Levacetylleucine (Aqneursa™, N-acetyl-L-leucine, NALL) is an acetylated derivative and pro-drug of L-leucine that is approved by the US FDA as monotherapy for Niemann–Pick disease type C (NPC). The acetyl modification confers a net negative charge at physiological pH, enabling uptake via monocarboxylate transporters (MCTs) and allowing efficient penetration of the blood–brain barrier and cellular entry. Intracellular hydrolysis by acylases produces L-leucine, which enhances mitochondrial bioenergetics and has been associated with improved lysosomal function and reductions in lysosomal lipid storage. Given the central role of lysosomal dysfunction in NPC, the effects of levacetylleucine on lysosomal regulatory pathways were investigated, focusing on Transcription Factor EB (TFEB), a master regulator of lysosome and autophagy gene networks (the CLEAR network).
The authors examined whether HeLa cells express the molecular machinery proposed to mediate levacetylleucine uptake and intracellular conversion. Using RT-qPCR, HeLa cells were shown to contain transcribed mRNA for multiple MCT isoforms (including MCT1) and for the aminoacylase ACY1, an enzyme known to hydrolyse N-acetyl groups from amino acids. Expression values were normalised to β-actin on a scale where β-actin equals 10,000 arbitrary units. These findings support the model that levacetylleucine can enter HeLa cells via MCTs and be deacetylated intracellularly to release L-leucine, which in turn can enter metabolic pathways that increase ATP synthesis and influence lysosomal clearance.
To assess direct effects on lysosomal regulation, the investigators quantified TFEB translocation from the cytoplasm to the nucleus—an established marker of TFEB activation. HeLa cells were transiently transfected with TFEB tagged with fluorescent proteins (TFEB-EGFP or TFEB-mScarlet3) and incubated with levacetylleucine (NALL). Imaging and colocalization analysis (Pearson’s Correlation Coefficient between TFEB fluorescence and nuclear stain) revealed that extracellular NALL induced nuclear TFEB in wild-type HeLa cells during an 18-hour incubation.
The response was concentration-dependent, with effects detectable at sub-millimolar concentrations consistent with plasma levels reported in mice dosed with therapeutic levacetylleucine. The onset of action was rapid: half-maximal nuclear translocation was reached within approximately 60 minutes at the tested concentration (2 mM). Nuclear localisation was quantified both as Pearson’s coefficient and as the percentage of cells with a Pearson coefficient > 0 (indicative of nuclear TFEB), with statistical significance assessed by ANOVA.
NPC1 gene dysfunction causes lysosomal lipid accumulation and associated cellular stress. The authors report that in NPC1-deficient (NPC1-/-) HeLa cells, TFEB is over-activated and enriched in the nucleus, consistent with lysosomal stress-driven activation of the TFEB pathway. Importantly, levacetylleucine produced a contrasting effect in this disease model: rather than further activating TFEB, the L-enantiomer reduced nuclear TFEB and restored a more normal cytoplasmic-to-nuclear balance. This bidirectional modulation—activation in wild-type cells and attenuation of over-activation in NPC1-/- cells—suggests a homeostasis-restoring mechanism of drug action at clinically relevant concentrations associated with lysosomal storage reduction.
The TFEB-modulatory effects described were stereospecific. The active form was the L-enantiomer (N-acetyl-L-leucine). The D-enantiomer and the racemate showed no effect on TFEB translocation in the assays reported, and the D-enantiomer displayed antagonistic properties. These observations indicate that the cellular effects on TFEB and lysosomal pathways depend on stereochemistry, aligning with the hypothesis that enzymatic deacetylation and downstream metabolic routing of L-leucine are required for activity.
Key experimental methods included HeLa cell culture and transient transfection of fluorescently tagged TFEB constructs; live-cell imaging with nuclear counterstains; quantification of TFEB nuclear translocation by Pearson’s correlation with nuclear stains; RT-qPCR for transporter and acylase mRNA expression (normalised to β-actin); Western blotting for NPC1 protein verification; immunofluorescent labelling of endogenous TFEB; and in-cell western assays to detect LAMP1 and TFEB/phospho-TFEB under treatment conditions. The NPC1 KO HeLa line was generated by CRISPR-Cas9 and verified as previously described.
These results identify a direct mechanism by which levacetylleucine can modulate lysosomal function via TFEB, complementing prior models in which intracellular L-leucine improves mitochondrial bioenergetics and indirectly benefits lysosomes. The bidirectional, stereospecific normalization of TFEB suggests a capacity to restore lysosomal homeostasis in conditions of either deficient or excessive TFEB activity. Given the role of TFEB dysregulation in multiple neurodegenerative and neurodevelopmental disorders, this mechanism provides a plausible basis for the therapeutic effects of levacetylleucine in NPC and supports continued investigation of its application across diverse neurological diseases.
This summary is restricted to findings reported in the source article. Detailed quantitative datasets, full time courses, additional mechanistic assays, and supporting figures are provided in the original manuscript and its Supporting Information, as cited by the authors. Where specific numeric results beyond those summarised (for example detailed fold-changes in gene expression or exact antagonist potency of the D-enantiomer) were not fully excerpted here, those details are reported in the primary publication. All relevant data are stated by the authors to be within the manuscript and supporting files.