Dysregulated Autophagy and Lysosome Function Are Linked to Exosome Production by Micro-RNA 155 in Alcoholic Liver Disease.

Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3-II levels. Alcohol reduced autophagy flux in vivo in chloroquine-treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol-related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy-related protein 7 (Atg7), proteins involved in phagophore-autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal-associated membrane protein 1 (LAMP1) and lysosomal-associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro-RNA 155 (miR-155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR-155-deficient mice were protected from alcohol-induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down-regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol-induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR-155 as a mediator of alcohol-related regulation of autophagy and exosome production in hepatocytes and macrophages.

Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2.

In light induced retinal degeneration (LIRD) photoreceptor cell death is mediated by caspase independent mechanisms. The activation of LEI/L-DNase II pathway in this model, is due to cathepsin D release from lysosomes, although the underlying mechanism remains poorly understood. In this paper we studied the involvement of calpains in lysosomal permeabilization. We investigated, for the first time, the calpain targets at lysosomal membrane level. We found that calpain 1 is responsible for lysosomal permeabilization by cleavage of the lysosomal associated membrane protein 2 (LAMP 2). Moreover, LAMP 2 degradation and lysosomal permeabilization were rescued by calpain inhibition and the use of MEF(-/-)lamp 2 cells indicates that the cleavage of LAMP 2A is essential for this permeabilization. Finally, we found that LAMP 2 is cleaved in LIRD, suggesting that the mechanism of calpain induced lysosomal permeabilization is not exclusive of a single cell death model. Overall, these data shed new light on understanding the mechanisms of lysosomal and caspase-independent cell death and point to the original targets for development of the new therapeutic protocols.

Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages.

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.

Lysosomal exocytosis: an important event during invasion of lamp deficient cells by extracellular amastigotes of Trypanosoma cruzi.

Trypanosoma cruzi is an obligate intracellular organism in vertebrate hosts. Lysosomes are involved in parasite invasion. LAMP-1 and LAMP-2 are the most abundant glycoproteins of the lysosomal membrane. This study is the first report on the invasion of T. cruzi extracellular amastigotes (EA) in single LAMP-1 or LAMP-2 knockouts, respectively, or in two independent LAMP-1/2 double-knockout cell lines. When compared to their respective wild type clones, the EA show higher infectivity in LAMP-2 knockouts, but no difference was seen in LAMP-1 knockout cells. Similarly, EA invasion rate was higher for one of the double knockout clones but not for the other. Higher lysosomal exocytosis correlated with a higher invasion rate and early lysosomal marker acquisition. These findings suggest that lysosomal exocytosis is important to EA cell invasion. Also, phagolysosome maturation in knockout cell lines differed from previous results revealing that EA enter cells by a mechanism other than receptor-mediated phagocytosis.

Small-Vessel Vasculopathy Due to Aberrant Autophagy in LAMP-2 Deficiency.

Lysosomal associated membrane protein 2 (LAMP2) is physiologically implicated in autophagy. A genetic LAMP2 defect causes Danon disease, which consists of two major phenotypes of myopathy and cardiomyopathy. In addition, arteriopathy may manifest on rare occasions but the pathological basis remains unknown. We encountered two Danon families that developed small-vessel vasculopathy in the coronary or cerebral arteries. To investigate the underlying mechanisms, we characterized the biological features of LAMP-2-deficient mice and cultured cells. LAMP-2-deficient mice at 9-24 months of age showed medial thickening with luminal stenosis due to proliferation of vascular smooth muscle cells (VSMC) in muscular arteries. Ultrastructural analysis of VSMC revealed various autophagic vacuoles scattered throughout the cytoplasm, suggesting impaired autophagy of long-lived metabolites and degraded organelles (i.e., mitochondria). The VSMC in Lamp2 null mice expressed more vimentin but less α-smooth muscle actin (α-SMA), indicating a switch from contractile to synthetic phenotype. Silencing of LAMP2 in cultured human brain VSMC showed the same phenotypic transition with mitochondrial fragmentation, enhanced mitochondrial respiration, and overproduction of reactive oxygen species (ROS). These findings indicate that LAMP-2 deficiency leads to arterial medial hypertrophy with the phenotypic conversion of VSMC, resulting from age-dependent accumulation of cellular waste generated by aberrant autophagy.

The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain.

