Markus Damme - Academia.edu (original) (raw)

Papers by Markus Damme

Biochemical Journal, 2009

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

Traffic, 2015

The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-... more 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. Recently, a putative M6P residue in a crystal structure of a recombinantely 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 results in M6P-dependent targeting of LIMP-2 to lysosomes. Since 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 β-glucocerebrosidase reach lysosomes independently of the M6P-pathway. In fibroblasts lacking either M6P receptors or the M6P-forming 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 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.

PLOS Genetics, 2015

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospina... more Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal PLOS Genetics |

Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver

PloS one, 2015

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been sho... more Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmpgt/gt mice (formerly known as Ncu-g1gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmpgt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmpgt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmpgt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmpgt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmpgt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmpgt/gt liver homogenates, compared to hepato...

Proceedings of the National Academy of Sciences

Deficiency of glycosaminoglycan (GAG) degradation causes a subclass of lysosomal storage disorder... more Deficiency of glycosaminoglycan (GAG) degradation causes a subclass of lysosomal storage disorders called mucopolysaccharidoses (MPSs), many of which present with severe neuropathology. Critical steps in the degradation of the GAG heparan sulfate remain enigmatic. Here we show that the lysosomal arylsulfatase G (ARSG) is the long-sought glucosamine-3-O-sulfatase required to complete the degradation of heparan sulfate. Arsg-deficient mice accumulate heparan sulfate in visceral organs and the central nervous system and develop neuronal cell death and behavioral deficits. This accumulated heparan sulfate exhibits unique nonreducing end structures with terminal N-sulfoglucosamine-3-O-sulfate residues, allowing diagnosis of the disorder. Recombinant human ARSG is able to cleave 3-O-sulfate groups from these residues as well as from an authentic 3-O-sulfated N-sulfoglucosamine standard. Our results demonstrate the key role of ARSG in heparan sulfate degradation and strongly suggest that A...

Autophagy in neuronal cells: general principles and physiological and pathological functions

Acta neuropathologica, 2015

Autophagy delivers cytoplasmic components and organelles to lysosomes for degradation. This pathw... more Autophagy delivers cytoplasmic components and organelles to lysosomes for degradation. This pathway serves to degrade nonfunctional or unnecessary organelles and aggregate-prone and oxidized proteins to produce substrates for energy production and biosynthesis. Macroautophagy delivers large aggregates and whole organelles to lysosomes by first enveloping them into autophagosomes that then fuse with lysosomes. Chaperone-mediated autophagy (CMA) degrades proteins containing the KFERQ-like motif in their amino acid sequence, by transporting them from the cytosol across the lysosomal membrane into the lysosomal lumen. Autophagy is especially important for the survival and homeostasis of postmitotic cells like neurons, because these cells are not able to dilute accumulating detrimental substances and damaged organelles by cell division. Our current knowledge on the autophagic pathways and molecular mechanisms and regulation of autophagy will be summarized in this review. We will describe...

The FEBS journal, 2009

The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix pr... more The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix protein that was initially identified in a screen for genes induced by retinoic acid. Recently, it has been spotlighted by several proteome analyses of the lysosomal compartment, but its cellular function and properties remain unknown to date. In this study, Scpep1 from mice was analysed with regard to its intracellular processing into a mature dimer consisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDa C-terminal fragment and the glycosylation status of the mature Scpep1 fragment. Although Scpep1 shares notable homology and a number of structural hallmarks with the well-described lysosomal carboxypeptidase protective protein/cathepsin A, the purified recombinant 55 kDa precursor and the homogenates of Scpep1-overexpressing cells do not show proteolytic activity or increased serine carboxypeptidase activity towards artificial serine carboxypeptidase substrates. Hence, we d...

Science (New York, N.Y.), Jan 27, 2014

Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its... more Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its broad tropism, chicken cells were reported 30 years ago to resist infection. We found that Lassa virus readily engaged its cell-surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1. Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1. A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection. The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo.

