CLN6 deficiency causes selective changes in the lysosomal protein composition.

Neuronal ceroid lipofuscinoses (NCLs) collectively account for the highest prevalence of inherited neurodegenerative diseases in childhood. This disease group is classified by the deposition of similar autofluorescence storage material in lysosomes that is accompanied by seizures, blindness and premature mortality in later disease stages. Defects in several genes affecting various proteins lead to NCL, one of them being CLN6, a transmembrane protein resident in the endoplasmic reticulum. Dysfunctionality of CLN6 causes variant late infantile NCL (vLINCL). The function of CLN6 and how its deficiency affects lysosomal integrity remains unknown. In this work, we performed a comparative proteomic analysis of isolated lysosomal fractions from liver tissue of nclf mice, a natural mouse model displaying a similar disease course than its human counterpart. We could identify a drastic reduction in the protein amounts of selected lysosomal proteins, amongst them several members of the NCL protein family. Most of these proteins were N-glycosylated, soluble hydrolases and their reduction in protein levels was verified by western blotting and enzymatic assays. Hereby we could directly link Cln6 dysfunction to changes in the lysosomal compartment and to other NCL forms.

Proteaphagy in Mammalian Cells Can Function Independent of ATG5/ATG7.

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), ß-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Because these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.

Systematic Comparison of Strategies for the Enrichment of Lysosomes by Data Independent Acquisition.

In mammalian cells, the lysosome is the main organelle for the degradation of macromolecules and the recycling of their building blocks. Correct lysosomal function is essential, and mutations in every known lysosomal hydrolase result in so-called lysosomal storage disorders, a group of rare and often fatal inherited diseases. Furthermore, it is becoming more and more apparent that lysosomes play also decisive roles in other diseases, such as cancer and common neurodegenerative disorders. This leads to an increasing interest in the proteomic analysis of lysosomes for which enrichment is a prerequisite. In this study, we compared the four most common strategies for the enrichment of lysosomes using data-independent acquisition. We performed centrifugation at 20,000 × g to generate an organelle-enriched pellet, two-step sucrose density gradient centrifugation, enrichment by superparamagnetic iron oxide nanoparticles (SPIONs), and immunoprecipitation using a 3xHA tagged version of the lysosomal membrane protein TMEM192. Our results show that SPIONs and TMEM192 immunoprecipitation outperform the other approaches with enrichment factors of up to 118-fold for certain proteins relative to whole cell lysates. Furthermore, we achieved an increase in identified lysosomal proteins and a higher reproducibility in protein intensities for label-free quantification in comparison to the other strategies.

The lysosomal transporter MFSD1 is essential for liver homeostasis and critically depends on its accessory subunit GLMP.

Lysosomes are major sites for intracellular, acidic hydrolase-mediated proteolysis and cellular degradation. The export of low-molecular-weight catabolic end-products is facilitated by polytopic transmembrane proteins mediating secondary active or passive transport. A number of these lysosomal transporters, however, remain enigmatic. We present a detailed analysis of MFSD1, a hitherto uncharacterized lysosomal family member of the major facilitator superfamily. MFSD1 is not N-glycosylated. It contains a dileucine-based sorting motif needed for its transport to lysosomes. Mfsd1 knockout mice develop splenomegaly and severe liver disease. Proteomics of isolated lysosomes from Mfsd1 knockout mice revealed GLMP as a critical accessory subunit for MFSD1. MFSD1 and GLMP physically interact. GLMP is essential for the maintenance of normal levels of MFSD1 in lysosomes and vice versa. Glmp knockout mice mimic the phenotype of Mfsd1 knockout mice. Our data reveal a tightly linked MFSD1/GLMP lysosomal membrane protein transporter complex.

Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

A mouse model for SPG48 reveals a block of autophagic flux upon disruption of adaptor protein complex five.

