N-acetyl-l-leucine lowers α-synuclein levels and improves synaptic function in Parkinson’s disease models
Pingping Song, Chuyu Chen, Rossella Franchini, Bryan Duong, Yi-Zhi Wang, Robert Coukos, Zhong Xie, Jeffrey N. Savas, Yueqin Zhou, Mariarita Bertoldi, D. James Surmeier, Loukia Parisiadou, Dimitri Krainc
Pingping Song, Chuyu Chen, Rossella Franchini, Bryan Duong, Yi-Zhi Wang, Robert Coukos, Zhong Xie, Jeffrey N. Savas, Yueqin Zhou, Mariarita Bertoldi, D. James Surmeier, Loukia Parisiadou, Dimitri Krainc
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Research Article Cell biology Neuroscience

N-acetyl-l-leucine lowers α-synuclein levels and improves synaptic function in Parkinson’s disease models

Abstract

N-acetyl-l-leucine (NALL), a derivative of the branched-chain amino acid leucine, has shown therapeutic potential for neurodegenerative diseases, including in prodromal stages of Parkinson’s disease (PD). However, the mechanism of its protective effects has been largely unknown. Using human induced pluripotent stem cell–derived dopaminergic neurons from patients carrying GBA1, LRRK2, or VPS35 mutations, as well as from sporadic PD cases, we found that NALL treatment markedly reduced Ser129 phosphorylated α-synuclein (pS129-syn). Discovery-based proteomic analysis revealed that NALL treatment upregulated lysosomal, mitochondrial, and synaptic proteins without inducing cytotoxicity. The reduction of pS129-syn was dependent on serine protease HTRA1, which was robustly induced by NALL. Moreover, NALL increased the expression of wild-type parkin in mutant dopaminergic neurons, leading to increased glycosylated dopamine transporter, elevated synaptic membrane-associated synaptojanin-1, and accelerated synaptic vesicle endocytosis, suggesting improved synaptic function. Furthermore, in LRRK2R1441C knockin mice, NALL administration decreased pS129-syn, elevated parkin levels, and ameliorated dopamine-dependent motor learning deficits. These findings highlight the therapeutic potential of NALL for PD by its protective effects on α-synuclein pathology and synaptic function in vulnerable dopaminergic neurons.

Authors

Pingping Song, Chuyu Chen, Rossella Franchini, Bryan Duong, Yi-Zhi Wang, Robert Coukos, Zhong Xie, Jeffrey N. Savas, Yueqin Zhou, Mariarita Bertoldi, D. James Surmeier, Loukia Parisiadou, Dimitri Krainc

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Figure 1

NALL leads to decreased pS129-syn in human dopaminergic neurons with GBA1 mutations.

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NALL leads to decreased pS129-syn in human dopaminergic neurons with GBA...
(A) Representative Western blot showing pS129-syn and total α-syn (Syn) levels following 30 days of treatment with increasing concentrations of NALL in the Triton-soluble fraction of GBA1 L444P mutant dopaminergic neurons. β-III-tubulin and GAPDH served as loading controls. (B) Quantification of pS129-syn (top) and total Syn (bottom) signals in A, normalized to β-III-tubulin and expressed relative to the 0 mM (DMSO) group (n = 3–4 independent experiments; 1-way ANOVA). (C) Western blot of pS129-syn and total Syn in the Triton-insoluble fraction of GBA1 L444P mutant neurons following NALL treatment; total protein staining served as a loading control. (D) Quantification of pS129-syn (top) and total Syn (bottom) signals in C, normalized to total protein and expressed relative to the 0 mM (DMSO) group (n = 3–4 independent experiments; 1-way ANOVA). (E–H) Similar analyses performed in GBA1 N370S mutant dopaminergic neurons. (E and G) Representative blots of soluble (E) and insoluble (G) fractions with corresponding quantifications (F and H), normalized and expressed relative to the 0 mM (DMSO) group (n = 3 independent experiments; 1-way ANOVA). (I) (Top) Schematic of the experimental design showing NALL treatment (10 mM) of GBA1 L444P mutant dopaminergic neurons at day 120 for 7, 14, or 21 days. (Bottom) Western blot of pS129-syn in the Triton-soluble fraction with or without NALL treatment. β-III-tubulin and GAPDH were used as loading controls. (J) Quantification of pS129-syn levels in I, normalized to β-III-tubulin and expressed relative to the nontreated (NT; DMSO) group (n = 3 independent experiments; 2-way ANOVA). (K) Western blot of pS129-syn in the Triton-insoluble fraction of GBA1 L444P neurons with or without NALL treatment; total protein staining was used as a loading control. (L) Quantification of pS129-syn in K, normalized to total protein and expressed relative to the NT (DMSO) group (n = 3 independent experiments; 2-way ANOVA). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.005.