Cell Biology 2 (CBG) Sbobine, A.Y. 2021-2022 PDF

01.12.2021 RETTA L.1 CELL BIOLOGY: AN INTRODUCTION Why do we have to study cell biology to become good medical doctors? WHAT IS A CELL? As we know, the cell is the smallest function...

01.12.2021 RETTA L.1 CELL BIOLOGY: AN INTRODUCTION Why do we have to study cell biology to become good medical doctors? WHAT IS A CELL? As we know, the cell is the smallest functional unit of life, if you consider life you have to start from cells, the basic unit of life. The cell theory, that dates back to 1838, says that “All living organisms are composed of one or more cells”, “All living cells arise from pre- existing cells by division” and “The cell is the fundamental unit of structure and function in all living organisms” (fig.1). Everything is related to cells, both to pathology and physiology as we will see. That’s why studying cell biology is very important to understand fig.1 future topics that we will consider during the course. CELL BIOLOGY: AN INTRODUCTION Talking about cell biology, we have to mention that there are different types of cells including eukaryotic and prokaryotic cells. 1665: HOOKE DISCOVERS THE CELL USING A RUDIMENTARY MICROSCOPE We started considering cells when the first observation happened, that dates back to 1665, when Robert Hooke discovered the first cell using a rudimentary microscope. We can see in the images a fig.2 representation of that microscope used by Robert Hooke (fig.2) HOW MANY CELLS ARE IN YOUR BODY? Cell is at the basis of life, so how many cells are present in our body? The human body contains around 37 trillion cells, some scientists tried to quantify this number of cells, some reports show that there are 50 billion fat cells, 2 billion heart muscle cells, etc... Each cell plays a fundamental role in the body, and inside every cell there is an intricated world. Do we only have human cells in our body? No, because, together with human cells, other cells are present. Indeed, the majority of cells present in our body is not human. The microbiome (bacterial cells, prokaryotic cells) present in our gut outnumbers human cells, there are more bacteria than human cells in our body, and they play fundamental roles both in physiology and pathology. If those cells are altered, if these bacteria making up the microbiome are altered, they could give rise to human diseases. They can as well contribute to the human health, and that’s very well established. One of the examples is the gut-brain axis, there is a functional connection between our gut and brain, thanks to the fundamental role of the microbiome that is even able to influence our brain, both in physiology and pathology. We will mention diseases resulting from alterations of the microbiome. The microbiome can also just contribute significantly to the pathogenesis of human diseases. Is fundamental to study cell biology. Cells are fundamental, we need to know how they work. Each cell has its own functions but still cooperates with other cells, 37 trillion cells cooperate for decades to give rise to the human body, instead of a chaotic war of selfish microbes. THE LONG CLIMB TO THE CURE OF A GENETIC DISORDER Now we understood why cell biology is important, and we can start to make an example of how scientists face a biological problem and solve it. So, we will start our course with an example related to a human genetic disease which is caused by a mutation of one or more genes. We know about genetic diseases, and we also know that in our body there are lots of genes. How many genes are present in our genome? 24000 approximately, they are less than expected. Before the sequencing of the human genome occurred, it was supposed to be around 70000, a lot more. Then we realised that 1  01.12.2021 RETTA L.1 they were actually less than we expected. There is a reason and explanation for this difference between the predicted number and the real number after the Human Genome Project. The relationship within genes and protein is not simple, it’s not only one gene and one protein, but one gene can produce a lot of proteins because of, for instance, alternative splicing. We know that genes are composed of different exons and introns, and because of splicing introns are removed and exons are joined together to form the mRNA and then code for proteins. Alternative splicing can take place, and different combinations of exons can occur and because of these combinations different proteins can derive from the single gene. Having 24000 genes does not mean that we have 24000 proteins, the number of proteins is much higher. There are genes that can make thousands of different isoforms, proteins that are similar but are not identical because of alternative splicing. Let’s go back on human genetic diseases. We will make an example related to a human genetic disease called Cerebral Cavernous Malformation (CCM), which is a cerebrovascular disease that affects brain capillaries and can cause severe clinical symptoms, including seizures, different neurological deficits and even death, because of intracerebral haemorrhages (ICH = bleeding within the brain tissue itself). The brain capillaries can break up and give rise to ICH, which can be fatal. This is not a very well-known human disease; it’s called Cerebral Cavernous Malformation (CCM). Cavernous Angioma and Cavernoma are different names referring to the same disease, and the most used one is CCM, because this a disease that affects brain vessels, but it is not a cancer disease, like haemangiomas (1). There is a similar disease indeed that is named haemangioma but compared to CCM, it is a cancer that affects vessels, in which the cells forming the vessels proliferate in an uncontrolled manner like a tumour, while Cavernous Angioma, CCM or Cavernoma is not a tumour, because it is not characterised by uncontrolled proliferation of cells that form the vessels. It is just a malformation, vessels are not very well constructed, and this is generally a congenital disease (2), but now there is clear evidence that shows it can also occur during adult life, these malformations for some reasons can occur during adult life. What is the reason of that? What is the mechanism underlying this disease like other diseases? 