Lysosomal Compartment Research Articles

En Route to Stabilized Compact Conformations of Single-Chain Polymeric Nanoparticles in Complex Media.

Precise control over the folding pathways of polypeptides using a combination of noncovalent and covalent interactions has evolved into a wide range of functional proteins with a perfectly defined 3D conformation. Inspired hereby, we develop a series of amphiphilic copolymers designed to form compact, stable, and structured single-chain polymeric nanoparticles (SCPNs) of defined size, even in competitive conditions. The SCPNs are formed through a combination of noncovalent interactions (hydrophobic and hydrogen-bonding interactions) and covalent intramolecular cross-linking using a light-induced [2 + 2] cycloaddition. By comparing different self-assembly pathways of the nanoparticles, we show that, like for proteins in nature, the order of events matters. When covalent cross-links are formed prior to the folding via hydrophobic and supramolecular interactions, larger particles with less structured interiors are formed. In contrast, when the copolymers first fold via hydrophobic and hydrogen-bonding interactions into compact conformations, followed by covalent cross-links, good control over the size of the SCPNs and microstructure of the hydrophobic interior is achieved. Such a structured SCPN can stabilize the solvatochromic dye benzene-1,3,5-tricarboxamide–Nile Red via molecular recognition for short periods of time in complex media, while showing slow exchange dynamics with the surrounding complex media at longer time scales. The SCPNs show good biocompatibility with cells and can carry cargo into the lysosomal compartments of the cells. Our study highlights the importance of control over the folding pathway in the design of stable SCPNs, which is an important step forward in their application as noncovalent drug or catalyst carriers in biological settings.

Photothermally responsive theranostic nanocomposites for near-infrared light triggered drug release and enhanced synergism of photothermo-chemotherapy for gastric cancer.

Near-infrared (NIR) photothermal therapy plays a critical role in the cancer treatment and diagnosis as a promising carcinoma treatment modalities nowadays. However, development of clinical application has been greatly limited due to the inefficient drug release and low tumor accumulation. Herein, we designed a NIR-light triggered indocyanine green (ICG)-based PCL core/P(MEO2MA-b-HMAM) shell nanocomposites (PPH@ICG) and evaluated their therapeutic effects in vitro and in vivo. The anticancer drug 5-fluorouracil (5Fu) and the photothermal agent ICG were loaded into a thermo-sensitive micelle (PPH@5Fu@ICG) by self-assembly. The nanoparticles formed were characterized using transmission electron microscopy, dynamic light scattering, and fluorescence spectra. The thermo-sensitive copolymer (PPH@5Fu@ICG) showed a great temperature-controlled drug release response with lower critical solution temperature. In vitro cellular uptake and TEM imaging proved that PPH@5Fu@ICG nanoparticles can home into the lysosomal compartments under NIR. Moreover, in gastric tumor-bearing nude mice, PPH@5Fu@ICG + NIR group exhibited excellent improvement in antitumor efficacy based on the NIR-triggered thermo-chemotherapy synergy, both in vitro and in vivo. In summary, the proposed strategy of synergistic photo-hyperthermia chemotherapy effectively reduced the 5Fu dose, toxic or side effect, which could serve as a secure and efficient approach for cancer theranostics.

Cardiac glycoside-mediated turnover of Na, K-ATPases as a rational approach to reducing cell surface levels of the cellular prion protein.

It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.

