Deacidification of endolysosomes by neuronal aging drives synapse loss.

Previously, we found that age-dependent accumulation of beta-amyloid is not sufficient to cause synaptic decline. Late-endocytic organelles (LEOs) may be driving synaptic decline as lysosomes (Lys) are a target of cellular aging and relevant for synapses. We found that LAMP1-positive LEOs increased in size and number and accumulated near synapses in aged neurons and brains. LEOs' distal accumulation might relate to the increased anterograde movement in aged neurons. Dissecting the LEOs, we found that late-endosomes accumulated while there are fewer terminal Lys in aged neurites, but not in the cell body. The most abundant LEOs were degradative Lys or endolysosomes (ELys), especially in neurites. ELys activity was reduced because of acidification defects, supported by the reduction in v-ATPase subunit V0a1 with aging. Increasing the acidification of aged ELys recovered degradation and reverted synaptic decline, while alkalinization or v-ATPase inhibition, mimicked age-dependent Lys and synapse dysfunction. We identify ELys deacidification as a neuronal mechanism of age-dependent synapse loss. Our findings suggest that future therapeutic strategies to address endolysosomal defects might be able to delay age-related synaptic decline.

New approaches to the control of chronic inflammatory diseases with a focus on the endolysosomal system of immune cells.

Chronic inflammation is implicated in many types of diseases, including cardiovascular, neurodegenerative, metabolic, and immune disorders. The search for therapeutic targets to control chronic inflammation often involves narrowing down the various molecules associated with pathology that have been discovered by various omics analyses. Herein, a different approach to identify therapeutic targets against chronic inflammation is proposed and one such target is discussed as an example. In chronically inflamed tissues, a large number of cells receive diverse proinflammatory signals, the intracellular signals are intricately integrated, and complicated intercellular interactions are orchestrated. This review focuses on effectively blocking this chaotic inflammatory signaling network via the endolysosomal system, which acts as a cellular signaling hub. In endolysosomes, the inflammatory signals mediated by pathogen sensors, such as Toll-like receptors, and the signals from nutrient and metabolic pathways are integrally regulated. Disruption of endolysosome signaling results in a strong anti-inflammatory effect by disrupting various signaling pathways, including pathogen sensor-mediated signals, in multiple immune cells. The endolysosome-resident amino acid transporter, solute carrier family 15 member 4 (SLC15A4), which plays an important role in the regulation of endolysosome-mediated signals, is a promising therapeutic target for several inflammatory diseases, including autoimmune diseases. The mechanisms by which SLC15A4 regulates inflammatory responses may provide a proof of concept for the efficacy of therapeutic strategies targeting immune cell endolysosomes.

mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Increasing the Survival of a Neuronal Model of Alzheimer's Disease Using Docosahexaenoic Acid, Restoring Endolysosomal Functioning by Modifying the Interactions between the Membrane Proteins C99 and Rab5.

Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.

TMEM175 downregulation participates in impairment of the autophagy related lysosomal dynamics following neonatal hypoxic-ischemic brain injury.

The mechanism underlying long-term cognitive impairment caused by neonatal hypoxic-ischemic brain injury (HIBI) remains unclear. Autophagy is a closely related mechanism and may play a role in this process. We aimed to investigate the role of lysosomal transmembrane protein 175 (TMEM175) in the autophagy-lysosome pathway in neonatal rats with HIBI. A neonatal rat model of HIBI was established, hypoxia was induced, followed by left common carotid artery ligation. Expression levels of TMEM175 and the corresponding proteins involved in autophagy flux and the endolysosomal system fusion process were measured. Rats were administered TMEM175 plasmid via intracerebroventricular injection to induce overexpression. Brain damage and cognitive function were then assessed. TMEM175 was downregulated in the hippocampal tissue, and the autophagy-lysosome pathway was impaired following HIBI in neonatal rats. Overexpression of TMEM175 significantly mitigated neuronal injury and improved long-term cognitive and memory function in neonatal rats with HIBI. We found that improvement in the autophagy-lysosome pathway and endolysosomal system homeostasis, which are TMEM175 related, occurred via regulation of lysosomal membrane dynamic fusion. TMEM175 plays a critical role in maintaining the autophagy-lysosome pathway and endolysosomal homeostasis, contributing to the amelioration of neuronal injury and impaired long-term cognitive function following neonatal HIBI.

Microglial LRRK2-mediated NFATc1 attenuates α-synuclein immunotoxicity in association with CX3CR1-induced migration and the lysosome-initiated degradation.

