@article{green_wall_weeks_mattingly_marsden_planchart_2023, title={Developmental cadmium exposure disrupts zebrafish vestibular calcium channels interfering with otolith formation and inner ear function}, volume={96}, ISSN={["1872-9711"]}, DOI={10.1016/j.neuro.2023.04.006}, abstractNote={Dizziness or balance problems are estimated to affect approximately 3.3 million children aged three to 17 years. These disorders develop from a breakdown in the balance control system and can be caused by anything that affects the inner ear or the brain, including exposure to environmental toxicants. One potential environmental toxicant linked to balance disorders is cadmium, an extremely toxic metal that occurs naturally in the earth's crust and is released as a byproduct of industrial processes. Cadmium is associated with balance and vestibular dysfunction in adults exposed occupationally, but little is known about the developmental effects of low-concentration cadmium exposure. Our findings indicate that zebrafish exposed to 10-60 parts per billion (ppb) cadmium from four hours post-fertilization (hpf) to seven days post-fertilization (dpf) exhibit abnormal behaviors, including pronounced increases in auditory sensitivity and circling behavior, both of which are linked to reductions in otolith growth and are rescued by the addition of calcium to the media. Pharmacological intervention shows that agonist-induced activation of the P2X calcium ion channel in the presence of cadmium restores otolith size. In conclusion, cadmium-induced ototoxicity is linked to vestibular-based behavioral abnormalities and auditory sensitivity following developmental exposure, and calcium ion channel function is associated with these defects.}, journal={NEUROTOXICOLOGY}, author={Green, Adrian J. and Wall, Alex R. and Weeks, Ryan D. and Mattingly, Carolyn J. and Marsden, Kurt C. and Planchart, Antonio}, year={2023}, month={May}, pages={129–139} }
 @article{hodorovich_harris_burton_neese_bieler_chudasama_marsden_2023, title={Effects of 4 testing arena sizes and 11 types of embryo media on sensorimotor behaviors in wild-type andchd7mutant zebrafish larvae}, url={https://doi.org/10.1101/2023.07.31.551330}, DOI={10.1101/2023.07.31.551330}, abstractNote={The larval zebrafish is a highly versatile model across research disciplines, and the expanding use of behavioral analysis has contributed to many advances in neuro-psychiatric, developmental, and toxicological studies, often through large-scale chemical and genetic screens. In the absence of standardized approaches to larval zebrafish behavior analysis, however, it is critical to understand the impact on behavior of experimental variables such as the size of testing arenas and the choice of embryo medium. Using a custom-built, modular high-throughput testing system, we examined the effects of 4 testing arena sizes and 11 types of embryo media on conserved sensorimotor behaviors in zebrafish larvae. Our data show that testing arena size impacts acoustic startle sensitivity and kinematics as well as spontaneous locomotion and thigmotaxis, with fish tested in larger arenas displaying reduced startle sensitivity and increased locomotion. We also find that embryo media can dramatically affect startle sensitivity, kinematics, habituation, and pre-pulse inhibition, as well as spontaneous swimming, turning, and overall activity. Common media components such as methylene blue and high calcium concentration consistently reduced startle sensitivity and locomotion. To further address how the choice of embryo medium can impact phenotype expression in zebrafish models of disease, we reared chd7 mutant larvae, a model of CHARGE syndrome with previously characterized morphological and behavioral phenotypes, in 5 different types of media and observed impacts on all phenotypes. By defining the effects of these key extrinsic factors on larval zebrafish behavior, these data can help researchers select the most appropriate conditions for their specific research questions, particularly for genetic and chemical screens.}, author={Hodorovich, Dana R. and Harris, Tiara Fryer and Burton, Derek and Neese, Katie and Bieler, Rachael and Chudasama, Vimal and Marsden, Kurt. C}, year={2023}, month={Aug} }
 @article{hodorovich_lindsley_berry_burton_marsden_2023, title={Morphological and sensorimotor phenotypes in a zebrafish CHARGE syndrome model are domain-dependent}, volume={1}, ISSN={["1601-183X"]}, url={https://doi.org/10.1111/gbb.12839}, DOI={10.1111/gbb.12839}, abstractNote={CHARGE syndrome is a heterogeneous disorder characterized by a spectrum of defects affecting multiple tissues and behavioral difficulties such as autism, attention-deficit/hyperactivity disorder, obsessive–compulsive disorder, anxiety, and sensory deficits. Most CHARGE cases arise from de novo, loss-of-function mutations in chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 is required for processes such as neuronal differentiation and neural crest cell migration, but how CHD7 affects neural circuit function to regulate behavior is unclear. To investigate the pathophysiology of behavioral symptoms in CHARGE, we established a mutant chd7 zebrafish line that recapitulates multiple CHARGE phenotypes including ear, cardiac, and craniofacial defects. Using a panel of behavioral assays, we found that chd7 mutants have specific auditory and visual behavior deficits that are independent of defects in sensory structures. Mauthner cell-dependent short-latency acoustic startle responses are normal in chd7 mutants, while Mauthner-independent long-latency responses are reduced. Responses to sudden decreases in light are also reduced in mutants, while responses to sudden increases in light are normal, suggesting that the retinal OFF pathway may be affected. Furthermore, by analyzing multiple chd7 alleles we observed that the penetrance of morphological and behavioral phenotypes is influenced by genetic background but that it also depends on the mutation location, with a chromodomain mutation causing the highest penetrance. This pattern is consistent with analysis of a CHARGE patient dataset in which symptom penetrance was highest in subjects with mutations in the CHD7 chromodomains. These results provide new insight into the heterogeneity of CHARGE and will inform future work to define CHD7-dependent neurobehavioral mechanisms.}, journal={GENES BRAIN AND BEHAVIOR}, author={Hodorovich, Dana R. R. and Lindsley, Patrick M. M. and Berry, Austen A. A. and Burton, Derek F. F. and Marsden, Kurt C. C.}, year={2023}, month={Jan} }