Massive Accumulation of Sphingomyelin Affects the Lysosomal and Mitochondria Compartments and Promotes Apoptosis in Niemann-Pick Disease Type A

Niemann-Pick type A disease (NPA) is a rare lysosomal storage disorder caused by mutations in the gene coding for the lysosomal enzyme acid sphingomyelinase (ASM). ASM deficiency leads to the consequent accumulation of its uncatabolized substrate, the sphingolipid sphingomyelin (SM), causing severe progressive brain disease. To study the effect of the aberrant lysosomal accumulation of SM on cell homeostasis, we loaded skin fibroblasts derived from a NPA patient with exogenous SM to mimic the levels of accumulation characteristic of the pathological neurons. In SM-loaded NPA fibroblasts, we found the blockage of the autophagy flux and the impairment of the mitochondrial compartment paralleled by the altered transcription of several genes, mainly belonging to the electron transport chain machinery and to the cholesterol biosynthesis pathway. In addition, SM loading induces the nuclear translocation of the transcription factor EB that promotes the lysosomal biogenesis and exocytosis. Interestingly, we obtained similar biochemical findings in the brain of the NPA mouse model lacking ASM (ASMKO mouse) at the neurodegenerative stage. Our work provides a new in vitro model to study NPA etiopathology and suggests the existence of a pathogenic lysosome-plasma membrane axis that with an impairment in the mitochondrial activity is responsible for the cell death.

Abstract 5130: Prognostic significance of loss of Cathepsin L expression in urinary bladder cancer progression

Abstract During 2021 there will be 83,730 new cases of urinary bladder cancer (UBC) in the US and 17,200 UBC deaths. Most UBC arise from the urothelial lining. UBC frequently recurs and is the most expensive cancer to treat due to the need for constant patient monitoring. New prognostic markers are needed to assist patient management. This study aims to determine if Cathepsin L is an independent prognostic factor after controlling for UBC Stage and Grade. We studied the interrelationship of Cathepsin L with Ki67, a prognostic marker in UBC. A clearer understanding of the biological mechanisms underlying UBC development and progression will lead to improved methods of UBC prognosis and suggest new therapies. Cathepsin L is a ubiquitously expressed cysteine endopeptidase capable of degrading enzymes, receptors, and transcription factors, and of activating enzymes, receptors, biologically active peptides, and pro-hormone processing by limited proteolysis. Subcellularly located in the endosomal lysosomal compartment, Cathepsin L is catalytically active at pH 3.0 - 6.5. This is a retrospective study of archived tissues and existing deidentified medical records and tumor registry information. Archived FFPE tissues were donated to Wood Hudson Cancer Research Laboratory by St. Elizabeth Healthcare (SEHC). The study was approved by the SEHC IRB. Tumors were from 41 women and 92 men. UBC were classified according to subtype, depth of invasion and grade by a Board Certified pathologist (LED). 36/62 (58.1%) of Stage 0 non-invasive UBC were Grade 1 or 2, and 26/62 (41.9%) were Grade 3 or 4. Similarly 39/90 papillary tumors (43.3%) were Grade 1 or 2 and 51/90 (36.7%) were Grade 3 or 4. In contrast 68/71 invasive UBC (Stages II, III and IV) were Grade 3 or 4. 39/90 papillary tumors were Grade 1 or 2. All non-papillary tumors were Grade 3 or 4. Ki 67 (clone MIB-1, Dako) and Cathepsin L (Abcam) expression were detected immunohistochemically. Cathepsin L was highly expressed in normal urothelium (p < 0.0001). In Stage 0, Cathepsin L expression in Grade 1,2,3 UBC was similar compared to expression in normal urothelium (p = 0.9070, 0.2560 and 0.0722). In Stage 0, Cathepsin L expression in Grade 4 UBC was significantly reduced compared to normal urothelium (p = 0.0011). Cathepsin L expression was significantly reduced in Grade 3 and 4 papillary tumors vs Grade 1 and 2 papillary tumors (p = 0.0002) and in Grade 3 and 4 non-papillary tumors vs Grade 3 and 4 papillary tumors (p = 0.0133). In each UBC subtype, grade or stage, when Cathepsin L expression was reduced cell proliferation as measured by Ki67 expression was increased and overall, this was significant (p < 0.0001). Significantly reduced Cathepsin L expression was associated with tumor recurrence and death from UBC (p = 0.03, p < 0.0001). These data suggest that when Cathepsin L is lost, intracellular levels of growth factor receptors such as EGFR may increase driving cell proliferation and UBC progression. Citation Format: Julia H. Carter, James A. Deddens, Michelle C. Robillard, Mariah K. Dooley, Daniel Stelzer, Zachary S. Taylor, Larry E. Douglass. Prognostic significance of loss of Cathepsin L expression in urinary bladder cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5130.