1. A haemangioma (he-man-jee-O-muh) is a bright red birthmark that shows up at birth or in the first or second week of life. It looks like a rubbery bump and is made up of extra blood vessels in the skin. A haemangioma can occur anywhere on the body, but most commonly appears on the face, scalp, chest or back. haemangiomas very rarely become cancerous, most do not require any medical treatment. 2. A congenital disorder is a condition that is present from birth. Congenital disorders can be inherited or caused by environmental factors. Their impact on a child's health and development isn't always severe, and sometimes it can be quite mild. Later we will talk about which are the characteristics of this disease, but for the moment let’s just keep it as an example related to human genetic diseases. This means that a gene mutation underlies this disease, but we have 24000 genes, how is it possible to identify the gene or the few genes that underlie a human genetic disease? This is very fig.3 difficult to do. Indeed, it’s shown here a stair of the path which scientists must follow in order to develop a cure for a human 2 01.12.2021 RETTA L.1 disease. There are different steps to find the cure. If we face a human disease, including human genetic diseases, first of all we have to identify the major cause, the genetic defect. The first step is to identify the gene that is altered, and, because it is altered, it causes the disease. Here (fig.3) is represented the amount of grants that an Italian charity foundation (Telethon) provided to scientists in order to study the different aspects of a human genetic disease, starting from the identification of the genetic causes up to the development of a therapeutic approach, a therapy. What are the steps? 1. The 1st is the identification of the genes that are associated with the human genetic disease. 2. The 2nd is to identify the function of this gene both in physiological and pathological conditions. Which are the altered mechanisms caused by the mutation of that gene? We can see here the amounts of grants that have been provided to scientists in order to study the different aspects of a human genetic disease. As we can notice, the majority of the grants were provided to study the 2nd step, which is the identification of the pathophysiological mechanism. Why did they do this? We can notice another aspect, the difference between the 2 decades: 1990s and 2000s. We can see that, as compared to the 90s, the amount of money provided for scientists to study human genetic diseases with the identification of causative genes was reduced in a decade, from 24% to 11%. Why? Because most genes were identified during this decade. It was not easy not only to the identify genes in general, but also the genes related to different diseases. How many human genetic diseases exist? 6000 genetic diseases have been identified. It cannot be 100000, why? Because the number of genes is 24000 and of course each gene can undergo different mutations, so different mutations can refer to the same genetic disease, they are related to the same gene. So human genetic disease cannot be higher than 24000. But not all the mutations cause genetic diseases. As we said before, the estimated amount of human genetic diseases is around 6000, still a high number. What is the most difficult step? The most difficult step in the stairs is the 2nd one, which is related to the characterisation of pathological and physiological functions of genes. So, it is very difficult to understand what is going on when a gene is muted. What are the relationships between the genes and the proteins? What are the mechanisms in which these proteins are involved? How functionally related are these proteins? It’s already very hard to imagine how cells cooperating. We can imagine inside a cell thousands of molecules, and how they interact and cooperate in order to make molecular mechanisms happen. What happens when one of these components is altered? That’s why, as we can see, almost half of the grants that are provided by charities is related (spent) on the second step. If you are able to identify the pathophysiological mechanisms that underlie a human genetic disease, then you can proceed to the next steps. 