LAMP2 Expression Research Articles

Entosis Acts as a Novel Way within Sertoli Cells to Eliminate Spermatozoa in Seminiferous Tubule.

The present study was designed to investigate the hypothesis that in vivo entosis is a novel pathway for eliminating spermatozoa in the seminiferous tubules (ST) during hibernation of the Chinese soft-shelled turtle. Western blot analysis revealed that the expression of LAMP1 in the testis was significantly higher during hibernation than that during non-hibernation. Immunohistochemistry reaction showed that LAMP1-positive substance was distributed within the Sertoli cells of the testis. Further examination by transmission electron microscopy (TEM), many degraded spermatozoa being enwrapped within large entotic vacuoles in Sertoli cells. The nucleus and the flagellum of the spermatozoa were shown to be decomposed and digested inside entotic vacuoles within Sertoli cells. More than two spermatozoa heads were always observed in each internalized vacuoles. Deserving note is that, a number of different autophagosomes, including initial autophagic vesicles and degradative autophagic vesicles were found inside the entotic vacuoles of the Sertoli cells during hibernation. At the end of hibernation, entotic vacuoles and their autophagosomes disappeared, and numerous large lipid droplets (LDs) appeared within the Sertoli cells. Adherens junctions were apparent between Sertoli cells and developing germ cells, which is the ultrastructural basis of entosis. Taken together, the results presented here show that in the turtle: (1) entosis with internal autophagosomes can take place within normal body cells during hibernation; (2) spermatozoa, as a highly differentiated cell can be internalized and degraded within Sertoli cell by entosis in vivo, which is in favor of the next reproductive cycle in the turtle.

Impaired mitophagy facilitates mitochondrial damage in Danon disease

RationaleLysosomal associated membrane protein type-2 (LAMP-2) is a highly conserved, ubiquitous protein that is critical for autophagic flux. Loss of function mutations in the LAMP-2 gene cause Danon disease, a rare X-linked disorder characterized by developmental delay, skeletal muscle weakness, and severe cardiomyopathy. We previously found that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from Danon patients exhibited significant mitochondrial oxidative stress and apoptosis. Understanding how loss of LAMP-2 expression leads to cardiomyocyte dysfunction and heart failure has important implications for the treatment of Danon disease as well as a variety of other cardiac disorders associated with impaired autophagy. ObjectiveElucidate the pathophysiology of cardiac dysfunction in Danon disease. Methods and resultsWe created hiPSCs from two patients with Danon disease and differentiated those cells into hiPSC-CMs using well-established protocols. Danon hiPSC-CMs demonstrated an accumulation of damaged mitochondria, disrupted mitophagic flux, depressed mitochondrial respiratory capacity, and abnormal gene expression of key mitochondrial pathways. Restoring the expression of LAMP-2B, the most abundant LAMP-2 isoform in the heart, rescued mitophagic flux as well as mitochondrial health and bioenergetics. To confirm our findings in vivo, we evaluated Lamp-2 knockout (KO) mice. Impaired autophagic flux was noted in the Lamp-2 KO mice compared to WT reporter mice, as well as an increased number of abnormal mitochondria, evidence of incomplete mitophagy, and impaired mitochondrial respiration. Physiologically, Lamp-2 KO mice demonstrated early features of contractile dysfunction without overt heart failure, indicating that the metabolic abnormalities associated with Danon disease precede the development of end-stage disease and are not merely part of the secondary changes associated with heart failure. ConclusionsIncomplete mitophagic flux and mitochondrial dysfunction are noted in both in vitro and in vivo models of Danon disease, and proceed overt cardiac contractile dysfunction. This suggests that impaired mitochondrial clearance may be central to the pathogenesis of disease and a potential target for therapeutic intervention.

Pathogenic Role of Associated Adherent-Invasive Escherichia coli in Crohn's Disease.

