Flashcards in Revision Cards Deck (181):
What are some of the biggest killers?
-Diabetes (dysfunctional glucose homeostasis)
-Heart Disease (plaque build-up in arteries)
-Chronic lower respiratory disease (airflow blockage)
-Cancer (uncontrolled growth and spread)
-Neurodegenerative disease (loss of nerves)
-Anxiety and depression (poorly characterised)
What is a model organism?
Non-human species which is studied as an in vivo model to understand aspects of human disease.
Why are specific model animals used?
-Mechanisms of gene action are conserved
-Easy genetic manipulation
-Transparent models allow real-time visualisation
-Help identify life-course genes/events
Why are animal models used?
-Understand variation between changes caused by disease progression and life course changes over time
- Genetically-tractable organisms that exist in large numbers and can be environmentally influenced (Gene X Environment)
-Sequencing and GWAS to understand mechanism and disease susceptibility [limited]
-Effect of new therapeutics in development
- Whole organism or system analysis
What are the limitations of GWAS?
These studies don't show when or where the gene is expressed, how gene is translated to protein or how the gene/protein might trigger, contribute or exacerbate the disease.
What do transgenic reporter lines enable?
Can allow for the real-time visualization of gene expression in a living organism in health and disease, or after drugs, genetic manipulation, environmental influence or insult. Follow expression over time and combine with cellular behavioral studies.
What can FACS do?
Fluorescence-activated cell sorting enables individual cells to be dissociated from the tissue and sorted according to their fluorescence by electrostatic deflection.
What are mice, drosophila and zebrafish usually used for?
-Identify molecular pathways in normal cells, tissues, organs
-Identify normal and aberrant cellular outputs
-Identify disease progression: early events and consequences
-High throughput screens for drugs and toxicity
What are mice and chicks usually used for?
Experimental embryology gain-of-function approaches
Informed regenerative stem cell medecine
What is Diabetes?
Metabolic disorder in which the body is unable to carefully control the levels of blood glucose in the body. Hyperglycemia due to insufficient insulin supply and/or insulin resistance.
What normally occurs in glucose homeostasis?
Stomach converts food substances to glucose. Glucose enters the blood stream. B-cells of the pancreas secrete insulin which encourages skeletal muscle tissue to take up excess glucose to restore normal blood glucose levels.
What is the difference between Type 1 and Type 2 diabetes?
- In T1D, patients don't have enough B-cells, make or secret enough insulin. Glucose doesn't get taken up.
-In T2D, patients have an normal initial mechanism of insulin production and secretion but body is resistant to its effects. Over time, B-cells become depleted.
How does insulin resistance cause loss of B-cells in T2D?
5-10 years before symptoms present, the body becomes resistant. Insulin levels rise to try and combat the increased glucose. Excessive levels of glucose and insulin cause the loss of B-cells. Insulin levels drop and hyperglycemia presents.
How is Type 2 Diabetes classified according to obesity?
Abdominal obesity along with two others factors: elevated BP, low HDL, elevated triglycerides or impaired fasting glucose.
How are T2D and obesity linked?
Obesity is highly associated with T2D. Unclear how it links because so many tissues are involved. Obesity is known to cause increased free fatty acids and insulin resistance but it is unknown which comes first.
How does changes in FFA and insulin resistance contribute to T2D?
Increased FFA and insulin resistance lead to increased levels of Apolipoprotein B and Hepatic Lipase. This leads to increased levels of triglycerides and small dense LDL and decreased HDL.
What causes hyperglycemia in T2D?
-Pancreas can no longer secrete sufficient insulin
-Dysfunction in liver glucose production
-Peripheral tissues are unable to uptake glucose
How does obesity cause the loss of adipocytes?
Obesity leads to the accumulation of fat in ectopic tissues such as the liver and skeletal muscle instead of in adipocytes. Imbalanced accumulation leads to a loss in the adipocytes.
How can mice be used to model the lack of adipocytes in obesity?
GM mice with a lack of adipose tissueare characterised by hyperphagia, hyperglycemia, insulin resistance and Type 2 Diabetes. Lack of adipose tissue means the mice are Leptin deficient. Suggests that primary cause is linked to brain dysfunction.
How can reporter lines be used to identify the cause of hyperglycemia?
Cross 3 reporter lines to make a fish in which you can see the pancreas, liver and adipose tissue. Feed high-fat diet and see which tissue falters first.
What is the biology behind reporter lines?
Depends on the fact that genes are deferentially transcribed in different tissues depending on their promoter/enhancer and all the cell-specific transcription factors.
How do you create reporter lines?
1) Cut off the coding part of the gene from the control regulatory elements.
2) Fuse liver-specific gene promoter to the coding sequences of genes that code for proteins that fluoresce when UV light is shone on them.
3) Make a transgenic mouse or fish in which this extra gene is incorporated. The reporter is only expressed in the liver at the time when the endogenous gene is expressed.
How can you measure proliferation in cells?
EDU or BrdU labelling intraperitoneal injection for body, intracerebroventricular for brain. Both are analogues of thymidine that have be modified so that they can be used to visualise proliferating cells.
-BrdU labelled with an anti-BrdU antibody
-EDU labelled with a click chemistry kit
How does EDU visualise proliferation?
1) Incubate cells with excess EDU
2) EDU is incorporated into the DNA after semi-conservative replication
3) Detection of newly synthesised DNA with a 'click' chemistry reaction that attaches EDU to the DNA within 30 minutes.
How to make a tissue-specific knockout?
1) Genetically modify the specific gene you want to KO so that it is flanked by 'lox' which are recognised by Cre recombinase. Make a transgenic line.
2) Identify a gene that is only transcribed in the specific tissue, clone the coding sequence for Cre recombinase downstream of the gene's promoter.
3) Cross these two lines. Whenever Cre is expressed, it will cut out the gene at the lox sites, no expression of the gene.
What is the current new idea for therapy for Type 1 and 2 Diabetes?
Both in type 1 and late-stage type 2 diabetes there is a depletion in the number of B cells. If you can restore the number of B cells, you could create a curative approach that increases life expectancy. This could be a human version of BefA but further study is needed.
What is the link between microbiota and B cells?
Hill et al found that a conserved protein from certain gut bacteria BefA is necessary for the pancreas to populate itself with a robust no. of B cells.
How did Hill et al (2016) establish a link between microbiota and B cells?
Hill et al (2016)
-Created a Tg(Ins:GFP) line in zebrafish to count the number of B cells. Found that after hatching, B cell numbers increased.
-In microbe-free environment, the number of B cells stayed the same as pre-hatching
-High levels of 'whole glucose' was found in MF
-Exposing MF fish to certain bacteria restores the MF B cells levels
-Using EDU, BefA protein causes B cells to proliferate and could restore B cell levels
How were rodents reveal the regenerative capacity of B cells?
Experimental ablation of B cells by chemical treatment or partial pancreatectomy in rodents displays significant recovery of the B cell mass.
This regenerative capacity could be exploited therapeutically.
How did Andersson et. al. (2012) test the drugs that could restore the numbers of B cells?
Andersson et. al. (2012)
Double transgenic Tg (ins: CFP-NTR) and Tg (ins:Kaede). CFP-NTR (nitroreductase enzyme) is cloned downstream of insulin promoter. When B cells are expressed, nitroreductase is also expressed which converts Metronidazole into a cytotoxic product, killing the B cells.
Then double-transgenic fish treated with MTZ were placed into 100s of wells to test over 7000 compounds. After washing away the MTZ and 2 days of recovery, B cell regeneration is easily quantified. Compound NECA showed a major increase in B cells.
