Compartmentalization proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation.

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL facilitate the elimination of damaged membranes, protein aggregates, membranous organelles and play an important role in calcium signaling. The specific role of EL in cardiac atrial fibrillation (AF) is not well understood. We isolated atrial EL organelles from AF goat biopsies and conducted a comprehensive integrated omics analysis to study the EL-specific proteins and pathways. We also performed electron tomography, protein and enzyme assays on these biopsies. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates, including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophagic-vacuole formation, irregularly shaped mitochondria, and glycogen deposition. Our results provide molecular information suggesting EL play a role in AF disease process over extended time frames.

The expanding boundaries of sphingolipid lysosomal storage diseases; insights from Niemann-Pick disease type C.

Lysosomal storage diseases are inborn errors of metabolism that arise due to loss of function mutations in genes encoding lysosomal enzymes, protein co-factors or lysosomal membrane proteins. As a consequence of the genetic defect, lysosomal function is impaired and substrates build up in the lysosome leading to 'storage'. A sub group of these disorders are the sphingolipidoses in which sphingolipids accumulate in the lysosome. In this review, I will discuss how the study of these rare lysosomal disorders reveals unanticipated links to other rare and common human diseases using Niemann-Pick disease type C as an example.

Cholera intoxication of human enteroids reveals interplay between decoy and functional glycoconjugate ligands.

Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.

NPC1 plays a role in the trafficking of specific cargo to melanosomes.

Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.

A Mouse Systems Genetics Approach Reveals Common and Uncommon Genetic Modifiers of Hepatic Lysosomal Enzyme Activities and Glycosphingolipids.

Identification of genetic modulators of lysosomal enzyme activities and glycosphingolipids (GSLs) may facilitate the development of therapeutics for diseases in which they participate, including Lysosomal Storage Disorders (LSDs). To this end, we used a systems genetics approach: we measured 11 hepatic lysosomal enzymes and many of their natural substrates (GSLs), followed by modifier gene mapping by GWAS and transcriptomics associations in a panel of inbred strains. Unexpectedly, most GSLs showed no association between their levels and the enzyme activity that catabolizes them. Genomic mapping identified 30 shared predicted modifier genes between the enzymes and GSLs, which are clustered in three pathways and are associated with other diseases. Surprisingly, they are regulated by ten common transcription factors, and their majority by miRNA-340p. In conclusion, we have identified novel regulators of GSL metabolism, which may serve as therapeutic targets for LSDs and may suggest the involvement of GSL metabolism in other pathologies.

Glb1 knockout mouse model shares natural history with type II GM1 gangliosidosis patients.

GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of ß-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1-/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1-/- mice die six weeks earlier than male Glb1-/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1-/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1-/- mice as seen in some patients. In addition, Glb1-/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1-/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1-/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.

Heat shock protein amplification improves cerebellar myelination in the Npc1nih mouse model.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare prematurely fatal lysosomal lipid storage disease with limited therapeutic options. The prominent neuropathological hallmarks include hypomyelination and cerebellar atrophy. We previously demonstrated the efficacy of recombinant human heat shock protein 70 (rhHSP70) in preclinical models of the disease. It reduced glycosphingolipid levels in the central nervous system (CNS), improving cerebellar myelination and improved behavioural phenotypes in Npc1nih (Npc1-/-) mice. Furthermore, treatment with arimoclomol, a well-characterised HSP amplifier, attenuated lysosomal storage in NPC patient fibroblasts and improved neurological symptoms in Npc1-/- mice. Taken together, these findings prompted the investigation of the effects of HSP amplification on CNS myelination. METHODS: We administered bimoclomol daily or rhHSP70 6 times per week to Npc1-/- (BALB/cNctr-Npc1m1N/J, also named Npc1nih) mice by intraperitoneal injection from P7 through P34 to investigate the impact on CNS myelination. The Src-kinase inhibitor saracatinib was administered with/without bimoclomol twice daily to explore the contribution of Fyn kinase to bimoclomol's effects. FINDINGS: Treatment with either bimoclomol or rhHSP70 improved myelination and increased the numbers of mature oligodendrocytes (OLs) as well as the ratio of active-to-inactive forms of phosphorylated Fyn kinase in the cerebellum of Npc1-/- mice. Additionally, treatment with bimoclomol preserved cerebellar weight, an effect that was abrogated when co-administered with saracatinib, an inhibitor of Fyn kinase. Bimoclomol-treated mice also exhibited increased numbers of immature OLs within the cortex. INTERPRETATION: These data increase our understanding of the mechanisms by which HSP70 regulates myelination and provide further support for the clinical development of HSP-amplifying therapies in the treatment of NPC. FUNDING: Funding for this study was provided by Orphazyme A/S (Copenhagen, Denmark) and a Pathfinder Award from The Wellcome Trust.

Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria.

Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease.

Anti-TNF therapy for inflammatory bowel disease in patients with neurodegenerative Niemann-Pick disease Type C.

Background:  Blockade of tumour necrosis factor (anti-TNF) is effective in patients with Crohn's Disease but has been associated with infection risk and neurological complications such as demyelination. Niemann-Pick disease Type C1 (NPC1) is a lysosomal storage disorder presenting in childhood with neurological deterioration, liver damage and respiratory infections. Some NPC1 patients develop severe Crohn's disease. Our objective was to investigate the safety and effectiveness of anti-TNF in NPC1 patients with Crohn's disease. Methods: Retrospective data on phenotype and therapy response were collected in 2019-2020 for the time period 2014 to 2020 from patients in the UK, France, Germany and Canada with genetically confirmed NPC1 defects and intestinal inflammation. We investigated TNF secretion in peripheral blood mononuclear cells treated with NPC1 inhibitor in response to bacterial stimuli . Results: NPC1 inhibitor treated peripheral blood mononuclear cells (PBMCs) show significantly increased TNF production after lipopolysaccharide or bacterial challenge providing a rationale for anti-TNF therapy. We identified 4 NPC1 patients with Crohn's disease (CD)-like intestinal inflammation treated using anti-TNF therapy (mean age of onset 8.1 years, mean treatment length 27.75 months, overall treatment period 9.25 patient years). Anti-TNF therapy was associated with reduced gastrointestinal symptoms with no apparent adverse neurological events. Therapy improved intestinal inflammation in 4 patients. Conclusions: Anti-TNF therapy appears safe in patients with NPC1 and is an effective treatment strategy for the management of intestinal inflammation in these patients.

Glucosamine amends CNS pathology in mucopolysaccharidosis IIIC mouse expressing misfolded HGSNAT.

The majority of mucopolysaccharidosis IIIC (MPS IIIC) patients have missense variants causing misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), which are potentially treatable with pharmacological chaperones. To test this approach, we generated a novel HgsnatP304L mouse model expressing misfolded HGSNAT Pro304Leu variant. HgsnatP304L mice present deficits in short-term and working/spatial memory 2-4 mo earlier than previously described constitutive knockout Hgsnat-Geo mice. HgsnatP304L mice also show augmented severity of neuroimmune response, synaptic deficits, and neuronal storage of misfolded proteins and gangliosides compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons further reduced levels of synaptic proteins. Memory deficits and majority of brain pathology were rescued in mice receiving HGSNAT chaperone, glucosamine. Our data for the first time demonstrate dominant-negative effects of misfolded HGSNAT Pro304Leu variant and show that they are treatable by oral administration of glucosamine. This suggests that patients affected with mutations preventing normal folding of the enzyme can benefit from chaperone therapy.

Current methods to analyze lysosome morphology, positioning, motility and function.

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Correlation of age of onset and clinical severity in Niemann-Pick disease type C1 with lysosomal abnormalities and gene expression.

Niemann-Pick disease type C1 (NPC1) is a rare, prematurely fatal lysosomal storage disorder which exhibits highly variable severity and disease progression as well as a wide-ranging age of onset, from perinatal stages to adulthood. This heterogeneity has made it difficult to obtain prompt diagnosis and to predict disease course. In addition, small NPC1 patient sample sizes have been a limiting factor in acquiring genome-wide transcriptome data. In this study, primary fibroblasts from an extensive cohort of 41 NPC1 patients were used to validate our previous findings that the lysosomal quantitative probe LysoTracker can be used as a predictor for age of onset and disease severity. We also examined the correlation between these clinical parameters and RNA expression data from primary fibroblasts and identified a set of genes that were significantly associated with lysosomal defects or age of onset, in particular neurological symptom onset. Hierarchical clustering showed that these genes exhibited distinct expression patterns among patient subgroups. This study is the first to collect transcriptomic data on such a large scale in correlation with clinical and cellular phenotypes, providing a rich genomic resource to address NPC1 clinical heterogeneity and discover potential biomarkers, disease modifiers, or therapeutic targets.

Vesicle cholesterol controls exocytotic fusion pore.

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Defective iron homeostasis and hematological abnormalities in Niemann-Pick disease type C1.

