bims-lycede 2025-04-06 papers

FEBS Open Bio. 2025 Apr 03.

A non-fluorescent immunohistochemistry method for measuring autophagy flux using MAP1LC3/LC3 and SQSTM1 as core markers.

Shahla Shojaei, Amir Barzegar Behrooz, Marco Cordani, Mahmoud Aghaei, Negar Azarpira, Daniel J Klionsky, Saeid Ghavami.

Macroautophagy/autophagy is a crucial cellular process for degrading and recycling damaged proteins and organelles, playing a significant role in diseases such as cancer and neurodegeneration. Evaluating autophagy flux, which tracks autophagosome formation, maturation, and degradation, is essential for understanding disease mechanisms. Current fluorescence-based methods are resource-intensive, requiring advanced equipment and expertise, limiting their use in clinical laboratories. Here, we introduce a non-fluorescent immunohistochemistry (IHC) method using MAP1LC3/LC3 and SQSTM1 as core markers for autophagy flux assessment. LC3 levels reflect autophagosome formation, whereas SQSTM1 degradation and a decrease in the number of its puncta indicate active flux (i.e., lysosomal turnover). We optimized chromogenic detection using diaminobenzidine (DAB) staining and developed a scoring system based on puncta number and the percentage of stained cells. This accessible, cost-effective method enables reliable autophagy quantification using a standard light microscope, bridging the gap between experimental research and clinical diagnostics. Our protocol allows accurate autophagy evaluation in fixed tissues, offering practical applications in biomedical research and clinical pathology assessment.

Keywords: autophagometer; autophagy flux measurement; cellular homeostasis analysis; chromogenic detection; cost‐effective autophagy assay; non‐fluorescent immunohistochemistry

DOI: https://doi.org/10.1002/2211-5463.70014

Elife. 2025 Apr 04. pii: RP103137. [Epub ahead of print]13

Small-molecule activation of TFEB alleviates Niemann-Pick disease type C via promoting lysosomal exocytosis and biogenesis.

Kaili Du, Hongyu Chen, Zhaonan Pan, Mengli Zhao, Shixue Cheng, Yu Luo, Wenhe Zhang, Dan Li.

Niemann-Pick disease type C (NPC) is a devastating lysosomal storage disease characterized by abnormal cholesterol accumulation in lysosomes. Currently, there is no treatment for NPC. Transcription factor EB (TFEB), a member of the microphthalmia transcription factors (MiTF), has emerged as a master regulator of lysosomal function and promoted the clearance of substrates stored in cells. However, it is not known whether TFEB plays a role in cholesterol clearance in NPC disease. Here, we show that transgenic overexpression of TFEB, but not TFE3 (another member of MiTF family) facilitates cholesterol clearance in various NPC1 cell models. Pharmacological activation of TFEB by sulforaphane (SFN), a previously identified natural small-molecule TFEB agonist by us, can dramatically ameliorate cholesterol accumulation in human and mouse NPC1 cell models. In NPC1 cells, SFN induces TFEB nuclear translocation via a ROS-Ca2+-calcineurin-dependent but MTOR-independent pathway and upregulates the expression of TFEB-downstream genes, promoting lysosomal exocytosis and biogenesis. While genetic inhibition of TFEB abolishes the cholesterol clearance and exocytosis effect by SFN. In the NPC1 mouse model, SFN dephosphorylates/activates TFEB in the brain and exhibits potent efficacy of rescuing the loss of Purkinje cells and body weight. Hence, pharmacological upregulating lysosome machinery via targeting TFEB represents a promising approach to treat NPC and related lysosomal storage diseases, and provides the possibility of TFEB agonists, that is, SFN as potential NPC therapeutic candidates.

Keywords: NPC1; TFEB agonists; cell biology; cholesterol accumulation; human; lysosome; mouse

DOI: https://doi.org/10.7554/eLife.103137

Biochem Cell Biol. 2025 Apr 01.

Lysosomal enzyme processing and trafficking in the social amoeba Dictyostelium discoideum.

Sean V Condie, William David Kim, Robert Joseph Huber.

Dictyostelium discoideum is a single-celled protist that undergoes multicellular development in response to nutrient deprivation. For close to a century, D. discoideum has been used as a model system for studying conserved cellular and developmental processes such as chemotaxis, cell adhesion, and cell differentiation. In the later decades of the 20th century, intensive research efforts examined the synthesis, trafficking, and activity of lysosomal enzymes in D. discoideum. Subsequent work has revealed that lysosomes are essential for all stages of the D. discoideum life cycle and the genome encodes dozens of homologs of human lysosomal enzymes, including those associated with lysosomal storage diseases. Additionally, protocols for examining the trafficking and activity of lysosomal enzymes in D. discoideum are well-established. Here, we provide a comprehensive up-to-date review that summarizes our current knowledge of lysosomal enzyme processing and trafficking in D. discoideum, with an eye towards re-establishing D. discoideum as a model eukaryote for studying the functions of conserved lysosomal enzymes and the pathways that regulate their trafficking.

DOI: https://doi.org/10.1139/bcb-2025-0062

Curr Opin Cell Biol. 2025 Mar 28. pii: S0955-0674(25)00043-2. [Epub ahead of print]94 102505

Structural insights into traffic through the Golgi complex.

Bryce A Brownfield, J Christopher Fromme.

The Golgi complex is the central sorting station of eukaryotic cells. Several unique trafficking pathways direct the transport of proteins between the Golgi and the endoplasmic reticulum, plasma membrane, and endolysosomal system. In this review we highlight several recent studies that use structural biology approaches to discover and characterize novel mechanisms cells use to control the flow of traffic through the Golgi. These studies provide important new insights into how activation of Arf and Rab GTPases is regulated, how cargo proteins are sorted during vesicle biogenesis, and how vesicle tethers identify their target compartments.

DOI: https://doi.org/10.1016/j.ceb.2025.102505