How are body-brain interactions affected in poor glucose homeostasis?
Likely that there is either abnormal signalling from the peripheral tissues like the gut, liver, endocrine pancreas and adipose tissue to the brain, indicating that there is insufficient fat increasing food intake OR abnormal signals from the brain to the tissues.
What is Leptin?
Hormone produced by adipose tissue to signal to the brain that status of the body's energy content. This tells it to eat more/less and exercise more/less.
How did Cohen et al (2001) link the leptin receptor to obesity?
Cohen et al (2001)
Mice lacking leptin are obese, diabetic, infertile and hypoactive. Immunohistochemistry shows that many tissues in the body express the leptin receptor. You can conditionally knockout the leptin receptor in different tissues. In a liver leptin receptor KO, the mice are normal. In a brain leptin receptor KO, obesity is induced. Intracerebroventricular administration of leptin in ob/ob mice causes reduction of body weight and food intake.
What are 2 major leptin-responsive neurons?
Animal model studies showed that within the brain the LepR neurons were in the hypothalamus in the arcuate nucleus.
-Leptin binds to Neuropeptide Y neurons and inhibits them. This coordinates action to stimulate food intake and reduce energy expediture.
-Leptin binds to Pomc neurons and stimulates them. Pomc is a neurohormone which acts to reduce food intake and increase energy expediture.
How are Pomc and NPY neurons generated?
Hypothalamic stem cells (Tanycytes) which have a radial glial character. Cell body lies at the 3rd ventricle and a lateral projection that is used as a scaffold. Tanycytes can divide to produce more of themselves or differentiated cells. The differentiated cells use the scaffold to migrate to the ventricular position.
It's likely that NPY and POMC neurons develop and differentiate to anticipate or respond to changes in glucose homeostasis.
What experiments were done to find that NPY and POMC neurons developed from stem and progenitor cells?
-Identify different populations that develop over time through markers. Stem cells express pea3, Glast1 and FGF10. Progenitors express Islet1.
-Lineage-trace the cells
-Identify the genes that maintain each cell.
-KO these genes at look at daughter cell differentiation
-Use the promoters of these genes to make cytotoxic transgenes to elemiate particular cell populations
How did Nasif et al (2015) link Islet1 to Pomc?
Nasif et al (2015)
Islet1 is well-known to be involved in motor neuron differentiation. Islet1 was seen to be expressed before Pomc because it is a progenitor marker. After that you see Islet1 and Pomc coexpressed.
Islet1 binds in vitro and in vivo to critical homeodomain binding DNA motifs of the Pomc enhancer. Without these binding sites (mutation in enhancers), there is no expression of Pomc. If you conditionally remove Islet1 from Pomc neurons, you impair Pomc expression and get hyperphagia and obesity.
How to perform lineage-tracing in an adult stem cell?
Cell-specific promoter identified and Cre-recombinase expressed downstream (Cre-ERT2) to create a transgenic mouse. This Cre is genetically modified to be fused to ERT2 which is a mutant oestrogen ligand-binding domain so that it is only activated when you add Tamoxifen.
Make a second transgenic mouse where a stable reporter is downstream of a constitutive reporter but seaparated by a floxed STOP sequence.
Breed mice and add tamoxifen at any stage of life to cut out STOP, allowing the reporter to be expressed in the cell and in all daughter cells.
How was lineage-tracing in adult stem cells used to find that tanycytes give rise to NPY and POMC neurons?
Identify that Glast1 is only expressed in tanycytes. Make Glast1:CreERT2 transgenic line and cross it with a RosaSTOPLacz trangenic line. Allow the mice to develop into adults and add tamoxifen at 6-8 weeks. Cre is activated, lox sites are removed and Lacz/GFP is expressed.
Using UV light or anti-GFP antibody you can visualise cells expressing Glast1. Leave for 9 months you cna see that a=tanycytes give rise to POMC and NPY neurons.
What did Scarlett et al (2016) find about intracerebroventricular injections FGF1 in ob/ob mice?
Models of T2D. You can see sustained diabetes remission unlike in intraperitoneal FGF1 infusion. Effects did not include changes in weight loss, hypoglycaemia, basal glucose production or glucose tolerance. Establishes a functional link between activation of 3rd ventricle tanycytes and diabetes remission induced by FGF10.
What is multi-morbidity?
Presence of two or more chronic non-communicable conditions like cardiovascular disease, cancer, chronic respiratory conditions and T2D. Whereby risk factors that affect one disease often affect other diseases too independent of age, nationality or class.
What increased risks do diabetics have?
Increased risk of CV disease in a Type 2 diabetic compared to a prediabetic or someone with normoglycaemia. Cardiometabolic risk factors tend to cluster and all effect one another.
Increased risk of breast and bowel cancer in a prediabetic.
What is insulin resistance?
Impaired response to the physological effects of insulin (Including those on glucose, lipid and protein metabolism) and the effects on vasular endothelial function.
What contributes to insulin resistance?
Some people have a genetic susceptibility to insulin resistance which can contribute to diabetes onset. Environmental factors like nutrition, obesity and physical inactivity can also contribute.
How does hyperinsulinemia contribute to poor vascular endothelial function?
Endothelial dysfunction refers to a maladapted endothelia phenotype charcaterised by reduced NO, increased oxidative stress, elevated expression of pro-inflammatory and pro-thrombotic factors and abnormal vasoreactivity. Insulin exerts vasodilator and vasoconstrictor actions which cause imbalance.
How can metabolic dysfunction lead to cancer?
1) Oestrogen - after menopause, oestrogen made by adipocytes can make cells multiple faster in the breasts and the womb, increasing risk of cancer.
2) Insulin and growth factors - excess fat can cause levels of insulin and other growth factors to rise, which tells cells to divide.
3) Inflammation - macrophages release cytokines which encourage cells to divide
Why is insulin linked to the cancer/aging?
Animal model studies show that the insulin pathway is an ancient, conserved signalling pathway fundamental to life and aging. Receptor and ligand have been conserved from hydra to mammals as well as the whole signalling transduction cascade. FOXO is the transcription factor at the end.
What is an environmental insult?
Environmental insult can be from the outside (like a mutagen) or can be within the cell or among the tissue. In cancer, it could be the environmental insult of inflammation or a genetic insult within a cancer cell.
How was Int1 discovered?
1000s of years ago 'fancy mice' were bred by the Chinese. Inbred mice were kept in Jackson labs in 1900s. In 1930s, female mice were found to be highly susceptible to mammary tumours.
In 1983, there was a retrovirus discovered in the mice that interfered with the mice's DNA machinery to insert itself upstream of a proto-oncogene and upregulate a gene. This gene was clones and found to be Int1 which was thought to be a secreted protein due to it's signal sequence. Very difficult to purify.
How was Wg discovered?
Nusslein-Volhard and Wieschaus performed a mutagenesis screen in Drosophila identifying patterning genes including segment polarity genes. One of these was Wingless.
What is Wnt1?
Wg had the same sequence homology to Int1. Thus, Wnt1 was born, which has adhesive, signalling and stem cell communication properties. Wnt signalling pathway was elucidated 1980-1995 using mice, drosophila and xenopus. Found that Wnt binds to Frizzled which causes B-catenin to become stabilized. Its accumulation in the nucleus allows it to activate target genes.
How can Wnt1 mutations lead to cancer?
Overexression of Wnt1 led to the development of mammary tumours in the mice. But in humans, 2 kinds of mutation. Activating mutations in protooncogene components of the Wnt pathway which mean that B-catenin is always stablised in the nucleus or when both copies of a repressor are lost and there are no breaks on the signalling, B-catenin is always in the nucleus.