3. The next step is starting to consider a therapeutic approach, and that is possible only if we understand the mechanism, otherwise how can we think of a therapeutic approach if you don’t know what you need to correct? If you don’t know the mechanism to identify and rescue the pathological phenotype you cannot do anything, but if you understand the mechanisms then you can start thinking about a therapeutic approach. Where can you start to develop a therapeutic approach? Which model can you use in order to develop the therapeutic approach? The 1st model is to use a cellular model of a human genetic disease. What is a cellular model of a human genetic disease? Could it consist of cells deriving from patients presenting the mutation? They are cells with the same mutation that cause the disease and we can study those cells to understand what is wrong in the genes because of the mutation. We can also cultivate these cells in vitro in a Petri dish, we can keep them in the laboratory and study them under the microscope and preforming biochemical experiments in order to characterise the proteins, and then eventually (because of the cellular models) you can understand the molecular mechanisms of the pathogenesis and develop a therapeutic approach to correct: if I’m a cell that at some point, because of the mutation of one gene, I become crazy and change my shapes and functions, then us as scientists can understand this changes and characterise, via a biochemical approach, under the microscope the defects and 3  01.12.2021 RETTA L.1 make sure the cell goes back to their physiological situation. Most alterations are related to the alternation of, for instance, proteins-proteins interactions. In order to allow mechanisms to work, proteins must interact with each other. For instance, an enzyme must interact with its substrate that can be a protein or can be phosphorylated. This phosphorylation can activate a downstream mechanism and so on. So, how is it possible to find two proteins interacting and discover that at some point they are not anymore interacting? You can follow this under the microscope. There are even experimental approaches based on the use of fluorescent proteins and GFP. In 2008 Roger Tsien, Martin Chalfie and Osamu Shimomura won the Nobel prize for discovering and developing GFP. So, thanks to GFP, it’s possible to monitor the dynamics of more labels. You can look at cellular models under the microscope, not only at cells dynamics but you can go inside and look at the dynamics of molecules by taking advantage of the fluorescent proteins, you can label different proteins with different fluorescent proteins (green, red, blue etc.) and then you will track and follow the protein because it’s labelled with a specific colour, and at some point you can see whether if they meet each other, interact with each other, if they separate, if they go in separate intracellular compartments. We can do all those things in a cellular model that is characterised by the mutation which is responsible for that disease. If you do not have the cells deriving from the patient you can take normal cells and cause the specific mutation: you have already identified the gene, you know how to manipulate the gene, you can cause a specific mutation, a targeted mutation on the gene of your interest. Hence you are able to create a cellular model. A cellular model can consist of cells deriving from the patient, or cells that you can manipulate by causing a specific genetic alternation by target mutagenesis (1). You can even mutate one nucleotide in a target manner. You can create these cellular models, and then you can develop therapeutic approach. If you understood the mechanisms underlying the disease, you have the cellular model and you can develop a therapeutic approach. For instance, you can think that maybe a compound can be effective in rescuing the pathological phenotype. How can you test this? You can check under the microscope: if the problem was the alternation of the cell morphology, by adding the candidate therapeutic compound, the cell should acquire the original phenotype. If for example the alteration affects the cytoskeleton, you can check this, you can see if the cytoskeleton alteration is rescued because of the treatment. If the treatment was effective in the cellular model, then the compound (which was effective) could be a potential candidate, so how do you develop this candidate drug? You can test its effectiveness in animal models. Before using this candidate as a real drug, you have to pass other two steps. 4. The 4th step is testing the potential candidates in animal models. Animal models could be the zebrafish, mice and other models that are currently used in the labs, in which you can cause again the genetic mutation by target mutagenesis, you can do this in an organism, you can cause germline mutation (2), so a mutation that will be present in all the cells. Consequently, in this manner you will generate a mouse model, which would develop the same genetic disease that affects humans. If the genetic disease is a vascular disease, maybe the mouse model will develop the same vascular disease. You can check it and analyse this model, you can observe whether this disease also occurs in the model, but you already know the mechanism and you can test the potential drug candidate in the mouse model. And in this manner if the drug candidate will be effective, then you can proceed to the final step. 5. The final step is the development of the clinical trial. Eventually, when you tested the effectiveness of the potential therapeutic compound in cellular models and mouse models (mice because they are mammalian, but other models can be used in order to understand more in detail the function of a gene in an organism inside cells). Eventually you will develop a clinical trial, and then if they will be effective the drug candidate will become a real drug. In this manner you developed a therapeutic approach for a human genetic disease. If you pass these crucial steps, then the others will be easier. Without understanding this mechanism, it 4 01.12.2021 RETTA L.1 will be not possible to develop the basic approach, unless you got lucky and used a compound by chance and found it effective. 