Zebrafish Model of MLL-Rearranged Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of leukemia. Standard treatment includes chemotherapy as well as stem cell transplantation, but for aging patients and those with impaired immune function these rigorous therapies are not always possible. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) exhibit far worse prognoses than patients without. Given these circumstances new therapies must be developed. Methods: Danio rerio (zebrafish) has emerged as a powerful model organism for investigating human blood malignancies due to the conservation of hematopoiesis between humans and zebrafish. We developed a transient transgenic model exhibiting AML characteristics by microinjecting single-cell zebrafish embryos with a tissue specific MLL-ENL expression construct. Results: We found that the expression of MLL-ENL induced a clustered expansion of MLL+ and pu.1+ myeloid cells on the yolk sac at 48 and 72 hours post fertilization (hpf). To characterize our transient AML model, we treated MLL-ENL expressing embryos with either one of or a combination of two drugs that are currently being used in human AML drug trials, Venetoclax and Flavopiridol. We found that treatment with either drug reduced the myeloid expansion induced by the expression of MLL-ENL, and that co-treatment reduced the observed myeloid expansion even further. Conclusions: Although further analysis is required, these data suggest that we successfully developed a transient transgenic AML model in zebrafish. Furthermore, these data suggest that Venetoclax and Flavopiridol co-treatment could yield better outcomes for AML patients than treatment with either drug individually.https://scholarscompass.vcu.edu/gradposters/1112/thumbnail.jp

The Human Postsynaptic Density Shares Conserved Elements with Proteomes of Unicellular Eukaryotes and Prokaryotes

The animal nervous system processes information from the environment and mediates learning and memory using molecular signaling pathways in the postsynaptic terminal of synapses. Postsynaptic neurotransmitter receptors assemble to form multiprotein complexes that drive signal transduction pathways to downstream cell biological processes. Studies of mouse and Drosophila postsynaptic proteins have identified key roles in synaptic physiology and behavior for a wide range of proteins including receptors, scaffolds, enzymes, structural, translational, and transcriptional regulators. Comparative proteomic and genomic studies identified components of the postsynaptic proteome conserved in eukaryotes and early metazoans. We extend these studies, and examine the conservation of genes and domains found in the human postsynaptic density with those across the three superkingdoms, archaeal, bacteria, and eukaryota. A conserved set of proteins essential for basic cellular functions were conserved across the three superkingdoms, whereas synaptic structural and many signaling molecules were specific to the eukaryote lineage. Genes involved with metabolism and environmental signaling in Escherichia coli including the chemotactic and ArcAB Two-Component signal transduction systems shared homologous genes in the mammalian postsynaptic proteome. These data suggest conservation between prokaryotes and mammalian synapses of signaling mechanisms from receptors to transcriptional responses, a process essential to learning and memory in vertebrates. A number of human postsynaptic proteins with homologs in prokaryotes are mutated in human genetic diseases with nervous system pathology. These data also indicate that structural and signaling proteins characteristic of postsynaptic complexes arose in the eukaryotic lineage and rapidly expanded following the emergence of the metazoa, and provide an insight into the early evolution of synaptic mechanisms and conserved mechanisms of learning and memory

Reciprocal regulation of microRNA and mRNA profiles in neuronal development and synapse formation.

BACKGROUND: Synapse formation and the development of neural networks are known to be controlled by a coordinated program of mRNA synthesis. microRNAs are now recognized to be important regulators of mRNA translation and stability in a wide variety of organisms. While specific microRNAs are known to be involved in neural development, the extent to which global microRNA and mRNA profiles are coordinately regulated in neural development is unknown. RESULTS: We examined mouse primary neuronal cultures, analyzing microRNA and mRNA expression. Three main developmental patterns of microRNA expression were observed: steady-state levels, up-regulated and down-regulated. Co-expressed microRNAs were found to have related target recognition sites and to be encoded in distinct genomic locations. A number of 43 differentially expressed miRNAs were located in five genomic clusters. Their predicted mRNA targets show reciprocal levels of expression. We identified a set of reciprocally expressed microRNAs that target mRNAs encoding postsynaptic density proteins and high-level steady-state microRNAs that target non-neuronal low-level expressed mRNAs. CONCLUSION: We characterized hundreds of miRNAs in neuronal culture development and identified three major modes of miRNA expression. We predict these miRNAs to regulate reciprocally expressed protein coding genes, including many genes involved in synaptogenesis. The identification of miRNAs that target mRNAs during synaptogenesis indicates a new level of regulation of the synapse

Recording long-term potentiation of synaptic transmission by three-dimensional multi-electrode arrays

BACKGROUND: Multi-electrode arrays (MEAs) have become popular tools for recording spontaneous and evoked electrical activity of excitable tissues. The majority of previous studies of synaptic transmission in brain slices employed MEAs with planar electrodes that had limited ability to detect signals coming from deeper, healthier layers of the slice. To overcome this limitation, we used three-dimensional (3D) MEAs with tip-shaped electrodes to probe plasticity of field excitatory synaptic potentials (fEPSPs) in the CA1 area of hippocampal slices of 129S5/SvEvBrd and C57BL/6J-Tyr(C-Brd )mice. RESULTS: Using 3D MEAs, we were able to record larger fEPSPs compared to signals measured by planar MEAs. Several stimulation protocols were used to induce long-term potentiation (LTP) of synaptic responses in the CA1 area recorded following excitation of Schäffer collateral/commissural fibres. Either two trains of high frequency tetanic stimulation or three trains of theta-burst stimulation caused a persistent, pathway specific enhancement of fEPSPs that remained significantly elevated for at least 60 min. A third LTP induction protocol that comprised 150 pulses delivered at 5 Hz, evoked moderate LTP if excitation strength was increased to 1.5× of the baseline stimulus. In all cases, we observed a clear spatial plasticity gradient with maximum LTP levels detected in proximal apical dendrites of pyramidal neurones. No significant differences in the manifestation of LTP were observed between 129S5/SvEvBrd and C57BL/6J-Tyr(C-Brd )mice with the three protocols used. All forms of plasticity were sensitive to inhibition of N-methyl-D-aspartate (NMDA) receptors. CONCLUSION: Principal features of LTP (magnitude, pathway specificity, NMDA receptor dependence) recorded in the hippocampal slices using MEAs were very similar to those seen in conventional glass electrode experiments. Advantages of using MEAs are the ability to record from different regions of the slice and the ease of conducting several experiments on a multiplexed platform which could be useful for efficient screening of novel transgenic mice