Medicine by Alexandros G. Sfakianakis: Monitoring Autophagy in Neurons

Τετάρτη 18 Ιουλίου 2018

Monitoring Autophagy in Neurons

By Christina Towers, PhD.

Autophagy is a critical cellular process used by most cells in the body to recycle nutrients and prevent harmful buildup of damaged proteins. It is particularly important in the brain, where a handful of pathologies have been linked to autophagy dysregulation. Conditional neuronal knock out of the core autophagy gene, ATG7, results in viable mice that eventually succumb to neurodegeneration accompanied by an accumulation of ubiquitin protein aggregates^{1, 2}. Likewise, decreased levels of functional autophagy have been linked to the three most common neurodegenerative diseases including Parkinson's, Huntington's and Alzheimer's disease³. Despite these findings, it has been a hurtle to find the most accurate and practical ways to measure autophagy in the brain, particularly in neurons.

Learn more about autophagy in neurodegeneration

The most common methods to measure autophagy in all cell types include monitoring autophagic flux by looking at LC3-II lipidation via western blot or analyzing LC3 lysosomal turn over via pH-sensitive tandem tagged-LC3, as well as autophagy specific cargo proteins like p62 and NBR1. Electron Microscopy is also commonly used to detect autophagy structures. While all of these methods can be used, there are some critical considerations to take into account when studying autophagy in the brain.

First, there are a variety of different types of cells in the central nervous system in addition to neurons, including supportive microglia cells and astrocytes as well as oligodendrocytes and endothelial cells. To study autophagy in neurons specially, neuronal cell lines (e.g., SH-SY5Y, Neuro2A, SK-N-SH, etc.) or primary cultured neurons are often used, which are not optimal as neither can fully polarize and primary cultures can only be maintained for 3-5 days. Nonetheless, autophagic flux can be measured in these systems by treatment of the cultured neurons with lysosomal inhibitors (e.g., Bafilomycin) followed by western blotting for LC3-II conversion. Interestingly, it has been reported that neurons maintain a high level of basal autophagy and lysosomal inhibitors cause only a slight increase in LC3-II but a more dramatic decrease in LC3-I. This is in contrast with what is often seen in autophagic flux experiments carried out in cancer cells.

Western Blot: LC3B Antibody (1251D) [NBP2-59800] - Total protein from HeLa and Neuro2A cells treated with or without 50 uM chloroquine for 24 hours was separated on a 4-15% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/ml anti-LC3 (1251d) in blocking buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Note the detection of LC3I and LC3II.

Second, cultured neurons are extremely sensitive and the dose and time course of lysosomal inhibition must be optimized to block LC3 turn over with minimal toxicity. The levels of cargo receptors like p62 and NBR1 may also be assessed via western blot to monitor flux. However, autophagic flux assays using flow cytometry to monitor pH-sensitive tandem constructs like mCherry-GFP-LC3 are not practical in these systems because these cells can be difficult to transfect and neuronal protrusions make these cells impossible to analyze in most flow cytometer machines.

Finally, one of the best methods to monitor autophagy in neurons is with transmission electron microscopy (TEM) using neurons cultured on glass cover slips. These images have enough resolution to identify double membrane autophagosomes and fused autolysosomes ⁴.

There is still much work to be done in the field of neuroscience to improve the methods to monitor autophagy: assays are desperately needed to understand the role of this relevant cellular process in the context of neurodegeneration.

Learn more about autophagy in neurodegeneration

Christina Towers, PhD
University of Colorado (AMC)
Dr. Towers studies the roles of autophagy, apoptosis and cell death in cancer.

References

Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, et al. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 2006;441(7095):880-4.
Karsli-Uzunbas G, Guo JY, Price S, Teng X, Laddha SV, Khor S, et al. Autophagy is required for glucose homeostasis and lung tumor maintenance. Cancer Discov. 2014;4(8):914-27.
Towers CG, Thorburn A. Therapeutic Targeting of Autophagy. EBioMedicine. 2016;14:15-23.
Benito-Cuesta I, Diez H, Ordonez L, Wandosell F. Assessment of Autophagy in Neurons and Brain Tissue. Cells. 2017;6(3).

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