MANIPULATING MITOCHONDRIAL REACTIVE OXYGEN SPECIES ALTERS SURVIVAL IN UNEXPECTED WAYS IN A DROSOPHILA MODEL OF NEURODEGENERATION.

Reactive oxygen species (ROS) are associated with aging and neurodegeneration, but the significance of this association remains obscure. Here, using a Drosophila model of age-related neurodegeneration, we probe this relationship in the pathologically relevant tissue, the brain, by quantifying three specific mitochondrial ROS and manipulating these redox species pharmacologically. Our goal is to ask whether pathology-associated changes in redox state are detrimental for survival, whether they may be beneficial responses, or whether they are simply covariates of pathology that do not alter viability. We find, surprisingly, that increasing mitochondrial H2O2 correlates with improved survival. We also find evidence that drugs that alter the mitochondrial glutathione redox potential modulate survival primarily through the compensatory effects they induce rather than through their direct effects on the final mitochondrial glutathione redox potential per se. We also find that the response to treatment with a redox-altering drug varies dramatically depending on the age at which the drug is administered, the duration of the treatment, and the genotype of the individual receiving the drug. These data have important implications for the design and interpretation of studies investigating the effect of redox state on health and disease as well as on efforts to modify the redox state to achieve therapeutic goals.

Changes in mitochondrial distribution occur at the axon initial segment in association with neurodegeneration in Drosophila.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic vs. axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity.

A new view of axon growth and guidance grounded in the stochastic dynamics of actin networks.

The mechanism of axon growth and guidance is a core, unsolved problem in neuroscience and cell biology. For nearly three decades, our view of this process has largely been based on deterministic models of motility derived from studies of neurons cultured in vitro on rigid substrates. Here, we suggest a fundamentally different, inherently probabilistic model of axon growth, one that is grounded in the stochastic dynamics of actin networks. This perspective is motivated and supported by a synthesis of results from live imaging of a specific axon growing in its native tissue in vivo, together with single-molecule computational simulations of actin dynamics. In particular, we show how axon growth arises from a small spatial bias in the intrinsic fluctuations of the axonal actin cytoskeleton, one that produces net translocation of the axonal actin network by differentially modulating local probabilities of network expansion versus compaction. We discuss the relationship between this model and current views of axon growth and guidance mechanism and demonstrate how it offers explanations for various longstanding puzzles in this field. We further point out the implications of the probabilistic nature of actin dynamics for many other processes of cell morphology and motility.

What do we mean by "aging"? Questions and perspectives revealed by studies in Drosophila.

What is the nature of aging, and how best can we study it? Here, using a series of questions that highlight differing perspectives about the nature of aging, we ask how data from Drosophila melanogaster at the organismal, tissue, cellular, and molecular levels shed light on the complex interactions among the phenotypes associated with aging. Should aging be viewed as an individual's increasing probability of mortality over time or as a progression of physiological states? Are all age-correlated changes in physiology detrimental to vigor or are some compensatory changes that maintain vigor? Why do different age-correlated functions seem to change at different rates in a single individual as it ages? Should aging be considered as a single, integrated process across the scales of biological resolution, from organismal to molecular, or must we consider each level of biological scale as a separate, distinct entity? Viewing aging from these differing perspectives yields distinct but complementary interpretations about the properties and mechanisms of aging and may offer a path through the complexities related to understanding the nature of aging.

Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila.

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Fringe-positive Golgi outposts unite temporal Furin 2 convertase activity and spatial Delta signal to promote dendritic branch retraction.

Golgi outposts (GOPs) in dendrites are known for their role in promoting branch extension, but whether GOPs have other functions is unclear. We found that terminal branches of Drosophila class IV dendritic arborization (C4da) neurons actively grow during the early third-instar (E3) larval stage but retract in the late third (L3) stage. Interestingly, the Fringe (Fng) glycosyltransferase localizes increasingly at GOPs in distal dendritic regions through the E3 to the L3 stage. Expression of the endopeptidase Furin 2 (Fur2), which proteolyzes and inactivates Fng, decreases from E3 to L3 in C4da neurons, thereby increasing Fng-positive GOPs in dendrites. The epidermal Delta ligand and neuronal Notch receptor, the substrate for Fng-mediated O-glycosylation, also negatively regulate dendrite growth. Fng inhibits actin dynamics in dendrites, linking dendritic branch retraction to suppression of the C4da-mediated thermal nociception response in late larval stages. Thus, Fng-positive GOPs function in dendrite retraction, which would add another function to the repertoire of GOPs in dendrite arborization.

Nucleation causes an actin network to fragment into multiple high-density domains.

