Serotonin receptor 2C regulates neurite growth and is necessary for normal retinal processing of visual information.

Serotonin (5HT) is present in a subpopulation of amacrine cells, which form synapses with retinal ganglion cells (RGCs), but little is known about the physiological role of retinal serotonergic circuitry. We found that the 5HT receptor 2C (5HTR2C) is upregulated in RGCs after birth. Amacrine cells generate 5HT and about half of RGCs respond to 5HTR2C agonism with calcium elevation. We found that there are on average 83 5HT+ amacrine cells randomly distributed across the adult mouse retina, all negative for choline acetyltransferase and 90% positive for tyrosine hydroxylase. We also investigated whether 5HTR2C and 5HTR5A affect RGC neurite growth. We found that both suppress neurite growth, and that RGCs from the 5HTR2C knockout (KO) mice grow longer neurites. Furthermore, 5HTR2C is subject to post-transcriptional editing, and we found that only the edited isoform's suppressive effect on neurite growth could be reversed by a 5HTR2C inverse agonist. Next, we investigated the physiological role of 5HTR2C in the retina, and found that 5HTR2C KO mice showed increased amplitude on pattern electroretinogram. Finally, RGC transcriptional profiling and pathways analysis suggested partial developmental compensation for 5HTR2C absence. Taken together, our findings demonstrate that 5HTR2C regulates neurite growth and RGC activity and is necessary for normal amplitude of RGC response to physiologic stimuli, and raise the hypothesis that these functions are modulated by a subset of 5HT+/ChAT-/TH+ amacrine cells as part of retinal serotonergic circuitry. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

The N-terminal Set-ß Protein Isoform Induces Neuronal Death.

Set-ß protein plays different roles in neurons, but the diversity of Set-ß neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-ß are altered in Alzheimer disease, cleavage of Set-ß leads to neuronal death after stroke, and the full-length Set-ß regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-ß isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-ß-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-ß transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-ß is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-ß isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-ß cleavage product can induce neuronal death.

Regulating Set-ß's Subcellular Localization Toggles Its Function between Inhibiting and Promoting Axon Growth and Regeneration.

The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-ß are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-ß also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-ß was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-ß suppressed, whereas Set-ß localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-ß to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-ß in primary neurons, raising the hypothesis that nuclear Set-ß may preferentially regulate gene expression whereas Set-ß at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-ß to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-ß differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation.

Elucidating functional context within microarray data by integrated transcription factor-focused gene-interaction and regulatory network analysis.

Microarrays do not yield direct evidence for functional connections between genes. However, transcription factors (TFs) and their binding sites (TFBSs) in promoters are important for inducing and coordinating changes in RNA levels, and thus represent the first layer of functional interaction. Similar to genes, TFs act only in context, which is why a TF/TFBS-based promoter analysis of genes needs to be done in the form of gene(TF)-gene networks, not individual TFs or TFBSs. In addition, integration of the literature and various databases (e.g. GO, MeSH, etc) allows the adding of genes relevant for the functional context of the data even if they were initially missed by the microarray as their RNA levels did not change significantly. Here, we outline a TF-TFBSs network-based strategy to assess the involvement of transcription factors in agonist signaling and demonstrate its utility in deciphering the response of human microvascular endothelial cells (HMEC-1) to leukemia inhibitory factor (LIF). Our strategy identified a central core of eight TFs, of which only STAT3 had previously been definitively linked to LIF in endothelial cells. We also found potential molecular mechanisms of gene regulation in HMEC-1 upon stimulation with LIF that allow for the prediction of changes of genes not used in the analysis. Our approach, which is readily applicable to a wide variety of expression microarray and next generation sequencing RNA-seq results, illustrates the power of a TF-gene networking approach for elucidation of the underlying biology.

Plant genome resources at the national center for biotechnology information.

The National Center for Biotechnology Information (NCBI) integrates data from more than 20 biological databases through a flexible search and retrieval system called Entrez. A core Entrez database, Entrez Nucleotide, includes GenBank and is tightly linked to the NCBI Taxonomy database, the Entrez Protein database, and the scientific literature in PubMed. A suite of more specialized databases for genomes, genes, gene families, gene expression, gene variation, and protein domains dovetails with the core databases to make Entrez a powerful system for genomic research. Linked to the full range of Entrez databases is the NCBI Map Viewer, which displays aligned genetic, physical, and sequence maps for eukaryotic genomes including those of many plants. A specialized plant query page allow maps from all plant genomes covered by the Map Viewer to be searched in tandem to produce a display of aligned maps from several species. PlantBLAST searches against the sequences shown in the Map Viewer allow BLAST alignments to be viewed within a genomic context. In addition, precomputed sequence similarities, such as those for proteins offered by BLAST Link, enable fluid navigation from unannotated to annotated sequences, quickening the pace of discovery. NCBI Web pages for plants, such as Plant Genome Central, complete the system by providing centralized access to NCBI's genomic resources as well as links to organism-specific Web pages beyond NCBI.