Methods for Monitoring Autophagy After Ischemic Stroke
At a glance
As a leading stroke research provider, Ace Therapeutics provides a variety of biochemical and cell biological methods for monitoring autophagy to characterize autophagic processes in experimental models of stroke. In addition, support studies on the exact role and molecular mechanisms of autophagy in ischemic stroke.
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Autophagy is involved in the neural death pathway after cerebral ischemia and plays a key role in brain pathology after ischemic stroke. Monitoring autophagy function is crucial for the development of autophagy-based therapies for ischemic stroke. To understand the role of autophagy, it is essential to detect the autophagic process and measure autophagic activity.
Morphological Methods for Monitoring Autophagy After Ischemic Stroke
Transmission electron microscopy (TEM) is a high-resolution imaging technique used to qualitatively examine autophagosomes within cells at high magnification. Since its introduction in the 1950s, TEM has been pivotal in the discovery of autophagy.
TEM can also effectively monitor both selective and non-selective autophagy. In selective autophagy, the cargo corresponds specifically to the targeted substrate, with bulk cytoplasm being largely excluded. Conversely, in non-selective autophagy, the autophagosome content mirrors the cytoplasm, containing similar ribosome densities and clearly identifiable, intact sequestered organelles. These features make TEM a powerful tool for both qualitative and quantitative analyses of autophagic structures and their changes.
Quantifying autophagic activity with TEM involves calculating the area or volume of autophagic vacuoles relative to the total cytoplasmic area or volume. This quantitative approach is unique to TEM and provides insight into the extent of autophagy. While software tools have streamlined this process, it still requires examining numerous images from different cells for comprehensive morphometric analysis.
Detection of Specific Markers of Autophagy After Ischemic Stroke
LC3 Detection and Quantification
LC3 (microtubule-associated protein 1 light chain 3) is a widely recognized protein marker for autophagy. Its role in the formation of autophagosomes makes it a reliable indicator for monitoring autophagic processes. Detecting LC3 provides a valuable means to assess and study autophagy in ischemic stroke. Techniques such as Western blot and immunofluorescence can be utilized to measure LC3 expression and localization, offering insights into the dynamics of autophagic activity during ischemic events.
Detection of LC3 by Western Blot
LC3 exists in different isoforms (LC3A, LC3B, LC3B2, and LC3C) and undergoes conversion between LC3-I and LC3-II, reflecting autophagic flux. It is generally believed that the transformation of LC3-I to LC3-II, or the increase of LC3-II content in western blot experiments represents the activation of autophagy, while the decrease of LC3-II content represents autophagic inhibition. Thus, an increase in LC3-II level has been considered a gold standard for demonstrating autophagic induction.
However, as LC3-II is subsequently degraded by autolyososomal proteases (a process referred to as autophagy flux), the increase in LC3-II by a test compound may not necessarily reflect autophagic activation but may also suggest inhibition in autophagic flux. To differentiate between autophagic induction and inhibition, inhibitors of lysosome-dependent degradation are used, such as Bafilomycin A1, E64d, pepstatin A, or leupeptin.
Other challenges include the presence of multiple LC3 isoforms, tissue-specific distributions, and the role of the GABARAP protein family, which can influence certain types of autophagy. Proper antibody selection and specificity are critical to ensure accurate interpretation of experimental results.
Detection of LC3 by Fluorescence Microscopy
Fluorescence microscopy is a classic method for detecting autophagy by visualizing LC3, either using antibodies against endogenous LC3 or GFP-tagged LC3 plasmids. The increase in GFP-LC3 puncta is an indicator of autophagy. Similar to Western blot, lysosomal inhibitors like bafilomycin A1 are used to differentiate between autophagy activation and flux inhibition.
GFP-LC3 can also be used to monitor colocalization with a target during autophagy-related processes, such as organelle degradation or microbial sequestration. Preincubating cells expressing GFP-LC3 with leupeptin, a lysosomal protease inhibitor, stabilizes the GFP-LC3 signal by preventing its degradation in autolysosomes during fluorescence microscopy.
