Automating the High-Throughput Screening of Protein-Based Optical Indicators and Actuators.

Over the last 25 years, protein engineers have developed an impressive collection of optical tools to interface with biological systems: indicators to eavesdrop on cellular activity and actuators to poke and prod native processes. To reach the performance level required for their downstream applications, protein-based tools are usually sculpted by iterative rounds of mutagenesis. In each round, libraries of variants are made and evaluated, and the most promising hits are then retrieved, sequenced, and further characterized. Early efforts to engineer protein-based optical tools were largely manual, suffering from low throughput, human error, and tedium. Here, we describe approaches to automating the screening of libraries generated as colonies on agar, multiwell plates, and pooled populations of single-cell variants. We also briefly discuss emerging approaches for screening, including cell-free systems and machine learning.

The MAP3Ks DLK and LZK Direct Diverse Responses to Axon Damage in Zebrafish Peripheral Neurons.

Mitogen-activated protein kinase kinase kinases (MAP3Ks) dual leucine kinase (DLK) and leucine zipper kinase (LZK) are essential mediators of axon damage responses, but their responses are varied, complex, and incompletely understood. To characterize their functions in axon injury, we generated zebrafish mutants of each gene, labeled motor neurons (MNs) and touch-sensing neurons in live zebrafish, precisely cut their axons with a laser, and assessed the ability of mutant axons to regenerate in larvae, before sex is apparent in zebrafish. DLK and LZK were required redundantly and cell autonomously for axon regeneration in MNs but not in larval Rohon-Beard (RB) or adult dorsal root ganglion (DRG) sensory neurons. Surprisingly, in dlk lzk double mutants, the spared branches of wounded RB axons grew excessively, suggesting that these kinases inhibit regenerative sprouting in damaged axons. Uninjured trigeminal sensory axons also grew excessively in mutants when neighboring neurons were ablated, indicating that these MAP3Ks are general inhibitors of sensory axon growth. These results demonstrate that zebrafish DLK and LZK promote diverse injury responses, depending on the neuronal cell identity and type of axonal injury.SIGNIFICANCE STATEMENT The MAP3Ks DLK and LZK are damage sensors that promote diverse outcomes to neuronal injury, including axon regeneration. Understanding their context-specific functions is a prerequisite to considering these kinases as therapeutic targets. To investigate DLK and LZK cell-type-specific functions, we created zebrafish mutants in each gene. Using mosaic cell labeling and precise laser injury we found that both proteins were required for axon regeneration in motor neurons but, unexpectedly, were not required for axon regeneration in Rohon-Beard or DRG sensory neurons and negatively regulated sprouting in the spared axons of touch-sensing neurons. These findings emphasize that animals have evolved distinct mechanisms to regulate injury site regeneration and collateral sprouting, and identify differential roles for DLK and LZK in these processes.

Dendrite regeneration in the vertebrate spinal cord.

Axon regeneration in response to injury has been documented in many animals over several hundred years. In contrast, how neurons respond to dendrite injury has been examined only in the last decade. So far, dendrite regeneration after injury has been documented in invertebrate model systems, but has not been assayed in a vertebrate. In this study, we use zebrafish motor neurons to track neurons after dendrite injury. We address two major gaps in our knowledge of dendrite regeneration: 1) whether post-synaptic dendrites can regenerate and 2) whether vertebrate dendrites can regenerate. We find that motor neurons survive laser microsurgery to remove one or all dendrites. Outgrowth of new dendrites typically initiated one to three days after injury, and a new, stable dendrite arbor was in place by five days after injury. We conclude that zebrafish motor neurons have the capacity to regenerate a new dendrite arbor.

Microtubule polarity is instructive for many aspects of neuronal polarity.

Many neurons in bilaterian animals are polarized with functionally distinct axons and dendrites. Microtubule polarity, microtubule stability, and the axon initial segment (AIS) have all been shown to influence polarized transport in neurons. Each of these cytoskeletal cues could act independently to control axon and dendrite identity, or there could be a hierarchy in which one acts upstream of the others. Here we test the hypothesis that microtubule polarity acts as a master regulator of neuronal polarity by using a Drosophila genetic background in which some dendrites have normal minus-end-out microtubule polarity and others have the axonal plus-end-out polarity. In these mosaic dendrite arbors, we found that ribosomes, which are more abundant in dendrites than axons, were reduced in plus-end-out dendrites, while an axonal cargo was increased. In addition, we determined that microtubule stability was different in plus-end-out and minus-end-out dendrites, with plus-end-out ones having more stable microtubules like axons. Similarly, we found that ectopic diffusion barriers, like those at the AIS, formed at the base of dendrites with plus-end-out regions. Thus, changes in microtubule polarity were sufficient to rearrange other cytoskeletal features associated with neuronal polarization. However, overall neuron shape was maintained with only subtle changes in branching in mosaic arbors. We conclude that microtubule polarity can act upstream of many aspects of intracellular neuronal polarization, but shape is relatively resilient to changes in microtubule polarity in vivo.

Microtubule organization of vertebrate sensory neurons in vivo.

Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance.

Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy.

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Neurons survive simultaneous injury to axons and dendrites and regrow both types of processes in vivo.

