Juvenile CLN3 disease is a lysosomal cholesterol storage disorder: similarities with Niemann-Pick type C disease.

BACKGROUND: The most common form of neuronal ceroid lipofuscinosis (NCL) is juvenile CLN3 disease (JNCL), a currently incurable neurodegenerative disorder caused by mutations in the CLN3 gene. Based on our previous work and on the premise that CLN3 affects the trafficking of the cation-independent mannose-6 phosphate receptor and its ligand NPC2, we hypothesised that dysfunction of CLN3 leads to the aberrant accumulation of cholesterol in the late endosomes/lysosomes (LE/Lys) of JNCL patients' brains. METHODS: An immunopurification strategy was used to isolate intact LE/Lys from frozen autopsy brain samples. LE/Lys isolated from samples of JNCL patients were compared with age-matched unaffected controls and Niemann-Pick Type C (NPC) disease patients. Indeed, mutations in NPC1 or NPC2 result in the accumulation of cholesterol in LE/Lys of NPC disease samples, thus providing a positive control. The lipid and protein content of LE/Lys was then analysed using lipidomics and proteomics, respectively. FINDINGS: Lipid and protein profiles of LE/Lys isolated from JNCL patients were profoundly altered compared to controls. Importantly, cholesterol accumulated in LE/Lys of JNCL samples to a comparable extent than in NPC samples. Lipid profiles of LE/Lys were similar in JNCL and NPC patients, except for levels of bis(monoacylglycero)phosphate (BMP). Protein profiles detected in LE/Lys of JNCL and NPC patients appeared identical, except for levels of NPC1. INTERPRETATION: Our results support that JNCL is a lysosomal cholesterol storage disorder. Our findings also support that JNCL and NPC disease share pathogenic pathways leading to aberrant lysosomal accumulation of lipids and proteins, and thus suggest that the treatments available for NPC disease may be beneficial to JNCL patients. This work opens new avenues for further mechanistic studies in model systems of JNCL and possible therapeutic interventions for this disorder. FUNDING: San Francisco Foundation.

Lysosomal phospholipase A2 contributes to the biosynthesis of the atypical late endosome lipid bis(monoacylglycero)phosphate.

The late endosome/lysosome (LE/Lys) lipid bis(monoacylglycero)phosphate (BMP) plays major roles in cargo sorting and degradation, regulation of cholesterol and intercellular communication and has been linked to viral infection and neurodegeneration. Although BMP was initially described over fifty years ago, the enzymes regulating its synthesis remain unknown. The first step in the BMP biosynthetic pathway is the conversion of phosphatidylglycerol (PG) into lysophosphatidylglycerol (LPG) by a phospholipase A2 (PLA2) enzyme. Here we report that this enzyme is lysosomal PLA2 (LPLA2). We show that LPLA2 is sufficient to convert PG into LPG in vitro. We show that modulating LPLA2 levels regulates BMP levels in HeLa cells, and affects downstream pathways such as LE/Lys morphology and cholesterol levels. Finally, we show that in a model of Niemann-Pick disease type C, overexpressing LPLA2 alleviates the LE/Lys cholesterol accumulation phenotype. Altogether, we shed new light on BMP biosynthesis and contribute tools to regulate BMP-dependent pathways.

Fast and Sensitive Method for the Determination of 17 Designer Benzodiazepines in Hair by Liquid Chromatography-Tandem Mass Spectrometry.

In recent years, identification and analysis of designer benzodiazepines have become a challenge in forensic toxicology. These substances are analogs of the classic benzodiazepines, but their pharmacology is not well known, and many of them have been associated with overdoses and deaths. As a result, there has been a surge in efforts to develop analytical methods to determine these compounds in different biological samples. Our aim was to develop and validate a fast, sensitive and specific method for determining 17 designer benzodiazepines (adinazolam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, N-desmethylclobazam, nifoxipam, nitrazolam, meclonazepam, pyrazolam and zolazepam) in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized, and a 20 mg aliquot was incubated in methanol in an ultrasound bath (1 h, 25°C). The supernatant was evaporated and reconstituted in 200 µL of mobile phase, and the extracts were filtered (nano-filter vials) before injection into LC-MS-MS. All analytes were eluted from the chromatographic column in 8 min, and two multiple-reaction monitoring (MRM) transitions were used to identify each compound. The limits of quantification were 5 or 25 pg/mg depending on the analyte, and the calibration functions were linear to 200 pg/mg. Imprecision was <19.2% (n = 15), and bias was from -13.7 to 18.3% (n = 15). All the analytes yielded high extraction efficiencies >70% and displayed ion suppression between -62.8% and -23.9% (n = 10). The method was applied to 19 authentic cases. Five samples were positive for flualprazolam ( 200 pg/mg) and/or etizolam (47.4-88.5 pg/mg). In conclusion, the present validated method has proven to be fast, sensitive, specific and capable of determining 17 designer benzodiazepines in hair using LC-MS-MS.