1. Mutagenesis is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory 2. A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. As we can see, still 50% of the money that is used to supporting researchers is related to studies being part of the 2nd step. But we can also notice that, about the other steps, the amount of invested money increased, because more studies are developing for new therapeutic approaches, which are very promising. This means that after understanding the genetic and molecular basis of diseases, many of these diseases can possibly be treated by these therapeutic approaches. CEREBRAL CAVERNOUS MALFORMATION CCM fig.4a fig.4b fig.4c Now let’s see the disease that we mentioned earlier. This slide is about the human genetic disease cerebral cavernous malformation (CCM). CCM is a disease that affects brain’s capillaries. We can see (fig.4a), in the images, normal capillaries and abnormal ones. The abnormal capillaries are enlarged and leaky, so they can give rise to haemorrhages. At first, they have an enhanced permeability, but eventually they can break up and give rise to haemorrhages. The diagnostic procedure that is used to diagnose this disease is MRI (Magnetic Resonance Imaging). MRI is the only diagnostic approach that is really effective for the diagnosis of this disease. Before the development of MRI, it wasn’t possible to identify this disease, which is one of the reasons why this disease is not very well known, after the 80s it has been possible to study it and make a precise diagnosis with the MRI. So, we can clearly see (fig.4b and fig.4c) the presence of lesions. There are 2 lesions, one is smaller, and the other is bigger. We can see a black ring around the lesion, which is related to haemorrhages, if you see black rings, it is because of haemosiderin deposit, which is indicative of a haemorrhagic event. They localise in different parts of the brain. Not only in the brain, they do not only affect the brain but also the spine, the retina and other organs, despite the fact that in other organs the symptoms are not so severe like in the Central Nervous System. The Central Nervous System is affected by this disease and the symptoms related to its alteration are more severe as compared to symptoms related to the formation of the lesions in other organs. They have been identified also in the liver, in the vertebrae, in the bones and in the skin. These lesions in the brain are therefore associated with lesions in the skin but not always of course, sometimes this association is possible, the presence of vascular lesions in the skin together with this lesion in the brain. 5  01.12.2021 RETTA L.1 As we can see in the slide we have a prevalence number, and it’s around 0.5%. What does this mean? What is the prevalence of a human disease? It means that, statistically, it affects one person out of 200 people. Every 200 people, there is statistically one person that is affected by this disease. We may think that it is not a rare disease, if a disease affects one person over 200 people it’s actually not that rare. But why is this disease not known? A reason is that it was only possible to start studying this kind of diseases in the 90s, even later. For instance, the first gene associated with this disease was identified in 1997, quite recently. There are 3 genes related to CCM, the first one was identified in 1997, the second one in 2004 – less than 20 years ago - and the third one was identified in 2005. That’s why medical doctors that got their degree in the early 2000s did not know about this disease, because simply it was not known, even though the prevalence is high. Why is there this discrepancy between the fact that the prevalence is high, but it’s not very well- known? Another explanation is that not all people affected by these lesions develop symptoms. In 70% of the cases these lesions remain asymptomatic, they are present, but people will never know unless they undergo MRI for other reasons, for car accidents for example. Because of MRI doctors find out that people resent such lesions, despite not presenting any symptoms because luckily the 70% of the cases are asymptomatic. However, 30% of the cases develop clinical symptoms. How can this occur? Why would a lesion, which was asymptomatic, give rise to clinical symptoms? Why do some people develop clinical symptoms and others do not, despite having the same lesions and disease? This is our research issue, which has been faced. The size of the lesions can be different, some of them are very small (millimeters), and others can be big (centimeters) as we can see in the slide. Symptomatic disease usually occurs between the third and the fourth decade of life, but it can occur in children as well, there are many cases of symptomatic CCM patients in children. CLINICAL SYMPTOMS What about the symptoms? We have a list of symptoms (fig.5), they consist of recurrent headaches, seizures, haemorrhages and neurological deficits (sensory, motor, visual, language, etc., fig.5 depending, for instance, on the localisation of the lesions). The symptoms can be