INTRODUCTION:Microglia have been implicated in the templated spread of tau aggregates in tauopathies through mouse studies. However, it is unclear whether these findings translate to human disease. METHODS:We challenged human induced pluripotent stem cell (iPSC)-derived microglia-like-cells (iMGL) with monomeric and fibrillar recombinant tau and tau purified from Alzheimer's patient brains, examining in detail the uptake, processing, release, and seeding of tau by microglia. RESULTS:iMGL take up tau via lipoprotein receptor-related protein 1 (LRP)1 and heparan sulfate proteoglycans, with leucine-rich repeat kinase 2 affecting LRP1 trafficking. Monomeric tau is digested effectively with minimal effects on iMGL, but recombinant or brain-derived tau fibrils induce chemokine/interferon response subtypes, alongside downregulation of homeostatic genes. Fibrillar tau is degradation-resistant, can escape into the cytoplasm, and becomes phosphorylated on two specific residues. iMGL release partially digested fibrillar tau, including in extracellular vesicles, visualized by cryo-electron microscopy, that seed aggregation in neurons. DISCUSSION:Our study reveals new insights into human microglial responses to tau, highlighting opportunities to limit pathogenic tau spread.

Targeted delivery systems offer a promising approach for selectively modulating cellular processes; yet the intracellular consequences of targeted nutrient delivery to trophoblast cells remain poorly defined. Here, we investigated a previously validated placenta-targeting peptide conjugated to liposomes encapsulating stable isotope-labelled L-arginine and L-lysine to examine cellular uptake and downstream molecular responses in a trophoblast-like cell model. Peptide-dependent uptake of fluorescently labelled liposomes was confirmed in BeWo cells, demonstrating selective internalisation compared with non-targeted controls. Encapsulation of isotope-labelled amino acids enabled direct quantification of intracellular delivery and incorporation into the cellular proteome using stable isotope labelling by amino acids in cell culture (SILAC). Quantitative proteomic analysis revealed coordinated changes in proteins associated with translation, metabolism, and nitric oxide synthase regulation following targeted liposomal uptake. Notably, V-type proton ATPase subunit G1 (ATP6V1G1) and large neutral amino acid transporter small subunit 1 (SLC7A5) showed increased incorporation of labelled amino acids and were independently validated by Western blotting. Together, these findings establish a proof-of-concept platform for targeted intracellular amino acid delivery to trophoblast-like cells and define the resulting proteomic responses. This work provides mechanistic insight into intracellular amino acid utilisation and a framework for future studies in placental cell biology.

DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helps maintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discovered that lysosomes process Topoisomerase 1-cleavage complexes (TOP1cc) DNA lesions in human cancer cell. TOP1 resolves DNA topological stress ahead of DNA replication and transcription. If not promptly repaired, TOP1cc hinder the progression of DNA replication and transcription fostering genomic instability. Thus, stabilising TOP1cc with TOP1 poisons, such as Camptothecin or its analogue, is highly effective and widely used in cancer therapy. Proteolysis of TOP1cc by the proteasome is described at excessively high doses of CPT that are not relevant in a clinical setting, calling into question the relevance of the proteasome role in TOP1cc repair for cancer therapy and patient response. Here we described that repair of TOP1cc induced by clinically relevant dose of Camptothecin depends on selective autophagy. Proteomics, sequencing, biochemistry techniques and live imaging all demonstrate the lysosomal uptake of TOP1cc upon induction of replication stress. Selective degradation of TOP1cc by autophagy directs DNA damage repair and cell survival. TOP1cc are exported from the nucleus to lysosomes through transient alteration of the nuclear envelope and independent of the proteasome. Mechanistically, the autophagy receptor TEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by the p97 ATPase and mediating the delivery of TOP1cc to lysosomes in an MRE11 nuclease and ATR kinase-dependent manner. We found an evolutionarily conserved role for selective autophagy in DNA damage repair that enables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients. Pauline Lascaux, Gwendoline Hoslett, Sara Tribble, Ivan Antičević, Cecile Otten, Ignacio Torrecilla, Yichen Zhao, Wei Song, Cristiano Peron, Giulio Deangeli, Enric Domingo, James Bancroft, Loïc Carrique, Errin Johnson, Alvin Wei Tian Ng, Joanne Ngeow, Nuno Raimundo, Tim Maughan, Marta Popović, Ira Milošević, and Kristijan Ramadan. TEX264 DRIVES SELECTIVE AUTOPHAGY OF DNA LESIONS TO PROMOTE DNA REPAIR AND CELL SURVIVAL [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P11.

