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Glaucoma treatments; are new developments hiding in plain sight?

Written by Olivia Kingston

Glaucoma is the leading cause of irreversible blindness worldwide (1). As patients age, cells in the eye gradually deteriorate, causing patients to eventually go completely blind. Scientists at Liverpool University believe that by replacing these non-working cells with working ones, vision loss may be prevented!

What’s happening in glaucoma?

how glaucoma works
Figure 1. Pathophysiological changes in the eye as a result of elevated pressure in glaucoma. A schematic diagram showing how elevated intra ocular pressure in glaucoma puts pressure on the lamina cribrosa, causing tissue deformation and damage to optic nerve axons. Image based upon description and diagrams in Quigley, 2011.

Our eyes are filled with fluid, known as aqueous humour, that is constantly filtered by a tissue called the trabecular meshwork (2). In glaucoma, the cells that make up this tissue decrease in numbers and those remaining don’t function as they should do (3). This prevents the fluid from moving through the meshwork as it becomes stiffer and blocked by debris, causing pressure in the eye to increase (4). This pressure damages axons of the optic nerves preventing signals that contain visual information being sent to the brain (5).

Treating Glaucoma Today

Currently glaucoma treatments involve laser treatments or surgery to create channels for the fluid to drain out of the eye and reduce pressure, which unfortunately aren’t always effective (6). If the lost cells of the trabecular meshwork in glaucoma patients could be replaced with healthy cells, then the trabecular meshwork may be able to function normally and regulate pressure in the eye.

schematic diagram illustrating the pathway of aqueous humour. Important in regulating pressure in the eye.
Figure 2. The main aqueous humour outflow pathway via the trabecular meshwork into Schlemm’s canal. A schematic diagram showing the outflow pathway of aqueous humour. Aqueous humour is created at the ciliary body and flows into the anterior chamber and through the trabecular meshwork. Image and information from in Goel et al., 2010.

Hiding in plain sight?

The problem lies in how to develop these working cells and get them to where they need to be? Well, recent scientific discoveries find that these cells may be “hiding in plain sight”.

By changing the environment in the trabecular meshwork, we may be able to make use of cells already present in the eye, that have a unique ability to develop into specialised cell types, stem cells (7). In the right conditions, these stem cells can be encouraged to grow into healthy and functioning cells that could aid in aqueous humour outflow. What scientists need to know, is what changes are needed to make this happen…and that’s what is currently being investigated at Liverpool University!

Think of it like gardening. Flower seeds buried deep in dry and old soil, won’t blossom anytime soon. But if you replace the soil with fresh, nutritious compost and plenty of water you’ll have a flourishing garden. By finding the right compost and supplying water, scientists can replace non-working cells with healthy ones, without having to transplant new cells into patients.

Olivia Kingston is a PhD student studying glaucoma.

References

  • Liu, B. et al. (2018) ‘Aging and ocular tissue stiffness in glaucoma’, Survey of Ophthalmology. Elsevier USA, pp. 56–74. doi: 10.1016/j.survophthal.2017.06.007.
  • Tamm, E. R. (2009) ‘The trabecular meshwork outflow pathways: Structural and functional aspects’, Experimental Eye Research. Academic Press, pp. 648–655. doi: 10.1016/j.exer.2009.02.007.
  • Liton, P. B. et al. (2005) ‘Cellular senescence in the glaucomatous outflow pathway’, Experimental Gerontology. NIH Public Access, 40(8–9), pp. 745–748. doi: 10.1016/j.exger.2005.06.005.
  • dysregulation in glaucoma’, Experimental Eye Research. Academic Press, pp. 112–125. doi: 10.1016/j.exer.2014.07.014.
  • Quigley, H. A. (2011) ‘Glaucoma’, The Lancet, 377(9774), pp. 1367–1377. doi: 10.1016/S0140-6736(10)61423-7.
  • Weinreb, R. N., Aung, T. and Medeiros, F. A. (2014) ‘The pathophysiology and treatment of glaucoma: A review’, JAMA – Journal of the American Medical Association. American Medical Association, pp. 1901–1911. doi: 10.1001/jama.2014.3192.
  • Yun, H. et al. (2016) ‘Stem cells in the trabecular meshwork for regulating intraocular pressure’, Journal of Ocular Pharmacology and Therapeutics. Mary Ann Liebert Inc., 32(5), pp. 253–260. doi: 10.1089/jop.2016.0005.
  • Goel et al. (2010) ‘Aqueous Humour Dynamics; A Review’, The Open Opthamology Journal. 4(1) pp 52-9. doi: 10.2174/1874364101004010052.

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Could Taking the Pee be the Future of Understanding Rare Genetic Kidney Diseases?

Written by Rebecca Dewhurst @becky_dewhurst from the @SayerLab

How can urine help us understand kidney diseases?

