Autophagy is a crucial cellular process that clears the cell of protein aggregates, toxins, and damaged cell products. Accumulation of toxins, damaged cell products and unwanted proteins has been proven to play a role in aging and many forms of disease and cancer. p62/SQSTM1, or sequestosome-1, is an autophagosome receptor that interacts with cargo tagged for degradation in order to turn on selective autophagy. p62/SQSTM1 binds LC3 through the LC3 interacting region (LIR), which is necessary for degradation of sequestosomes. p62/SQSTM1 is also required for the formation and degradation of polyubiquitin-containing bodies in autophagy. Outside of p62/SQSTM1’s role in autophagy, it has also been implicated in cell signaling that impacts differentiation, apoptosis and immune response. Here we take a closer look at the proteins in the brain that may be required for autophagy, as well as defective autophagy in neurons, with the use of a p62/SQSTM1 mouse monoclonal antibody.
Immunocytochemistry/Immunofluorescence: p62/SQSTM1 Antibody (2C11) [H00008878-M01] - Analysis of p62 in SH-SY5Y cells using anti-p62/SQSTM1 antibody. Red - p62 puncta; Blue - nuclear DAPI; Green - Cytoskeleton. Image from verified customer review.
To begin, Wang et al used a p62/SQSTM1 antibody to investigate the role of 14-3-3-tau in the regulation of autophagy. While it is established that Beclin-1 plays a large role in autophagic processes, the regulation of Beclin-1 is understudied. Due to the indication of Beclin-1 as an E2F target, 14-3-3-tau came into play as a regulator of E2F stability. First, Wang et al established a U2OS cell line that expressed siRNA against 14-3-3-tau, which showed a significant decrease in Beclin-1 transcript expression. With this information, the behavior of autophagy as it relates to 14-3-3-tau was examined. Using a p62/SQSTM1 antibody in western blot on U2OS cells, they found that conducting amino acid starvation with Earle’s balanced salt solution (EBSS) yielded a degradation of p62/SQSTM1. This finding was coupled with an increase in LC3-II. Furthermore, introduction of bafilomycin AI, which prevents autophagic degradation, caused both LC3-II and p62/SQSTM1 expression levels to stabilize. Overall, this study used a p62/SQSTM1 antibody to demonstrate a critical role for 14-3-3-tau in autophagy through the regulation of Beclin-1.
Next, Ferguson et al used a p62/SQSTM1 antibody to show that mice deficient in PI(3,5)P2 have defective autophagic processes in their neurons and astrocytes. For starters, mutations that affect the conversion of PI3P to PI(3,5)P2 have been implicated in ALS and Charcot-Marie-Tooth Disease. In order to further examine this correlation, neurons and astrocytes with mutations in the PI(5,3)P2 regulatory complex were generated. A p62/SQSTM1 antibody was used in western blot to assess the amount of p62 in these null mice. This study showed a 3-fold increase in p62 in the mutant mouse brain over the wild-type brain. Furthermore, elevated LC3-11 was found after using an LC3 antibody in western blot, which is also indicative of a defective autophagosome. In order to determine whether elevated p62 in the mutant mice brains was due to association with ubiquitinated proteins, brain slices were immunostained with both a p62/SQSTM1 antibody and an ubiquitin antibody. This experiment did in fact find co-localization of p62 and ubiquitin. Lastly, a p62/SQSTM1 antibody and a GFAP antibody were used to show that there was indeed increased p62 expression and p62/GFAP co-localization in mutant astrocytes. All in all, these findings show a strong role for deficient PI(5,3)P2 in blocking the normal function of autophagy in neurons and astrocytes.
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