Utilizing RNA sequencing to identify gene expression markers of stroke-causing thrombi origin: A pilot study.

INTRODUCTION: Stroke embolic source have an unknown origin in 30-40% of cases. Mechanical thrombectomy for acute large vessel occlusion stroke has provided us with a method to directly retrieve the thrombi from patients for analysis. By collecting stroke-causing thrombi from known sources, we can then use high-throughput RNA sequencing (RNAseq) technology to directly measure the gene expression signatures of these clots. This may allow us to identify genetic markers to predict the cause of cryptogenic embolism. METHODS: This is a prospective study in which RNAseq was used to analyze cerebral thrombi retrieved by mechanical thrombectomy devices in acute ischemic stroke patients. Samples were separated into two groups based on known stroke thrombus etiology, including Carotid group (patients with ipsilateral >70% carotid stenosis) and Atrial fibrillation (AF) group (patients with atrial fibrillation). Gene expression was compared by RNAseq analysis between the groups. RESULTS: From October 2016 to September 2017, 8 thrombi (4 in Carotid group, 4 in Afib group) were included in this study. There were 131 genes that were significantly up- or down-regulated between the two groups defined as a false discovery rate ≤ 0.05 and a fold change ≥ 2. Twenty-six genes were selected as candidate gene biomarkers based on the criteria in the methods section. Candidate genes HSPA1B, which encodes a heatshock protein, and GPRC5B, which encodes a G-protein, showed the greatest fold differences in expression between the two groups. CONCLUSION: This study has shown that RNA sequencing of acute ischemic stroke thrombi is feasible and indentified potential novel biomarkers for identifying stroke-causing thrombi origin, especially in cryptogenic stroke.

The Interdisciplinary Stem Cell Institute's Use of Food and Drug Administration-Expanded Access Guidelines to Provide Experimental Cell Therapy to Patients With Rare Serious Diseases.

The U.S. Food and Drug Administration (FDA) provides guidance for expanded access to experimental therapies, which in turn plays an important role in the Twenty-first Century Cures Act mandate to advance cell-based therapy. In cases of incurable diseases where there is a lack of alternative treatment options, many patients seek access to cell-based therapies for the possibility of treatment responses demonstrated in clinical trials. Here, we describe the use of the FDA's expanded access to investigational new drug (IND) to address rare and emergency conditions that include stiff-person syndrome, spinal cord injury, traumatic brain stem injury, complex congenital heart disease, ischemic stroke, and peripheral nerve injury. We have administered both allogeneic bone marrow-derived mesenchymal stem cell (MSC) and autologous Schwann cell (SC) therapy to patients upon emergency request using Single Patient Expanded Access (SPEA) INDs approved by the FDA. In this report, we present our experience with 10 completed SPEA protocols.

The unfulfilled gene and nervous system development in Drosophila.

The unfulfilled gene of Drosophila encodes a member of the NR2E subfamily of nuclear receptors. Like related members of the NR2E subfamily, UNFULFILLED is anticipated to function as a dimer, binding to DNA response elements and regulating the expression of target genes. The UNFULFILLED protein may be regulated by ligand-binding and may also be post-transcriptionally modified by sumoylation and phosphorylation. unfulfilled mutants display a range of aberrant phenotypes, problems with eclosion and post-eclosion behaviors, compromised fertility, arrhythmicity, and a lack of all adult mushroom body lobes. The locus of the fertility problem has not been determined. The behavioral arrhythmicity is due to the unfulfilled-dependent disruption of gene expression in a set of pacemaker neurons. The eclosion and the mushroom body lobe phenotypes of unfulfilled mutants are the result of developmental problems associated with failures in axon pathfinding or re-extension. Interest in genes that act downstream of unfulfilled has resulted in the identification of a growing number of unfulfilled interacting loci, providing the first glimpse into the composition of unfulfilled-dependent gene networks. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

unfulfilled interacting genes display branch-specific roles in the development of mushroom body axons in Drosophila melanogaster.

