Impact of BECLIN1 haploinsufficiency on goblet cell function and susceptibility to colitis

Impact of BECLIN1 haploinsufficiency on goblet cell function and susceptibility to colitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impact of BECLIN1 haploinsufficiency on goblet cell function and susceptibility to colitis Erinna Lee, Juliani Juliani, Sharon Tran, Tiffany Harris, Sarah Ellis, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7567745/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract BECLIN1 is a central regulator of autophagy and endocytic trafficking essential for epithelial homeostasis. While complete intestinal epithelial loss of BECLIN1 causes fatal enteritis, the consequence of its partial loss in the gut remains unclear. Given that BECLIN1 expression can vary in human disease, we investigated whether reduced BECLIN1 is sufficient to impair gut barrier function. Heterozygous Becn1 deletion ( Becn1 +/− ) in the mouse intestinal epithelium caused subtle but functionally important defects, including shortened small intestines, reduced colonic crypt length, altered epithelial architecture, and loss of goblet cells with reduced mucin production, particularly in mature goblet cells. These changes occurred despite preservation of basal autophagy, implicating trafficking-related functions. Supporting this conclusion, Becn1 +/− intestinal epithelial cells showed modest increases in RAB5 + ve vesicles, redistribution of E-CADHERIN with F-actin along lateral membranes, increased apico-basal cell length and reduced basal width. Following dextran sulfate sodium (DSS) treatment, Becn1 +/− mice exhibited greater weight loss, higher disease activity, more severe histological colitis score, and disproportionate loss of neutral mucins, with inflammation confined to the mucosa. Goblet cell dysfunction likely underpinned these barrier defects. These findings establish that BECLIN1 insufficiency destabilises epithelial organisation and barrier defence, thereby sensitising the gut to inflammatory challenge and further positioning BECLIN1 as a key determinant of intestinal homeostasis. Biological sciences/Cell biology/Autophagy/Macroautophagy Health sciences/Medical research/Preclinical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Genome-wide association studies have identified polymorphisms in autophagy regulators, such as ATG16L1 and IRGM , as strongly associated with inflammatory bowel disease (IBD) ( 1 – 7 ). These findings highlight the importance of autophagy in intestinal homeostasis and implicate its disruption in IBD pathogenesis. Functional studies in mouse and cell-based models have revealed both epithelial-intrinsic roles, including maintenance of barrier integrity and cell survival ( 2 , 8 – 16 ), and immune-mediated extrinsic roles, such as regulating inflammatory responses and microbial balance ( 17 – 21 ). Recent studies have shown that the prototypical autophagy regulator BECLIN1 is critical for intestinal homeostasis. Constitutive activation of BECLIN1-dependent autophagy alleviates goblet cell ER stress, thickening the mucus barrier and protecting against chemical- and infection-driven inflammation ( 11 ). Beyond autophagy, BECLIN1 regulates endocytic trafficking, maintaining barrier integrity via correct junctional protein localisation, cytoskeletal organisation, and epithelial remodelling ( 22 – 24 ). Complete intestinal epithelial deletion of BECLIN1 is rapidly fatal due to severe enterocolitis with extensive epithelial cell loss, impaired specialised IEC functions, inflammation, and barrier breakdown ( 23 ). BECLIN1 undergoes extensive post-translational modifications that fine-tune its activity and interactions ( 25 – 28 ). In diseases, however, it is reduced BECLIN1 expression rather than mutation that contributes to pathogenesis ( 29 – 31 ), with changes arising from altered protein interactions, cleavage, epigenetic regulation, or monoallelic deletion ( 32 – 35 ). Despite this, the consequences of partial BECLIN1 loss in the intestinal epithelium remain poorly understood. Here, we demonstrate that partial loss of BECLIN1 impairs goblet cell function and increases susceptibility to inflammation. By dissecting its roles in endocytic trafficking, cytoskeletal organisation and mucin secretion, we provide new insights into how BECLIN1 insufficiency destabilises epithelial integrity and compromises barrier defence. MATERIALS AND METHODS Mice All mouse strains used in this study were bred on the C57BL/6 J background. Becn1 tm1b(KOMP)Wtsi mice were purchased from the European Conditional Mouse Mutagenesis Program (EUCOMM). Becn1 fl/fl mice were generated by breeding Becn1tm1b(KOMP)Wtsi mice onto CAG-FLPe mice. Becn1 +/+ ;, Becn1 +/fl ;, and Becn1 fl/fl ; Vil1-CreERT2 Cre/+ mice were then subsequently generated by breeding Becn1 +/+ , Becn1 fl/+ and Becn1 fl/fl mice to the Vil1-CreERT2 mice ( 36 ). Mice were housed at the La Trobe Animal Research and Teaching Facility (LARTF, La Trobe University, VIC, Australia) under Specific Pathogen Free (SPF) conditions. All experiments performed were approved by the La Trobe University animal ethics committees (approvals AEC18024, AEC18036) in accordance with the Australian code for the care and use of animals for scientific purposes. All research with these mice has complied with all relevant ethical regulations for animal use. To induce gene deletion, male and female mice aged six weeks or older, were selected indiscriminately and intraperitoneally injected with 4 mg tamoxifen (T5648, Sigma-Aldrich, St. Louis, Missouri, USA) in sunflower seed oil (25007, Sigma-Aldrich, St. Louis, Missouri, USA), delivered as one 200 µl injection per day of a 10 mg/ml stock, over two consecutive days. Mice were humanely euthanised by CO 2 asphyxiation. Dextran Sulphate Sodium (DSS) treatment Following Tamoxifen treatment to induce gene deletion, mice were administered DSS (2% (w/v), MP Biomedicals, Irvine, California, USA) in autoclaved drinking water provided ad libitum for 5 days. Control groups received autoclaved drinking water without DSS for the same duration. During the experimental period, mice were monitored daily and weighed. The average daily intake of 2% (w/v) DSS water was calculated by measuring the weight difference of drinking bottles at the start and end of the experiment and dividing by the number of treatment days. The disease activity index (DAI) was assessed using a cumulative scoring system based on parameters outlined in Table 1 , including weight loss, stool consistency and the presence of hematochezia (presence of blood in stool). At the experimental endpoint, mice were humanely euthanised using CO 2 asphyxiation. The small and large intestine were harvested, and their lengths were measured prior to rolling and fixation in 10% (v/v) neutral buffered formalin for 48 hours. Table 1 Scoring system for the evaluation of colitis severity in mice based on weight loss, stool consistency and haematochezia parameters. Score Weight loss (%) Stool consistency Haematochezia 0 Normal Normal No blood 1 20 Liquid, sticky or unable to defecate after 5 min Intestinal organoid culture Organoids were established by culturing crypt-enriched fractions from the duodenum of untreated mice as described previously ( 23 ). To induce Becn1 deletion, organoids were seeded into media containing 200 nM 4-hydroxytamoxifen (4-HT) (H7904, Sigma-Aldrich, St. Louis, Missouri, USA) for three days and then maintained as per normal. For organoid re-passaging experiments, 4-HT-treated organoids were passaged at day 7 and maintained as above. Intestinal epithelial cell isolation The mouse small intestine was opened longitudinally, rinsed with Dulbecco’s Phosphate Buffered Saline (dPBS), and incubated in dPBS + 15 mM EDTA for 15 minutes at 37°C with agitation. Dissociation of IECs was achieved by vortexing. The cell suspension was then washed once with ice-cold dPBS, and the cell pellet snap-frozen on dry ice and stored at − 80°C until use. Histology, immunohistochemistry and organoid whole-mount immunofluorescence Preparation of intestinal “Swiss-rolls”, haematoxylin and eosin (H&E), Periodic acid-Schiff, Alcian blue (PAS-AB) and Ki67 staining were all performed as described previously ( 23 ). All slides were scanned using the Aperio AT2 (Leica, Wetzlar, Germany) and images captured and analysed using Aperio ImageScope v12.4.2.5010 and Fiji ImageJ. Whole-mount staining of intestinal organoids was performed as previously described ( 22 ). Immunoblotting of mouse tissues Preparation of mouse cell lysates and immunoblotting was performed as described previously ( 23 ). Primary antibodies were: Beclin1, 1:500 (3495, Cell Signaling Technology, Danvers, Massachusetts, USA); p62/SQSTM1, 1:500 (5114, Cell Signaling Technology, Danvers, Massachusetts, USA); LC3B, 1:500 (NB100-2220, Novus Biologicals, Centennial Colorado, USA); β-Actin, 1:5 000 (A2228, Sigma-Aldrich, St. Louis, Missouri, USA); GAPDH, 1:5 000 (MA5-15738, Invitrogen, Waltham, Massachusetts, USA). Secondary antibodies used were: Donkey anti-Rabbit IgG, 1:10 000 (NA943V, GE Healthcare, Chicago, Illinois, USA); Goat anti-Mouse IgG, 1:10 000 (A0168, Sigma-Aldrich, St. Louis, Missouri, USA). RESULTS Generation of mice with inducible monoallelic deletion of Becn1 in the intestinal epithelium We previously reported that homozygous Becn1 deletion in the intestinal epithelium results in rapid, fatal enterocolitis, with severe epithelial disruption ( 23 ). To model partial BECLIN1 loss, we generated mice in which monoallelic Becn1 deletion could be induced in the intestinal epithelium using mice heterozygous for the Becn1 floxed allele ( Becn1 fl/+ ) and carrying the Vil1-Cre ERT2 transgene ( Becn1 fl/+ ; Vil1-CreERT2 Cre/+ ). Littermates lacking the floxed allele ( Becn1 +/+ ) but positive for Vil1-CreERT2 Cre/+ ( Becn1 +/+ ; Vil1-CreERT2 Cre/+ ) served as controls. Intraperitoneal Tamoxifen injections induced deletion in the small intestine (duodenum, jejunum, ileum) and colon of Becn1 fl/+ ; Vil1-CreERT2 Cre/+ mice, confirmed genomically and by protein analysis showing approximately 50% reduced BECLIN1 levels (Fig. 1 A, Supplementary Fig. 1A). Tamoxifen-treated Becn1 +/+ ; Vil1-CreERT2 Cre/+ and Becn1 fl/+ ; Vil1-CreERT2 Cre/+ mice are referred to as Becn1 +/+ and Becn1 +/− mice respectively throughout. Mice with reduced BECLIN1 have shortened small intestines and reduced crypt lengths without other overt phenotypes At 7 days post-BECLIN1 deletion, there were no significant body weight differences between Becn1 +/+ and Becn1 +/− mice (Supplementary Fig. 1B), unlike the marked weight loss previously seen with homozygous Becn1 deletion in the intestinal epithelium ( 23 ). Despite appearing grossly normal, Becn1 +/− mice displayed significantly shorter small intestines than Becn1 +/+ controls, while colon lengths were unchanged (Fig. 1 B). Histological examination by H&E staining revealed largely normal small and large intestinal morphology in Becn1 +/− mice (Fig. 1 C). At this time point, distal colonic crypt length (Fig. 1 D) and width (Supplementary Fig. 1C) did not differ between genotypes. However, by 14- and 35-days post deletion, Becn1 +/− mice exhibited significantly shorter colonic crypts and reduced crypt volume fraction ( 37 ) compared to controls (Fig. 1 D). Observation over one month post deletion also revealed a persistently shorter small intestine (Supplementary Fig. 1D) without weight loss (Supplementary Fig. 1B), and otherwise normal intestinal morphology (Supplementary Fig. 1E). These phenotypes contrasted sharply with Becn1 −/− mice, where small intestines were shortened, swollen, and lytic, with villus stunting along the entire length within 7 days of gene deletion (Figs. 1 B, C) ( 23 ). The need to euthanise Becn1 ⁻/⁻ mice within 7 days precluded investigation of equivalent colonic changes. In summary, apart from reduced small intestinal and colonic crypt lengths, heterozygous Becn1 deletion does not cause overt abnormalities under basal conditions up to one month post deletion, in contrast to its homozygous loss ( 23 ). Intestinal epithelial cells with reduced BECLIN1 levels display no disruption to basal autophagy, differentiation, or proliferation Monoallelic Becn1 deletion caused minimal changes in basal autophagy, with total P62 and LC3B levels comparable to wild-type IECs (Fig. 1 E). In contrast, Becn1 −/− IECs showed pronounced P62 and LC3B accumulation (Fig. 1 E) ( 23 ). We previously identified an essential role for BECLIN1 in endocytic trafficking in intestinal-derived organoids ( 22 , 23 ). To test whether monoallelic Becn1 loss induced similar defects, we generated organoids from Becn1 +/+ ; Becn1 fl/+ ; and Becn1 fl/fl ; Vil1-CreERT2 Cre/+ mice. Treatment with 4-hydroxytamoxifen (4-HT) led to reduced BECLIN1 expression in Becn1 fl/+ organoids which did not impact basal autophagy (as with intestinal epithelial cells from mice), unlike homozygous deletion (Supplementary Figs. 2A, B). At 7 days post 4-HT treatment, Becn1 −/− organoids showed significantly reduced budding crypt formation (Fig. 1 F) ( 23 ), whereas Becn1 +/− organoids formed similar crypt-like projections as Becn1 +/+ organoids, indicating normal IEC proliferation and differentiation (Fig. 1 F). Re-passaging of Becn1 +/+ and Becn1 +/− yielded mature organoids by Day 5, with similar numbers of budding crypts, further indicating minimal defects in proliferation or differentiation (Fig. 1 G). This is consistent with Ki67 staining of intestinal tissues from Becn1 +/− mice showing no proliferation differences, including at the crypt bases (Supplementary Fig. 2C). In contrast, surviving Becn1 −/− organoids failed to generate viable organoid structures upon re-passaging, producing significantly fewer crypt-like projections by Day 5 (Fig. 1 G). Thus, reduced BECLIN1 expression does not impair basal autophagy, survival, differentiation, or proliferation of IECs. Intestinal epithelial cells with reduced BECLIN1 levels display changes in early endocytic trafficking and E-CADHERIN distribution BECLIN1 loss has been reported to affect endocytic trafficking in other tissues, similar to the effects of homozygous loss in the gut ( 22 , 23 , 38 – 42 ). To determine whether this also occurs following heterozygous loss in intestinal epithelial cells, we examined Becn1 +/− organoids and found a modest but significant increase in cytoplasmic RAB5 + ve early endosomes (Figs. 2 A, B, Supplementary Fig. 3) though without the aberrant enlargement seen in Becn1 −/− organoids (Figs. 2 A, C). As the junctional protein E-CADHERIN can become trapped within enlarged RAB5 + ve early endosomes in Becn1 −/− IECs (Figs. 2 A, D, E) ( 23 ), we examined its localisation in Becn1 +/− IECs. Here, E-CADHERIN levels were increased