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By Christina Towers, PhD.
Lysosomes are highly acidic organelles that are critical for cellular function and indispensable for degradative pathways like autophagy and endocytosis. There are a number of different diseases that have been associated with lysosomal dysfunction, the most detrimental being neurological disorders including Huntington's Disease, Alzheimer's disease, and Parkinson's disease. Other rare neurological diseases like Niemen-Pick disease Type C (NPC) have been directly linked to familial-mutations in endolysosomal genes. All of these disorders are attributed to a buildup of the detrimental lysosomal targeted proteins that cannot be degraded when the lysosome is dysfunctional.
A host of proteins and lipids are involved in the endocytic and autophagic pathways, however recent studies have highlighted the importance of the class III phosphatidylinositol-3-kinase (PI3K- III)/VPS34 signaling pathway as the main culprit in lysosomal dysfunction. VPS34 is an upstream regulator of these pathways and interacts with the Beclin 1 complex to generate PI3P on either pre-autophagosomal membranes or early endosomes depending on other specific complex components. Cytosolic PI3P interacting proteins are recruited to these membrane structures and regulate autophagosome biogenesis and maturation as well as endosomal mobility and fusion.
A recent high profile publication by Miranda et. al., published in Nature Communications this year, reports that inhibition of VPS34 in neurons can affect lysosomal degradation, autophagy, lipid metabolism as well as endolysosomal membrane integrity. The authors used the newly identified potent pharmacological inhibitor of VPS34, called VPS34IN1 to assess the role of VPS34 in lysosomal function, autophagy induction, and endosomal trafficking.
| Autophagy Inhibitors | |
|---|---|
| Pharmacological Compound | Mode of Action |
| 3-Methyladenine | AMPK inhibitor |
| (±)-Bay K 8644 | L-type Ca2+ channel activator |
| Spautin 1 | USP10 and USP13 inhibitor |
| LY 294002 *,a Wortmannina |
VPS34 inhibitor |
| MRT 67307 MRT 68921 |
ULK inhibitor |
Available at Tocris: www.tocris.com/autophagy
High resolution confocal imaging of p62, LC3, and LAMP-1 showed a block in autophagy initiation after VPS34 inhibition. The authors also used mRNA and protein analysis as well as confocal imaging to show that VPS34IN1 induced early endosomal abnormalities. These results lead the authors to test the effects of VPS34 inhibition on broader, secondary lipid dysfunction, via lipid chromatography–mass spectrometry (LC-MS) which revealed that sphingolipids are the class of lipids most altered in cultured cortical neurons following VPS34IN1 treatment.
Arguably, one of the most interesting findings of these studies was the identification of exosomes enriched with the amyloid precursor protein C-terminal fragments (APP-CTFs) as well as other undigested lysosomal substrates including sphingolipids and phospholipid bis(mono-acylglycero)phosphate (BMP). An unexpected finding, as these lipids are usually found on internal endolysosomal vesicles. The results suggest that neurons can compensate for lysosomal dysfunction by upregulating exosomal trafficking.
The question remains if these compensatory mechanisms could be utilized clinically to help eliminate toxic proteins and prevent pathological build up as a potential treatment for these horrific neurodegenerative diseases. At the very least the authors propose that exosome released and circulating APP-CTFs and BMP could be used as a biomarker for diagnosis of lysosomal diseases. Nonetheless, these studies have identified a new mechanism of homeostatic control linking lysosomal dysfunction and exosomal trafficking, and opened the door for a host of new questions including:
Learn more about Autophagy and Neurodegeneration
Christina Towers, PhD
University of Colorado (AMC)
Dr. Towers studies the roles of autophagy, apoptosis and cell death in cancer.
References
Platt, F. M. (2014). Sphingolipid lysosomal storage disorders. Nature, 510(7503) https://doi.org/10.1038/nature13476
Miranda, A. M., Lasiecka, Z. M., Xu, Y., Neufeld, J., Shahriar, S., Simoes, S., … Di Paolo, G. (2018). Neuronal lysosomal dysfunction releases exosomes harboring APP C-terminal fragments and unique lipid signatures. Nature Communications, 9(1). https://doi.org/10.1038/s41467-017-02533-w
