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Reduction of nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43–related neurodegeneration
Leon Tejwani, … , Paymaan Jafar-Nejad, Janghoo Lim
Leon Tejwani, … , Paymaan Jafar-Nejad, Janghoo Lim
Published June 29, 2023
Citation Information: J Clin Invest. 2023;133(16):e138207. https://doi.org/10.1172/JCI138207.
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Research Article Genetics Neuroscience Article has an altmetric score of 19

Reduction of nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43–related neurodegeneration

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Abstract

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding transactive response DNA-binding protein 43 kDa (TDP-43), account for less than 1% of all ALS cases, TDP-43–positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS–causing (fALS-causing) mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer’s disease, and Parkinson’s disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a function of nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and life span deficits in 2 distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared through the autophagy/lysosome pathway, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.

Authors

Leon Tejwani, Youngseob Jung, Hiroshi Kokubu, Sowmithra Sowmithra, Luhan Ni, Changwoo Lee, Benjamin Sanders, Paul J. Lee, Yangfei Xiang, Kimberly Luttik, Armand Soriano, Jennifer Yoon, Junhyun Park, Hannah H. Ro, Hyoungseok Ju, Clara Liao, Sofia Massaro Tieze, Frank Rigo, Paymaan Jafar-Nejad, Janghoo Lim

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Figure 1

Reduction of Nlk increases lysosome biogenesis in vitro.

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Reduction of Nlk increases lysosome biogenesis in vitro.
(A) Schematic o...
(A) Schematic of dual-guide RNA targeting of Cas9 nickase (Cas9n) to Nlk for generation of isogenic Nlk KON 2a cells and NLK+/- hiPSCs. (B and C) Enrichment analyses for all significantly differentially expressed genes from RNA-Seq of isogenic WT and Nlk KO N2a cells. Significant enrichment scores of 1.3 (FDR adjusted P value q < 0.05) are designated by vertical red lines. (D) Heatmap of significantly upregulated lysosomal genes from RNA-Seq (n = 3 biological replicates). (E and F) Western blot validation (E) confirmed RNA-Seq results, quantified in F (n = 3 biological replicates). Blot lanes separated by the dotted line were run on lysates from distinct experimental replicates and normalized to their respective housekeeping proteins. (G and H) Representative LysoTracker images of N2a cells (G), quantified in H, demonstrated increased lysosome number in Nlk KO cells (WT, n = 61 cells; Nlk KO, n = 57 cells). (I and J) Representative images of functional lysosomes in N2a cells incubated with DQ BSA (I), quantified in J (WT, n = 50 cells; Nlk KO, n = 50 cells). (K and L) CLEAR-luciferase assay in Nlk KO N2a cells (K) or WT N2a cells transfected with WT or KN Nlk (L) demonstrated Nlk kinase activity suppresses CLEAR network transcription (K, n = 10 replicates from 2 different Nlk KO clones; L, n = 7 replicates from 2 experimental repeats). (M and N) Western blots (M) showed increased LC3-II/LC3-I and decreased p62 levels in Nlk KO N2a cells, quantified in N (n = 4 biological replicates). (O) Representative immunostaining images of hiPSC-derived motor neurons. (P) qPCR showing NLK+/– iPSC–derived motor neurons expressed higher levels of CTSA, CTSD, LAMP1, and LAMP2 compared with isogenic controls (n = 3 biological replicates from distinct motor neuron differentiations). Two-tailed t tests (F, H, J, K, N, and P) or 1-way ANOVA (L) analyses were performed, and data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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