Mutations are implicated in a variety of human cancers. Mostly commonly mutations in APC lead to colorectal cancer.
How can B-catenin be visualised?
You can make anibodies to detect stablised B-catenin in the nucleus. Animal models have allowed the identification of a 'biomarker' to detect an early-stage cancer (adenoma). Now used in humans to detect cancer. More stablised B-catenin will precede any change in morphology.
How have transgenic mice been used to trace Wnt-responding cells?
Gene called Axin is usually turned on by Wnt signalling. Generating mouse transgenic lines whether by Axin promoter is fused to LacZ, means when Axin is expressed so is LacZ. LacZ blue can be visualised by anti-LacZ stain to visualise Wnt signalling. Can be used to trace Wnt-responding cells and examine their origin and fate with or without insult.
Found that most cells responding to Wnt are stem cells involved in growth and development.
How have transgenic mice been used to track CBC stem cells?
Use Cre-ERT2 to lineage-trace Lgr5+ CBC cells shows that the Lgr+ CBC cells are stem cells (self-renew, multipotent).
What did Clevers and Nusse (2014) find out about the intestinal stem cell niche?
Clevers and Nusse (2014)
One of the most well-defined stem cell niches is the mammalian gut crypt. At the distal end, there are stem cell residing, and differentiating cells at the proximal sustained by Notch. Lgr5 is a G-protein coupled receptor expressed by crypt base columnar (CBC) cells which are pluripotent and long-lived stem cells. They give rise to all differentiated cell types of the intestinal epithelium. Trans-ampliying cells emerge from the stem cells and differentiate over 2-3 days into one of the four principal epithelial cell lineages of the intestine.
How can genetically modified mice be used to aid in human cancer research?
- Identify oncogenes and tumour suppressor genes and how they control signalling and act in cancer
-Predict which genes might give rise to cancers if mutated
-Impact on diagnostics and treatment
-Understand how stem cells can go out of control
How are stem cells linked to new therapy for breast cancer?
Cancers are present in mixed populations of stem cells and progenitors either of which can overproliferate. Some breast cancers are resistant to commonly-used anti-oestrogen therapies. New rationale is to treat these patients with a combination of anti-oestrogen to target progenitors and anti-Notch/Wnt/Hh to target breast cancer stem cells.
Can we replicate human diseases in animals, particularly CV disease?
No, but we can model some disease processes and elements involved.
There appears to be enough shared mechanisms in zebrafish to justify their use in CV disease. Can cure CV disease in zebrafish but this has not yet translated to humans.
What is congenital heart disease?
Common form of heart disease (1% of all live births, more common in spontaneously aborted fetuses).
Septal defects, aortic or pulmonary stenosis, AV canal defects, Fallot's tetralogy abnormalities and single gene defects are the most common.
Often a feature of genetic diseases through single gene mutations and chromosomal abnormalities but not generally known.
Can reduce life expectancy.
What are the features of human cardiovascular diseases?
Many 'preventable' elements.
Usually manifest in 40s, 50s, 60s.
Maternal factors like nutrition have an effect on CV development.
Low birth weight associated with a risk of heart attack, diabetes and hypertension in adult life.
Adult 'lifestyle choices' programmed in childhood.
How can normal parts of the heart go wrong?
MYOCARDIUM (leads to heart failure)
Reduced ability of heart to pump blood sufficiently. Caused by prior MI, hypertension, valve disease, alcohol, genetic disease. Symptoms of breathlessness and swelling.
VALVES (leads to valve disease)
Stenosis or leaking/regurgitation of heart valve caused by deposits of calcium. Congenital or acquired. Some genetic contribution.
CORONARY ARTERY DISEASE
Biggest cause of death. Blockage of coronary artery caused by progressive atherosclerosis leading to thrombosis, visualized using injections of X-ray contrast in angiograms.
Assessed using electrocardiogram to measure Q-T interval. Normal cardiac cycle tightly controls atrial and ventricular contraction (depolarisation). Genetic predisposition.
What are the disadvantages of current CV disease models?
Only partial success in modelling disease, good for artherosclerosis, moderate for hypertension, poor for valve disease.
Difficult to genetically manipulate.
Very difficult to image the vasculature.
How to visualise the vasculature when looking at animal models of CV disease?
In non-transparent organisms, a technique is needed to see blood vessels. Angiography involves the injection of x-ray opaque dye followed by an MRI. This takes 4 hours, often kills the mouse and you can't see the capillaries.
Confocal/fluorescent microscopy in transgenic lines can image blood or endothelium. Digital motion angiography shows the sum of subtracted frames. Much quicker and allows for investigation of physiology and anatomy.
What CV diseases have been modelled in Zebrafish?
Beis et al (2005)
Genetic dilated cardiomyopathy - easy to model, identify common familial mutation, KO in zebrafish. Informs human screening.
Cardiac defects - Fish have simple single circulation. But patterning signalling are similar.
Coarctation of aorta - narrowing of aorta. Rare condition. Look for genes in aortic formation. KO for phenotype.
Heart regeneration - After a MI, damage is permanent, but adult fish can regenerate their hearts.
Thrombosis - Can label thrombocytes to assess clotting.
Drug-induced long QT syndrome - Identify genetic KO that predispose to defects in heart rhythms. Submerse zebrafish in different drugs.
What are the advantages of the zebrafish?
Cheaper, smaller, genetically tractable.
Able to oxygenate via diffusion.
Vascular visualisation is much easier.
What are the weaknesses of Zebrafish?
Small size limits some techniques.
Genetically less similar to humans than mammals.
Rapid development - moving baseline.
No spontaneous cardiovascular diseases similar to humans and can't give them CV disease.
How is inflammation linked to progression of disease?
Cancer Research (2013) Initially Rudolf Virchow
Inflammation is thought to increase the progression of cardiovascular disease, cancer and Type 2 diabetes.
What did Myant et al (2013) find that showed that inflammation lead to malignant colon cancer?
Myant et al (2013)
Constitutively active Wnt signalling leads to increased active Rac1, leading to hyperproliferation. Rac mediates this effect via ROS and NF-kB, indicating a role for inflammation in progression of a benign to a malignant tumour.
NF-kB is pro-inflammatory factor that positively regulates the expression of proinflammatory genes including cytokines and chemokines.
ROS are key signalling molecules that play an important role in the progression of inflammatory disorders.
How does inflammation encourage tumour growth?
As a tumour grows. its demand for nutrients, oxygen outstrips its supply. Cancer cells begin to secrete proinflammatory signals, including cytokines which will attract more inflammatory cells.
Macrophages invade the tumour, and begin to secrete even more cytokines that kick start angiogenesis (growth of new capillaries). Inflammatory signals and cells also help break down extracellular matrix, to promote metastasis.
How was Cyclopamine found to antagonise Smo?
1940-50s: Pregnant sheep eating corn-lillies gave birth to cyclopic lambs.
1960-70s: Cyclopamine identified.
1970-80s: Hh gene identified that when mutated would make drosophila curled and spikey.
1980s: Vertebrate homologues Shh cloned and characterised - expressed down the midline.
1990s: Hh/Shh pathway was characterised. Ptc inhibits Smo, activation of Smo needed for activation of Shh pathway.
1990s: Shh -/- KO leads to holoprosencephaly in mice and humans.
In the 1990-2000s how did scientists' view of Shh's role change?
It was seen that shh signalling is usually at high levels suring building of the tissue but when its finished there are low levels negatively regulated at many end stages.
