We are studying autophagy regulation in a neurodegenerative disease. We have found a decrease of LC3-B and an increment of p62 immunolabelling in affected brains. How do you interpret these results in relation to autophagy regulation?
The first and foremost thing is to nail down whether you observe an active autophagy flux. Merely analysing the level of p62 and LC3II will not lead to a definite answer. You need to analyse LC3II and p62 with and without BafilomycinA1 or Chloroquine which will prevent autophagosomes to mature into autolysosmes. If there is an active autophagy flux, you will observe increased level of LC3II or p62 after chloroquine or BafilomycinA1 treatment.
However, let you observe an active autophagy flux, then how can you interpret your data. You are assuming that as you are observing elevated p62 level there is a chance that autophagy is blocked. However, p62 level is not always a good measure for autophagy. In different context the mRNA level of p62 can change and that might reflect a change in your p62 protein level . Therefore, you can analyse the mRNA level of p62 to pinpoint your conclusion. So, the next thing is how you can observe less LC3II punctae if there is an active autophagy flux. In basal condition if some stress situation accelerates autophagy flux then LC3II will be turned over more rapidly, and you may observe a decrease in LC3II + punctae. However, you need to perform a co-immunostaining for an autophagosomal marker (LC3II) and a lysosomal/autolysosomal marker (LAMP1). If you observe a decrease in LC3II + punctae but a concomitant increase in LAMP1+ punctae, you can interpret that there is an accelarated autophagy flux which is turning over LC3II more rapidly.
Another suggestion is you might perform western blots to analyse LC3II to LC3I ratio and p62 level. Quantifying your data from a western blot often give you much precise results rather than quantifying your immunofluorescence data.
Thank you for your help @Nilay Nandi.
We can't add BafilomycinA1 or Chloroquine because we are analysing tissues. However, we could perform IHC for LAMP1 and western blott if we have enough tissue.
Inmaculada Martín-Burriel Is it possible for you to use an animal model of that neurodegenerative disease to get more definitive answers and dig deep into the mechanism?
Nilay Nandi Yes, I am already using an animal model for prion diseases but the role of autophagy is not clear in the neuropathology of the diseas is not clear either.
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