Biochemical and Biophysical Research Communications, 2015

The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor ... more The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase b-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

Acta Neuropathologica, 2014

grN-associated FTlD-TDP (FTlD-TDP/grN), but also those which are characteristic for NCl and lysos... more grN-associated FTlD-TDP (FTlD-TDP/grN), but also those which are characteristic for NCl and lysosomal impairment. In Grn(−/−) mice the lysosomal proteins cathepsin D (CTSD), lAMP (lysosomal-associated membrane protein) 1 and the NCl storage components saposin D and subunit c of mitochondrial ATP synthase (SCMAS) were all found to be elevated. Moreover, these mice display increased levels of transmembrane protein (TMeM) 106B, a lysosomal protein known as a risk factor for FTlD-TDP pathology. In line with a potential pathological overlap of FTlD and NCl, Ctsd(−/−) mice, a model for NCl, show elevated levels of the FTlD-associated proteins grN and TMeM106B. In addition, pathologically phosphorylated TDP-43 occurs in Ctsd(−/−) mice to a similar extent as in Abstract Heterozygous loss-of-function mutations in the progranulin (GRN) gene and the resulting reduction of grN levels is a common genetic cause for frontotemporal lobar degeneration (FTlD) with accumulation of TAr DNA-binding protein (TDP)-43. recently, it has been shown that a complete grN deficiency due to a homozygous GRN loss-of-function mutation causes neuronal ceroid lipofuscinosis (NCl), a lysosomal storage disorder. These findings suggest that lysosomal dysfunction may also contribute to some extent to FTlD. Indeed, Grn(−/−) mice recapitulate not only pathobiochemical features of Electronic supplementary material The online version of this article (

PLoS ONE, 2012

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompa... more 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. Citation: Thelen M, Damme M, Schweizer M, Hagel C, Wong AMS, et al. (2012) Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis. PLoS ONE 7(4): e35493.

Correction: Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

PLoS ONE, 2012

Neurobiology of Disease, 2014

Molecular and Cellular Biology, 2012

Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient recept... more Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5 ؊/؊ ) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5 ؊/؊ mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products.

Molecular and Cellular Biology, 2010

␣-Mannosidosis is caused by the genetic defect of the lysosomal ␣-D-mannosidase (LAMAN), which is... more ␣-Mannosidosis is caused by the genetic defect of the lysosomal ␣-D-mannosidase (LAMAN), which is involved in the breakdown of free ␣-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with ␣-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with ␣-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human ␣-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparaginelinked oligosaccharides on native lysosomal proteins.

Journal of Neuropathology and Experimental Neurology, 2011

α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyllinked... more α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyllinked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy (ERT) that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term ERT partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides and cholesterol. The present study demonstrates cerebellar alterations in αmannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures.

Disease Models & Mechanisms, 2014

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biologica... more Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1 gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1 gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1 gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1 gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage. . Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflamed liver. Histochem. Cell Biol. 135, 305-315. Barrett, A. J. (1980). Fluorimetric assays for cathepsin B and cathepsin H with methylcoumarylamide substrates. Biochem. J. 187, 909-912. . Transcriptional profiling reveals novel markers of liver fibrogenesis: gremlin and insulin-like growth factor-binding proteins. . (2003b). Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis. J. Clin. Invest. 112, 152-159. . Cholesteryl ester storage disease: pathologic changes in an affected fetus. Am. J. Med. Genet. 26, 689-698. Engelhardt, N. V., Factor, V. M., Yasova, A. K., Poltoranina, V. S., Baranov, V. N. and Lasareva, M. N. (1990). Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes. Differentiation 45, 29-37. Engelhardt, N. V., Factor, V. M., Medvinsky, A. L., Baranov, V. N., Lazareva, M. N. and Poltoranina, V. S. (1993). Common antigen of oval and biliary epithelial cells (A6) is a differentiation marker of epithelial and erythroid cell lineages in early development of the mouse. Differentiation 55, 19-26.

Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice

Brain, 2012

Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of ... more Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, β-hexosaminidase, α-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease.