Several lines of evidence suggest that adherent-invasive Escherichia coli (AIEC) strains play an important role in Crohn's disease (CD). The objective of this study was to investigate the pathogenic role of two AIEC strains, LF82 and O83:H1, in CD patients. Organ cultures of colonic biopsies from patients were set up to assess the effects of LF82 and O83:H1 on the expression of CEACAM6, LAMP1, HLA-DR, ICAM1 by immunohistochemistry and of IL-8, IFNʏ, and TNF-α genes by RT-PCR. Moreover, on Caco2 cells, we analyzed the cell cycle, the expression of MGMT and DNMT1 genes, and DNA damage induced by LF82 and O83:H1, by FACS, RT-PCR, and DAPI staining, respectively. Epithelial and lamina propria mononuclear cells (LPMNC) expression of CEACAM6 and LAMP1 were higher in biopsies cultured in the presence of both O83:H1 and LF82 than in biopsies cultured with non-pathogenic E. coli. Both AIEC strains induced increased expression of ICAM-1 on blood vessels and HLA-DR on LPMNC. We observed higher levels of TNF-α, IFN-γ, and IL-8 transcripts in biopsies cultured with both AIEC strains than in those cultured with NP. Both LF82 and O83:H1, block the cell cycle into S phase, inducing DNA damage, and modulate the expression of DNMT1 and MGMT genes. Our data suggest that LF82 and 083:H1 strains of E. coli are able to increase in CD colonic biopsies the expression of all the pro-inflammatory cytokines and all the mucosal immune markers investigated. J. Cell. Physiol. 232: 2860-2868, 2017. © 2016 Wiley Periodicals, Inc.

Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemia–reperfusion brain injury

Cerebral ischemia leads to astrocyte’s activation and glial scar formation. Glial scar can inhibit axonal regeneration during the recovery phase. It has demonstrated that sevoflurane has neuroprotective effects against ischemic stroke, but its effects on ischemia-induced formation of astrogliosis and glial scar are unknown. This study was designed to investigate the effect of sevoflurane postconditioning on astrogliosis and glial scar formation in ischemic stroke model both in vivo and in vitro. The results showed that 2.5% of sevoflurane postconditioning could significantly reduce infarction volume and improve neurologic deficits. And it could also decrease the expression of the glial scar marker glial fibrillary acidic protein (GFAP), neurocan and phosphacan in the peri-infarct region and markedly reduce the thickness of glial scar after ischemia/reperfusion (I/R). Consistent with the in vivo data, in the oxygen and glucose deprivation/reoxygenation (OGD/Re) model, sevoflurane postconditioning could protect astrocyte against OGD/Re-induced injury, decrease the expression of GFAP, neurocan and phosphacan. Further studies demonstrated that sevoflurane postconditioning could down-regulate the expression of Lamp1 and active cathepsin B, and block I/R or OGD/Re-induced release of cathepsin B from the lysosomes into cytoplasm. In order to confirm whether inhibition of cathepsin B could attenuate the formation of glial scar, we used cathepsin B inhibitor CA-074Me as a positive control. The results showed that inhibition of cathepsin B could decrease the expression of GFAP, neurocan and phosphacan. Taken together, sevoflurane postconditioning can attenuate astrogliosis and glial scar formation after ischemic stroke, associating with inhibition of the activation and release of lysosomal cathepsin B.

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy

Non-thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti-proliferative efficacy. Interestingly, NTP demonstrated greater selective anti-proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti-proliferative effect of NTP was enhanced by pre-incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM-H2DCFDA, MitoSOX and C11-BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor-1 and increased ferritin-H and -L chain expression that was correlated with decreased iron-regulatory protein expression and RNA-binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)-selective probe, HMRhoNox-M, and was consistent with autophagic-induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid-phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B-II expression, which co-localized with LAMP1, indicating autophagolysosome formation. Autophagic-induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid-phase endocytosis, lysosome biogenesis and autophagy.

Human vaginal epithelial cells augment autophagy marker genes in response to Candida albicans infection.

Autophagy plays an important role in clearance of intracellular pathogens. However, no information is available on its involvement in vaginal infections such as vulvo-vaginal candidiasis (VVC). VVC is intimately associated with the immune status of the human vaginal epithelial cells (VECs). The objective of our study is to decipher if autophagy process is involved during Candida albicans infection of VECs. In this study, C. albicans infection system was established using human VEC line (VK2/E6E7). Infection-induced change in the expression of autophagy markers like LC3 and LAMP-1 were analyzed by RT-PCR, q-PCR, Western blot, immunofluorescence and transmission electron microscopy (TEM) studies were carried out to ascertain the localization of autophagosomes. Multiplex ELISA was carried out to determine the cytokine profiles. Analysis of LC3 and LAMP-1 expression at mRNA and protein levels at different time points revealed up-regulation of these markers 6hours post C. albicans infection. LC3 and LAMP-1 puncti were observed in infected VECs after 12hours. TEM studies showed C. albicans entrapped in autophagosomes. Cytokines-TNF-α and IL-1β were up-regulated in culture supernatants of VECs at 12hours post-infection. The results suggest that C. albicans invasion led to the activation of autophagy as a host defense mechanism of VECs.