The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (lysosomal-associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. Here we report that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCAA30P). Using KFERQ-tagged fluorescent biosensors, we observed that LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body, but did not increase this process under starvation conditions. Noteworthy, we found that neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection. ABBREVIATIONS: Act5C: actin 5C; a.E.: after eclosion; Atg5: autophagy-related 5; Atg8a/LC3: autophagy-related 8a; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; elav: embryonic lethal abnormal vision; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; GABARAP: GABA typeA receptor-associated protein; Hsc70-4: heat shock protein cognate 4; HSPA8/Hsc70: heat shock protein family A (Hsp70) member 8; LAMP2: lysosomal associated membrane protein 2; MDA: malondialdehyde; PA-mCherry: photoactivable mCherry; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PD: Parkinson disease; Ref(2)P/p62: refractory to sigma P; ROS: reactive oxygen species; RpL32/rp49: ribosomal protein L32; RT-PCR: reverse transcription polymerase chain reaction; SING: startle-induced negative geotaxis; SNCA/α-synuclein: synuclein alpha; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; UAS: upstream activating sequence.

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 ßIII tubulin (ßIII 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.

Chaperone-mediated autophagy in the kidney: the road more traveled.

Chaperone-mediated autophagy (CMA) is a lysosomal proteolytic pathway in which cytosolic substrate proteins contain specific chaperone recognition sequences required for degradation and are translocated directly across the lysosomal membrane for destruction. CMA proteolytic activity has a reciprocal relationship with macroautophagy: CMA is most active in cells in which macroautophagy is least active. Normal renal proximal tubular cells have low levels of macroautophagy, but high basal levels of CMA activity. CMA activity is regulated by starvation, growth factors, oxidative stress, lipids, aging, and retinoic acid signaling. The physiological consequences of changes in CMA activity depend on the substrate proteins present in a given cell type. In the proximal tubule, increased CMA results from protein or calorie starvation and from oxidative stress. Overactivity of CMA can be associated with tubular lysosomal pathology and certain cancers. Reduced CMA activity contributes to protein accumulation in renal tubular hypertrophy, but may contribute to oxidative tissue damage in diabetes and aging. Although there are more questions than answers about the role of high basal CMA activity, this remarkable feature of tubular protein metabolism appears to influence a variety of chronic diseases.

Autophagy in the stress-induced myocardium.

Cardiovascular disease is a leading cause of death worldwide, particularly in Western societies. During an ischaemic insult, ventricular pressure from the heart is diminished as a result of cardiac myocyte death by necrosis and apoptosis. Autophagy is a process whereby cells catabolise intracellular proteins in order to generate ATP in times of stress such as nutrient starvation and hypoxia. Emerging evidence suggests that autophagy plays a positive role in cardiac myocyte survival during periods of cellular stress performing an important damage limitation function. By promoting cell survival, cardiac myocyte loss is reduced thereby minimising the potential of heart failure. In contrast, it has been reported that autophagy can also be a form of cell death. By considering the various animal models of autophagy, we examine the role of the Signal Transducers and Activator of Transcription (STAT) proteins in the autophagic response. Additionally we review the role of the tumour suppressor, p53 and its family member p73 and their potential role in the autophagic response.

Impaired phagosomal maturation in neutrophils leads to periodontitis in lysosomal-associated membrane protein-2 knockout mice.

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal disease.

LAMP proteins are required for fusion of lysosomes with phagosomes.

Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other.

LAMP-2 deficient mice show depressed cardiac contractile function without significant changes in calcium handling.

OBJECTIVE: Mutations in the highly glycosylated lysosome associated membrane protein-2 (LAMP-2) cause, as recently shown, familial Danon disease with mental retardation, mild myopathy and fatal cardiomyopathy. Extent and basis of the contractile dysfunction is not completely understood. METHODS: In LAMP-2 deficient mice, we investigated cardiac function in vivo using Doppler-echocardiography and contractile function in vitro in isolated myocardial trabeculae. RESULTS: LAMP-2 deficient mice displayed reduced ejection fraction (EF) (58.9+/-3.4 vs. 80.7+/-5.1%, P<0.05) and reduced cardiac output (8.3+/-3.1 vs. 14.7+/-3.6 ml/min, P<0.05) as compared to wild-type controls. Isolated multicellular muscle preparations from LAMP-2 deficient mice confirmed depressed force development (3.2+/-0.6 vs. 8.4+/-0.9 mN/mm2, P<0.01). All groups showed similar force-frequency behaviour when normalised to baseline force. Post-rest potentiation was significantly depressed at intervals>15 s in LAMP-2 deficient mice (P<0.05). Although attenuated in absolute force development, the normalised inotropic response to increased calcium and beta-adrenoreceptor stimulation was unaltered. Electron microscopic analysis revealed autophagic vacuoles in LAMP-2 deficient cardiomyocytes. Protein analysis showed unaltered levels of SERCA2a, calsequestrin and phospholamban. CONCLUSIONS: Cardiac contractile function in LAMP-2 deficient mice as a model for Danon disease is significantly attenuated. The occurrence of autophagic vacuoles in LAMP-2 deficient myocytes is likely to be causal for the depressed contractile function resulting in an attenuated cardiac pump reserve.