Investigating the role of Hedgehog/GLI1 signaling in glioblastoma cell response to temozolomide.

Resistance to chemotherapy substantially hinders successful glioblastoma (GBM) treatment, contributing to an almost 100% mortality rate. Resistance to the frontline chemotherapy, temozolomide (TMZ), arises from numerous signaling pathways that are deregulated in GBM, including Hedgehog (Hh) signaling. Here, we investigate suppression of Hh signaling as an adjuvant to TMZ using U87-MG and T98G cell lines as in vitro models of GBM. We found that silencing GLI1 with siRNA reduces cell metabolic activity by up to 30% in combination with TMZ and reduces multidrug efflux activity by 2.5-fold. Additionally, pharmacological GLI inhibition modulates nuclear p53 levels and decreases MGMT expression in combination with TMZ. While we surprisingly found that silencing GLI1 does not induce apoptosis in the absence of TMZ co-treatment, we discovered silencing GLI1 without TMZ co-treatment induces senescence as evidenced by a significant 2.3-fold increase in senescence associated β-galactosidase staining, and this occurs in a loss of PTEN-dependent manner. Finally, we show that GLI inhibition increases apoptosis in glioma stem-like cells by up to 6.8-fold in combination with TMZ, and this reduces the size and number of neurospheres grown from glioma stem-like cells. In aggregate, our data warrant the continued investigation of Hh pathway inhibitors as adjuvants to TMZ chemotherapy and highlight the importance of identifying signaling pathways that determine whether co-treatment will be successful

Recovery of burrowing behavior after spinal cord injury in the larval sea lamprey

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Katz, H. R., Fouke, K. E., Losurdo, N. A., & Morgan, J. R. Recovery of burrowing behavior after spinal cord injury in the larval sea lamprey. Biological Bulletin, 239(3), (2020): 174-182, doi:10.1086/711365.Following traumatic spinal cord injury, most mammalian species are unable to achieve substantial neuronal regeneration and often experience loss of locomotor function. In contrast, larval sea lampreys (Petromyzon marinus) spontaneously recover normal swimming behaviors by 10–12 weeks post-injury, which is supported by robust regeneration of spinal axons. While recovery of swimming behavior is well established, the lamprey’s ability to recover more complex behaviors, such as burrowing, is unknown. Here we evaluated the lamprey’s ability to burrow into a sand substrate over the typical time course of functional recovery (1–11 weeks post-injury). Compared to uninjured control lampreys, which burrow rapidly and completely, spinal-transected animals did not attempt burrowing until 2 weeks post-injury; and they often did not succeed in fully covering their entire body in the sand. Burrowing behavior gradually improved over post-injury time, with most animals burrowing partially or completely by 9–11 weeks post-injury. Burrowing behavior has two components: the initial component that resembles swimming with propagated body undulations and the final component that pulls the tail under the sand. While the duration of the initial component did not differ between control and spinal-transected animals across the entire recovery period, the duration of the final component in spinal-transected animals was significantly longer at all time points measured. These data indicate that, after spinal cord injury, lampreys are able to recover burrowing behaviors, though some deficits persist.We thank Eduardo Guadarrama for performing lamprey transection surgeries and Dr. Eric D. Tytell (Tufts University) for valuable discussion and feedback, as well the Marine Biological Laboratory for providing funding support. NAL was funded in part by a National Science Foundation-sponsored Research Experiences for Undergraduates (REU) program at the Marine Biological Laboratory: “Biological Discovery in Woods Hole” (grant 1659604; PIs: A. Mensinger, V. Martinez Acosta)

Dynamics of Myoblast Transplantation Reveal a Discrete Minority of Precursors with Stem Cell–like Properties as the Myogenic Source

Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47–57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769–779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell–like characteristics

Regenerative capacity in the lamprey spinal cord is not altered after a repeated transection

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 14(1), (2019):e0204193, doi: 10.1371/journal.pone.0204193.The resilience of regeneration in vertebrates is not very well understood. Yet understanding if tissues can regenerate after repeated insults, and identifying limitations, is important for elucidating the underlying mechanisms of tissue plasticity. This is particularly challenging in tissues, such as the nervous system, which possess a large number of terminally differentiated cells and often exhibit limited regeneration in the first place. However, unlike mammals, which exhibit very limited regeneration of spinal cord tissues, many non-mammalian vertebrates, including lampreys, bony fishes, amphibians, and reptiles, regenerate their spinal cords and functionally recover even after a complete spinal cord transection. It is well established that lampreys undergo full functional recovery of swimming behaviors after a single spinal cord transection, which is accompanied by tissue repair at the lesion site, as well as axon and synapse regeneration. Here we begin to explore the resilience of spinal cord regeneration in lampreys after a second spinal transection (re-transection). We report that by all functional and anatomical measures tested, lampreys regenerate after spinal re-transection just as robustly as after single transections. Recovery of swimming, synapse and cytoskeletal distributions, axon regeneration, and neuronal survival were nearly identical after spinal transection or re-transection. Only minor differences in tissue repair at the lesion site were observed in re-transected spinal cords. Thus, regenerative potential in the lamprey spinal cord is largely unaffected by spinal re-transection, indicating a greater persistent regenerative potential than exists in some other highly regenerative models. These findings establish a new path for uncovering pro-regenerative targets that could be deployed in non-regenerative conditions.The authors would like to thank Dr. Cristina Roman-Vendrell and Louie Kerr, Director of the Central Microscopy Facility at the MBL, for technical support. We also thank Dr. Juan Diaz-Quiroz for helpful comments on the manuscript. EG was supported in part by an NSF REU Award (#1659604: Biological Discovery in Woods Hole at the Marine Biological Laboratory)

