dOCRL maintains immune cell quiescence by regulating endosomal traffic.

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

Cellular Uptake of A Taurine-Modified, Ester Bond-Decorated D-Peptide Derivative via Dynamin-Based Endocytosis and Macropinocytosis.

Most of the peptides used for promoting cellular uptake bear positive charges. In our previous study, we reported an example of taurine (bearing negative charges in physiological conditions) promoting cellular uptake of D-peptides. Taurine, conjugated to a small D-peptide via an ester bond, promotes the cellular uptake of this D-peptide. Particularly, intracellular carboxylesterase (CES) instructs the D-peptide to self-assemble and to form nanofibers, which largely disfavors efflux and further enhances the intracellular accumulation of the D-peptide, as supported by that the addition of CES inhibitors partially impaired cellular uptake of this molecule in mammalian cell lines. Using dynamin 1, 2, and 3 triple knockout (TKO) mouse fibroblasts, we demonstrated that cells took up this molecule via macropinocytosis and dynamin-dependent endocytosis. Imaging of Drosophila larval blood cells derived from endocytic mutants confirmed the involvement of multiple endocytosis pathways. Electron microscopy (EM) indicated that the precursors can form aggregates on the cell surface to facilitate the cellular uptake via macropinocytosis. EM also revealed significantly increased numbers of vesicles in the cytosol. This work provides new insights into the cellular uptake of taurine derivative for intracellular delivery and self-assembly of D-peptides.

Active Probes for Imaging Membrane Dynamics of Live Cells with High Spatial and Temporal Resolution over Extended Time Scales and Areas.

Despite the advancement of molecular imaging techniques, there is an unmet need for probes for direct imaging of membrane dynamics of live cells. Here we report a novel type of active (or enzyme responsive) probes to directly image membrane dynamics of live cells with high spatial and temporal resolution over extended time scales and areas. Because lipid rafts enrich cholesterols and GPI-anchored enzymes (e.g., ectophosphatases), we design probes that consist of an enzymatic trigger, a fluorophore, and a cholesterol that are affinitive to the cell membrane. Being water-soluble and as the substrate of ectophosphatase, these cell compatible probes preferentially and rapidly assemble in plasma membrane, exhibit strong fluorescence, work at micromolar concentrations, and easily achieve high resolution monitoring of nanoscale heterogeneity in membranes of live cells, the release of exosomes, and the membrane dynamics of live cells. This work provides a facile means to link membrane dynamics and heterogeneity to cellular processes for understanding the interactions between membranes and proteins.

Essential roles for stat92E in expanding and patterning the proximodistal axis of the Drosophila wing imaginal disc.

The Drosophila wing imaginal disc is subdivided along the proximodistal axis into the distal pouch, the hinge, the surrounding pleura, and the notum. While the genetic pathways that specify the identity of each of these domains have been well studied, the mechanisms that coordinate the relative expansion of these domains are not well understood. Here we investigated the role of the stat92E signal transducer and activator of transcription in wing proximodistal development. We find that stat92E is active ubiquitously in early wing imaginal discs, where it acts to inhibit the induction of ectopic wing fields. Subsequently, stat92E activity is down regulated in the notum and distal pouch. These dynamics coincide with and contribute to the proportional subdivision and expansion of these primordia. As development proceeds, stat92E activity becomes restricted to the hinge, where it promotes normal expansion of the hinge, and restricts expansion of the notum. We also find that stat92E is required autonomously to specify dorsal pleura identity and inhibit notum identity to properly subdivide the body wall. Our data suggest that stat92E activity is regulated along the proximodistal axis to pattern this axis and control the relative expansion of the pouch, hinge, and notum.

ESCRT disruption provides evidence against transsynaptic signaling functions for extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

ESCRT disruption provides evidence against trans-synaptic signaling via extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types, including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating endosomal sorting complex required for transport (ESCRT) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo evenness interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell-autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

An approach for quantitative mapping of synaptic periactive zone architecture and organization.