The alkalinizing, lysosomotropic agent ML-9 induces a pH-dependent depletion of ER Ca2+ stores in cellulo

ML-9 elicits a broad spectrum of effects in cells, including inhibition of myosin light chain kinase, inhibition of store-operated Ca2+ entry and lysosomotropic actions that result in prostate cancer cell death. Moreover, the compound also affects endoplasmic reticulum (ER) Ca2+ homeostasis, although the underlying mechanisms remain unclear. We found that ML-9 provokes a rapid mobilization of Ca2+ from ER independently of IP3Rs or TMBIM6/Bax Inhibitor-1, two ER Ca2+-leak channels. Moreover, in unidirectional 45Ca2+ fluxes in permeabilized cells, ML-9 was able to reduce ER Ca2+-store content. Although the ER Ca2+ store content was decreased, ML-9 did not directly inhibit SERCA's ATPase activity in vitro using microsomal preparations. Consistent with its chemical properties as a cell-permeable weak alkalinizing agent (calculated pKa of 8.04), ML-9 provoked a rapid increase in cytosolic pH preceding the Ca2+ efflux from the ER. Pre-treatment with the weak acid 3NPA blunted the ML-9-evoked increase in intracellular pH and subsequent ML-9-induced Ca2+ mobilization from the ER. This experiment underpins a causal link between ML-9's impact on the pH and Ca2+ dynamics. Overall, our work indicates that the lysosomotropic drug ML-9 may not only impact lysosomal compartments but also have severe impacts on ER Ca2+ handling in cellulo.

Antibiotic-Loaded Amphiphilic Chitosan Nanoparticles Target Macrophages and Kill an Intracellular Pathogen.

In this work, levofloxacin (LVX), a third-generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan-g-poly(methyl methacrylate) produced by self-assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non-crosslinked nanoparticles display a size of 29nm and a zeta-potential of +36mV, while the crosslinked counterparts display 45nm and +24mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH-S and the human bronchial epithelial cell line BEAS-2B in vitro. Internalization events are detected after 10min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX-loaded nanoformulations (50% w/w drug content) is assessed in MH-S and BEAS-2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.

MAPK15 protects from oxidative stress-dependent cellular senescence by inducing the mitophagic process.

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1‐dependent PRKN Ser108 phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15‐dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age‐associated human diseases.

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.

C9ORF72-derived poly-GA DPRs undergo endocytic uptake in iAstrocytes and spread to motor neurons.

Dipeptide repeat (DPR) proteins are aggregation-prone polypeptides encoded by the pathogenic GGGGCC repeat expansion in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic β-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

SHIP-2 inhibits human microglia-like cell function in a TREM2 independent manner

Abstract Microglia are a crucial component of the immune response in neurodegenerative diseases including Alzheimer’s Disease (AD). The Triggering Receptor Expressed on Myeloid Cells #2 (TREM2) mediates microglial phagocytosis and migration, and promotes metabolic homeostasis. Src homology domain-containing inositol 5-phosphatase 1 (SHIP-1) inhibits TREM2 in myeloid cells like macrophages and osteoclasts. SHIP-2, a paralog of SHIP-1, inhibits other myeloid cell receptors including Macrophage Colony-Stimulating Factor (M-CSF). We hypothesize that SHIP-2, inhibits TREM2-mediated microglial functions. To test our hypothesis, we studied the effect of pharmacological inhibition of SHIP-2 (and SHIP-1) in the function of wild-type (HMC3WT) and Trem2−/− human microglia-like cells (HMC3T2KO), which predominantly express SHIP-2 compared to SHIP-1. HMC3WT displayed superior phagocytic ability of oligomerized amyloid β and apoptotic neurons, have a larger lysosomal compartment, and have increased mitochondrial activity compared to HMC3T2KO. Treatment with a pan SHIP-1/2 inhibitor (SHIPi) K161 significantly improved the phagocytic uptake of fluorescently tagged oligomerized Aβ42 in both HMC3WT and HMC3T2KO. K161 treatment resulted in an enlarged lysosomal compartment and increased mitochondrial activity in both HMC3WT and HMC3T2KO. Our data suggests that, in cases of low SHIP-1 expression, pan SHIP inhibition improves microglia-like cell functions independently of TREM2. Further studies to look for SHIP2-TREM2 interactions and the effect of SHIP-1 expression in combination with SHIPi treatment in HMC3 cell function are underway. SHIPi are non-toxic in HCM3 cells and may offer potential treatment options for AD. Supported by grants from NIH (1RF1AG059717-01)