at apical and lateral membranes (Figs. 2 F, G) and in the cytoplasm (Fig. 2 D), but notably, co-localisation with RAB5 was unchanged at all subcellular sites (Figs. 2 E, H, I). Thus, reduced BECLIN1 alters early endosomal dynamics, but E-CADHERIN localisation changes appear independent of RAB5-mediated trafficking. Monoallelic Becn1 deletion alters F-actin organisation and intestinal epithelial cell architecture As homozygous BECLIN1 loss disrupts the cytoskeleton and compromises the intestinal epithelial barrier ( 22 ), we examined whether altered F-actin organisation, critical for adhesion and endocytosis ( 43 , 44 ) contributes to the E-CADHERIN changes in Becn1 +/− organoids. Here, Becn1 +/− IECs displayed significantly increased F-actin and co-localised E-CADHERIN along the lateral membrane but not apical membrane (Figs. 3 A-E), consistent with an F-actin-driven “cadherin flow” mechanism, whereby cadherins are transported along remodelling actin networks ( 45 ), potentially contributing to altered E-CADHERIN localisation (Figs. 2 A, E). Notably, E-CADHERIN was redistributed within the lateral membrane toward apicolateral junctions, possibly to maintain cell-cell contacts and preserve epithelial integrity. Given this increased lateral membrane localisation of both E-CADHERIN and F-actin in Becn1 +/− IECs (Fig. 3 E), we assessed cell morphology and found Becn1 +/− organoids had a longer apico-basal axis and shorter basal IEC width (Figs. 3 A, F, G), likely contributing to the shorter small intestinal length (Fig. 1 B). Reduced BECLIN1 therefore alters adhesion dynamics, cytoskeletal organisation, and epithelial architecture. Reduced BECLIN1 levels in intestinal epithelial cells increases susceptibility to DSS-induced colitis To test whether the changes in endocytic trafficking, intercellular adhesion, and cytoskeleton architecture observed in Becn1 +/− mice increase susceptibility to inflammation, 7- to 14-week-old mice were treated with dextran sulphate sodium (DSS) to induce colitis, a widely used model of IBD pathogenesis ( 46 ). Following Tamoxifen-induced gene deletion, mice received either 2% DSS in drinking water for 5 days or normal water (Fig. 4 A). There were no genotype-dependent differences in starting body weight or DSS-water consumption (Supplementary Figs. 4A, B). At endpoint, both DSS-treated genotypes displayed expected colitic changes, including darker, reddish areas in the colon consistent with hyperemia/inflammation (Figs. 4 B, C) and colon shortening with swelling and constriction (Figs. 4 C, D), but gross colonic changes were similar (Figs. 4 B-D). The small intestines of Becn1 +/− mice were significantly shorter than Becn1 +/+ controls regardless of DSS treatment (Supplementary Fig. 4C). Both genotypes lost weight after DSS exposure, but this was greater in Becn1 +/− mice (Fig. 4 E). They also developed more severe diarrhea, with a higher proportion producing pasty to liquid stools (Fig. 4 F), though hematochezia did not differ (Fig. 4 G). Importantly, the composite disease activity index (DAI), incorporating weight loss, stool consistency, and rectal bleeding (Table 1 ) ( 47 , 48 ), was significantly higher in Becn1 +/− mice, indicating greater colitis severity (Fig. 4 H). Histological assessment of DSS-treated colons was performed using a blinded scoring system evaluating crypt architecture, inflammatory cell infiltration, and epithelial integrity, with the composite score reported as the histological colitis score (HCS) ( 49 ). Untreated mice of both genotypes had minimal mucosal damage and low histological colitis scores (HCS) (Figs. 4 I, J, K). DSS treatment increased the HCS scores in both genotypes, but Becn1 +/− mice scored approximately 2.2-fold higher (Fig. 4 K), with more severe epithelial injury and crypt attenuation, epithelial loss, and immune infiltration (Figs. 4 I, J). These mice also had more regions of epithelial erosion (Fig. 4 L), characterised by complete crypt loss and dense localised immune cell infiltration. Lymphoid follicle counts and sizes did not differ between groups (Supplementary Figs. 4D, E), indicating that inflammation was largely confined to the epithelial layer. Hence, monoallelic Becn1 loss worsens DSS-induced colitis, indicating increased sensitivity to intestinal inflammation (Figs. 4 E-L). Monoallelic Becn1 deletion causes goblet cell defects and compromises mucosal immunity under basal conditions Goblet cells are secretory IECs specialised for mucus production, forming a critical barrier against pathogens and mechanical damage ( 50 ). Disruption of this barrier is a hallmark of chronic inflammatory conditions, including IBD, often arising from goblet cell loss or defects in mucin formation, storage, or secretion ( 50 – 53 ). Periodic-Acid-Schiff (PAS) and Alcian Blue (AB) staining revealed a significant reduction in PAS-AB staining in the distal colon (Figs. 5 A, B), but not the proximal colon (Fig. 5 B), of untreated Becn1 +/− mice compared to untreated Becn1 +/+ mice (Figs. 5 A, B), suggesting either fewer goblet cells and/or reduced mucin production ( 13 ). As goblet cell maturation involves migration to the crypt surface, we also quantified goblet cells in the lower (immature) and upper (mature) colonic crypt compartments (Fig. 5 C, Supplementary Fig. 5) ( 13 , 55 ). PAS-AB + ve mucin staining was reduced in both lower and upper crypt compartments, but more so in the latter (50% versus 15%) (Fig. 5 C). This maturation defect was further supported by the enlarged cytoplasmic mucin (theca) areas in remaining mature goblet cells of untreated Becn1 +/− mice compared with Becn1 +/+ mice (Fig. 5 D), consistent with impaired mucin secretion. These results show that reduced BECLIN1 results in goblet cell loss and defective mucin production, potentially compromising mucosal barrier function even under basal conditions. Reduction of BECLIN1 levels exacerbates DSS-induced barrier dysfunction We next investigated goblet cell responses in DSS-treated Becn1 +/+ and Becn1 +/− mice. The epithelial damage in both DSS-treated Becn1 +/+ and Becn1 +/− mice (Figs. 4 I-L) was accompanied by a significant reduction in PAS-AB + ve mucin area in the distal colon compared with their respective untreated controls (Figs. 5 A, B), consistent with the distal colon being the primary site of DSS-induced injury ( 54 ). The epithelial damage in the distal colon was further exacerbated in DSS-treated Becn1 +/− mice, where the PAS-AB + ve mucin area was significantly reduced when compared with DSS-treated Becn1 +/+ mice (Figs. 5 A, B). We also assessed PAS-AB staining across the entire colon, which revealed a loss of PAS + ve neutral mucins in Becn1 +/− mice compared to Becn1 +/+ in the untreated groups, with no significant change in AB + ve acidic mucins (Fig. 5 E). Notably, following DSS treatment, Becn1 +/− mice exhibited a loss of both neutral and acidic mucins, consistent with the exacerbated epithelial damage observed (Figs. 5 E, 4 I-L). This reveals a vulnerability of the mucosal barrier when BECLIN1 is reduced, reinforcing its role in epithelial homeostasis. The selective baseline loss of neutral mucins, and subsequent loss of both mucin types after DSS treatment, highlights the critical importance of neutral mucins in protecting against DSS-induced colitis. DISCUSSION We previously identified an essential role for BECLIN1 in maintaining intestinal homeostasis and barrier integrity mediated through its autophagy and endocytic trafficking functions ( 22 , 23 ). Given that homozygous Becn1 deletion causes fatal enteritis in adult mice ( 23 ), we investigated whether partial reduction, mimicking clinically relevant haploinsufficiency, also impairs intestinal function. Unlike homozygous BECLIN1 deletion, monoallelic loss did not trigger overt spontaneous disease. However, Becn1 +/− mice did display subclinical abnormalities, including shortened small intestines, reduced distal colonic crypt length, and goblet cell loss – hallmarks of compromised epithelial integrity. These changes occurred despite minimal changes in basal autophagy, suggesting BECLIN1’s trafficking and epithelial organisation roles may be more critical in this context ( 22 , 23 ). Altered tissue architecture was marked by increased apico-basal length, reduced basal width, and redistribution of E-CADHERIN and F-actin along the lateral membranes, likely reflecting cytoskeletal remodelling. These features mirror BECLIN1-null IECs, where E-CADHERIN becomes trapped in early endosomes ( 22 , 23 ). While cytoplasmic RAB5 + ve vesicles were modestly increased, early endosome enlargement was absent in Becn1 +/− cells, suggesting a threshold effect in trafficking disruption. Increased co-localisation of E-CADHERIN with lateral F-actin in Becn1 +/− IECs may stabilise junctional integrity under impaired trafficking ( 43 , 44 ), though altered F-actin organisation can also impair IEC contractility, crypt architecture ( 56 ), and mucin granule exocytosis ( 57 , 58 ). Goblet cells were disproportionately affected, with reduced numbers, altered maturation (larger thecae), and diminished mucin staining, especially in the upper crypt. This aligns with the known role of autophagy in promoting goblet cell function via ER stress alleviation and mucin handling ( 11 ). Following DSS challenge, Becn1 +/− mice failed to mount the same level of compensatory mucin response as wild-type controls, showing depletion of both neutral and acidic mucins. The baseline vulnerability due to selective mucin loss, compounded by the broad depletion after DSS treatment, highlights the critical role of neutral mucins in maintaining the buffering capacity of the mucus barrier. Loss of the major secreted mucin, MUC2, similarly causes spontaneous colitis in mice ( 53 ) and comparable mucin shifts occur in human IBD ( 59 – 64 ). Together, our findings suggest that beyond its canonical autophagy role, BECLIN1 integrates trafficking, cytoskeletal dynamics, and mucosal defence. The heightened DSS susceptibility of Becn1 +/− mice, despite inflammation remaining confined to the mucosa, supports a barrier-specific defect consistent with goblet cell dysfunction and epithelial disorganisation. Although IBD GWAS have not identified BECLIN1 single nucleotide polymorphisms (SNPs), emerging evidence implicates both IEC-intrinsic and -extrinsic mechanisms that lower BECLIN1 levels, thereby exacerbating colitis. For instance, cyclic GMP-AMP synthase (cGAS) promotes autophagy in IECs through interaction with BECLIN1, and its loss worsens colitis ( 65 ). Similarly, the m 6 A nuclear reader, YTHDC1, stabilises Becn1 mRNA and is downregulated in DSS colitis and in IBD patient microbiota ( 35 , 66 ). Moreover, while no IBD-associated SNPs have been identified within Becn1 itself, variants in upstream regulators such as STAT3, a transcriptional regulator of Becn1 , have been implicated in IBD pathogenesis ( 67 – 69 ). Caspase-mediated cleavage of BECLIN1 during apoptosis may also contribute to reduced protein levels in inflamed tissue, amplifying epithelial injury ( 70 , 71 ). Looking forward, resources such as atlases of IBD patient samples ( 72 , 73 ) provide opportunities to examine BECLIN1 expression at endoscopically defined disease margins. Expanding clinical analyses will be critical to determine whether there are changes in BECLIN1 levels across broader clinical cohorts and to assess its potential as a biomarker of IBD pathogenesis and severity. In summary, partial BECLIN1 reduction compromises epithelial homeostasis and exacerbates inflammation in the absence of complete autophagy loss. These findings establish BECLIN1 as a critical epithelial defence regulator. Future studies should explore whether restoring BECLIN1 levels or mimicking its trafficking-related functions could preserve barrier integrity and enable patient stratification for precision therapeutic interventions in IBD. Declarations CONFLICT OF INTEREST J.M.Murphy, A.L.S., K.M.P., and S.N.Y. contribute to, or have contributed to, the development of necroptosis pathway inhibitors in collaboration with Anaxis Pharma Pty. Ltd. J.M.Murphy has also received research funding from Anaxis Pharma Pty. Ltd. All other authors declare no competing interests. AUTHOR CONTRIBUTIONS J.J., S.T., designed, performed, and analysed experiments and wrote the paper; T.J.H., Y.W.N.R., S.L.E., A.H.A-A, K.P., S.N.Y., M.E., D.B., C.M.R., R.N., L.J.J., designed, performed experiments, analysed and interpreted data; P.D.C., K.D., B.T.K., A.S.Y., J.M.M., B.C., A.L.S., J.M.Murphy analysed and interpreted data; W.D.F., E.F.L. designed the project, analysed, interpreted data, and wrote the paper. All authors commented on the manuscript. ACKNOWLEDGEMENTS We acknowledge scholarship support for J.J. (La Trobe Graduate Research Scholarship and Full Fee Research Scholarship), S.T. (La Trobe University Research Training Program Scholarship) and A.H.A.-A (University of Melbourne Australian Commonwealth Government Research Training Program, Crohn’s and Colitis Australia (IBD PhD Scholarship), Avant (Doctors in Training Scholarship), and the Gastroenterological Society of Australia (Celltrion IBD Fellowship). We are grateful to the Australian Research Council for grant support to E.F.L., W.D.F., J.M.Mariadason (DP190102612); the National Health and Medical Research Council to A.L.S. (2002965, 2011584), K.D. and A.S.Y. (2010704, 1136592), PDC (2008909), J.M.Murphy (1172929, 2034104); NHMRC IRIISS and the Victorian Government Operational Infrastructure Support Scheme; the Kenneth Rainin Foundation to J.M.Murphy, B.C., A.L.S. and A.H.A.-A; the US DOD (HT94252310088) to A.S.Y.; and the Victorian Cancer Agency to E.F.L. (MCRF19045) for fellowship support. AVAILABILITY OF DATA AND MATERIALS Correspondence and requests for materials should be addressed to Erinna F. Lee or Walter D. Fairlie. References Henckaerts L, Cleynen I, Brinar M, John JM, Van Steen K, Rutgeerts P, et al. Genetic variation in the autophagy gene ULK1 and risk of Crohn's disease. Inflamm Bowel Dis. 2011;17(6):1392–7. Lassen KG, Kuballa P, Conway KL, Patel KK, Becker CE, Peloquin JM, et al. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc Natl Acad Sci U S A. 2014;111(21):7741–6. Parkes M, Barrett JC, Prescott NJ, Tremelling M, Anderson CA, Fisher SA, et al. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility. Nat Genet. 2007;39(7):830–2. Prescott NJ, Fisher SA, Franke A, Hampe J, Onnie CM, Soars D, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn's disease and is independent of CARD15 and IBD5. Gastroenterology. 2007;132(5):1665–71. Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39(5):596–604. Tran S, Juliani J, Fairlie WD, Lee EF. The emerging roles of autophagy in intestinal epithelial cells and its links to inflammatory bowel disease. Biochem Soc Trans. 