Endolysosomal System Research Articles

Neurodegenerative Lysosomal Storage Disorders: TPC2 Comes to the Rescue!

Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca2+ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca2+-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo.

Loss of endosomal exchanger NHE6 leads to pathological changes in tau in human neurons.

SummaryDisruption of endolysosomal and autophagy-lysosomal systems is increasingly implicated in neurodegeneration. Sodium-proton exchanger 6 (NHE6) contributes to the maintenance of proper endosomal pH, and loss-of function mutations in the X-linked NHE6 lead to Christianson syndrome (CS) in males. Neurodegenerative features of CS are increasingly recognized, with postmortem and clinical data implicating a role for tau. We generated cortical neurons from NHE6 knockout (KO) and isogenic wild-type control human induced pluripotent stem cells. We report elevated phosphorylated and sarkosyl-insoluble tau in NHE6 KO neurons. We demonstrate that NHE6 KO leads to lysosomal and autophagy dysfunction involving reduced lysosomal number and protease activity, diminished autophagic flux, and p62 accumulation. Finally, we show that treatment with trehalose or rapamycin, two enhancers of autophagy-lysosomal function, each partially rescue this tau phenotype. We provide insight into the neurodegenerative processes underlying NHE6 loss of function and into the broader role of the endosome-lysosome-autophagy network in neurodegeneration.

Alpha-Synuclein: The Spark That Flames Dopaminergic Neurons, In Vitro and In Vivo Evidence.

Mitochondria, α-syn fibrils and the endo-lysosomal system are key players in the pathophysiology of Parkinson’s disease. The toxicity of α-syn is amplified by cell-to-cell transmission and aggregation of endogenous species in newly invaded neurons. Toxicity of α-syn PFF was investigated using primary cultures of dopaminergic neurons or on aged mice after infusion in the SNpc and combined with mild inhibition of GBA. In primary dopaminergic neurons, application of α-syn PFF induced a progressive cytotoxicity associated with mitochondrial dysfunction, oxidative stress, and accumulation of lysosomes suggesting that exogenous α-syn reached the lysosome (from the endosome). Counteracting the α-syn endocytosis with a clathrin inhibitor, dopaminergic neuron degeneration was prevented. In vivo, α-syn PFF induced progressive neurodegeneration of dopaminergic neurons associated with motor deficits. Histology revealed progressive aggregation of α-syn and microglial activation and accounted for the seeding role of α-syn, injection of which acted as a spark suggesting a triggering of cell-to-cell toxicity. We showed for the first time that a localized SNpc α-syn administration combined with a slight lysosomal deficiency and aging triggered a progressive lesion. The cellular and animal models described could help in the understanding of the human disease and might contribute to the development of new therapies.

627 Inhibition of PAI-1 blocks PD-L1 endocytosis and improves the response of melanoma cells to immune checkpoint blockade

Immune checkpoint molecules, especially PD-1 and its ligand PD-L1, act as a major mechanism of cancer immune evasion. Although anti–PD-1/PD-L1 monotherapy increases therapeutic efficacy in melanoma treatment, only a subset of patients exhibits long-term tumor remission, and the underlying mechanism of resistance to PD-1/PD-L1 inhibitors remains unclear. In this study, we demonstrated that cell surface retention of PD-L1 is inversely correlated with PAI-1 expression in vitro, in vivo, and in clinical specimens. Moreover, extracellular PAI-1 induced the internalization of surface-expressed PD-L1 by triggering clathrin-mediated endocytosis. The endocytosed PD-L1 was transported to lysosomes for degradation by endolysosomal systems, resulting in the reduction of surface PD-L1. Notably, inhibition of PAI-1 by pharmacological inhibitor with tiplaxtinin led to elevated PD-L1 expression on the plasma membrane, both in vitro and in vivo. Strikingly, targeting PAI-1 by tiplaxtinin treatment synergizes with anti–PD-L1 immune checkpoint blockade therapy in a syngeneic murine model of melanoma. Our findings demonstrate a role for PAI-1 activity in immune checkpoint modulation by promoting surface PD-L1 for lysosomal degradation and provides an insight into the combination of PAI-1 inhibition and anti–PD-L1 immunotherapy as a promising therapeutic regimen for melanoma treatment.