connected to the localisation and depends also on the size of this lesions: the bigger the lesions are, the more probability there is to give rise to the symptoms. The number of lesions can vary from 1 (there are patients affected only by one lesion) up to 700. There are patients that have been identified with 700 lesions, and some of these people affected by this disease with a high number of lesions, they do not develop any symptom. CENTRAL CAVERNOUS MALFORMATION (CCM) Because symptoms, beside all the aspects we mentioned, also depend on the presence of an haemorrhage or not, even a small haemorrhage could be sufficient to give rise to symptoms. How can some lesions develop toward a haemorrhagic state and others remain quiescent? If the most severe symptoms are related to haemorrhagic events, how can these events take place? What’s wrong? CLINICAL SYMPTOMS Cerebral haemorrhages of course are the most severe clinical symptom, sometimes haemorrhages can be fatal. CCM MAR ARISE THROUGHOUT THE CNS So, we said that these lesions can be localised in different parts of the CNS. We can see that some of them are bigger. So, in the slide (fig.6) we can see a patient at the current time. This patient had a surgical intervention to remove a cavernous malformation, via neurosurgery. Neurosurgery is the only therapeutic approach available so far to treat this disease, and pharmacological approaches are under 6 01.12.2021 RETTA L.1 development. As we can see they are very big, and they are localised in the brain stem. This localization in the brain stem is very risky and can give rise to serious clinical symptoms, and on the other hand these malformations are difficult to be removed by surgical intervention, because of the localisation. fig.6 SPINAL CORD AND CEREBELLUM CCM MAY ALSO OCCUR 7  01.12.2021 RETTA L.1 Lesions can affect all the CNS, including the cerebellum, we can see the lesions in the cerebellum as well in the slide (fig.7). We can see the lesion of the spinal cord. This is a spinal cord CCM lesion which is removed by surgery, and they are referred as Mulberry-like vascular lesions, compared to CCM lesions (they are similar). Mulberry-like vascular lesions are related to capillaries. fig.7 CCM CAN BE ASSOCIATED WITH RETINAL AND CUTANEOUS ANGIOMAS As we said sometimes there is an association with the hyperkeratotic cutaneous capillary-venous malformation (HCCVM), some patients also present this (fig.8). We can see that sometimes CCM lesions can affect the retina (fig.9). The spectrum of the localisation of these lesions is quite wide. fig.8 fig.9 CCM LESIONS CAN INCREASE IN SIZE AND NUMBER OVER TIME 8 01.12.2021 RETTA L.1 As we said, the size also can be different. Some of them are small (fig.10), some of them are big, as shown there (fig.11) is a big lesion that already caused haemorrhage, it’s a very severe haemorrhagic event. The lesions can vary in number, the haemorrhagic events can be different as well. What we can add is that these lesions, besides being focal (they occur somewhere but not everywhere), they can have a dynamic nature. Despite the original thoughts, which were only about congenital genetic diseases, now it’s clear that these lesions can also develop during adult life de novo. Most of them remain clinically silent, but others can give rise to serious clinical symptoms at any age. fig.10 fig.11 PROGNOSTIC BIOMARKERS AND PHARMACOLOGICAL THERAPIES REMAIN TO BE DEFINED What about therapeutic approaches? We will see the basic pathogenic mechanisms that are associated to this disease, but in general, how do clinicians face this disease? What are the therapeutic approaches available to treat this disease? The only therapeutic approach that is really effective now is the neurosurgical removal of accessible lesions. Of course, they must be accessible for neurosurgery, if they are located in areas that are difficult to access by neurosurgery, most likely it won’t be possible to treat it. So, this is a surgical removal of a CCM (fig.12), in the image we can see a real CCM that happens to be removed. What about the pharmacological approach? Are there pharmacological approaches available right now? Just by referring to the steps (fig.3) of the stairs, we are now at the last step. Some clinical trials are on the way to test the fig.12 potential candidates. MECHANISMS OF CCM PATHOGENESIS We have two last important questions: - Why, where and when CCM forms? - Why CCMs are focal and dynamic? These are the fundamental questions of cell biology. You can always ask these questions while facing biological issues: why, where and when this occurs and does not develop in other places, and why do they develop at some point during people lives. If we were able to answer these questions, then we would solve that biological issue, just by providing answers to these questions. Now we will introduce this slide (fig.13). It refers to the three genes that are fig.13 identified and associated to CCM (the first one identified in 1997, the second in 2004, the third one in 2005). They have been mapped, two of them are present on chromosome number 7, one on the short arm and the other on the long arm. The third one is localised on the long arm of chromosome number 3. So, there have been mapped and named CCM1, CCM2 and CCM3 since the disease is CCM. The gene that is more often mutated is the “CCM1”, which is also called “KRIT1” (KRIT1 is the name of its protein). So, in 1997 this story took place. 9