SHIP1 modulation and proteome characterization of microglia.

Understanding microglial states in the aging brain has become crucial, especially with the discovery of numerous Alzheimer's disease (AD) risk and protective variants in genes such as INPP5D and TREM2, which are essential to microglia function in AD. Here we present a thorough examination of microglia-like cells and primary mouse microglia at the proteome and transcriptome levels to illuminate the roles these genes and the proteins they encode play in various cell states. First, we compared the proteome profiles of wildtype and INPP5D (SHIP1) knockout primary microglia.

Common Assays in Mammalian Golgi Studies.

The Golgi is a complex structure characterized by stacks of tightly aligned flat cisternae. In mammalian cells, Golgi stacks often concentrate in the perinuclear region and link together to form a ribbon. This structure is dynamic to accommodate continuous cargo flow in and out of the Golgi in both directions and undergoes morphological changes under physiological and pathological conditions.

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin.

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease-related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA-binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress.

GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion.

Golgi Structure and Function in Health, Stress, and Diseases.

The Golgi apparatus is a central intracellular membrane-bound organelle with key functions in trafficking, processing, and sorting of newly synthesized membrane and secretory proteins and lipids. To best perform these functions, Golgi membranes form a unique stacked structure. The Golgi structure is dynamic but tightly regulated; it undergoes rapid disassembly and reassembly during the cell cycle of mammalian cells and is disrupted under certain stress and pathological conditions.

New Insights Into the Golgi Stacking Proteins.

The Golgi stacking proteins, GRASP55 and GRASP65, are best known for their roles in Golgi structure formation. These peripheral Golgi proteins form -oligomers that hold the flat cisternal membranes into stacks. Depletion of both GRASP proteins in cells disrupts the Golgi stack structure, increases protein trafficking, but impairs accurate glycosylation, and sorting.

GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion.

It has been indicated that the Golgi apparatus contributes to autophagy, but how it is involved in autophagosome formation and maturation is not well understood. Here we show that amino acid starvation causes -Golgi derived membrane fragments to colocalize with autophagosomes. Depletion of the Golgi stacking protein GORASP2/GRASP55, but not GORASP1/GRASP65, increases both MAP1LC3 (LC3)-II and SQSTM1/p62 levels.

GRASP depletion-mediated Golgi destruction decreases cell adhesion and migration via the reduction of α5β1 integrin.

The Golgi apparatus is a membrane-bound organelle that serves as the center for trafficking and processing of proteins and lipids. To perform these functions, the Golgi forms a multilayer stacked structure held by GRASP55 and GRASP65 trans-oligomers and perhaps their binding partners. Depletion of GRASP proteins disrupts Golgi stack formation and impairs critical functions of the Golgi, such as accurate protein glycosylation and sorting.