Background: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium. The lysosomal system regulates key aspects of iron homeostasis, which prompted us to investigate whether there are hematological abnormalities and iron metabolism defects in NPC1. Methods: Iron-related hematological parameters, systemic and tissue metal ion and relevant hormonal and proteins levels, expression of specific pro-inflammatory mediators and erythrophagocytosis were evaluated in an authentic mouse model and in a large cohort of NPC patients. Results: Significant changes in mean corpuscular volume and corpuscular hemoglobin were detected in Npc1 -/- mice from an early age. Hematocrit, red cell distribution width and hemoglobin changes were observed in late-stage disease animals. Systemic iron deficiency, increased circulating hepcidin, decreased ferritin and abnormal pro-inflammatory cytokine levels were also found. Furthermore, there is evidence of defective erythrophagocytosis in Npc1 -/- mice and in an in vitro NPC1 cellular model. Comparable hematological changes, including low normal serum iron and transferrin saturation and low cerebrospinal fluid ferritin were confirmed in NPC1 patients. Conclusions: These data suggest loss of iron homeostasis and hematological abnormalities in NPC1 may contribute to the pathophysiology of this disease.

Glycosphingolipid metabolism and its role in ageing and Parkinson's disease.

It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson's disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.

GM1 Gangliosidosis-A Mini-Review.

GM1 gangliosidosis is a progressive, neurosomatic, lysosomal storage disorder caused by mutations in the GLB1 gene encoding the enzyme ß-galactosidase. Absent or reduced ß-galactosidase activity leads to the accumulation of ß-linked galactose-containing glycoconjugates including the glycosphingolipid (GSL) GM1-ganglioside in neuronal tissue. GM1-gangliosidosis is classified into three forms [Type I (infantile), Type II (late-infantile and juvenile), and Type III (adult)], based on the age of onset of clinical symptoms, although the disorder is really a continuum that correlates only partially with the levels of residual enzyme activity. Severe neurocognitive decline is a feature of Type I and II disease and is associated with premature mortality. Most of the disease-causing ß-galactosidase mutations reported in the literature are clustered in exons 2, 6, 15, and 16 of the GLB1 gene. So far 261 pathogenic variants have been described, missense/nonsense mutations being the most prevalent. There are five mouse models of GM1-gangliosidosis reported in the literature generated using different targeting strategies of the Glb1 murine locus. Individual models differ in terms of age of onset of the clinical, biochemical, and pathological signs and symptoms, and overall lifespan. However, they do share the major abnormalities and neurological symptoms that are characteristic of the most severe forms of GM1-gangliosidosis. These mouse models have been used to study pathogenic mechanisms, to identify biomarkers, and to evaluate therapeutic strategies. Three GLB1 gene therapy trials are currently recruiting Type I and Type II patients (NCT04273269, NCT03952637, and NCT04713475) and Type II and Type III patients are being recruited for a trial utilizing the glucosylceramide synthase inhibitor, venglustat (NCT04221451).

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue.

The importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Identification of genetic modifiers of murine hepatic ß-glucocerebrosidase activity.

The acid ß-glucocerebrosidase (GCase) enzyme cleaves glucosylceramide into glucose and ceramide. Loss of function variants in the gene encoding for GCase can lead to Gaucher disease and Parkinson's disease. Therapeutic strategies aimed at increasing GCase activity by targeting a modulating factor are attractive and poorly explored. To identify genetic modifiers, we measured hepatic GCase activity in 27 inbred mouse strains. A genome-wide association study (GWAS) using GCase activity as a trait identified several candidate modifier genes, including Dmrtc2 and Arhgef1 (p=2.1x10-7), and Grik5 (p=2.1x10-7). Bayesian integration of the gene mapping with transcriptomics was used to build integrative networks. The analysis uncovered additional candidate GCase regulators, highlighting modules of the acute phase response (p=1.01x10-8), acute inflammatory response (p=1.01x10-8), fatty acid beta-oxidation (p=7.43x10-5), among others. Our study revealed previously unknown candidate modulators of GCase activity, which may facilitate the design of therapies for diseases with GCase dysfunction.

An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.

Acetylation turns leucine into a drug by membrane transporter switching.

Small changes to molecules can have profound effects on their pharmacological activity as exemplified by the addition of the two-carbon acetyl group to make drugs more effective by enhancing their pharmacokinetic or pharmacodynamic properties. N-acetyl-D,L-leucine is approved in France for vertigo and its L-enantiomer is being developed as a drug for rare and common neurological disorders. However, the precise mechanistic details of how acetylation converts leucine into a drug are unknown. Here we show that acetylation of leucine switches its uptake into cells from the L-type amino acid transporter (LAT1) used by leucine to organic anion transporters (OAT1 and OAT3) and the monocarboxylate transporter type 1 (MCT1). Both the kinetics of MCT1 (lower affinity compared to LAT1) and the ubiquitous tissue expression of MCT1 make it well suited for uptake and distribution of N-acetyl-L-leucine. MCT1-mediated uptake of a N-acetyl-L-leucine as a prodrug of leucine bypasses LAT1, the rate-limiting step in activation of leucine-mediated signalling and metabolic process inside cells such as mTOR. Converting an amino acid into an anion through acetylation reveals a way for the rational design of drugs to target anion transporters.