The templated spread of tau aggregates in tauopathies has been attributed to neuron-to- neuron spread, but microglia have also been implicated through mouse studies. Here we examine in detail the uptake, processing, release and seeding of tau using human iPS- derived microglia (iMGL). We show that tau is taken up by iMGL via LRP1 and heparan sulfate proteoglycans, with a role for LRRK2 in LRP1 trafficking, and that phagocytosed fibrils can escape into the cytoplasm. Monomeric tau has minimal effects on iMGL, but recombinant or brain-derived tau fibrils induce a shift towards chemokine and interferon response subtypes, alongside downregulation of homeostatic and MHC genes. Endogenous tau protein is undetectable in iMGL, and monomeric internalised tau is digested to completion, but fibrillar tau is more resistant to degradation and becomes phosphorylated on two specific residues. Finally, fibrillar tau is released by iMGL, visualized within extracellular vesicles by cryo-EM, and can seed tau aggregation in downstream neurons. ### Competing Interest Statement W.A.M. is an academic founder, shareholder, and scientific advisor for TRIMTECH Therapeutics. T.A.D. is an employee and minor shareholder of Eli Lilly and Company. A.H. was supported by a research grant to S.A.C. from Eli Lilly and Company.

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the dysferlin (DysF) domain on the Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo. The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that the ER-vacuole MCS requires a Pex30 C-terminal domain of unknown function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

Neisseria gonorrhoeae is a human-specific pathogen that causes the important sexually transmitted infection, gonorrhoea, an inflammatory condition of the genitourinary tract. The bacterium is closely related to the meningococcus, a leading cause of bacterial meningitis. Both these invasive bacterial species undergo autolysis when in the stationary phase of growth. Autolysis is a form of programmed cell death (PCD) which is part of the life cycle of remarkably few bacteria and poses an evolutionary conundrum as altruistic death provides no obvious benefit for single-celled organisms. Here, we searched for genes present in these 2 invasive species but not in other members of the Neisseria genus. We identified a ~3.4 kb horizontally acquired region, we termed the nap island, which is largely restricted to the gonococcus and meningococcus. The nap island in the gonococcus encodes 3 cationic, bacteriocin-like peptides which have no detectable antimicrobial activity. Instead, the gonococcal Neisseria autolysis peptides (Naps) promote autolytic cell death when bacteria enter the stationary phase of growth. Furthermore, strains lacking the Naps exhibit reduced autolysis in assays of PCD. Expression of Naps is likely to be phase variable, explaining how PCD could have arisen in these important human pathogens. NapC also induces lysis of human cells, so the peptides are likely to have multiple roles during colonisation and disease. The acquisition of the nap island contributed to the emergence of PCD in the gonococcus and meningococcus and potentially to the appearance of invasive disease in Neisseria spp.

DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helps maintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discovered that lysosomes process topoisomerase 1 cleavage complexes (TOP1cc) DNA lesions in vertebrates. Selective degradation of TOP1cc by autophagy directs DNA damage repair and cell survival at clinically relevant doses of topoisomerase 1 inhibitors. TOP1cc are exported from the nucleus to lysosomes through a transient alteration of the nuclear envelope and independent of the proteasome. Mechanistically, the autophagy receptor TEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by the p97 ATPase and mediating the delivery of TOP1cc to lysosomes in an MRE11-nuclease- and ATR-kinase-dependent manner. We found an evolutionarily conserved role for selective autophagy in DNA repair that enables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients.

Loss-of-function mutations in the TLDc family of proteins cause a range of severe childhood-onset neurological disorders with common clinical features that include cerebellar neurodegeneration, ataxia and epilepsy. Of these proteins, oxidation resistance 1 (OXR1) has been implicated in multiple cellular pathways related to antioxidant function, transcriptional regulation and cellular survival; yet how this relates to the specific neuropathological features in disease remains unclear. Here, we investigate a range of loss-of-function mouse model systems and reveal that constitutive deletion of Oxr1 leads to a rapid and striking neuroinflammatory response prior to neurodegeneration that is associated with lysosomal pathology. We go on to show that neuroinflammation and cell death in Oxr1 knockouts can be completely rescued by the neuronal expression of Oxr1, suggesting that the phenotype is driven by the cell-intrinsic defects of neuronal cells lacking the gene. Next, we generate a ubiquitous, adult inducible knockout of Oxr1 that surprisingly displays rapid-onset ataxia and cerebellar neurodegeneration, establishing for the first time that the distinctive pathology associated with the loss of Oxr1 occurs irrespective of developmental stage. Finally, we describe two new homozygous human pathogenic variants in OXR1 that cause neurodevelopmental delay, including a novel stop-gain mutation. We also compare functionally two missense human pathogenic mutations in OXR1, including one newly described here, that cause different clinical phenotypes but demonstrate partially retained neuroprotective activity against oxidative stress. Together, these data highlight the essential role of Oxr1 in modulating neuroinflammatory and lysosomal pathways in the mammalian brain and support the hypothesis that OXR1 protein dosage may be critical for pathological outcomes in disease.

