Lysosomes Of Fibroblasts Research Articles

Late-onset Krabbe disease presenting as spastic paraplegia - implications of GCase and CTSB/D.

Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). Additionally to cerebral MRI, protein structural analyses of the β-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of β-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.

Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function.

Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.

Graphene flakes for enhanced delivery of the enzyme to the lysosomes of patient-derived fibroblasts: Bio-persistence and kinetics of substrate degradation

Graphene flakes for enhanced delivery of the enzyme to the lysosomes of patient-derived fibroblasts: Bio-persistence and kinetics of substrate degradation

Restoration of β-GC trafficking improves the lysosome function in Gaucher disease.

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic routes. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher disease (GD), the lysosomes accumulate glucosylceramide because of low β-glucocerebrosidase (β-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant β-GC to lysosomes may improve the lysosome function in GD. RNAi screen using high throughput based β-GC activity assay followed by reporter trafficking assay utilizing β-GC-mCherry led to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the β-GC activity by increasing the folding and trafficking of Gaucher mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their β-GC activity upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity is an alternative therapeutic strategy to restore the lysosome function in GD.

Effect of rapamycin on mitochondria and lysosomes in fibroblasts from patients with mtDNA mutations.

Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, patients with MELAS treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in patients with MELAS are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared with control, healthy individuals. We observed that mitochondria from patients were fragmented, had a threefold decline in the average speed of motility, a twofold reduced mitochondrial membrane potential, and a 1.5- to 2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared with healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 h resulted in increased mitochondrial respiration compared with control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.

Nanoscopic quantification of sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells derived from patients with mitochondrial diseases

SLC25A46 mutations have been found to lead to mitochondrial hyper-fusion and reduced mitochondrial respiratory function, which results in optic atrophy, cerebellar atrophy, and other clinical symptoms of mitochondrial disease. However, it is generally believed that mitochondrial fusion is attributable to increased mitochondrial oxidative phosphorylation (OXPHOS), which is inconsistent with the decreased OXPHOS of highly-fused mitochondria observed in previous studies. In this paper, we have used the live-cell nanoscope to observe and quantify the structure of mitochondrial cristae, and the behavior of mitochondria and lysosomes in patient-derived SLC25A46 mutant fibroblasts. The results show that the cristae have been markedly damaged in the mutant fibroblasts, but there is no corresponding increase in mitophagy. This study suggests that severely damaged mitochondrial cristae might be the predominant cause of reduced OXPHOS in SLC25A46 mutant fibroblasts. This study demonstrates the utility of nanoscope-based imaging for realizing the sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells, which may be particularly valuable for the quick evaluation of pathogenesis of mitochondrial morphological abnormalities.

MFSD12 mediates the import of cysteine into melanosomes and lysosomes.

Dozens of genes contribute to the vast variation in human pigmentation. Many of these encode proteins that localize to the melanosome, the lysosome-related organelle that synthesizes pigment, but have unclear functions 1,2. Here, we describe the MelanoIP method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use it to study MFSD12, a transmembrane protein of unknown molecular function that when suppressed causes darker pigmentation in mice and humans 3,4. We find that MFSD12 is required to maintain normal levels of cystine, the oxidized dimer of cysteine, in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation 5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes, and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal storage disease caused by inactivation of the lysosomal cystine exporter CTNS (Cystinosin)7–9. Thus, MFSD12 is an essential component of the long-sought cysteine importer for melanosomes and lysosomes.

Biodistribution and intracellular localization of hyaluronan and its nanogels. A strategy to target intracellular S. aureus in persistent skin infections

Intracellular pathogens are a critical challenge for antimicrobial therapies. Staphylococcus aureus (S. aureus) causes approximately 85% of all skin and soft tissue infections in humans worldwide and more than 30% of patients develop chronic or recurrent infections within three months, even after appropriate antibacterial therapies. S. aureus is also one of the most common bacteria found in chronic wounds. Recent evidences suggest that S. aureus is able to persist within phagolysosomes of skin cells (i.e. keratinocytes, phagocytic cells), being protected from both the immune system and a number of antimicrobials. To overcome these limits, nano-formulations that enable targeted therapies against intracellular S. aureus might be developed. Herein, the biodistribution and intracellular localisation of hyaluronan (HA) and HA-based nanoparticles (nanogels, NHs) are investigated, both after intravenous (i.v.) injections (in mice) and topical administrations (in ex vivo human skin). Results indicate HA and NHs accumulate especially in skin and liver of mice after i.v. injection. After topical application on human skin explants, no penetration of both HA and NHs was detected in skin with intact stratum corneum. By contrast, in barrier-disrupted human skin (with partial removal and loosening of stratum corneum), HA and NHs penetrate to the viable epidermis and are taken up by keratinocytes. In mechanically produced wounds (skin without epidermis) they accumulate in wound tissue and are taken up by dermis cells, e.g. fibroblasts and phagocytic cells. Interestingly, in all cases, the cellular uptake is CD44-mediated. In vitro studies confirmed that after CD44-mediated uptake, both HA and NHs accumulate in lysosomes of dermal fibroblasts and macrophages, as previously reported for keratinocytes. Finally, the colocalisation between intracellular S. aureus and HA or NHs is demonstrated, in macrophages. Altogether, for the first time, these results strongly suggest that HA and HA-based NHs can provide a targeted therapy to intracellular S. aureus, in persistent skin or wound infections.