Following exocytosis at active zones, synaptic vesicle membranes and membrane-bound proteins must be recycled. The endocytic machinery that drives this recycling accumulates in the periactive zone (PAZ), a region of the synapse adjacent to active zones, but the organization of this machinery within the PAZ, and how PAZ composition relates to active zone release properties, remains unknown. The PAZ is also enriched for cell adhesion proteins, but their function at these sites is poorly understood. Here, using Airyscan and stimulated emission depletion imaging of Drosophila synapses, we develop a quantitative framework describing the organization and ultrastructure of the PAZ. Different endocytic proteins localize to distinct regions of the PAZ, suggesting that subdomains are specialized for distinct biochemical activities, stages of membrane remodeling, or synaptic functions. We find that the accumulation and distribution of endocytic but not adhesion PAZ proteins correlate with the abundance of the scaffolding protein Bruchpilot at active zones-a structural correlate of release probability. These data suggest that endocytic and exocytic activities are spatially correlated. Taken together, our results identify novel relationships between the exocytic and endocytic apparatus at the synapse and provide a new conceptual framework to quantify synaptic architecture.

An autoinhibitory clamp of actin assembly constrains and directs synaptic endocytosis.

Synaptic membrane-remodeling events such as endocytosis require force-generating actin assembly. The endocytic machinery that regulates these actin and membrane dynamics localizes at high concentrations to large areas of the presynaptic membrane, but actin assembly and productive endocytosis are far more restricted in space and time. Here we describe a mechanism whereby autoinhibition clamps the presynaptic endocytic machinery to limit actin assembly to discrete functional events. We found that collective interactions between the Drosophila endocytic proteins Nwk/FCHSD2, Dap160/intersectin, and WASp relieve Nwk autoinhibition and promote robust membrane-coupled actin assembly in vitro. Using automated particle tracking to quantify synaptic actin dynamics in vivo, we discovered that Nwk-Dap160 interactions constrain spurious assembly of WASp-dependent actin structures. These interactions also promote synaptic endocytosis, suggesting that autoinhibition both clamps and primes the synaptic endocytic machinery, thereby constraining actin assembly to drive productive membrane remodeling in response to physiological cues.

Neurons constantly talk to each other by sending chemical signals across the tiny gap, or 'synapse', that separates two cells. While inside the emitting cell, these molecules are safely packaged into small, membrane-bound vessels. Upon the right signal, the vesicles fuse with the external membrane of the neuron and spill their contents outside, for the receiving cell to take up and decode. The emitting cell must then replenish its vesicle supply at the synapse through a recycling mechanism known as endocytosis. To do so, it uses dynamically assembling rod-like 'actin' filaments, which work in concert with many other proteins to pull in patches of membrane as new vesicles. The proteins that control endocytosis and actin assembly abound at neuronal synapses, and, when mutated, are linked to many neurological diseases. Unlike other cell types, neurons appear to 'pre-deploy' these actin-assembly proteins to synaptic membranes, but to keep them inactive under normal conditions. How neurons control the way this machinery is recruited and activated remains unknown. To investigate this question, Del Signore et al. conducted two sets of studies. First, they exposed actin to several different purified proteins in initial 'test tube' experiments. This revealed that, depending on the conditions, a group of endocytosis proteins could prevent or promote actin assembly: assembly occurred only if the proteins were associated with membranes. Next, Del Signore et al. mutated these proteins in fruit fly larvae, and performed live cell microscopy to determine their impact on actin assembly and endocytosis. Consistent with the test tube findings, endocytosis mutants had more actin assembly overall, implying that the proteins were required to prevent random actin assembly. However, the same mutants had reduced levels of endocytosis, suggesting that the proteins were also necessary for productive actin assembly. Together, these experiments suggest that, much like a mousetrap holds itself poised ready to spring, some endocytic proteins play a dual role to restrain actin assembly when and where it is not needed, and to promote it at sites of endocytosis. These results shed new light on how neurons might build and maintain effective, working synapses. Del Signore et al. hope that this knowledge may help to better understand and combat neurological diseases, such as Alzheimer's, which are linked to impaired membrane traffic and cell signalling.

