Endolysosomal Compartments Research Articles

Pharmacological inhibition reveals participation of the endocytic compartment in positive feedback IL-6 secretion in human skeletal myotubes

IL-6 is an important cytokine involved in metabolic, immunological, and cell-fate responses. It is released upon stimulation by skeletal muscle cells through partially characterized mechanisms. In some cell types, IL-6 has been reported to activate a positive feedback loop involving endocytic vesicles, but evidence is mostly based on transcription and signal transduction mechanisms and is very scarce in muscle cells. Our aim was to directly demonstrate the presence of positive feedback in the ATP-induced release of IL-6 into the supernatant of human skeletal muscle cultures. The total release (production) of IL-6 was reduced for higher volumes of supernatant, when the secreted IL-6 molecules are more diluted, and enhanced when the supernatant volume was lower. In addition, secretion was impaired both by tocilizumab, a blocker of human IL-6 receptors, and by the soluble form of the receptor. The secretion in response to ATP was also inhibited by treatment with the endocytosis inhibitor dynasore, and by disruption of the acidic gradient of the endocytic compartment using different methods (chloroquine, NH4Cl or monensin). IL-6 secretion was also impaired by NED-19, a specific inhibitor of the two pore channels receptor mediating Ca2+ release from the endolysosomal compartment. IL-6 and ATP increased IL-6 mRNA levels, an effect blocked by tocilizumab. Altogether, our results demonstrate that ATP-secreted IL-6 activates a positive loop based on IL-6 receptors, endocytosis, two pore channels and IL-6 transcription. Given the importance of muscle IL-6 as a systemic regulator and as an inflammatory mediator, our study can help to understand muscle pathophysiology.

Cryosectioning and immunofluorescence of C. elegans reveals endogenous polyphosphate in intestinal endo-lysosomal organelles

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C.elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment.

Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood-brain barrier integrity

The neurovascular unit (NVU) is a complex multicellular structure that helps maintain cerebral homeostasis and blood-brain barrier (BBB) integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration, the underlying molecular mechanisms remain unclear. Here, we use zebrafish embryos carrying a mutation in Scavenger Receptor B2, a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells (RGCs), to investigate the interplay among different NVU components. Through live imaging and genetic manipulations, we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling, thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling, leading to severe vascular defects, hemorrhages, and a leaky BBB. Altogether, our findings provide insights into NVU formation and function and offer avenues for investigating diseases involving white matter defects and vascular abnormalities.

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

High‐Load Core@Shell Nanocarriers with Irinotecan and 5‐Fluorouracil for Combination Chemotherapy in Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer type and second leading cause of cancer‐related deaths worldwide, requiring novel drug‐delivery concepts. ITC@ZrO(TocP)/ZrO(FdUMP) core@shell nanocarriers (designated ITC‐FdUMP‐NC) with the clinically relevant chemotherapeutics irinotecan (ITC) and fluoro‐2′‐deoxyuridine‐5′‐phosphate (FdUMP) (active derivative of 5′‐fluorouracil/5‐FU) are a new type of nanocarrier with high drug payload (22 wt% of lipophilic ITC: particle core; 10 wt% of hydrophilic FdUMP: particle shell). The nanocarriers are tested in different CRC cell lines, a normal cell line, and rectal cancer patient‐derived organoids (PDOs). Fluorescence‐labeled nanocarriers show efficient uptake by all CRC cells and allow to distinctly track the intracellular trafficking toward endolysosomal compartments. Although free chemotherapeutic drugs exhibit a greater potency in 2D cell cultures, ITC‐FdUMP‐NC demonstrate equivalent cytotoxic efficacies as the freely dissolved drugs in the more complex 3D rectal cancer PDOs. The sustained drug‐release profile of the nanocarriers contrasts favorably with conventional free drugs, potentially enhancing the therapeutic outcome in vivo. With a chemotherapeutic cocktail comparable to the clinically applied FOLFIRI (ITC + 5‐FU), the ITC‐FdUMP‐NC represent a novel type of nanocarrier with high anti‐tumor effect and high drug payload, offering a promising strategy to circumvent chemoresistance and to improve therapy efficacy in vivo with less side effects.

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

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

Evaluation of neuroretina following i.v. or intra-CSF AAV9 gene replacement in mice with MPS IIIA, a childhood dementia.

Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is a childhood dementia caused by inherited mutations in the sulfamidase gene. At present, there is no treatment and children with classical disease generally die in their late teens. Intravenous or intra-cerebrospinal fluid (CSF) injection of AAV9-gene replacement is being examined in human clinical trials; evaluation of the impact on brain disease is an intense focus; however, MPS IIIA patients also experience profound, progressive photoreceptor loss, leading to night blindness. To compare the relative efficacy of the two therapeutic approaches on retinal degeneration in MPS IIIA mice. Neonatal mice received i.v. or intra-CSF AAV9-sulfamidase or vehicle and after 20 weeks, biochemical and histological evaluation of neuroretina integrity was carried out. Both treatments improved central retinal thickness; however, in peripheral retina, outer nuclear layer thickness and photoreceptor cell length were only significantly improved by i.v. gene replacement. Further, normalization of endo-lysosomal compartment size and microglial morphology was only observed following intravenous gene delivery. Confirmatory studies are needed in adult mice; however, these data indicate that i.v. AAV9-sulfamidase infusion leads to superior outcomes in neuroretina, and cerebrospinal fluid-delivered AAV9 may need to be supplemented with another therapeutic approach for optimal patient quality of life.

Bis-benzylisoquinoline alkaloids inhibit African swine fever virus internalization and replication by impairing late endosomal/lysosomal function.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly infectious disease afflicting domestic pigs and wild boars. It exhibits an alarming acute infection fatality rate of up to 100%. Regrettably, no commercial vaccines or specific drugs for combating this disease are currently available. This study evaluated the anti-ASFV activities in porcine alveolar macrophages, 3D4/21 cells, and PK-15 cells of four bis-benzylisoquinoline alkaloids (BBAs): cepharanthine (CEP), tetrandrine, fangchinoline, and iso-tetrandrine. Furthermore, we demonstrated that CEP, which exhibited the highest selectivity index (SI = 81.31), alkalized late endosomes/lysosomes, hindered ASFV endosomal transport, disrupted virus uncoating signals, and thereby inhibited ASFV internalization. Additionally, CEP disrupted ASFV DNA synthesis, leading to the inhibition of viral replication. Moreover, berbamine was labeled with NBD to synthesize a fluorescent probe to study the cellular location of these BBAs. By co-staining with Lyso-Tracker and lysosome-associated membrane protein 1, we demonstrated that BBAs target the endolysosomal compartments for the first time. Our data together indicated that BBAs are a class of natural products with significant inhibitory effects against ASFV infection. These findings suggest their potential efficacy as agents for the prevention and control of ASF, offering valuable references for the identification of potential drug targets.IMPORTANCEThe urgency and severity of African swine fever (ASF) underscore the critical need for effective interventions against this highly infectious disease, which poses a grave threat to domestic pigs and wild boars. Our study reveals the potent anti-African swine fever virus (ASFV) efficacy of bis-benzylisoquinoline alkaloids (BBAs), particularly evident in the absence of progeny virus production under a 5 µM concentration treatment. The structural similarity among cepharanthine, tetrandrine, fangchinoline, and iso-tetrandrine, coupled with their analogous inhibitory stages and comparable selectivity indexes, strongly suggests a shared antiviral mechanism within this drug category. Further investigation revealed that BBAs localize to lysosomes and inhibit the internalization and replication of ASFV by disrupting the endosomal/lysosomal function. These collective results have profound implications for ASF prevention and control, suggesting the potential of the investigated agents as prophylactic and therapeutic measures. Furthermore, our study offers crucial insights into identifying drug targets and laying the groundwork for innovative interventions.

Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling.

Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

Cutting Edge: ATP13A2 Is an Endolysosomal Regulator of TLR9/7 Activation in Human Plasmacytoid Dendritic Cells.

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.

Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

PROPPINs and membrane fission in the endo-lysosomal system.

PROPPINs constitute a conserved protein family with multiple members being expressed in many eukaryotes. PROPPINs have mainly been investigated for their role in autophagy, where they co-operate with several core factors for autophagosome formation. Recently, novel functions of these proteins on endo-lysosomal compartments have emerged. PROPPINs support the division of these organelles and the formation of tubulo-vesicular cargo carriers that mediate protein exit from them, such as those generated by the Retromer coat. In both cases, PROPPINs provide membrane fission activity. Integrating information from yeast and human cells this review summarizes the most important molecular features that allow these proteins to facilitate membrane fission and thus provide a critical element to endo-lysosomal protein traffic.