Hereditary spastic paraplegia is a spastic gait disorder that arises from degeneration of corticospinal axons. The subtype SPG48 is associated with mutations in the zeta subunit of the adaptor protein complex five (AP5). AP5 function and the pathophysiology of SPG48 are only poorly understood. Here, we report an AP5 zeta knockout mouse, which shows an age-dependent degeneration of corticospinal axons. Our analysis of knockout fibroblasts supports a trafficking defect from late endosomes to the transGolgi network and reveals a structural defect of the Golgi. We further show that both autophagic flux and the recycling of lysosomes from autolysosomes were impaired in knockout cells. In vivo, we observe an increase of autophagosomes and autolysosomes and, at later stages, the accumulation of intracellular waste in neurons. Taken together, we propose that loss of AP5 function blocks autophagy and thus leads to the aberrant accumulation of autophagic cargo, which finally results in axon degeneration.

Disease-Linked Glutarylation Impairs Function and Interactions of Mitochondrial Proteins and Contributes to Mitochondrial Heterogeneity.

Lysine glutarylation (Kglu) of mitochondrial proteins is associated with glutaryl-CoA dehydrogenase (GCDH) deficiency, which impairs lysine/tryptophan degradation and causes destruction of striatal neurons during catabolic crisis with subsequent movement disability. By investigating the role of Kglu modifications in this disease, we compared the brain and liver glutarylomes of Gcdh-deficient mice. In the brain, we identified 73 Kglu sites on 37 mitochondrial proteins involved in various metabolic degradation pathways. Ultrastructural immunogold studies indicated that glutarylated proteins are heterogeneously distributed in mitochondria, which are exclusively localized in glial cells. In liver cells, all mitochondria contain Kglu-modified proteins. Glutarylation reduces the catalytic activities of the most abundant glutamate dehydrogenase (GDH) and the brain-specific carbonic anhydrase 5b and interferes with GDH-protein interactions. We propose that Kglu contributes to the functional heterogeneity of mitochondria and may metabolically adapt glial cells to the activity and metabolic demands of neighboring GCDH-deficient neurons.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

Loss of CLN7 results in depletion of soluble lysosomal proteins and impaired mTOR reactivation.

Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses. Here, we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild-type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct neuronal ceroid lipofuscinosis disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared with controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease.

SILAC-Based Comparative Proteomic Analysis of Lysosomes from Mammalian Cells Using LC-MS/MS.

Mass spectrometry-based proteomics of lysosomal proteins has led to significant advances in understanding lysosomal function and pathology. The ever-increasing sensitivity and resolution of mass spectrometry in combination with labeling procedures which allow comparative quantitative proteomics can be applied to shed more light on the steadily increasing range of lysosomal functions. In addition, investigation of alterations in lysosomal protein composition in the many lysosomal storage diseases may yield further insights into the molecular pathology of these disorders. Here, we describe a protocol which allows to determine quantitative differences in the lysosomal proteome of cells which are genetically and/or biochemically different or have been exposed to certain stimuli. The method is based on stable isotope labeling of amino acids in cell culture (SILAC). Cells are exposed to superparamagnetic iron oxide particles which are endocytosed and delivered to lysosomes. After homogenization of cells, intact lysosomes are rapidly enriched by passing the cell homogenates over a magnetic column. Lysosomes are eluted after withdrawal of the magnetic field and subjected to mass spectrometry.

Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes.

Disruption of the autophagy-lysosome pathway is involved in neuropathology of the nclf mouse model of neuronal ceroid lipofuscinosis.

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration.

High expression of disease-related Cln6 in the cerebral cortex, purkinje cells, dentate gyrus, and hippocampal ca1 neurons.

Mutations in the CLN6 gene cause a variant form of late infantile neuronal ceroid lipofuscinosis, a relentless neurodegenerative disease that is inherited as an autosomal recessive trait in humans and in the naturally occurring nclf mouse strain. The CLN6 protein is localized in the endoplasmic reticulum, but it has an unknown function. To develop a molecular understanding of neurodegeneration induced by mutations in CLN6, we examined the spatial and temporal distribution of Cln6 mRNA expression in murine brain. By using Northern blot and tissue qPCR array techniques, a single Cln6 transcript was detected throughout the adult brain, with greatest expression in the cerebellum and hypothalamus. Real-time qPCR showed 2.4-4-fold increases in Cln6 mRNA levels in the cortex and cerebellum during the first 28 days of life, with less prominent enhancement of expression in the hippocampus. In situ hybridization analyses demonstrated Cln6 expression in brainstem, dentate gyrus, and hippocampal neurons of newborn P0 mice. From P14 onward, Cln6 expression is widely distributed throughout the brain and is most prominent in cells of cortical layers II-VI, the Purkinje cell layer, dentate gyrus, and hippocampal CA1 region of adult mice. In different regions of the brain in P0 and P28 nclf mice, the Cln6 mRNA abundance was reduced by 30-40% compared with control mice. These findings implicate Cln6 in the survival and maturation of specific neuronal populations during development and make it possible to compare regional Cln6 expression with the distribution of subsequent pathology.