Synucleinopathies refer to a range of neurodegenerative diseases caused by abnormal α-synuclein (α-Syn) deposition, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Their pathogenesis is strongly linked to microglial dysfunction and neuroinflammation, which involves the leucine-rich-repeat kinase 2 (LRRK2)-regulated nuclear factor of activated T-cells (NFAT). Of the NFAT family, NFATc1 has been found to be increasingly translocated into the nucleus in α-syn stimulation. However, the specific role of NFATc1-mediated intracellular signaling in PD remains elusive in regulating microglial functions. In the current study, we crossbred LRRK2 or NFATc1 conditional knockout mice with Lyz2Cre mice to generate mice with microglia-specific deletion of LRRK2 or NFATc1, and by stereotactic injection of fibrillary α-Syn, we generated PD models in these mice. We found that LRRK2 deficiency enhanced microglial phagocytosis in the mice after α-Syn exposure and that genetic inhibition of NFATc1 markedly diminished phagocytosis and α-Syn elimination. We further demonstrated that LRRK2 negatively regulated NFATc1 in α-Syn-treated microglia, in which microglial LRRK2-deficiency facilitated NFATc1 nuclear translocation, CX3CR1 upregulation, and microglia migration. Additionally, NFATc1 translocation upregulated the expression of Rab7 and promoted the formation of late lysosomes, resulting in α-Syn degradation. In contrast, the microglial NFATc1 deficiency impaired CX3CR1 upregulation and the formation of Rab7-mediated late lysosomes. These findings highlight the critical role of NFATc1 in modulating microglial migration and phagocytosis, in which the LRRK2-NFATc1 signaling pathway regulates the expression of microglial CX3CR1 and endocytic degradative Rab7 to attenuate α-synuclein immunotoxicity.

Autophagy and the endolysosomal system in presynaptic function.

The complex morphology of neurons, the specific requirements of synaptic neurotransmission and the accompanying metabolic demands create a unique challenge for proteostasis. The main machineries for neuronal protein synthesis and degradation are localized in the soma, while synaptic junctions are found at vast distances from the cell body. Sophisticated mechanisms must, therefore, ensure efficient delivery of newly synthesized proteins and removal of faulty proteins. These requirements are exacerbated at presynaptic sites, where the demands for protein turnover are especially high due to synaptic vesicle release and recycling that induces protein damage in an intricate molecular machinery, and where replacement of material is hampered by the extreme length of the axon. In this review, we will discuss the contribution of the two major pathways in place, autophagy and the endolysosomal system, to presynaptic protein turnover and presynaptic function. Although clearly different in their biogenesis, both pathways are characterized by cargo collection and transport into distinct membrane-bound organelles that eventually fuse with lysosomes for cargo degradation. We summarize the available evidence with regard to their degradative function, their regulation by presynaptic machinery and the cargo for each pathway. Finally, we will discuss the interplay of both pathways in neurons and very recent findings that suggest non-canonical functions of degradative organelles in synaptic signalling and plasticity.

Glycans in autophagy, endocytosis and lysosomal functions.

Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal proteins and in lysosomal functions. Moreover, we review evidence on the regulation of endocytosis and autophagy by glycans. Finally, we discuss the emerging concept that cytosolic exposure of luminal glycans, and their detection by endogenous lectins, provides a mechanism for the surveillance of the integrity of the endolysosomal compartments, and serves their eventual repair or disposal.

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing.

Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic.

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network.

Acute Pancreatitis: A Multifaceted Set of Organelle and Cellular Interactions.

Acute pancreatitis is an inflammatory disorder of the exocrine pancreas associated with tissue injury and necrosis. The disease can be mild, involving only the pancreas, and resolve spontaneously within days or severe, with systemic inflammatory response syndrome-associated extrapancreatic organ failure and even death. Importantly, there are no therapeutic agents currently in use that can alter the course of the disease. This article emphasizes emerging findings that stressors (environmental and genetic) that cause acute pancreatitis initially cause injury to organelles of the acinar cell (endoplasmic reticulum, mitochondria, and endolysosomal-autophagy system), and that disorders in the functions of the organelles lead to inappropriate intracellular activation of trypsinogen and inflammatory pathways. We also review emerging work on the role of damage-associated molecular patterns in mediating the local and systemic inflammatory response in addition to known cytokines and chemokine pathways. In the review, we provide considerations for correction of organelle functions in acute pancreatitis to create a discussion for clinical trial treatment and design options.

The human ABCB6 protein is the functional homologue of HMT-1 proteins mediating cadmium detoxification.

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.

Bidirectional traffic between the Golgi and the endosomes - machineries and regulation.

The bidirectional transport between the Golgi complex and the endocytic pathway has to be finely regulated in order to ensure the proper delivery of newly synthetized lysosomal enzymes and the return of sorting receptors from degradative compartments. The high complexity of these routes has led to experimental difficulties in properly dissecting and separating the different pathways. As a consequence, several models have been proposed during the past decades. However, recent advances in our understanding of endosomal dynamics have helped to unify these different views. We provide here an overview of the current insights into the transport routes between Golgi and endosomes in mammalian cells. The focus of the Commentary is on the key molecules involved in the trafficking pathways between these intracellular compartments, such as Rab proteins and sorting receptors, and their regulation. A proper understanding of the bidirectional traffic between the Golgi complex and the endolysosomal system is of uttermost importance, as several studies have demonstrated that mutations in the factors involved in these transport pathways result in various pathologies, in particular lysosome-associated diseases and diverse neurological disorders, such as Alzheimer's and Parkinson's disease.