Dysregulation of the endomembrane homeostasis triggers vascular smooth muscle cell phenotype switching

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft Background Cardiovascular diseases are a leading cause of mortality globally, with cardiovascular calcification as a prominent predictor and contributor. Notably, microcalcifications might cause atherosclerotic plaque rupture, leading to myocardial infarction. Calcified plaque remodeling accompanies phenotypic modulation of vascular smooth muscle cells (SMCs). Previously, we showed that extracellular vesicles (EVs) released from calcifying SMCs are pivotal for microcalcification formation. EVs originate from the endolysosomal system. The lipid kinase FYVE-type zinc finger containing phosphoinositide kinase (PIKfyve) plays a role in the endolysosomal maturation. Here, we investigated how modulation of the endolysosomal system intersects with SMC phenotype regulation. Methods and Results In calcified arteries and SMCs, we observed increased PIKfyve expression. Inhibition of PIKfyve with the small molecule Apilimod inhibited osteogenic differentiation of SMCs in an in vitro calcification model. Apilimod reduced tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA (-79%, p<0.01), protein (-96%, p<0.01) and activity (-92%, p<0.01) levels. Additionally, matrix mineralization and collagen network formation are inhibited. SMC-secreted EVs exhibited a lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Transcriptome analyses revealed that the anti-calcifying effect is accompanied by a decreased expression of contractile SMC genes. Validation by RT-PCR supported that SMC-specific markers (ACTA2, SM22) and the key SMC transcription factor MYOCD were downregulated, while the expression of macrophage-like SMC markers (LGALS3, CD68) was induced. On the other hand, Apilimod increased the expression of adipogenic transcription factors (CEBPA, PPARG) and genes of the cholesterol and fatty acid metabolism pathways (SREBF2, HMGCR, FABP3, CD36) and induced the nuclear translocation of PPARγ, the main regulator of adipogenic differentiation. Fluorescence-based assays revealed increased fatty acid and disrupted low-density lipoprotein (LDL) uptake in Apilimod-treated SMCs. In Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks and treated with Apilimod for 5 weeks, we found differentially distributed plaque lipid depositions detected by Oil Red O staining. Conclusion Disrupting the endolysosomal maturation by PIKfyve inhibition ameliorates SMC calcification and induces a phenotypic adipogenic-like, pro-inflammatory adaption. Our data elucidates the importance of SMC phenotype monitoring during therapeutic interference. The exact lineage characteristics of these SMCs remain to be further investigated.

Functional Characterization of CLCN4 Variants Associated With X-Linked Intellectual Disability and Epilepsy.

Early/late endosomes, recycling endosomes, and lysosomes together form the endo-lysosomal recycling pathway. This system plays a crucial role in cell differentiation and survival, and dysregulation of the endo-lysosomal system appears to be important in the pathogenesis of neurodevelopmental and neurodegenerative diseases. Each endo-lysosomal compartment fulfils a specific function, which is supported by ion transporters and channels that modify ion concentrations and electrical gradients across endo-lysosomal membranes. CLC-type Cl–/H+ exchangers are a group of endo-lysosomal transporters that are assumed to regulate luminal acidification and chloride concentration in multiple endosomal compartments. Heterodimers of ClC-3 and ClC-4 localize to various internal membranes, from the endoplasmic reticulum and Golgi to recycling endosomes and late endosomes/lysosomes. The importance of ClC-4-mediated ion transport is illustrated by the association of naturally occurring CLCN4 mutations with epileptic encephalopathy, intellectual disability, and behavioral disorders in human patients. However, how these mutations affect the expression, subcellular localization, and function of ClC-4 is insufficiently understood. We here studied 12 CLCN4 variants that were identified in patients with X-linked intellectual disability and epilepsy and were already characterized to some extent in earlier work. We analyzed the consequences of these mutations on ClC-4 ion transport, subcellular trafficking, and heterodimerization with ClC-3 using heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings. The mutations led to a variety of changes in ClC-4 function, ranging from gain/loss of function and impaired heterodimerization with ClC-3 to subtle impairments in transport functions. Our results suggest that even slight functional changes to the endosomal Cl–/H+ exchangers can cause serious neurological symptoms.

