Lysosomal Membrane Protein LAMP-1 Research Articles

Lysosomal membrane proteins LAMP1 and LIMP2 are segregated in the Golgi apparatus independently of their clathrin adaptor binding motif.

To reach the lysosome, lysosomal membrane proteins (LMPs) are translocated in the endoplasmic reticulum after synthesis and then transported to the Golgi apparatus. The existence of a direct transport from the Golgi apparatus to the endosomes but also of an indirect route through the plasma membrane has been described. Clathrin adaptor binding motifs contained in the cytosolic tail of LMPs have been described as key players in their intracellular trafficking. Here we used the RUSH assay to synchronize the biosynthetic transport of multiple LMPs. After exiting the Golgi apparatus, RUSH-synchronized LAMP1 was addressed to the cell surface both after overexpression or at endogenous level. Its YXXΦ motif was not involved in the transport from the Golgi apparatus to the plasma membrane but in its endocytosis. LAMP1 and LIMP2 were sorted from each other after reaching the Golgi apparatus. LIMP2 was incorporated in punctate structures for export from the Golgi apparatus from which LAMP1 is excluded. LIMP2-containing post-Golgi transport intermediates did not rely neither on its adaptor binding signal nor on its C-terminal cytoplasmic domain.

Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.

New insights into the role of endosomal proteins for African swine fever virus infection.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.

Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism.

This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan‐reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image‐based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy.

Defective Autophagic Degradation in Aged D2‐mdx Diaphragms

Duchenne muscular dystrophy (DMD) is a progressive, fatal, muscle degenerative disease caused by the absence of dystrophin, which follows an X‐linked recessive inheritance pattern. Among other cellular dysfunctions, impaired autophagy has been identified as a consequence of dystrophin deficiency in humans. Accordingly, we hypothesized that degradation of autophagosomes would be impaired in muscles of D2‐mdx mice, an emerging DMD model. To test this hypothesis, diaphragm muscles from 11 month old D2‐mdx (diseased) and DBA (healthy) mice were collected, protein extracted, and western blot analysis was performed. Data were compared with a Student’s t‐test and significance was established as p<0.05. Among the markers of upstream activation of autophagy, total AMPK was increased by 3.5‐fold, phosphorylated (p)‐AMPK was similar, and p‐AMPK/AMPK was decreased by 80% in dystrophic muscle compared to healthy muscle. Similarly, total ULK1 was increased 1.7‐fold in muscle from D2‐mdx compared to DBA, while p‐ULK1 and p‐ULK1/ULK1 were similar between groups. Total Beclin1 was increased 10‐fold and p‐Beclin1 was increased 4‐fold, resulting in a 70% reduction in p‐Beclin1/Beclin1 in dystrophic muscle compared to healthy muscle. Total mTOR, p‐mTOR, and p‐mTOR/mTOR were similar between groups. Autophagosome formation proteins ATG7, ATG16L‐1, ATG12/5 complex, LC3A/B‐I and LC3A/B‐II were increased 2–3‐fold in D2‐mdx compared to DBA, however, the ratio of LC3A/B‐II/I was similar between groups. Relative protein abundance of p62 was increased 2‐fold in D2‐mdx compared to DBA. The relative amounts of the lysosomal membrane proteins LAMP1 and LAMP2 were similar between groups, suggesting similar lysosomal content in diseased and healthy muscles. These data appear to indicate decreased autophagosome degradation in dystrophic muscle compared to healthy. Given the similar lysosome content, autophagic dysfunction in D2‐mdx mice may be due to decreased lysosome fusion or function.Support or Funding InformationSupported in part by Ryan’s Quest, Michael’s cause and Parent Project Muscular Dystrophy (01297)

Expression of Lysosomal Membrane Proteins LAMP1, TPC1 and TPC2 in Acute Myeloid Leukemia Cells and Its Clinical Significance

To investigate the relationship between the expression of lysosomal membrane proteins LAMP1, TPC1 and TPC2 in acute myeloid leukemia (AML) cells and clinical indications of AML and to explore the possible role in the genesis and development of AML and clinical significance. Real-time quantitative PCR was used to detect the mRNA expression of LAMP1, TPC1 and TPC2 in AML cell lines (HL-60, NB4) and 57 patients with acute myeloid leukemia (including 44 initially treated patients and 13 relapsed and refractory patients). The relationship of mRNA expression levels with clinical indicators and post-chemotherapy remission was analyzed. Compared with CD34+ hematopoietic stem cells (HSC), the expression levels of LAMP1 and TPC1 in AML cell lines HL-60 and NB4 significantly increased, while the expression level of TPC2 was not significantly different. The expression levels of LAMP1, TPC1 and TPC2 in bone marrow mononuclear cells (BMMNC) of AML patients were higher than those in normal human BMMNC (P<0.05), and the expression levels of LAMP1, TPC1 and TPC2 in CD34+ primary AML cells(CD34+ primary cells in the patient's bone marrow >90%) were also high. There was no significant difference in the expression of LAMP1, TPC1 and TPC2 between CD34+HSC of patients with AML and relapsed/refractory patients (P>0.05). No correlation was found between age, sex and genotype and expression of membrane proteins (P>0.05). The expression levels of LAMP1 and TPC1 positively correlated with the number of white blood cells in peripheral blood of patients (P<0.01). LAMP1 and TPC2 were found to be associated with remission after a course of chemotherapy in newly diagnosed patients. Initially treated patients with high expression of LAMP1 in the bone marrow not easily relieved after one course of chemotherapy. Patients with high expression of TPC2 in the bone marrow more likely to be relieved after one course of chemotherapy. The mRNA of the three membrane proteins are highly expressed in AML patients, and LAMP1 and TPC1 are risk factors for AML disease progression. High expression of TPC2 is beneficial for chemotherapy of patients with newly diagnosed AML.

