Cellular responses to DNA damage: from molecular insights to new medicines

Speaker: Steve Jackson
Department: Gurdon institute, University of Cambridge
Subject: Cellular responses to DNA damage
Location: Erasmus MC Rotterdam

Date: 27 September 2016
Author: Gabriele Kockelkoren

On average, 2×1021  lesions occur per day in each person. These lesions can be caused by errors in the replication pathway or exposure to radiation. Luckily, cells have mechanisms to repair the lesions. Different types of lesions can be distinguished: single-strand breaks (SSBs), double-strand breaks (DSBs), insertions, deletions and mismatches. These can all be repaired by different kinds of DNA repair mechanisms. Each mechanism is dedicated to the reparation of a certain lesion. In most cases the DNA is repaired rapidly and with high precision/accuracy. These pathways are very fascinating to work on, but we also know that defects in the pathways give rise to a wide range of pathology. These errors may lead to the development of cancer, premature aging, infertility or immune-deficiency. In addition to DNA repair by protein complexes, the cell reacts on the repair machinery by induction of repairing factors, transcriptional regulation, cell cycle control and apoptosis or renascence. This cascade of reaction is known as the DNA damage response (DDR).

Steve Jackson from the University of Cambridge is interested in studying the moving field of DNA damage repair, especially from a therapeutic perspective. Nowadays, the field of DNA damage and its repair is utmost interesting, as many new tools and techniques are being developed which have never been used before. These tools give us new insights in the cellular machinery and its complex behaviour. Almost 500 proteins are directly connected to the repair system. This gives in advantage in treatment for pathologies caused by defects in these pathways, as there are many druggable targets to inhibit the various DNA repair systems.

Genes work together with other genes and they are connected to the phenotypes and affect diseases. Phenotypes caused by a gene defect are strongly affected by the actions of other genes and by environmental factors. If we think about genetic interactions we see that some genes have few interactions, while others have high interactive abilities in different biological parts, while being involved in separate pathways. This gives a strong distinction between genetic enhancers (synthetic lethality) and suppressors (synthetic viability).

In 1997 Steve Jackson founded his own company, named KuDOS. The main goal of the company was to create inhibitors for the DNA repair pathways. By inhibiting the DNA repair pathways you:

  1. Enhance efficacy and reduce side-effects of radiotherapy and chemotherapy.
  2. Create selective activity on cancers by targeting differences between cancerous and normal cells.

The most promising compound which has been developed by KuDOS is a poly ADP-ribose polymerase (PARP) inhibitor called olaparib/LynparzaTM. PARP is a DNA damage activator enzyme, as PARP promotes single-strand break repair and base excision repair. By inhibiting PARP, the repair is slowed down and the PARP molecule remains trapped on the DNA. The inhibition of PARP in healthy cells for a short period is not deleterious, however cancer cells are very sensitive to the inhibition of PARP. Steve Jackson used the BRCA1 and BRCA2 mutations as an example. Cancer cells with these mutations are not able to repair DSBs and by addition of the inhibition of PARP, the cells will be exposed to too much damage. As they are not able to repair this, cell death is the final option.

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Figure 1: Suppressing PARP activity prevents SSB repair via the BER pathway, but other DNA repair pathways such as HR and NHEJ simply take over. However, if PARP inhibitors are used against tumours in which there is already a DNA repair defect, the combination drives synthetic lethality. Source: http://www.onclive.com/publications/oncology-live/2013/august-2013/new-life-for-parp-inhibitors-emerging-agents-leave-mark-at-asco#sthash.Prd7k8XI.dpuf

In December 2014 the use of Olaparib has received the European and FDA approval for ovarian cancer associated with BRCA1/2. This is the first in class drug targeting PARP. KuDOS is working on other potential drugs for clinical trial.

It is very interesting to see how an idea can grow into a successful business, where it all starts in the lab. Steve Jackson also showed us the difficulties of being a scientist and proving that your method, is the one working for many patients.