Autophagy regulates transforming growth factor β signaling and receptor trafficking

Transforming growth factor beta (TGFβ) stimulates tumorigenesis by inducing epithelial to mesenchymal transition (EMT) and cell migration. TGFβ signaling is regulated by the endocytosis of cell surface receptors and their subcellular trafficking into the endo-lysosomal system. Here we investigated how autophagy, a cellular quality control network that delivers material to lysosomes, regulates TGFβ signaling pathways that induce EMT and cell migration. We impaired autophagy in non-small cell lung cancer cells using chloroquine, spautin-1, ULK-101, or small interfering RNA (siRNA) targeting autophagy-related gene (ATG)5 and ATG7 and observed that inhibiting autophagy results in a decrease in TGFβ1-dependent EMT transcription factor and cell marker expression, as well as attenuated stress fiber formation and cell migration. This correlated with decreased internalization of cell surface TGFβ receptors and their trafficking to early/late endosomal and lysosomal compartments. The effects of autophagy inhibition on TGFβ signaling were investigated by Smad2/Smad3 phosphorylation and cellular localization using western blotting, subcellular fractionation, and immunofluorescence microscopy. We observed that inhibiting autophagy decreased the amount and timeframe of Smad2/Smad3 signaling. Taken together, our results suggest that inhibiting autophagy attenuates pro-tumorigenic TGFβ signaling by regulating receptor trafficking, resulting in impaired Smad2/Smad3 phosphorylation and nuclear accumulation.

Phosphoinositides as membrane organizers.

Phosphoinositides are signalling lipids derived from phosphatidylinositol, a ubiquitous phospholipid in the cytoplasmic leaflet of eukaryotic membranes. Initially discovered for their roles in cell signalling, phosphoinositides are now widely recognized as key integrators of membrane dynamics that broadly impact on all aspects of cell physiology and on disease. The past decade has witnessed a vast expansion of our knowledge of phosphoinositide biology. On the endocytic and exocytic routes, phosphoinositides direct the inward and outward flow of membrane as vesicular traffic is coupled to the conversion of phosphoinositides. Moreover, recent findings on the roles of phosphoinositides in autophagy and the endolysosomal system challenge our view of lysosome biology. The non-vesicular exchange of lipids, ions and metabolites at membrane contact sites in between organelles has also been found to depend on phosphoinositides. Here we review our current understanding of how phosphoinositides shape and direct membrane dynamics to impact on cell physiology, and provide an overview of emerging concepts in phosphoinositide regulation.

Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway.

Down Syndrome Fetal Fibroblasts Display Alterations of Endosomal Trafficking Possibly due to SYNJ1 Overexpression

Endosomal trafficking is essential for cellular homeostasis. At the crossroads of distinct intracellular pathways, the endolysosomal system is crucial to maintain critical functions and adapt to the environment. Alterations of endosomal compartments were observed in cells from adult individuals with Down syndrome (DS), suggesting that the dysfunction of the endosomal pathway may contribute to the pathogenesis of DS. However, the nature and the degree of impairment, as well as the timing of onset, remain elusive. Here, by applying imaging and biochemical approaches, we demonstrate that the structure and dynamics of early endosomes are altered in DS cells. Furthermore, we found that recycling trafficking is markedly compromised in these cells. Remarkably, our results in 18–20 week-old human fetal fibroblasts indicate that alterations in the endolysosomal pathway are already present early in development. In addition, we show that overexpression of the polyphosphoinositide phosphatase synaptojanin 1 (Synj1) recapitulates the alterations observed in DS cells, suggesting a role for this lipid phosphatase in the pathogenesis of DS, likely already early in disease development. Overall, these data strengthen the link between the endolysosomal pathway and DS, highlighting a dangerous liaison among Synj1, endosomal trafficking and DS.