Inhibitors of cytochrome c release with therapeutic potential for Huntington's disease.

Release of mitochondrial cytochrome c resulting in downstream activation of cell death pathways has been suggested to play a role in neurologic diseases featuring cell death. However, the specific biologic importance of cytochrome c release has not been demonstrated in Huntington's disease (HD). To evaluate the role of cytochrome c release, we screened a drug library to identify new inhibitors of cytochrome c release from mitochondria. Drugs effective at the level of purified mitochondria were evaluated in a cellular model of HD. As proof of principle, one drug was chosen for in depth evaluation in vitro and a transgenic mouse model of HD. Our findings demonstrate the utility of mitochondrial screening to identify inhibitors of cell death and provide further support for the important functional role of cytochrome c release in HD. Given that many of these compounds have been approved by the Food and Drug Administration for clinical usage and cross the blood-brain barrier, these drugs may lead to trials in patients.

Therapeutic attenuation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no current therapy preventing cumulative neuronal loss. There is substantial evidence that mitochondrial dysfunction, oxidative stress, and associated caspase activity underlie the neurodegeneration observed. One potential drug therapy is the potent free radical scavenger and antioxidant cystamine, which has demonstrated significant clinical potential in models of neurodegenerative disorders and human neurological disease. This study examined the oral efficacy of cystamine in the MPTP and 6-hydroxydopamine neurotoxin models of PD. The neuroprotective effects of cystamine treatment significantly ameliorated nigral neuronal loss, preserved striatal dopaminergic projections, and improved striatal dopamine and metabolite levels, as compared to MPTP alone. Cystamine normalized striatal 8-hydroxy-2'-deoxyguanosine levels and ATP concentrations, consistent with reduced oxidative stress and improved mitochondrial function. Cystamine also protected against MPTP-induced mitochondrial loss, as identified by mitochondrial heat shock protein 70 and superoxide dismutase 2, with concomitant reductions in cytochrome c and caspase-3 activities. The neuroprotective value of cystamine was confirmed in the 6-hydroxydopamine model. Together these findings show cystamine's therapeutic benefit to reduce neuronal loss through attenuation of oxidative stress and mitochondrial dysfunction, providing the rationale for human clinical trials in PD patients.

Combined riluzole and sodium phenylbutyrate therapy in transgenic amyotrophic lateral sclerosis mice.

Recent evidence suggests that transcriptional dysregulation may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). The histone deacetylase inhibitor, sodium phenylbutyrate (NaPB), is neuroprotective and corrects aberrant gene transcription in ALS mice and has recently been shown to be safe and tolerable in ALS patients while improving hypoacetylation. Since many patients are already on riluzole, it is important to ensure that any proposed therapy does not result in negative synergy with riluzole. The combined treatment of riluzole and NaPB significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice beyond either agent alone. Combination therapy increased survival by 21.5%, compared to the separate administration of riluzole (7.5%) and NaPB (12.8%), while improving both body weight loss and grip strength. The data show that the combined treatment was synergistic. In addition, riluzole/NaPB treatment ameliorated gross lumbar and ventral horn atrophy, attenuated lumbar ventral horn neuronal cell death, and decreased reactive astrogliosis. Riluzole/NaPB administration increased acetylation at H4 and increased NF-kappaB p50 translocation to the nucleus in G93A mice, consistent with a therapeutic effect. These data suggest that NaPB may not interfere with the pharmacologic action of riluzole in ALS patients.

The enemy of my enemy: PTEN and PLCXD collude to fight endosomal PtdIns(4,5)P2.

Loss of the phosphoinositide 5-phosphatase OCRL causes accumulation of PtdIns(4,5)P2 on membranes and, ultimately, Lowe syndrome. In this issue, Mondin et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201805155) discover that a surprising partnership between PTEN and the phospholipase PLCXD can compensate for OCRL to suppress endosomal PtdIns(4,5)P2 accumulation.