2000s: Key idea emerges that although initially involved as a patterning protein, many of the genes activated by Shh signalling are genes involved with proliferation and stem/progenitor cell renewal e.g. PTCH1, CycD1, Myc, Bcl-2, NANOG, Sox2
How was Shh linked to cancer?
It was predicted that cancer may develop in places where Shh is involved in development and then downregulated. Or in places where Shh levels are normally held at particular levels and in careful balance e.g. in a stem cell niche
What is the stem cell niche?
Skin cells are continually generated through life from a skin stem cell. Shh is expressed in the skin stem cell niche. It's levels are carefully regulated (by Wnt, BMP) to achieve the correct balance of stem cells, proliferating progenitors and differentiating cells. Proliferating cells in the deep dermis express Shh pathway components.
How was Smo linked to Basal cell carcinoma?
In 1998 USA demonstrated that activating Smo mutations can lead to BCC (most common skin cancer).
In 2000-2005 Shh was confirmed to be pathologically relevant to 90% of BCCs.
In activating Smo mutations, there is upregulation of GliA leading to an abundance of GliA in the deep dermis of BCC tumours. Similar to abundance of B-catenin in Wnt signalling pathway.
What Ptc and Smo mutations cause BCC?
Normally Ptc represses Smo. Only need 1 mutation for Smo because it is a protooncogene. 2 mutations are required for Ptc because it is a tumour suppressor gene. Predicted that a LOF mutation of Ptc will lead to BCC.
How did Erivedge come about?
Taipale et al (2000) and Rimkus et al (2016)
It was thought that cyclopamine could reverse the oncogenic action of mutated Smo by competitively antagonising Smo, causing the transcription factors Gli1 and Gli2 to remain inactive.
Curis Inc. patented 'Preventing cell proliferation using Hh antagonists'.
Curis and Genentech preclinically developed components that inhibit the Hh pathway, identified Vismodegib, very similar to cyclopamine.
Phase I and Phase II studies performed in BCC patients.
Erivedge submitted as a FDA-approved drug, and then licensed.
BUT it costs $7,500 monthly so is not cost-effective. Market competition should bring this down. But there is a major 20 yr time lag between first identification and nitty gritty discussion of cost.
What is Melanoma?
Skin cancer (5th most common cancer in the UK).
BCC, SCC are 20x more frequent but is still clinically prevelent.
Ptc (Hh activation) are initiator mutations.
Early on it can be treated but no treatment for when it spreads into the dermis/epidermis.
Emerges from pigment cells that grow out from moles (nevi).
What are the risk factors for Melanoma?
-Age: increase in cumulative mutations
-Sex: males have increased risk may be due to increased somatic mutation rate or hormones
-Skin type: light skins has 2.4% increased risk
-Sunburn/UV exposure: burning increases risk
-Genetic mutations that predispose patients to an increased risk.
What genetic mutations increase your risk of melanoma?
Melancortin receptor - gives phenotype of red hair and sensitive skin. Common in places with low sun exposure.
DNA repair - leads to Xeroderma pigmentosum. Problems with repairing UV-induced DNA damage.
CDKNZA codes for p14ARF and p16INKa which work in the cell cycle, preventing division of cells with DNA damage.
Rare promoter mutations that activate TERT expression, which elongates telomere length.
What other mutations accumulate in melanoma?
Different stages of melanoma were screened for mutations. It was found that there are always initiator mutations leading to MAPK pathway activation.
BRAFV600E - single point mutation -
Later on when the tumour is progressing...
SWI/SNF (chromatin remodelling)
CDKN2A (cell cycle)
Inactivation of p53 and PTEN
Where does melanomagenesis come from? Davies et al (2002)
Davies et al (2002)
Nervus/mole already has increased RAS activity. Activating BRAF mutations already present in 60% of nevi. This can develop into melanoma. BRAF V00E mutation present in more than 75% of melanomas.
Which model systems are used to study melanomas and associated pathways?
Core cell cycle machinery - Yeast
Upstream signalling like EGF/RAS/MAPK, Hh, Wnt controls cell division/differentiation/death - Drosophila, C.elegans
Tumour growth and metastasis requires angiogenesis/lymph vessels so need vertebrates - Mice (close to humans, precision genetics, KOs, xenografts) or Fish (large scale, optic clarity, fast genetics).
What are mice xenografts/xenotransplants?
Xenotransplanation involves the transplantation of one species' cells, tisssues or organs into another. Usually human cell lines are tranplanted into immunocompromised nude mice which provides a far better model of tumour growth than in vitro. However, the mice do not have any immune response so must be kept in a carefully controlled environment. Tumours use and interact with immune responses so it is not a perfect model.
How can CRISPR technology be used to create a genetic model of a BRAF melanoma mouse?
Tyr:Cre-ERT2:Braf CA/+;PTEN lox/lox
Tyrosinase promoter is only expressed in melanocytes.
Cre-ERT2 restricts Cre recombinase action until Tamoxifen is added.
Braf CA is cre-activatable constitutive active form of Braf.
PTEN lox is an allele of PTEN than can be inactivated by Cre mediated excision.
Therefore in the presence of Tamoxifen, Cre-ERT2 is activated and it moves into the nucleus to perform its recombinase function. It causes Braf to become constitutively active and simultaneously inactivates PTEN. This will only happen in melanocytes where the Tyr promoter is active.
How did Yang et al (2010) use mice in melanoma drug research?
Yang et al (2010)
Braf was discovered as a major driver behind Melanoma. RG7204, a selective BRAF V600E inhibitor was developed which displays anti-tumour activity.
Initially they looked at its activity in IN VITRO cell lines but they are very individualistic. Switched to nude mouse xenotransplant models and used three different cell lines. Found that tumour volume decreased and mouse survival increased with addition of the drug.
Taken through clinical trials, but found to develop resistance in human patients.
How did White et al (2011) use zebrafish to screen for drugs that interfere with melanocyte precursor formation?
Yang et al (2010)
Give fish melanoma using a transgenic mitfa:braf(V600E) in a p53 -/- background to establish rapid and reliable tumours.
Perform a chemical screen of 2000 bioactive compounds that inhibit crestin (marker for neural crest progenitors) and lineage during embryogenesis.
NSC210027 was found to block formation of neural crest progenitors, similar to Leflunomide.
Leuflunomide does inhibit human melanoma growth but is more potent WITH PLX4720.
Strong response found in mice xenotransplants.
Clinical trials in humans underway.
How did Ceol et al (2011) use zebrafish to idenitify genes that lead to melanoma progression/metastasis?
Ceol et al (2011)
The genes that cause oncogenic progression are poorly understood. A large region in chromosome 1 was found to promote malignant progression of melanoma. Using a melanoma assay transgenic mifta:braf (V600E) fish in a p53-/- background they induce rapid and reliable tumours. Found that removing mitfa gene could reverse tumour.
Mitfa -/-, p53 -/- and mitfa:Braf fish were tested. 50 different plasmids were created, each containing a different gene from chromsome 1 region. Injected into the fish, some will be taken up. Mitfa with SETDB1 gene (methyl transferase) showed enhanced cancer formation compared to mitfa alone. Therefore an important gene in melanoma progression, a inhibitor could be created.
What is the uprising of inflammaging?
Non-communicable diseases that harass inflammation are now the greatest killers, replacing infectious diseases.
Diseases such as heart disease, stroke, COPD, Neurodegenerative disease, Musculoskeletal disease.
As we age, we lose the balance and become prone to inflammation AND infection.
What are the characteristics of inflammation?
Plus loss of function
What can haemopoietic stem cells become?