Structural and functional insights into the interaction and targeting hub TMD0 of the polypeptide transporter TAPL

The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.

The Major Lysosomal Membrane Proteins LAMP-1 and LAMP-2 Participate in Differentiation of C2C12 Myoblasts.

Lysosomes are organelles that play a crucial role in the degradation of endocytosed molecules, phagocytosed macromolecules and autophagic substrates. The membrane of lysosomes contains several highly glycosylated membrane proteins, and lysosome-associated membrane protein (LAMP)-1 and LAMP-2 account for a major portion of the lysosomal membrane glycoproteins. Although it is well known that LAMP-2 deficiency causes Danon disease, which is characterized by cardiomyopathy, myopathy and mental retardation, the roles of lysosomal membrane proteins including LAMP-1 and LAMP-2 in myogenesis are not fully understood. In this study, to understand the role of LAMP proteins in the course of differentiation of myoblasts into myotubes, we used C2C12 myoblasts and found that the protein and mRNA levels of LAMP-1 and LAMP-2 were increased in the course of differentiation of C2C12 myoblasts into myotubes. Then, we investigated the effects of LAMP-1 or LAMP-2 knockdown on C2C12 myotube formation, and found that LAMP-1 or LAMP-2 depletion impaired the differentiation of C2C12 myoblasts and reduced the diameter of C2C12 myotubes. LAMP-2 knockdown more severely impaired C2C12 myotube formation compared with LAMP-1 knockdown, and knockdown of LAMP-1 did not exacerbate the suppressive effects of LAMP-2 knockdown on C2C12 myotube formation. In addition, knockdown of LAMP-1 or LAMP-2 decreased the expression levels of myogenic regulatory factors, MyoD and myogenin. These results demonstrate that both LAMP-1 and LAMP-2 are involved in C2C12 myotube formation and LAMP-2 may contribute dominantly to it.

A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of β-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo.

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 μm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

Apoptotic Photoreceptor Loss and Altered Expression of Lysosomal Proteins in thenclfMouse Model of Neuronal Ceroid Lipofuscinosis

Mutations in the CLN6 gene cause variant late-infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder clinically characterized by progressive loss of vision, dementia, seizures, and early death. Here, we analyzed the time course of photoreceptor loss and the role of lysosomes in nclf mice, an animal model of the human CLN6 disease. Labeling of apoptotic cells, activated astrocytes, and Müller cells, and expression analyses of glial fibrillary acidic protein, rhodopsin, and lysosomal proteins were performed on nclf mice during the course of retinal degeneration. In addition, the distribution and variability of storage material was examined at the ultrastructural level. Progressive apoptotic loss of photoreceptor cells was observed in nclf mice, resulting in reduction of the outer nuclear layer to approximately 3 rows of photoreceptor cells at 9 months of age. Onset of reactive gliosis was observed in 1-month-old nclf mice. Ultrastructural analysis revealed lysosomal storage material containing curvilinear and fingerprint-like inclusions in various retinal cell types. Expression levels of soluble mannose 6-phosphate-containing lysosomal enzymes, such as cathepsin D and the lysosomal membrane protein Lamp1, were increased in retinal cells of nclf mice. Accumulation of heterogeneous nondegraded macromolecules in dysfunctional lysosomes and autolysosomes impairs photoreceptor cells, ultimately leading to early-onset apoptotic death with subsequent activation of astrocytes and Müller cells in the retina of nclf mice. The defined steps of photoreceptor degeneration suggest that nclf mice might serve as an ideal animal model for experimental therapeutic approaches aimed at attenuating vision loss in neuronal ceroid lipofuscinosis.

Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR

The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His10-tag and containing a codon optimized AT-rich sequence.

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.