 

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Synaptic Origins of Cerebellar disease

Speaker: Roy Sillitoe
Department: Jan and Duncan Neurological Institute, Baylor College of Medicine
Subject: Synaptic Origins of Cerebellar disease
Location: Erasmus MC

Date: 6 June 2016
Author: Gabriele Kockelkoren

In the spirit of sharing unpublished work, Roy Sillitoe shares his knowledge on the cerebellum synapses. One of the models he is intrigued is, is how in the cerebellum it is all wired up. What are the electro physical consequences of this wiring and what happens when the wiring is not according to plan? Cerebellar damage causes serious consequences during development, which turn out to be catastrophic. Roy Sillitoe looks into these cellular and circuit consequences. How does this relatively simple circuit give rise to such great variety of diseases? From a molecular perspective, it is not as easy as it may look like, as the cerebellum is wired into highly complex topographic circuits that control behaviour.

The cerebellum is a region in the brain, which is involved in coordination, fine-tuning and motor learning. An important cell type and the most dominant one in the cerebellum, are Purkinje cells. These neurons have parallel and climbing fibers through which they receive input. The inferior olive provides the cells with input. Purkinje cells have two types of action potentials: single and complex. In this way the Purkinje cells provide feedback for motor coordination.

Over the years, his group has been intrigued in the effects within topographic zones and effects on the cerebellar circuits, when confronted to damage. To understand these connections how these connections develop, they use mouse models. In the lab they try to target the synapses.

In the time Roy Sillitoe has worked in Oxford, he tried to understand the human genetic disease Dystonia. Patients with Dystonia present involuntary sustained muscle co-contractions. This disease can be inherited or acquired. In Oxford he studied the effect of mutated proteins. These proteins incorrectly move throughout the cell, which is seen as the most probable outcome for the disease. Most of the mouse models that are based on the human genetic studies, do not display dystonic postures. However, mouse models were very informative to his group, as Sillitoe and colleagues used a mouse who displays Dystonia. Connectivity between circuits was severely reduced in mice which had Dystonia. So the conclusion could be drawn that there was something wrong at the circuit level. Recent evidence suggests that the cerebellum is not only involved in Dystonia, but it can instigate Dystonia as well.

cerebellum

Figure 1:The inferior olive inputs sensory information to the cerebellum. The Purkinje cells communicate the output. Source: http://www.neuronbank.org/wiki/index.php/Inferior_Olivary_Neurons

Sillitoe and his colleagues have been wondering whether the disease is caused by errors in the input signals. However, trying to manipulate cerebellar connections is not a trivial task. The inferior olive has a powerful and unique excitatory connection to the cerebellum. This excitation can be tracked by measuring activity of the inferior olive. This activity results in spikes of potential. Sillitoe wants to answer the question: Can we use the inferior olive to cerebellum circuitry as in inroad for designing a more flexible model for understanding dystonia? The goal is to generate a mouse model that exhibits Dystonia and understand what is happening in such mice.

They started with the hypothesis that selectively silencing developing olivo-cerebellar synapses will cause circuit malformations that induce Dystonia in mice. However, cutting the olive is not possible, as this would be catastrophic for the development of the mouse. It is however difficult to target the inferior olive because its gene expression pattern overlaps with connected regions. Therefore they have been working with the genes Ptf1a and Vglut2, which are heavily expressed in inferior olive and cerebellum. This way they made use of an unique intersection of gene expression in the inferior olive. This caused the inferior olivary neurons to release empty vesicles and thus no response is triggered. This created mice exhibiting symptoms of Dystonia. The mice were studied using electromyography (EMG), measuring local field potential (LFP) and single-unit recordings.

As a way to cure the disease, the Sillitoe lab has been looking at DBS (Deep brain stimulation) and saw that for a short period of time, the mouse regained normal movement. However, as soon as the effect stopped, the mouse showed Dystonia symptoms all over again.

This presentation was quite difficult to understand, as it contained many medical terms that are supposed to be known to the public. It is of course nice to be able to recognize and understanding gene editing machinery, however that is a very small part of a great and complex research field.

 

The many layers of the Neocortex

Speaker: Randy M. Bruno
Department: Neuroscience and Zuckerman Institute of Columbia University
Subject: The many layers of the Neocortex
Location: Erasmus MC

Date: 5 October 2015
Author: Gabriele Kockelkoren

 

The neocortex is the top layer of the cerebral hemispheres, 2-4 mm thick, and made up of six layers, labelled I to VI (with VI being the innermost and I being the outermost). It is involved in higher functions such as sensory perception, generation of motor commands, spatial reasoning, conscious thought, and in humans, language. In Figure 1 the neocortex is shown. Each of the three brains is connected by nerves to the other two, but each seems to operate as its own brain system with distinct capacities.

