Dynamics and distribution of endosomes and lysosomes in dendrites.

All cells are filled with membrane-bound organelles which are responsible for the synthesis and transport as well as degradation of membrane proteins. The localization of these organelles inside cells is highly regulated. The regulation of organelle positioning has been widely studied in many cell types. In neurons, organelle positioning and its regulation is of particular interest because of the enormous size of neurons and the high spatial heterogeneity of different functional domains, such as axons, proximal and distal portions of dendrites, and synapses. We will discuss new discoveries with regard to the dynamic positioning of endosomes and lysosomes between soma and along dendrites. Just as the "how" of dynamic endosome/lysosome positioning is still being investigated, the "why" is also being explored. An exciting possibility is that synaptic activity influences organelle behaviors. We will discuss what is currently known about the how and the why of endosome/lysosome dynamics in dendrites.

De Novo Design of A Membrane-Anchored Probe for Multidimensional Quantification of Endocytic Dynamics.

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis of intracellular nutrients and other functions. To study the dynamics of endocytic pathway, a membrane-anchored pH probe, ECGreen, is synthesized to visualize endocytic vesicles under structured illumination microscopy (SIM), a super-resolution technology. Being sensitive to acidity with increasing fluorescence at low pH, ECGreen can differentiate early and late endosomes as well as endolysosomes. Meanwhile, membrane anchoring not only improves the durability of ECGreen, but also provides an excellent anti-photobleaching property for long-time imaging with SIM. Moreover, by taking these advantages of ECGreen, a multidimensional analysis model containing spatial, temporal, and pH information is successfully developed for elucidating the dynamics of endocytic vesicles and their interactions with mitochondria during autophagy, and reveals a fast conversion of endosomes near the plasma membrane.

Pharmacological Activation of cGAS for Cancer Immunotherapy.

When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition.

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson's disease. Here, we study the basic neurobiology of VPS35 and Parkinson's disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

Exosome Traceability and Cell Source Dependence on Composition and Cell-Cell Cross Talk.

Exosomes are small vesicles with an average diameter of 100 nm that are produced by many, if not all, cell types. Exosome cargo includes lipids, proteins, and nucleic acids arranged specifically in the endosomes of donor cells. Exosomes can transfer the donor cell components to target cells and can affect cell signaling, proliferation, and differentiation. Important new information about exosomes' remote communication with other cells is rapidly being accumulated. Recent data indicates that the results of this communication depend on the donor cell type and the environment of the host cell. In the field of cancer research, major questions remain, such as whether tumor cell exosomes are equally taken up by cancer cells and normal cells and whether exosomes secreted by normal cells are specifically taken up by other normal cells or also tumor cells. Furthermore, we do not know how exosome uptake is made selective, how we can trace exosome uptake selectivity, or what the most appropriate methods are to study exosome uptake and selectivity. This review will explain the effect of exosome source and the impact of the donor cell growth environment on tumor and normal cell interaction and communication. The review will also summarize the methods that have been used to label and trace exosomes to date.

JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes.

Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

Protective genes and pathways in Alzheimer's disease: moving towards precision interventions.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder that is characterized by neurodegeneration, cognitive impairment, and an eventual inability to perform daily tasks. The etiology of Alzheimer's is complex, with numerous environmental and genetic factors contributing to the disease. Late-onset AD is highly heritable (60 to 80%), and over 40 risk loci for AD have been identified via large genome-wide association studies, most of which are common variants with small effect sizes. Although these discoveries have provided novel insight on biological contributors to AD, disease-modifying treatments remain elusive. Recently, the concepts of resistance to pathology and resilience against the downstream consequences of pathology have been of particular interest in the Alzheimer's field as studies continue to identify individuals who evade the pathology of the disease even into late life and individuals who have all of the neuropathological features of AD but evade downstream neurodegeneration and cognitive impairment. It has been hypothesized that a shift in focus from Alzheimer's risk to resilience presents an opportunity to uncover novel biological mechanisms of AD and to identify promising therapeutic targets for the disease. This review will highlight a selection of genes and variants that have been reported to confer protection from AD within the literature and will also discuss evidence for the biological underpinnings behind their protective effect with a focus on genes involved in lipid metabolism, cellular trafficking, endosomal and lysosomal function, synaptic function, and inflammation. Finally, we offer some recommendations in areas where the field can rapidly advance towards precision interventions that leverage the ideas of protection and resilience for the development of novel therapeutic strategies.