Microautophagy catabolizes lipid droplets in liver hepatocytes via the small GTPase Rab5 and is connected to alcoholic fatty liver

Introduction and ObjectiveAlcohol consumption has increased dramatically in recent years, representing a significant challenge to human health in metabolic disease. The leading cause of alcohol‐related death is alcohol‐associated liver disease (AALD), which is present at early stages (i.e. steatosis) in 90% of heavy drinkers. During early AALD, liver hepatocytes accumulate large stores of triglycerides in the form of lipid droplets (LDs). Under normal conditions, degradative lysosomes form direct contacts with LDs and catabolize lipid via microlipophagy, a specialized autophagic pathway utilizing the lipid‐degrading enzyme lysosomal acid lipase (LAL). However, the trafficking mechanisms that govern LD‐lysosome contacts during microlipophagy are poorly understood. Several promising candidates exist within the Rab family of small GTPases, which are well‐known to direct the trafficking of intracellular vesicles and LDs. Moreover, previous studies suggest that Rab proteins play central roles in LD catabolism. Thus, the OBJECTIVE of this study was to investigate novel Rab proteins that might be impaired by EtOH leading to altered LD dynamics and microautophagy.Materials and MethodsTo this end, we utilized RNAi gene silencing, subcellular fractionation, Western blot analysis, and high‐resolution microscopy to define Rabs that might target LDs to lysosomes during microlipophagy. To mimic alcoholic fatty liver, cellular and animal models of alcohol consumption were also used. Changes in LD‐lysosome interaction were measured by quantitative confocal microscopy following pharmacological inhibition of LAL by LAListat.ResultsFirst, confocal microscopy revealed that GFP‐Rab5, an early endosomal Rab GTPase, localizes to hepatocellular LDs consistent with proteomic and Western blot analyses showing abundant levels of Rab5 in biochemically isolated LDs. Importantly, siRNA knockdown of Rab5 in cultured hepatocytes showed substantial increases in total LD area (2‐fold) and LD number (1.6‐fold), and also blocked the catabolism of LDs in lipid‐loaded cells. Finally, Rab5 knockdown nearly completely abolished microlipophagy as measured by fluorescent LD accumulation in lysosomes following LAListat treatment. The dissociation of LDs from lysosomes in Rab5 knockdown cells was strikingly similar to the defect observed in EtOH‐fed hepatocytes. Together, these findings suggest that Rab5 is a key mediator of targeting LDs to the lysosome compartment for catabolism and may be a target of EtOH insult.Significance/ImplicationThis study sheds light on new molecular mechanisms of microlipophagy that are affected by chronic alcohol consumption. These mechanisms may represent promising avenues for future therapeutic development toward mitigating hepatic steatosis and slowing the progression of AALD.

Extralysosomal cathepsin B in central nervous system: Mechanisms and therapeutic implications.

