In vivo imaging of calcium dynamics in zebrafish hepatocytes

Hepatocytes were the first cell-type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a non-invasive manner due to the optical inaccessibility of the mammalian liver. Here we take advantage of the transparency of the zebrafish larvae to develop a setup that allows in vivo imaging of calcium flux in zebrafish hepatocytes at cellular resolution. Using this, we provide quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo, which are lost upon starvation. Feeding recommences calcium waves in the liver, but in a spatially restricted manner. Further, ethanol treatment as well as cell ablation induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, while the latter leads to a single calcium spike. Overall, we demonstrate the presence of oscillations, waves and spikes in vivo. Thus, our study introduces a platform for observing diverse calcium dynamics while maintaining the native environment of the liver, which will help investigations into the dissection of molecular mechanisms supporting the intra- and intercellular calcium signaling in the liver.

Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia

ABSTRACT Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Three Birds with one stone: A single AIEgen for dual-organelle imaging, cell viability evaluation and photodynamic cancer cell ablation

Fully understanding the relationship among various organelles and cell viability is great important to clarify the mechanism of cancer diagnosis and treatment. However, the development of a single fluorescent probe...

An Investigation for Large Volume, Focal Blood-Brain Barrier Disruption with High-Frequency Pulsed Electric Fields

The treatment of CNS disorders suffers from the inability to deliver large therapeutic agents to the brain parenchyma due to protection from the blood-brain barrier (BBB). Herein, we investigated high-frequency pulsed electric field (HF-PEF) therapy of various pulse widths and interphase delays for BBB disruption while selectively minimizing cell ablation. Eighteen male Fisher rats underwent craniectomy procedures and two blunt-tipped electrodes were advanced into the brain for pulsing. BBB disruption was verified with contrast T1W MRI and pathologically with Evans blue dye. High-frequency irreversible electroporation cell death of healthy rodent astrocytes was investigated in vitro using a collagen hydrogel tissue mimic. Numerical analysis was conducted to determine the electric fields in which BBB disruption and cell ablation occur. Differences between the BBB disruption and ablation thresholds for each waveform are as follows: 2-2-2 μs (1028 V/cm), 5-2-5 μs (721 V/cm), 10-1-10 μs (547 V/cm), 2-5-2 μs (1043 V/cm), and 5-5-5 μs (751 V/cm). These data suggest that HF-PEFs can be fine-tuned to modulate the extent of cell death while maximizing peri-ablative BBB disruption. Furthermore, numerical modeling elucidated the diffuse field gradients of a single-needle grounding pad configuration to favor large-volume BBB disruption, while the monopolar probe configuration is more amenable to ablation and reversible electroporation effects.

The characterisation and application of bacterial nitroreductase enzymes

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

The characterisation and application of bacterial nitroreductase enzymes

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish. There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels. The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>

Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish. There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels. The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>

Neural Progenitor Cells Expressing Herpes Simplex Virus-Thymidine Kinase for Ablation Have Differential Chemosensitivity to Brivudine and Ganciclovir

Neural progenitor cell (NPC) transplants are a promising therapy for treating spinal cord injury (SCI), however, their long-term role after engraftment and the relative contribution to ongoing functional recovery remains a key knowledge gap. Selective human cell ablation techniques, currently being developed to improve the safety of progenitor cell transplant therapies in patients, may also be used as tools to probe the regenerative effects attributable to individual grafted cell populations. The Herpes Simplex Virus Thymidine Kinase (HSV-TK) and ganciclovir (GCV) system has been extensively studied in the context of SCI and broader CNS disease. However, the efficacy of brivudine (BVDU), another HSV-TK prodrug with potentially reduced bystander cytotoxic effects and in vivo toxicity, has yet to be investigated for NPC ablation. In this study, we demonstrate successful generation and in vitro ablation of HSV-TK-expressing human iPSC-derived NPCs with a &gt;80% reduction in survival over controls. We validated an HSV-TK and GCV/BVDU synergistic system with iPSC-NPCs using an efficient gene-transfer method and in vivo ablation in a translationally relevant model of SCI. Our findings demonstrate enhanced ablation efficiency and reduced bystander effects when targeting all rapidly dividing cells with combinatorial GCV and BVDU treatment. However, for use in loss of function studies, BVDU alone is optimal due to reduced nonselective cell ablation.

Demyelination induces transcriptional reprogramming in proprioceptive and Aβ rapidly adapting low-threshold-mechanoreceptor neurons.

Schwann cells, the main glial cell in the peripheral nervous system (PNS), ensheath bundles of small unmyelinated axons or form myelin on larger axons. PNS injuries initiate transcriptional reprograming in both Schwann cells and sensory neurons that promotes regeneration. While the factors that initiate the transcriptional reprograming in Schwann cells are well characterized, the full range of stimuli that initiate this reprograming in sensory neurons remain elusive. Here, using a genetic model of Schwann cell ablation, we find that Schwann cell loss results in transient PNS demyelination without overt axonal loss. By profiling sensory ganglia at single-cell resolution we show that this demyelination induces transcriptional reprogramming preferably in proprioceptive and Aβ RA-LTMR neurons. Transcriptional reprogramming is assumed to be a cell-autonomous response of sensory neurons to mechanical axonal injury. By identifying similar reprogramming in non-injured, demyelinated neurons, our study suggests that this reprogramming represents a non-cell-autonomous transcriptional response of sensory neurons to the loss of axon-Schwann cell interactions.