Among the most devastating human diseases is a group of rare genetic disorders called lysosomal storage disorders (LSDs). They are so rare, in fact, that charities and patient advocate groups representing these different diseases have come together to form a collaborative to improve treatments. LSDs are caused by inactivating mutations in proteins that normally contribute to the maintenance of a cell’s health.
Animal cells have multiple ways of dealing with old and defective cellular components, one of which is the so-called autophagy pathway. In this pathway (see the schematic below), a specialised organelle called the autophagosome engulfs old proteins and even whole organelles, including mitochondria. The autophagosome then fuses with other organelles called lysosomes. Lysosomes are acidic compartments containing enzymes that degrade macro-molecules, including proteins, lipids, and complex sugars. Once the macro-molecules are degraded, their component parts can be exported and recycled.
The faulty proteins that cause LSDs are usually lysosomal enzymes. Consequently, the unifying feature of these diseases is that cells in a patient’s body heap up the cellular “rubbish” that should be degraded by the lysosomes. The lysosomes also grow larger and more numerous. [For a recent review on LSDs, see Parenti et al, 2021.]
Fucosidosis is a specific LSD caused by inactivating mutations in the gene FUCA1, which encodes the lysosomal enzyme α-L-fucosidase 1 (FUCA1). FUCA1 is necessary for breaking down complex sugars by cleaving off a sugar molecule called fucose from larger macro-molecules, including proteins and lipids. Fewer than 150 cases of fucosidosis have been described in the biomedical literature (see Stepien et al, 2020). However, patients with homozygous FUCA1 mutations generally die of their disease, and its secondary complications, by the age of 30. The main symptoms are decreased mobility as well as developmental and neurological abnormalities; this is thought to be because nerve cells are particularly dependent on the degradation of proteins and organelles by lysosomes. Before our new open-access study there was only a limited amount of research on fucosidosis, so there were many questions about how the inactivation of FUCA1 leads to the disease.
A former post-doc in our lab at the CRUK Beatson Institute, Alice Baudot, started this work on fucosidosis because the lab has an interest in the autophagy pathway, and especially in its roles during cancer development. However, as so often is the case, the research led away from the initial question and Alice developed a new mouse model of fucosidosis.
Alice first observed what happens to mice lacking the FUCA1 protein. She found that both male and female mice without FUCA1 started showing signs of motor and neurological deficiencies during adulthood, and they did not survive as long as wild-type control mice. Examination of tissues from the FUCA1 knockout mice revealed that cells in multiple organs (including the brain, liver, pancreas and kidney) had accumulated large lysosomes – a process called vacuolation – and that these organs were less healthy than wild-type organs. The livers of FUCA1 knockout mice were also bigger than those of wild-type mice; this is a feature that has also been observed in some fucosidosis patients. Furthermore, when I studied the tissues from these mice, I noticed that they had accumulated mitochondria in various regions of the brain, suggesting that they were not being cleared by autophagy.
Following this examination of the mouse phenotype, Alice started conducting experiments on cells from these mice, either with or without the FUCA1 protein. It became clear quickly that the FUCA1 knockout cells had abnormal levels of various proteins involved in autophagy.
Following on from Alice’s experiments I tried to identify in more detail exactly how FUCA1 loss was affecting the autophagy pathway. We found that, although it looked like the autophagy process had gone into overdrive in the knockout cells, autophagy was actually stalling. Instead of going through the five-step process depicted in the schematic above, the whole procedure was stuck at various points. Maybe unsurprisingly, the lysosomes themselves were less able to digest proteins and sugars. Alice found that FUCA1 was needed to control other digestive enzymes, so that losing FUCA1 led to a domino effect of faulty enzymes in the lysosomes. The build-up of cellular components in the lysosomes caused a backlog in autophagy, thus slowing down the flux of autophagy cargo to the lysosomes.
Interestingly, I identified a specific step in the process that was going wrong, namely the fusion of autophagosomes with lysosomes (step 3 in the schematic above). The figure below shows a fluorescent microscopy image of a FUCA1 wild-type mouse cell (left): the lysosome is labelled in green (Lamp2 protein) and you can see that the autophagosome in purple (LC3 protein) is physically inside the lysosome, indicating successful autophagosome-lysosome fusion. In the FUCA1 knockout cell (right) the lysosome and autophagosome are next to each other and not fusing. I analysed a number of both wild-type and knockout cells and observed this pattern over and over again. Somehow – although we did not figure out exactly how – losing FUCA1 interferes with this fusion process, contributing to the overall abnormalities of autophagy.
One avenue of work that could be explored next is to find out whether treating the fucosidosis cells/mice with certain drugs could “force” or improve the flux of cargo through the autophagy pathway. This may alleviate some of the toxicity resulting from the build-up of cellular material. However, one major difficulty in treating this disease is that it affects all cells of the body to some extent, and therefore the drugs would need to reach all of them, and especially the cells in the brain. Although this kind of basic/discovery research will not, in the near future, lead to new treatment options for patients I think Alice and I have contributed a small piece to the puzzle of why fucosidosis is such a devastating disease.
Baudot Alice D.*, Wang Victoria M.-Y.*, Leach Josh D., O’Prey Jim, Long Jaclyn S., Paulus-Hock Viola, Lilla Sergio, et al. ‘Glycan Degradation Promotes Macroautophagy’. Proceedings of the National Academy of Sciences 119, no. 26 (28 June 2022): e2111506119. https://doi.org/10.1073/pnas.2111506119.
Parenti G, Medina DL, Ballabio A. The rapidly evolving view of lysosomal storage diseases. EMBO Molecular Medicine 13, no. 2 (5 Feb 2021):e12836. https://10.15252/emmm.202012836
Stepien KM, Ciara E, Jezela-Stanek A. ‘Fucosidosis—Clinical Manifestation, Long-Term Outcomes, and Genetic Profile—Review and Case Series’. Genes 11, no. 11 (2020). https://doi.org/10.3390/genes11111383.