Size- and Shape-controlled Biodegradable Polymer Brushes Based on l-Amino Acid for Intracellular Drug Delivery and Deep-Tissue Penetration.

We report size- and shape-controlled polymer brushes based on l-amino acid bioresource and study the role of polymer topology on the enzymatic biodegradation and deep-tissue penetration under in vitro and in vivo. For this purpose, l-tyrosine-based propargyl-functionalized monomer is tailor-made and polymerized via solvent-free melt polycondensation strategy to yield hydrophobic and clickable biodegradable poly(ester-urethane)s. Postpolymerization click chemistry strategy is applied to make well-defined amphiphilic one-dimensional rodlike and three-dimensional spherical polymer brushes by merely varying the lengths of PEG-azides in the reaction. These core-shell polymer brushes are found to be nontoxic and nonhemolytic and capable of loading clinical anticancer drug doxorubicin and deep-tissue penetrable near-infrared biomarker IR-780. In vitro enzymatic drug-release kinetics and lysotracker-assisted real-time live-cell confocal bioimaging revealed that the rodlike polymer brush is superior than its spherical counterparts for faster cellular uptake and enzymatic biodegradation at the endolysosomal compartments to release DOX at the nucleus. Further, in vivo live-animal bioimaging by IVIS technique established that the IR-780-loaded rodlike polymer brush exhibited efficient deep-tissue penetration ability and emphasized the importance of polymer brush topology control for biological activity. Polymer brushes exhibit good stability in the blood plasma for more than 72 h, they predominately accumulate in the digestive organs like liver and kidney, and they are less toxic to heart and brain tissues. IVIS imaging of cryotome tissue slices of organs confirmed the deep-penetrating ability of the polymer brushes. The present investigation opens opportunity for bioderived and biodegradable polymer brushes as next-generation smart drug-delivery scaffolds.

Zwitterionic Strategy to Stabilize Self-Immolative Polymer Nanoarchitecture under Physiological pH for Drug Delivery In Vitro and In Vivo.

The major bottleneck in using polymer nanovectors for biomedical application, particularly those based on self-immolative poly(amino ester) (PAE), lies in their uncontrolled autodegradation at physiological pH before they can reach the intended target. Here, an elegant triblock-copolymer strategy is designed to stabilize the unstable PAE chains via zwitterionic interactions under physiological pH (pH 7.4) and precisely program their enzyme-responsive biodegradation specifically within the intracellular compartments, ensuring targeted delivery of the cargoes. To achieve this goal, biodegradable polycaprolactone (PCL) platform is chosen, and structure-engineered several di- and triblock architectures to arrive the precise macromolecular geometry. The hydrophobic-PCL core and hydrophilic anionic-PCL block at the periphery shield PAEs against autodegradation, thereby ensuring stability under physiological pH in PBS, FBS, cell culture medium and bloodstream. The clinical anticancer drug doxorubicin and deep-tissue penetrable near-infrared IR-780 biomarker is encapsulated to study their biological actions by in vitro live cancer cells and in vivo bioimaging in live animals. These zwitterions are biocompatible, nonhemolytic, and real-time in vitro live-cell confocal studies have confirmed their internalization and enzymatic biodegradation in the endo-lysosomal compartments to deliver the payload. In vivo bioimaging establishes their prolonged blood circulation for over 72h, and the biodistribution analysis reveals the accumulation of nanoparticles predominantly in the excretory organs.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