Impact of resistance training on the autophagy-inflammation-apoptosis crosstalk in elderly subjects.

Aging is associated with a decline in autophagy and a state of low-grade inflammation which further affects apoptosis and autophagy. Importantly, these alterations could reverse with regular physical activity. This study assessed the effects of a resistance exercise training program on autophagy, NLRP3 inflammasome, and apoptosis in peripheral blood mononuclear cells (PBMCs) from old subjects. Twenty-six healthy women and men (age, 69.6±1.5 yr) were randomized to a training (TG) or a control (CG) group. TG performed an 8-week resistance training program, while CG followed their daily routines. Protein expression of beclin-1, Atg12, Atg16 and LAMP-2 increased following the training program, while expression of p62/SQSTM1 and phosphorylation of ULK-1 at Ser757 were significantly lower. Resistance exercise also induced a decrease in NLRP3 expression and in the caspase-1/procaspase-1 ratio. Expression of Bcl-2 and Bcl-xL, as well as the Bad/BcL-2 ratio were reduced, and there was a significant decrease in the protein content of caspase-3. The results obtained seem to indicate that 8-week resistance training stimulates autophagy, prevents NLRP3 inflammasome activation, and reduces apoptosis in PBMCs from elderly subjects. These data could have a significant impact in prevention and rehabilitation programs currently employed in elderly population.

PCSK9-related LDL-C value is correlated to Rab5 isoform expression level

Circulating LDL metabolism is mostly related to the LDL internalization and intracellular lysosome-related degradation events. In this way, the competitive biologic agents and vesicular membrane-integrated or assembled proteins are important functioning factors. The aim of this study was to investigate the relationships between Rab5, Rab7, Lamp1 and Lamp2 gene expression levels, circulating PCSK9 value and, serum LDL-C level. One hundred ten subjects were recruited from healthy adults. Lipid profile was measured by laboratory routine techniques. PCSK9 value was measured with ELISA Kit. The Rab5, Rab7, Lamp1 and Lamp2 gene expression levels of buffy coat samples were measured using Real-Time qPCR technique. Serum LDL-C level was correlated to Rab5 expression level (r 0.25, p 0.02) and circulating PCSK9 value (r 0.3, p 0.003). Although PCSK9 elevated positively with LDL-C value but the Rab5 expression level increased up to 75centiles. Furthermore, multiple regression analyses showed that the Rab5 expression level is significantly correlated to LDL-C value (p 0.025, β 0.003), age (p 0.037, β −0.006) and Lamp1 expression (p 0.001, β 0.079) while the Rab7 expression level did not relate to the study factors except for Lamp1 (p 0.011, β 0.053). The results proposed that the LDL-C-correlated Rab5 expression level may be a primary cellular response due to reduced PCSK9-associated LDL influx. Furthermore, the LDL-C value was not correlated to Lamp1 and Rab7 expression levels. Our data suggested that the vesicular structures may be reduced with age.

ABCB1 2677G>T/A variant enhances chemosensitivity to anti-cancer agents acting on microtubule dynamics through LAMP1 inhibition

Overexpression of ABCB1 associated with single nucleotide variants in cancers was reported to encode a protein responsible for drug resistance. We studied chemosensitivity-related genes associated with ABCB1 2677G>T/A variant. The associated genes were identified based on the results of the significance analysis of microarray, and then prediction accuracy was evaluated using the prediction analysis of microarray. Functional assay of the selected gene was performed by using siRNA and drug accumulation study.A higher frequency of chemoresistance to microtubule-modulating agents was found in cell lines with wild-type ABCB1 compared to cell lines with 2677G>T/A ABCB1 variant. Based on the pharmacogenetic association study with 2677 variant, we identified seven genes that could predict chemosensitivity to microtubule dynamics modulators. The classification accuracy with these seven genes was 90.0%, and the predicted probability was 0.73. LAMP1 was the only gene that was commonly related to chemosensitivity. LAMP1 expression levels were relatively higher in chemoresistant ABCB1 wild-type compared to chemosensitive polymorphic cells. But, there was no difference in ABCB1 expression levels between the two groups. Following LAMP1 siRNA, chemosensitivity was restored due to increased intracellular drug accumulation in wild type cell line. In conclusion, ABCB1 2677G>T/A variant enhances chemosensitivity on microtubule dynamics through LAMP1 inhibition.