Neurons extend dendrites and axons to receive and send signals. If either type of process is removed, the cell cannot function. Rather than undergoing cell death, some neurons can regrow axons and dendrites. Axon and dendrite regeneration have been examined separately and require sensing the injury and reinitiating the correct growth program. Whether neurons in vivo can sense and respond to simultaneous axon and dendrite injury with polarized regeneration has not been explored. To investigate the outcome of simultaneous axon and dendrite damage, we used a Drosophila model system in which neuronal polarity, axon regeneration, and dendrite regeneration have been characterized. After removal of the axon and all but one dendrite, the remaining dendrite was converted to a process that had a long unbranched region that extended over long distances and a region where shorter branched processes were added. These observations suggested axons and dendrites could regrow at the same time. To further test the capacity of neurons to implement polarized regeneration after axon and dendrite damage, we removed all neurites from mature neurons. In this case a long unbranched neurite and short branched neurites were regrown from the stripped cell body. Moreover, the long neurite had axonal plus-end-out microtubule polarity and the shorter neurites had mixed polarity consistent with dendrite identity. The long process also accumulated endoplasmic reticulum at its tip like regenerating axons. We conclude that neurons in vivo can respond to simultaneous axon and dendrite injury by initiating growth of a new axon and new dendrites.

Endosomal Wnt signaling proteins control microtubule nucleation in dendrites.

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.

The receptor tyrosine kinase Ror is required for dendrite regeneration in Drosophila neurons.

While many regulators of axon regeneration have been identified, very little is known about mechanisms that allow dendrites to regenerate after injury. Using a Drosophila model of dendrite regeneration, we performed a candidate screen of receptor tyrosine kinases (RTKs) and found a requirement for RTK-like orphan receptor (Ror). We confirmed that Ror was required for regeneration in two different neuron types using RNA interference (RNAi) and mutants. Ror was not required for axon regeneration or normal dendrite development, suggesting a specific role in dendrite regeneration. Ror can act as a Wnt coreceptor with frizzleds (fzs) in other contexts, so we tested the involvement of Wnt signaling proteins in dendrite regeneration. We found that knockdown of fz, dishevelled (dsh), Axin, and gilgamesh (gish) also reduced dendrite regeneration. Moreover, Ror was required to position dsh and Axin in dendrites. We recently found that Wnt signaling proteins, including dsh and Axin, localize microtubule nucleation machinery in dendrites. We therefore hypothesized that Ror may act by regulating microtubule nucleation at baseline and during dendrite regeneration. Consistent with this hypothesis, localization of the core nucleation protein γTubulin was reduced in Ror RNAi neurons, and this effect was strongest during dendrite regeneration. In addition, dendrite regeneration was sensitive to partial reduction of γTubulin. We conclude that Ror promotes dendrite regeneration as part of a Wnt signaling pathway that regulates dendritic microtubule nucleation.

Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.

Nicotinamide is an endogenous agonist for a C. elegans TRPV OSM-9 and OCR-4 channel.

TRPV ion channels are directly activated by sensory stimuli and participate in thermo-, mechano- and chemo-sensation. They are also hypothesized to respond to endogenous agonists that would modulate sensory responses. Here, we show that the nicotinamide (NAM) form of vitamin B3 is an agonist of a Caenorhabditis elegans TRPV channel. Using heterologous expression in Xenopus oocytes, we demonstrate that NAM is a soluble agonist for a channel consisting of the well-studied OSM-9 TRPV subunit and relatively uncharacterized OCR-4 TRPV subunit as well as the orthologous Drosophila Nan-Iav TRPV channel, and we examine stoichiometry of subunit assembly. Finally, we show that behaviours mediated by these C. elegans and Drosophila channels are responsive to NAM, suggesting conservation of activity of this soluble endogenous metabolite on TRPV activity. Our results in combination with the role of NAM in NAD+ metabolism suggest an intriguing link between metabolic regulation and TRPV channel activity.

Bidirectional Homeostatic Regulation of a Depression-Related Brain State by Gamma-Aminobutyric Acidergic Deficits and Ketamine Treatment.

BACKGROUND: Major depressive disorder is increasingly recognized to involve functional deficits in both gamma-aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission. To elucidate the relationship between these phenotypes, we used GABAA receptor γ2 subunit heterozygous (γ2(+/-)) mice, which we previously characterized as a model animal with construct, face, and predictive validity for major depressive disorder. METHODS: To assess possible consequences of GABAergic deficits on glutamatergic transmission, we quantitated the cell surface expression of N-methyl-D-aspartate (NMDA)-type and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors and the function of synapses in the hippocampus and medial prefrontal cortex of γ2(+/-) mice. We also analyzed the effects of an acute dose of the experimental antidepressant ketamine on all these parameters in γ2(+/-) versus wild-type mice. RESULTS: Modest defects in GABAergic synaptic transmission of γ2(+/-) mice resulted in a strikingly prominent homeostatic-like reduction in the cell surface expression of NMDA-type and AMPA-type glutamate receptors, along with prominent functional impairment of glutamatergic synapses in the hippocampus and medial prefrontal cortex. A single subanesthetic dose of ketamine normalized glutamate receptor expression and synaptic function of γ2(+/-) mice to wild-type levels for a prolonged period, along with antidepressant-like behavioral consequences selectively in γ2(+/-) mice. The GABAergic synapses of γ2(+/-) mice were potentiated by ketamine in parallel but only in the medial prefrontal cortex. CONCLUSIONS: Depressive-like brain states that are caused by GABAergic deficits involve a homeostatic-like reduction of glutamatergic transmission that is reversible by an acute, subanesthetic dose of ketamine, along with regionally selective potentiation of GABAergic synapses. The data merge the GABAergic and glutamatergic deficit hypotheses of major depressive disorder.