 

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Figure 1: The brain can be subdivided into three main parts: the neocortex, the limbic brain and the brain stem. Source: https://bookofthrees.com/triune-brain/

 The conventional model states that axons coming from the thalamus reach to layer four of the neocortex. The cells of the neocortex transduce the signal to layers II and III, which subsequently activate layers V and VI. The latter layers function as feedback mechanisms to the outer world.

Randy Bruno is particularly interested in the whisker system of rodents and sensory perception. This system resembles the sensitive human skin. Axons from the thalamus are projected into every layer of the cortex. Bruno and colleagues observed that axons from the thalamus branch into layer IV, and in some cases in layer V and VI. Randy Bruno’s lab intends to analyse how and why this occurs? What is the function of this shortcut?

The first step in answering these questions relies in recording the reaction of neurons from each layer after applying a stimulus. Rats were used to do these experiments. By analysing the results, it has been found that cells in the IVth layer respond prior to cells in layer II and III. This is in line with the conventional concept of the signal processing pathway. However, in half of the tracked neuron cells derived from the Vth layer cells depolarised at the same time or even before the layer IV cells. Concerning the other half of the layer V cells, these reacted after layer IV, but prior to cells coming from layer II and III.

Furthermore experiments have been conducted with mice to see the relevance of the IVth layer compared to the Vth layer. A patch was inserted in the IVth layer of the neocortex, which registers all activity and action potential in the layer. Additionally a pipe filled with ACSF (artificial cerebrospinal fluid) is inserted in layer IV. Hereafter the ACSF is switched for an anaesthetic, lidocaine. This results in no action potential. The same procedure is done for a patch in layer V (ACSF is still in layer IV). Lidocaine is used to set all action potential to zero in layer four and the behaviour of cells in layer five was observed. The signal was normal, indicating that the layer five response does not require layer four.

After thus finding, Bruno’s group dedicated research to knowing the difference in the function of cells in different layers of the neocortex. Therefore they looked at the response of mice neurons by applying stimuli to the whiskers of mice in different directions. The neurons they were interested in, do not respond often to the stimuli, therefor they solely looked at the voltage and not at peaks above the threshold. This enabled finding a pattern of stimuli which is required to get a maximum response. For layer IV, V and VI cells the optimized stimulus gave a better response than a random stimulus. For the layers II and III there was no difference in response.

I liked Mr. Bruno’s talk very much, as it showed research that goes against common ideas and this is very valuable to see. It was difficult to understand many technical terms in the presentation, as I lack the knowledge of this medical jargon. Therefor it is very difficult to interact in discussions in neuroscience related talks.

Speaker: Wei-Feng Xue
Department: School of Biosciences, University of Kent
Subject: Nano-scale properties of the amyloid life-cycle
Location: TU Delft

Date: 12 May 2015
Author: Gabriele Kockelkoren

 

Wei-Feng  Xue leads a research group at the school of Biosciences at the University of Kent. Its work focusses of the mechanisms of amyloid assembly. In his lab a multidisciplinary approach can be found, physics and biology are combined in numerous ways. With great enthusiasm and a constant smile on his face, this seminar was extremely pleasant to attend.

The lab focusses on the life-cycle of amyloid. Amyloids are fibrillary structures formed out of peptides, these are aggregates. These structures are nanometers wide and micrometers long. Amyloids have disease-associated properties. Examples of these diseases are Alzheimer, Parkinson and Diabetes. In terms of disease, amyloids are interesting for studying their formation, growth, interaction and toxicity to cells. Next to amyloids, prions exist. Prions are transmissible amyloids associated with diseases, like the Mad-Cow disease and Creutzfeldt-Jakob disease. So the main difference is their propagation and transmission. The border between amyloid and prions disease is quite blurred, that is because some diseases have the tendency to transmit themselves. Xue tries to find out what makes amyloids transmissible or not.  Amyloids fibres are potential candidates for high-performance nanomaterials due to their strong mechanical strength and great elasticity. Amyloid have a beta structure sheet going perpendicular to the fibre structure and are formed out of a great variety of proteins.

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Figure 1: AFM image of amyloid filaments. Source: https://nanotechlab.physik.unibas.ch/NSLBildergallerie.html

Biophysics behind amyloid formation is extremely complex. Amyloids are known in a great variety of species. All amyloid species have a resembling free energy diagram in formation. To be able to fold the ‘pre-amyloids’ have to cross a free energy barrier. This suggests that their formation is extremely slow. Of-course from a disease point of view this is very favourable, as it takes a long time to get the disease.