 @article{hodorovich_lindsley_berry_burton_marsden_2022, title={Morphological and sensorimotor phenotypes in a zebrafish CHARGE syndrome model are domain-dependent}, url={https://doi.org/10.1101/2022.07.14.499979}, DOI={10.1101/2022.07.14.499979}, abstractNote={CHARGE syndrome is a rare disorder characterized by a spectrum of defects affecting multiple tissues and behavioral difficulties such as autism, attention-deficit/hyperactivity disorder, obsessive-compulsive disorder, anxiety, and sensory deficits. Most CHARGE cases arise from de novo, loss-of-function mutations in a master transcriptional regulator, chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 regulates key neurodevelopmental factors and is required for neural processes including neuronal differentiation and neural crest cell migration, but how CHD7 affects neural circuit function to regulate behavior is unclear. To investigate the pathophysiology of behavioral symptoms in CHARGE, we established a mutant chd7 zebrafish line using CRISPR/Cas9 that recapitulates multiple CHARGE phenotypes. Using a panel of behavioral assays, we find that chd7 mutants have specific auditory and visually driven behavioral deficits that are independent of defects in sensory structures, implicating chd7 in the regulation of underlying brain circuits. Furthermore, by analyzing multiple chd7 alleles we show that the penetrance of morphological and behavioral phenotypes depends on the mutation location. These results provide novel insight into the heterogeneity of CHARGE syndrome and will inform future work to define mechanisms of CHD7-dependent neurobehavioral symptoms.}, author={Hodorovich, Dana R. and Lindsley, Patrick M. and Berry, Austen A. and Burton, Derek F. and Marsden, Kurt C.}, year={2022}, month={Jul} }
 @article{martin_bereman_marsden_2022, title={The Cyanotoxin 2,4-DAB Reduces Viability and Causes Behavioral and Molecular Dysfunctions Associated with Neurodegeneration in Larval Zebrafish}, volume={40}, ISSN={["1476-3524"]}, url={https://doi.org/10.1007/s12640-021-00465-4}, DOI={10.1007/s12640-021-00465-4}, abstractNote={Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's disease. While the cyanotoxin β-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA, including 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether 2,4-DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB; however, only modulated startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB's effects, we analyzed the protein profile of larval zebrafish exposed to 500 µM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.}, number={2}, journal={NEUROTOXICITY RESEARCH}, publisher={Springer Science and Business Media LLC}, author={Martin, Rubia M. and Bereman, Michael S. and Marsden, Kurt C.}, year={2022}, month={Jan} }
 @article{meserve_nelson_marsden_hsu_echeverry_jain_wolman_pereda_granato_2021, title={A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1}, volume={17}, ISSN={["1553-7404"]}, url={https://doi.org/10.1371/journal.pgen.1008943}, DOI={10.1371/journal.pgen.1008943}, abstractNote={The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.}, number={6}, journal={PLOS GENETICS}, publisher={Public Library of Science (PLoS)}, author={Meserve, Joy H. and Nelson, Jessica C. and Marsden, Kurt C. and Hsu, Jerry and Echeverry, Fabio A. and Jain, Roshan A. and Wolman, Marc A. and Pereda, Alberto E. and Granato, Michael}, editor={Moens, CeciliaEditor}, year={2021}, month={Jun} }
 @article{martin_bereman_marsden_2021, title={BMAA and MCLR Interact to Modulate Behavior and Exacerbate Molecular Changes Related to Neurodegeneration in Larval Zebrafish}, volume={179}, ISSN={["1096-0929"]}, url={https://doi.org/10.1093/toxsci/kfaa178}, DOI={10.1093/toxsci/kfaa178}, abstractNote={Exposure to toxins produced by cyanobacteria (ie, cyanotoxins) is an emerging health concern due to their increasing prevalence and previous associations with neurodegenerative diseases including amyotrophic lateral sclerosis. The objective of this study was to evaluate the neurotoxic effects of a mixture of two co-occurring cyanotoxins, β-methylamino-l-alanine (BMAA) and microcystin leucine and arginine (MCLR), using the larval zebrafish model. We combined high-throughput behavior-based toxicity assays with discovery proteomic techniques to identify behavioral and molecular changes following 6 days of exposure. Although neither toxin caused mortality, morphological defects, nor altered general locomotor behavior in zebrafish larvae, both toxins increased acoustic startle sensitivity in a dose-dependent manner by at least 40% (p < .0001). Furthermore, startle sensitivity was enhanced by an additional 40% in larvae exposed to the BMAA/MCLR mixture relative to those exposed to the individual toxins. Supporting these behavioral results, our proteomic analysis revealed a 4-fold increase in the number of differentially expressed proteins in the mixture-exposed group. Additionally, prediction analysis reveals activation and/or inhibition of 8 enriched canonical pathways (enrichment p-value < .01; z-score≥|2|), including ILK, Rho Family GTPase, RhoGDI, and calcium signaling pathways, which have been implicated in neurodegeneration. We also found that expression of TDP-43, of which cytoplasmic aggregates are a hallmark of amyotrophic lateral sclerosis pathology, was significantly upregulated by 5.7-fold following BMAA/MCLR mixture exposure. Together, our results emphasize the importance of including mixtures of cyanotoxins when investigating the link between environmental cyanotoxins and neurodegeneration as we reveal that BMAA and MCLR interact in vivo to enhance neurotoxicity.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, publisher={Oxford University Press (OUP)}, author={Martin, Rubia M. and Bereman, Michael S. and Marsden, Kurt C.}, year={2021}, month={Feb}, pages={251–261} }
