The Role of Autophagy in Neurodegeneration

Humzah Khan - Sep. 11, 2024 - 5 min read - #Science

Autophagy, an essential cellular process responsible for the degradation and recycling of subcellular structures, plays a critical role in maintaining cellular homeostasis. An increasing number of studies highlight the importance of autophagy, especially in the central nervous system where its dysfunction has been linked to many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Amyotrophic Lateral Sclerosis (ALS). This article explores what autophagy is, its importance and the potential it has to positively impact neurodegeneration.

What is autophagy?

Autophagy is a mechanism which breaks down less useful cellular components, repurposing the raw materials to create more functional and essential molecules. An example of this is breaking down a dysfunctional protein, and using the amino acids to create a new, useful enzyme. There are several types of autophagy, the three major being macroautophagy, microautophagy and chaperone-mediated autophagy. This survival mechanism is initiated due to a drop in ATP and an increase in AMP, sensed by AMP kinase which activates ULK1, thus triggering the process of autophagy. mTOR is also a protein which regulates autophagy by being a nutrient sensor; if excess nutrients are present, mTOR will inhibit ULK1 by phosphorylating it, therefore preventing autophagy. In the reverse scenario, a limitation of nutrients is sensed by AMP kinase which will activate mTOR inhibitors, consequently promoting autophagy.

Figure 1 “The process of macroautophagy. (a) Schematic representation of the autophagic process, and (b) cartoon illustration of the fluorescence signal of a cell expressing GFP-LC3B and stained with LysoTracker Red.”

Neurodegenerative diseases and Autophagy

A key pathology of many neurodegenerative disorders is the aggregation of proteins, therefore harnessing the ability to control autophagy via these (and many more) regulators may lead to significant progress towards understanding neurological degradation. Increasing studies have shown that autophagy declines with age. Therefore, the impairment of intracellular quality control exacerbates the accumulation of aggregate prone (potentially toxic) proteins and organelle dysfunction, which may also contribute to neural damage. Autophagy helps maintain synapses by clearing out damaged synaptic proteins; synaptic dysfunction generally increases with time, which is an early feature of many neurodegenerative diseases such as cognitive impairment in Alzheimer’s and motor dysfunction in Parkinson’s. Impairment of the autophagy pathway has been linked to many diseases shown in Figure 1, highlighting the important link between the two.

Figure 2

Blue: dysfunction associated with autophagosome formation

Red: dysfunction associated with transportation to lysosomes

Green: dysfunction associated with lysosomes

Measuring autophagic flux and its challenges

LC3-I is an important marker and involved in the early stages of autophagy. It is converted to LC3-II during the elongation and maturation stages, LC3-II being a membrane bound marker. LC3-II plays a key role in autophagosome expansion and closure, marking autophagosomes for fusion with lysosomes where engulfed material is degraded. The conversion of LC3-I to LC3-II can be used to measure autophagic flux, defined as a measure of autophagic degradation activity, using tests such as western blots and immunofluorescence microscopy.

A western blot analysis can reveal semi-quantitative results on the proportion of LC3-I/II, thus indirectly signifying the formation of autolysosomes. Western blotting is a very common assay and therefore offers reproducible data, which is an important aspect of research. However, western blots can be riddled with challenges due to its many steps and potential to make mistakes. This assay technique captures autophagic activity at a single time point, thus making it difficult to capture flux over time without creating multiple blots. Furthermore, western blots often have high variability and find it difficult to differentiate between small changes in LC3-II levels.

Another assay to measure autophagic flux is mCherry-GFP-LC3 with Flow Cytometry. Flow cytometry can analyse thousands of cells individually in a short amount of time, providing robust statistical data. Additionally, The mCherry-GFP-LC3 system allows for the differentiation between autophagosomes and autolysosomes based on the ratio of red (mCherry) to green (GFP) fluorescence. GFP is quenched in the acidic environment of the autolysosome, so a shift in the GFP/mCherry ratio indicates autophagosome maturation. Despite this, flow cytometry is a more technically demanding assay and more expensive compared to western blotting. An error in this assay could also be bleed-through, where GFP partially overlaps with mCherry which can result in incorrect measurements or misleading conclusions. Different cell types also differ in autophagy, for example their pH or LC3 processing. This may make it difficult to standardise the mCherry-GFP-LC3 assay, as it can affect the sensitive fluorescence.

Different sections of the autophagy process can be isolated and tested, often through the use of inhibitors. For example, chloroquine or bafilomycin A1 block lysosomal degradation and are often used to measure the total autolysosomes formed, rather than only the remaining undegraded autolysosomes. This is particularly useful in neurodegenerative disease research as Figure 2 displays how smaller sections of the autophagy process are impaired rather than the entire process, therefore a more accurate investigation can be undertaken, by isolating and experimenting on the affected segment.

Concluding remarks

The intricate relationship between neurodegenerative diseases and autophagy underscores the critical role of autophagic flux in maintaining neural health. Dysfunctional autophagy contributes significantly to the accumulation of misfolded or aggregation of proteins and damaged organelles; hallmarks of neurodegenerative disorders like Alzheimer’s, Parkinson’s, and ALS. By understanding the regulation of autophagy, researchers can explore novel therapeutic strategies aimed at either enhancing autophagic activity to clear toxic aggregates or tempering excessive autophagy to prevent cell death. Although methods of autophagic flux pose challenges, these techniques allow for a more nuanced exploration of autophagy. Continued advancement of these assays along with an improved understanding of autophagy has the potential to drive groundbreaking treatments. Ultimately, targeting autophagy in neurodegenerative diseases holds great promise, and the exponential-like-interest in autophagy over the last few decades gives hope for a potential therapy for currently incurable conditions. Further research is needed to understand this mechanism and translate findings into clinical practice, with the potential to improve outcomes in neurodegenerative diseases.

Figure 1 Source:

du Toit, A., Hofmeyr, J. S., Gniadek, T. J., & Loos, B. (2018). Measuring autophagosome flux. Autophagy, 14(6), 1060–1071.

https://doi.org/10.1080/15548627.2018.1469590

Figure 2 Source:

Rubinsztein, D (2022). David Rubinsztein, Cambridge | Autophagy and Neurodegeneration. 10:57-11:57.

https://www.youtube.com/watch?v=0WJ4UoR0R-I&ab_channel=ForesightInstitute

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