Inorganic polyphosphate (polyP) is a ubiquitous polymer that controls fundamental processes. To overcome the absence of a genetically tractable mammalian model, we developed an inducible mammalian cell line expressing Escherichia coli polyphosphate kinase 1 (EcPPK1). Inducing EcPPK1 expression prompted polyP synthesis, enabling validation of polyP analytical methods. Virtually all newly synthesized polyP accumulates within the nucleus, mainly in the nucleolus. The channeled polyP within the nucleolus results in the redistribution of its markers, leading to altered rRNA processing. Ultrastructural analysis reveals electron-dense polyP structures associated with a hyper-condensed nucleolus resulting from an exacerbation of the liquid-liquid phase separation (LLPS) phenomena controlling this membraneless organelle. The selective accumulation of polyP in the nucleoli could be interpreted as an amplification of polyP channeling to where its physiological function takes place. Indeed, quantitative analysis of several mammalian cell lines confirms that endogenous polyP accumulates within the nucleolus.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons, which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS, and TDP-43 modulates several mitochondrial transcripts. In this study, we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons, linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected, which correlated with downregulation of the motor protein complex, DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport, which can be partially rescued by DCTN1 overexpression.

Journal Article Elemental Maps to Dye for: Energy Dispersive X-ray Spectrometry Facilitates a Better Understanding of the Contrast Mechanisms in Common Electron Microscopy Stains Get access Louise Hughes, Louise Hughes Oxford Instruments NanoAnalysis, High Wycombe, UK Corresponding author Louise.Hughes@oxinst.com Search for other works by this author on: Oxford Academic Google Scholar Pedro Machado, Pedro Machado Oxford Instruments NanoAnalysis, High Wycombe, UK Search for other works by this author on: Oxford Academic Google Scholar Errin Johnson Errin Johnson The William Dunn School of Pathology, The University of Oxford, Oxford, UK Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1185–1186, https://doi.org/10.1093/micmic/ozad067.610 Published: 22 July 2023

The chloroplast is the most prominent member of a diverse group of plant organelles called the plastids, and it is characterized by its vital role in photosynthesis.1,2,3 Most of the ∼3,000 different proteins in chloroplasts are synthesized in the cytosol in precursor (preprotein) form, each with a cleavable transit peptide.4,5,6,7,8 Preproteins are imported via translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively.9,10,11,12,13 Discovery of the chloroplast-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) demonstrated that the nucleocytosolic ubiquitin-proteasome system (UPS) targets the TOC apparatus to dynamically control protein import and chloroplast biogenesis in response to developmental and environmental cues. The relevant UPS pathway is termed chloroplast-associated protein degradation (CHLORAD).14,15,16 Two homologs of SP1 exist, SP1-like1 (SPL1) and SPL2, but their roles have remained obscure. Here, we show that SP1 is ubiquitous in the Viridiplantae and that SPL2 and SPL1 appeared early during the evolution of the Viridiplantae and land plants, respectively. Through genetic and biochemical analysis, we reveal that SPL1 functions as a negative regulator of SP1, potentially by interfering with its ability to catalyze ubiquitination. In contrast, SPL2, the more distantly related SP1 homolog, displays partial functional redundancy with SP1. Both SPL1 and SPL2 modify the extent of leaf senescence, like SP1, but do so in diametrically opposite ways. Thus, SPL1 and SPL2 are bona fide CHLORAD system components with negative and positive regulatory functions that allow for nuanced control of this vital proteolytic pathway.