2023;51(2):811–26. Wellcome Trust Case Control C. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007;447(7145):661–78. Benjamin JL, Sumpter R, Jr., Levine B, Hooper LV. Intestinal epithelial autophagy is essential for host defense against invasive bacteria. Cell Host Microbe. 2013;13(6):723–34. Cadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, Lennerz JK, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature. 2008;456(7219):259–63. Cadwell K, Patel KK, Komatsu M, Virgin HWt, Stappenbeck TS. A common role for Atg16L1, Atg5 and Atg7 in small intestinal Paneth cells and Crohn disease. Autophagy. 2009;5(2):250–2. Naama M, Telpaz S, Awad A, Ben-Simon S, Harshuk-Shabso S, Modilevsky S, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress. Cell Host Microbe. 2023;31(3):433–46 e4. Nighot PK, Blikslager AT. Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin. Am J Physiol Cell Physiol. 2012;302(1):C178-87. Patel KK, Miyoshi H, Beatty WL, Head RD, Malvin NP, Cadwell K, et al. Autophagy proteins control goblet cell function by potentiating reactive oxygen species production. EMBO J. 2013;32(24):3130–44. Saha K, Subramenium Ganapathy A, Wang A, Michael Morris N, Suchanec E, Ding W, et al. Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux. J Crohns Colitis. 2023;17(3):433–49. Wittkopf N, Gunther C, Martini E, Waldner M, Amann KU, Neurath MF, et al. Lack of intestinal epithelial atg7 affects paneth cell granule formation but does not compromise immune homeostasis in the gut. Clin Dev Immunol. 2012;2012:278059. Zhao Z, Fux B, Goodwin M, Dunay IR, Strong D, Miller BC, et al. Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host Microbe. 2008;4(5):458–69. Cooney R, Baker J, Brain O, Danis B, Pichulik T, Allan P, et al. NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nat Med. 2010;16(1):90–7. Martin PK, Marchiando A, Xu R, Rudensky E, Yeung F, Schuster SL, et al. Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota. Nat Microbiol. 2018;3(10):1131–41. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature. 2008;456(7219):264–8. Sanjuan MA, Dillon CP, Tait SW, Moshiach S, Dorsey F, Connell S, et al. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007;450(7173):1253–7. Strisciuglio C, Duijvestein M, Verhaar AP, Vos AC, van den Brink GR, Hommes DW, et al. Impaired autophagy leads to abnormal dendritic cell-epithelial cell interactions. J Crohns Colitis. 2013;7(7):534–41. Juliani J, Sharon T, J. HT, Peter DC, L. ES, A. GP, et al. BECLIN-1 is essential for the maintenance of gastrointestinal epithelial integrity by regulating endocytic trafficking, F-actin organization, and lysosomal function. Autophagy Reports. 2025;4(1):2484494. Tran S, Juliani J, Harris TJ, Evangelista M, Ratcliffe J, Ellis SL, et al. BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking. Commun Biol. 2024;7(1):209. Wong M, Ganapathy AS, Suchanec E, Laidler L, Ma T, Nighot P. Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1. Am J Physiol Cell Physiol. 2019;316(5):C753-C65. Hill SM, Wrobel L, Rubinsztein DC. Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation. Cell Death Differ. 2019;26(4):617–29. Lee EF, Perugini MA, Pettikiriarachchi A, Evangelista M, Keizer DW, Yao S, et al. The BECN1 N-terminal domain is intrinsically disordered. Autophagy. 2016;12(3):460–71. Lee EF, Smith NA, Soares da Costa TP, Meftahi N, Yao S, Harris TJ, et al. Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1. Autophagy. 2019;15(5):785–95. Tran S, Fairlie WD, Lee EF. BECLIN1: Protein Structure, Function and Regulation. Cells. 2021;10(6). Gong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, et al. Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells. Oncogene. 2013;32(18):2261-72, 72e 1–11. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999;402(6762):672–6. Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, et al. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. J Clin Invest. 2008;118(6):2190–9. Li Z, Chen B, Wu Y, Jin F, Xia Y, Liu X. Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors. BMC Cancer. 2010;10:98. Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003;112(12):1809–20. Rohn TT, Wirawan E, Brown RJ, Harris JR, Masliah E, Vandenabeele P. Depletion of Beclin-1 due to proteolytic cleavage by caspases in the Alzheimer's disease brain. Neurobiol Dis. 2011;43(1):68–78. Zhou L, Zhang L, Lv Y, Qian J, Huang L, Qu C. YTHDC1 inhibits autophagy-dependent NF-kappaB signaling by stabilizing Beclin1 mRNA in macrophages. J Inflamm (Lond). 2024;21(1):22. el Marjou F, Janssen KP, Chang BH, Li M, Hindie V, Chan L, et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis. 2004;39(3):186–93. DeRoche TC, Xiao SY, Liu X. Histological evaluation in ulcerative colitis. Gastroenterol Rep (Oxf). 2014;2(3):178–92. Matthew-Onabanjo AN, Janusis J, Mercado-Matos J, Carlisle AE, Kim D, Levine F, et al. Beclin 1 Promotes Endosome Recruitment of Hepatocyte Growth Factor Tyrosine Kinase Substrate to Suppress Tumor Proliferation. Cancer Res. 2020;80(2):249–62. McKnight NC, Zhong Y, Wold MS, Gong S, Phillips GR, Dou Z, et al. Beclin 1 is required for neuron viability and regulates endosome pathways via the UVRAG-VPS34 complex. PLoS Genet. 2014;10(10):e1004626. Noguchi S, Honda S, Saitoh T, Matsumura H, Nishimura E, Akira S, et al. Beclin 1 regulates recycling endosome and is required for skin development in mice. Commun Biol. 2019;2:37. Rohatgi RA, Janusis J, Leonard D, Bellve KD, Fogarty KE, Baehrecke EH, et al. Beclin 1 regulates growth factor receptor signaling in breast cancer. Oncogene. 2015;34(42):5352–62. Wijshake T, Zou Z, Chen B, Zhong L, Xiao G, Xie Y, et al. Tumor-suppressor function of Beclin 1 in breast cancer cells requires E-cadherin. Proc Natl Acad Sci U S A. 2021;118(5). Apodaca G. Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton. Traffic. 2001;2(3):149–59. Vasioukhin V, Bauer C, Yin M, Fuchs E. Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell. 2000;100(2):209–19. Woichansky I, Beretta CA, Berns N, Riechmann V. Three mechanisms control E-cadherin localization to the zonula adherens. Nat Commun. 2016;7:10834. Mizoguchi A. Animal models of inflammatory bowel disease. Prog Mol Biol Transl Sci. 2012;105:263–320. Park YH, Kim N, Shim YK, Choi YJ, Nam RH, Choi YJ, et al. Adequate Dextran Sodium Sulfate-induced Colitis Model in Mice and Effective Outcome Measurement Method. J Cancer Prev. 2015;20(4):260–7. Reehorst CM, Nightingale R, Luk IY, Jenkins L, Koentgen F, Williams DS, et al. EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis. Development. 2021;148(12). Garcia-Hernandez V, Neumann PA, Koch S, Lyons R, Nusrat A, Parkos CA. Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model. J Vis Exp. 2021(168). Gustafsson JK, Johansson MEV. The role of goblet cells and mucus in intestinal homeostasis. Nat Rev Gastroenterol Hepatol. 2022;19(12):785–803. Buisine MP, Desreumaux P, Leteurtre E, Copin MC, Colombel JF, Porchet N, et al. Mucin gene expression in intestinal epithelial cells in Crohn's disease. Gut. 2001;49(4):544–51. Choi WT, Salomao M, Zhao L, Alpert L, Setia N, Liao X, et al. Hypermucinous, Goblet Cell-Deficient and Crypt Cell Dysplasias in Inflammatory Bowel Disease are Often Associated with Flat/Invisible Endoscopic Appearance and Advanced Neoplasia on Follow-Up. J Crohns Colitis. 2022;16(1):98–108. Van der Sluis M, De Koning BA, De Bruijn AC, Velcich A, Meijerink JP, Van Goudoever JB, et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology. 2006;131(1):117–29. Laroui H, Ingersoll SA, Liu HC, Baker MT, Ayyadurai S, Charania MA, et al. Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon. PLoS One. 2012;7(3):e32084. Gersemann M, Becker S, Kubler I, Koslowski M, Wang G, Herrlinger KR, et al. Differences in goblet cell differentiation between Crohn's disease and ulcerative colitis. Differentiation. 2009;77(1):84–94. Hinnant T, Ning W, Lechler T. Compartment specific responses to contractility in the small intestinal epithelium. PLoS Genet. 2024;20(3):e1010899. Ehre C, Rossi AH, Abdullah LH, De Pestel K, Hill S, Olsen JC, et al. Barrier role of actin filaments in regulated mucin secretion from airway goblet cells. Am J Physiol Cell Physiol. 2005;288(1):C46-56. Oliver MG, Specian RD. Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion. Am J Physiol. 1990;259(6 Pt 1):G991-7. Campbell BJ, Yu LG, Rhodes JM. Altered glycosylation in inflammatory bowel disease: a possible role in cancer development. Glycoconj J. 2001;18(11–12):851–8. Corfield AP, Myerscough N, Bradfield N, Corfield Cdo A, Gough M, Clamp JR, et al. Colonic mucins in ulcerative colitis: evidence for loss of sulfation. Glycoconj J. 1996;13(5):809–22. Larsson JM, Karlsson H, Crespo JG, Johansson ME, Eklund L, Sjovall H, et al. Altered O-glycosylation profile of MUC2 mucin occurs in active ulcerative colitis and is associated with increased inflammation. Inflamm Bowel Dis. 2011;17(11):2299–307. McGuckin MA, Linden SK, Sutton P, Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Microbiol. 2011;9(4):265–78. Raouf AH, Tsai HH, Parker N, Hoffman J, Walker RJ, Rhodes JM. Sulphation of colonic and rectal mucin in inflammatory bowel disease: reduced sulphation of rectal mucus in ulcerative colitis. Clin Sci (Lond). 1992;83(5):623–6. Van Klinken BJ, Van der Wal JW, Einerhand AW, Buller HA, Dekker J. Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis. Gut. 1999;44(3):387–93. Khan S, Mentrup HL, Novak EA, Siow VS, Wang Q, Crawford EC, et al. Cyclic GMP-AMP synthase contributes to epithelial homeostasis in intestinal inflammation via Beclin-1-mediated autophagy. FASEB J. 2022;36(5):e22282. Ge X, Xue G, Ding Y, Li R, Hu K, Xu T, et al. The Loss of YTHDC1 in Gut Macrophages Exacerbates Inflammatory Bowel Disease. Adv Sci (Weinh). 2023;10(14):e2205620. Ferguson LR, Han DY, Fraser AG, Huebner C, Lam WJ, Morgan AR, et al. Genetic factors in chronic inflammation: single nucleotide polymorphisms in the STAT-JAK pathway, susceptibility to DNA damage and Crohn's disease in a New Zealand population. Mutat Res. 2010;690(1–2):108–15. Miao LJ, Huang FX, Sun ZT, Zhang RX, Huang SF, Wang J. Stat3 inhibits Beclin 1 expression through recruitment of HDAC3 in nonsmall cell lung cancer cells. Tumour Biol. 2014;35(7):7097–103. Tang Y, Tan SA, Iqbal A, Li J, Glover SC. STAT3 Genotypic Variant rs744166 and Increased Tyrosine Phosphorylation of STAT3 in IL-23 Responsive Innate Lymphoid Cells during Pathogenesis of Crohn's Disease. J Immunol Res. 2019;2019:9406146. Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC. Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett. 2009;274(1):95–100. Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, et al. Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis. 2010;1(1):e18. Chiou S, Al-Ani AH, Pan Y, Patel KM, Kong IY, Whitehead LW, et al. An immunohistochemical atlas of necroptotic pathway expression. EMBO Mol Med. 2024;16(7):1717–49. Pang J, Al-Ani AH, Patel KM, Young SN, Kong I, Chen J-j, et al. A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease. bioRxiv. 2024:2024.11.13.623307. Additional Declarations There is no duality of interest Supplementary Files JulianietalSubmittedCDDiffSuppInformation.docx Supplementary Information JulianietalSubmittedCDDiffSuppInformationOriginalWB.pdf Supplementary Information Original Western blots JulianietalSubmittedCDDiffSuppFigures.pdf Supplementary Figures Cite Share Download PDF Status: Under Review Version 1 posted Unknown event 03 Nov, 2025 Editorial decision: Reject after peer review 30 Sep, 2025 Review # 1 received at journal 30 Sep, 2025 Review # 2 received at journal 21 Sep, 2025 Reviewer # 2 agreed at journal 15 Sep, 2025 Reviewer # 1 agreed at journal 12 Sep, 2025 Reviewers invited by journal 12 Sep, 2025 Submission checks completed at journal 09 Sep, 2025 Editor assigned by journal 08 Sep, 2025 First submitted to journal 08 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7567745","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":514224937,"identity":"f9ef02ae-80c1-4311-bea0-0e4dd5c2a432","order_by":0,"name":"Erinna Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYBAC+wbGBhCdwMDAfPAAiGUAInjwaDFggGthSzgAoojQAgFAtTwGRGqRPtz2uKCGIYGfvefDgZ8/7smZszcwPnjbxiBvcACHX/gS241nHGNIkOw5u+FgT0KxsWXPAWbDuW0MhhtwaDHgYWyT5mFjSDC4kbvhAE9CQuKGGwls0rxtDIz4tfxjSLC//+bBwT8gLfcfsP8GarHHqwWoIMFAgofhMMQWBjZmoEgifi19EgkSZ9IMDsukJQD9ktgsOeecRPJMXN7vYX8mzfPNpp6//fDDh29sEoAhdvjghzdlNrZ9OLRAgQQyBxy5EtgVjoJRMApGwSggCgAAJdFZ8/4SZdkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1255-9808","institution":"La Trobe University / La Trobe Institute for Molecular Science","correspondingAuthor":true,"prefix":"","firstName":"Erinna","middleName":"","lastName":"Lee","suffix":""},{"id":514224938,"identity":"12114eee-02f3-4b16-b3b4-9339b6eb862f","order_by":1,"name":"Juliani Juliani","email":"","orcid":"","institution":"La Trobe University / La Trobe Institute for Molecular Science","correspondingAuthor":false,"prefix":"","firstName":"Juliani","middleName":"","lastName":"Juliani","suffix":""},{"id":514224939,"identity":"807d3c51-d750-4309-ab50-b71051dba6df","order_by":2,"name":"Sharon Tran","email":"","orcid":"","institution":"Olivia Newton-John Cancer Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sharon","middleName":"","lastName":"Tran","suffix":""},{"id":514224940,"identity":"02de4cdd-2227-4d09-9cfa-3faa979e1012","order_by":3,"name":"Tiffany Harris","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tiffany","middleName":"","lastName":"Harris","suffix":""},{"id":514224941,"identity":"c2e39f49-0567-412e-a238-a344f8c4d521","order_by":4,"name":"Sarah Ellis","email":"","orcid":"https://orcid.org/0000-0002-5772-6051","institution":"Peter MacCallum Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Ellis","suffix":""},{"id":514224942,"identity":"0cc5f0f9-4e59-46ed-8a06-5d1402d3da47","order_by":5,"name":"Aysha Al-Ani","email":"","orcid":"","institution":"Royal Melbourne Hospital","correspondingAuthor":false,"prefix":"","firstName":"Aysha","middleName":"","lastName":"Al-Ani","suffix":""},{"id":514224943,"identity":"a5f326d7-9266-48f7-9d1d-5ede0fe6100b","order_by":6,"name":"Yashodha Wijebandara","email":"","orcid":"","institution":"La Trobe University / La Trobe Institute for Molecular Science","correspondingAuthor":false,"prefix":"","firstName":"Yashodha","middleName":"","lastName":"Wijebandara","suffix":""},{"id":514224944,"identity":"f36f15d2-6f87-4d37-a0c5-59c9c1bea331","order_by":7,"name":"Komal Patel","email":"","orcid":"","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Komal","middleName":"","lastName":"Patel","suffix":""},{"id":514224945,"identity":"1bec633c-3502-4641-9174-ce8e05f2cc76","order_by":8,"name":"Samuel