Progranulin and GPNMB: interactions in endo-lysosome function and inflammation in neurodegenerative disease.

BACKGROUND: Alterations in progranulin (PGRN) expression are associated with multiple neurodegenerative diseases (NDs), including frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), and lysosomal storage disorders (LSDs). Recently, the loss of PGRN was shown to result in endo-lysosomal system dysfunction and an age-dependent increase in the expression of another protein associated with NDs, glycoprotein non-metastatic B (GPNMB). MAIN BODY: It is unclear what role GPNMB plays in the context of PGRN insufficiency and how they interact and contribute to the development or progression of NDs. This review focuses on the interplay between these two critical proteins within the context of endo-lysosomal health, immune function, and inflammation in their contribution to NDs. SHORT CONCLUSION: PGRN and GPNMB are interrelated proteins that regulate disease-relevant processes and may have value as therapeutic targets to delay disease progression or extend therapeutic windows.

Emerging autophagic endo-lysosomal targets in the management of Parkinson's disease.

Synucleopathies, specifically Parkinson's disease, are still incurable and available therapeutic options are scarce and symptomatic. The autophagy-lysosomal-endosomal system is an indigenous mechanism to manage the proteome. Excess/misfolded protein accumulation activates this system, which degrades the undesired proteins via lysosomes. Cells also eliminate these proteins by releasing them into the extracellular space via exosomes. However, the sutophagy-lysosomal-endosomal system becomes unfunctional in Parkinson's disease and there is accumulation and spread of pathogenic alpha-synuclein. Neuronal degeneration results Owing to pathogenic alpha-synuclein. Thus, the autophagy-lysosomal-endosomal system could be a promising target for neuroprotection. In the present review, we discuss the autophagy-lysosomal-endosomal system as an emerging target for the management of Parkinson's disease. Modulation of these targets associated with the autophagy-lysosomal-endosomal system can aid in clearing pathogenic alpha-synuclein and prevent the degeneration of neurons.

Cellular endo-lysosomal dysfunction in the pathogenesis of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), an increasingly devastating human disorder, is characterized by intrahepatic fat accumulation. Although important progress has been made in understanding NAFLD, the fundamental mechanisms involved in the pathogenesis of NAFLD have not been fully explained. The endo-lysosomal trafficking network is central to lipid metabolism, protein degradation and signal transduction, which are involved in a variety of diseases. In recent years, many genes and pathways in the endo-lysosomal trafficking network and involved in lysosomal biogenesis have been associated with the development and progression of NAFLD. Mutations of these genes and impaired signalling lead to dysfunction in multiple steps of the endo-lysosomal network (endocytic trafficking, membrane fusion and lysosomal degradation), resulting in the accumulation of pathogenic proteins. In this review, we will focus on how alterations in these genes and pathways affect endo-lysosomal trafficking as well as the pathophysiology of NAFLD.

Endo-lysosomal dysfunction in neurodegenerative diseases: opinion on current progress and future direction in the use of exosomes as biomarkers.

Over the past two decades, increased research has highlighted the connection between endosomal trafficking defects and neurodegeneration. The endo-lysosomal network is an important, complex cellular system specialized in the transport of proteins, lipids, and other metabolites, essential for cell homeostasis. Disruption of this pathway is linked to a wide range of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and frontotemporal dementia. Furthermore, there is strong evidence that defects in this pathway create opportunities for diagnostic and therapeutic intervention. In this Opinion piece, we concisely address the role of endo-lysosomal dysfunction in five neurodegenerative diseases and discuss how future research can investigate this intracellular pathway, including extracellular vesicles with a specific focus on exosomes for the identification of novel disease biomarkers. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Functional characterization of endo-lysosomal compartments by correlative live-cell and volume electron microscopy.

Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce "functional correlative microscopy" (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures.

Pathways of integrins in the endo-lysosomal system.

In this review, we present recent scientific advances about integrin trafficking in the endo-lysosomal system. In the last few years, plenty of new information has emerged about the endo-lysosomal system, integrins, and the mechanism, how exactly the intracellular trafficking of integrins is regulated. We review the internalization and recycling pathways of integrins, and we provide information about the possible ways of lysosomal degradation through the endosomal and autophagic system. The regulation of integrin internalization and recycling proved to be a complex process worth studying. Trafficking of integrins, together with the regulation of their gene expression, defines cellular adhesion and cellular migration through bidirectional signalization and ligand binding. Thus, any malfunction in this system can potentially (but not necessarily) lead to tumorigenesis or metastasis. Hence, extensive examinations of integrins in the endo-lysosomal system raise the possibility to identify potential new medical targets. Furthermore, this knowledge can also serve as a basis for further determination of integrin signaling- and adhesion-related processes.