Biochemical Journal, 2009

Biochemical Journal, 2011

Disrupted in renal carcinoma 2" (DIRC2) has been initially identified as a breakpoint spanning ge... more Disrupted in renal carcinoma 2" (DIRC2) has been initially identified as a breakpoint spanning gene in a chromosomal translocation putatively associated with the development of renal cancer. The DIRC2 protein belongs to the major facilitator superfamily (MFS) and has been previously detected by organellar proteomics as a tentative constituent of lysosomal membranes. In the present study, lysosomal residence of overexpressed as well as endogenous DIRC2 was shown by several approaches. DIRC2 is proteolytically processed into a N-glycosylated N-terminal and a non-glycosylated C-terminal fragment, respectively. Proteolytic cleavage occurs in lysosomal compartments and critically depends on the activity of cathepsin L which was found to be indispensable for this process in murine embryonic fibroblasts. The cleavage site within DIRC2 was mapped between amino acid residues 214 and 261 using internal epitope tags and is presumably located within the tentative fifth intralysosomal loop assuming the typical MFS topology. Lysosomal targeting of DIRC2 was demonstrated to be mediated by a N-terminal dileucine motif. By disrupting this motif, DIRC2 can be redirected to the plasma membrane. Finally, in a whole-cell electrophysiological assay based on heterologous expression of the targeting mutant at the plasma membrane of Xenopus oocytes, the application of a complex metabolic mixture evokes an outward current associated with the surface expression of full-length DIRC2. Taken together, these data strongly support the idea that DIRC2 is an electrogenic lysosomal metabolite transporter which is subjected to and presumably modulated by limited proteolytic processing.

Biochemical Journal, 2009

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

Traffic, 2015

The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-... more 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. Recently, a putative M6P residue in a crystal structure of a recombinantely 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 results in M6P-dependent targeting of LIMP-2 to lysosomes. Since 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 β-glucocerebrosidase reach lysosomes independently of the M6P-pathway. In fibroblasts lacking either M6P receptors or the M6P-forming 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 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.

PLOS Genetics, 2015

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospina... more Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal PLOS Genetics |

Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver

PloS one, 2015

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been sho... more Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmpgt/gt mice (formerly known as Ncu-g1gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmpgt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmpgt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmpgt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmpgt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmpgt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmpgt/gt liver homogenates, compared to hepato...

Proceedings of the National Academy of Sciences

Deficiency of glycosaminoglycan (GAG) degradation causes a subclass of lysosomal storage disorder... more Deficiency of glycosaminoglycan (GAG) degradation causes a subclass of lysosomal storage disorders called mucopolysaccharidoses (MPSs), many of which present with severe neuropathology. Critical steps in the degradation of the GAG heparan sulfate remain enigmatic. Here we show that the lysosomal arylsulfatase G (ARSG) is the long-sought glucosamine-3-O-sulfatase required to complete the degradation of heparan sulfate. Arsg-deficient mice accumulate heparan sulfate in visceral organs and the central nervous system and develop neuronal cell death and behavioral deficits. This accumulated heparan sulfate exhibits unique nonreducing end structures with terminal N-sulfoglucosamine-3-O-sulfate residues, allowing diagnosis of the disorder. Recombinant human ARSG is able to cleave 3-O-sulfate groups from these residues as well as from an authentic 3-O-sulfated N-sulfoglucosamine standard. Our results demonstrate the key role of ARSG in heparan sulfate degradation and strongly suggest that A...

Autophagy in neuronal cells: general principles and physiological and pathological functions

Acta neuropathologica, 2015

Autophagy delivers cytoplasmic components and organelles to lysosomes for degradation. This pathw... more Autophagy delivers cytoplasmic components and organelles to lysosomes for degradation. This pathway serves to degrade nonfunctional or unnecessary organelles and aggregate-prone and oxidized proteins to produce substrates for energy production and biosynthesis. Macroautophagy delivers large aggregates and whole organelles to lysosomes by first enveloping them into autophagosomes that then fuse with lysosomes. Chaperone-mediated autophagy (CMA) degrades proteins containing the KFERQ-like motif in their amino acid sequence, by transporting them from the cytosol across the lysosomal membrane into the lysosomal lumen. Autophagy is especially important for the survival and homeostasis of postmitotic cells like neurons, because these cells are not able to dilute accumulating detrimental substances and damaged organelles by cell division. Our current knowledge on the autophagic pathways and molecular mechanisms and regulation of autophagy will be summarized in this review. We will describe...