Abstract 2605: STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells

Abstract Dipeptidyl Peptidase 4 (DPP4/CD26) is a membrane glycoprotein involved in particular chemotactic and inflammatory responses, via its N-terminal protein cleavage enzymatic activities. DPP4 expression is found in various cell types, in certain subsets of lymphocytes and frequently overexpressed in primary tumors and metastases where its role remains still poorly understood. Recently, DPP4 was identified as a novel biomarker of senescent cells. In cancers, the harmful effect of senescent tumor cells is well recognized and mediated by the local production of pro-inflammatory cytokines, soluble immunosuppressive factors and metalloproteinases, called ‘SASP’ phenotype which could be induced by many chemotherapy and radiation regimens. This deleterious microenvironment promotes tumor growth, invasion and neoangiogenesis which can lead to treatment resistance, relapses and poor prognosis in many cancers, especially at advanced stages. STX-1 is a leading first-in-class ADC platform based on a proprietary anti-DPP4 mAb generated after a screen between normal and senescence-induced cells identifying DPP4 as a candidate for cancer targeting. Other biomarkers of interest were also identified as being selectively expressed for further preclinical evaluation. This IgG1/k mAb has potent in vitro activities on DPP4+ cells: i) in a bleomycin-induced senescent fibroblasts model; ii) on DPP4-expressing and chemotherapy- or radiotherapy-induced senescent human tumor cell lines with overexpression of DPP4; iii) binding of the mAb to its target results in its internalization into the endolysosomal compartment of cells. The first formulations of STX-1 are undergoing preclinical evaluation in vitro and in vivo and consist of a classical maleimide-based conjugation, an enzymatically cleavable cathepsin-dipeptide linker comprising a self-immolative spacer and either inhibitor of tubulin (MMAE/DAR4) or topoisomerase I inhibitor (exatecan/DAR8) payloads. Plasma stability and affinity, dose-response potency and permeability bystander activity on human tumor cell lines were evaluated in vitro. Preliminary efficacy is demonstrated in vivo on tumor xenograft models with various DPP4 expressions (constitutive or chemo/radio-induced to senescence) based on a 21-28 days weekly infusion schedule (3 or 5 µg/kg). The indications selected for further therapeutic development are prostate, liver and colorectal carcinoma models. Data on antitumor efficacy, tolerability and pharmacokinetics will be presented. Further studies will include optimization of the best STX-1 formulation, NHP studies and GMP scale-up with planned IND submission expected in Q3-2024. Exploiting the targeting of senescent cells in cancer based on selected biomarkers such as DPP4 constitutes a new therapeutic avenue to counteract tumor dormancy, recurrences and resistance to conventional chemo/radiotherapies. Citation Format: Eric Angevin, Virginie Lelarge, Remi Capelle, Justine Choeur, Thierry Mathieu, Benjamin Le Calvé. STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2605.

Near-infrared light-activated smart nanogels for remotely controlled cytochrome c release and photodynamic therapy

The utilization of reactive oxygen species (ROS) generated through photodynamic therapy (PDT) for remote-controlled release of protein drugs at specific sites using near-infrared light represents a groundbreaking development in protein delivery system innovation. To illustrate this principle, we engineered near-infrared light-activated smart nanogels (NASNs) by chemically cross-linking ROS-responsive linkages within hyaluronic acid-graft-methoxy poly(ethylene glycol)-diethylenetriamine-grafted-chlorine e6 (Ce6) polymer (HA-PEG-Dien-g-Ce6) and carboxylated thioketal (TK) serving as a cross-linker to enable PDT-driven cytochrome c (CC) liberation. The NASNs demonstrated substantial loading capacity and exceptional stability across diverse biological settings, attributed to the robust nature of the TK bond. Notably, CC-loaded NASNs effectively penetrated A549 cells via CD44-mediated endocytosis and rapidly escaped from the endo-lysosomal compartment. Upon light activation, the Ce6 produced ROS, leading to TK bond disruption in NASNs, and consequent release of encapsulated CC. This precise, photo-induced PDT mechanism and CC liberation markedly enhanced antitumor efficacy while maintaining minimal toxicity, establishing a solid foundation for remote, as-needed protein delivery systems and potential integration with additional therapeutic modalities.

The Cl- transporter ClC-7 is essential for phagocytic clearance by microglia.

Microglia, professional phagocytic cells of the brain, rely upon the appropriate activation of lysosomes to execute their immune and clearance functions. Lysosomal activity is, in turn, modulated by a complex network of over 200 membrane and accessory proteins that relay extracellular cues to these key degradation centers. The ClC-7 chloride (Cl-)-proton (H+) antiporter (also known as CLCN7) is localized to the endolysosomal compartments and mutations in CLCN7 lead to osteopetrosis and neurodegeneration. Although the functions of ClC-7 have been extensively investigated in osteoclasts and neurons, its role in microglia in vivo remains largely unexamined. Here, we show that microglia and embryonic macrophages in zebrafish clcn7 mutants cannot effectively process extracellular debris in the form of apoptotic cells and β-amyloid. Despite these functional defects, microglia develop normally in clcn7 mutants and display normal expression of endosomal and lysosomal markers. We also find that mutants for ostm1, which encodes the β-subunit of ClC-7, have a phenotype that is strikingly similar to that of clcn7 mutants. Together, our observations uncover a previously unappreciated role of ClC-7 in microglia and contribute to the understanding of the neurodegenerative phenotypes that accompany mutations in this channel.