Cytotoxic Granule Trafficking and Fusion in Synaptotagmin7-Deficient Cytotoxic T Lymphocytes.

Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8+ T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.

A DNA nanomachine chemically resolves lysosomes in live cells.

Lysosomes are multifunctional, subcellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimer's disease, Parkinson's disease and rare lysosomal storage diseases, but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome subpopulations with different physical characteristics including dynamics, morphology or cellular localization. Here, we chemically resolve lysosome subpopulations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single-lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from healthy individuals shows two main lysosome populations, one of which is absent in primary cells derived from patients with Niemann-Pick disease. When patient cells are treated with relevant therapeutics, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia.

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

Mannose 6-phosphate-independent Lysosomal Sorting of LIMP-2.

The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for β-glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP-2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP-2 with the cation-independent M6P receptor (MPR) results in M6P-dependent targeting of LIMP-2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P-dependent delivery of LIMP-2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP-2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P-forming N-acetylglucosamine (GlcNAc)-1-phosphotransferase, LIMP-2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP-2 levels within lysosomes purified from liver of wild-type (wt) and GlcNAc-1-phosphotransferase-defective mice. Heterologous expression of the luminal domain of LIMP-2 in wild-type, LIMP-2-deficient and GlcNAc-1-phosphotransferase-defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP-2. Finally, cathepsin Z, a known GlcNAc-1-phosphotransferase substrate, but not LIMP-2, could be precipitated with M6P-specific antibodies. These data prove M6P-independent lysosomal sorting of LIMP-2 and subsequently GC in vivo.

MCP-1 downregulates MMP-9 export via vesicular redistribution to lysosomes in rat portal fibroblasts.

Portal fibroblasts (PF) are one of the two primary cell types contributing to the myofibroblast population of the liver and are thus essential to the pathogenesis of liver fibrosis. Monocyte chemoattractant protein‐1 (MCP‐1) is a known profibrogenic chemokine that may be of particular importance in biliary fibrosis. We examined the effect of MCP‐1 on release of matrix metalloproteinase‐9 (MMP‐9) by rat PF. We found that MCP‐1 blocks PF release of MMP‐9 in a posttranslational fashion. We employed an optical and electron microscopic approach to determine the mechanism of this downregulation. Our data demonstrated that, in the presence of MCP‐1, MMP‐9‐containing vesicles were shunted to a lysosome‐like compartment. This is the first report of a secretory protein to be so regulated in fibrogenic cells.

Ocular and histologic findings in a series of children with infantile pompe disease treated with enzyme replacement therapy.

To report the ophthalmologic and histologic findings in a series of children with infantile Pompe disease treated with enzyme replacement therapy (ERT). Records of children with infantile Pompe disease treated with ERT who had at least one complete ophthalmic examination and the ocular histopathology of children with infantile Pompe disease who were treated with ERT were reviewed. The patients' clinical history, including external ocular examination, ocular alignment and motility, dilated fundus examination, and cycloplegic refraction, was evaluated. A literature review was performed for ophthalmologic findings in infantile Pompe disease using PubMed. The clinical findings of 13 children were included and the ocular histopathology of 3 children with infantile Pompe disease who were treated with ERT were reviewed. Forty-six percent (6 of 13) had bilateral ptosis, 23% (3 of 13) had strabismus, 62% (8 of 13) had myopia, and 69% (9 of 13) had astigmatism. On histologic examination, there was vacuolar myopathy affecting the extraocular muscles, ciliary body, and iris smooth muscle and glycogen accumulation in corneal endothelial, lens epithelium, and retinal ganglion cells, and within lysosomes of scleral fibroblasts. It is important that ophthalmic providers are aware of the high prevalence of myopia, astigmatism, and ptosis in children with infantile Pompe disease treated with ERT because they are potentially amblyogenic but treatable factors.

