Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.

Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

The vascular endothelial growth factor receptors (VEGFRs) can shape the neovascular phenotype of vascular endothelial cells when translocated to the nucleus, however the spatial and temporal changes in the intracellular distribution and translocation of VEGFRs to the nucleus and the organelles involved in this process is unclear. This study reports the effect of exogenous VEGF on translocation of VEGFRs and organelles in micro- and macrovascular endothelial cells. We showed that VEGF is responsible for: a rapid and substantial nuclear translocation of VEGFRs; VEGFR1 and VEGFR2 exhibit distinct spatial, temporal and structural translocation characteristics both in vitro and in vivo and this determines the nuclear VEGFR1:VEGFR2 ratio which differs between microvascular and macrovascular cells; VEGFR2 nuclear translocation is associated with the endosomal pathway transporting the receptor from Golgi in microvascular endothelial cells; and an increase in the volume of intracellular organelles. In conclusion, the nuclear translocation of VEGFRs is both receptor and vessel (macro versus micro) dependent and the endosomal pathway plays a key role in the translocation of VEGFRs to the nucleus and the subsequent export to the lysosomal system. Modulating VEGF-mediated VEGFR1 and VEGFR2 intracellular transmigration pathways may offer an alternative for the development of new anti-angiogenic therapies.

Host Serine Proteases: A Potential Targeted Therapy for COVID-19 and Influenza

The ongoing pandemic illustrates limited therapeutic options for controlling SARS-CoV-2 infections, calling a need for additional therapeutic targets. The viral spike S glycoprotein binds to the human receptor angiotensin-converting enzyme 2 (ACE2) and then is activated by the host proteases. Based on the accessibility of the cellular proteases needed for SARS-S activation, SARS-CoV-2 entrance and activation can be mediated by endosomal (such as cathepsin L) and non-endosomal pathways. Evidence indicates that in the non-endosomal pathway, the viral S protein is cleaved by the furin enzyme in infected host cells. To help the virus enter efficiently, the S protein is further activated by the serine protease 2 (TMPRSS2), provided that the S has been cleaved by furin previously. In this review, important roles for host proteases within host cells will be outlined in SARS-CoV-2 infection and antiviral therapeutic strategies will be highlighted. Although there are at least five highly effective vaccines at this time, the appearance of the new viral mutations demands the development of therapeutic agents. Targeted inhibition of host proteases can be used as a therapeutic approach for viral infection.

Characterization of RNA in Extracellular Vesicles

Extracellular vesicles (EVs) are important players in the communication between different kinds of cells by delivering their content, consisting of different types of RNA, proteins, bioactive lipids, or signaling nucleotides, into their target cells. Several types of EVs are distinguished: (1) exosomes with sizes from 30 to 150 nm originate from the endosomal pathway and form intracellular multivesicular bodies (MVBs), which fuse to the plasma membrane before their secretion. (2) EVs with sizes ranging from 100 to 1000 nm in diameter are formed during cell surface budding. (3) Apoptotic bodies with diameters from 500 to 2000 nm are released from blebbing of the cell membrane of apoptotic cells. It is well established that various RNA molecules such as coding RNAs and noncoding RNAs (long noncoding RNAs, microRNAs, circular RNAs, and rRNAs) are present in different amounts in EVs depending on the type and origin of EV. Here we will give an overview of methods to isolate different types of EVs and to quantify and characterize different RNA species.

Identification of selected genes associated with the SARS-CoV-2: a therapeutic approach and disease severity

Background The ongoing pandemic of COVID-19 viruses takes its sole origin from the Wuhan Huanan seafood market, China. The first case was recorded as viral pneumonia and later became a worldwide pandemic (officially declared by WHO on March 11, 2020). Main body SARS-CoV-2 is an extremely infectious and transferrable virus that develops severe conditions like respiratory syndrome, high blood pressure and weakens the immune system. Coronavirus falls under the Coronaviridae family and Beta coronavirus genus. Affected individuals will encounter problems starting with fever followed by severe complications like SARS, ARDS, and many others. These SARS-CoV and MERS-CoV enter the host cells by the endosomal pathway, and about 16 non-structural proteins are involved in assembling the viral RNA synthesis complex. They possess a positive-sense single-stranded RNA, and about four major genes are mainly associated with the development of ASRD, SARS, and other respiratory problems. Conclusion Susceptibility of these four major genes such as ACE2, IL-2, 7 and 10, TNF, and VEGF is associated with COVID-19. This highlights the identification of the above-mentioned genes that can be used as potential biomarkers for early diagnosis and targeted drug delivery for treating the SARS-CoV-2 neurological symptoms and reducing inflammation in the brain.

Identification of a new, Rab14-dependent, endo-lysosomal pathway

Cells can endocytose material from the surrounding environment. Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Several endocytosis pathways have been described (e.g. clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway). Besides possible recycling routes to the plasma membrane and various organelles, these pathways all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs) used in research and clinic, we have discovered a second endocytic pathway that moves material to late endosomes/lysosomes and that is fully independent of Rab5 and Rab7 but requires the Rab14 protein. This newly identified pathway differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already and is not affected by a series of compounds that inhibit the Rab5-dependent pathway. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins. Rab14 is expressed by the last eukaryotic common ancestor. The Rab14-dependent pathway may therefore correspond to a primordial endosomal pathway taken by cationic cargos.

NECAB2 orchestrates an endosomal pathway of mitochondrial quality control at striatal synapses

SummarySynaptic signaling depends on ATP generated by mitochondria. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction and thus requires efficient mitochondrial quality control. We found that the neuronal calcium-binding protein NECAB2 ensures synaptic function in the striatum by increasing mitochondrial efficiency. NECAB2 associates with early endosomes and mitochondria at striatal synapses. Loss of NECAB2 dysregulates proteins of the endosomal ESCRT machinery and oxidative phosphorylation. Mitochondria from NECAB2-deficient mice are more abundant but less efficient. These mitochondria exhibit increased respiration and superoxide production but produce less ATP. This accumulation of faulty mitochondria is caused by a defective assembly of mitochondria with early endosomes in response to oxidative stress. Impairment of this mechanism causes loss of striatal synapses and behavioral dysfunctions such as reduced motivation and altered sensory gating. NECAB2 therefore orchestrates an endosomal pathway of mitochondrial quality control important for striatal function.

Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

The microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

Invariant chain regulates endosomal fusion and maturation through an interaction with the SNARE Vti1b

ABSTRACTThe invariant chain (Ii, also known as CD74) is a multifunctional regulator of adaptive immune responses and is responsible for sorting major histocompatibility complex class I and class II (MHCI and MHCII, respectively) molecules, as well as other Ii-associated molecules, to a specific endosomal pathway. When Ii is expressed, endosomal maturation and proteolytic degradation of proteins are delayed and, in non-antigen presenting cells, the endosomal size increases, but the molecular mechanisms underlying this are not known. We identified that a SNARE, Vti1b, is essential for regulating these Ii-induced effects. Vti1b binds to Ii and is localized at the contact sites of fusing Ii-positive endosomes. Furthermore, truncated Ii lacking the cytoplasmic tail, which is not internalized from the plasma membrane, relocates Vti1b to the plasma membrane. Knockout of Ii in an antigen-presenting cell line was found to speed up endosomal maturation, whereas silencing of Vti1b inhibits the Ii-induced maturation delay. Our results suggest that Ii, by interacting with the SNARE Vti1b in antigen-presenting cells, directs specific Ii-associated SNARE-mediated fusion in the early part of the endosomal pathway that leads to a slower endosomal maturation for efficient antigen processing and MHC antigen loading.