RTKN-1/Rhotekin shields endosome-associated F-actin from disassembly to ensure endocytic recycling.

Cargo sorting and the subsequent membrane carrier formation require a properly organized endosomal actin network. To better understand the actin dynamics during endocytic recycling, we performed a genetic screen in C. elegans and identified RTKN-1/Rhotekin as a requisite to sustain endosome-associated actin integrity. Loss of RTKN-1 led to a prominent decrease in actin structures and basolateral recycling defects. Furthermore, we showed that the presence of RTKN-1 thwarts the actin disassembly competence of UNC-60A/cofilin. Consistently, in RTKN-1-deficient cells, UNC-60A knockdown replenished actin structures and alleviated the recycling defects. Notably, an intramolecular interaction within RTKN-1 could mediate the formation of oligomers. Overexpression of an RTKN-1 mutant form that lacks self-binding capacity failed to restore actin structures and recycling flow in rtkn-1 mutants. Finally, we demonstrated that SDPN-1/Syndapin acts to direct the recycling endosomal dwelling of RTKN-1 and promotes actin integrity there. Taken together, these findings consolidated the role of SDPN-1 in organizing the endosomal actin network architecture and introduced RTKN-1 as a novel regulatory protein involved in this process.

Preclinical modeling of chronic inhibition of the Parkinson's disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo.

The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.

ALS2 regulates endosomal trafficking, postsynaptic development, and neuronal survival.

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. Here, we demonstrate that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). We further demonstrate that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival.

Rab14/MACF2 complex regulates endosomal targeting during cytokinesis.

Abscission is a complex cellular process that is required for mitotic division. It is well established that coordinated and localized changes in actin and microtubule dynamics are vital for cytokinetic ring formation, as well as establishment of the abscission site. Actin cytoskeleton reorganization during abscission would not be possible without the interplay between Rab11- and Rab35-containing endosomes and their effector proteins, whose roles in regulating endocytic pathways at the cleavage furrow have now been studied extensively. Here, we identified Rab14 as a novel regulator of cytokinesis. We demonstrate that depletion of Rab14 causes either cytokinesis failure or significantly prolongs division time. We show that Rab14 contributes to the efficiency of recruiting Rab11-endosomes to the thin intracellular bridge (ICB) microtubules and that Rab14 knockout leads to inhibition of actin clearance at the abscission site. Finally, we demonstrate that Rab14 binds to microtubule minus-end interacting MACF2/CAMSAP3 complex and that this binding affects targeting of endosomes to the ICB microtubules. Collectively, our data identified Rab14 and MACF2/CAMSAP3 as proteins that regulate actin depolymerization and endosome targeting during cytokinesis.

The role of the multifunctional antimicrobial peptide melittin in gene delivery.

Melittin is vital for the endosomal escape of nanoparticles, but its excessive cytotoxicity in mammalian cells limits its value as a potential therapeutic agent. Several novel analogs of melittin have been optimized and characterized to establish a non-toxic melittin-based gene delivery system, in which the sequences of the melittin peptides were altered to reduce their cytotoxic activity. This review focusses on the involvement of melittin in nanoparticle endosomal escape and on the construction of melittin conjugates to boost gene delivery. Endosomal escape mechanisms for melittin, as well as the development of melittin as a therapeutic agent and its potential applications in nanomedicine, are discussed.

Ponesimod suppresses hepatitis B virus infection by inhibiting endosome maturation.