CD4+ T cell-mediated cytotoxicity eliminates primary tumor cells in metastatic melanoma through high MHC class II expression and can be enhanced by inhibitory receptor blockade.

Metastatic melanoma is a rapidly progressing disease with high mortality rate and limited treatment options. Immunotherapy based on tumor-targeting cytotoxic T cell responses represents a promising strategy. To assist in its development, we examined the possibility and efficacy of using CD4+ cytotoxic T cells. The regulatory mechanisms controlling CD4+ T cell-mediated cytotoxicity were also investigated. We found that naturally occurring granzyme B and perforin-expressing CD4+ cytotoxic T cells can be recovered from metastatic melanoma patients at significantly elevated frequencies compared to those from healthy controls. These CD4+ cytotoxic T cells were also capable of killing autologous tumor cells harvested from metastatic melanoma, independent of CD8+ T cells or any other cell types. However, several restricting factors were observed. First, the cytolytic activity by CD4+ T cells required high MHC class II expression on melanoma cells, which was not satisfied in a subset of melanomas. Second, the granzyme B and perforin release by activated CD4+ cytotoxic T cells was reduced after coculturing with autologous melanoma cells, characterized by low LAMP-1 expression and low granzyme B and perforin secretion in the supernatant. This suggested that inhibitory mechanisms were present to suppress CD4+ cytotoxic T cells. Indeed, blockade of PD-1 and CTLA-4 had increased the cytolytic activity of CD4+ T cells but was only effective in MHC class II high but not MHC class II low melanomas. Together, our study showed that CD4+ T cell-mediated cytotoxicity could eliminate primary melanoma cells but the efficacy depended on MHC class II expression.

Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells.

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer.

LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes.

ABSTRACTAutophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2­-double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression.

Abstract 1198: Characterization of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 expressed at the surface of tumor cells

Abstract Lysosome-associated membrane protein 1 (LAMP1), one of the most abundant protein expressed at the membrane of lysosomes, is a type I transmembrane protein involved in the maintenance of lysosome membrane integrity and phagolysosome formation. In activated lymphocytes LAMP1 is described as a marker of degranulation. Unexpectedly, by immunizing mice with a colon patient-derived xenograft (PDX) followed by a screening of monoclonal antibodies (mAb) by immunohistochemistry (IHC) for selection of antibodies that specifically stain tumor plasma membrane and de-orphaning by Immunoprecipitation-Mass spectrometry, we identified LAMP1 as the target of several antibodies. One of them, Ab-1, showed binding to the luminal domain of human LAMP1 with nM affinity. Crystal structure of its Fab with LAMP1 extracellular domain, showed that the epitope was non-linear, not a glycotope and spanned between position 29 to195. LAMP1 expression was further documented by IHC with Ab-1, showing limited cell surface expression in normal tissues while moderate to high plasma membrane expression was found in a number of breast, including TNBC, colorectal, gastric, prostate, lung and ovary tumors. The humanized Ab-1 mAb, humAb-1, was shown to display rapid cycling after binding to LAMP1 at the surface of colo205 cell line, allowing internalization and processing of a number of LAMP1/antibody complex 10 folds higher than the number of LAMP1 molecules at the cell surface. humAb-1 was conjugated to DM4 maytansinoid derivative using an SPDB cleavable linker to generate a new antibody-drug conjugate, SAR428926, for the treatment of patients with cell surface LAMP1-positive tumors. Conjugated and naked antibody displayed similar affinities for LAMP1. SAR428926 killed tumor cell lines (engineered to express cell surface LAMP1) in the sub-nM range. In contrast, no target-mediated cytotoxicity was observed when SAR428926 was incubated with normal cells, including resting or activated lymphocytes. PDX models reflecting the pattern and level of LAMP1 expression at the surface of human tumors were selected to evaluate SAR428926 in vivo efficacy. Outstanding in vivo activity was observed in different indications, including TNBC, lung and colon PDXs, with complete regressions after a single administration at 5 mg/kg. These encouraging preclinical data have prompted the initiation of IND-enabling studies with the goal to progress humAb-1-ADC to the clinic in patients with tumors expressing LAMP1 at the cell surface. The First-In-Human trial has been initiated in October 2015. Citation Format: Yves Baudat, Beatrice Cameron, Tarik Dabdoubi, Anne-Marie Lefebvre, Ana Merino-Trigo, Corinne Thomas, Veronique Pecheux, Bruno Genet, Loreley Calvet, Lydia Blot, Magali Mathieu, Laurence Gauzy, Laurence Berthou-Soulie, Catherine Prades, Celine Amara, Manoel Nunes, Christophe Henry, Cecile Combeau, Francis Blanche, Jean-Francois Mayaux, Carlos Garcia-Echeverria, Souad Naimi, Veronique Blanc. Characterization of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 expressed at the surface of tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1198.

