• Journal of Life Science
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• v.34 no.9
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• pp.609-619
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• 2024

Neurodegenerative diseases are marked by the accumulation of toxic misfolded proteins in neurons. Therefore, strategies for the effective prevention and clearance of aggregates are crucial for therapeutic interventions. Cytoplasmic dynein plays a crucial role in the clearance of aggregates by transporting them to the cell center, where lysosomes are enriched and the aggregates undergo extensive autophagic degradation. Previously, we reported evidence for the activation of dynein by N-acetylglucosamine kinase (NAGK) and Reln. In the present study, we explored the effects of NAGK and Reln upregulation on the clearance of aggregates. To upregulate NAGK and Reln genes in HEK293T cells (a human embryonic kidney cell line), CRISPR/dCas9 activation systems (CASs) were used with specific plasmids encoding target-specific 20 nt guide RNA. The effects of this genetic modulation were analyzed in Huntington's disease cellular models, including HEK293T cells and primary mouse cortical cells, where external mutant huntingtin (mHtt, Q74) aggregates were induced. The results showed that the CAS activation of NAGK or Reln, or their combination, significantly reduced the proportion of cells with Q74 aggregates (aggresomes). This effect was reversed by Ciliobrevin D (a dynein inhibitor) and chloroquine (an autophagy inhibitor), indicating the role of dynein-mediated autophagy in aggregate clearance. These findings provide the basis for therapeutic strategies aimed at enhancing neuronal health through targeted gene activation.

• Anatomy and Cell Biology
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• v.56 no.1
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• pp.94-108
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• 2023

Cancer cell heterogeneity is a serious problem in the control of tumor progression because it can cause chemoresistance and metastasis. Heterogeneity can be generated by various mechanisms, including genetic evolution of cancer cells, cancer stem cells (CSCs), and niche heterogeneity. Because the genetic heterogeneity of CSCs has been poorly characterized, the genetic mutation status of CSCs was examined using Exome-Seq and RNA-Seq data of liver cancer. Here we show that different surface markers for liver cancer stem cells (LCSCs) showed a unique propensity for genetic mutations. Cluster of differentiation 133 (CD133)-positive cells showed frequent mutations in the IRF2, BAP1, and ERBB3 genes. However, leucine-rich repeat-containing G protein-coupled receptor 5-positive cells showed frequent mutations in the CTNNB1, RELN, and ROBO1 genes. In addition, some genetic mutations were frequently observed irrespective of the surface markers for LCSCs. BAP1 mutations was frequently observed in CD133-, CD24-, CD13-, CD90-, epithelial cell adhesion molecule-, or keratin 19-positive LCSCs. ASXL2, ERBB3, IRF2, TLX3, CPS1, and NFATC2 mutations were observed in more than three types of LCSCs, suggesting that common mechanisms for the development of these LCSCs. The present study provides genetic heterogeneity depending on the surface markers for LCSCs. The genetic heterogeneity of LCSCs should be considered in the development of LCSC-targeting therapeutics.