 @article{lasseigne_echeverry_ijaz_michel_martin_marsh_trujillo_marsden_pereda_miller_2021, title={Electrical synaptic transmission requires a postsynaptic scaffolding protein}, volume={10}, ISSN={["2050-084X"]}, url={https://doi.org/10.7554/eLife.66898}, DOI={10.7554/eLife.66898}, abstractNote={Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses.Neurons ‘talk’ with each another at junctions called synapses, which can either be chemical or electrical. Communication across a chemical synapse involves a ‘sending’ neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron. These complex junctions involve a large number of well-studied molecular actors. Electrical synapses, on the other hand, are believed to be simpler. There, neurons are physically connected via channels formed of ‘connexin’ proteins, which allow electrically charged ions to flow between the cells. However, it is likely that other proteins help to create these structures. In particular, recent evidence shows that without a structurally supporting ‘scaffolding’ protein called ZO1, electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish. As their name implies, scaffolding proteins help cells organize their internal structure, for example by anchoring other molecules to the cell membrane. By studying electrical synapses in zebrafish, Lasseigne, Echeverry, Ijaz, Michel et al. now show that these structures are more complex than previously assumed. In particular, the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization, these junctions can be asymmetric, like their chemical cousins. The results also show that ZO1 must be present for connexins to gather at electrical synapses, whereas the converse is not true. This suggests that when a new electrical synapse forms, ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells. In many animals with a spine, electrical synapses account for about 20% of all neural junctions. Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses, such as epilepsy.}, journal={ELIFE}, author={Lasseigne, Abagael M. and Echeverry, Fabio A. and Ijaz, Sundas and Michel, Jennifer Carlisle and Martin, E. Anne and Marsh, Audrey J. and Trujillo, Elisa and Marsden, Kurt C. and Pereda, Alberto E. and Miller, Adam C.}, year={2021}, month={Apr} }
 @article{kikel-coury_green_nichols_zellmer_pai_hedlund_marsden_smith_2021, title={Pioneer Axons Utilize a Dcc Signaling-Mediated Invasion Brake to Precisely Complete Their Pathfinding Odyssey}, volume={41}, ISSN={["1529-2401"]}, DOI={10.1523/JNEUROSCI.0212-21.2021}, abstractNote={Axons navigate through the embryo to construct a functional nervous system. A missing part of the axon navigation puzzle is how a single axon traverses distinct anatomic choice points through its navigation. The dorsal root ganglia (DRG) neurons experience such choice points. First, they navigate to the dorsal root entry zone (DREZ), then halt navigation in the peripheral nervous system to invade the spinal cord, and then reinitiate navigation inside the CNS. Here, we used time-lapse super-resolution imaging in zebrafish DRG pioneer neurons to investigate how embryonic axons control their cytoskeleton to navigate to and invade at the correct anatomic position. We found that invadopodia components form in the growth cone even during filopodia-based navigation, but only stabilize when the axon is at the spinal cord entry location. Further, we show that intermediate levels of DCC and cAMP, as well as Rac1 activation, subsequently engage an axon invasion brake. Our results indicate that actin-based invadopodia components form in the growth cone and disruption of the invasion brake causes axon entry defects and results in failed behavioral responses, thereby demonstrating the importance of regulating distinct actin populations during navigational challenges. <b>SIGNIFICANCE STATEMENT</b> Correct spatiotemporal navigation of neuronal growth cones is dependent on extracellular navigational cues and growth cone dynamics. Here, we link <i>dcc</i>-mediated signaling to actin-based invadopodia and filopodia dynamics during pathfinding and entry into the spinal cord using an <i>in vivo</i> model of dorsal root ganglia (DRG) sensory axons. We reveal a molecularly-controlled brake on invadopodia stabilization until the sensory neuron growth cone is present at the dorsal root entry zone (DREZ), which is ultimately essential for growth cone entry into the spinal cord and behavioral response.}, number={31}, journal={JOURNAL OF NEUROSCIENCE}, author={Kikel-Coury, Nina L. and Green, Lauren A. and Nichols, Evan L. and Zellmer, Abigail M. and Pai, Sanjana and Hedlund, Sam A. and Marsden, Kurt C. and Smith, Cody J.}, year={2021}, month={Aug}, pages={6617–6636} }
 @article{martin_bereman_marsden_2021, title={The cyanotoxin 2,4-DAB enhances mortality and causes behavioral and molecular dysfunctions associated with neurodegeneration in larval zebrafish}, volume={10}, url={https://doi.org/10.1101/2021.10.13.464292}, DOI={10.1101/2021.10.13.464292}, abstractNote={Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's disease. While the cyanotoxin beta-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA including 2,4-diaminobutyric acid (2,4-DAB), and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB, however, only modulated the startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB's effects, we analyzed the protein profile of larval zebrafish exposed to 500 uM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.}, publisher={Cold Spring Harbor Laboratory}, author={Martin, Rubia M. and Bereman, Michael S. and Marsden, Kurt C.}, year={2021}, month={Oct} }
 @article{meserve_nelson_marsden_hsu_echeverry_jain_wolman_pereda_granato_2020, title={A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1}, volume={6}, url={https://doi.org/10.1101/2020.06.19.161240}, DOI={10.1101/2020.06.19.161240}, abstractNote={Abstract The acoustic startle response is an evolutionary conserved avoidance behavior. Disruptions in startle behavior, in particular startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. This identified mutants in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 acts independently of supraspinal inputs to regulate locomotion, suggesting its site of action is within spinal circuitry. Moreover, we show that Kv1.1 protein is mis-localized in dolk mutants, suggesting they act in a common genetic pathway to regulate movement magnitude. Combined, our results identify a diverse set of eight genes all associated with human disorders that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway. Author summary Underlying all animal behaviors are neural circuits, which are controlled by numerous molecular pathways that direct neuron development and activity. To identify and study these molecular pathways that control behavior, we use a simple vertebrate behavior, the acoustic startle response, in the larval zebrafish. In response to an intense noise, larval zebrafish will quickly turn and swim away to escape. From a genetic screen, we have identified a number of mutants that behave in abnormal ways in response to an acoustic stimulus. We cloned these mutants and identified eight genes that regulate startle behavior. All eight genes are associated with human disorders, and here we focus on two genes, dolk and kcna1a , encoding Dolk, a key regulator of protein glycosylation, and the potassium channel Kv1.1, respectively. We demonstrate that loss of dolk or kcna1a causes larval zebrafish to perform exaggerated swim movements and that Dolk is required for Kv1.1 protein localization to axons of neurons throughout the nervous system, providing strong evidence that dolk and kcna1a act in a common molecular pathway. Combined, our studies provide new insights into the genetic regulation of startle behavior.}, publisher={Cold Spring Harbor Laboratory}, author={Meserve, Joy H. and Nelson, Jessica C. and Marsden, Kurt C. and Hsu, Jerry and Echeverry, Fabio A. and Jain, Roshan A. and Wolman, Marc A. and Pereda, Alberto E. and Granato, Michael}, year={2020}, month={Jun} }