The mushroom body (MB) of Drosophila melanogaster is an organized collection of interneurons that is required for learning and memory. Each of the three subtypes of MB neurons, γ, α'/ß', and α/ß, branch at some point during their development, providing an excellent model in which to study the genetic regulation of axon branching. Given the sequential birth order and the unique patterning of MB neurons, it is likely that specific gene cascades are required for the different guidance events that form the characteristic lobes of the MB. The nuclear receptor UNFULFILLED (UNF), a transcription factor, is required for the differentiation of all MB neurons. We have developed and used a classical genetic suppressor screen that takes advantage of the fact that ectopic expression of unf causes lethality to identify candidate genes that act downstream of UNF. We hypothesized that reducing the copy number of unf-interacting genes will suppress the unf-induced lethality. We have identified 19 candidate genes that when mutated suppress the unf-induced lethality. To test whether candidate genes impact MB development, we performed a secondary phenotypic screen in which the morphologies of the MBs in animals heterozygous for unf and a specific candidate gene were analyzed. Medial MB lobes were thin, missing, or misguided dorsally in five double heterozygote combinations (;unf/+;axin/+, unf/+;Fps85D/+, ;unf/+;Tsc1/+, ;unf/+;Rheb/+, ;unf/+;msn/+). Dorsal MB lobes were missing in ;unf/+;DopR2/+ or misprojecting beyond the termination point in ;unf/+;Sytß double heterozygotes. These data suggest that unf and unf-interacting genes play specific roles in axon development in a branch-specific manner.

Netrin-guided accessory cell morphogenesis dictates the dendrite orientation and migration of a Drosophila sensory neuron.

Accessory cells, which include glia and other cell types that develop in close association with neurons, have been shown to play key roles in regulating neuron development. However, the underlying molecular and cellular mechanisms remain poorly understood. A particularly intimate association between accessory cells and neurons is found in insect chordotonal organs. We have found that the cap cell, one of two accessory cells of v'ch1, a chordotonal organ in the Drosophila embryo, strongly influences the development of its associated neuron. As it projects a long dorsally directed cellular extension, the cap cell reorients the dendrite of the v'ch1 neuron and tows its cell body dorsally. Cap cell morphogenesis is regulated by Netrin-A, which is produced by epidermal cells at the destination of the cap cell process. In Netrin-A mutant embryos, the cap cell forms an aberrant, ventrally directed process. As the cap cell maintains a close physical connection with the tip of the dendrite, the latter is dragged into an abnormal position and orientation, and the neuron fails to undergo its normal dorsal migration. Misexpression of Netrin-A in oenocytes, secretory cells that lie ventral to the cap cell, leads to aberrant cap cell morphogenesis, suggesting that Netrin-A acts as an instructive cue to direct the growth of the cap cell process. The netrin receptor Frazzled is required for normal cap cell morphogenesis, and mutant rescue experiments indicate that it acts in a cell-autonomous fashion.

The unfulfilled gene is required for the development of mushroom body neuropil in Drosophila.

BACKGROUND: The mushroom bodies (MBs) of Drosophila are required for complex behaviors and consist of three types of neurons, gamma, alpha'/beta' and alpha/beta. Previously, roles for transcription factors in MB neuronal differentiation have only been described for a subset of MB neurons. We are investigating the roles of unfulfilled (unf; HR51, CG16801) in MB development. unf encodes a nuclear receptor that is orthologous to the nuclear receptors fasciculation of axons defective 1 (FAX-1) of the nematode and photoreceptor specific nuclear receptor (PNR) of mammals. Based on our previous observations that unf transcripts accumulate in MB neurons at all developmental stages and the presence of axon pathfinding defects in fax-1 mutants, we hypothesized that unf regulates MB axon growth and pathfinding. RESULTS: We show that unf mutants exhibit a range of highly penetrant axon stalling phenotypes affecting all neurons of the larval and adult MBs. Phenotypic analysis of unfX1 mutants revealed that alpha'/beta' and alpha/beta neurons initially project axons but stall prior to the formation of medial or dorsal MB lobes. unfZ0001 mutants form medial lobes, although these axons fail to branch, which results in a failure to form the alpha or alpha' dorsal lobes. In either mutant background, gamma neurons fail to develop larval-specific dorsal projections. These mutant gamma neurons undergo normal pruning, but fail to re-extend axons medially during pupal development. unfRNAi animals displayed phenotypes similar to those seen in unfZ0001 mutants. Unique asymmetrical phenotypes were observed in unfX1/unfZ0001 compound heterozygotes. Expression of UAS-unf transgenes in MB neurons rescues the larval and adult unf mutant phenotypes. CONCLUSIONS: These data support the hypothesis that unf plays a common role in the development of all types of MB neurons. Our data indicate that unf is necessary for MB axon extension and branching and that the formation of dorsal collaterals is more sensitive to the loss of unf function than medial projections. The asymmetrical phenotypes observed in compound heterozygotes support the hypothesis that the earliest MB axons may serve as pioneers for the later-born MB neurons, providing evidence for pioneer MB axon guidance in post-embryonic development.

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo.

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.