Golgi requires a new casting in the screenplay of mucopolysaccharidosis II cytopathology.

Lysosome (L), a hydrolytic compartment of the endo-lysosomal system (ELS), plays a central role in the metabolic regulation of eukaryotic cells. Furthermore, it has a central role in the cytopathology of several diseases, primarily in lysosomal storage diseases (LSDs). Mucopolysaccharidosis II (MPS II, Hunter disease) is a rare LSD caused by idunorate-2-sulphatase (IDS) enzyme deficiency. To provide a new platform for drug development and clarifying the background of the clinically observed cytopathology, we established a human in vitro model, which recapitulates all cellular hallmarks of the disease. Some of our results query the traditional concept by which the storage vacuoles originate from the endosomal system and suggest a new concept, in which endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and RAB2/LAMP positive Golgi (G) vesicles play an initiative role in the vesicle formation. In this hypothesis, Golgi is not only an indirectly affected organelle but enforced to be the main support of vacuole formation. The purposes of this minireview are to give a simple guide for understanding the main relationships in ELS, to present the storage vacuoles and their relation to ELS compartments, to recommend an alternative model for vacuole formation, and to place the Golgi in spotlight of MPS II cytopathology.

Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the Aß fly model and the AßPP-BACE1 fly model. In the Aß fly model, the Aß peptide is fused to a secretion sequence and directly overexpressed. In the AßPP-BACE1 model, human AßPP and human BACE1 are expressed in the fly, resulting in in vivo production of Aß peptides and other AßPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the Aß fly model, this correlates with high Aß1-42 levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the AßPP-BACE1 fly model, neuronal cell death correlates with low Aß1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of AßPP/Aß antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the AßPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.

Focusing on cellular biomarkers: The endo-lysosomal pathway in Down syndrome.

Down syndrome (DS) is the most frequent chromosomal disorder. It is caused by the triplication of human chromosome 21, leading to increased dosage of a variety of genes including APP (Amyloid Precursor Protein). Mainly for this reason, individuals with DS are at high risk to develop Alzheimer's disease (AD). Extensive literature identified various morphological and molecular abnormalities in the endo-lysosomal pathway both in DS and AD. Most studies in this field investigated the causative role of APP (Amyloid Precursor Protein) in endo-lysosomal dysfunctions, thus linking phenotypes observed in DS and AD. In DS context, several lines of evidence and emerging hypotheses suggest that other molecular players and pathways may be implicated in these complex phenotypes. In this review, we outline the normal functioning of endosomal trafficking and summarize the research on endo-lysosomal dysfunction in DS in light of AD findings. We emphasize the role of genes of chromosome 21 implicated in endocytosis to explain endosomal abnormalities and set the limitations and perspectives of models used to explore endo-lysosomal dysfunction in DS and find new biomarkers. The review highlights the complexity of endo-lysosomal dysfunction in DS and suggests directions for future research in the field.

Vesicular Transport Machinery in Brain Endothelial Cells: What We Know and What We Do not.

The vesicular transport machinery regulates numerous essential functions in cells such as cell polarity, signaling pathways, and the transport of receptors and their cargoes. From a pharmaceutical perspective, vesicular transport offers avenues to facilitate the uptake of therapeutic agents into cells and across cellular barriers. In order to improve receptor-mediated transcytosis of biologics across the blood-brain barrier and into the diseased brain, a detailed understanding of intracellular transport mechanisms is essential. The vesicular transport machinery is a highly complex network and involves an array of protein complexes, cytosolic adaptor proteins, and the subcellular structures of the endo-lysosomal system. The endo-lysosomal system includes several types of vesicular entities such as early, late, and recycling endosomes, exosomes, ectosomes, retromer-coated vesicles, lysosomes, trans-endothelial channels, and tubules. While extensive research has been done on the trafficking system in many cell types, little is known about vesicular trafficking in brain endothelial cells. Consequently, assumptions on the transport system in endothelial cells are based on findings in polarised epithelial cells, although recent studies have highlighted differences in the endothelial system. This review highlights aspects of the vesicular trafficking machinery in brain endothelial cells, including recent findings, limitations, and opportunities for further studies.

Extracellular Vesicles and Damage-Associated Molecular Patterns: A Pandora's Box in Health and Disease.