For advanced analysis of autophagic flux, tandem fluorescent-tagged LC3 constructs like mRFP-GFP-LC3 or mCherry-EGFP-LC3 are widely used. These constructs leverage the pH sensitivity of GFP (quenched in acidic autolysosomes) and the stability of mRFP/mCherry in acidic environments. In such experiments, yellow puncta indicate autophagosomes (co-localization of green and red fluorescence), while red puncta represent autolysosomes, allowing precise differentiation and detailed analysis of autophagic flux. The tandem mRFP/mCherry-GFP reporter method is highly advantageous as it enables simultaneous evaluation of autophagy induction and flux under native conditions, eliminating the need for drug treatments and providing reliable insights into autophagic dynamics.
Fig. 1 Monitoring of GF-fluorescent signal with the observation of in vivo imaging over the head of the ischemic hemisphere. (Stanzione, et al., 2024)
Detection of LC3 by Flow Cytometry
Flow cytometry is a high-throughput method for detecting autophagosomes, complementing microscopy by enabling the analysis of large cell populations, including non-adherent cells like blood monocytes. It allows the quantification of autophagic flux by measuring changes in GFP-LC3 fluorescence.
When autophagy is induced, GFP-LC3 is degraded in autolysosomes, resulting in a measurable decrease in total cellular GFP fluorescence. This decrease is used as an indicator of autophagic activity. Flow cytometry provides robust and precise data, especially in heterogeneous cell populations or conditions that disrupt normal autophagic processes.
Detection of Additional Autophagy-related Markers
Several other proteins, in addition to LC3, can be used to monitor different stages of autophagy. Some core autophagy proteins include Atg12, Atg5, Beclin1, and DRAM1. An increase in the mRNA and protein levels of these autophagy-related genes and proteins often coincides with the induction of autophagy. These changes can be measured using real-time qPCR or northern blotting to assess gene expression and western blotting with specific antibodies to measure protein levels. While useful for detecting autophagy-related processes, gene and protein expression markers may not always provide a complete picture of autophagic activity.
SQSTM1/p62 Degradation and Related LC3 Binding Protein Turnover Assays for Monitoring Autophagy After Ischemic Stroke
SQSTM1/p62 is a selective autophagy receptor that facilitates the degradation of ubiquitinated proteins by linking them to LC3, a core component of the autophagy machinery. The interaction results in the transfer of the targeted substrates into autophagosomes, which later fuse with lysosomes to form autolysosomes for degradation. Phosphorylation at Ser403 of p62 regulates the clearance of ubiquitinated proteins. Anti-phosphorylated p62 antibodies can detect this modification, offering a tool to study autophagic activity.
Detection of the Degradation of Long-lived Proteins for Monitoring Autophagy After Ischemic Stroke
Long-lived proteins and some organelles within cells are primarily degraded via autophagy. Monitoring the degradation of these proteins offers a more reliable assessment of autophagic flux than merely counting autophagy lysosomes. Observing autophagic protein degradation is an established autophagy dynamic quantitative analysis method, which generally uses radioactive amino acids, such as 35S methionine, 3H-leucine, 14C-leucine, and 14C-valine before inducing autophagy and monitoring the release of radioactivity as a measure of degradation of long-lived proteins upon induction of autophagy.
If the amino acids released from the degradation of long-lived proteins are not properly excreted from the cells after activating autophagic flux, they could be resynthesized or contribute to new protein synthesis within the cells. This could introduce experimental artifacts or skew results, necessitating careful controls to monitor this process.
Keima Assay for Detection of Autophagy After Ischemic Stroke
Keima is a fluorescent protein derived from coral, which is both acid-stable and resistant to lysosomal proteases. It undergoes a reversible color change in response to acidic pH, which makes it useful for studying autophagic processes, particularly in lysosomal environments. Keima provides advantages over the RFP-GFP-LC3 system because it does not depend on the LC3 lipidation process, making it useful for detecting autophagy independent of the Atg5 conjugation system. This allows it to be applied in scenarios where the traditional LC3 system may not work or where autophagy occurs through different pathways. Keima has been successfully used to study mitophagy, the selective degradation of mitochondria via autophagy.
- Tian, F., et al. (2010). In vivo imaging of autophagy in a mouse stroke model. Autophagy, 6(8), 1107-1114.
- Mizushima, N. (2004). Methods for monitoring autophagy. The international journal of biochemistry & cell biology, 36(12), 2491-2502.