Secretory autophagy maintains proteostasis upon lysosome inhibition.

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.

The Endolysosomal System: The Acid Test for SARS-CoV-2.

This review aims to describe and discuss the different functions of the endolysosomal system, from homeostasis to its vital role during viral infections. We will initially describe endolysosomal system’s main functions, presenting recent data on how its compartments are essential for host defense to explore later how SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) and other coronaviruses subvert these organelles for their benefit. It is clear that to succeed, pathogens’ evolution favored the establishment of ways to avoid, escape, or manipulate lysosomal function. The unavoidable coexistence with such an unfriendly milieu imposed on viruses the establishment of a vast array of strategies to make the most out of the invaded cell’s machinery to produce new viruses and maneuvers to escape the host’s defense system.

The Dictyostelium Model for Mucolipidosis Type IV.

Mucolipidosis type IV, a devastating neurological lysosomal disease linked to mutations in the transient receptor potential channel mucolipin 1, TRPML1, a calcium permeable channel in the membranes of vesicles in endolysosomal system. TRPML1 function is still being elucidated and a better understanding of the molecular pathogenesis of Mucolipidosis type IV, may facilitate development of potential treatments. We have created a model to study mucolipin function in the eukaryotic slime mould Dictyostelium discoideum by altering expression of its single mucolipin homologue, mcln. We show that in Dictyostelium mucolipin overexpression contributes significantly to global chemotactic calcium responses in vegetative and differentiated cells. Knockdown of mucolipin also enhances calcium responses in vegetative cells but does not affect responses in 6–7 h developed cells, suggesting that in developed cells mucolipin may help regulate local calcium signals rather than global calcium waves. We found that both knocking down and overexpressing mucolipin often, but not always, presented the same phenotypes. Altering mucolipin expression levels caused an accumulation or increased acidification of Lysosensor Blue stained vesicles in vegetative cells. Nutrient uptake by phagocytosis and macropinocytosis were increased but growth rates were not, suggesting defects in catabolism. Both increasing and decreasing mucolipin expression caused the formation of smaller slugs and larger numbers of fruiting bodies during multicellular development, suggesting that mucolipin is involved in initiation of aggregation centers. The fruiting bodies that formed from these smaller aggregates had proportionately larger basal discs and thickened stalks, consistent with a regulatory role for mucolipin-dependent Ca2+ signalling in the autophagic cell death pathways involved in stalk and basal disk differentiation in Dictyostelium. Thus, we have provided evidence that mucolipin contributes to chemotactic calcium signalling and that Dictyostelium is a useful model to study the molecular mechanisms involved in the cytopathogenesis of Mucolipidosis type IV.

The Many Faces of G Protein-Coupled Receptor 143, an Atypical Intracellular Receptor.