Young","email":"","orcid":"https://orcid.org/0009-0001-6543-1851","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Young","suffix":""},{"id":514224946,"identity":"5f9fd2c4-bef8-4137-82d8-fc57a6574257","order_by":9,"name":"Marco Evangelista","email":"","orcid":"","institution":"Olivia Newton-John Cancer Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Evangelista","suffix":""},{"id":514224947,"identity":"712e9962-cb61-4c96-9c29-b23afb2be9ca","order_by":10,"name":"David Baloyan","email":"","orcid":"","institution":"Olivia Newton-John Cancer Research Institute","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Baloyan","suffix":""},{"id":514224948,"identity":"be023e45-9140-41e8-89c2-3bce2a8a5d18","order_by":11,"name":"Camilla Reehorst","email":"","orcid":"","institution":"Olivia Newton-John Cancer Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Camilla","middleName":"","lastName":"Reehorst","suffix":""},{"id":514224949,"identity":"dbef77cb-fe33-4f3e-bf50-02590e9de14a","order_by":12,"name":"Rebecca Nightingale","email":"","orcid":"","institution":"Olivia Newton-John Cancer Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Nightingale","suffix":""},{"id":514224950,"identity":"0b1f4b96-75ec-43fa-bb77-0cf0aeff6ed3","order_by":13,"name":"Laura Jenkins","email":"","orcid":"","institution":"Olivia Newton-John Cancer Wellness \u0026 Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Jenkins","suffix":""},{"id":514224951,"identity":"4567b2e1-13af-476e-847a-f660709d511f","order_by":14,"name":"Peter De Cruz","email":"","orcid":"","institution":"Austin Health","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"De Cruz","suffix":""},{"id":514224952,"identity":"da95716d-efd2-49bb-b505-b9f25d491d2a","order_by":15,"name":"Kinga Duszyc","email":"","orcid":"","institution":"The University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Kinga","middleName":"","lastName":"Duszyc","suffix":""},{"id":514224953,"identity":"3e7df502-1096-40c2-a01c-51914b2e5974","order_by":16,"name":"Benjamin Kile","email":"","orcid":"","institution":"Garvan Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Kile","suffix":""},{"id":514224954,"identity":"a41239fb-4d48-4cdb-9868-fa83728dd5ba","order_by":17,"name":"Alpha Yap","email":"","orcid":"","institution":"Institute for Molecular Bioscience\u2028The University of Queensland\u2028St Lucia QLD 4072\u2028Australia.","correspondingAuthor":false,"prefix":"","firstName":"Alpha","middleName":"","lastName":"Yap","suffix":""},{"id":514224955,"identity":"b39f9c6c-ceb4-45e6-9ea3-05c5cc5d2cda","order_by":18,"name":"John Mariadason","email":"","orcid":"https://orcid.org/0000-0001-9123-7684","institution":"Olivia Newton-John Cancer Wellness \u0026 Research Centre","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Mariadason","suffix":""},{"id":514224956,"identity":"9c05fdf7-b943-4cf8-a82a-7f8035ba7498","order_by":19,"name":"Britt Christensen","email":"","orcid":"","institution":"Royal Melbourne Hospital","correspondingAuthor":false,"prefix":"","firstName":"Britt","middleName":"","lastName":"Christensen","suffix":""},{"id":514224957,"identity":"16de08da-e04f-4578-822e-2bc78fe13b1d","order_by":20,"name":"Andre Samson","email":"","orcid":"https://orcid.org/0000-0002-0637-2716","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Andre","middleName":"","lastName":"Samson","suffix":""},{"id":514224958,"identity":"2b7cb804-9013-4669-8920-37904a19a9f9","order_by":21,"name":"James Murphy","email":"","orcid":"https://orcid.org/0000-0003-0195-3949","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Murphy","suffix":""},{"id":514224959,"identity":"27f726b7-3b55-4ff6-a39f-7b8d1a40b11c","order_by":22,"name":"Walter Fairlie","email":"","orcid":"https://orcid.org/0000-0002-2498-1160","institution":"Olivia Newton-John Cancer Wellness \u0026 Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Walter","middleName":"","lastName":"Fairlie","suffix":""}],"badges":[],"createdAt":"2025-09-08 22:25:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7567745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7567745/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91706730,"identity":"1a2beab5-cca9-4fbd-a92b-02a3e9350ec3","added_by":"auto","created_at":"2025-09-19 11:41:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2225521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonoallelic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBecn1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion induces mild intestinal epithelial alterations under basal conditions, distinct from the severe defects observed with homozygous \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBecn1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion. (A) \u003c/strong\u003eRepresentative Western blots (top) showing BECLIN1 levels in the indicated tissues, with densitometry quantification (bottom). Data represent a minimum of \u003cem\u003en = \u003c/em\u003e5 animals per genotype from three independent experiments. GAPDH was used as a loading control. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative images of intestinal tracts of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice (top) with the measurements of intestinal length at Day 7 post Tamoxifen administration (bottom). Data represent \u003cem\u003en\u003c/em\u003e \u0026gt; 12 animals per genotype from \u003cem\u003en\u003c/em\u003e \u0026gt; 3 independent experiments \u003cstrong\u003e(C) \u003c/strong\u003eH\u0026amp;E-stained FFPE sections of the intestinal tracts from \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Becn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. Scale bars = 100 μm. Data represent \u003cem\u003en\u003c/em\u003e \u0026gt; 6 biologically independent mice of each genotype from \u003cem\u003en\u003c/em\u003e = 3 independent experiments. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of distal colon crypt length in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice at days 7, 14 and 35 post-tamoxifen administration. Only visible full-length crypts were included in the analysis, with a minimum of \u003cem\u003en \u0026gt;\u003c/em\u003e 5 crypts measured per mouse and averaged. Data represent \u003cem\u003en \u0026gt; \u003c/em\u003e4 animals per genotype from \u003cem\u003en = \u003c/em\u003e3 independent experiments. \u003cstrong\u003e(E)\u003c/strong\u003e Western blot showing markers of basal autophagy across different sections of the gastrointestinal tract in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. The duodenum of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice was included as a positive control for defective autophagy. β-ACTIN was used as a loading control. \u003cstrong\u003e(F)\u003c/strong\u003e Representative phase contrast microscopy images of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e organoids at Day 7 post-4-HT treatment and \u003cstrong\u003e(G)\u003c/strong\u003e re-passaged surviving \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e organoids at Day 5, along with respective quantification of the number of budding crypt-like projections per organoid. Scale bar = 100 µm. Data in (F) and (G) were obtained from \u003cem\u003en = \u003c/em\u003e20 organoids per genotype across \u003cem\u003en = \u003c/em\u003e3 independent experiments. Graphs show the mean ± S.E.M. Statistical significance was determined by unpaired (Student’s) t-test, except in (F) and (G) where ordinary one-way ANOVA was used. SI: small intestine. Stom: stomach. Duo: duodenum. Jej: jejunum. Ile: ileum. Col: Colon. MW: molecular weight. 4-HT: 4-hydroxytamoxifen. ns: not significant (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/5013217af4e53b3003b2c7e3.png"},{"id":91706998,"identity":"e1bf8cb4-723e-4ae4-90cf-917a7545ae42","added_by":"auto","created_at":"2025-09-19 11:49:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2227190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBECLIN1 reduction alters E-CADHERIN trafficking in a RAB5-independent manner. (A) \u003c/strong\u003eRepresentative whole-mount immunofluorescence staining for RAB5 (red), E-CADHERIN (green) and DAPI (blue) in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Becn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and Becn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eorganoids. Increased cytoplasmic RAB5 is indicated by white arrows. Aberrant E-CADHERIN localisation is denoted by white (apical), red (lateral), and purple (cytoplasmic) open arrowheads. Scale bar = 5 μm. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of cytoplasmic RAB5 pixels per cell. \u003cstrong\u003e(C)\u003c/strong\u003e Measurement of the average size of RAB5\u003csup\u003e+ve\u003c/sup\u003e vesicles. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of cytoplasmic E-CADHERIN pixels per cell, along with its \u003cstrong\u003e(E) \u003c/strong\u003eco-localisation with cytoplasmic RAB5, measured using Pearson’s correlation coefficient. Quantification of \u003cstrong\u003e(F)\u003c/strong\u003e apical and \u003cstrong\u003e(G)\u003c/strong\u003e lateral E-CADHERIN pixels in intestinal epithelial cells. Measurement of the degree of colocalisation between RAB5 and E-CADHERIN on the \u003cstrong\u003e(H)\u003c/strong\u003e apical and \u003cstrong\u003e(I)\u003c/strong\u003e lateral membrane of intestinal epithelial cells. Data are representative of at least \u003cem\u003en = 3 \u003c/em\u003edifferent slices per organoid and of at least \u003cem\u003en = \u003c/em\u003e3 biologically independent organoids. For each z-section, at least \u003cem\u003en \u003c/em\u003e= 3 individual IECs with clear apical to basal delineation were used. Graphs indicate the mean ± S.E.M. Statistical significance was determined using unpaired (Student’s) t-test. ns = not significant (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/1d98ae7657057cd418ac18ef.png"},{"id":91707891,"identity":"7ecd76eb-5550-4c6b-ae6b-afe5fbff595c","added_by":"auto","created_at":"2025-09-19 11:57:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2809452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonoallelic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBecn1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion alters F-actin organisation in intestinal epithelial cells. (A)\u003c/strong\u003e Representative whole-mount immunofluorescence staining for F-actin (red), E-CADHERIN (green) and DAPI (blue) in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, Becn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and Becn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eorganoids. Increased lateral F-actin in\u003cem\u003e Becn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eorganoids is indicated by white arrowheads. Scale bar = 5 μm. Quantification of \u003cstrong\u003e(B)\u003c/strong\u003e apical and \u003cstrong\u003e(C)\u003c/strong\u003e lateral F-actin signals per cell. Colocalisation between F-actin and E-CADHERIN on the \u003cstrong\u003e(D)\u003c/strong\u003e apical and \u003cstrong\u003e(E)\u003c/strong\u003e lateral membranes of intestinal epithelial cells, assessed using Pearson’s correlation coefficient. \u003cstrong\u003e(F)\u003c/strong\u003e Measurement of cell length (apical to basal membrane) and \u003cstrong\u003e(G)\u003c/strong\u003e cell width (lateral to lateral membrane). Data are representative of at least \u003cem\u003en =\u003c/em\u003e 3 different z-slices per organoid and of at least \u003cem\u003en =\u003c/em\u003e 3 biologically independent organoids. For each z-slice, at least \u003cem\u003en = \u003c/em\u003e3 individual cells with clear apical to basal delineation were used for quantification. Graphs indicate the mean ± S.E.M. Statistical significance was determined using unpaired (Student’s) t-test for all graphs. ns = not significant (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/b1412dc00860aae169079ca2.png"},{"id":91706732,"identity":"45fbaadf-1396-46ec-873a-bca13fa65e68","added_by":"auto","created_at":"2025-09-19 11:41:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2564109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonoallelic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBecn1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion exacerbates colitis severity following Dextran Sulphate Sodium administration. (A) \u003c/strong\u003eSchematic diagram depicting the timeline of Tamoxifen-induced \u003cem\u003eBecn1\u003c/em\u003e gene deletion followed by 5 days of 2% Dextran Sulphate Sodium (DSS) treatment. Image created in BioRender.com. \u003cstrong\u003e(B)\u003c/strong\u003e Representative images of abdominal necropsy and \u003cstrong\u003e(C) \u003c/strong\u003eintestinal tracts of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice, who received normal drinking water (untreated) or 2% DSS drinking water, at endpoint. Red arrowheads and black open arrowheads highlight points of colon constriction and swelling, respectively.\u0026nbsp; \u003cstrong\u003e(D)\u003c/strong\u003e Colon lengths of mice from each treatment group at endpoint. \u003cstrong\u003e(E)\u003c/strong\u003e Changes in body weight of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice during the 2% DSS treatment period, normalised to body weight at the start of DSS treatment (day 9). \u003cstrong\u003e(F)\u003c/strong\u003e Diarrheal and \u003cstrong\u003e(G)\u003c/strong\u003e haematochezia scores of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice who received either normal drinking water or 2% DSS drinking water. Scores were determined based on parameters outlined in Table 1. \u003cstrong\u003e(H)\u003c/strong\u003e Disease Activity Index (DAI) score of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+ \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice receiving either normal drinking water or 2% DSS drinking water, calculated by summing the scores of the parameters outlined in Table 1. \u003cstrong\u003e(I) \u003c/strong\u003eH\u0026amp;E-stained FFPE distal colon sections from \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice who received normal drinking water (untreated) or 2% DSS drinking water (2% DSS). The black double-headed arrow denotes the muscularis mucosae, and goblet cells are indicated by *. Immune cell infiltration in the submucosa and mucosa is marked by black and red open arrowheads, respectively. A magnified area (indicated by the box) provides a closer view of the mucosa, illustrating the increased immune infiltration in DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(J) \u003c/strong\u003ePercentage of injured or inflamed epithelium, normalised to the length of the muscularis mucosae, in the colon of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice receiving either normal drinking water or 2% DSS drinking water. \u003cstrong\u003e(K)\u003c/strong\u003e The histological colitis scores (HCS) of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice with and without DSS treatment was calculated based on described methods (49). \u003cstrong\u003e(L)\u003c/strong\u003e Percentage of eroded epithelium, normalised to the length of the muscularis mucosae, in the colon of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice receiving either normal drinking water or 2% DSS drinking water. Data are representative of at least \u003cem\u003en = \u003c/em\u003e9 biologically independent mice from \u003cem\u003en = \u003c/em\u003e3 independent experiments. Graphs indicate the ± S.E.M. Statistical significance was determined using ordinary one-way ANOVA except in (E) where changes in body weight was determined using two-way ANOVA with Tukey’s post-hoc test.