Abstract 1197: Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors

Abstract Lysosome-associated membrane protein 1 (LAMP1) is involved in the maintenance of lysosome membrane integrity and phagolysosome formation. LAMP1 shows very limited expression at the cell surface in normal tissues while, a moderate to high expression was found in a number of solid tumors. We generated SAR428926, an antibody drug conjugate (ADC) comprising an anti-LAMP1 humanized monoclonal antibody conjugated via a cleavable SPDB linker, to the maytansinoid derivative DM4, a tubulin interfering molecule. Here we report the annotation of LAMP1 expression in a large panel of human tumors and correlate it to the in vivo efficacy of SAR428926 in a panel of over 50 human patient-derived xenograft (PDX) solid tumors. Indications of interest were identified by immunohistochemistry (IHC) conducted on a large panel of frozen tumor samples using the murine version of the therapeutic anti-human LAMP1 antibody. The prevalence of tumor samples with positive membrane staining was 49% in breast invasive lobular or ductal carcinoma, and in particular in a TNBC subset (87%), 25% in gastric adenocarcinoma, 52% in colon/rectum adenocarcinoma, 22% in lung adenocarcinoma, 20% in lung squamous cell carcinoma, 25% in prostate adenocarcinoma and 31% in ovary adenocarcinoma. In vivo efficacy of SAR428926 was evaluated in PDX models engrafted subcutaneously into immunocompromised SCID mice. PDX models retain the molecular diversity, cellular heterogeneity, and histology typically seen in patient tumors and offer a distinct advantage over cell line models. The PDX models used in the study, which include colon, breast, lung, prostate, gastric and ovarian cancer, were selected by IHC based on LAMP1 expression levels distribution and the frequency of LAMP1 membrane positive cells, which varied between 5% and 100% of the tumor cells, allowing coverage of a wide variety of cases. Outstanding efficacy with complete regressions was observed at 5 mg/kg iv single administration, a dose that gives an exposure tolerated in toxicology species. Efficacy was also observed at lower doses of 2.5 mg/kg and 1.25 mg/kg after single administration. Our results show that while the presence of the antigen is required as there is no efficacy in LAMP1-negative PDX, there is no linear correlation between the level of antigen expression and the antitumor activity across the panel of PDX tested. In addition to membrane LAMP1 expression, a key driver for efficacy was the intrinsic sensitivity of the PDX models to DM4. These results highlight the potential of ADCs that target human cancers expressing membrane LAMP1 at the surface of tumor cells. These encouraging preclinical data have prompted us to initiate IND-enabling studies with the goal to progress anti LAMP1-ADC to the clinic in patients with solid tumors expressing LAMP1 at the cell surface. The phase I study started in October 2015. Citation Format: Loreley Calvet, Anne-Marie Lefebvre, Celine Nicolazzi, Lydia Blot, Corinne Thomas, Yves Baudat, Beatrice Cameron, Carlos Garcia-Echeverria, Jean-Francois Mayaux, Veronique Blanc, Cecile Combeau, Souad Naimi, Sukhvinder Sidhu. Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1197.

