Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino aciddependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.Peer reviewe

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites

Degeneration of Photoreceptor Cells in Arylsulfatase G-Deficient Mice

Kruszewski K, Luellmann-Rauch R, Dierks T, Bartsch U, Damme M. Degeneration of Photoreceptor Cells in Arylsulfatase G-Deficient Mice. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE. 2016;57(3):1120-1131.PURPOSE. Retinal degeneration is a common feature of several lysosomal storage disorders, including the mucopolysaccharidoses, a group of metabolic disorders that is characterized by widespread accumulation of glycosaminoglycans due to lysosomal enzyme dysfunction. We used a new mouse model of mucopolysaccharidosis IIIE to study the effect of Arylsulfatase G (ARSG) deficiency on retina integrity. METHODS. The retina of Arsg knockout mice aged 1 to 24 months was studied by immunohistochemistry and Western blot analysis. Electron microscopic analyses were performed on retinas from 15- and 22-month-old animals. Photoreceptor and microglia cell numbers and retina thickness were determined to quantitatively characterize retinal degeneration in ARSG-deficient mice. RESULTS. Arsg knockout mice showed a progressive degeneration of photoreceptor cells starting between 1 and 6 months of age, resulting in the loss of more than 50% of photoreceptor cells in 24-month-old mice. Photoreceptor loss was accompanied by reactive astrogliosis, reactive microgliosis that was evident in the outer but not inner retina, and elevated expression levels of some lysosomal proteins. Electron microscopic analyses of retinas revealed no evidence for the presence of storage vacuoles. Of note, expression of ARSG protein in wild-type mice was detectable only in the RPE which, however, appeared morphologically unaffected in knockout mice at the electron microscopic level. CONCLUSIONS. To our knowledge, this is the first study demonstrating that ARSG deficiency results in progressive photoreceptor degeneration and dysregulation of various lysosomal proteins

A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease

Fucosidosis is a rare lysosomal storage disorder caused by the inherited deficiency of the lysosomal hydrolase α-L-fucosidase, which leads to an impaired degradation of fucosylated glycoconjugates. Here, we report the generation of a fucosidosis mouse model, in which the gene for lysosomal α-L-fucosidase (Fuca1) was disrupted by gene targeting. Homozygous knockout mice completely lack α-L-fucosidase activity in all tested organs leading to highly elevated amounts of the core-fucosylated glycoasparagine Fuc(α1,6)-GlcNAc(β1-N)-Asn and, to a lesser extent, other fucosylated glycoasparagines, which all were also partially excreted in urine. Lysosomal storage pathology was observed in many visceral organs, such as in the liver, kidney, spleen and bladder, as well as in the central nervous system (CNS). On the cellular level, storage was characterized by membrane-limited cytoplasmic vacuoles primarily containing water-soluble storage material. In the CNS, cellular alterations included enlargement of the lysosomal compartment in various cell types, accumulation of secondary storage material and neuroinflammation, as well as a progressive loss of Purkinje cells combined with astrogliosis leading to psychomotor and memory deficits. Our results demonstrate that this new fucosidosis mouse model resembles the human disease and thus will help to unravel underlying pathological processes. Moreover, this model could be utilized to establish diagnostic and therapeutic strategies for fucosidosis.status: publishe

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

Lopez DM, Thelen M, Stahl F, et al. The lysosomal transporter MFSD1 is essential for liver homeostasis and critically depends on its accessory subunit GLMP. ELIFE. 2019;8: e50025.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

Chronic enzyme replacement therapy ameliorates neuropathology in alpha-mannosidosis mice

OBJECTIVE: The lysosomal storage disease alpha-mannosidosis is caused by the deficiency of the lysosomal acid hydrolase alpha-mannosidase (LAMAN) leading to lysosomal accumulation of neutral mannose-linked oligosaccharides throughout the body, including the brain. Clinical findings in alpha-mannosidosis include skeletal malformations, intellectual disabilities and hearing impairment. To date, no curative treatment is available. We previously developed a beneficial enzyme replacement therapy (ERT) regimen for alpha-mannosidase knockout mice, a valid mouse model for the human disease. However, humoral immune responses against the injected recombinant human alpha-mannosidase (rhLAMAN) precluded long-term studies and chronic treatment. METHODS: Here, we describe the generation of an immune-tolerant alpha-mannosidosis mouse model that allowed chronic injection of rhLAMAN by transgenic expression of a catalytically inactive variant of human LAMAN in the knockout background. RESULTS: Chronic ERT of rhLAMAN revealed pronounced effects on primary substrate storage throughout the brain, normalization of lysosomal enzyme activities and morphology as well as a decrease in microglia activation. The positive effect of long-term ERT on neuronal lysosomal function was reflected by an improvement of cognitive deficits and exploratory activity. in vivo and in vitro uptake measurements indicate rapid clearance of rhLAMAN from circulation and a broad uptake into different cell types of the nervous system. INTERPRETATION: Our data contribute to the understanding of neurological disorders treatment by demonstrating that lysosomal enzymes such as rhLAMAN can penetrate into the brain and is able to ameliorate neuropathology.status: publishe

The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons

Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons.status: publishe

The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons

Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons

LIMP-2 expression is critical for beta-glucocerebrosidase activity and alpha-synuclein clearance

Rothaug M, Zunke F, Mazzulli JR, et al. LIMP-2 expression is critical for beta-glucocerebrosidase activity and alpha-synuclein clearance. Proceedings of the National Academy of Sciences. 2014;111(43):15573-15578.Mutations within the lysosomal enzyme beta-glucocerebrosidase (GC) result in Gaucher disease and represent a major risk factor for developing Parkinson disease (PD). Loss of GC activity leads to accumulation of its substrate glucosylceramide and alpha-synuclein. Since lysosomal activity of GC is tightly linked to expression of its trafficking receptor, the lysosomal integral membrane protein type-2 (LIMP-2), we studied alpha-synuclein metabolism in LIMP-2-deficient mice. These mice showed an alpha-synuclein dosage-dependent phenotype, including severe neurological impairments and premature death. In LIMP-2-deficient brains a significant reduction in GC activity led to lipid storage, disturbed autophagic/lysosomal function, and alpha-synuclein accumulation mediating neurotoxicity of dopaminergic (DA) neurons, apoptotic cell death, and inflammation. Heterologous expression of LIMP-2 accelerated clearance of overexpressed alpha-synuclein, possibly through increasing lysosomal GC activity. In surviving DA neurons of human PD midbrain, LIMP-2 levels were increased, probably to compensate for lysosomal GC deficiency. Therefore, we suggest that manipulating LIMP-2 expression to increase lysosomal GC activity is a promising strategy for the treatment of synucleinopathies

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) is involved in lysosomal cholesterol export

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol-like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.status: publishe