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder often characterized by severe neurodevelopmental abnormalities and neuro-retinal degeneration. Mutations in the TRPML1 gene are causative for MLIV. We used lead optimization strategies to identify--and MLIV patient fibroblasts to test--small-molecule activators for their potential to restore TRPML1 mutant channel function. Using the whole-lysosome planar patch-clamp technique, we found that activation of MLIV mutant isoforms by the endogenous ligand PI(3,5)P2 is strongly reduced, while activity can be increased using synthetic ligands. We also found that the F465L mutation renders TRPML1 pH insensitive, while F408Δ impacts synthetic ligand binding. Trafficking defects and accumulation of zinc in lysosomes of MLIV mutant fibroblasts can be rescued by the small molecule treatment. Collectively, our data demonstrate that small molecules can be used to restore channel function and rescue disease associated abnormalities in patient cells expressing specific MLIV point mutations.

Insulin receptor antibody-sulfamidase fusion protein penetrates the primate blood-brain barrier and reduces glycosoaminoglycans in Sanfilippo type A cells.

Mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also called sulfamidase, cause accumulation of lysosomal inclusion bodies in the brain of children born with mucopolysaccharidosis type IIIA, also called Sanfilippo type A syndrome. Enzyme replacement therapy with recombinant SGSH does not treat the brain because the enzyme is a large molecule drug that does not cross the blood–brain barrier (BBB). A BBB-penetrating form of SGSH was produced by re-engineering the enzyme as an IgG fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb domain of the HIRMAb–SGSH fusion protein acts as a molecular Trojan horse to ferry the fused enzyme across the BBB. The HIRMAb–SGSH was produced in stably transfected host cells and purified to homogeneity by protein A chromatography. The fusion protein reacted with antibodies against either human IgG or SGSH on Western blotting. High affinity binding to the HIR was retained following SGSH fusion to the HIRMAb, with an EC50 of 0.33 ± 0.05 nM in an HIR binding ELISA. The SGSH enzyme activity of the HIRMAb–SGSH fusion protein was 4712 ± 388 units/mg protein based on a two-step fluorometric enzyme assay. The HIRMAb–SGSH was taken up by lysosomes in MPSIIIA fibroblasts, and treatment of these cells with the fusion protein caused an 83% reduction in sulfate incorporation into lysosomal glycosoaminoglycans. The HIRMAb–SGSH fusion protein was radiolabeled with the [125I]-Bolton–Hunter reagent and injected intravenously in the Rhesus monkey. The brain uptake of the fusion protein was high, ∼1% injected dose/brain. Calculations, based on this level of brain uptake, suggest normalization of brain SGSH enzyme activity is possible following administration of therapeutic doses of the fusion protein. These studies describe a novel IgG–SGSH fusion protein that is a new noninvasive treatment of the brain in MPS type IIIA.

Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis

Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

Tfeb Functions As a Tumor Suppressor That Harnesses Myc-Induced Lymphomagenesis By Provoking Senescence and Metabolic Catastrophe