GPCRs transform extracellular stimuli into a physiological response by activating an intracellular signaling cascade initiated via binding to G proteins. Orphan G protein-coupled receptors (GPCRs) hold the potential to pave the way for development of new, innovative therapeutic strategies. In this review we will introduce G protein-coupled receptor 143 (GPR143), an enigmatic receptor in terms of classification within the GPCR superfamily and localization. GPR143 has not been assigned to any of the GPCR families due to the lack of common structural motifs. Hence we will describe the most important motifs of classes A and B and compare them to the protein sequence of GPR143. While a precise function for the receptor has yet to be determined, the protein is expressed abundantly in pigment producing cells. Many GPR143 mutations cause X-linked Ocular Albinism Type 1 (OA1, Nettleship-Falls OA), which results in hypopigmentation of the eyes and loss of visual acuity due to disrupted visual system development and function. In pigment cells of the skin, loss of functional GPR143 results in abnormally large melanosomes (organelles in which pigment is produced). Studies have shown that the receptor is localized internally, including at the melanosomal membrane, where it may function to regulate melanosome size and/or facilitate protein trafficking to the melanosome through the endolysosomal system. Numerous additional roles have been proposed for GPR143 in determining cancer predisposition, regulation of blood pressure, development of macular degeneration and signaling in the brain, which we will briefly describe as well as potential ligands that have been identified. Furthermore, GPR143 is a promiscuous receptor that has been shown to interact with multiple other melanosomal proteins and GPCRs, which strongly suggests that this orphan receptor is likely involved in many different physiological actions.

Lysosomes and lysosome-related organelles in immune responses.

The catabolic, degradative capacity of the endo‐lysosome system is put to good use in mammalian immune responses as is their recently established status as signaling platforms. From the ‘creative destruction’ of antigenic and ‘self’ material for antigen presentation to T cells to the re‐purposing of lysosomes as toxic exocytosable lysosome‐related organelles (granules) in leukocytes such as CD8 T cells and eosinophils, endo‐lysosomes are key players in host defense. Signaled responses to some pathogen products initiate in endo‐lysosomes and these organelles are emerging as important in distinct ways in the unique immunobiology of dendritic cells. Potential self‐inflicted toxicity from lysosomal and granule proteases is countered by expression of serpin and cystatin family members.

Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila

ABSTRACT The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes. Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.

Synaptic Vesicle Recycling and the Endolysosomal System: A Reappraisal of Form and Function.

The endolysosomal system is present in all cell types. Within these cells, it performs a series of essential roles, such as trafficking and sorting of membrane cargo, intracellular signaling, control of metabolism and degradation. A specific compartment within central neurons, called the presynapse, mediates inter-neuronal communication via the fusion of neurotransmitter-containing synaptic vesicles (SVs). The localized recycling of SVs and their organization into functional pools is widely assumed to be a discrete mechanism, that only intersects with the endolysosomal system at specific points. However, evidence is emerging that molecules essential for endolysosomal function also have key roles within the SV life cycle, suggesting that they form a continuum rather than being isolated processes. In this review, we summarize the evidence for key endolysosomal molecules in SV recycling and propose an alternative model for membrane trafficking at the presynapse. This includes the hypotheses that endolysosomal intermediates represent specific functional SV pools, that sorting of cargo to SVs is mediated via the endolysosomal system and that manipulation of this process can result in both plastic changes to neurotransmitter release and pathophysiology via neurodegeneration.

Multi-transcriptomic analysis points to early organelle dysfunction in human astrocytes in Alzheimer's disease