In the lab they find conditions to get up this amyloid-formation kinetics to form them more fast. When monomers are put together in solution, it takes a long time before nucleation starts. This waiting time is called the lag phase. The lag phase is followed by an exponential growth until a plateau is reached, this is the case for amyloids. In the case of prions the lag time is skipped because preformed polymers are trans missed. After this first nucleation step, seeding succeeds. Seeding is the process were polymers that have been formed fragment. New polymers form out of the fragmented pieces. The real interesting question is why some amyloid are not really transmissible. In living systems there are a lot of ways to go into amyloid formation. They are formed out of oligomers that proliferate through secondary pathways.

Fig 1 v4.

Figure2: The amyloid life cycle. An amyloid is depicted as a circle, the soluble monomeric protein as parallelograms. The main processes in amyloid assembly are given by the red arrows, primary nucleation by the purple arrow and secondary nucleation by the blue and orange arrow. Source:  Wei-Feng Xue, Nucleation: The Birth of a New Protein Phase, Biophysical Journal, Volume 109, Issue 10, 17 November 2015, Pages 1999-2000, ISSN 0006-3495, http://dx.doi.org/10.1016/j.bpj.2015.10.011.

Xue tends to talk about the life-cycle of amyloid assembly. He sees it as a circular process with a positive feedback-loop. From primary nucleation (de novo), there are secondary pathways (fragmentation and secondary nucleation). This forms new entities and seeds of amyloids. According to Xue, it resembles population dynamics.

His lab is interested in the structure of fibres, however not at atomic level. They have found that the fibril size and shape defines their biological activities, even though at atomic level they may seem fairly similar. Actually, size and shape gives a wide variety of responses. The amyloid fibrils are highly stable and can resist to chemicals, high concentrations of detergents and high temperatures.  In the lab they look at how they interact with vessels and  are interested are size distribution, persistence length, force resistance. AFM is used as a structural tool at mesoscopic length scale to look at how fibres twist and behave.

I enjoyed this talk very much and it was really nice for Nanobiology students. Kent is an enthusiastic speaker who inspires scientists around the world.

 

Illuminating biology at the nanoscale with single-molecule and super-resolution imaging.

Speaker: Xiaowei Zhuang
Department: Chemistry Department of Harvard University
Subject: Microscopy imaging
Location: TU Delft

Date: 12 January 2017
Author: Gabriele Kockelkoren

At the Dies Natalis of the TU Delft, Xiaowei Zhuang is introduced to the public by Chirlmin Joo. Xiaowei Zhuang is principal investigator of a lab and professor at Harvard who contributed greatly to the design of the well-known techniques STORM and FRET. As a leading example of innovation and success in her field, she shared her valuable research.

The presentation starts with a short voyage back in history, leading to the first microscope ever created by Anthonie van Leeuwenhoek. This first ‘microscope’ consisted of a very tiny lens which allowed the visualization of bacteria and sperm cells. Since then, many new insights and techniques have arisen, which grant the possibility to ‘unravel’ even more details of the microscopic and nanoscopic world.

In order to image a complex system of proteins that give rise to cell life through collaboration, you require:  nanometer scale resolution, molecular specificity and dynamic imaging. The greatest problem is the diffraction limit for most conventional microscopes. In conventional fluorescence microscopy where all fluorophores in the sample are fluorescent, their diffraction limited images overlap, creating a smooth but blurred picture. This has been circumvented by the use of SIM and STORM. Xiaowei Zhuang developed the STORM technique.  STORM  is a type of super-resolution optical microscopy technique based on stochastic switching of single-molecule fluorescence signal. STORM/PALM utilizes fluorescent probes that can switch between fluorescent and dark states so that in every snapshot, only a small, optically resolvable fraction of the fluorophores is detected. This enables determining their positions with high precision from the centre positions of the fluorescent spots. With multiple snapshots of the sample, each capturing a random subset of the fluorophores, a final super-resolution image can be reconstructed from the accumulated positions.

 xiaowei-1

Figure 1: The principal of STORM. Stochastically fluorophores are excited. The overlap of all figures, results in a super-resolution image of the locations of the fluorophores. Source: http://huanglab.ucsf.edu/STORM.html

