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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 , 3 – 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. The lysosomal transmembrane protein CLN3 is required for the lysosomal clearance of glycerophosphodiesters in mice and in human cells, suggesting that the loss of CLN3 causes Batten disease in children due to defects in glycerophospholipid metabolism.

The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo -inositol oxygenases that convert myo -inositol into d -glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo -inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen. IMPORTANCE The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. Our recent studies revealed that inositol utilization by the fungus promotes C. neoformans development in nature and invasion of the central nervous system during infection. The mechanisms of the inositol regulation in fungal virulence remain incompletely understood. In this study, we found that C. neoformans has three genes encoding myo -inositol oxygenase, a key enzyme in the inositol catabolic pathway. Expression of these genes is highly induced by inositol, and they are highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. Our mutagenesis analysis indeed demonstrates that inositol catabolism is required for C. neoformans virulence by altering the growth and structure of polysaccharide capsule, a major virulence factor. Considering the abundance of free inositol and inositol-related metabolites in the brain, our study reveals an important mechanism of host inositol-mediated fungal pathogenesis for this neurotropic fungal pathogen.

Primary central nervous system lymphoma (PCNSL) is a rare and aggressive extranodal lymphoma, which is distinct from nodal diffuse large B-cell lymphoma (DLBCL) in clinical presentation, molecular pathology, treatment, and outcomes. This study employed metabolomics and proteomics to characterize the molecular features of cerebrospinal fluid (CSF) sample from PCNSL, aiming to identify novel diagnostic, therapeutic, and prognostic markers for PCNSL. For metabolomics, CSF samples were obtained from 6 patients with PCNSL and 13 patients with primary nodal DLBCL. The liquid chromatography-mass spectrometric (LC/MS) method was used to detect the metabolites of CSF. Comparative investigations were performed to identify the distinct metabolic products and pathways. For proteomics, TMT-labeled quantitative proteomics was used to analyze CSF samples from 3 PCNSL patients at three stages: before treatment, during disease remission, and during disease progression. Protein screening and enrichment analysis were performed to obtain the protein markers and pathways. 183 elevated and 314 decreased metabolites were found in the CSF of PCNSL, compared to primary nodal DLBCL. 14 differential metabolites were identified (VIP > 1, P < 0.05), including adenine, ribitol, myo-inositol, d-xylitol, hypoxanthine, D-mannose, gluconic acid, etc. The metabolites participated in 31 metabolic pathways, including pentose and glucuronate interconversions, valine, leucine, and isoleucine biosynthesis, galactose, pentose phosphate, ascorbate and aldarate, fructose and mannose, glyoxylate and dicarboxylate, and glycine, serine, and threonine pathways, etc. All differential metabolites exhibited high diagnostic values with area under the curve (AUC) ≥ 0.8. Compared to the untreated group, 14 differentially expressed proteins were found in disease remission group; Compared to the untreated group, 10 differentially expressed proteins were found in disease progression group; Compared to disease remission group, 18 differentially expressed proteins were found in disease progression group. Differentially expressed proteins were enriched in cornified envelope formation, estrogen signaling pathway, metabolic pathway, and protein digestion and absorption. These multi-omic data indicated the distinct metabolic and proteomic phenotypes in PCNSL patients. The analysis of differential metabolites and proteins is expected to offer diagnostic value for PCNSL and contribute to the exploration of the underlying mechanisms of PCNSL.