Everyday thousands of healthy kidney cells are shed into our urine. Under the right conditions, these cells, known as urine-derived renal epithelial cells, or hURECs for short, can be collected as a liquid biopsy, and used to study a wide range of rare genetic kidney diseases without a traditional painful invasive biopsy.

isolating kidney cells from urine to study kidney diseases
Figure 1. Simplified method of isolating and culturing human urine-derived renal epithelial cells (hURECs).

What do the Kidneys do?

The kidneys are bean shaped organs found towards the back of the upper abdomen involved in the ultrafiltration of our blood. Our kidneys are vital in maintaining appropriate water levels, and produce waste products like urea which is excreted in urine.

Who is affected by kidney disease?

In the UK alone, one in ten people have Chronic Kidney Disease (CKD); this equates to around 3 million patients in total (1). There are a number of reasons for the onset of kidney disease, including poor control of blood sugar levels in diabetes, high blood pressure or inherited causes.

What diseases can we study using these cells?

In our Newcastle University Renal Genetics group, we predominantly study a group of diseases known as ciliopathies, which occur due to cilia defects. Found on the surface of almost all cells, cilia are finger-like protrusions acting like radio antennae feeding information back to the control centre of the cell, known as the nucleus. Ciliopathies can affect a number of organ systems, including the eyes, brain, liver and kidneys. Examples of ciliopathies affecting the kidneys include Joubert Syndrome, Oral-facial-digital Syndrome, Nephronophthisis and Autosomal Dominant Polycystic Kidney Disease, all of which we can study using hURECs.

Figure 2. Basic structure of a primary cilium. Cilia are finger-like protrusions which are found on the surface of almost all cells, and are involved in key cellular signalling processes. Ciliopathies can result in extra-long, short, or curly cilia. Figure adapted from (2).

How have these cells been used?

For many years, the Newcastle University Renal Genetics group we have been using hURECs to further understand how and why kidney disease develops in ciliopathies like Joubert Syndrome (3; 4) and Nephronophthisis (5). These hURECs allow us to investigate both the phenotype (characteristics that we can see) and the genotype (the genetic blueprint including genes and DNA) which are involved in renal ciliopathies, giving much desired answers to patients and their families.

Remarkably, hURECs have been used in our hands to generate 3D cell models known as organoids which can be considered as ‘kidneys in a dish’. These renal organoids, known as tubuloids (6) or nephrospheres (7), have allowed for more complex kidney disease modelling.

Figure 3. Images of human urine-derived renal epithelial cells (hURECs) from Wild Type and Joubert Syndrome urine samples. Cell nuclei are shown in blue with cilia shown in green. Image taken, with permission from  (4).

What does this mean for patients?

One of the main benefits of using hURECs is that we can gain a kidney organ specific snapshot of how the renal cilia are affected, which can sometimes be missed using other cell types like fibroblasts, which are derived from skin biopsies. Urine sample collection in order to grow hURECs is also quick, easy and most importantly pain free, meaning multiple samples can be taken.  Exciting opportunities in the development of patient-specific hUREC generated disease models highlight why urine samples, a waste product, may hold the key to developing our understanding of kidney diseases.  

References

1. Kidney Research UK. Annual Reports and Accounts. Kidney Research UK. [Online] 2020. [Cited: 26 06 2020.] https://kidneyresearchuk.org/about-us/annual-reports/.

2. Ciliopathies: an expanding disease spectrum. Waters, A M and Beales, P L. 7, 2011, Pediatric Nephrology, Vol. 26, pp. 1039-1056.

3. A human patient-derived cellular model of Joubert syndrome reveals ciliary defects which can be rescued with targeted therapies. Srivastava, S, et al. 23, 2017, Human Molecular Genetics, Vol. 26, pp. 4657-4667.

4. Targeted exon skipping of a CEP290 mutation rescues Joubert syndrome phenotypes in vitro and in a murine model. Ramsbottom, S A, et al. 49, 2018, PNAS, Vol. 115, pp. 12489-12494.

5. Human urine-derived renal epithelial cells provide insights into kidney-specific alternate splicing variants. Molinari, E, et al. 2018, European Journal of Human Genetics, Vol. 26, pp. 1791-1796.

6. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Schutgens, F, et al. 2019, Nature Biotechnology, Vol. 37, pp. 303-313.

7. Urinary nephrospheres indicate recovery from acute kidney injury in renal allograft recipients – a piolet study. Knafl, D, et al. 251, 2019, BMC Nephrology, Vol. 20.

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‘Humanised’ worms for the discovery of new anti-epileptic drugs

Written by Lucy Job: https://www.linkedin.com/in/lucyjob/

Caenorhabditis elegans is a type of tiny soil-dwelling worm, found to accept certain human genes into their own genome [1]. These ‘humanised’ worms are a very useful tool to study human disease, with researchers at the University of Liverpool now using them to study human epilepsy and discover new anti-epileptic drugs [1] [2].

“C. elegans, model organism in life sciences” by ZEISS Microscopy

What is epilepsy?

Epilepsy is one of the most common conditions affecting the brain, shaping the lives of an estimated 50 million people worldwide [3]. In the UK alone, there are an estimated 600,000 epilepsy patients, which is almost 1 in every 100 people, with 87 people diagnosed every day [4]. There are a lot of different causes of epilepsy, and it can affect people of all ages [5].