Haemopoietic stem cells can remain as stem cells or develop into a multipotential stem cell. This can differentiate into a lymphoid progenitor cell or a myeloid progenitor cell.
What are neutrophils?
Fastest cell in the body, rapid migrate around
Contains granules which contain ROS and toxic proteases.
Hunts bacteria, may have to digest tissue to reach it.
Once bacteria is engulfed by phagosome, it injects its toxic materials.
Short half-life (24-48 hours) and killed in chemo.
They transmigrate to insults.
After destroying bacteria, they die by apoptosis or are recycled inside macrophages in the bone marrow.
What are the current models used in neutrophil study?
In vitro - neutrophils die very quickly in cell monoculture
Mouse - difficult to visualise inflammation
Zebrafish - contain polymorphic nuclei, primary and secondary granules, MPO, NADPH and make NETS (neutrophil extracellular traps) like humans.
Can show whole animal inflammation
Increased numbers in response to infection
How was neutrophil lifespan measured?
In the past, peripheral blood neutrophils have been used to measure their half life, shown to be about 6-8 hours which is an underestimate. This was done by labelling the neutrophils, putting them back into the body and measuring the amount of label at different points.
What is hypoxia's relationship with inflammation?
Hypoxia delays neutrophil apoptosis in vitro .
Hypoxia signalling is necessary for robust anti-bacterial responses.
Activation of hypoxia signallnig causes more inflammation in mouse models.
All cells make hypoxia-inducible factor alpha which is hydroxylated and broken down when conditions are normal. It is very difficult to induce whole animal hypoxia. DMOG is a chemical used to suppress neutrophil apoptosis by allowing HIF-a to carry out its transcriptional action and increase inflammation.
How was zebrafish used to find Tanshinone IIA?
Take zebrafish, remove tailfin, inducing high levels of inflammation. Screened for drugs to reduce inflammation. Tanshinone IIA was found to accelerate human neutrophil apoptosis and increase reverse migration. It is unknown how this happens. This suggests that the survival signals from within the neutrophils may be retention signals.
How can the neutrophil kinase be used for screening of anti-inflammatory drugs?
The human neutrophil kinome is well characterised and contains druggable kinases that transduce survival signals for the neutrophils. 38 kinase inhibitors were screened in zebrafish. ErbBs were shown to have a high number of hits, structurally related receptor tyrosine kinases. Known to play an essential role in regulation of cell proliferation, differentiation, apoptosis, metabolism etc. Inhibitors and mAb of these already used in cancer treatment, ErbB inhibitors could be a potential anti-inflammatory.
Why are 'simple' tissues easy to research?
Simple tissues are made up of very few different types of cell and often have the ability to regenerate through life e.g pancreas, skin, heart. The quantity of cells makes them easier to interrogate, understand and manipulate in health and disease.
Why is the nervous system difficult to study?
Very complex. Composed of many more different types of cells. Much more spatially integrated with one another - difficult to access. Small quantity of each cell type.
How can we use developmental biology to study the decode the nervous system's complexity?
We can harness our understanding of developmental neurobiology to direct the differentiation of defined neurons/CNS from what we've learnt of cell specification in animal models. We can use this to increase numbers of specific cells/cells in complicated tissue.
What have we learnt from animal models about the development of the nervous system?
-Spinal cord motor neurons develop in the ventral part of the forming spinal cord and hindbrain in the posterior neuroaxis.
-To induce posterior spinal cord tissue, you have to induce neural tissue and then posterise it by a retinoic acid concentration
-Notochord and floorplate create shh which establishes a concentration gradient. Cells exposed to specific concentrations will develop into motor neurons.
What is motor neuron disease?
AKA Amyotrophic lateral sclerosis
A neurodegenerative disease where motor neurons die. Initiates muscle wasting due to lack of muscle stimulation. Swallowing and breathing become affected. The primary cause of nerve and support cell degeneration is unknown. Further research is needed to develop an effective therapeutic.
How did Wichterle et al (2002) cause motor neurons to become differentiated in culture?
From a fertilized egg, you have the trophectoderm and the inner cell mass. You can culture and suspense the inner cell mass to obtain embryonic stem cells, creating an embryoid body. Different embryoid bodies can be suspensed in different antibiotics. Exposure to retinoic acid can cause stem cells to express posterior markers which would usually display anterior markers.
After cells have been RA-treated, posteriorised embryoid bodies expressing Pax6, Nkx2.3 and Olig2 are exposed to a specific concentration of Shh causing them to differentiate into motor neurons.
How can you check that these culture grown motor neurons are bone fide motor neurons?
In embryos, motor neurons express the transcription factors Is11 and HB9. Treatment of embryoid bodies with RA/Shh causes induction of Hb9+ motor neurons. They also express additional properties that define motor neurons. This shows that you can effectively cuase stem cells to differentiate as real motor neurons.
What is the major issue growing motor neurons from embryoid bodies?
You wouldn't be able to transplant them back into a host. There would be a mixed population, only 10% would be motor neurons. Even with exposure to correct morphogens, you can't get a pure population of motor neurons. You also want to be able to track their activity.
How did Miles et al (2004) obtain a pure motor neuron population from embryonic stem cells?
-Find a motor neuron specific transcription factor, in this case HB9. Subclone the HB9 promoter upstream of GFP,
-Breed male and female transgenic mice. In pregnant mother, remove inner cell mass from embryo.
-Isolate ES cells from the HB9-eGFP transgenic mouse. Neuralise, posteriorise and treat the embryoid bodies with Shh to get motor neurons.
-Shine fluorescent light and MNs can be visualised.
-This allows you to follow MN in vivo or can select for a pure population of MN using a FACS machine.
-You can transplant MN which will become integrated into host chick spinal cord and take normal axon trajectories.
What are the short and long term goals for advances in motor neuron disease using the stem-cell differentiation of motor neurons?
-You can complete drug screens to find drugs specific to motor neurons
-Enable the derivation of human ES cells and their differentiation to motor neurons
-Push pluripotent cells to motor neuron fate ex vivo
-Find the differences between MN from pluripotent cells taken from a healthy person and a MND patient
-Establish which of the many integrated cell tyrpes in the nervous system goes wrong first in MND.
Make dishes of each cell type and recombine them to give healthy motor neurons.
What are stem cells and pluripotent cells?
Pluripotent cells can differentiate into any cell fate including germ cells. Stem cell also has the ability to self-renew as well as differentiate into specialised cells. Usually reside in a stem cell niche.
e.g. haematopoietic, skin, adipose stem cells etc
Important for development and homeostasis
When do stem cells first appear in mouse embryos and what do they do?
Day 0 egg gets fertilised. Day 4/5 embryo is implanted. Pluripotent stem cells are found in pre and post implanted epiblast. At day 6/7, cells begin to differentiate and specialise. Germ layers ectoderm, mesoderm and endoderm which lead to skin, CNS, PNS; blood, heart, kidney; liver and gut.
What define pluripotent cells in the early embryo?
To define these cells markers such as Nanog, Oct4 and Sox2 can be used in in situ hybridisation. Combined expression of these transcription factors defines pluirpotent cells.
If you graft cells into a recipient mouse, you give rise to tetratocarcinomas whereas non-pluripotent cells only give rise to small tumours or none at all.
What are the challenges associated with studying embryos and how can these be tackled?
Embryos are difficult to study because of their small size, in utero development and the ethical issues.
Can study them by capturing pluripotent cells in a petri dish and/or initiating in vitro modelling of embryonic development.
How can you capture pluripotent cells in vitro?