The FEBS journal, 2009

The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix pr... more The retinoid-inducible serine carboxypeptidase 1 (Scpep1; formerly RISC) is a lysosomal matrix protein that was initially identified in a screen for genes induced by retinoic acid. Recently, it has been spotlighted by several proteome analyses of the lysosomal compartment, but its cellular function and properties remain unknown to date. In this study, Scpep1 from mice was analysed with regard to its intracellular processing into a mature dimer consisting of a 35 kDa N-terminal fragment and a so far unknown 18 kDa C-terminal fragment and the glycosylation status of the mature Scpep1 fragment. Although Scpep1 shares notable homology and a number of structural hallmarks with the well-described lysosomal carboxypeptidase protective protein/cathepsin A, the purified recombinant 55 kDa precursor and the homogenates of Scpep1-overexpressing cells do not show proteolytic activity or increased serine carboxypeptidase activity towards artificial serine carboxypeptidase substrates. Hence, we d...

Science (New York, N.Y.), Jan 27, 2014

Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its... more Lassa virus spreads from a rodent to humans and can lead to lethal hemorrhagic fever. Despite its broad tropism, chicken cells were reported 30 years ago to resist infection. We found that Lassa virus readily engaged its cell-surface receptor α-dystroglycan in avian cells, but virus entry in susceptible species involved a pH-dependent switch to an intracellular receptor, the lysosome-resident protein LAMP1. Iterative haploid screens revealed that the sialyltransferase ST3GAL4 was required for the interaction of the virus glycoprotein with LAMP1. A single glycosylated residue in LAMP1, present in susceptible species but absent in birds, was essential for interaction with the Lassa virus envelope protein and subsequent infection. The resistance of Lamp1-deficient mice to Lassa virus highlights the relevance of this receptor switch in vivo.

Biochemical and Biophysical Research Communications, 2015

The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor ... more The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase b-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

Acta Neuropathologica, 2014

grN-associated FTlD-TDP (FTlD-TDP/grN), but also those which are characteristic for NCl and lysos... more grN-associated FTlD-TDP (FTlD-TDP/grN), but also those which are characteristic for NCl and lysosomal impairment. In Grn(−/−) mice the lysosomal proteins cathepsin D (CTSD), lAMP (lysosomal-associated membrane protein) 1 and the NCl storage components saposin D and subunit c of mitochondrial ATP synthase (SCMAS) were all found to be elevated. Moreover, these mice display increased levels of transmembrane protein (TMeM) 106B, a lysosomal protein known as a risk factor for FTlD-TDP pathology. In line with a potential pathological overlap of FTlD and NCl, Ctsd(−/−) mice, a model for NCl, show elevated levels of the FTlD-associated proteins grN and TMeM106B. In addition, pathologically phosphorylated TDP-43 occurs in Ctsd(−/−) mice to a similar extent as in Abstract Heterozygous loss-of-function mutations in the progranulin (GRN) gene and the resulting reduction of grN levels is a common genetic cause for frontotemporal lobar degeneration (FTlD) with accumulation of TAr DNA-binding protein (TDP)-43. recently, it has been shown that a complete grN deficiency due to a homozygous GRN loss-of-function mutation causes neuronal ceroid lipofuscinosis (NCl), a lysosomal storage disorder. These findings suggest that lysosomal dysfunction may also contribute to some extent to FTlD. Indeed, Grn(−/−) mice recapitulate not only pathobiochemical features of Electronic supplementary material The online version of this article (

PLoS ONE, 2012

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompa... more 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. Citation: Thelen M, Damme M, Schweizer M, Hagel C, Wong AMS, et al. (2012) Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis. PLoS ONE 7(4): e35493.