The WAVE Regulatory Complex and Branched F-Actin Counterbalance Contractile Force to Control Cell Shape and Packing in the Drosophila Eye.

Contractile forces eliminate cell contacts in many morphogenetic processes. However, mechanisms that balance contractile forces to promote subtler remodeling remain unknown. To address this gap, we investigated remodeling of Drosophila eye lattice cells (LCs), which preserve cell contacts as they narrow to form the edges of a multicellular hexagonal lattice. We found that during narrowing, LC-LC contacts dynamically constrict and expand. Similar to other systems, actomyosin-based contractile forces promote pulses of constriction. Conversely, we found that WAVE-dependent branched F-actin accumulates at LC-LC contacts during expansion and functions to expand the cell apical area, promote shape changes, and prevent elimination of LC-LC contacts. Finally, we found that small Rho GTPases regulate the balance of contractile and protrusive dynamics. These data suggest a mechanism by which WAVE regulatory complex-based F-actin dynamics antagonize contractile forces to regulate cell shape and tissue topology during remodeling and thus contribute to the robustness and precision of the process.

Combination therapy using minocycline and coenzyme Q10 in R6/2 transgenic Huntington's disease mice.

Huntington's disease (HD) is a fatal neurodegenerative disorder of genetic origin with no known therapeutic intervention that can slow or halt disease progression. Transgenic murine models of HD have significantly improved the ability to assess potential therapeutic strategies. The R6/2 murine model of HD, which recapitulates many aspects of human HD, has been used extensively in pre-clinical HD therapeutic treatment trials. Of several potential therapeutic candidates, both minocycline and coenzyme Q10 (CoQ10) have been demonstrated to provide significant improvement in the R6/2 mouse. Given the specific cellular targets of each compound, and the broad array of abnormalities thought to underlie HD, we sought to assess the effects of combined minocycline and CoQ10 treatment in the R6/2 mouse. Combined minocycline and CoQ10 therapy provided an enhanced beneficial effect, ameliorating behavioral and neuropathological alterations in the R6/2 mouse. Minocycline and CoQ10 treatment significantly extended survival and improved rotarod performance to a greater degree than either minocycline or CoQ10 alone. In addition, combined minocycline and CoQ10 treatment attenuated gross brain atrophy, striatal neuron atrophy, and huntingtin aggregation in the R6/2 mice relative to individual treatment. These data suggest that combined minocycline and CoQ10 treatment may offer therapeutic benefit to patients suffering from HD.

Dose ranging and efficacy study of high-dose coenzyme Q10 formulations in Huntington's disease mice.

There is substantial evidence that a bioenergetic defect may play a role in the pathogenesis of Huntington's Disease (HD). A potential therapy for remediating defective energy metabolism is the mitochondrial cofactor, coenzyme Q10 (CoQ10). We have reported that CoQ10 is neuroprotective in the R6/2 transgenic mouse model of HD. Based upon the encouraging results of the CARE-HD trial and recent evidence that high-dose CoQ10 slows the progressive functional decline in Parkinson's disease, we performed a dose ranging study administering high levels of CoQ10 from two commercial sources in R6/2 mice to determine enhanced efficacy. High dose CoQ10 significantly extended survival in R6/2 mice, the degree of which was dose- and source-dependent. CoQ10 resulted in a marked improvement in motor performance and grip strength, with a reduction in weight loss, brain atrophy, and huntingtin inclusions in treated R6/2 mice. Brain levels of CoQ10 and CoQ9 were significantly lower in R6/2 mice, in comparison to wild type littermate control mice. Oral administration of CoQ10 elevated CoQ10 plasma levels and significantly increased brain levels of CoQ9, CoQ10, and ATP in R6/2 mice, while reducing 8-hydroxy-2-deoxyguanosine concentrations, a marker of oxidative damage. We demonstrate that high-dose administration of CoQ10 exerts a greater therapeutic benefit in a dose dependent manner in R6/2 mice than previously reported and suggest that clinical trials using high dose CoQ10 in HD patients are warranted.