Plasma PCSK9 level affects passively LAMP-2 expression; an evidence of transcription network

Free cholesterol accumulation in lysosomes is related to highly glycosylated membrane-protein functions, endogenous cholesterol synthesis and, LDL influx. The LAMP-1 and LAMP-2 are two lysosome proteins involved in cholesterol transport. The study aim was to investigate the associations between LAMP-2 expression level, LAMP-2/LAMP-1 expression ratio, blood PCSK9 protein and LDL-C values. One hundred twenty six healthy subjects were selected during a medical interview. The biochemical parameters were measured using routine laboratory techniques. The plasma PCSK9 level was identified by ELISA method. The LAMP-1/LAMP-2 expression levels were estimated by Real time qPCR technique. The PCSK9 and LAMP-2 expression networks were designed with Cytoscape software and, were enriched using transcription factor databases. Linear correlations were observed between PCSK9 and LDL-C levels (r=0.34; p=0.002) and, TC/HDL-C ratio (r=0.35; p=0.001). The LAMP-2 expression level and LAMP-2/LAMP-1 expression ratio were inversely related to LDL-C values (p=0.02). The transcription factors were compartmented on the merged network and were strictly controlled the PCSK9 expression. The results suggested that the decreased LDL influx due to the increase of plasma PCSK9 level attenuates the LAMP-2 gene expression level. Furthermore, the merged network supported our findings that the LAMP-2 expression is not in relation to transcription factors involved with PCSK9 expression.

58-F, a flavanone from Ophiopogon japonicus, prevents hepatocyte death by decreasing lysosomal membrane permeability.

Lysosome membrane permeabilization (LMP) has been implicated in cell death. In the present study, we investigated the relationship between cell death and H2O2-/CCl4-induced LMP in hepatocytes in vitro and following acute liver injury in vivo. The key finding was that H2O2 triggered LMP by oxidative stress, as evidenced by a suppression of LAMP1 expression, a reduction in LysoTracker Green and AO staining, and the leakage of proton and cathepsin B/D from the lysosome to the cytoplasm, resulting in cell death. CCl4 also triggered hepatocyte death by decreasing lysosome LAMP1 expression and by inducing the accumulation of products of peroxidative lipids and oxidized proteins. Furthermore, a novel compound 5,8-dimethoxy-6-methyl-7-hydroxy-3-3(2-hydroxy-4-methoxybenzyl) chroman-4-one (58-F) was extracted from Ophiopogon japonicus and served as a potential therapeutic drug. In vivo and in vitro results showed that 58-F effectively rescued hepatocytes by decreasing LMP and by inducing lysosomal enzyme translocation to the cytosol.