Sterile inflammation develops as part of an innate immunity response to molecules released upon tissue injury and collectively indicated as damage-associated molecular patterns (DAMPs). While coordinating the clearance of potential harmful stimuli, promotion of tissue repair, and restoration of tissue homeostasis, a hyper-activation of such an inflammatory response may be detrimental. The complex regulatory pathways modulating DAMPs generation and trafficking are actively investigated for their potential to provide relevant insights into physiological and pathological conditions. Abnormal circulating extracellular vesicles (EVs) stemming from altered endosomal-lysosomal system have also been reported in several age-related conditions, including cancer and neurodegeneration, and indicated as a promising route for therapeutic purposes. Along this pathway, mitochondria may dispose altered components to preserve organelle homeostasis. However, whether a common thread exists between DAMPs and EVs generation is yet to be clarified. A deeper understanding of the highly complex, dynamic, and variable intracellular and extracellular trafficking of DAMPs and EVs, including those of mitochondrial origin, is needed to unveil relevant pathogenic pathways and novel targets for drug development. Herein, we describe the mechanisms of generation of EVs and mitochondrial-derived vesicles along the endocytic pathway and discuss the involvement of the endosomal-lysosomal in cancer and neurodegeneration (i.e., Alzheimer's and Parkinson's disease).

Mitochondrial Dysfunction, Oxidative Stress, and Neuroinflammation: Intertwined Roads to Neurodegeneration.

Oxidative stress develops as a response to injury and reflects a breach in the cell's antioxidant capacity. Therefore, the fine-tuning of reactive oxygen species (ROS) generation is crucial for preserving cell's homeostasis. Mitochondria are a major source and an immediate target of ROS. Under different stimuli, including oxidative stress and impaired quality control, mitochondrial constituents (e.g., mitochondrial DNA, mtDNA) are displaced toward intra- or extracellular compartments. However, the mechanisms responsible for mtDNA unloading remain largely unclear. While shuttling freely within the cell, mtDNA can be delivered into the extracellular compartment via either extrusion of entire nucleoids or the generation and release of extracellular vesicles. Once discarded, mtDNA may act as a damage-associated molecular pattern (DAMP) and trigger an innate immune inflammatory response by binding to danger-signal receptors. Neuroinflammation is associated with a large array of neurological disorders for which mitochondrial DAMPs could represent a common thread supporting disease progression. The exploration of non-canonical pathways involved in mitochondrial quality control and neurodegeneration may unveil novel targets for the development of therapeutic agents. Here, we discuss these processes in the setting of two common neurodegenerative diseases (Alzheimer's and Parkinson's disease) and Down syndrome, the most frequent progeroid syndrome.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells.

BACKGROUND: Brain endothelial cell-based in vitro models are among the most versatile tools in blood-brain barrier research for testing drug penetration to the central nervous system. Transcytosis of large pharmaceuticals across the brain capillary endothelium involves the complex endo-lysosomal system. This system consists of several types of vesicle, such as early, late and recycling endosomes, retromer-positive structures, and lysosomes. Since the endo-lysosomal system in endothelial cell lines of in vitro blood-brain barrier models has not been investigated in detail, our aim was to characterize this system in different models. METHODS: For the investigation, we have chosen two widely-used models for in vitro drug transport studies: the bEnd.3 mouse and the hCMEC/D3 human brain endothelial cell line. We compared the structures and attributes of their endo-lysosomal system to that of primary porcine brain endothelial cells. RESULTS: We detected significant differences in the vesicular network regarding number, morphology, subcellular distribution and lysosomal activity. The retromer-positive vesicles of the primary cells were distinct in many ways from those of the cell lines. However, the cell lines showed higher lysosomal degradation activity than the primary cells. Additionally, the hCMEC/D3 possessed a strikingly unique ratio of recycling endosomes to late endosomes. CONCLUSIONS: Taken together our data identify differences in the trafficking network of brain endothelial cells, essentially mapping the endo-lysosomal system of in vitro blood-brain barrier models. This knowledge is valuable for planning the optimal route across the blood-brain barrier and advancing drug delivery to the brain.

The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro.

Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-µ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

Stuck in traffic: an emerging theme in diseases of the nervous system.

The past decade has seen an explosion of DNA sequencing activities and many mutations and genetic variances underlying neurological and neurodegenerative diseases have been determined. This wealth of genetic data is now placed in molecular pathways revealing the nodes that underlie the disrupted processes. Many mutations in neurological diseases affect proteins controlling endosomal/lysosomal transport. Although the age of onset of these diseases range from juvenile [i.e., Niemann-Pick type C (NPC) and Charcot-Marie-Tooth (CMT) disease] to late onset (Parkinson's and Alzheimer's disease), deregulation of endosomal transport is a common theme. This review summarizes how elucidating the genetic basis for the various neurological diseases has advanced our understanding of the endo-lysosomal system and why the various mutations all translate into similar disease phenotypes.