STORM has proven to be widely applicable, from single cells and sperm tail to neuron networks. During the talk Xiaowei Zhuang highlighted one application in her own lab. Shown in Figure 2 is actin in axons. The actin filaments are depicted as the blue stripes. In conventional microscopy techniques these filaments are not visible. The observation can be made that the rings of actin we see are extremely regularly spaced. This can also be seen in the periodic autocorrelation of peaks and defined Fourier peaks. This periodicity equals about 180nm-190nm.

fig2N copy

Figure 2: A. STORM image of actin rings on axon. B. Fourier transform to detect the most frequent periodicity. C. Histogram of measured spacing values (nm). Source Xu et al. Science 2013

This indicates the presence of a spacer that needs to be 180nm long and interacts with actin. This brought to spectrin tetramers. Regarding the functional role of the pattern, not much is known yet. It makes the axon very robust and still flexible. Furthermore, it is thought that the structure is important to maintain the integrity of the axon under stress.

The second part of Zhuang’s talk focussed on imaging of the transcriptome. The transcriptome is the total of all RNAs present in a cell. By understanding and analysing spatially-resolved single-cell transcriptomics, new insights can be gained in the subcellular organization of the transcriptome and of the spatial organization of transcriptome in tissue.  For transcriptome imaging, the lab started looking at FISH. Here the FISH probe is bound to the RNA. To distinguish thousands of RNA species only one type of RNAs are labelled in the first image and activated, then a second category is activated and so the cycles continues. Each RNA has a binary code to which it is connected. So in 16 rounds of imaging, you can distinguish 2^16=65535 RNAs. FISH is very accurate as it has a 5% error for 1 bit.

I have enjoyed this talk very much, as Xiaowei Zhuang shows innovation in all her projects. This innovation and out-of-box thinking, brought her to great success. She is an inspirational scientist for every Nanobiology student.

Dissection back-to-front cell polarity with optogenetics

Speaker: Mathieu Coppey
Department: Physics and Chemistry Department, Marie Curie Institute Paris                       Subject: Optogenetics
Location: TU Delft

Date: 16 September 2016

Author: Gabriele Kockelkoren

Mathieu Copey works in the Physics and Chemistry department of the Marie Curie Institute in Paris, France. The general motivation and driving force in his research is understanding the various scales that govern life. By understanding them all, insights can be gained in the behaviour and mechanisms of cells. One of the main interests of the his lab is cell polarity and ways to manipulate cell polarity. Cell polarity is the asymmetric organisation of several cellular components, including its plasma membrane, cytoskeleton or organelles. This asymmetry can be used for specialised functions, such as maintaining a barrier within an epithelium or transmitting signals in neurons. Polarity occurs at many scales in biology. The two main projects in his lab are: 1. Manipulation of protein distribution: light-gated dimerize for plasma membrane localization. 2. RhoA signalling perturbation and back to front polarity.

In order to manipulate cell polarity, Mathieu Coppey uses optogenetics.  The optogenetics method the lab applies for manipulation makes use of the CRY2/CIBN dimerizer. In this technique the CRY2 protein is labelled with mCherry and CIBN is a protein labelled with GFP. By illuminating the sample with blue light CRY2-mCherry will bind to CIBN-GFP which is attached to the plasma membrane (Figure 1). Excitation with blue light is done using TIRF microscopy and only a small part of the cell can be excited.

 

Figure2

 

Figure 1: By excitation with blue light CRY2 binds to CIBN. Source: Kennedy, M.J., Hughes, R.M., Peteya, L.A., Schwartz, J.W., Ehlers, M.D. & Tucker, C.L. Nature Mehods 7, 973-975 (2010)

By succeeding in the use of light to manipulate the cell polarization, they introduced Rho GTPase to the system. Rho GTPase signalling controls cell morphology and migration. Cdc42  is a Rho GTPase. Rho GTPases gives the possibility to activate other proteins at the location of excitation. The Rho GTPase used in the experiments is Cdc42, which works as a switch that is active when bound to GTP and inactive when bound to GDP. Its activation occurs via ITSN (intersectin), which has the catalytic domain DHPH. This domain is fused to the CRY2-mCherry construct and the CIBN-GFP construct. Through this construction excitation by blue light causes the DHPH-CRY2-mCherry construct to bind the DHPH-CIBN-GFP at the plasma membrane. Cdc42 influences movements within the cell and as a result only the Cdc42 which is nearby the plasma membrane is activated. This results in the polarization of the cells and controlled migration by blue light excitation.