The phenotypic transformation of astrocytes in Alzheimer's disease (AD) is still not well understood. Recent analyses based on single-nucleus RNA sequencing of postmortem Alzheimer's disease (AD) samples are limited by the low number of sequenced astrocytes, small cohort sizes, and low number of differentially expressed genes detected. To optimize the detection of astrocytic genes, we employed a novel strategy consisting of the localization of pre-determined astrocyte and neuronal gene clusters in publicly available whole-brain transcriptomes. Specifically, we used cortical transcriptomes from 766 individuals, including cognitively normal subjects (Controls), and people diagnosed with mild cognitive impairment (MCI) or dementia due to AD. Samples came from three independent cohorts organized by the Mount Sinai Hospital, the Mayo Clinic, and the Religious Order Study/Memory and Aging Project (ROSMAP). Astrocyte- and neuron-specific gene clusters were generated from human brain cell-type specific RNAseq data using hierarchical clustering and cell-type enrichment scoring. Genes from each cluster were manually annotated according to cell-type specific functional Categories. Gene Set Variation Analysis (GSVA) and Principal Component Analysis (PCA) were used to establish changes in these functional categories among clinical cohorts. We highlight three novel findings of the study. First, individuals with the same clinical diagnosis were molecularly heterogeneous. Particularly in the Mayo Clinic and ROSMAP cohorts, over 50% of Controls presented down-regulation of genes encoding synaptic proteins typical of AD, whereas 30% of patients diagnosed with dementia due to AD presented Control-like transcriptomic profiles. Second, down-regulation of neuronal genes related to synaptic proteins coincided, in astrocytes, with up-regulation of genes related to perisynaptic astrocytic processes (PAP) and down-regulation of genes encoding endolysosomal and mitochondrial proteins. Third, down-regulation of astrocytic mitochondrial genes inversely correlated with the disease stages defined by Braak and CERAD scoring. Finally, we interpreted these changes as maladaptive or adaptive from the point of view of astrocyte biology in a model of the phenotypical transformation of astrocytes in AD. The main prediction is that early malfunction of the astrocytic endolysosomal system, associated with progressive mitochondrial dysfunction, contribute to Alzheimer's disease. If this prediction is correct, therapies preventing organelle dysfunction in astrocytes may be beneficial in preclinical and clinical AD.

Plasma Metabolite Signature Classifies Male LRRK2 Parkinson's Disease Patients.

Parkinson’s disease (PD) is a progressive neurodegenerative disease, causing loss of motor and nonmotor function. Diagnosis is based on clinical symptoms that do not develop until late in the disease progression, at which point the majority of the patients’ dopaminergic neurons are already destroyed. While many PD cases are idiopathic, hereditable genetic risks have been identified, including mutations in the gene for LRRK2, a multidomain kinase with roles in autophagy, mitochondrial function, transcription, molecular structural integrity, the endo-lysosomal system, and the immune response. A definitive PD diagnosis can only be made post-mortem, and no noninvasive or blood-based disease biomarkers are currently available. Alterations in metabolites have been identified in PD patients, suggesting that metabolomics may hold promise for PD diagnostic tools. In this study, we sought to identify metabolic markers of PD in plasma. Using a 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) NMR spectroscopy metabolomics platform coupled with machine learning (ML), we measured plasma metabolites from approximately age/sex-matched PD patients with G2019S LRRK2 mutations and non-PD controls. Based on the differential level of known and unknown metabolites, we were able to build a ML model and develop a Biomarker of Response (BoR) score, which classified male LRRK2 PD patients with 79.7% accuracy, 81.3% sensitivity, and 78.6% specificity. The high accuracy of the BoR score suggests that the metabolomics/ML workflow described here could be further utilized in the development of a confirmatory diagnostic for PD in larger patient cohorts. A diagnostic assay for PD will aid clinicians and their patients to quickly move toward a definitive diagnosis, and ultimately empower future clinical trials and treatment options.

The ins and outs of virus trafficking through acidic Ca2+ stores

Many viruses exploit host-cell Ca2+ signaling processes throughout their life cycle. This is especially relevant for viruses that translocate through the endolysosomal system, where cellular infection is keyed to the microenvironment of these acidic Ca2+ stores and Ca2+-dependent trafficking pathways. As regulators of the endolysosomal ionic milieu and trafficking dynamics, two families of endolysosomal Ca2+-permeable cation channels – two pore channels (TPCs) and transient receptor potential mucolipins (TRPMLs) – have emerged as important host-cell factors in viral entry. Here, we review: (i) current evidence implicating Ca2+ signaling in viral translocation through the endolysosomal system, (ii) the roles of these ion channels in supporting cellular infection by different viruses, and (iii) areas for future research that will help define the potential of TPC and TRPML ligands as progressible antiviral agents.