Absence of p55 TNF receptor reduces atherosclerosis, but has no major effect on angiotensin II induced aneurysms in LDL receptor deficient mice.

BACKGROUND: The aim of the current study was to investigate the role of p55 TNF Receptor (p55 TNFR), the main signaling receptor for the pro-inflammatory cytokine tumor necrosis factor (TNF), in the development of two vascular disorders: atherosclerosis and angiotensin (Ang) II-induced abdominal aortic aneurysms (AAA). METHODOLOGY/PRINCIPAL FINDINGS: p55 TNFR deficient mice were crossed to an LDL receptor deficient background and were induced for the development of either atherosclerosis or AngII-induced AAA, and compared to littermate controls, wild-type for p55 TNFR expression. p55 TNFR deficient mice developed 43% smaller atherosclerotic lesions in the aortic sinuses compared to controls. Moreover, expression of CD68, a macrophage specific marker, exhibited a 50% reduction in the aortic arches. Decreased atherosclerosis correlated with a strong down-regulation in the expression of adhesion molecules, such as VCAM-1 and ICAM-1, by p55 TNFR deficient endothelium. In addition, expression levels of the pro-inflammatory cytokines and chemokines TNF, IL-6, MCP-1 and RANTES were significantly reduced in aortas of p55 TNFR deficient mice. In contrast, in the AngII-induced model of AAA, p55 TNFR deficiency correlated with a slight trend towards increased aneurismal lethality, but the incidence of aortic rupture due to a dissecting aneurysm, and the expansion of the suprarenal aorta were not significantly different compared to controls. CONCLUSION/SIGNIFICANCE: We found that p55 TNFR expression promotes atherosclerosis, among other mechanisms, by enhancing expression of endothelial adhesion molecules, while it seems to have no major role in the development of AngII-induced AAA.

P55 tumour necrosis factor receptor in bone marrow-derived cells promotes atherosclerosis development in low-density lipoprotein receptor knock-out mice.

AIMS: Tumour necrosis factor (TNF) is a pivotal pro-inflammatory cytokine with a clear pathogenic role in many chronic inflammatory diseases, and p55 TNF receptor (TNFR) mediates the majority of TNF responses. The aim of the current study was to investigate the role of p55 TNFR expression in bone marrow-derived cells, in atherosclerotic lesion development. METHODS AND RESULTS: Irradiated low-density lipoprotein receptor knock-out mice were reconstituted with either p55 TNFR knock-out or control haematopoietic stem cells to generate chimeras deficient or wild-type for p55 TNFR specifically in bone marrow-derived cells, including macrophages. Upon high fat feeding, p55 TNFR knock-out transplanted mice developed smaller atherosclerotic lesions. These lesions were characterized by the presence of smaller foam cells and a reduced macrophage foam cell area. They did not differ in other compositional characteristics as determined by quantification of inflammatory T-cell and neutrophil influx, apoptotic and necrotic cell death, and collagen content. In vitro studies confirmed a significant defect in modified lipoprotein endocytosis by p55 TNFR knock-out macrophages due to reduced scavenger receptor class A expression. Interestingly, plasma cytokine/chemokine profile analysis indicated that monocyte chemoattractant protein-1 (MCP-1) levels, a major chemokine involved in atherogenesis, were consistently and significantly lower in p55 TNFR knock-out transplanted mice compared with controls, before and after high fat feeding. CONCLUSION: p55 TNFR expression in bone marrow-derived cells contributes to the development of atherosclerosis by enhancing lesional foam cell formation and by promoting the expression of pro-atherosclerotic chemokines such as MCP-1.