 @article{martin_bereman_marsden_2020, title={Exposure to a mixture of BMAA and MCLR synergistically modulates behavior in larval zebrafish while exacerbating molecular changes related to neurodegeneration}, volume={7}, url={https://doi.org/10.1101/2020.07.15.205617}, DOI={10.1101/2020.07.15.205617}, abstractNote={Abstract Exposure to toxins produced by cyanobacteria (i.e., cyanotoxins) is an emerging health concern due to their increased occurrence and previous associations with neurodegenerative disease including amyotrophic lateral sclerosis (ALS). The objective of this study was to evaluate the neurotoxic effects of a mixture of two co-occurring cyanotoxins, β-methylamino-L-alanine (BMAA) and microcystin leucine and arginine (MCLR), using the larval zebrafish model. We combined high-throughput behavior based toxicity assays with discovery proteomic techniques to identify behavioral and molecular changes following 6 days of exposure. While neither toxin caused mortality, morphological defects, or altered general locomotor behavior in zebrafish larvae, both toxins increased acoustic startle sensitivity in a dose-dependent manner by at least 40% (p<0.0001). Furthermore, startle sensitivity was enhanced by an additional 40% in larvae exposed to the BMAA/MCLR mixture relative to those exposed to the individual toxins. Supporting these behavioral results, our proteomic analysis revealed a 4-fold increase in the number of differentially expressed proteins (DEPs) in the mixture exposed group. Additionally, prediction analysis reveals activation and/or inhibition of 8 enriched canonical pathways (enrichment p-value<0.01; z-score≥|2|), including ILK, Rho Family GTPase, RhoGDI, and calcium signaling pathways, which have been implicated in neurodegeneration. We also found that expression of TDP-43, of which cytoplasmic aggregates are a hallmark of ALS pathology, was significantly upregulated by 5.7-fold following BMAA/MCLR mixture exposure. Together, our results emphasize the importance of including mixtures of cyanotoxins when investigating the link between environmental cyanotoxins and neurodegeneration as we reveal that BMAA and MCLR interact in vivo to enhance neurotoxicity.}, publisher={Cold Spring Harbor Laboratory}, author={Martin, Rubia M. and Bereman, Michael S. and Marsden, Kurt C.}, year={2020}, month={Jul} }
 @article{bremer_marsden_miller_granato_2019, title={The ubiquitin ligase PHR promotes directional regrowth of spinal zebrafish axons}, volume={2}, ISSN={["2399-3642"]}, url={https://doi.org/10.1038/s42003-019-0434-2}, DOI={10.1038/s42003-019-0434-2}, abstractNote={Abstract To reconnect with their synaptic targets, severed axons need to regrow robustly and directionally along the pre-lesional trajectory. While mechanisms directing axonal regrowth are poorly understood, several proteins direct developmental axon outgrowth, including the ubiquitin ligase PHR (Mycbp2). Invertebrate PHR also limits regrowth of injured axons, whereas its role in vertebrate axonal regrowth remains elusive. Here we took advantage of the high regrowth capacity of spinal zebrafish axons and observed robust and directional regrowth following laser transection of spinal Mauthner axons. We found that PHR directs regrowing axons along the pre-lesional trajectory and across the transection site. At the transection site, initial regrowth of wild-type axons was multidirectional. Over time, misdirected sprouts were corrected in a PHR-dependent manner. Ablation of cyfip2 , known to promote F-actin-polymerization and pharmacological inhibition of JNK reduced misdirected regrowth of PHR-deficient axons, suggesting that PHR controls directional Mauthner axonal regrowth through cyfip2 - and JNK-dependent pathways.}, number={1}, journal={COMMUNICATIONS BIOLOGY}, publisher={Springer Science and Business Media LLC}, author={Bremer, Juliane and Marsden, Kurt C. and Miller, Adam and Granato, Michael}, year={2019}, month={May} }
 @article{marsden_jain_wolman_echeverry_nelson_hayer_miltenberg_pereda_granato_2018, title={A Cyfip2-Dependent Excitatory Interneuron Pathway Establishes the Innate Startle Threshold}, volume={23}, ISSN={["2211-1247"]}, url={http://europepmc.org/articles/PMC6642828}, DOI={10.1016/j.celrep.2018.03.095}, abstractNote={Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process.}, number={3}, journal={CELL REPORTS}, author={Marsden, Kurt C. and Jain, Roshan A. and Wolman, Marc A. and Echeverry, Fabio A. and Nelson, Jessica C. and Hayer, Katharina E. and Miltenberg, Ben and Pereda, Alberto E. and Granato, Michael}, year={2018}, month={Apr}, pages={878–887} }
 @article{jain_wolman_marsden_nelson_shoenhard_echeverry_szi_bell_skinner_cobbs_et al._2018, title={A Forward Genetic Screen in Zebrafish Identifies the G-Protein-Coupled Receptor CaSR as a Modulator of Sensorimotor Decision Making}, volume={28}, ISSN={["1879-0445"]}, url={http://europepmc.org/articles/PMC5940496}, DOI={10.1016/j.cub.2018.03.025}, abstractNote={Animals continuously integrate sensory information and select contextually appropriate responses. Here, we show that zebrafish larvae select a behavioral response to acoustic stimuli from a pre-existing choice repertoire in a context-dependent manner. We demonstrate that this sensorimotor choice is modulated by stimulus quality and history, as well as by neuromodulatory systems-all hallmarks of more complex decision making. Moreover, from a genetic screen coupled with whole-genome sequencing, we identified eight mutants with deficits in this sensorimotor choice, including mutants of the vertebrate-specific G-protein-coupled extracellular calcium-sensing receptor (CaSR), whose function in the nervous system is not well understood. We demonstrate that CaSR promotes sensorimotor decision making acutely through Gαi/o and Gαq/11 signaling, modulated by clathrin-mediated endocytosis. Combined, our results identify the first set of genes critical for behavioral choice modulation in a vertebrate and reveal an unexpected critical role for CaSR in sensorimotor decision making.}, number={9}, journal={CURRENT BIOLOGY}, author={Jain, Roshan A. and Wolman, Marc A. and Marsden, Kurt C. and Nelson, Jessica C. and Shoenhard, Hannah and Echeverry, Fabio A. and Szi, Christina and Bell, Hannah and Skinner, Julianne and Cobbs, Emilia N. and et al.}, year={2018}, month={May}, pages={1357-+} }