I particularly enjoyed this talk as it combines innovation of techniques with the field of biology and physics. In other words, a perfect talk for a Nanobiology student.

 

CRISPR-based interference in Prokaryotes from exploration to exploitation

Speaker: John van der Oost
Department: Microbiology, Wageningen University
Subject: CRISPR-Cas system
Location: TU Delft

Date: 12 November 2015
Author: Gabriele Kockelkoren

 

Professor John van der Oost works in the laboratories of Microbiology of Wageningen UR and is extremely interested in the CRISPR system. Originally having a background in microbiology, John van der Oost is very fascinated by bacteria. The CRISPR system, subject of this BN talk, is owned by 85% of the archaea and by 40% of the bacteria.

Normally viruses infect prokaryotes, then the viral replication cycle occurs and new viral phages are formed which can infect new prokaryotes. This cycle repeats itself over and over again. However, the potential hosts have evolutionary developed skills to overcome this. These established mechanisms are: inhibition of absorption, inhibition of DNA injection, degradation of DNA and abortive infection systems. In 1987 researchers in a Japanese lab described a remarkable repetitive structure in the DNA of bacteria. These repeat were interspaced by variable spaces, these spaces are called spacers. However, at that time its function was not known. This unknown structure was later called CRISPR, which is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats. So a new defence mechanism was found.

It was found that the repetitive stretches are co-localised with the repetitive genes and that many CRISPR spacers are homologous to viruses or plasmids. Next to the CRISPR loci, the CRISPR associated (Cas) genes are located. Therefore, the whole pathway is often referred to as the CRISPR-Cas9 pathway. By experimental evidence it provides an adaptable and inheritable immunity.

The process of the CRISPR-Cas9 system can be divided into three main stages. The first stage is the spacer acquisition state. When an infection occurs and this infection does not end successfully for the virus, then the foreign DNA of the virus, will be cut into pieces. Parts of this DNA will be incorporated as spacers in the DNA of the host as a spacer. This way the CRISPR system has time to acquire an additional unit in its CRISPR. During the second stage the Cas genes are transcribed and pre-crRNA (CRISPR pre-RNA) is formed. Afterwards the pre-crRNA is changed into mature crRNA. The third and final stage is the target interference. This stage involves activation and targeting of Cas-proteins and crRNA for neutralisation of viral DNA.

It is essential for this process to be very specific. There has to be a clear discrimination between the sequences of the host and those of the virus. As the sequences contain both the DNA of the bacterium itself and of the virus, looking only at the sequence is not enough. Hence the discrimination is made due to the protospacer adjacent motif, called PAM. This PAM is only found the DNA of the host. If the motif is present, only then the process starts attacking the other system. This forms a very subtle, but important difference. There is also the Seed sequence which serves as a second control point. The Seed sequence may not have any mismatches. If any mismatches are found, then the crRNA will not bind.

 

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Figure 1: General overview of CRISPR-Cas-system. Source: https://www.horizondiscovery.com/about-us/our-science/CRISPR

There is a huge diversity in the CRISPR-Cas systems. This diversity is mainly related to the CRISPR-associated proteins and genes. The types of CRISPR systems can be divided into three main categories and several subtypes. The type I and III are very similar and belong to Class1. Type II and the more recently discovered type V belong to Class2. The Class1 system relies on the Cascade system, where different Cas genes work together forming a complex named CRISPR-associated complex for antiviral defence (Cascade). Researching Class1 in the E. Coli system, it is observed that when all 8 Cas proteins are expressed, the results are not successful. However, by expressing 6 genes with designed CRISPR with spacers which correspond to a certain phage lambda sequence, immunity is produced. While looking at the Cas proteins, Cas1 and Cas2 are most likely involved in the acquisition of spacers. As Cas1 and 2 make up a complex and bind to dsDNA, they recognize the PAM sequence.  Cas 3 appears to be a helicase-nuclease hybrid, which is recruited by a conformational change of the Cascade due to an increasing density of the nucleic acid. Together with Cas6, Cascade is responsible for the pre-crRNA processing. As aforementioned, Class1 contains the type I and type III complex. The type III targets RNA rather than DNA.  In Class 2 no Cascade is present, there is only a single protein which is Cas9 for Type II. Type II is remarkable for two aspects: First the use of tracrRNA, which stands for trans-activating CRISPR RNA and secondly, the nuclease which is used to cut the DNA is RNaseIII. Most recently a new type has been discovered, type V, also called Cpf1. This system also has one single unit like Cas9. The difference is that there is no tracrRNA and the PAM is at the 5’-side instead of the 3’-side. Furthermore, type V uses a non-Cas RNase and the dsDNA break that it makes is staggered. In all the other types this break is cut.