\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/77ec05e3db79d00e4e06162c.png"},{"id":91706738,"identity":"fef5b884-e759-4ad8-bceb-9f2487a32d9b","added_by":"auto","created_at":"2025-09-19 11:41:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2667992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonoallelic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBecn1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeletion disrupts goblet cell function and predisposes to DSS-induced colitis. (A)\u003c/strong\u003e Representative PAS-AB-stained distal colon sections from untreated and DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. Scale bar = 100 μm. Black arrowheads depicts enlarged goblet cells\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of PAS-AB\u003csup\u003e+ve\u003c/sup\u003e area (measured per µm of muscularis mucosae) normalised to gross colon length in the distal and proximal colon of untreated and DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of PAS-AB\u003csup\u003e+ve\u003c/sup\u003e area in the lower half and upper half of distal colon crypts, as delineated in Supplementary Figure 5, in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of goblet cell theca size in the upper half of distal colonic crypts of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice. \u003cstrong\u003e(E)\u003c/strong\u003e Graphical representation of acidic (AB\u003csup\u003e+ve\u003c/sup\u003e), neutral (PAS\u003csup\u003e+ve\u003c/sup\u003e), and total (Merge) mucins in untreated and DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice. Scale bars = 1 mm. The areas of acidic (AB\u003csup\u003e+ve\u003c/sup\u003e) and neutral (PAS\u003csup\u003e+ve\u003c/sup\u003e) mucus were quantified per µm of muscularis mucosae and normalised to gross total colon length. Data represent n \u0026gt; 5 biologically independent mice from \u003cem\u003en = \u003c/em\u003e2\u003cem\u003e \u003c/em\u003eindependent experiments. Only animals that received DSS treatment from the same lot number were included for analysis. Graphs show the ± S.E.M. Statistical significance was determined by unpaired (Student’s) t-test. For crypt-specific quantifications in (C), full-length intact crypts with at least three contiguous intact crypts were analysed, with \u003cem\u003en \u0026gt; \u003c/em\u003e3 crypts analysed per animal. The average goblet cell theca area in (D) was obtained by measuring \u0026gt;100 goblet cells per animal. DSS: dextran sulphate sodium. PAS: periodic acid Schiff. AB: alcian blue. ns: not significant (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/e1df784416529ec7fd37633f.png"},{"id":92514570,"identity":"59d235ca-0baf-40a3-b775-11cba6437058","added_by":"auto","created_at":"2025-09-30 13:59:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13886021,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/ef1fb54b-987e-4294-80c4-76339d36236c.pdf"},{"id":91706733,"identity":"afbabebf-bbf8-4f23-899b-4be67b22288d","added_by":"auto","created_at":"2025-09-19 11:41:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25195,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"JulianietalSubmittedCDDiffSuppInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/1338e434277f635de073bfe2.docx"},{"id":91707004,"identity":"4b21538e-14fb-4342-9ad1-6983ed47a6d1","added_by":"auto","created_at":"2025-09-19 11:49:16","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5739356,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information Original Western blots\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffSuppInformationOriginalWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/2a8ef6a6ec94c454250daa13.pdf"},{"id":91706750,"identity":"5a7b07ed-de1d-46a9-b5f8-b0c94393425a","added_by":"auto","created_at":"2025-09-19 11:41:16","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13493567,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figures\u003c/p\u003e","description":"","filename":"JulianietalSubmittedCDDiffSuppFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7567745/v1/534afab61165a77ac901e51c.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Impact of BECLIN1 haploinsufficiency on goblet cell function and susceptibility to colitis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGenome-wide association studies have identified polymorphisms in autophagy regulators, such as \u003cem\u003eATG16L1\u003c/em\u003e and \u003cem\u003eIRGM\u003c/em\u003e, as strongly associated with inflammatory bowel disease (IBD) (\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These findings highlight the importance of autophagy in intestinal homeostasis and implicate its disruption in IBD pathogenesis. Functional studies in mouse and cell-based models have revealed both epithelial-intrinsic roles, including maintenance of barrier integrity and cell survival (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and immune-mediated extrinsic roles, such as regulating inflammatory responses and microbial balance (\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent studies have shown that the prototypical autophagy regulator BECLIN1 is critical for intestinal homeostasis. Constitutive activation of BECLIN1-dependent autophagy alleviates goblet cell ER stress, thickening the mucus barrier and protecting against chemical- and infection-driven inflammation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Beyond autophagy, BECLIN1 regulates endocytic trafficking, maintaining barrier integrity via correct junctional protein localisation, cytoskeletal organisation, and epithelial remodelling (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Complete intestinal epithelial deletion of BECLIN1 is rapidly fatal due to severe enterocolitis with extensive epithelial cell loss, impaired specialised IEC functions, inflammation, and barrier breakdown (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBECLIN1 undergoes extensive post-translational modifications that fine-tune its activity and interactions (\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In diseases, however, it is reduced BECLIN1 expression rather than mutation that contributes to pathogenesis (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), with changes arising from altered protein interactions, cleavage, epigenetic regulation, or monoallelic deletion (\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Despite this, the consequences of partial BECLIN1 loss in the intestinal epithelium remain poorly understood. Here, we demonstrate that partial loss of BECLIN1 impairs goblet cell function and increases susceptibility to inflammation. By dissecting its roles in endocytic trafficking, cytoskeletal organisation and mucin secretion, we provide new insights into how BECLIN1 insufficiency destabilises epithelial integrity and compromises barrier defence.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eAll mouse strains used in this study were bred on the C57BL/6 J background. Becn1\u003csup\u003etm1b(KOMP)Wtsi\u003c/sup\u003e mice were purchased from the European Conditional Mouse Mutagenesis Program (EUCOMM). Becn1\u003csup\u003efl/fl\u003c/sup\u003e mice were generated by breeding Becn1tm1b(KOMP)Wtsi mice onto CAG-FLPe mice. \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e;, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/fl\u003c/em\u003e\u003c/sup\u003e;, and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e mice were then subsequently generated by breeding \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice to the Vil1-CreERT2 mice (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMice were housed at the La Trobe Animal Research and Teaching Facility (LARTF, La Trobe University, VIC, Australia) under Specific Pathogen Free (SPF) conditions. All experiments performed were approved by the La Trobe University animal ethics committees (approvals AEC18024, AEC18036) in accordance with the Australian code for the care and use of animals for scientific purposes. All research with these mice has complied with all relevant ethical regulations for animal use. To induce gene deletion, male and female mice aged six weeks or older, were selected indiscriminately and intraperitoneally injected with 4 mg tamoxifen (T5648, Sigma-Aldrich, St. Louis, Missouri, USA) in sunflower seed oil (25007, Sigma-Aldrich, St. Louis, Missouri, USA), delivered as one 200 \u0026micro;l injection per day of a 10 mg/ml stock, over two consecutive days. Mice were humanely euthanised by CO\u003csub\u003e2\u003c/sub\u003e asphyxiation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDextran Sulphate Sodium (DSS) treatment\u003c/h3\u003e\n\u003cp\u003eFollowing Tamoxifen treatment to induce gene deletion, mice were administered DSS (2% (w/v), MP Biomedicals, Irvine, California, USA) in autoclaved drinking water provided \u003cem\u003ead libitum\u003c/em\u003e for 5 days. Control groups received autoclaved drinking water without DSS for the same duration. During the experimental period, mice were monitored daily and weighed. The average daily intake of 2% (w/v) DSS water was calculated by measuring the weight difference of drinking bottles at the start and end of the experiment and dividing by the number of treatment days. The disease activity index (DAI) was assessed using a cumulative scoring system based on parameters outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, including weight loss, stool consistency and the presence of hematochezia (presence of blood in stool). At the experimental endpoint, mice were humanely euthanised using CO\u003csub\u003e2\u003c/sub\u003e asphyxiation. The small and large intestine were harvested, and their lengths were measured prior to rolling and fixation in 10% (v/v) neutral buffered formalin for 48 hours.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eScoring system for the evaluation of colitis severity in mice based on weight loss, stool consistency and haematochezia parameters.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eScore\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeight loss (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStool consistency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHaematochezia\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo blood\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVisible blood in rectum\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePasty, semiformed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVisible blood on fur\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLiquid, sticky or unable to defecate after 5 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eIntestinal organoid culture\u003c/h3\u003e\n\u003cp\u003eOrganoids were established by culturing crypt-enriched fractions from the duodenum of untreated mice as described previously (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). To induce \u003cem\u003eBecn1\u003c/em\u003e deletion, organoids were seeded into media containing 200 nM 4-hydroxytamoxifen (4-HT) (H7904, Sigma-Aldrich, St. Louis, Missouri, USA) for three days and then maintained as per normal. For organoid re-passaging experiments, 4-HT-treated organoids were passaged at day 7 and maintained as above.\u003c/p\u003e\n\u003ch3\u003eIntestinal epithelial cell isolation\u003c/h3\u003e\n\u003cp\u003eThe mouse small intestine was opened longitudinally, rinsed with Dulbecco\u0026rsquo;s Phosphate Buffered Saline (dPBS), and incubated in dPBS\u0026thinsp;+\u0026thinsp;15 mM EDTA for 15 minutes at 37\u0026deg;C with agitation. Dissociation of IECs was achieved by vortexing. The cell suspension was then washed once with ice-cold dPBS, and the cell pellet snap-frozen on dry ice and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e\n\u003ch3\u003eHistology, immunohistochemistry and organoid whole-mount immunofluorescence\u003c/h3\u003e\n\u003cp\u003ePreparation of intestinal \u0026ldquo;Swiss-rolls\u0026rdquo;, haematoxylin and eosin (H\u0026amp;E), Periodic acid-Schiff, Alcian blue (PAS-AB) and Ki67 staining were all performed as described previously (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). All slides were scanned using the Aperio AT2 (Leica, Wetzlar, Germany) and images captured and analysed using Aperio ImageScope v12.4.2.5010 and Fiji ImageJ. Whole-mount staining of intestinal organoids was performed as previously described (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblotting of mouse tissues\u003c/h2\u003e\u003cp\u003ePreparation of mouse cell lysates and immunoblotting was performed as described previously (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Primary antibodies were: Beclin1, 1:500 (3495, Cell Signaling Technology, Danvers, Massachusetts, USA); p62/SQSTM1, 1:500 (5114, Cell Signaling Technology, Danvers, Massachusetts, USA); LC3B, 1:500 (NB100-2220, Novus Biologicals, Centennial Colorado, USA); β-Actin, 1:5 000 (A2228, Sigma-Aldrich, St. Louis, Missouri, USA); GAPDH, 1:5 000 (MA5-15738, Invitrogen, Waltham, Massachusetts, USA). Secondary antibodies used were: Donkey anti-Rabbit IgG, 1:10 000 (NA943V, GE Healthcare, Chicago, Illinois, USA); Goat anti-Mouse IgG, 1:10 000 (A0168, Sigma-Aldrich, St. Louis, Missouri, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eGeneration of mice with inducible monoallelic deletion of\u003c/b\u003e \u003cb\u003eBecn1\u003c/b\u003e \u003cb\u003ein the intestinal epithelium\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe previously reported that homozygous \u003cem\u003eBecn1\u003c/em\u003e deletion in the intestinal epithelium results in rapid, fatal enterocolitis, with severe epithelial disruption (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). To model partial BECLIN1 loss, we generated mice in which monoallelic \u003cem\u003eBecn1\u003c/em\u003e deletion could be induced in the intestinal epithelium using mice heterozygous for the \u003cem\u003eBecn1\u003c/em\u003e floxed allele (\u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e and carrying the \u003cem\u003eVil1-Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e transgene (\u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e). Littermates lacking the floxed allele (\u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e) but positive for \u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e) served as controls. Intraperitoneal Tamoxifen injections induced deletion in the small intestine (duodenum, jejunum, ileum) and colon of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e mice, confirmed genomically and by protein analysis showing approximately 50% reduced BECLIN1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1A). Tamoxifen-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e mice are referred to as \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice respectively throughout.