Phenology of Drosophila species across a temperate growing season and implications for behavior

Data have been deposited in Dryad, https://doi.org/10.5061/dryad.1bc102k.Drosophila community composition is complex in temperate regions with different abundance of flies and species across the growing season. Monitoring Drosophila populations provides insights into the phenology of both native and invasive species. Over a single growing season, we collected Drosophila at regular intervals and determined the number of individuals of the nine species we found in Kansas, USA. Species varied in their presence and abundance through the growing season with peak diversity occurring after the highest seasonal temperatures. We developed models for the abundance of the most common species, Drosophila melanogaster, D. simulans, D. algonquin, and the recent invasive species, D. suzukii. These models revealed that temperature played the largest role in abundance of each species across the season. For the two most commonly studied species, D. melanogaster and D. simulans, the best models indicate shifted thermal optima compared to laboratory studies, implying that fluctuating temperature may play a greater role in the physiology and ecology of these insects than indicated by laboratory studies, and should be considered in global climate change studies.Kansas State Biology Graduate Student Association Research GrantKU EEB GRF 210508

The Synaptic Vesicle Cycle Revisited:New Insights into the Modes and Mechanisms

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 39(42), (2019): 8209-8216, doi:10.1523/JNEUROSCI.1158-19.2019.Neurotransmission is sustained by endocytosis and refilling of synaptic vesicles (SVs) locally within the presynapse. Until recently, a consensus formed that after exocytosis, SVs are recovered by either fusion pore closure (kiss-and-run) or clathrin-mediated endocytosis directly from the plasma membrane. However, recent data have revealed that SV formation is more complex than previously envisaged. For example, two additional recycling pathways have been discovered, ultrafast endocytosis and activity-dependent bulk endocytosis, in which SVs are regenerated from the internalized membrane and synaptic endosomes. Furthermore, these diverse modes of endocytosis appear to influence both the molecular composition and subsequent physiological role of individual SVs. In addition, previously unknown complexity in SV refilling and reclustering has been revealed. This review presents a modern view of the SV life cycle and discusses how neuronal subtype, physiological temperature, and individual activity patterns can recruit different endocytic modes to generate new SVs and sculpt subsequent presynaptic performance.This work was supported by: Schram-Stiftung T287/25457 and Deutsche Forschungsgemeinschaft (Emmy Noether Young Investigator Award MI-1702/1 to I.M.); the Wellcome Trust (204954/Z/16/Z to M.A.C.); the National Science Foundation (1727260 to S.W.), the National Institutes of Health (NINDS DP2 NS111133 and R01 NS105810 to S.W.); the McKnight Foundation (S.W.); the Sloan Foundation (S.W.); and the National Institutes of Health (NINDS/NIA R01 NS078165 to J.R.M. and NIMH R01 MH066198 to Dr. Ege Kavalali, which supports N.L.C.). We thank Dragomir Milovanovic for helpful comments on this manuscript.2020-04-1

Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Pattanayak, R., Underwood, R., Crowley, M. R., Crossman, D. K., Morgan, J. R., & Yacoubian, T. A. Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse. Scientific Reports, 12(1), (2022): 9837, https://doi.org/10.1038/s41598-022-13557-8.We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870–61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.This study was supported by NIH [R56NS115767 (TAY), RF1NS115767-01A1 (TAY), P50NS108675 (TAY), and NINDS/NIA RF1NS078165 (JRM)]

A Novel Educational Module to Teach Neural Circuits for College and High School Students: NGSS-Neurons, Genetics, and Selective Stimulations

This report introduces various approaches to target defined neural pathways for stimulation and to address the effect of particular neural circuits on behavior in a model animal, the fruit fly (Drosophila melanogaster). The objective of this novel educational module described can be used to explain and address principle concepts in neurobiology for high school and college level students. A goal of neurobiology is to show how neural circuit activity controls corresponding behavior in animals. The fruit fly model system provides powerful genetic tools, such as the UAS-Gal4 system, to manipulate expression of non-native proteins in various populations of defined neurons: glutamergic, serotonergic, GABAergic, and cholinergic. The exhibited behaviors in the examples we provide allows teachers and students to address questions from behaviors to details at a cellular level. We provided example sets of data, obtained in a research lab, as well as ideas on ways to present data for participants and instructors. The optogenetic tool, channelrhodpsin 2 (ChR2), is employed to increase the activity of each population of neurons in a spatiotemporal controlled manner in behaving larvae and adult flies. Various behavioral assays are used to observe the effect of a specific neuron population activation on crawling behavior in larvae and climbing behavior in adult flies. Participants using this module become acquainted with the actions of different neurotransmitters in the nervous system. A pre- and post- assessment survey on the content is provided for teachers, as templates, to address learning of content and concepts