Known causes of epilepsy, adapted from the World Health Organisation Infographics of Epilepsy [5]

Those with epilepsy suffer from recurrent seizures, which is caused by sudden bursts of electrical activity within the brain. The most well-known seizure type affects movement, causing muscle jerking, twitching, and/or weakness. Lesser known seizures affect the person’s awareness to their surroundings and can cause changes in sensation or emotion [6].

Currently, there are over 20 anti-epileptic drugs available with many different drug targets [7]. These drugs try to stop the sudden uncontrolled bursts of electrical activity within the brain. However, these anti-epileptic drugs do not work for one third of patients, named ‘refractory epilepsy’, with the reason for this not fully understood [8]. Therefore, new drugs are desperately needed for those patients with refractory epilepsy.

Why worms for drug development?

Making new drugs is controversial as testing on mammals, like mice and primates, has many ethical and financial issues. As these worms are invertebrates, less complex animals, they do not require extensive ethical training for use [9]. Also, these worms have a short reproduction time (~3 days) and lifespan (~3 weeks) and do not need expensive equipment take care of them, making them a much cheaper alternative [10]. Therefore, it is beneficial to test new drugs on less complex animals first, and then move onto more complex animals later in the drug development process. However, it is hard to justify using tiny soil-dwelling worms to test drugs for humans when we are so different [9].

Humanising the worm

To tackle this, researchers led by Professor Alan Morgan at the University of Liverpool have created humanised worms to study severe forms of epilepsy, such as Ohtahara syndrome [1]. This was achieved, in part, using the highly specialised gene editing process CRIPSR-Cas9 and which is used to insert, delete, or substitute target genes, to fix or introduce gene mutations [11].

Brief summary of CRISPR-Cas9. The Cas9 enzyme cuts the target genetic code, causing a DNA break. Once broken, a DNA repair process, leads to the desired insertion, deletions, or substitution at the target site.

GABA is a neurotransmitter (a biological chemical) which can prevent sudden electrical activity in the human brain. Studies by multiple different research teams have found GABA mutations (more specifically GABAA receptors) in patients with a spectrum of epileptic encephalopathies, such as Lennox-Gastaut syndrome and West syndrome [12].

In this project we aim to introduce mutated human GABAA receptor genes found in epilepsy patients into worms. We will then induce seizures in the worms and test a range of new anti-seizure drugs to see which compounds are most effective. Therefore, we will be able to assess the effect the new anti-seizure drugs may have in humans with the same genetic mutations.

Humanised worms used to study anti-epileptic drugs at the University of Liverpool.

Using these humanised worms as a first step in drug development is beneficial as it gives researchers the opportunity to fast-track the selection process, whittling down large numbers of drug candidates to a select few in a short space of time [2].

References

1.              Zhu, B., et al., Functional analysis of epilepsy-associated variants in STXBP1/Munc18-1 using humanized Caenorhabditis elegans. Epilepsia, 2020. 61(4): p. 810-821.

2.              Wong, S.Q., et al., A Caenorhabditis elegans assay of seizure-like activity optimised for identifying antiepileptic drugs and their mechanisms of action. J Neurosci Methods, 2018. 309: p. 132-142.

3.              Organization, W.H., Epilepsy: a public health imperative. 2019: World Health Organization,.

4.              Action, E. What is epilepsy? 2019 2019 [cited 2020 29.06.2020]; Available from: https://www.epilepsy.org.uk/info/what-is-epilepsy.

5.              Organization, W.H. Infographics on epilepsy. 2016-2017  [cited 2020 29.06.2020]; Available from: https://www.who.int/mediacentre/infographic/mental-health/epilepsy/en/.

6.              NHS. Symptoms – Epilepsy. 2017  [cited 2020 29.06.2020]; Available from: https://www.nhs.uk/conditions/epilepsy/symptoms/.

7.              Loscher, W., et al., New avenues for anti-epileptic drug discovery and development. Nat Rev Drug Discov, 2013. 12(10): p. 757-76.

8.              Kwan, P. and M.J. Brodie, Early identification of refractory epilepsy. N Engl J Med, 2000. 342(5): p. 314-9.

9.              Cunliffe, V.T., et al., Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure, 2015. 24: p. 44-51.

10.            Chen, X., et al., Using C. elegans to discover therapeutic compounds for ageing-associated neurodegenerative diseases. Chem Cent J, 2015. 9: p. 65.

11.            Zhang, F., Y. Wen, and X. Guo, CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet, 2014. 23(R1): p. R40-6.

12.            O’Reilly, L.P., et al., C. elegans in high-throughput drug discovery. Adv Drug Deliv Rev, 2014. 69-70: p. 247-53.

13.            Hernandez, C.C. and R.L. Macdonald, A structural look at GABAA receptor mutations linked to epilepsy syndromes. Brain Res, 2019. 1714: p. 234-247.

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