-Place ES cell sample on a layer of feeder cells (irradiated stromal cells derived from later ES cells which produce chemicals that support ES cell growth).
This causes the ES cells to self-renew.
-After they have divided a few times, disaggregate and re-plate them.
-Critical signals (Mouse: LIF, BMP; Human: FGF2, TGFB) required to maintain cells in self-renewing undifferentiated state by replacing the feeders.
How can ES cells contribute to normal development in mice?
You can reintroduce GFP-positive ES cells into normal blastocytes and they will contribute to normal development. You can then visualise the lineage of the stem cells by looking at GFP fluorescence with UV light.
How can you reprogram adult somatic cells to a pluripotent cell fate?
Take somatic cells from an adult and introduce and culture them in the presence pluripotency factors (Oct4, Sox2, cMyc, KIf4, Nanog) that encourage self-renewal.
How can you recapitulate normal development of cells in vitro?
Remove signals that keep stem cells self-renewing and add specific signals over time in combination to give rise to cell of interest.
The resultant cell must express the right markers (gene expression) and have the correct functionality.
This can be achieved in a 3D or a 2D manner.
How do you carry out 3D in vitro differentiation?
Remove the signals that keep the cells in an undifferentiated state (BMP/LIF for mice, FGF2/TGFB for humans).
Grow in aggregates (embryoid bodies) in presence or absence of signals.
Embryoid bodies contain many differentiated cell types.
Advantage: Recapitulates embryonic environment more accurately
Disadvantage: Difficult to observe/dissect the role of individual signals. Leaves lots to chance. Chaotic.
What is a Axin-LacZ reporter line?
Axin2 is turned on when Wnt signalling is active, particularly in the primative streak.
Lacz is cloned upstream of Axin2 to give blue colour when it is transcriptionally active.
This allows visualisation of stem cells which there is increased Wnt signalling.
What are cerebral organoids?
ES cells are cultured under the right conditions to produce cerebral tissue structures. Look for expression of the right markers at the right place. Recapturisation of neurons and neural precursors.
Cardiomyocytes that can beat can alos be generated from embryoid bodies.
How do you carry out 2D in vitro differentiation?
Plate a defined number of cells on the right subtrate/ECM.
Remove signals that keep cells in a undifferentiated state (BMP/LIF for mice, FGF2/TGFB for humans).
Grow in defined medium with appropriate amounts of signals (e.g. FGF, Wnt etc).
Advantage: More tractable system for live imaging, easier to test the role of specific signals.
Disadvantage: Loss of cell interactions that may occur in vivo.
What is Microcephaly?
Neurodevelopmental disorder in which infants are born with an abnormally small brain. Due to various autosomal recessive mutations.
Symptoms include neurological defects and seizures.
Mouse mutant failed to recapitulate the condition.
How can cerebral organoids be used to recapitulate microcephaly?
Take skin fibroblast from an affected individual which will carry the CDK5RAP2 gene. Using Oct4, Sox2, KIf4 and Myc, force the cells to become pluripotent stem cells to generate cerebral organoids. Create cerebral organoids from healthy patients using healthy skin fibroblast sample. It was found that there was a lower expression of markers and a small organoid was formed from the microcephaly patient.
This method has also been used in ALS, heart disease, Alzheimer's disease and Parkinson's disease.
How was Microcephaly linked to the Zika Virus Infection?
Increase in microcephaly cases linked it to Zika virus outbreak. Cerebral organoids were cultured with and without ZKV. ZKV+ organoid showed a smaller size due to cell death (Using Caspase 3 marker). Likely that Zika virus is killing fetal brain cells. Emricasan, a drug that blocks cell death, was added which decreased ZKV-induced cell death.
What are the benefits and challenges involved with using organoids to model human disease?
Fatehullah et al (2016) In vitro 3D organoid models facilitate an accurate study of a range of in vivo biological processes including tissue renewal, stem cell/niche functions and tissue responses to drugs, mutation or damage.
-Multiple genetic causes (e.g. autism spectrum disorder)
-Complex phenotypes (e.g. cleft palate)
-Late onset (e.g. Parkinson's disease)
-Lack of effecient differentiation protocols (e.g. blood cell lineages)
How is cell replacement being used in Parkinson's disease?
Fox et al (2014) Differentiated PSC-derived cells will facilitate treatment of many diseases.
PD is affecting 1 in 500. Symptoms include tremor, slowness of movement, rigidity, dementia, anxiety.
Progressive loss of dominergic neurons in substantia nigra.
Transplant h-ES-cell derived dominergic neurons into model of Parkinson's disease into the substantia nigra. They express tyrosine hydroxylase, an enzyme involved with the synthesis of dopamine. Cell replacement facilitates significant improvement in mouse models of PD, displaying reversal of symtoms.
What are the challenges involved with cell replacement?
Unsure whether you need progenitors or mature differentiated cells for transplantation.
Immune rejection/development of carcinoma.
Unsure whether repair occurs through replacement or regeneration.
Unsure whether positional identity is important and whether the cells are plastic.
What are mental health conditions?
Influence the health of the mind, body and nervous system.
10% of the global burden of disease.
Leading cause of disability worldwide.
Diseases include depression, anxiety disorders, schizophrenia, bipolar disorder, alcohol and drug use disorders and mental disorders of childhood.
There are huge treatment gaps for neuropsychiatric disorders, patients do not receive adequate or effective interventions. Used to face discrimination.
What do grand challenges research in the context of mental health?
Grand challenges allow for funding in specific areas of scientific research.
In the context of mental health, grand challenges need to...
-Identify modifible social and biological risk factors across the life course
-Collect global data and establish systems for collecting surveillance data
-Understnad adaptive, normative and resilient responses to daily life stress
What are GWAS?
Genome-wide association studies
Vast genomic data sets from vast numbers of individuals. Looking for small changes in base sequence (SNPs) and the major human disease traits in phenotype that this might correlate with. Used to identify risk genes that correlate with disease. This can then be modelled in animals.
What genetic investigation was used before GWAS?
Prior to GWAS, genetic risk factors were identified by analysing the genomes of families which are inbred. Looking for common themes between genome and phenotype.
How was DISC1 discovered?
'Disrupted in Schizophrenia 1' (DISC1) was first identified through genetic analysis of an inbred family with high-risk mental problems. Led to the conclusion that DISC1 gene is a genetic risk factor for schizophrenia and other mental health problems.
Social and environmental factors appear to act as triggers for the phenotype e.g. lack of sleep, stress, substance abuse.
Why are mental health issues so difficult to research?
There are many variables likely to be involved. The way they present is dependent on genetic basis and their environmental experience. Difficult to dissect mechanism and identify primary cause.
This is why it's important to using high numbers in data so that robust conclusions can be drawn (Zebrafish and Drosophila are useful).
How was location of DISC1 expression modelled in zebrafish?
In the zebrafish embryo, disc1 mRNA is first expressed in the developing hypothalamus. Furthermore, analysis of disc1 expression with other markers showed that disc1 is expressed in hypothalamic stem/progenitor cells, and incidentally is probably regulated by Wnt signalling.
What do we know of the link between the hypothalamus and the stress regulatory pathway?
Hypothalamus is an ancient, conserved, central regulator of homeostasis.
Through other work we know that hypothalamic stem/progenitor cells give rise to neurons that are involved in the 'stress regulatory pathway'. These neurons include Pomc, Sf1 and crh neurons.
Usually in response to stress, the hypothalamus releases corticotropin-releasing hormone, which causes the Pituitary gland to release adrenocorticotrophic hormone which causes the adrenal gland to release cortisol into circulation initiating a stress response.