 @article{miller_whitebirch_shah_marsden_granato_o'brien_moens_2017, title={A genetic basis for molecular asymmetry at vertebrate electrical synapses}, volume={1}, DOI={10.1101/102319}, abstractNote={Abstract Neural network function is based upon the patterns and types of connections made between neurons. Neuronal synapses are adhesions specialized for communication and they come in two types, chemical and electrical. Communication at chemical synapses occurs via neurotransmitter release whereas electrical synapses utilize gap junctions for direct ionic and metabolic coupling. Electrical synapses are often viewed as symmetrical structures, with the same components making both sides of the gap junction. By contrast, we show that a broad set of electrical synapses in zebrafish, Danio rerio , require two gap-junction-forming Connexins for formation and function. We find that one Connexin functions presynaptically while the other functions postsynaptically in forming the channels. We also show that these synapses are required for the speed and coordination of escape responses. Our data identify a genetic basis for molecular asymmetry at vertebrate electrical synapses and show they are required for appropriate behavioral performance.}, publisher={Cold Spring Harbor Laboratory}, author={Miller, Adam C and Whitebirch, Alex C and Shah, Arish N and Marsden, Kurt C and Granato, Michael and O'Brien, John and Moens, Cecilia B}, year={2017}, month={Jan} }
 @article{wolman_jain_marsden_bell_skinner_hayer_hogenesch_granato_2015, title={A Genome-wide Screen Identifies PAPP-AA-Mediated IGFR Signaling as a Novel Regulator of Habituation Learning}, volume={85}, DOI={10.1016/j.neuron.2015.02.025}, abstractNote={Habituation represents a fundamental form of learning, yet the underlying molecular genetic mechanisms are not well defined. Here we report on a genome-wide genetic screen, coupled with whole-genome sequencing, that identified 14 zebrafish startle habituation mutants including mutants of the vertebrate-specific gene pregnancy-associated plasma protein-aa (pappaa). PAPP-AA encodes an extracellular metalloprotease known to increase IGF bioavailability, thereby enhancing IGF receptor signaling. We find that pappaa is expressed by startle circuit neurons, and expression of wild-type but not a metalloprotease-inactive version of pappaa restores habituation in pappaa mutants. Furthermore, acutely inhibiting IGF1R function in wild-type reduces habituation, while activation of IGF1R downstream effectors in pappaa mutants restores habituation, demonstrating that pappaa promotes learning by acutely and locally increasing IGF bioavailability. In sum, our results define the first functional gene set for habituation learning in a vertebrate and identify PAPPAA-regulated IGF signaling as a novel mechanism regulating habituation learning.}, number={6}, journal={Neuron}, publisher={Elsevier BV}, author={Wolman, Marc A. and Jain, Roshan A. and Marsden, Kurt C. and Bell, Hannah and Skinner, Julianne and Hayer, Katharina E. and Hogenesch, John B. and Granato, Michael}, year={2015}, month={Mar}, pages={1200–1211} }
 @article{wolman_jain_marsden_bell_skinner_hayer_hogenesch_granato_2015, title={A Genome-wide Screen Identifies PAPP-AA-Mediated IGFR Signaling as a Novel Regulator of Habituation Learning}, volume={87}, DOI={10.1016/j.neuron.2015.08.009}, abstractNote={(Neuron 85, 1200–1211; March 18, 2015) In the original publication, Figure 6 did not accurately reflect the pappaap170 genotypes analyzed for habituation. The corrected version of the figure is shown below, and this has now been corrected in the article online. A Genome-wide Screen Identifies PAPP-AA-Mediated IGFR Signaling as a Novel Regulator of Habituation LearningWolman et al.NeuronMarch 5, 2015In BriefThrough a forward genetic screen in zebrafish, Wolman et al. isolate the first functional vertebrate gene set for habituation learning and identify pregnancy-associated plasma protein aa (PAPP-AA)-regulated IGF signaling as a novel mechanism underlying habituation learning. Full-Text PDF Open Archive}, number={4}, journal={Neuron}, publisher={Elsevier BV}, author={Wolman, Marc A. and Jain, Roshan A. and Marsden, Kurt C. and Bell, Hannah and Skinner, Julianne and Hayer, Katharina E. and Hogenesch, John B. and Granato, Michael}, year={2015}, month={Aug}, pages={906–907} }
 @article{marsden_granato_2015, title={In Vivo Ca2+ Imaging Reveals that Decreased Dendritic Excitability Drives Startle Habituation}, volume={13}, DOI={10.1016/j.celrep.2015.10.060}, abstractNote={Exposure to repetitive startling stimuli induces habitation, a simple form of learning. Despite its simplicity, the precise cellular mechanisms by which repeated stimulation converts a robust behavioral response to behavioral indifference are unclear. Here, we use head-restrained zebrafish larvae to monitor subcellular Ca(2+) dynamics in Mauthner neurons, the startle command neurons, during startle habituation in vivo. Using the Ca(2+) reporter GCaMP6s, we find that the amplitude of Ca(2+) signals in the lateral dendrite of the Mauthner neuron determines startle probability and that depression of this dendritic activity rather than downstream inhibition mediates glycine and N-methyl-D-aspartate (NMDA)-receptor-dependent short-term habituation. Combined, our results suggest a model for habituation learning in which increased inhibitory drive from feedforward inhibitory neurons combined with decreased excitatory input from auditory afferents decreases dendritic and Mauthner neuron excitability.}, number={9}, journal={Cell Reports}, publisher={Elsevier BV}, author={Marsden, Kurt C. and Granato, Michael}, year={2015}, pages={1733–1740} }
 @article{butler_iben_marsden_epstein_granato_weinstein_2015, title={SNPfisher: tools for probing genetic variation in laboratory-reared zebrafish}, volume={142}, DOI={10.1242/dev.118786}, abstractNote={Single nucleotide polymorphisms (SNPs) are the benchmark molecular markers for modern genomics. Until recently, relatively few SNPs were known in the zebrafish genome. The use of next-generation sequencing for the positional cloning of zebrafish mutations has increased the number of known SNP positions dramatically. Still, the identified SNP variants remain under-utilized, owing to scant annotation of strain specificity and allele frequency. To address these limitations, we surveyed SNP variation in three common laboratory zebrafish strains using whole-genome sequencing. This survey identified an average of 5.04 million SNPs per strain compared with the Zv9 reference genome sequence. By comparing the three strains, 2.7 million variants were found to be strain specific, whereas the remaining variants were shared among all (2.3 million) or some of the strains. We also demonstrate the broad usefulness of our identified variants by validating most in independent populations of the same laboratory strains. We have made all of the identified SNPs accessible through ‘SNPfisher’, a searchable online database (snpfisher.nichd.nih.gov). The SNPfisher website includes the SNPfisher Variant Reporter tool, which provides the genomic position, alternate allele read frequency, strain specificity, restriction enzyme recognition site changes and flanking primers for all SNPs and Indels in a user-defined gene or region of the zebrafish genome. The SNPfisher site also contains links to display our SNP data in the UCSC genome browser. The SNPfisher tools will facilitate the use of SNP variation in zebrafish research as well as vertebrate genome evolution.