615. Choroid Plexus-Targeted Viral Gene Therapy for Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) represent a category of approximately 60 inherited neurometabolic illnesses with considerable morbidity and mortality in children and adults. The burden of LSD is high due to the chronic progressive decline in the neurocognitive function of affected individuals that profoundly limits their societal integration. Current therapy for LSDs has focused on recombinant enzyme replacement therapy (ERT), which while promising, is not ideal for numerous reasons, including the short half-lives of ERT with weekly or monthly administration, potential immune responses against ERT may also reduce the effectiveness for the treatment and inefficient passage through the blood-brain barrier. The choroid plexuses are vascularized structures that project into the cerebrospinal fluid (CSF) and feature specialized polarized epithelia derived from neuroectoderm that are post-mitotic, i.e., do not undergo turnover, and produce CSF by transporting water and ions into the brain ventricles. We hypothesized that remodeling these epithelia to secrete missing lysosomal enzymes by one-time administration of a recombinant AAV vector with selective tropism for choroid plexus (e.g., serotype 5) would be an attractive strategy for long-term treatment of LSDs. Potentially this approach would result in steady secretion of the missing enzyme into the CSF, which normally carries molecules throughout the ventricular system into the subarachnoid space, and ultimately deliver enzyme to the entire brain. The cross-correction phenomenon in many LSDs would provide a further advantage. To evaluate this hypothesis in preclinical animal models, we chose two prototypical LSDs, α-mannosidosis and mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B syndrome). We cloned the respective cDNAs (human (h) LAMAN and NAGLU) into rAAV shuttle plasmids and generated high titer of rAAV5 expressing the enzymes. We administered viral particles to the lateral cerebroventricles sof homozygous mutant mice on day 2 or 3 of life at doses of 5 × 109 or 5 × 1010 vector genomes. In the LAMAN deficient mice, we documented dose-dependent transduction and hLAMAN mRNA expression confined to the choroid plexuses of rAAV5-treated animals. Brain biochemical analyses at 1, 2 and 6 months post-treatment documented sustained, highly statistically significant increases of LAMAN enzyme activity in the brain globally (olfactory bulb, cerebral cortex, cerebellum, brainstem). By 8 months of age, untreated mutant mice showed prominent lysosomal vacuoles in hippocampal neurons, in contrast to rAAV5-hLAMAN treated mutants for which brain histopathology was comparable to wild-type. Lysosomal associated membrane protein 1 (Lamp1) levels were normalized in AAV5-hLAMAN treated mutant brain. In MPS IIIB mutant mice, levels of NAGLU enzyme activity were 2-8 fold higher in brain sections six weeks after rAAV5-hNAGLU treatment compared to normal controls. β-Hexosaminidase activity, which is elevated in MPS IIIB, was reduced to heterozygote carrier levels. Tissue evaluations by immunohistochemistry showed robust hNAGLU expression in the choroid plexus epithelia. Lamp1 expression was significantly reduced in hippocampus and frontal cortex of treated mice. The approach outlined herein challenges existing treatment modalities for LSDs, by exploiting the ability of choroid plexus-targeted gene therapy to restore missing or defective lysosomal enzymes at concentrations and distributions in CSF suitable for disease correction. The potential impact on clinical practice in the field of LSD is high since, if these results extend to larger animal disease models, nonhuman primates, and human subjects, the largest current barriers to health for affected patients would be circumvented.

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.

Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis

Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis.The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD.

Abstract 12746: Impaired Autophagic Flux in Ischemic/Reperfused Diabetic Heart and Its Reversal by a Novel Orally Active Adiponectin Receptor Agonist via AMPK-Mediated Autophagosome Formation and AMPK-Independent Autophagosome Clearance

Autophagy is increasingly recognized as an important regulator of myocardial ischemia/reperfusion (MI/R) injury. However, whether and how autophagy is altered in the ischemic/reperfused diabetic heart remains unknown. Normal control (NC), high-fat-diet-induced diabetic (HFD), and adiponectin (APN) knockout (APNKO) mice were subjected to MI/R (30 minutes/3-24 hours) and treated with vehicle or AdipoRon (50 mg/kg gavage, 10 minutes before MI; or 10 mg/kg IP, 10 min before R). Compared to NC mice, autophagosome formation was significantly inhibited and autophagosome clearance was nearly completely blocked in the HFD heart after MI/R (P<0.01). APNKO largely reproduced the phenotypic alterations observed in the HFD heart, with both autophagosome formation and clearance inhibited after MI/R (P<0.01 vs. WT). Treatment of HFD and APKNO mice with AdipoRon stimulated autophagosome formation, increased autophagosome clearance, reduced infarct size, and improved cardiac function (P<0.01). Mechanistically, AdipoRon caused significant phosphorylation of AMPK, BeclineT119, and VPS34S16, and enhanced expression of lysosome protein LAMP2. Compound C pretreatment abolished AdipoRon-induced BeclineT119 and VPS34S164 phosphorylation without altering AdipoRon-induced LAMP2 expression. In vitro AdipoRon treatment of adult wild type cardiomyocytes enhanced phosphorylation of AMPK, BeclineT119, and VPS34S164. These effects are lost in cardiomyocytes isolated from cardiomyocyte-specific AMPK dominant negative mice (AMPK-DN). In contrast, AdipoRon-stimulated LAMP2 expression was preserved in AMPK-DN cardiomyocytes. Collectively, these results demonstrated for the first time that hypoadiponectinemia during diabetic conditions impairs autophagic flux in cardiomyocytes after MI/R via marked inhibition of both autophagosome clearance and formation. Administration of an orally active APN receptor agonist increased autophagosome formation via AMPK activation, and stimulated autophagosome clearance in an AMPK-independent fashion. Improving autophagic flux by restoring APN signaling systems might be a novel intervention against MI/R injury in diabetic conditions.