The discovery of novel antivirals to treat hepatitis B virus (HBV) infection is urgently needed, as the currently available drugs mainly target viral proteins at replication step, whereas host factors also play significant roles in HBV infection. Although numerous studies have reported candidate drugs for HBV treatment, there remains a need to find a new drug that may target other steps of the HBV life cycle. In this study, by drug screening of a 533 G-protein-coupled receptors (GPCRs)-associated compound library, we identified ponesimod, a selective agonist of sphingosine-1-phosphate receptor 1 (S1P1), as a drug candidate for the suppression of HBV infection. However, the anti-HBV effect of ponesimod is independent of S1P1 and other sphingosine-1-phosphate receptors (S1PRs). Treatment with ponesimod at an early step of infection but not at a post-entry step significantly reduced the HBV relaxed circular DNA (rcDNA) level in a dose-dependent manner. Ponesimod treatment did not inhibit attachment, binding, or internalization of HBV particles via endocytosis through an interaction with sodium taurocholate cotransporting polypeptide (NTCP) or epidermal growth factor receptor (EGFR). Importantly, during the transportation of HBV particles to the nucleus, co-localization of HBV with early endosomes but not with late endosomes and lysosomes was induced by the treatment with ponesimod, suggesting that ponesimod interferes with the conversion of early endosomes to late endosomes without significant damage to cellular growth. Conclusion: Ponesimod is a promising anti-HBV drug targeting the endosome maturation of HBV. This finding can be applied to the development of novel antivirals that target the trafficking pathway of HBV particles.

Connecting the dots: combined control of endocytic recycling and degradation.

Endocytosis is an essential process where proteins and lipids are internalised from the plasma membrane in membrane-bound carriers, such as clathrin-coated vesicles. Once internalised into the cell these vesicles fuse with the endocytic network where their contents are sorted towards degradation in the lysosome or recycling to their origin. Initially, it was thought that cargo recycling is a passive process, but in recent years the identification and characterisation of specialised recycling complexes has established a hitherto unthought-of level of complexity that actively opposes degradation. This review will summarise recent developments regarding the composition and regulation of the recycling machineries and their relationship with the degradative pathways of the endosome.

Implications of Altered Endosome and Lysosome Biology in Space Environments.

Space exploration poses multiple challenges for mankind, not only on a technical level but also to the entire physiology of the space traveller. The human system must adapt to several environmental stressors, microgravity being one of them. Lysosomes are ubiquitous to every cell and essential for their homeostasis, playing significant roles in the regulation of autophagy, immunity, and adaptation of the organism to changes in their environment, to name a few. Dysfunction of the lysosomal system leads to age-related diseases, for example bone loss, reduced immune response or cancer. As these conditions have been shown to be accelerated following exposure to microgravity, this review elucidates the lysosomal response to real and simulated microgravity. Microgravity activates the endo-lysosomal system, with resulting impacts on bone loss, muscle atrophy and stem cell differentiation. The investigation of lysosomal adaptation to microgravity can be beneficial in the search for new biomarkers or therapeutic approaches to several disease pathologies on earth as well as the potential to mitigate pathophysiology during spaceflight.

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

LRRK2 and the Endolysosomal System in Parkinson's Disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson's disease (PD), with pathogenic mutations enhancing LRRK2 kinase activity. There is a growing body of evidence indicating that LRRK2 contributes to neuronal damage and pathology both in familial and sporadic PD, making it of particular interest for understanding the molecular pathways that underlie PD. Although LRRK2 has been extensively studied to date, our understanding of the seemingly diverse functions of LRRK2 throughout the cell remains incomplete. In this review, we discuss the functions of LRRK2 within the endolysosomal pathway. Endocytosis, vesicle trafficking pathways, and lysosomal degradation are commonly disrupted in many neurodegenerative diseases, including PD. Additionally, many PD-linked gene products function in these intersecting pathways, suggesting an important role for the endolysosomal system in maintaining protein homeostasis and neuronal health in PD. LRRK2 activity can regulate synaptic vesicle endocytosis, lysosomal function, Golgi network maintenance and sorting, vesicular trafficking and autophagy, with alterations in LRRK2 kinase activity serving to disrupt or regulate these pathways depending on the distinct cell type or model system. LRRK2 is critically regulated by at least two proteins in the endolysosomal pathway, Rab29 and VPS35, which may serve as master regulators of LRRK2 kinase activity. Investigating the function and regulation of LRRK2 in the endolysosomal pathway in diverse PD models, especially in vivo models, will provide critical insight into the cellular and molecular pathophysiological mechanisms driving PD and whether LRRK2 represents a viable drug target for disease-modification in familial and sporadic PD.

The Small GTPase Rab5c Exerts Bi-Function in Singapore Grouper Iridovirus Infections and Cellular Responses in the Grouper, Epinephelus coioides.

The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides, and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus, and Homo sapiens, respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions.