A piece of the puzzle

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.

Overview of autophagy: without the degradative enzyme FUCA1 the process stalls at multiple points (red crosses), including autophagosome-lysosome fusion, lysosomal degradation and recycling, as well as overall autophagic flux. Image created using the free version of BioRender.

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.

Zooming in on a FUCA1 wild-type mouse cell: green protein represents a lysosome after fusing with the autophagosome labelled in purple. The histogram shows how the purple protein is inside the lysosome.
Copied from Baudot & Wang et al, 2022, Fig. S5

Zooming in on a FUCA1 knockout mouse cell: green protein represents a lysosome located next to an autophagosome labelled in purple; they haven’t fused. The histogram shows them as separate organelles.
Copied from Baudot & Wang et al, 2022, Fig. S5

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.

References:
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.

First PhD Checkpoint

In December, we – the (mostly) young and innocent first-year PhD students at the Francis Crick Institute – gave our first formal talks. Each student had to present the outline of their project to all the other students in a ten minute slot. This was probably intended mostly for our own benefit to ensure that we had at least a rough idea of what we will be working on for the foreseeable future. Here I’d just like to mention a few of the talks that I found particularly interesting, but it’s worth saying that I thought the overall level of presentations was very high and the questions we ended up asking each other were well thought through. Overall a very enjoyable experience.

1. To begin with there were a couple of talks from students in the same lab studying the interactions of cancerous cells with the immune system. In particular, they are trying to find out how dendritic cells – cells that normally alert effector cells of the immune system that something is wrong (e.g. an infection is happening) – can sense the presence of dead/dying tumour cells and relay this information to so-called T cells. The two students are looking at both the molecular mechanism by which this happens, but also whether precursors of dendritic cells in the bone marrow have similar abilities.

2. A few students in the programme are working on mathematical/computational projects and will never have to wear a lab coat. For example, one lab is interested in understanding how non-cancerous cells near a tumour interact with the cancer cells and influence their ability to move. To do this one can mathematically model the movement patterns of the “cancer-associated fibroblasts” and how they interact with extracellular proteins to form tracks for the cancer cells to move along. In the simplest terms (and that’s the only level at which I understand this), the model relies on the Morse potential, which is normally used to understand how atoms interact but can be scaled up to model interacting cells. Here is a video of a fibroblast interacting with a breast cancer cell; the accompanying text is maybe overly simplistic, but you get the gist:

   Another student is studying how sheets of cells move together, both during embryonic development and tumour formation. This relies (roughly) on modelling cells as polygonal shapes that stick together via their vertices. Yet another “dry” project is investigating how cancers evolve over their lifetime: this is done by collecting DNA sequencing data from cancer cells at various stages of their development and inferring which changes happened when.

3. Since it is generally the metastases that are the deadly part of cancers it is important to  understand how cells move. There is, of course, a lot of information about this already but here the aim is to find out more about how different cancer cells (e.g. breast cancer and skin cancer) share certain features in their movement patterns.

4. Not all labs in the institute study cancer. Some labs focus on basic research using yeast as a model organism. Both yeast cells and our cells contain a lot of DNA that is not translated into protein; for a long time all this DNA was termed “junk” and nobody bothered with it too much. It is becoming increasingly clear that this so-called non-coding DNA can still play various roles in the cell and some of these may be deleterious. Therefore one student is studying how cells prevent the activation (transcription) of some of these stretches of DNA.

5. Another major branch of the institute deals with infectious diseases and the immune system more broadly. Two talks that I enjoyed on this front were given by students again working in the same lab. They are studying “neutrophil extracellular traps” (NETs), which I had never heard of before and sound quite cool. Neutrophils are a cell type of the immune system and are the first to react to infectious agents. By releasing very broad-acting antimicrobials they try to quell an infection in its infancy, but by doing so they also cause the four main symptoms of inflammation: pain, heat, redness and swelling. NETs are made of DNA and proteins from the neutrophils and are sticky. One of the students is looking into how NETs can exacerbate atherosclerosis, while the other is finding out how NETs physically trap invaders, such as the fungus Candida albicans.

6. Lastly, and because it would pain me not to mention CRISPR, one student is trying to find a way to control the sex ratio of offspring in laboratory animals, specifically mice. While at first glance this might seem dangerous or cruel, it is actually part of an effort to reduce, replace and refine the use of animals in research. For example, if you are studying prostate cancer or ovarian cancer half of the experimental animals born are completely useless and end up being “wasted”. At the moment, some agriculture relies on physically sorting sperm cells into those carrying X or Y chromosomes and using mainly those with X chromosomes for in vitro fertilisation (because far fewer male animals are needed). Although this is a very accurate method it is expensive and time-consuming. Since CRISPR is precise and can be genetically encoded it would virtually work by itself once established.

This is by no means an exhaustive list of the topics covered by our projects, but hopefully it’s an interesting glimpse into what we are currently spending (almost all?) of our brains and energy trying to figure out.

These talks will be complemented with a so-called thesis committee meeting later this month: here each student will present a very similar talk to three professors or group leaders, who will be advising the project from an outside perspective. Hopefully being locked in with three clever and knowledgeable people will conjure up constructive criticism as well as (even more) new ideas!

A bit more pancreas

Last time there was an introduction to the overall anatomy of the pancreas, its basic functions and some of its diseases. Today we will go into a bit more depth but still keep it relatively simple.

Firstly, what does it look like inside a pancreas when one zooms in a bit more? In the following diagram the exocrine cells are shown in red – these are divided into:

• Acinar cells. They are found in clusters that sometimes resemble bunches of grapes. In the diagram the blue spots represent the nuclei (where DNA is kept). However, these are actually found on the side of the acinar cells that does not face into the duct (i.e. the basal side; the apical side faces into the duct/lumen).
• Ductal cells. These line the ducts through which the digestive enzymes – produced by the acinar cells – pass.
• Centroacinar cells. A small group of cells that reside at the boundary of an acinus and a duct.

Diagram of exocrine and endocrine parts of the pancreas – copied directly from Wikipedia here

The endocrine portion of the pancreas, on the other hand, is shown in turquoise. These cells are organised into islets and secrete the hormones they produce directly into the bloodstream. They consist of:

• Alpha cells. Produce the hormone glucagon, which opposes insulin and raises blood glucose levels.
• Beta cells. Produce the hormone insulin, which decreases blood glucose levels by stimulating the uptake of glucose into muscle and fat cells.
• Delta cells. Produce the hormone somatostatin, which opposes a lot of other hormones in the body and, in particular, can slow down digestion.
• Gamma cells. Produce so-called pancreatic polypeptide, which regulates secretion of other hormones in the pancreas.
• Epsilon cells. Produce the hormone ghrelin, which is also known as the “hunger hormone” and signals to the brain that the stomach is empty.

Overall the two parts of the pancreas perform complementary roles: the exocrine pancreas helps to physically digest food coming from the stomach, whereas the endocrine pancreas makes sure that the glucose released into the blood due to the digestive processes is used/stored properly.

Secondly, people who are not scientists sometimes ask me how we actually study both normal and disease processes in the lab. Focussing on pancreatic ductal adenocarcinoma (PDAC), here is a brief overview of the systems used in the lab to gain a better understanding of what is going on in human disease.

1. The easiest system to use are so-called (pancreatic cancer) cell lines – these are cells that were once taken from a patient and then cultured in the lab (in vitro). So although they are originally from a very relevant biological setting, the continuous culturing in the lab means that they are no longer physiological. For example, they grow in flat sheets on plastic trays, do not interact with any other cells and often have mutations that go above and beyond what is found in a real cancer. However, they allow relatively quick, controlled experiments to be conducted and are therefore still indispensable.
2. More recently there has been a surge in research involving (pancreatic) organoids. These are mini-organs that are functionally closer to the real organ being studied and also have three dimensional structures that are more physiological. However, they need to be kept in relatively specialised conditions and are a little bit more tricky to manipulate than the cell lines.
3. Since it is impossible (for a lot of reasons, including ethical ones) to conduct experiments on humans, cancer biologists most often turn to mice since they are also mammals and are genetically similar to us – we share approximately 90% of our DNA sequences. Mouse models of cancer allow complicated, intricate and biologically relevant experiments to be carried out, but they do take more time and need to be planned very carefully. Guerra & Barbacid (2013) have written a review specifically on PDAC mouse models.
4. Data from human cancer biopsies. Increasingly, as sequencing whole genomes is becoming cheaper and easier to do, labs use DNA sequencing data from patients to compare some of their results from the mouse experiments. Biopsies can also be used to look at the histology (micro-anatomy) of certain stages or types of cancer in humans.

In the end, these techniques are used together, since all of them have strengths and weaknesses, allowing (hopefully) the most accurate description of what is going on in a certain (disease) scenario.

Lastly, what exactly do we use all these methods and systems for; what are the outstanding questions surrounding PDAC? Well, there are quite a few, but here are some of the more common ones: Why do most PDACs arise in the head and not the tail of the pancreas? Why are they so prone to metastasise (spread through the body)? How can we detect PDAC sooner and, importantly, distinguish it from inflammation of the pancreas? Where exactly (which cells) do PDACs come from? What roles do stem cells play in all of this? (If you have the answers to any of these questions, please do not hesitate to get in touch.)

Reference:

Guerra C, Barbacid M (2013) Genetically engineered mouse models of pancreatic adenocarcinoma. Mol Oncol 7: 232-247

An Introduction to the Pancreas and its Cancer(s)

Given that the four-year PhD I am about to embark on is being partly funded by Cancer Research UK (CRUK), why not start with a short video they have put together containing ten things about that pancreatic cancer that you may (not) have known:

Firstly, is there anything more to its anatomy than “being a small organ in the abdomen”? Why yes, funny you should ask. The pancreas reaches up to 15 cm in length in humans and is a J-shaped structure, apparently a bit like a hockey stick. The organ is not symmetric, rather it is made of a head (left), body (middle) and tail (right), as shown below. In the head the main pancreatic duct merges with the common bile duct before they join into the duodenum, where food is further digested after it leaves the stomach.

Cartoon illustration of a human pancreas – copied directly from Hezel et al, 2006

As is often the case in biology – and this applies from the level of individual proteins to a whole organism – there is an intricate relationship between structure and function. The pancreas has two main functions: firstly, it contains endocrine cells that produce hormones, such as insulin, to regulate glucose balance (or homeostasis). Secondly, it contains exocrine cells, which make up the majority of the organ, that produce enzymes to aid food digestion. These two categories of cells therefore work together and coordinate the body’s metabolic response to food consumption.

Not all animals have pancreases as complex as those found in mammals since they have evolved over time. Worms, for instance, have some specialised gut cells that produce a substance similar to insulin. Ancient sharks and some fish contain organs in which the exo- and endocrine cells are found together. Fruit flies astonishingly produce insulin in their brains and, in particular, those cells develop from a completely different tissue in the embryo – called the ectoderm – than in other animals, where the pancreas stems from the endoderm. In amphibians and mammals the anatomy becomes more complex with characteristic “islets” of endocrine cells. (See Stanger & Hebrok, 2013 for more details.)

Since I am the daughter of two doctors I am usually interested in the pathological side of human biology. What sort of diseases befall the pancreas? Possibly the first that comes to mind is (type I, auto-immune type) diabetes in which the pancreas fails to make enough insulin because the body is attacking its own beta cells that normally produce insulin. Other than that the pancreas can of course become inflamed like any other organ and this is known as pancreatitis. It is caused, among other things, by high doses of alcohol. Lastly, the pancreas can become cancerous.

Although there are a few different types of pancreatic cancer the focus will lie on pancreatic ductal adenocarcinoma (PDAC) since it is both the most frequent and also the best understood type. It is one of the deadliest types of cancer because it often goes unnoticed until the tumour has disseminated (or metastasised) in the body – fewer than 10% of people who are diagnosed survive for longer than five years.

Therefore it is important to find out more about PDAC. But for the moment there is a lot we do know, decades of hard work that are often frustratingly glossed over. For instance, the genetic mutations that start and maintain PDACs are well known. Among them are activating mutations in a famous oncogene – a gene whose overactivation contributes to cell proliferation and cancer – called KRAS, and deletions of tumour suppressors – those genes/proteins that act as checks and balances and stop cells from overproliferating – such as p16 and p53. Moreover, it is well known when these mutations occur during the development of the cancer. The following image shows how some PDACs form: from healthy tissue to slightly abnormal tissue until it invades into the rest of the body.

Histology of normal (left), dysplastic (middle) and malignant (right) pancreatic tissue – copied directly from Bardeesy & DePinho, 2002

As for treatment, the most effective method, if successful, is surgery. However, this often involves removing the head of the pancreas as well as parts of the duodenum and this, in turn, means that the stomach has to be surgically joined directly to subsequent parts of the digestive tract, making it a complex procedure. In addition, patients receive radio- and/or chemotherapy, which “targets” most proliferating cells. Targeted therapies that are specific to the cancer cells, as opposed to healthily dividing cells, are, as far as I am aware, not particularly established yet for pancreatic cancer.

For surgery to be effective the cancer needs to be detected at earlier stages. In other words, better diagnosis procedures need to be developed. Recently, two papers attracted some attention because they uncovered methods that could potentially make diagnoses easier using either blood (Melo et al, 2015) or urine (Radon et al, 2015) samples. However, as CRUK rightly point out, these techniques need to be refined for them to be able to tell true cancers from chronic pancreas infections.

And on that cheery note… Next time you can look forward to learning a bit more about how PDAC is studied in the lab and pancreatic anatomy in slightly more detail.

References:

Bardeesy N, DePinho RA (2002) Pancreatic cancer biology and genetics. Nat Rev Cancer 2: 897-909

Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, DePinho RA (2006) Genetics and biology of pancreatic ductal adenocarcinoma. Genes & Development 20: 1218-1249

Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R (2015) Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523: 177-182

Radon TP, Massat NJ, Jones R, Alrawashdeh W, Dumartin L, Ennis D, Duffy SW, Kocher HM, Pereira SP, Guarner  L, Murta-Nascimento C, Real FX, Malats N, Neoptolemos J, Costello E, Greenhalf W, Lemoine NR, Crnogorac-Jurcevic T (2015) Identification of a Three-Biomarker Panel in Urine for Early Detection of Pancreatic Adenocarcinoma. Clinical Cancer Research 21: 3512-3521

Stanger BZ, Hebrok M (2013) Control of Cell Identity in Pancreas Development and Regeneration. Gastroenterology 144: 1170-1179

More Fluorescent Proteins

Although I’m approximately two years late in reporting about this discovery I still think it’s pretty cool. In 2013 Kumagai et al. for the first time discovered a fluorescent protein in a vertebrate, the Unagi eel. Until then fluorescent proteins had only been found in invertebrates, such as reef corals and the jellyfish Aequorea victoria, where the famous green fluorescent protein originally came from. My attention was drawn to this finding in a brief article in the Chemistry World journal, in which the author claims that, “Unagi’s status as a culinary delicacy means you’re more likely to encounter these eels in a restaurant than a lab”. [Picture copied directly from the article link.]

Apart from (presumably) tasting good and looking pretty, the fluorescent protein – called UnaG – from Unagi eels may be able to form the basis for a diagnostic test for liver disease. UnaG only fluoresces when bound to bilirubin, which is a break-down product of haem, the molecule that carries oxygen in the blood. Livers with impaired function have difficulty further processing the bilirubin before it is excreted, leading to a build-up of bilirubin in the body, and in extreme cases to jaundice. So the intensity of UnaG fluorescence can be used as a read-out for how badly the liver is damaged.

And now for something completely different: next time I’ll be writing about what it’s like to be an intern at the journal eLife!

Reference:

Kumagai A, Ando R, Miyatake H, Greimel P, Kobayashi T, Hirabayashi Y, Shimogori T, Miyawaki A A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 153: 1602-1611