Impaired autophagy and organellar dysfunction in pancreatitis

Recent findings from our group, obtained on experimental in vivo and ex vivo models of pancreatitis, reveal that this disease causes a profound dysfunction of key cellular organelles, lysosomes and mitochondria. We found that autophagy, the main cellular degradative, lysosome-driven process, is activated but also impaired in acute pancreatitis because of its' inefficient progression/resolution (flux) resulting from defective function of lysosomes. One mechanism underlying the lysosomal dysfunction in pancreatitis is abnormal processing (maturation) and activation of cathepsins, major lysosomal hydrolases; another is a decrease in pancreatic levels of key lysosomal membrane proteins LAMP-1 and LAMP-2. Our data indicate that lysosomal dysfunction plays an important initiating role in pancreatitis pathobiology. The impaired autophagy mediates vacuole accumulation in acinar cells; furthermore, the abnormal maturation and activation of cathepsins leads to increase in intra-acinar trypsin, the hallmark of pancreatitis; and LAMP-2 deficiency causes inflammation and acinar cell necrosis. Thus, the autophagic and lysosomal dysfunctions mediate key pathologic responses of pancreatitis. On the other hand, we showed that pancreatitis causes acinar cell mitochondria depolarization, mediated by the permeability transition pore (PTP). Genetic (via deletion of cyclophilin D) inactivation of PTP prevents mitochondrial depolarization and greatly ameliorates the pathologic responses of pancreatitis. Further, our data suggest that mitochondrial damage, by stimulating autophagy, increases the demand for efficient lysosomal degradation and therefore aggravates the pathologic consequences of lysosomal dysfunction. Thus, the combined autophagic, lysosomal and mitochondrial dysfunctions are key to the pathogenesis of pancreatitis.

Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis

Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood-borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well-characterized antibodies to axonal antigens, that axonal damage is associated with HLA-DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA-DR(+) cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF-L), present in white and gray matter, was observed in human microglia. The number of NF-L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF-L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF-L is taken up and degraded by the cell. In vivo, NF-L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA-DR(+) cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.

Interactions between Magnetic Nanowires and Living Cells: Uptake, Toxicity, and Degradation

We report on the uptake, toxicity, and degradation of magnetic nanowires by NIH/3T3 mouse fibroblasts. Magnetic nanowires of diameters 200 nm and lengths between 1 and 40 μm are fabricated by controlled assembly of iron oxide (γ-Fe(2)O(3)) nanoparticles. Using optical and electron microscopy, we show that after 24 h incubation the wires are internalized by the cells and located either in membrane-bound compartments or dispersed in the cytosol. Using fluorescence microscopy, the membrane-bound compartments were identified as late endosomal/lysosomal endosomes labeled with lysosomal associated membrane protein (Lamp1). Toxicity assays evaluating the mitochondrial activity, cell proliferation, and production of reactive oxygen species show that the wires do not display acute short-term (<100 h) toxicity toward the cells. Interestingly, the cells are able to degrade the wires and to transform them into smaller aggregates, even in short time periods (days). This degradation is likely to occur as a consequence of the internal structure of the wires, which is that of a noncovalently bound aggregate. We anticipate that this degradation should prevent long-term asbestos-like toxicity effects related to high aspect ratio morphologies and that these wires represent a promising class of nanomaterials for cell manipulation and microrheology.

The effects of lysosomal and proteasomal inhibitors on abnormal forms of prion protein degradation in murine macrophages

It has been reported that macrophages degrade infectious forms of prion protein (PrP(Sc) ). In order to investigate the mechanisms underlying PrP(Sc) degradation in macrophages, the effects of lysosomal and proteasomal inhibitors on macrophage cell lines which were incubated with scrapie-affected brain homogenate were studied. PrP(Sc) degradation was inhibited in the presence of both proteasomal and lysosomal inhibitors. Indirect fluorescence assays to determine the cellular localization of PrP(Sc) were undertaken. PrP(Sc) colocalized with the lysosomal membrane protein Lamp-1 and ubiquitin, a protein that is related to the proteasome. The present data indicate that macrophages might degrade PrP(Sc) via the lysosomal and proteasomal pathways.

Valosin containing protein associated inclusion body myopathy: abnormal vacuolization, autophagy and cell fusion in myoblasts

Inclusion body myopathy associated with Paget’s disease and frontotemporal dementia (IBMPFD) is caused by mutations in the valosin containing protein (VCP) gene. The disease is associated with progressive proximal muscle weakness, inclusions and vacuoles in muscle fibers, malfunction in the bone remodeling process resulting in Paget’s disease, and premature frontotemporal dementia. VCP is involved in several cellular processes related to the endoplasmic reticulum associated degradation of proteins. To understand the pathological mechanisms underlying the myopathy in IBMPFD, we have studied the cellular consequences of VCP mutations in human primary myoblasts. Our results revealed that patients’ myoblasts accumulate large vacuoles. Lysosomal membrane proteins Lamp1 and Lamp2 show increased molecular weights in patients’ myoblasts due to differential N-glycosylation. Additionally, mutant myoblasts show increased autophagy when cultured in the absence of nutrients, as well as defective cell fusion and increased apoptosis. Our results elucidate that VCP mutations result in disturbances in several cellular processes, which will help us in the understanding of the pathological mechanisms resulting in muscle weakness and other features of VCP associated disease.

Candida albicansactively modulates intracellular membrane trafficking in mouse macrophage phagosomes

The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.

Neuraminidase 1 Is a Negative Regulator of Lysosomal Exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.