Actin networks rely on nucleation mechanisms to generate new filaments because spontaneous nucleation is kinetically disfavored. Branching nucleation of actin filaments by actin-related protein (Arp2/3), in particular, is critical for actin self-organization. In this study, we use the simulation platform for active matter MEDYAN to generate 2000 s long stochastic trajectories of actin networks, under varying Arp2/3 concentrations, in reaction volumes of biologically meaningful size (>20 µm3). We find that the dynamics of Arp2/3 increase the abundance of short filaments and increases network treadmilling rate. By analyzing the density fields of F-actin, we find that at low Arp2/3 concentrations, F-actin is organized into a single connected and contractile domain, while at elevated Arp2/3 levels (10 nM and above), such high-density actin domains fragment into smaller domains spanning a wide range of volumes. These fragmented domains are extremely dynamic, continuously merging and splitting, owing to the high treadmilling rate of the underlying actin network. Treating the domain dynamics as a drift-diffusion process, we find that the fragmented state is stochastically favored, and the network state slowly drifts toward the fragmented state with considerable diffusion (variability) in the number of domains. We suggest that tuning the Arp2/3 concentration enables cells to transition from a globally coherent cytoskeleton, whose response involves the entire cytoplasmic network, to a fragmented cytoskeleton, where domains can respond independently to locally varying signals.

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones.

Extensive studies of growing axons have revealed many individual components and protein interactions that guide neuronal morphogenesis. Despite this, however, we lack any clear picture of the emergent mechanism by which this nanometer-scale biochemistry generates the multimicron-scale morphology and cell biology of axon growth and guidance in vivo. To address this, we studied the downstream effects of the Abl signaling pathway using a computer simulation software (MEDYAN) that accounts for mechanochemical dynamics of active polymers. Previous studies implicate two Abl effectors, Arp2/3 and Enabled, in Abl-dependent axon guidance decisions. We now find that Abl alters actin architecture primarily by activating Arp2/3, while Enabled plays a more limited role. Our simulations show that simulations mimicking modest levels of Abl activity bear striking similarity to actin profiles obtained experimentally from live imaging of actin in wild-type axons in vivo. Using a graph theoretical filament-filament contact analysis, moreover, we find that networks mimicking hyperactivity of Abl (enhanced Arp2/3) are fragmented into smaller domains of actin that interact weakly with each other, consistent with the pattern of actin fragmentation observed upon Abl overexpression in vivo. Two perturbative simulations further confirm that high-Arp2/3 actin networks are mechanically disconnected and fail to mount a cohesive response to perturbation. Taken together, these data provide a molecular-level picture of how the large-scale organization of the axonal cytoskeleton arises from the biophysics of actin networks.

The Unfolded Protein Responses in Health, Aging, and Neurodegeneration: Recent Advances and Future Considerations.

Aging and age-related neurodegeneration are both associated with the accumulation of unfolded and abnormally folded proteins, highlighting the importance of protein homeostasis (termed proteostasis) in maintaining organismal health. To this end, two cellular compartments with essential protein folding functions, the endoplasmic reticulum (ER) and the mitochondria, are equipped with unique protein stress responses, known as the ER unfolded protein response (UPR ER ) and the mitochondrial UPR (UPR mt ), respectively. These organellar UPRs play roles in shaping the cellular responses to proteostatic stress that occurs in aging and age-related neurodegeneration. The loss of adaptive UPR ER and UPR mt signaling potency with age contributes to a feed-forward cycle of increasing protein stress and cellular dysfunction. Likewise, UPR ER and UPR mt signaling is often altered in age-related neurodegenerative diseases; however, whether these changes counteract or contribute to the disease pathology appears to be context dependent. Intriguingly, altering organellar UPR signaling in animal models can reduce the pathological consequences of aging and neurodegeneration which has prompted clinical investigations of UPR signaling modulators as therapeutics. Here, we review the physiology of both the UPR ER and the UPR mt , discuss how UPR ER and UPR mt signaling changes in the context of aging and neurodegeneration, and highlight therapeutic strategies targeting the UPR ER and UPR mt that may improve human health.

Common features of aging fail to occur in Drosophila raised without a bacterial microbiome.

Lifespan is limited both by intrinsic decline in vigor with age and by accumulation of external insults. There exists a general picture of the deficits of aging, one that is reflected in a pattern of age-correlated changes in gene expression conserved across species. Here, however, by comparing gene expression profiling of Drosophila raised either conventionally, or free of bacteria, we show that ∼70% of these conserved, age-associated changes in gene expression fail to occur in germ-free flies. Among the processes that fail to show time-dependent change under germ-free conditions are two aging features that are observed across phylogeny, declining expression of stress response genes and increasing expression of innate immune genes. These comprise adaptive strategies the organism uses to respond to bacteria, rather than being inevitable components of age-dependent decline. Changes in other processes are independent of the microbiome and can serve as autonomous markers of aging of the individual.

A polymer index-matched to water enables diverse applications in fluorescence microscopy.

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

Htt is a repressor of Abl activity required for APP-induced axonal growth.

