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Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus 1 . Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs) 2-4 . For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal β-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.

HSD17B1 is a steroid metabolising enzyme. We have previously generated knockout mice that had the entire coding region of Hsd17b1 replaced with lacZ-neo cassette (Hsd17b1-LacZ/Neo mice). This resulted in a 90% reduction of HSD17B1 activity, associated with severe subfertility in the knockout females. The present study indicates that Hsd17b1-LacZ/Neo male mice have a metabolic phenotype, including reduced adipose mass, increased lean mass and lipid accumulation in the liver. During the characterisation of this metabolic phenotype, it became evident that the expression of the Naglu gene, located closely upstream of Hsd17b1, was severely reduced in all tissues analysed. Similar results were obtained from Hsd17b1-LacZ mice after removing the neo cassette from the locus or by crossing the Hsd17b1-LacZ/Neo mice with transgenic mice constitutively expressing human HSD17B1. The deficiency of Naglu caused the accumulation of glycosaminoglycans in all studied mouse models lacking the Hsd17b1 gene. The metabolic phenotypes of the Hsd17b1 knockout mouse models were recapitulated in Naglu knockout mice. Based on the data we propose that the Hsd17b1 gene includes a regulatory element controlling Naglu expression and the metabolic phenotype in mice lacking the Hsd17b1 genomic region is caused by the reduced expression of Naglu rather than the lack of Hsd17b1.

Lysosomal storage diseases are inborn errors of metabolism, the hallmark of which is the accumulation, or storage, of macromolecules in the late endocytic system. They are monogenic disorders that occur at a collective frequency of 1 in 5,000 live births and are caused by inherited defects in genes that mainly encode lysosomal proteins, most commonly lysosomal enzymes. A subgroup of these diseases involves the lysosomal storage of glycosphingolipids. Through our understanding of the genetics, biochemistry and, more recently, cellular aspects of sphingolipid storage disorders, we have gained insights into fundamental aspects of cell biology that would otherwise have remained opaque. In addition, study of these disorders has led to significant progress in the development of therapies, several of which are now in routine clinical use. Emerging mechanistic links with more common diseases suggest we need to rethink our current concept of disease boundaries.

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.

Storage diseases because of lysosomal enzyme deficiencies may be treated by the transplantation of cells that secrete the enzyme which is deficient in patients. One can expect that increasing the amount of secreted enzymes will improve the therapy efficacy. Secretion of lysosomal enzymes can be enhanced by reducing the mannose 6-phosphate receptor involved in the lysosomal sorting of newly synthesized lysosomal enzymes. For this purpose, we have constructed hammerhead ribozymes targeting the mRNA of the large murine mannose 6-phosphate receptor (M6PR300). In vitro ribozymes cleave M6PR300 RNA fragments efficiently with cleavage rates of 69-93% after 3 h of incubation. Ribozymes were cloned into an expression vector in which they are integrated into the VaI adenovirus RNA to increase stability and in which they are transcribed from an RNA polymerase III promoter. These plasmids were transiently transfected into BHK cells to investigate in vivo activity. Two ribozymes reduce efficiently the levels of murine M6PR300 mRNA in transient transfection experiments to 42-45%. This correlates with the reduction of M6PR300 biosynthesis, which is reduced also to 37% of normal. We can also demonstrate that the reduction in M6PR300 is sufficient to increase a lysosomal enzyme secretion.

Despite disappointments with early clinical studies, there is continued interest in the development of gene therapy for the group of metabolic diseases referred to as lysosomal storage disorders (LSDs). The LSDs are monogenic and several small and large, representative animal models of the human diseases are available. Further, the successful reconstitution of only low and unregulated tissue levels of the affected lysosomal enzymes are expected to be sufficient to correct the disease at least in the case of some of the LSDs. For these reasons, they are perceived as good models for the evaluation of different gene delivery vectors and of different strategies for treating chronic genetic diseases by gene transfer. In this review, we will highlight the progress that has been made over the past 2 years in preclinical research for this group of disorders and speculate on future prospects.

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danon's disease is associated with the accumulation of autophagic material in striated myocytes.

Deficiency of lysosomal protective protein/cathepsin A in humans is the primary cause of galactosialidosis, a lysosomal storage disease characterized by combined deficiency of beta-galactosidase and neuraminidase. We have investigated 20 galactosialidosis patients and nine of their obligate heterozygous parents. A group of 12 patients with the early infantile type of the disease exhibited practically complete absence of cathepsin A activity, whereas eight patients with either the late infantile or the juvenile/adult type had 2-5% residual activity. Highest levels (5%) were present in two patients with milder clinical manifestations and later onset of the disease. In most fibroblast strains, beta-galactosidase activity was 10-15% of normal levels, whereas neuraminidase was reduced to less than 4%. Interestingly, a substantial residual activity (10%) of the latter enzyme was detected in the patient with the mildest phenotype and the highest cathepsin A activity. Heterozygous values for cathepsin A were reduced on average to half of normal levels. However, in two cell strains, the activity was far below control range, and in these cases, neuraminidase activity was severely depressed. Finally, we showed that cathepsin A had considerable activity in chorionic villi and amniocytes, but was deficient in amniocytes from a pregnancy with an affected fetus, indicating the relevance of cathepsin A assay for prenatal diagnosis of galactosialidosis.