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMice with reduced BECLIN1 have shortened small intestines and reduced crypt lengths without other overt phenotypes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 7 days post-BECLIN1 deletion, there were no significant body weight differences between \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;1B), unlike the marked weight loss previously seen with homozygous \u003cem\u003eBecn1\u003c/em\u003e deletion in the intestinal epithelium (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Despite appearing grossly normal, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed significantly shorter small intestines than \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls, while colon lengths were unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Histological examination by H\u0026amp;E staining revealed largely normal small and large intestinal morphology in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At this time point, distal colonic crypt length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and width (Supplementary Fig.\u0026nbsp;1C) did not differ between genotypes. However, by 14- and 35-days post deletion, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited significantly shorter colonic crypts and reduced crypt volume fraction (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Observation over one month post deletion also revealed a persistently shorter small intestine (Supplementary Fig.\u0026nbsp;1D) without weight loss (Supplementary Fig.\u0026nbsp;1B), and otherwise normal intestinal morphology (Supplementary Fig.\u0026nbsp;1E). These phenotypes contrasted sharply with \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, where small intestines were shortened, swollen, and lytic, with villus stunting along the entire length within 7 days of gene deletion (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The need to euthanise \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e⁻/⁻\u003c/sup\u003e mice within 7 days precluded investigation of equivalent colonic changes. In summary, apart from reduced small intestinal and colonic crypt lengths, heterozygous \u003cem\u003eBecn1\u003c/em\u003e deletion does not cause overt abnormalities under basal conditions up to one month post deletion, in contrast to its homozygous loss (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntestinal epithelial cells with reduced BECLIN1 levels display no disruption to basal autophagy, differentiation, or proliferation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMonoallelic \u003cem\u003eBecn1\u003c/em\u003e deletion caused minimal changes in basal autophagy, with total P62 and LC3B levels comparable to wild-type IECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In contrast, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs showed pronounced P62 and LC3B accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). We previously identified an essential role for BECLIN1 in endocytic trafficking in intestinal-derived organoids (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). To test whether monoallelic \u003cem\u003eBecn1\u003c/em\u003e loss induced similar defects, we generated organoids from \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e; and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eVil1-CreERT2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre/+\u003c/em\u003e\u003c/sup\u003e mice. Treatment with 4-hydroxytamoxifen (4-HT) led to reduced BECLIN1 expression in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e organoids which did not impact basal autophagy (as with intestinal epithelial cells from mice), unlike homozygous deletion (Supplementary Figs.\u0026nbsp;2A, B).\u003c/p\u003e\u003cp\u003eAt 7 days post 4-HT treatment, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids showed significantly reduced budding crypt formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), whereas \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids formed similar crypt-like projections as \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e organoids, indicating normal IEC proliferation and differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Re-passaging of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e yielded mature organoids by Day 5, with similar numbers of budding crypts, further indicating minimal defects in proliferation or differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). This is consistent with Ki67 staining of intestinal tissues from \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showing no proliferation differences, including at the crypt bases (Supplementary Fig.\u0026nbsp;2C). In contrast, surviving \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids failed to generate viable organoid structures upon re-passaging, producing significantly fewer crypt-like projections by Day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Thus, reduced BECLIN1 expression does not impair basal autophagy, survival, differentiation, or proliferation of IECs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntestinal epithelial cells with reduced BECLIN1 levels display changes in early endocytic trafficking and E-CADHERIN distribution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBECLIN1 loss has been reported to affect endocytic trafficking in other tissues, similar to the effects of homozygous loss in the gut (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40 CR41\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). To determine whether this also occurs following heterozygous loss in intestinal epithelial cells, we examined \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids and found a modest but significant increase in cytoplasmic RAB5\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e early endosomes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, Supplementary Fig.\u0026nbsp;3) though without the aberrant enlargement seen in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C). As the junctional protein E-CADHERIN can become trapped within enlarged RAB5\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e early endosomes in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, D, E) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), we examined its localisation in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs. Here, E-CADHERIN levels were increased at apical and lateral membranes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G) and in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), but notably, co-localisation with RAB5 was unchanged at all subcellular sites (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, H, I). Thus, reduced BECLIN1 alters early endosomal dynamics, but E-CADHERIN localisation changes appear independent of RAB5-mediated trafficking.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMonoallelic\u003c/b\u003e \u003cb\u003eBecn1\u003c/b\u003e \u003cb\u003edeletion alters F-actin organisation and intestinal epithelial cell architecture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs homozygous BECLIN1 loss disrupts the cytoskeleton and compromises the intestinal epithelial barrier (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), we examined whether altered F-actin organisation, critical for adhesion and endocytosis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) contributes to the E-CADHERIN changes in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids. Here, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs displayed significantly increased F-actin and co-localised E-CADHERIN along the lateral membrane but not apical membrane (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E), consistent with an F-actin-driven \u0026ldquo;cadherin flow\u0026rdquo; mechanism, whereby cadherins are transported along remodelling actin networks (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), potentially contributing to altered E-CADHERIN localisation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, E). Notably, E-CADHERIN was redistributed within the lateral membrane toward apicolateral junctions, possibly to maintain cell-cell contacts and preserve epithelial integrity. Given this increased lateral membrane localisation of both E-CADHERIN and F-actin in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), we assessed cell morphology and found \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e organoids had a longer apico-basal axis and shorter basal IEC width (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, F, G), likely contributing to the shorter small intestinal length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Reduced BECLIN1 therefore alters adhesion dynamics, cytoskeletal organisation, and epithelial architecture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eReduced BECLIN1 levels in intestinal epithelial cells increases susceptibility to DSS-induced colitis\u003c/h3\u003e\n\u003cp\u003eTo test whether the changes in endocytic trafficking, intercellular adhesion, and cytoskeleton architecture observed in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice increase susceptibility to inflammation, 7- to 14-week-old mice were treated with dextran sulphate sodium (DSS) to induce colitis, a widely used model of IBD pathogenesis (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Following Tamoxifen-induced gene deletion, mice received either 2% DSS in drinking water for 5 days or normal water (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). There were no genotype-dependent differences in starting body weight or DSS-water consumption (Supplementary Figs.\u0026nbsp;4A, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt endpoint, both DSS-treated genotypes displayed expected colitic changes, including darker, reddish areas in the colon consistent with hyperemia/inflammation (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C) and colon shortening with swelling and constriction (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D), but gross colonic changes were similar (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). The small intestines of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were significantly shorter than \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls regardless of DSS treatment (Supplementary Fig.\u0026nbsp;4C). Both genotypes lost weight after DSS exposure, but this was greater in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). They also developed more severe diarrhea, with a higher proportion producing pasty to liquid stools (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), though hematochezia did not differ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Importantly, the composite disease activity index (DAI), incorporating weight loss, stool consistency, and rectal bleeding (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), was significantly higher in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, indicating greater colitis severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eHistological assessment of DSS-treated colons was performed using a blinded scoring system evaluating crypt architecture, inflammatory cell infiltration, and epithelial integrity, with the composite score reported as the histological colitis score (HCS) (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Untreated mice of both genotypes had minimal mucosal damage and low histological colitis scores (HCS) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J, K). DSS treatment increased the HCS scores in both genotypes, but \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice scored approximately 2.2-fold higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), with more severe epithelial injury and crypt attenuation, epithelial loss, and immune infiltration (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J). These mice also had more regions of epithelial erosion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), characterised by complete crypt loss and dense localised immune cell infiltration. Lymphoid follicle counts and sizes did not differ between groups (Supplementary Figs.\u0026nbsp;4D, E), indicating that inflammation was largely confined to the epithelial layer. Hence, monoallelic \u003cem\u003eBecn1\u003c/em\u003e loss worsens DSS-induced colitis, indicating increased sensitivity to intestinal inflammation (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-L).