}, number={8}, journal={Development}, publisher={The Company of Biologists}, author={Butler, M. G. and Iben, J. R. and Marsden, K. C. and Epstein, J. A. and Granato, M. and Weinstein, B. M.}, year={2015}, month={Mar}, pages={1542–1552} }
 @article{perni_marsden_escobar_hollingworth_baylor_franzini-armstrong_2015, title={Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle}, volume={145}, DOI={10.1085/jgp.201411303}, abstractNote={The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca(2+) release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as "junctional feet" (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation-contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca(2+) sparks, which are elementary SR Ca(2+) release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca(2+) sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere(-1) s(-1). The few Ca(2+) sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca(2+) sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca(2+) flux underlying Ca(2+) sparks detected in fully differentiated frog and fish fibers.}, number={3}, journal={The Journal of General Physiology}, publisher={Rockefeller University Press}, author={Perni, Stefano and Marsden, Kurt C. and Escobar, Matias and Hollingworth, Stephen and Baylor, Stephen M. and Franzini-Armstrong, Clara}, year={2015}, month={Feb}, pages={173–184} }
 @article{perni_marsden_escobar_hollingworth_baylor_franzini-armstrong_2015, title={Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle}, volume={145}, DOI={10.1085/jgp.20141130302112015c}, abstractNote={The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca2+ sparks, which are elementary SR Ca2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere−1 s−1. The few Ca2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca2+ flux underlying Ca2+ sparks detected in fully differentiated frog and fish fibers.}, number={3}, journal={The Journal of General Physiology}, publisher={Rockefeller University Press}, author={Perni, Stefano and Marsden, Kurt C. and Escobar, Matias and Hollingworth, Stephen and Baylor, Stephen M. and Franzini-Armstrong, Clara}, year={2015}, month={Feb}, pages={253–253} }
 @article{casimiro_sossa_uzunova_beattie_marsden_carroll_2011, title={mGluR and NMDAR activation internalize distinct populations of AMPARs}, volume={48}, DOI={10.1016/j.mcn.2011.07.007}, abstractNote={Activation of metabotropic- (mGluRs) or NMDA-type glutamate receptors (NMDARs) each can induce long-term depression (LTD) of synaptic transmission in CA1 hippocampal neurons. These two forms of LTD are triggered by diverse signaling pathways yet both are expressed by the internalization of AMPA-type glutamate receptors (AMPARs). An unanswered question remains as to whether the convergence of the mGluR and NMDAR signaling pathways on AMPAR endocytosis renders these two forms of plasticity functionally equivalent, with both pathways inducing endocytosis of the same population of synaptic AMPARs. We now report evidence that these pathways couple to the endocytosis of distinct populations of AMPARs defined by their mobility in the membrane surface. NMDAR activation enhances removal of surface AMPARs that rapidly cycle into and out of the membrane surface, while activation of mGluRs with DHPG results in the internalization of a non-mobile population of AMPARs. Glutamate Receptor Interacting Proteins 1 and 2 (GRIP1/2) play a key role in defining the non-cycling receptor population. GRIP1/2 knockdown with siRNA increases the proportion of rapidly cycling surface AMPARs and inhibits mGluR- but not NMDAR-mediated AMPAR internalization. Additionally, we find that mGluR activation dissociates surface AMPARs from GRIP1/2 while stimulation of NMDARs elicits the loss of membrane receptors not bound to GRIP1/2. We propose that these two receptor pathways can drive the endocytosis of distinct populations of AMPARs: NMDARs activation induces the endocytosis of rapidly cycling surface AMPARs not directly associated with GRIP1/2 while mGluR activation induces the endocytosis of non-cycling GRIP-bound surface AMPARs.}, number={2}, journal={Molecular and Cellular Neuroscience}, publisher={Elsevier BV}, author={Casimiro, Tanya M. and Sossa, Kenneth G. and Uzunova, Genoveva and Beattie, Jennifer B. and Marsden, Kurt C. and Carroll, Reed C.}, year={2011}, month={Oct}, pages={161–170} }
 @article{marsden_shemesh_bayer_carroll_2010, title={Selective translocation of Ca2+/calmodulin protein kinase II  (CaMKII ) to inhibitory synapses}, volume={107}, DOI={10.1073/pnas.1010346107}, abstractNote={Ca 2+ /Calmodulin protein kinase IIα (CaMKIIα) has a central role in regulating neuronal excitability. It is well established that CaMKIIα translocates to excitatory synapses following strong glutamatergic stimuli that induce NMDA-receptor (NMDAR)-dependent long-term potentiation in CA1 hippocampal neurons. We now show that CaMKIIα translocates to inhibitory but not excitatory synapses in response to more moderate NMDAR-activating stimuli that trigger GABA A -receptor (GABA A R) insertion and enhance inhibitory transmission. Such moderate NMDAR activation causes Thr286 autophosphorylation of CaMKIIα, which our results demonstrate is necessary and sufficient, under basal conditions, to localize CaMKIIα at inhibitory synapses and enhance surface GABA A R expression. Although stronger glutamatergic stimulation coupled to AMPA receptor insertion also elicits Thr286 autophosphorylation, accumulation of CaMKIIα at inhibitory synapses is prevented under these conditions by the phosphatase calcineurin. This preferential targeting of CaMKIIα to glutamatergic or GABAergic synapses provides neurons with a mechanism whereby activity can selectively potentiate excitation or inhibition through a single kinase mediator.}, number={47}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Marsden, K. C. and Shemesh, A. and Bayer, K. U. and Carroll, R. C.}, year={2010}, month={Nov}, pages={20559–20564} }