Cathepsin B (CatB) is a typical cysteine lysosomal protease involved in a variety of physiologic and pathological processes. It is expressed in most cell types and is primarily localized within subcellular endosomal and lysosomal compartments. Emerging scientific evidence indicates that lysosomal leaked CatB is involved in mitochondrial stress, inflammasome activation, and nuclear senescence, but without the acidic environment. CatB is also secreted as a myokine, which is involved in muscle‐brain cross talk and neuronal dendritic remodeling. Lysosomal‐leaked and cellular‐secreted CatB functions are dependent on its enzymatic activity at a neutral pH. In the present review, we summarize the available experimental evidence that mechanistically links extralysosomal CatB to physiological and pathological functions in central nervous system, and their potential for use in therapeutic approaches.

Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

BackgroundDespite the incorporation of novel therapeutics, advanced triple negative breast cancer (TNBC) still represents a relevant clinical problem. Considering this, as well as the clinical efficacy of antibody-drug conjugates (ADCs), we aimed at identifying novel ADC targets that could be used to treat TNBC.MethodsTranscriptomic analyses were performed on TNBC and normal samples from three different studies. Plasma membrane proteins of three cell lines representative of the TNBC subtype were identified by cell surface biotinylation or plasma membrane isolation, followed by analyses of cell surface proteins using the Surfaceome online tool. Immunofluorescence and western studies were used to characterize the action of a CD98hc-directed ADC, which was prepared by in house coupling of emtansine to an antibody that recognized the ectodomain of CD98hc. Xenografted TNBC cells were used to analyze the antitumoral properties of the anti-CD98hc ADC.ResultsComparative genomic studies between normal breast and TNBC tissues, together with proteomic and bioinformatic analyses resulted in the elaboration of a catalog of potential ADC targets. One of them, the CD98hc transmembrane protein, was validated as an ADC target. An antibody recognizing the ectodomain of CD98hc efficiently internalized and reached the lysosomal compartment. An emtansine-based ADC derived from such antibody was prepared and showed antitumoral properties in TNBC in vitro and in vivo models. Mechanistically, the anti-CD98hc ADC blocked cell cycle progression, that was followed by cell death caused by mitotic catastrophe.ConclusionsThis work describes a list of potential ADC targets in TNBC and validates one of them, the transmembrane protein CD98hc. The studies presented here also demonstrate the robustness of the multiomic approach herewith described to identify novel potential ADC targets.

SPG15 protein deficits are at the crossroads between lysosomal abnormalities, altered lipid metabolism and synaptic dysfunction.

Hereditary spastic paraplegia type 15 (HSP15) is a neurodegenerative condition caused by the inability to produce SPG15 protein, which leads to lysosomal swelling. However, the link between lysosomal aberrations and neuronal death is poorly explored. To uncover the functional consequences of lysosomal aberrations in disease pathogenesis, we analyze human dermal fibroblasts from HSP15 patients as well as primary cortical neurons derived from an SPG15 knockout (KO) mouse model. We find that SPG15 protein loss induces defective anterograde transport, impaired neurite outgrowth, axonal swelling and reduced autophagic flux in association with the onset of lysosomal abnormalities. Additionally, we observe lipid accumulation within the lysosomal compartment, suggesting that distortions in cellular lipid homeostasis are intertwined with lysosomal alterations. We further demonstrate that SPG15 KO neurons exhibit synaptic dysfunction, accompanied by augmented vulnerability to glutamate-induced excitotoxicity. Overall, our study establishes an intimate link between lysosomal aberrations, lipid metabolism and electrophysiological impairments, suggesting that lysosomal defects are at the core of multiple neurodegenerative disease processes in HSP15.

Transcellular propagation of fibrillar \u03b1-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent

Parkinson’s disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

Internalization and trafficking of CSPG-bound recombinant VAR2CSA lectins in cancer cells

Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.

CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Silica Nanoparticle Internalization Improves Chemotactic Behaviour of Human Mesenchymal Stem Cells Acting on the SDF1α/CXCR4 Axis.

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo.

Improved Cathepsin Probes for Sensitive Molecular Imaging.

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.