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αß neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αß neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.

Phosphorylation and Proteolytic Cleavage of Notch in Canonical and Noncanonical Notch Signaling.

The Notch signaling pathway seems deceptively simple, with its key feature being a direct connection between extracellular signal and transcriptional output without the need for an extended chain of protein intermediaries as required by so many other signaling paradigms. However, this apparent simplicity hides considerable complexity. Consistent with its central role in many aspects of development, Notch signaling has an extensive collection of mechanisms that it employs alongside of its core transcriptional machinery. These so-called noncanonical Notch pathways diversify the potential outputs of Notch, and allow it to coordinate regulation of many aspects of the biology of cells. Here we will review noncanonical Notch signaling with special attention to the role of posttranslational modifications of Notch. We will also consider the importance of coordinating the activity of gene expression with regulation of cell morphology in biological processes, including axon guidance and other morphological events during embryogenesis.

Dynamic morphogenesis of a pioneer axon in Drosophila and its regulation by Abl tyrosine kinase.

The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. We here dissect this process using live imaging of the TSM1 axon of the developing Drosophila wing. We find that the growth cone is almost purely filopodial, and that it extends by a protrusive mode of growth. Quantitative analysis reveals two separate groups of growth cone properties that together account for growth cone structure and dynamics. The core morphological features of the growth cone are strongly correlated with one another and define two discrete morphs. Genetic manipulation of a critical mediator of axon guidance signaling, Abelson (Abl) tyrosine kinase, shows that while Abl weakly modulates the ratio of the two morphs it does not greatly change their properties. Rather, Abl primarily regulates the second group of properties, which report the organization and distribution of actin in the growth cone and are coupled to growth cone velocity. Other experiments dissect the nature of that regulation of actin organization and how it controls the spatial localization of filopodial dynamics and thus axon extension. Together, these observations suggest a novel, probabilistic mechanism by which Abl biases the stochastic fluctuations of growth cone actin to direct axon growth and guidance.

Abl signaling directs growth of a pioneer axon in Drosophila by shaping the intrinsic fluctuations of actin.

The fundamental problem in axon growth and guidance is understanding how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. Live imaging of the TSM1 axon of the developing Drosophila wing has shown that the essential role of the core guidance signaling molecule, Abelson (Abl) tyrosine kinase, is to modulate the organization and spatial localization of actin in the advancing growth cone. Here, we dissect in detail the properties of that actin organization and its consequences for growth cone morphogenesis and motility. We show that advance of the actin mass in the distal axon drives the forward motion of the dynamic filopodial domain that defines the growth cone. We further show that Abl regulates both the width of the actin mass and its internal organization, spatially biasing the intrinsic fluctuations of actin to achieve net advance of the actin, and thus of the dynamic filopodial domain of the growth cone, while maintaining the essential coherence of the actin mass itself. These data suggest a model whereby guidance signaling systematically shapes the intrinsic, stochastic fluctuations of actin in the growth cone to produce axon growth and guidance.

Reduced autophagy efficiency induces innate immune activation leading to neurodegeneration.

Macroautophagy/autophagy and innate immunity are central processes in neurodegeneration, but it has been unclear whether they work independently or in combination to assault the neuron. We recently demonstrated that reduced efficiency of autophagy causes hyperactivation of innate immunity, which in turn is necessary and sufficient for loss of dopaminergic neurons in a Cdk5-mediated model of degeneration in Drosophila. Genetically restoring autophagy, or reducing innate immune activation, rescues the dopaminergic neuron loss that occurs due to altered Cdk5 activity. This work revealed an alliance of innate immunity and autophagy that causes neuron loss in Cdk5-mediated neurodegeneration.

A role for actomyosin contractility in Notch signaling.

BACKGROUND: Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling. RESULTS: To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems-as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture. CONCLUSIONS: Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.

Expression of a Fragment of Ankyrin 2 Disrupts the Structure of the Axon Initial Segment and Causes Axonal Degeneration in Drosophila.

Neurodegenerative stimuli are often associated with perturbation of the axon initial segment (AIS), but it remains unclear whether AIS disruption is causative for neurodegeneration or is a downstream step in disease progression. Here, we demonstrate that either of two separate, genetically parallel pathways that disrupt the AIS induce axonal degeneration and loss of neurons in the central brain of Drosophila. Expression of a portion of the C-terminal tail of the Ank2-L isoform of Ankyrin severely shortens the AIS in Drosophila mushroom body (MB) neurons, and this shortening occurs through a mechanism that is genetically separate from the previously described Cdk5α-dependent pathway of AIS regulation. Further, either manipulation triggers morphological degeneration of MB axons and is accompanied by neuron loss. Taken together, our results are consistent with the hypothesis that disruption of the AIS is causally related to degeneration of fly central brain neurons, and we suggest that similar mechanisms may contribute to neurodegeneration in mammals.