 @article{weinger_omari_marsden_raine_shafit-zagardo_2009, title={Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions}, volume={175}, DOI={10.2353/ajpath.2009.080807}, abstractNote={Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer (∼150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity. Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer (∼150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity. Multiple sclerosis (MS) is a debilitating white matter disease of the central nervous system (CNS). Although much of the evidence from animal models and MS suggests it to be an autoimmune disorder mediated by TH-1 type T cells,1Sospedra M Martin R Immunology of multiple sclerosis.Annu Rev Immunol. 2005; 23: 683-747Crossref PubMed Scopus (1780) Google Scholar other possible causes include genetic and environmental factors, antibody-dependent cytotoxicity, and bacterial and viral infections that may mediate altered protein expression resulting in inflammation, axonal and oligodendrocyte damage, demyelination and CNS scarring.2Noseworthy JH Lucchinetti C Rodriguez M Weinshenker BG Multiple sclerosis.N Engl J Med. 2000; 343: 938-952Crossref PubMed Scopus (3016) Google Scholar Growth and survival factors that protect against axonal and oligodendrocyte damage or loss, and dampen the inflammatory response are actively being pursued for MS therapy.2Noseworthy JH Lucchinetti C Rodriguez M Weinshenker BG Multiple sclerosis.N Engl J Med. 2000; 343: 938-952Crossref PubMed Scopus (3016) Google Scholar, 3Fontoura P Steinman L Miller A Emerging therapeutic targets in multiple sclerosis.Curr Opin Neurol. 2006; 19: 260-266Crossref PubMed Scopus (28) Google Scholar, 4Frohman EM Racke MK Raine CS Multiple sclerosis–the plaque and its pathogenesis.N Engl J Med. 2006; 354: 942-955Crossref PubMed Scopus (1375) Google Scholar, 5Raine CS The Norton Lecture: a review of the oligodendrocyte in the multiple sclerosis lesion.J Neuroimmunol. 1997; 77: 135-152Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 6Franklin RJ Why does remyelination fail in multiple sclerosis?.Nat Rev Neurosci. 2002; 3: 705-714Crossref PubMed Scopus (672) Google Scholar One growth factor associated with oligodendrocyte maturation, survival and dampening the immune response is growth-arrest specific protein 6 (Gas6). 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Chronic active MS lesions are characterized by ongoing demyelination, astrogliosis, macrophage and lymphocyte infiltration, astroglial hypertrophy, and oligodendrocyte hyperplasia.46Raine CS Multiple sclerosis: clinical and pathogenic basis.in: Raine CS McFarland HF Tourtellotte WW Chapman & Hall Medical, 1997: 151-171Google Scholar Chronic silent MS lesions are characterized by the absence of actively infiltrating and inflammatory cells, oligodendrocyte loss and no evidence of ongoing demyelination.46Raine CS Multiple sclerosis: clinical and pathogenic basis.in: Raine CS McFarland HF Tourtellotte WW Chapman & Hall Medical, 1997: 151-171Google Scholar, 47Lassmann H Raine CS Antel J Prineas JW Immunopathology of multiple sclerosis: report on an international meeting held at the Institute of Neurology of the University of Vienna.J Neuroimmunol. 1998; 86: 213-217Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 48Cannella B Raine CS Multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation.Ann Neurol. 2004; 55: 46-57Crossref PubMed Scopus (117) Google Scholar In this study, we investigated in chronic active and chronic silent MS lesions whether increased expression of soluble Axl and Mer was associated with increased expression of the MMPs ADAM17 and ADAM10, similar to previous studies that showed an association between increased ADAM17 and ADAM10 with TNFα in the CNS of MS patients.37Kieseier BC Pischel H Neuen-Jacob E Tourtellotte WW Hartung HP ADAM-10 and ADAM-17 in the inflamed human CNS.Glia. 2003; 42: 398-405Crossref PubMed Scopus (50) Google Scholar, 38Guo L Eisenman JR Mahimkar RM Peschon JJ Paxton RJ Black RA Johnson RS A proteomic approach for the identification of cell-surface proteins shed by metalloproteases.Mol Cell Proteomics. 2002; 1: 30-36Crossref PubMed Scopus (79) Google Scholar We also investigated whether in lesions increased soluble Axl and Mer was associated with decreased Gas6, resulting in loss of the beneficial effects from activating membrane-bound Axl and Mer receptors. Cryostat sections and protein homogenates were prepared from nine MS cases; two primary progressive and seven secondary progressive, in total containing six chronic active and eight chronic silent lesions. Tissue sections and homogenates from cerebral white matter of three cases of other neurological disease (OND) included olivopontocerebellar degeneration, amyotrophic lateral sclerosis, and stroke. Tissue from three non-neurological subjects, and five normal appearing white matter sections from MS brains were classified as normal (Table 1). There were no histological differences between tissue from non-neurological subjects and normal appearing white matter; therefore, material from these subjects were grouped.Table 1Summary of Cases Utilized for Immunohistochemistry and ImmunoblottingCase no.DiagnosisDisease duration (yr)Sex/age (yr)Cause of death1PPMS8F/31Respiratory failure2PPMS20F/39Respiratory failure3SPMS11F/38Bronchopneumonia4SPMS20F/45Bronchopneumonia5SPMS20F/47Cardiac arrest6SPMS21F/56Cardiac arrest7SPMS15M/46Cardiac arrest8SPMS23M/67Cardiac arrest9SPMS16M/61Cardiac arrest10OPCD4M/31Bronchopneumonia11ALS5F/49Bronchopneumonia12Stroke12 hoursF/80Stroke13Non-neurologicaln/aM/19Cardiac arrest/obesity14Non-neurologicaln/aM/40Adult respiratory distress15Non-neurologicaln/aF/80Metastatic cancerPPMS, primary progressive multiple sclerosis; SPMS, secondary progressive multiple sclerosis; OPCD, olivopontocerebellar degeneration; ALS, amyotrophic lateral sclerosis; n/a, not applicable. Open table in a new tab PPMS, primary progressive multiple sclerosis; SPMS, secondary progressive multiple sclerosis; OPCD, olivopontocerebellar degeneration; ALS, amyotrophic lateral sclerosis; n/a, not applicable. Ten-micrometer frozen sections fixed in acetone were quenched for endogenous peroxidase activity and blocked for 1 hour with 10% normal goat serum. Sections were then incubated with primary antibodies against Gas6 (catalog AF885), Axl (MAB154), Mer (MAB8911), and Tyro3 (MAB859; R&D Systems; Minneapolis, MN) or Gas6 polyclonal antibody (generated in the laboratory of Dr. Anne Prieto, Indiana University), diluted in 2% normal goat serum in 1X Tris-buffered saline (TBS) overnight at 4°C. Immunoreacted