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMonoallelic\u003c/b\u003e \u003cb\u003eBecn1\u003c/b\u003e \u003cb\u003edeletion causes goblet cell defects and compromises mucosal immunity under basal conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGoblet cells are secretory IECs specialised for mucus production, forming a critical barrier against pathogens and mechanical damage (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Disruption of this barrier is a hallmark of chronic inflammatory conditions, including IBD, often arising from goblet cell loss or defects in mucin formation, storage, or secretion (\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Periodic-Acid-Schiff (PAS) and Alcian Blue (AB) staining revealed a significant reduction in PAS-AB staining in the distal colon (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), but not the proximal colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), of untreated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to untreated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), suggesting either fewer goblet cells and/or reduced mucin production (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). As goblet cell maturation involves migration to the crypt surface, we also quantified goblet cells in the lower (immature) and upper (mature) colonic crypt compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;5) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). PAS-AB\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e mucin staining was reduced in both lower and upper crypt compartments, but more so in the latter (50% \u003cem\u003eversus\u003c/em\u003e 15%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This maturation defect was further supported by the enlarged cytoplasmic mucin (theca) areas in remaining mature goblet cells of untreated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared with \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), consistent with impaired mucin secretion. These results show that reduced BECLIN1 results in goblet cell loss and defective mucin production, potentially compromising mucosal barrier function even under basal conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eReduction of BECLIN1 levels exacerbates DSS-induced barrier dysfunction\u003c/h2\u003e\u003cp\u003eWe next investigated goblet cell responses in DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. The epithelial damage in both DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L) was accompanied by a significant reduction in PAS-AB\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e mucin area in the distal colon compared with their respective untreated controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), consistent with the distal colon being the primary site of DSS-induced injury (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The epithelial damage in the distal colon was further exacerbated in DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, where the PAS-AB\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e mucin area was significantly reduced when compared with DSS-treated \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). We also assessed PAS-AB staining across the entire colon, which revealed a loss of PAS\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e neutral mucins in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e in the untreated groups, with no significant change in AB\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e acidic mucins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Notably, following DSS treatment, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited a loss of both neutral and acidic mucins, consistent with the exacerbated epithelial damage observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L). This reveals a vulnerability of the mucosal barrier when BECLIN1 is reduced, reinforcing its role in epithelial homeostasis. The selective baseline loss of neutral mucins, and subsequent loss of both mucin types after DSS treatment, highlights the critical importance of neutral mucins in protecting against DSS-induced colitis.\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe previously identified an essential role for BECLIN1 in maintaining intestinal homeostasis and barrier integrity mediated through its autophagy and endocytic trafficking functions (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Given that homozygous \u003cem\u003eBecn1\u003c/em\u003e deletion causes fatal enteritis in adult mice (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), we investigated whether partial reduction, mimicking clinically relevant haploinsufficiency, also impairs intestinal function.\u003c/p\u003e\u003cp\u003eUnlike homozygous BECLIN1 deletion, monoallelic loss did not trigger overt spontaneous disease. However, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did display subclinical abnormalities, including shortened small intestines, reduced distal colonic crypt length, and goblet cell loss \u0026ndash; hallmarks of compromised epithelial integrity. These changes occurred despite minimal changes in basal autophagy, suggesting BECLIN1\u0026rsquo;s trafficking and epithelial organisation roles may be more critical in this context (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Altered tissue architecture was marked by increased apico-basal length, reduced basal width, and redistribution of E-CADHERIN and F-actin along the lateral membranes, likely reflecting cytoskeletal remodelling. These features mirror BECLIN1-null IECs, where E-CADHERIN becomes trapped in early endosomes (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile cytoplasmic RAB5\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e vesicles were modestly increased, early endosome enlargement was absent in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cells, suggesting a threshold effect in trafficking disruption. Increased co-localisation of E-CADHERIN with lateral F-actin in \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e IECs may stabilise junctional integrity under impaired trafficking (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), though altered F-actin organisation can also impair IEC contractility, crypt architecture (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e), and mucin granule exocytosis (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGoblet cells were disproportionately affected, with reduced numbers, altered maturation (larger thecae), and diminished mucin staining, especially in the upper crypt. This aligns with the known role of autophagy in promoting goblet cell function via ER stress alleviation and mucin handling (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Following DSS challenge, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice failed to mount the same level of compensatory mucin response as wild-type controls, showing depletion of both neutral and acidic mucins. The baseline vulnerability due to selective mucin loss, compounded by the broad depletion after DSS treatment, highlights the critical role of neutral mucins in maintaining the buffering capacity of the mucus barrier. Loss of the major secreted mucin, MUC2, similarly causes spontaneous colitis in mice (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) and comparable mucin shifts occur in human IBD (\u003cspan additionalcitationids=\"CR60 CR61 CR62 CR63\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTogether, our findings suggest that beyond its canonical autophagy role, BECLIN1 integrates trafficking, cytoskeletal dynamics, and mucosal defence. The heightened DSS susceptibility of \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, despite inflammation remaining confined to the mucosa, supports a barrier-specific defect consistent with goblet cell dysfunction and epithelial disorganisation.\u003c/p\u003e\u003cp\u003eAlthough IBD GWAS have not identified BECLIN1 single nucleotide polymorphisms (SNPs), emerging evidence implicates both IEC-intrinsic and -extrinsic mechanisms that lower BECLIN1 levels, thereby exacerbating colitis. For instance, cyclic GMP-AMP synthase (cGAS) promotes autophagy in IECs through interaction with BECLIN1, and its loss worsens colitis (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Similarly, the m\u003csup\u003e6\u003c/sup\u003eA nuclear reader, YTHDC1, stabilises \u003cem\u003eBecn1\u003c/em\u003e mRNA and is downregulated in DSS colitis and in IBD patient microbiota (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Moreover, while no IBD-associated SNPs have been identified within \u003cem\u003eBecn1\u003c/em\u003e itself, variants in upstream regulators such as STAT3, a transcriptional regulator of \u003cem\u003eBecn1\u003c/em\u003e, have been implicated in IBD pathogenesis (\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Caspase-mediated cleavage of BECLIN1 during apoptosis may also contribute to reduced protein levels in inflamed tissue, amplifying epithelial injury (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLooking forward, resources such as atlases of IBD patient samples (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e) provide opportunities to examine BECLIN1 expression at endoscopically defined disease margins. Expanding clinical analyses will be critical to determine whether there are changes in BECLIN1 levels across broader clinical cohorts and to assess its potential as a biomarker of IBD pathogenesis and severity.\u003c/p\u003e\u003cp\u003eIn summary, partial BECLIN1 reduction compromises epithelial homeostasis and exacerbates inflammation in the absence of complete autophagy loss. These findings establish BECLIN1 as a critical epithelial defence regulator. Future studies should explore whether restoring BECLIN1 levels or mimicking its trafficking-related functions could preserve barrier integrity and enable patient stratification for precision therapeutic interventions in IBD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\u003cp\u003eJ.M.Murphy, A.L.S., K.M.P., and S.N.Y. contribute to, or have contributed to, the development of necroptosis pathway inhibitors in collaboration with Anaxis Pharma Pty. Ltd. J.M.Murphy has also received research funding from Anaxis Pharma Pty. Ltd. All other authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\u003cp\u003eJ.J., S.T., designed, performed, and analysed experiments and wrote the paper; T.J.H., Y.W.N.R., S.L.E., A.H.A-A, K.P., S.N.Y., M.E., D.B., C.M.R., R.N., L.J.J., designed, performed experiments, analysed and interpreted data; P.D.C., K.D., B.T.K., A.S.Y., J.M.M., B.C., A.L.S., J.M.Murphy analysed and interpreted data; W.D.F., E.F.L. designed the project, analysed, interpreted data, and wrote the paper. All authors commented on the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eWe acknowledge scholarship support for J.J. (La Trobe Graduate Research Scholarship and Full Fee Research Scholarship), S.T. (La Trobe University Research Training Program Scholarship) and A.H.A.-A (University of Melbourne Australian Commonwealth Government Research Training Program, Crohn\u0026rsquo;s and Colitis Australia (IBD PhD Scholarship), Avant (Doctors in Training Scholarship), and the Gastroenterological Society of Australia (Celltrion IBD Fellowship). We are grateful to the Australian Research Council for grant support to E.F.L., W.D.F., J.M.Mariadason (DP190102612); the National Health and Medical Research Council to A.L.S. (2002965, 2011584), K.D. and A.S.Y. (2010704, 1136592), PDC (2008909), J.M.Murphy (1172929, 2034104); NHMRC IRIISS and the Victorian Government Operational Infrastructure Support Scheme; the Kenneth Rainin Foundation to J.M.Murphy, B.C., A.L.S. and A.H.A.-A; the US DOD (HT94252310088) to A.S.Y.; and the Victorian Cancer Agency to E.F.L. (MCRF19045) for fellowship support.\u003c/p\u003e\u003ch2\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/h2\u003e\u003cp\u003eCorrespondence and requests for materials should be addressed to Erinna F. Lee or Walter D. Fairlie.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHenckaerts L, Cleynen I, Brinar M, John JM, Van Steen K, Rutgeerts P, et al. Genetic variation in the autophagy gene ULK1 and risk of Crohn's disease. Inflamm Bowel Dis. 2011;17(6):1392\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLassen KG, Kuballa P, Conway KL, Patel KK, Becker CE, Peloquin JM, et al. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc Natl Acad Sci U S A. 2014;111(21):7741\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParkes M, Barrett JC, Prescott NJ, Tremelling M, Anderson CA, Fisher SA, et al. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility. Nat Genet. 2007;39(7):830\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrescott NJ, Fisher SA, Franke A, Hampe J, Onnie CM, Soars D, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn's disease and is independent of CARD15 and IBD5. Gastroenterology. 2007;132(5):1665\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39(5):596\u0026ndash;604.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTran S, Juliani J, Fairlie WD, Lee EF. The emerging roles of autophagy in intestinal epithelial cells and its links to inflammatory bowel disease. Biochem Soc Trans. 2023;51(2):811\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWellcome Trust Case Control C. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007;447(7145):661\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBenjamin JL, Sumpter R, Jr., Levine B, Hooper LV. Intestinal epithelial autophagy is essential for host defense against invasive bacteria. Cell Host Microbe. 2013;13(6):723\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, Lennerz JK, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature. 2008;456(7219):259\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCadwell K, Patel KK, Komatsu M, Virgin HWt, Stappenbeck TS. A common role for Atg16L1, Atg5 and Atg7 in small intestinal Paneth cells and Crohn disease. Autophagy. 2009;5(2):250\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaama M, Telpaz S, Awad A, Ben-Simon S, Harshuk-Shabso S, Modilevsky S, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress. Cell Host Microbe. 2023;31(3):433\u0026ndash;46 e4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNighot PK, Blikslager AT. Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin. Am J Physiol Cell Physiol. 2012;302(1):C178-87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel KK, Miyoshi H, Beatty WL, Head RD, Malvin NP, Cadwell K, et al. Autophagy proteins control goblet cell function by potentiating reactive oxygen species production. EMBO J. 2013;32(24):3130\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaha K, Subramenium Ganapathy A, Wang A, Michael Morris N, Suchanec E, Ding W, et al. Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux. J Crohns Colitis. 2023;17(3):433\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWittkopf N, Gunther C, Martini E, Waldner M, Amann KU, Neurath MF, et al. Lack of intestinal epithelial atg7 affects paneth cell granule formation but does not compromise immune homeostasis in the gut. Clin Dev Immunol. 2012;2012:278059.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao Z, Fux B, Goodwin M, Dunay IR, Strong D, Miller BC, et al. Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host Microbe. 2008;4(5):458\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCooney R, Baker J, Brain O, Danis B, Pichulik T, Allan P, et al. NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nat Med. 2010;16(1):90\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartin PK, Marchiando A, Xu R, Rudensky E, Yeung F, Schuster SL, et al. Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota. Nat Microbiol. 2018;3(10):1131\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature. 2008;456(7219):264\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanjuan MA, Dillon CP, Tait SW, Moshiach S, Dorsey F, Connell S, et al. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007;450(7173):1253\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStrisciuglio C, Duijvestein M, Verhaar AP, Vos AC, van den Brink GR, Hommes DW, et al. Impaired autophagy leads to abnormal dendritic cell-epithelial cell interactions. J Crohns Colitis. 2013;7(7):534\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJuliani J, Sharon T, J. HT, Peter DC, L. ES, A. GP, et al. BECLIN-1 is essential for the maintenance of gastrointestinal epithelial integrity by regulating endocytic trafficking, F-actin organization, and lysosomal function. Autophagy Reports. 2025;4(1):2484494.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTran S, Juliani J, Harris TJ, Evangelista M, Ratcliffe J, Ellis SL, et al. BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking. Commun Biol. 2024;7(1):209.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong M, Ganapathy AS, Suchanec E, Laidler L, Ma T, Nighot P. Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1. Am J Physiol Cell Physiol. 2019;316(5):C753-C65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHill SM, Wrobel L, Rubinsztein DC. Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation. Cell Death Differ. 2019;26(4):617\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee EF, Perugini MA, Pettikiriarachchi A, Evangelista M, Keizer DW, Yao S, et al. The BECN1 N-terminal domain is intrinsically disordered. Autophagy. 2016;12(3):460\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee EF, Smith NA, Soares da Costa TP, Meftahi N, Yao S, Harris TJ, et al. Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1. Autophagy. 2019;15(5):785\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTran S, Fairlie WD, Lee EF. BECLIN1: Protein Structure, Function and Regulation. Cells. 2021;10(6).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, et al. Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells. Oncogene. 2013;32(18):2261-72, 72e 1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999;402(6762):672\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, et al. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. J Clin Invest. 2008;118(6):2190\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Chen B, Wu Y, Jin F, Xia Y, Liu X. Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors. BMC Cancer. 2010;10:98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003;112(12):1809\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRohn TT, Wirawan E, Brown RJ, Harris JR, Masliah E, Vandenabeele P. Depletion of Beclin-1 due to proteolytic cleavage by caspases in the Alzheimer's disease brain. Neurobiol Dis. 2011;43(1):68\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou L, Zhang L, Lv Y, Qian J, Huang L, Qu C. YTHDC1 inhibits autophagy-dependent NF-kappaB signaling by stabilizing Beclin1 mRNA in macrophages. J Inflamm (Lond). 2024;21(1):22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eel Marjou F, Janssen KP, Chang BH, Li M, Hindie V, Chan L, et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis. 2004;39(3):186\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeRoche TC, Xiao SY, Liu X. Histological evaluation in ulcerative colitis. Gastroenterol Rep (Oxf). 2014;2(3):178\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatthew-Onabanjo AN, Janusis J, Mercado-Matos J, Carlisle AE, Kim D, Levine F, et al. Beclin 1 Promotes Endosome Recruitment of Hepatocyte Growth Factor Tyrosine Kinase Substrate to Suppress Tumor Proliferation. Cancer Res. 2020;80(2):249\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcKnight NC, Zhong Y, Wold MS, Gong S, Phillips GR, Dou Z, et al. Beclin 1 is required for neuron viability and regulates endosome pathways via the UVRAG-VPS34 complex. PLoS Genet. 2014;10(10):e1004626.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNoguchi S, Honda S, Saitoh T, Matsumura H, Nishimura E, Akira S, et al. Beclin 1 regulates recycling endosome and is required for skin development in mice. Commun Biol. 2019;2:37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRohatgi RA, Janusis J, Leonard D, Bellve KD, Fogarty KE, Baehrecke EH, et al. Beclin 1 regulates growth factor receptor signaling in breast cancer. Oncogene. 2015;34(42):5352\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWijshake T, Zou Z, Chen B, Zhong L, Xiao G, Xie Y, et al. Tumor-suppressor function of Beclin 1 in breast cancer cells requires E-cadherin. Proc Natl Acad Sci U S A. 2021;118(5).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eApodaca G. Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton. Traffic. 2001;2(3):149\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVasioukhin V, Bauer C, Yin M, Fuchs E. Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell. 2000;100(2):209\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWoichansky I, Beretta CA, Berns N, Riechmann V. Three mechanisms control E-cadherin localization to the zonula adherens. Nat Commun. 2016;7:10834.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMizoguchi A. Animal models of inflammatory bowel disease. Prog Mol Biol Transl Sci. 2012;105:263\u0026ndash;320.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark YH, Kim N, Shim YK, Choi YJ, Nam RH, Choi YJ, et al. Adequate Dextran Sodium Sulfate-induced Colitis Model in Mice and Effective Outcome Measurement Method. J Cancer Prev. 2015;20(4):260\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReehorst CM, Nightingale R, Luk IY, Jenkins L, Koentgen F, Williams DS, et al. EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis. Development. 2021;148(12).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarcia-Hernandez V, Neumann PA, Koch S, Lyons R, Nusrat A, Parkos CA. Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model. J Vis Exp. 2021(168).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGustafsson JK, Johansson MEV. The role of goblet cells and mucus in intestinal homeostasis. Nat Rev Gastroenterol Hepatol. 2022;19(12):785\u0026ndash;803.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuisine MP, Desreumaux P, Leteurtre E, Copin MC, Colombel JF, Porchet N, et al. Mucin gene expression in intestinal epithelial cells in Crohn's disease. Gut. 2001;49(4):544\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi WT, Salomao M, Zhao L, Alpert L, Setia N, Liao X, et al. Hypermucinous, Goblet Cell-Deficient and Crypt Cell Dysplasias in Inflammatory Bowel Disease are Often Associated with Flat/Invisible Endoscopic Appearance and Advanced Neoplasia on Follow-Up. J Crohns Colitis. 2022;16(1):98\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan der Sluis M, De Koning BA, De Bruijn AC, Velcich A, Meijerink JP, Van Goudoever JB, et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology. 2006;131(1):117\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLaroui H, Ingersoll SA, Liu HC, Baker MT, Ayyadurai S, Charania MA, et al. Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon. PLoS One. 2012;7(3):e32084.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGersemann M, Becker S, Kubler I, Koslowski M, Wang G, Herrlinger KR, et al. Differences in goblet cell differentiation between Crohn's disease and ulcerative colitis. Differentiation. 2009;77(1):84\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHinnant T, Ning W, Lechler T. Compartment specific responses to contractility in the small intestinal epithelium. PLoS Genet. 2024;20(3):e1010899.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEhre C, Rossi AH, Abdullah LH, De Pestel K, Hill S, Olsen JC, et al. Barrier role of actin filaments in regulated mucin secretion from airway goblet cells. Am J Physiol Cell Physiol. 2005;288(1):C46-56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOliver MG, Specian RD. Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion. Am J Physiol. 1990;259(6 Pt 1):G991-7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampbell BJ, Yu LG, Rhodes JM. Altered glycosylation in inflammatory bowel disease: a possible role in cancer development. Glycoconj J. 2001;18(11\u0026ndash;12):851\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorfield AP, Myerscough N, Bradfield N, Corfield Cdo A, Gough M, Clamp JR, et al. Colonic mucins in ulcerative colitis: evidence for loss of sulfation. Glycoconj J. 1996;13(5):809\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLarsson JM, Karlsson H, Crespo JG, Johansson ME, Eklund L, Sjovall H, et al. Altered O-glycosylation profile of MUC2 mucin occurs in active ulcerative colitis and is associated with increased inflammation. Inflamm Bowel Dis. 2011;17(11):2299\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcGuckin MA, Linden SK, Sutton P, Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Microbiol. 2011;9(4):265\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaouf AH, Tsai HH, Parker N, Hoffman J, Walker RJ, Rhodes JM. Sulphation of colonic and rectal mucin in inflammatory bowel disease: reduced sulphation of rectal mucus in ulcerative colitis. Clin Sci (Lond). 1992;83(5):623\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Klinken BJ, Van der Wal JW, Einerhand AW, Buller HA, Dekker J. Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis. Gut. 1999;44(3):387\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan S, Mentrup HL, Novak EA, Siow VS, Wang Q, Crawford EC, et al. Cyclic GMP-AMP synthase contributes to epithelial homeostasis in intestinal inflammation via Beclin-1-mediated autophagy. FASEB J. 2022;36(5):e22282.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGe X, Xue G, Ding Y, Li R, Hu K, Xu T, et al. The Loss of YTHDC1 in Gut Macrophages Exacerbates Inflammatory Bowel Disease. Adv Sci (Weinh). 2023;10(14):e2205620.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerguson LR, Han DY, Fraser AG, Huebner C, Lam WJ, Morgan AR, et al. Genetic factors in chronic inflammation: single nucleotide polymorphisms in the STAT-JAK pathway, susceptibility to DNA damage and Crohn's disease in a New Zealand population. Mutat Res. 2010;690(1\u0026ndash;2):108\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiao LJ, Huang FX, Sun ZT, Zhang RX, Huang SF, Wang J. Stat3 inhibits Beclin 1 expression through recruitment of HDAC3 in nonsmall cell lung cancer cells. Tumour Biol. 2014;35(7):7097\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang Y, Tan SA, Iqbal A, Li J, Glover SC. STAT3 Genotypic Variant rs744166 and Increased Tyrosine Phosphorylation of STAT3 in IL-23 Responsive Innate Lymphoid Cells during Pathogenesis of Crohn's Disease. J Immunol Res. 2019;2019:9406146.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC. Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett. 2009;274(1):95\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, et al. Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis. 2010;1(1):e18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChiou S, Al-Ani AH, Pan Y, Patel KM, Kong IY, Whitehead LW, et al. An immunohistochemical atlas of necroptotic pathway expression. EMBO Mol Med. 2024;16(7):1717\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePang J, Al-Ani AH, Patel KM, Young SN, Kong I, Chen J-j, et al. A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease. bioRxiv. 2024:2024.11.13.623307.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7567745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7567745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBECLIN1 is a central regulator of autophagy and endocytic trafficking essential for epithelial homeostasis. While complete intestinal epithelial loss of BECLIN1 causes fatal enteritis, the consequence of its partial loss in the gut remains unclear. Given that BECLIN1 expression can vary in human disease, we investigated whether reduced BECLIN1 is sufficient to impair gut barrier function. Heterozygous \u003cem\u003eBecn1\u003c/em\u003e deletion (\u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) in the mouse intestinal epithelium caused subtle but functionally important defects, including shortened small intestines, reduced colonic crypt length, altered epithelial architecture, and loss of goblet cells with reduced mucin production, particularly in mature goblet cells. These changes occurred despite preservation of basal autophagy, implicating trafficking-related functions. Supporting this conclusion, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003eintestinal epithelial cells showed modest increases in RAB5\u003csup\u003e+\u0026thinsp;ve\u003c/sup\u003e vesicles, redistribution of E-CADHERIN with F-actin along lateral membranes, increased apico-basal cell length and reduced basal width. Following dextran sulfate sodium (DSS) treatment, \u003cem\u003eBecn1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited greater weight loss, higher disease activity, more severe histological colitis score, and disproportionate loss of neutral mucins, with inflammation confined to the mucosa. Goblet cell dysfunction likely underpinned these barrier defects. These findings establish that BECLIN1 insufficiency destabilises epithelial organisation and barrier defence, thereby sensitising the gut to inflammatory challenge and further positioning BECLIN1 as a key determinant of intestinal homeostasis.\u003c/p\u003e","manuscriptTitle":"Impact of BECLIN1 haploinsufficiency on goblet cell function and susceptibility to colitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 11:41:11","doi":"10.21203/rs.3.rs-7567745/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Cell Death \u0026 Disease","date":"2025-11-03T05:15:42+00:00","index":"","fulltext":""},{"type":"decision","content":"Reject after peer review","date":"2025-09-30T13:40:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-30T05:39:29+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-22T03:39:11+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-15T05:58:55+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-12T15:38:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-09-12T13:17:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T11:29:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T22:20:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2025-09-08T22:20:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a2ef2c28-c1f1-45ca-9359-716811f985c1","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54633434,"name":"Biological sciences/Cell biology/Autophagy/Macroautophagy"},{"id":54633435,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2025-11-07T16:31:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-19 11:41:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7567745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7567745","identity":"rs-7567745","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}