Yanzhuang Wang

The COVID-19 pandemic has impacted millions of people worldwide, and recent studies have shown that SARS-CoV-2 infection can lead to an Alzheimer's-like neuropathological and biomarker phenotype, as well as clinical symptoms of "brain fog". This raises an intriguing question: "How and where might the molecular pathways underlying SARS-CoV-2 infection and Alzheimer's disease (AD) converge?" One common feature of both SARS-CoV-2 infection and AD is the alteration of the endomembrane system, particularly the fragmentation of the Golgi apparatus. In this review article, we summarize the existing literature on SARS-CoV-2 infection biology and speculate about the potential mechanisms linking Golgi defects, SARS-CoV-2 infection, and neurodegeneration.

The COVID-19 pandemic is caused by the enveloped virus SARS-CoV-2. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Blocking endoplasmic reticulum (ER) to Golgi trafficking dramatically inhibits SARS-CoV-2 assembly and secretion without reducing viral genome replication. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion without affecting viral entry, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to modulate viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

The Golgi apparatus plays a crucial role in the delivery of lysosomal enzymes. Golgi reassembly stacking proteins, GRASP55 and GRASP65, are vital for maintaining Golgi structure and function. GRASP55 depletion results in the missorting and secretion of the lysosomal enzyme cathepsin D, though the mechanisms remain unclear. In this study, we conducted secretomic analyses of GRASP55 knockout cells and found a significant increase in lysosome-associated proteins in the extracellular medium. Using the lysosomal beta-hexosaminidase subunit alpha (HEXA) as a model, we found that GRASP55 depletion disrupted normal trafficking and processing of HEXA, resulting in increased secretion of the immature (pro-form) HEXA into the extracellular milieu, along with decreased levels of the mature form and enzymatic activity within the cell. GRASP55 depletion significantly reduced the complex formation between HEXA and mannose 6-phosphate (M6P) receptors (MPR), despite no overall change in MPR expression. Finally, we found there was a notable reduction in the expression of GNPTAB, leading to a reduction in M6P modification of HEXA, hindering its efficient targeting to lysosomes. These findings reveal the role of GRASP55 in regulating lysosomal enzyme dynamics, emphasizing its role in the sorting and trafficking of lysosomal proteins.

The Golgi apparatus plays a crucial role in the delivery of lysosomal enzymes. Golgi Reassembly Stacking Proteins, GRASP55 and GRASP65, are vital for maintaining Golgi structure and function. GRASP55 depletion results in the missorting and secretion of the lysosomal enzyme Cathepsin D (Xiang et al ., 2013), though the mechanisms remain unclear. In this study, we conducted secretomic analyses of GRASP55 knockout (KO) cells and found a significant increase in lysosome-associated proteins in the extracellular medium. Using the lysosomal beta-hexosaminidase subunit alpha (HEXA) as a model, we found that GRASP55 depletion disrupted normal trafficking and processing of HEXA, resulting in increased secretion of the immature (pro-form) HEXA into the extracellular milieu, along with decreased levels of the mature form and enzymatic activity within the cell. GRASP55 depletion significantly reduced the complex formation between HEXA and mannose-6-phosphate (M6P) receptors (MPR), despite no overall change in MPR expression. And finally, we found there was a notable reduction in the expression of GNPTAB, leading to a reduction in M6P modification of HEXA, hindering its efficient targeting to lysosomes. These findings reveal the role of GRASP55 in regulating lysosomal enzyme dynamics, emphasizing its role in the sorting and trafficking of lysosomal proteins.

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately 9 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 μg/mL, 5.61 μg/mL and 178 μg/mL. Remarkably, the 178 μg/mL eHsp90α matches the previously reported 100–300 μg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data.

The Golgi apparatus plays a crucial role in lysosome biogenesis and the delivery of lysosomal enzymes, essential for maintaining cellular homeostasis and ensuring cell survival. Deficiencies in Golgi structure and function can profoundly impact lysosomal homeostasis, leading to various lysosomal storage diseases and neurodegenerative disorders. In this review, we highlight the role of the Golgi Reassembly Stacking Proteins (GRASPs) in the formation and function of the Golgi apparatus, emphasizing the current understanding of the association between the Golgi apparatus, lysosomes, and lysosomal storage diseases. Additionally, we discuss how Golgi dysfunction leads to the secretion of lysosomal enzymes. This review aims to serve as a concise resource, offering insights into Golgi structure, function, disease-related defects, and their consequential effects on lysosomal biogenesis and function. By highlighting Golgi defects as an underappreciated contributor to lysosomal dysfunction across various diseases, we aim to enhance comprehension of these intricate cellular processes.

In this chapter, we provide a detailed guide for the application of commonly used small molecules to study Golgi structure and function in vitro. Furthermore, we have curated a concise, validated list of endomembrane markers typically used in downstream assays to examine the consequent effect on the Golgi via microscopy and western blot after drug treatment. This chapter will be useful for researchers beginning their foray into the field of intracellular trafficking and Golgi biology. Key words inhibitors markers small molecules antibodies lectin dyes probes immunofluorescence immunoblot