During peptidoglycan recycling (PR) bacteria can recover extracellular fragments of peptidoglycan (PGN) liberated by peptidoglycan turnover (PT) during cell growth and division, and reuse them in cell wall biosynthesis or central carbon metabolism. In Gram-negative bacteria, PR has been well studied, and functions in the induction of resistance to certain classes of antibiotics, and in host-pathogen interaction. However, while Gram-negative cell envelope architecture allows for highly efficient PR, Gram-positive bacteria, which lack an outer cell membrane and are instead enclosed by a glycopolymer layer, can shed large quantities of PGN-derived material to the external environment during growth. Nonetheless, the occurrence of PR was recently demonstrated in several Gram-positive bacteria, including the Gram-positive bacterial pathogen Staphylococcus aureus , and its potential adaptive functions are largely unexplored. Given the known roles of PR in Gram-negative bacteria, and that Gram-positive bacteria include several important human pathogens, we asked what role PR may play during Gram-positive pathogen-host interaction. Usingthe model insect host Drosophila melanogaster, we demonstrate that S. aureus mutants impaired in extracellular PGN hydrolysis (Δ atl ) and PGN fragment uptake (Δ murP ) show differential virulence compared to their wild-type counterpart. This was linked to increased activation of the D. melanogaster Toll-cascade by spent supernatant from the Δ atl mutant. Thus, we propose that S. aureus , and potentially other Gram-positive bacteria, may use extracellular PGN degradation during PT to simultaneously process PGN fragments for recycling and for immune evasion, while recovery and/or metabolism of peptidoglycan fragments during PR may play more subtle roles in determining virulence. Author summary PGN is a key component of the bacterial cell wall, forming a stress-bearing sacculus surrounding the cell and providing cell shape. During growth and division, the sacculus is dynamically degraded and remodelled to ensure daughter cell separation, resulting in PT. PGN fragments released during PT can be recovered and reutilised by the cell during PR. In Gram-negative pathogens, PR is linked to antibiotic resistance, virulence and modulation of host immune recognition. In Gram-positive bacteria, PR was only recently observed. Here, we explore the roles of PT and PR in host-pathogen interaction in S. aureus, a Gram-positive pathogen of significant clinical relevance. Disruption of PT in S. aureus affected host-pathogen interaction through altering host recognition of shed PGN fragments and PR through modulation of PGN fragment recovery. This improves our understanding of the biology of this important pathogen and may aid development of novel therapeutic approaches to treat S. aureus infections.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte‐specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti‐inflammatory energy substrates. Instead, Atg7 ‐deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2‐mediated upregulation of Ephx1 . This shift reduced secretion of IL‐10 from adipose tissues, which was dependent on the cytochrome P450‐EPHX pathway, and lowered circulating levels of IL‐10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat‐gut crosstalk through an autophagy‐dependent regulation of anti‐inflammatory oxylipins via the cytochrome P450‐EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

ABSTRACT:Traumatic peripheral nerve injuries are at high risk of neuropathic pain for which novel effective therapies are urgently needed. Preclinical models of neuropathic pain typically involve irreversible ligation and/or nerve transection (neurotmesis). However, translation of findings to the clinic has so far been unsuccessful, raising questions on injury model validity and clinically relevance. Traumatic nerve injuries seen in the clinic commonly result in axonotmesis (ie, crush), yet the neuropathic phenotype of "painful" nerve crush injuries remains poorly understood. We report the neuropathology and sensory symptoms of a focal nerve crush injury using custom-modified hemostats resulting in either complete ("full") or incomplete ("partial") axonotmesis in adult mice. Assays of thermal and mechanically evoked pain-like behavior were paralleled by transmission electron microscopy, immunohistochemistry, and anatomical tracing of the peripheral nerve. In both crush models, motor function was equally affected early after injury; by contrast, partial crush of the nerve resulted in the early return of pinprick sensitivity, followed by a transient thermal and chronic tactile hypersensitivity of the affected hind paw, which was not observed after a full crush injury. The partially crushed nerve was characterized by the sparing of small-diameter myelinated axons and intraepidermal nerve fibers, fewer dorsal root ganglia expressing the injury marker activating transcription factor 3, and lower serum levels of neurofilament light chain. By day 30, axons showed signs of reduced myelin thickness. In summary, the escape of small-diameter axons from Wallerian degeneration is likely a determinant of chronic pain pathophysiology distinct from the general response to complete nerve injury.

Volume electron microscopy (vEM) is a group of techniques that reveal the 3D ultrastructure of cells and tissues through continuous depths of at least 1 micrometer. A burgeoning grassroots community effort is fast building the profile and revealing the impact of vEM technology in the life sciences and clinical research.

Aims Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. Methods and results Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Conclusions Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Aims Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study. Methods and results Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial (RA) appendage and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy. All RA- and LV-IMCAs from organ donors and pigs developed circa 25% MT. In contrast, 44% of human RA-IMCAs from 88 patients with heart valve disease had poor (<10%) MT yet retained cell viability and an ability to raise cytoplasmic Ca2+ in response to vasoconstrictor agents. Comparing across human heart chambers and species, we found that based on patient medical history and six tests, the strongest predictor of poor MT in IMCAs was increased expression of the synthetic marker caldesmon relative to the contractile marker SM-myosin heavy chain. In addition, high resolution imaging revealed a distinct layer of longitudinally aligned SMCs between ECs and radial SMCs, and we show poor MT was associated with disruptions in these cellular alignments. Conclusion These data demonstrate the first use of atrial and ventricular biopsies from patients and pigs to reveal that impaired coronary MT reflects a switch of viable SMCs towards a synthetic phenotype, rather than a loss of SMC viability. These arteries represent a model for further studies of coronary microvascular contractile dysfunction.

Heavy metal staining of biological material remains an important topic for electron microscopists, particularly with the rise of three-dimensional (3D) scanning electron microscopy (SEM) techniques and the ultrastructural study of large tissue volumes, which are often not compatible with cryo-imaging methods. There are a huge variety of stains that have been used to generate contrast in biological samples and many publications have gone into detail about how to apply these stains and what structures they bind or have affinity to in different sample types.