The Contributions of the Endolysosomal Compartment and Autophagy to APOEɛ4 Allele-Mediated Increase in Alzheimer's Disease Risk.

Apolipoprotein E4 (APOE4), although yet-to-be fully understood, increases the risk and lowers the age of onset of Alzheimer's disease (AD), which is the major cause of dementia among elderly individuals. The endosome-lysosome and autophagy pathways, which are necessary for homeostasis in both neurons and glia, are dysregulated even in early AD. Nonetheless, the contributory roles of these pathways to developing AD-related pathologies in APOE4 individuals and models are unclear. Therefore, this review summarizes the dysregulations in the endosome-lysosome and autophagy pathways in APOE4 individuals and non-human models, and how these anomalies contribute to developing AD-relevant pathologies. The available literature suggests that APOE4 causes endosomal enlargement, increases endosomal acidification, impairs endosomal recycling, and downregulates exosome production. APOE4 impairs autophagy initiation and inhibits basal autophagy and autophagy flux. APOE4 promotes lysosome formation and trafficking and causes ApoE to accumulate in lysosomes. APOE4-mediated changes in the endosome, autophagosome and lysosome could promote AD-related features including Aβ accumulation, tau hyperphosphorylation, glial dysfunction, lipid dyshomeostasis, and synaptic defects. ApoE4 protein could mediate APOE4-mediated endosome-lysosome-autophagy changes. ApoE4 impairs vesicle recycling and endosome trafficking, impairs the synthesis of autophagy genes, resists being dissociated from its receptors and degradation, and forms a stable folding intermediate that could disrupt lysosome structure. Drugs such as molecular correctors that target ApoE4 molecular structure and enhance autophagy may ameliorate the endosome-lysosome-autophagy-mediated increase in AD risk in APOE4 individuals.

Control of the Assembly and Disassembly of Spherical Nucleic Acids Is Critical for Enhanced Gene Silencing.

Spherical nucleic acids─nanospheres with nucleic acids on their corona─have emerged as a promising class of nanocarriers, aiming to address the shortcomings of traditional nucleic therapeutics, namely, their poor stability, biodistribution, and cellular entry. By conjugating hydrophobic monomers to a growing nucleic acid strand in a sequence-defined manner, our group has developed self-assembled spherical nucleic acids (SaSNAs), for unaided, enhanced gene silencing. By virtue of their self-assembled nature, SaSNAs can disassemble under certain conditions in contrast to covalent or gold nanoparticle SNAs. Gene silencing involves multiple steps including cellular uptake, endosomal escape, and therapeutic cargo release. Whether assembly vs disassembly is advantageous to any of these steps has not been previously studied. In this work, we modify the DNA and hydrophobic portions of SaSNAs and examine their effects on stability, cellular uptake, and gene silencing. When the linkages between the hydrophobic units are changed from phosphate to phosphorothioate, we find that the SaSNAs disassemble better in endosomal conditions and exhibit more efficacious silencing, despite having cellular uptake similar to that of their phosphate counterparts. Thus, disassembly in the endolysosomal compartments is advantageous, facilitating the release of the nucleic acid cargo and the interactions between the hydrophobic units and endosomal lipids. We also find that SaSNAs partially disassemble in serum to bind albumin; the disassembled, albumin-bound strands are less efficient at cellular uptake and gene silencing than their assembled counterparts, which can engage scavenger receptors for internalization. When the DNA portion is cross-linked by G-quadruplex formation, disassembly decreases and cellular uptake significantly increases. However, this does not translate to greater gene silencing, again illustrating the need for disassembly of the SaSNAs when they are in the endosome. This work showcases the advantages of the dual nature of SaSNAs for gene silencing, requiring extracellular assembly and disassembly inside the cell compartments.