Can we sustain our stress response?
Cortisol release in response to stress affects the whole body (heart, liver, muscles etc) which induces a high-alert pathway. This CANNOT be sustained so normally there is a negative feedback loop to prevent the over-release of corticotrophin-releasing hormone and adrenocorticotrophic hormone, thereby reducing cortisol release.
What were the zebrafish disc1 mutants that were found?
2 point mutations LII5 and Y472
No full length mRNA or protein
What did Eachus et al (2017) find about the consequences of the zebrafish disc1 mutations?
Eachus et al (2017)
Disc1 mutants show a less well pronounced T-shape in the hypothalamus. Stem and progenitor cells differentiate prematurely and exhaust their supply. Hypothalamic neurons especially those involved with stress homeostasis differentiate abnormally.
In Disc1 mutant there are fewer Pomc neurons but more sf1 and crh neurons, giving the wrong balance. This data was generated from large numbers of zebrafish and analysis of whole hypothalamic region.
How did Eachus et al (2017) test Disc1 mutant zebrafish for their stress response pathway?
Larval and adult fish showed an abnormal stress response as measured through behavioural assays and neuroendocrine function.
It is known that NaCl and Schreckstoff (fish-produced chemical that alerts other fish to danger) can be used to initiate a stress response which is measured with a visual tracker camera. In a disc1 mutant there is no stress response.
You can then measure cortisol levels after the stress response. Cortisol and ff1b (sf1) upregulated in WT alarmed fish but not in disc1 mutants. There was no change in cortisol levels in mutants.
This suggests that in Disc1 humans, there is failure to mount a proper stress pathway. Unknown whether this is a cause of bipolar disease.
What determines whether we are asleep or awake?
Current models suggest it is a function of two interacting processes: a poorly understood sleep homeostatic drive (sleep load) and a better-understood circadian sleep.
What is sleep load?
Sleep load is low after waking up and high just before sleeping.
Asleep: when there is greatest difference between sleep load and circadian clock
Awake: when these two coincide
What did Hastings (1998) say about what the circadian clock is?
Circadian rhythms are 24 hr rhythms in physiology and behaviour generated by molecular clocks (counting 24 hours in cells) which serve to coordinate internal time with the external world. Life has evolved around day (warmth, sunlight, UV) and night (none of the above).
Circadian clocks can anticipate the regular 24hr environmental changes and establish endogenous 24hr rhythms to ensure appropriate/optimised output to the time window each day.
What are the bodily differences between day and night?
Daytime: Fastest increase in BP, high alertness, best coordination, fastest reaction times, highest body temperature
Nightime: Highest BP, Melatonin secretion, Deep sleep, Lowest body temperature
How are the molecular clocks of individual cells controlled?
Most cells in the body possess a molecular clock and are maintained in synchrony by a master clock located in the suprachiasmatic nuclei of the hypothalamus.
Regulates plasma melatonin, core body temperature and plasma cortisol, all of which regulate major body function.
What controls the circadian clock genetically?
Thousands of clock-controlled genes that orchestrate the oscillation of tissue-specific metabolic and physiological functions. Between 2-30% of each tissues transcriptome displays a circadian rhythm.
How is circadian rhythm being linked to mental health?
KEY RESEARCH Q: Do sleep and circadian rhythm disruption due to mutations in circadian and clock-controlled genes or social health reasons contribute to the development of a range of disorders including mental illness?
What are the mechanisms underlying circadian regulation?
Hall, Rosbash and Young (Nobel Prize 2017)
Hall, Rosbash and Young (Nobel Prize 2017)
Cell autonomous transcription-translation feedback loops. Transcription factors e.g. CLOCK, CLK, BMAL1 drive the expression of other genes e.g. Period (per1/2) and Cryptochrome (Cry1/2). After time for transcription and translation has passed, their protein products in turn feedback to inhibit CLOCK and BMAL1. This leads to less Period transcription, less Period protein, breaking the cycle,
This cycle takes 24 hours. Many more genes have been discovered through drosophila then mouse.
Environmental inputs can entrain the clock. For instance, light can activate the protein product of the cryptochrome cry gene and promote its binding to TIM, leading to its degradation in the proteosome.
What is the evidence for intrinsic clock disruption in mental health disorders?
Roybal et al (2007) Landgraf et al (2016) Wulff et al (2012) Lamont et al (2007)
Patients with neuropsychiatric diseases such as bipolar disorder, schizophrenia and depression exhibit sleep and circadian rhythm disruption. These patients show dysregulation of multiple circadian outputs and of the core molecular clock.
Fibroblasts isolated from schizophrenic patients show a loss of rhythmicity in CRY1 and PER1 expression and theur peripheral blood leukocytes have decreased and/or disrupted diurnal expression of CLOCK/PER1/2/3/CRY1 and a functional clock homologue of NPAS2 in comparison to healthy controls. GWAS and SNP analyses show that gene encoding the core components of the molecular clock have been associated with mental health disorders.
Is disruption of the molecular clock cause or consequence of mental health disorders?
Many animal model studies show that disruption of the molecular clock are not just consequences of neuropsychiatric illness but instead form a part of a directional feedback loop with neuropsychiatric disease perturbations in one exacerbate in the other.
Do social/behaviour aspects lead to sleep/mental health problems?
Intrinsic clock has evolved over millions/billions of years to be in perfect synchrony with the 24hr extrinsic light-dark clock. If this is lost, does it affect hormonal/physiological events and consequently health?
How is the intrinsic clock involved with metabolism as a key physiological process?
As the mechanisms behind the intrinsic clock become clearer, elucidates the link between the clock and a physiological process like metabolism. There are metabolic target genes under the control of the clock. This is helping us to understand the central role of the clock genes in many aspects of disease including metabolic syndrome, neurodegenerative diseases and immune system diseases.
What is Melatonin?
Ubiquitous in nature - phylogenetically ancient in biological signalling mechanisms.
Primary function is a potent antioxidant, but also involved in circadian rhythm regulation (produced in pineal gland), sleep propensity, blood pressure regulation, immune function, detoxification of free radicals and regulation of bicarbonate secretion in GI tract.
Why is Melatonin a better anti-oxidant than most?
Devoid of pro-oxidant side effects
Scavenging of free-radicals
Upregulation of antioxidant enzymes
Direct inhibition of free radical formation
How is melatonin linked to mental health disorders?
Animal models suggest that it is critical to clear free radicals and that failure to do so can cause mental health and neurodegenerative diseases. Could be a potential therapeutic. Incidentally many traditional chinese medicine contain melatonin used for age-related diseases.
How is core clock machinery involved with chemotherapy?
Kang et al (2009) and Sancar et al (2015)
Clock evolved to help organisms protect themselves against UV-induced DNA damage. Particular molecules involved with DNA repair are expressed at highest levels in late afternoon and lowest at night. Chemotherapy works by promoting DNA damage of cancer cells. Theory is to give chemo when body is least able to function optimally in repairing this damage.
Which drugs require which receptors?
Opiates: u opoid receptors
Nicotine: acetylcholine receptors
Cannabis: cannabinoid receptors
Alcohol: major being GABA (gamma-aminobutyric) and NMDA (N-methyl-d-aspartate) receptors
Drugs of abuse: act secondarily through dopamine and mesolimbic 'reward' system
What is the most harmful drug?
In 2013, 8,500 alcohol-related deaths in the UK
Huge social and healthcare burden
What is the dopamine and mesolimbic 'reward' system?
Positive reinforcing of natural rewards: food, sex and social interactions release dopamine from ventral tegmental area (VTA)
What are the contributing factors to drug addiction?