Metabolic reprogramming is a hallmark of cancer. MYC oncoproteins orchestrate the control of many aspects of metabolism, including mitochondrial biogenesis, glycolysis and glutaminolysis. This response allows the highly proliferative cancer cell to meet the increased demands for macromolecules and energy. Here we report that MYC promotes anabolism by actively suppressing the autophagy-lysosomal catabolic pathway. Notably, disabling this response by reactivating catabolism provokes metabolic catastrophe and senescence in MYC-driven lymphomas.Gene expressing profiling of B220+ B cells from bone marrow of wild type (Wt) and pre-cancerous Eµ-Myc mice, and of Eµ-Myc lymphoma, demonstrated that genes involved in autophagy and lysosomal biogenesis are suppressed in MYC-expressing B cells. This response was due to MYC, as activation of the c-MYC transgene in human P493-6 B cells suppressed the expression of autophagy and lysosomal biogenesis genes. Further, this response was regulated by physiological cues that control endogenous c-Myc expression, as it was evident in naïve mouse splenic B cells treated with mitogen and in cytokine-treated hematopoietic cells. Finally, the suppression of autophagy-lysosomal genes was a hallmark of human malignancies with MYC involvement, including Burkitt lymphoma and colorectal cancer. Importantly, these changes in autophagy-lysosomal gene expression have functional consequences, as there are marked decreases in lysosomes in MYC-expressing versus control mouse embryonic fibroblasts and primary B cells, and in P493-6 B cells following MYC activation. Finally, consistent with an impaired autophagy-lysosomal pathway, there were marked increases in the levels of p62/Sequestrin, a receptor for ubiquitylated cargo on autophagosomes that is normally degraded by the autophagosomal pathway, in premalignant and neoplastic B220+ Eµ-Myc B cells.A master regulator of autophagy and lysosomal biogenesis is TFEB that, like MYC, functions as a basic helix-loop-helix leucine zipper transcription factor. Our studies suggest that MYC can block TFEB function at three levels. First, TFEB expression is suppressed in human malignancies with MYC involvement, suggesting that in some contexts MYC may repress TFEB transcription. Second, MYC can repress TFEB transcription targets by competing with TFEB for binding to autophagy-lysosome gene targets. Specifically, the DNA recognition sequence of TFEB (CLEAR sequence) overlaps with the E-Box sequence of MYC, and chromatin immunoprecipitation analyses showed that MYC bound to these recognition elements in select TFEB targets and repressed their expression. Finally, MYC-expressing B cells display activation of mTORC1, which phosphorylates TFEB and blocks its nuclear localization. Specifically, MYC-expressing B cells have marked increases in the phosphorylation of the mTORC1 substrates S6K and 4EBP1, and show cytoplasmic localization of TFEB.Given these findings, we hypothesized that MYC-transformed lymphoma cells might be sensitive to activating this catabolic pathway. We tested this hypothesis in Eµ-Myc;Rosa26-rtTa lymphoma cells that express the reverse tetracycline transactivator. These lymphoma cells were transduced with a pTight doxycycline- (Dox)-inducible retrovirus that expresses a constitutively active (mTORC1-resistant and nuclear) form of TFEB (TFEB-S211A) and the effects of the induction of TFEB were monitored. Importantly, our analyses established that the induction of TFEB-S211A markedly impaired the proliferation and tumorigenic potential of Eµ-Myc lymphoma. Mechanistically, this proliferative arrest was associated with the induction of the senescence program, and with an increased extracellular acidification rate. Strikingly, this metabolic shift was associated with reductions in oxygen consumption rate and reduced numbers of mitochondria. Therefore, TFEB functions as a tumor suppressor that harnesses MYC-induced tumorigenesis by inducing senescence and disabling mitochondrial anabolic pathways that are needed to sustain the growth of malignant cells. Disclosures:No relevant conflicts of interest to declare.

Time-dependent decrement of dermal gadolinium deposits and significant improvement of skin symptoms in a patient with nephrogenic systemic fibrosis after temporary renal failure

Nephrogenic systemic fibrosis (NSF) represents a rare fibrosing disorder occurring after administration of gadolinium-containing contrast agents during renal insufficiency. In order to prove the effect of gadolinium elimination on clinical signs, we identified and quantified gadolinium in skin biopsies of a 62-year-old patient with NSF with regard to improving skin lesions after recovery of renal function. Gadolinium deposits were visualized and identified in NSF skin biopsies by light microscopy and transmission electron microscopy (EM) and by scanning EM. Inductively coupled plasma-mass spectrometry (ICPMS) was used for quantifying gadolinium concentration. Transmission EM studies revealed electron-dense material in connective matrix around blood vessels and inside lysosomes of histiocytes and fibroblasts. A remarkable reduction of gadolinium deposits was observed in transmission EM and scanning EM and confirmed by ICPMS in follow-up biopsies. After spontaneous recovery of renal function, his skin induration improved notably over the next 2 years. The reduction of clinical and histomorphological signs of NSF correlated with decreasing gadolinium concentration in skin biopsies within 3 years. Our study suggests a possible pathogenetic mechanism of NSF including a chance for recovery after elimination of gadolinium and reduced histamine liberation by mast cells.

The Thr224Asn mutation in the VPS45 gene is associated with the congenital neutropenia and primary myelofibrosis of infancy

AbstractSevere congenital neutropenia as well as primary myelofibrosis are rare in infancy. Elucidation of the underlying mechanism is important because it extends our understanding of the more common adult forms of these disorders. Using homozygosity mapping followed by exome sequencing, we identified a Thr224Asn mutation in the VPS45 gene in infants from consanguineous families who suffered from life-threatening neutropenia, which was refractory to granulocyte CSF, from defective platelet aggregation and myelofibrosis. The mutation segregated in the families, was not present in controls, affected a highly conserved codon, and apparently destabilized the Vps45 protein, which was reduced in the patients' leukocytes. Introduction of the corresponding mutation into yeast resulted in reduced cellular levels of Vps45 and also of the cognate syntaxin Tlg2, which is required for membrane traffic through the endosomal system. A defect in the endosomal-lysosomal pathway, the homologous system in humans, was suggested by the absence of lysosomes in the patients' fibroblasts and by the depletion of α granules in their platelets. Importantly, accelerated apoptosis was observed in the patients' neutrophils and bone marrow. This is the first report of a Vps45-related disease in humans, manifesting by neutropenia, thrombasthenia, myelofibrosis, and progressive bone marrow failure.