sections were developed by incubating with biotinylated secondary antibodies, followed by Vectastain avidin-biotin complex solution (Vector Laboratories; Burlingame, CA), and 3,3′-diaminobenzidine (DAB) solution (KPL, Gaithersburg, MD). As negative controls, sections were incubated with isotype-matched irrelevant antibodies (BD Bioscience; San Diego, CA), at the same concentration as the primary antibody, or with carrier buffer alone. Double-label immunohistochemistry was performed to confirm the identity of the cells expressing Axl. Sections were stained with the Axl primary antibody and developed with DAB as described above. Sections were then washed twice with 1X TBS, permeabilized in 0.25% Triton X-100 in 1X TBS for 30 minutes at 22°C, washed an additional two times with 1X TBS, and incubated with the astrocyte specific marker, glial fibrillary acidic protein (1:300; Biogenex; San Ramon, CA) polyclonal antibody (pAb) or the microglia specific marker, Iba-1 (1:400, Wako; Richmond, VA) pAb in 1X TBS plus 5% bovine serum albumin, overnight at 4°C. Alternatively, after development with DAB and subsequent washes, sections were incubated with 10 μg/ml Proteinase K (Sigma, Saint Louis, MO) in 20 mmol/L Tris pH 7.5 for 15 minutes at 37°C; washed in 0.1% Triton X-100 in 1X TBS for 30 minutes at 22°C; and blocked in 5% goat serum/0.1% Triton X-100/5% milk in 1X TBS for 1 hour at 22°C. These alternatively treated sections were incubated with the oligodendrocyte-specific marker, platelet-derived growth factor receptor α (PDGFRα, 1:100) overnight at 4°C. The PDGFRα pAb was generated in the laboratory of Dr. William Stallcup at the Burnham Institute for Medical Research (La Jolla, CA). Following incubation with primary antibodies, the sections were washed three times in 1X TBS and incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit secondary antibody (1:300; Southern Biotechnology; Birmingham, AL), for 1 hour at 22°C. One Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium AP (BCIP/NB-AP) substrate tablet (Sigma) was dissolved in 10 ml of ddH20 (plus 1 μmol/liter levimasole (Sigma)). Sections were incubated with BCIP/NB-AP for 30 minutes at 22°C, washed three times with 1X TBS, dehydrated in 80%/95%/100% ethanol for 5 minutes each, followed by xylene for 10 minutes, and mounted under Permount (Fisher Scientific; Fair Lawn, NJ). Total protein was extracted from fresh frozen brain autopsy tissue from chronic active MS, chronic silent MS, OND, and normal cases as previously described. Except where noted in the figure legends, 80 μg of protein were loaded in 1X final concentration loading buffer containing 2% sodium dodecyl sulfate, 0.017% bromophenol blue dye, and 0.28 mol/L β-mercaptoethanol (load dye) and separated in a 10% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis.49Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar, 50Towbin H Staehelin T Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44893) Google Scholar, 51Zamora-Leon SP Bresnick A Backer JM Shafit-Zagardo B Fyn phosphorylates human MAP-2c on tyrosine 67.J Biol Chem. 2005; 280: 1962-1970Crossref PubMed Scopus (15) Google Scholar Following electrophoresis, proteins were transferred to nitrocellulose. Blots were incubated with 5% nonfat dry milk and 5% goat serum in 1X TBS for 1 hour at room temperature. After blocking, membranes were incubated with respective primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Primary monoclonal antibodies (mAb) and pAb’s included: Axl mAb (IgG1, 1:250), Tyro3 mAb (IgG1, 1:500), Mer mAb (IgG2b, 1:500), R&D Gas6 pAb (1:100), Prieto Gas6 pAb (1:8000), Furin pAb (1:250, made in the laboratory of Dr. Dennis Shields, Albert Einstein College of Medicine), ADAM10 pAb (1:1000, eBioscience; San Diego, CA), ADAM17 pAb (1:1000, eBioscience), and β-actin mAb (IgG2A, 1:5000; Sigma, St. Louis, MO). Secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA) included: goat α-rabbit IgG (1:10,000), and goat α-mouse IgG1 (1:10,000), IgG2A (1:10,000), and IgG2b (1:10,000). Visualization of all secondary antibodies was by enhanced chemiluminescence (GE HealthCare; Piscataway, NJ). A peptide-N-glycosidase F (PNGaseF) kit (QAbio; Palm Desert, CA) was used to assess glycosylation of ADAM17. The volume of 40 μg of protein homogenate from normal samples was adjusted to 35 μl, and 10 μl of 5 × 250 mmol/L sodium phosphate, pH 7.5 (reaction buffer), and 2.5 μl of 2% sodiu}, number={1}, journal={The American Journal of Pathology}, publisher={Elsevier BV}, author={Weinger, Jason G. and Omari, Kakuri M. and Marsden, Kurt and Raine, Cedric S. and Shafit-Zagardo, Bridget}, year={2009}, month={Jul}, pages={283–293} }
 @article{marsden_beattie_friedenthal_carroll_2007, title={NMDA Receptor Activation Potentiates Inhibitory Transmission through GABA Receptor-Associated Protein-Dependent Exocytosis of GABAA Receptors}, volume={27}, DOI={10.1523/jneurosci.4433-07.2007}, abstractNote={The trafficking of postsynaptic AMPA receptors (AMPARs) is a powerful mechanism for regulating the strength of excitatory synapses. It has become clear that the surface levels of inhibitory GABA A receptors (GABA A Rs) are also subject to regulation and that GABA A R trafficking may contribute to inhibitory plasticity, although the underlying mechanisms are not fully understood. Here, we report that NMDA receptor activation, which has been shown to drive excitatory long-term depression through AMPAR endocytosis, simultaneously increases expression of GABA A Rs at the dendritic surface of hippocampal neurons. This NMDA stimulus increases miniature IPSC amplitudes and requires the activity of Ca 2+ calmodulin-dependent kinase II and the trafficking proteins N -ethylmaleimide-sensitive factor, GABA receptor-associated protein (GABARAP), and glutamate receptor interacting protein (GRIP). These data demonstrate for the first time that endogenous GABARAP and GRIP contribute to the regulated trafficking of GABA A Rs. In addition, they reveal that the bidirectional trafficking of AMPA and GABA A receptors can be driven by a single glutamatergic stimulus, providing a potent postsynaptic mechanism for modulating neuronal excitability.}, number={52}, journal={Journal of Neuroscience}, publisher={Society for Neuroscience}, author={Marsden, K. C. and Beattie, J. B. and Friedenthal, J. and Carroll, R. C.}, year={2007}, pages={14326–14337} }
 @article{clarke_buskey_marsden_2004, title={Effects of water motion and prey behavior on zooplankton capture by two coral reef fishes}, volume={146}, DOI={10.1007/s00227-004-1528-y}, number={6}, journal={Marine Biology}, publisher={Springer Nature}, author={Clarke, R. D. and Buskey, E. J. and Marsden, K. C.}, year={2004}, pages={1145–1155} }