α-Synuclein-independent histopathological and motor deficits in mice lacking the endolysosomal Parkinsonism protein Atp13a2.

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.

Endolysosomal dysfunction in Parkinson's disease: Recent developments and future challenges.

Increasingly, genetic, cell biological, and in vivo work emphasizes the role of the endolysosomal system dysfunction in Parkinson's disease pathogenesis. Yet many questions remain about the mechanisms by which primary endolysosomal dysfunction causes PD as well as how the endolysosomal system interacts with α-synuclein-mediated neurotoxicity. We recently described a new mouse model of parkinsonism in which loss of the endolysosomal protein Atp13a2 causes behavioral, neuropathological, and biochemical changes similar to those present in human subjects with ATP13A2 mutations. In this Scientific Perspectives, we revisit the evidence implicating the endolysosomal system in PD, current hypotheses of disease pathogenesis, and how recent studies refine these hypotheses and raise new questions for future research. © 2016 International Parkinson and Movement Disorder Society.

Dendrimer-like Hierarchical Framework Nucleic Acid for Real-Time Imaging of Intracellular Trafficking.

Framework nucleic acids (FNAs) represent a new type of DNA-based nanomaterials and possess great potentials in biosensing, bioimaging, and molecular delivery. Hierarchical DNA nanostructures that consist of multiple FNA monomers increase the capacity for drug delivery and multifunctional modification. However, there are relatively few studies devoted to the behavior and regulation of hierarchical FNAs in living cells, impeding their further applications. Herein, we constructed a dendritic nanostructure with five tetrahedral DNA nanocages and characterized the real-time internalization, inter-organelle trafficking, and exocytosis in living mammalian cells. In comparison to FNA monomers, FNA dendrimers exhibit increased endocytosis and prolonged cellular retention. Single-particle tracking on hundreds of FNA dendrimers exhibits no interference on the mobility or kinetics of subcellular organelles, implying that FNAs as well as their higher-order derivatives are ideal intracellular imaging probes and nanocarriers. Our study validates the suitability and superiority of hierarchical DNA nanostructures as high-valency scaffolds for biomedical applications.

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

The critical role of the endolysosomal system in cerebral ischemia.

Cerebral ischemia is a serious disease that triggers sequential pathological mechanisms, leading to significant morbidity and mortality. Although most studies to date have typically focused on the lysosome, a single organelle, current evidence supports that the function of lysosomes cannot be separated from that of the endolysosomal system as a whole. The associated membrane fusion functions of this system play a crucial role in the biodegradation of cerebral ischemia-related products. Here, we review the regulation of and the changes that occur in the endolysosomal system after cerebral ischemia, focusing on the latest research progress on membrane fusion function. Numerous proteins, including N-ethylmaleimide-sensitive factor and lysosomal potassium channel transmembrane protein 175, regulate the function of this system. However, these proteins are abnormally expressed after cerebral ischemic injury, which disrupts the normal fusion function of membranes within the endolysosomal system and that between autophagosomes and lysosomes. This results in impaired "maturation" of the endolysosomal system and the collapse of energy metabolism balance and protein homeostasis maintained by the autophagy-lysosomal pathway. Autophagy is the final step in the endolysosomal pathway and contributes to maintaining the dynamic balance of the system. The process of autophagosome-lysosome fusion is a necessary part of autophagy and plays a crucial role in maintaining energy homeostasis and clearing aging proteins. We believe that, in cerebral ischemic injury, the endolysosomal system should be considered as a whole rather than focusing on the lysosome. Understanding how this dynamic system is regulated will provide new ideas for the treatment of cerebral ischemia.

Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson's disease.

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

Vps33B controls Treg cell suppressive function through inhibiting lysosomal nutrient sensing complex-mediated mTORC1 activation.

The suppressive function of regulatory T (Treg) cells is tightly controlled by nutrient-fueled mechanistic target of rapamycin complex 1 (mTORC1) activation, yet its dynamics and negative regulation remain unclear. Here we show that Treg-specific depletion of vacuolar protein sorting 33B (Vps33B) in mice results in defective Treg cell suppressive function and acquisition of effector phenotype, which in turn leads to disturbed T cell homeostasis and boosted antitumor immunity. Mechanistically, Vps33B binds with lysosomal nutrient-sensing complex (LYNUS) and promotes late endosome and lysosome fusion and clearance of the LYNUS-containing late endosome/lysosome, and therefore suppresses mTORC1 activation. Vps33B deficiency in Treg cells results in disordered endosome lysosome fusion, which leads to accumulation of LYNUS that causes elevated mTORC1 activation and hyper-glycolytic metabolism. Taken together, our study reveals that Vps33B maintains Treg cell suppressive function through sustaining endolysosomal homeostasis and therefore restricting amino acid-licensed mTORC1 activation and metabolism.