Chronology of behavioral symptoms and neuropathological sequela in R6/2 Huntington's disease transgenic mice.

Genetic murine models play an important role in the study of human neurological disorders by providing accurate and experimentally accessible systems to study pathogenesis and to test potential therapeutic treatments. One of the most widely employed models of Huntington's disease (HD) is the R6/2 transgenic mouse. To characterize this model further, we have performed behavioral and neuropathological analyses that provide a foundation for the use of R6/2 mice in preclinical therapeutic trials. Behavioral analyses of the R6/2 mouse reveal age-related impairments in dystonic movements, motor performance, grip strength, and body weight that progressively worsen until death. Significant neuropathological sequela, identified as increasing marked reductions in brain weight, are present from 30 days, whereas decreased brain volume is present from 60 days and decreased neostriatal volume and striatal neuron area, with a concomitant reduction in striatal neuron number, are present at 90 days of age. Huntingtin-positive aggregates are present at postnatal day 1 and increase in number and size with age. Our findings suggest that the R6/2 HD model exhibits a progressive HD-like behavioral and neuropathological phenotype that more closely corresponds to human HD than previously believed, providing further assurance that the R6/2 mouse is an appropriate model for testing potential therapies for HD.

Drosophila comes of age as a model system for understanding the function of cytoskeletal proteins in cells, tissues, and organisms.

For the last 100 years, Drosophila melanogaster has been a powerhouse genetic system for understanding mechanisms of inheritance, development, and behavior in animals. In recent years, advances in imaging and genetic tools have led to Drosophila becoming one of the most effective systems for unlocking the subcellular functions of proteins (and particularly cytoskeletal proteins) in complex developmental settings. In this review, written for non-Drosophila experts, we will discuss critical technical advances that have enabled these cell biological insights, highlighting three examples of cytoskeletal discoveries that have arisen as a result: (1) regulation of Arp2/3 complex in myoblast fusion, (2) cooperation of the actin filament nucleators Spire and Cappuccino in establishment of oocyte polarity, and (3) coordination of supracellular myosin cables. These specific examples illustrate the unique power of Drosophila both to uncover new cytoskeletal structures and functions, and to place these discoveries in a broader in vivo context, providing insights that would have been impossible in a cell culture model or in vitro. Many of the cellular structures identified in Drosophila have clear counterparts in mammalian cells and tissues, and therefore elucidating cytoskeletal functions in Drosophila will be broadly applicable to other organisms.

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms.

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C(2)H(2) zinc finger genes, which has been implicated in notum growth and patterning. Here we show that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. Our data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior-posterior (AP) axis using both autonomous and non-autonomous mechanisms.

Modulation of nucleosome dynamics in Huntington's disease.

Transcriptional dysregulation and aberrant chromatin remodeling are central features in the pathology of Huntington's disease (HD). In order to more fully characterize these pathogenic events, an assessment of histone profiles and associated gene changes were performed in transgenic N171-82Q (82Q) and R6/2 HD mice. Analyses revealed significant chromatin modification, resulting in reduced histone acetylation with concomitant increased histone methylation, consistent with findings observed in HD patients. While there are no known interventions that ameliorate or arrest HD progression, DNA/RNA-binding anthracyclines may provide significant therapeutic potential by correcting pathological nucleosome changes and realigning transcription. Two such anthracyclines, chromomycin and mithramycin, improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. There was a significant shift in the balance between methylation and acetylation in treated HD mice to that found in wild-type mice, resulting in greater acetylation of histone H3 at lysine 9 and promoting gene transcription. Gene expression profiling in anthracycline-treated HD mice showed molecular changes that correlate with disease correction, such that a subset of downregulated genes were upregulated with anthracycline treatment. Improved nucleosomal dynamics were concurrent with a significant improvement in the behavioral and neuropathological phenotype observed in HD mice. These data show the ability of anthracycline compounds to rebalance epigenetic histone modification and, as such, may provide the rationale for the design of human clinical trials in HD patients.