RSP3 gene consists of eight exons and seven introns and spans over 230 kb. The genomic organisations of the human and rat RSP3 genes are similar although they span approximately 23 and 53 kb, respectively. This is in contrast to the Chlamydomonas RSP3 gene, where RSP3 was first isolated, which consists of four exons and three introns and spans approximately 2.7 kb. Despite these differences, the nucleotide and amino acid sequences upstream of, and throughout the RPII-binding domain of RSP3 are highly conserved between all the above-mentioned species. Mouse RSP3 mRNA was restricted to the developing neocortex, hippocampus and cerebellum during the stages when these structures are known to contain large numbers of migratory neurons. Gene expression studies suggest that RSP3 function is consistent with a locomotory role for this protein in migrating young neurons. In addition, expression of RSP3 mRNA in adult neurons point to additional, though still unknown functions. Our data provides the first evidence for the expression of radial spoke proteins in higher eukaryotes, and provides a biological framework for how these proteins may participate in microtubule sliding and neuronal migration in the embryonic brain.

Genomic organisation and nervous system expression of radial spoke protein 3.

The telencephalon develops from a seemingly homogeneous population of precursor cells that generates neurons with divergent properties and cell fates. An important question concerns the genetic control of cell states belonging to different generations. Mph2 was identified by serial analysis of gene expression as a differentially expressed gene during cortical development. Belonging to the Polycomb group, Mph2 may be involved in transcriptional repression of cell states from one generation to the next. Here, we use in situ hybridization to determine the spatial localization of Mph2 expression in the developing nervous system. Mph2 expression evident in proliferating precursor cell populations in the developing mouse brain such as the ventricular zones of the cortex and ganglionic eminences as well as in mature structures such as the granule cell layer of the adult dentate gyrus and cerebellum indicates a role for Mph2 in both the developing and adult brain.

Mph2 expression in germinal zones of the mouse brain.

Manual neuron tracing is a very labor-intensive task. In the drug screening context, the sheer number of images to process means that this approach is unrealistic. Moreover, the lack of reproducibility, objectivity, and auditing capability of manual tracing is limiting even in the context of smaller studies. We have developed fast, sensitive, and reliable algorithms for the purpose of detecting and analyzing neurites in cell cultures, and we have integrated them in software called HCA-Vision, suitable for the research environment. We validate the software on images of cortical neurons by comparing results obtained using HCA-Vision with those obtained using an established semi-automated tracing solution (NeuronJ). The effect of the Sez-6 deletion was characterized in detail. Sez-6 null neurons exhibited a significant increase in neurite branching, although the neurite field area was unchanged due to a reduction in mean branch length. HCA-Vision delivered considerable speed benefits and reliable traces.

Automated analysis of neurite branching in cultured cortical neurons using HCA-Vision.

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.

Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons.

Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development.

Seizure-related gene 6 (Sez-6) in amacrine cells of the rodent retina and the consequence of gene deletion.

Reelin is an important protein that is indispensable for cortical lamination. In the absence of Reelin, cortical layers fail to form due to inappropriate neuron migration and positioning. The inversion of cortical layers is attributed to failure of neurons to migrate past earlier-generated neurons although how Reelin-insufficiency causes this is unclear. The issue is complicated by recent studies showing that very little Reelin is required for cortical layering. To test how variation in the number of Reelin-producing cells is linked to cortical lamination, we have employed Reelin(+/+) <--> Reelin(-/-) chimeras in which the number of Reelin-expressing neurons is adjusted. We found that the Reeler phenotype was rescued in chimeras with a large contribution of Reelin(+/+) neurons; conversely in chimeras with a weak contribution by Reelin(+/+) neurons, the mutant phenotype remained. However, increasing the number of Reelin(+/+) neurons beyond an unknown threshold resulted in partial rescue, with the formation of a correctly layered secondary cortex lying on top of an inverted mutant cortex. Therefore, the development of cortical layers in the correct order requires a minimal level of Reelin protein to be present although paradoxically, this is insufficient to prevent the simultaneous formation of inverted cortical layers in the same hemisphere.

Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras.

Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.

Jenny M. Gunnersen

Department of Anatomy & Neuroscience,
Florey Institute of Neuroscience & Mental Health

The University of Melbourne

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Jenny M. Gunnersen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Anatomy & Neuroscience,Florey Institute of Neuroscience & Mental Health

The University of Melbourne

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Department of Anatomy & Neuroscience,
Florey Institute of Neuroscience & Mental Health