Correction: Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

PLoS ONE, 2012

Neurobiology of Disease, 2014

Molecular and Cellular Biology, 2012

Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient recept... more Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5 ؊/؊ ) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5 ؊/؊ mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products.

Molecular and Cellular Biology, 2010

␣-Mannosidosis is caused by the genetic defect of the lysosomal ␣-D-mannosidase (LAMAN), which is... more ␣-Mannosidosis is caused by the genetic defect of the lysosomal ␣-D-mannosidase (LAMAN), which is involved in the breakdown of free ␣-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with ␣-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with ␣-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human ␣-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparaginelinked oligosaccharides on native lysosomal proteins.

Journal of Neuropathology and Experimental Neurology, 2011

α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyllinked... more α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyllinked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy (ERT) that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term ERT partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides and cholesterol. The present study demonstrates cerebellar alterations in αmannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures.

Disease Models & Mechanisms, 2014

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biologica... more Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1 gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1 gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1 gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1 gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage. . Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflamed liver. Histochem. Cell Biol. 135, 305-315. Barrett, A. J. (1980). Fluorimetric assays for cathepsin B and cathepsin H with methylcoumarylamide substrates. Biochem. J. 187, 909-912. . Transcriptional profiling reveals novel markers of liver fibrogenesis: gremlin and insulin-like growth factor-binding proteins. . (2003b). Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis. J. Clin. Invest. 112, 152-159. . Cholesteryl ester storage disease: pathologic changes in an affected fetus. Am. J. Med. Genet. 26, 689-698. Engelhardt, N. V., Factor, V. M., Yasova, A. K., Poltoranina, V. S., Baranov, V. N. and Lasareva, M. N. (1990). Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes. Differentiation 45, 29-37. Engelhardt, N. V., Factor, V. M., Medvinsky, A. L., Baranov, V. N., Lazareva, M. N. and Poltoranina, V. S. (1993). Common antigen of oval and biliary epithelial cells (A6) is a differentiation marker of epithelial and erythroid cell lineages in early development of the mouse. Differentiation 55, 19-26.

Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice

Brain, 2012

Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of ... more Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, β-hexosaminidase, α-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease.

Biochemical Journal, 2009

Biochemical Journal, 2011

Disrupted in renal carcinoma 2" (DIRC2) has been initially identified as a breakpoint spanning ge... more Disrupted in renal carcinoma 2" (DIRC2) has been initially identified as a breakpoint spanning gene in a chromosomal translocation putatively associated with the development of renal cancer. The DIRC2 protein belongs to the major facilitator superfamily (MFS) and has been previously detected by organellar proteomics as a tentative constituent of lysosomal membranes. In the present study, lysosomal residence of overexpressed as well as endogenous DIRC2 was shown by several approaches. DIRC2 is proteolytically processed into a N-glycosylated N-terminal and a non-glycosylated C-terminal fragment, respectively. Proteolytic cleavage occurs in lysosomal compartments and critically depends on the activity of cathepsin L which was found to be indispensable for this process in murine embryonic fibroblasts. The cleavage site within DIRC2 was mapped between amino acid residues 214 and 261 using internal epitope tags and is presumably located within the tentative fifth intralysosomal loop assuming the typical MFS topology. Lysosomal targeting of DIRC2 was demonstrated to be mediated by a N-terminal dileucine motif. By disrupting this motif, DIRC2 can be redirected to the plasma membrane. Finally, in a whole-cell electrophysiological assay based on heterologous expression of the targeting mutant at the plasma membrane of Xenopus oocytes, the application of a complex metabolic mixture evokes an outward current associated with the surface expression of full-length DIRC2. Taken together, these data strongly support the idea that DIRC2 is an electrogenic lysosomal metabolite transporter which is subjected to and presumably modulated by limited proteolytic processing.