-Genetics: sensitivity and development of tolerance
-Social environment: past and present
-Personality and personal history e.g trauma
Are drosophila a good model of drug addiction?
We share about 500 million genes. 50% of Drosophila genes are present in humans. 75% of human genes implicated in disease are present in drosophila.
Develop quickly: 10 days at 25C, 25 days at 18C
Have catecholamine reuptake transporters, nicotinic AchR, ligand and voltage gated ion channels and G protein activated K channel.
GABA, NMDA, and dopamine receptors.
No opoid receptors or cannaboid receptors.
How does the "booze-o-mat" work?
Uses a container with drosophila in, where the air is infused with ethanol fumes. The movement in the flies is recorded by a camera with and without ethanol. With ethanol, there is increased locomotion before they become unconscious. The time between high activity and sedation can be used to determine a fly's sensitivity to the ethanol.
How does the "inebriometer" work?
A chamber which within it contains a series of steps, through which the drosophila can move. Air infused with ethanol can be released. When the flies are sober they can easier move up and down the steps. When they are too drunk they fall out the bottom. This can be used to measure elution time for flies.
Sensitive flies will elute faster, the more resistant will stay in for longer.
Mutant and transgenic flies exist for almost every gene in Drosophila genome (can irradiate them to induce mutagenesis), these behaviours can be linked to specific genes involved.
What are the 2 independent genetic factors contributing to drug addiction?
Sensitivity and development of tolerance
How did Schuckit (1994) link ethanol sensitivity with future alcoholism?
Low sensitivity to modest doses of ethanol are associated with a significant increase in the risk of future alcoholism, increasing the chances a person will drink more heavily and more often.
What did Moore et al. (1998) discover about the cheapdate mutant?
A mutant discovered in drosophila using an inebriometer that induces a higher sensitivity to alcohol. When ethanol is supplied to both chambers, one with WT and the other 'cheapdate', cheapdate flies become sedated very quickly.
Cheapdate is an allele of amnesiac, a neuropeptide that activates the cAMP pathway. Neuropeptide binds to receptor, which activates heterotrimeric G protein complex. Ga subunit then exchanges GDP for GTP and is released from the complex. Then it binds to adenylyl cyclase which converts ATP to cAMP leading to activation of PKA. PKA phosphorylates transcription factors including CREB, changing gene expression.
How were other mutations found in the drosophila cAMP pathway which affect alcohol sensitivity?
Rutabaga is the adenylyl cyclase in the cAMP pathway, DCO is the PKA catalytic subunit and dunce is the cAMP phosphodiesterase which inactivates cAMP.
Mutations in rutabaga and DCO similar to cheapdate, giving drosophila lower sensitivity. Therefore, this shows that the activity of cAMP pathway decreases alcohol sensitivity.
What is the development of tolerance?
Tolerance is acquired resistance to the physiological and behavioural effects of the drug. May contribute to addiction.
Normal after the first exposure, time before sedation is 20 mins, after a second time it will be increased. Higher doses are needed to achieve the same outcome.
How was development of tolerance measured in drosophila?
Using the "inebriometer" expose the flies to ethanol and then allow them to rest for a couple of hours. Expose them a second time and measure their elution time in both cases. Experiment shows that after second exposure they have a greater time before sedation occurs, showing that they are less sensitive to ethanol after a second exposure.
What is hangover mutant?
In Hangover mutants there is no hangover protein and the drosophila have reduced tolerance.
What is the hangover protein?
Hangover encodes a Zn-finger protein which binds RNA of dunce (cAMP phosphodiesterase that activates cAMP) and stabilises it. More stable RNA, more translation, more protein. Increase in dunce protein, moves out of the nucleus. Dunce inhibits hangover. Likely to be expressed in the brain, visualised using in situ hybridisation.
Balance between 'hangover' and 'dunce' expression determines the degree of tolerance.
What is aging?
Progressive, irreversible decline in organismal performance. Nobody dies of old age, aging leads to the decline of one or more of the following systems resulting in malignant cancers, heart and lung problems, Alzheimer's or Parkinson's.
Can we prevent aging?
Maximum lifespan of a human is thought to be 125 years old.
It is possible to increase lifespan if we know the factors influencing ageing...
-Environment - diet, lifestyle, exposure to external factors
-Genetics - genotype at birth and accumulation of mutations
What does a model organism for aging need to be?
Small, easy to culture
Short lifespan - drosophila (2 months), mice (2-3 years)
Controllable environment (food intake)
Genetic screens and short lifespan makes drosophila a powerful tool for modelling aging.
How can we measure aging?
No direct method of measuring ageing. We measure age at death and use demographics to gather large cohorts of identical animals to compare and analyse median lifespan.
What is the insulin pathway?
Insulin is a hormone that promotes the uptake of glucose from blood for storage. Insulin binds to insulin receptor which induces a cascade of activation. Chico becomes activated which activates PI3 Kinase, converting PIP2 to PIP3. This activates Protein Kinase B. PKB inhbits FOXO which inhibitis cell number or activates SSk which increases cell size.
How did Clancy et al (2001) link the insulin pathway to aging?
First protein found to regulate lifespan was Chico.
Half dosage of chico was given to males and females separately because they have different lifespans. They saw that the drosophila had increased lifespan.
They then mutated/overexpressed different genetic components surrounding Chico.
dPTEN inhibits activity of insulin pathway, so overexpression dPTEN leads to inactivation of the insulin pathway.
What conclusions were made about how insulin controls life-span?
Decreased activity of the insulin pathway/insulin growth factor/mTOR leads to increased activity of FOXO, SIRT AMPK which leads to increased autophagy and DNA repair, decreasing the amount of oxidative stress. This leads to increased health and longevity.
What is the link between dietary restriction and lifespan?
Oldest known proven method to extend lifespan. Modest dietary restriction extends lifespan across species. Too little or too much decreases lifespan, whilst intermediate increases lifespan in drosophila. It is not required throughout life, dietary restriction reduces mortality risk at any time of life.
Also true for mice. CALORIE (Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy) is the current research into humans.
How does dietary restriction increase life-span?
Dietary restriction increase HSF/HSP70 activity, decreased insulin and inflammation which all lead to increased autophagy and DNA repair, reducing oxidative stress. Reduced inflammation also reduces risk of cancer. DR can also reduce adiposity and increase lipid profile to decrease risk of CV disease. All of these things increase health and longevity. Therefore, dietary restriction can improve healthy aging by reducing prevalence of age-related disease.
How is the population aging?
In all countries, the average age in population is increasing. Average age in UK in 1960 was 71 years old, but is now 80 years old in 2010. 18% of people were over 65 in 2016, predicted to be 24% in 2040. Age is the main risk factor for death and all the main diseases.
What is Alzheimer's disease and how are Drosophila involved?
Neurodegeneration, neuronal cell death and tissue atrophies due to accumulation of B-amyloid peptide.
Overexpression of B-amyloid peptide in Drosophila is neurotoxic. This makes it possible to screen for genes that reduce neurotoxicity using drosophila.
How are drosophila used to screen for genes that can cause Alzheimer's?
Using a UAS/Gal4 system. A promoter of a gene expressed in a particular tissue regulates the expresion of Gal4. In another fly, a construct is made with UAS upstream of the gene of interest, in this case B-amyloid. The flies are bred together and in the offspring, when Gal4 is produced it will bind to UAS and the expression of B-amyloid will be increased.
Overexpression of drosophila insulin degrading enzyme (IDE) showed reduced levels of B-amyloid and increased lifespan.