The applications of the CRISPR-Cas system are great. Not only is it an engineering tool for creating viral immunity for bacteria, but also viruses can be made that target “bad” bacteria. Ultimately, CRISPR-Cas can be used as a powerful genome engineering tool which can lead to great consequences for human evolution.

 

Material-cell interactions

Speaker: Christine Payne
Department: School of Chemistry and Biochemistry, Georgia Tech
Subject: Material-cell interactions
Location: TU Delft

Date: 15 October 2015
Author: Gabriele Kockelkoren

 

Material-Cell Interactions

The goal of research in the Payne Lab at Georgia Tech is to understand the underlying molecular mechanisms by which cells interact with materials. This talk was divided into the two main research topics of her lab: nanoparticles interactions and conducting polymers. In her research the use of fluorescence microscopy for cellular interactions to get the spatial form of the cell is essential. Professor Payne makes also use of spectroscopy at the molecular level and calorimetry.

Nanoparticles interactions

Nanoparticles can be used for cellular applications as drug delivery of imagining and sensing. Her lab focuses more on these last two applications. As her lab is interested in the interactions of nanoparticles with cells, cells are cultured in a aqueous solution of  salts, amino acids and glucose. In the medium there is also the serum FBS. How surface of nanoparticles affects binding of FBS. They have one cationic nanoparticles and one anionic nanoparticles. They use a nanoparticle washing procedure. At some wash you do not see the protein anymore. They found albumin on the surface. It didn’t matter what the surface charge was, both experiments gave the same result for albumin presence.

NIHMS394753.html

These images are made using fluorescence microscopy. The left part of the image shows cationic nanoparticles and the right part anionic nanoparticles. Some cells are placed in only MEM, others are placed in a MEM medium including  a 10% FBS solution. Cationic NPs are amine-modified and anionic NPs are carboxylate-modified.  Source: Fleischer CC, Payne CK. Nanoparticle surface charge mediates the cellular receptors used by protein-nanoparticle complexes. J Phys Chem B. 2012;116:8901–8907.

The Pain-lab continued its researched the effects of FBS on the binding properties of NPs at the cell. For this they have used ionic and cationic NPs. To look at the interactions at the cellular level epi-fluorescent microscopy was used. Cellular binding is a function of the initial NP charge. If nanoparticles are initially cationic, the no particles can be observed. If a certain percentage of FBS is present, then NPs are to be seen. The experiments showed that for anionic NPs, FBS inhibits the binding of nanoparticles. For this experiment special NP-washing procedures have been used. To check their results, they compared these to experiments with BSA. The identical results were found. It can be seen that NPs are competing with the free proteins for the binding site. Complexes that are formed from anionic NPs use native protein receptors. Furthermore it is shown that increasing the FBS concentration, decreases the binding affinity. In summary, this experiment showed that cationic NPs with BSA use a different cellular receptor than BSA, while anionic use the same receptor. The lab had already hypothesized  which receptor this could be. To test whether indeed anionic NPs bind to this specific receptor, a competition essay  between polyinosinic acid which binds the receptor and NPs was done. Using CD spectrometry it was checked whether there where structural changes in the protein when it was bound to the cell surface. For anionic NPs we see no change, however for ionic NPs a clear shift can be seen in spectrometry. These experiments led to the conclusion that the protein corona structure determines which cell surface receptors can be used for the binding of NPs.

Conducting polymers

In the second part of the seminar conducting polymer-cell interactions were highlighted. Mrs Payne and her colleagues want to synthesize PEDOT:PSS in cells, this is the biomolecular synthesis of conducting polymers. PEDOT:PSS are highly conductive polymers made out of the PEDOT monomer and the PSS monomer.  Peroxisomes control intracellular reactions and are organelles where fatty acids are degraded in the cell. Most importantly, PEDOT:PSS is synthesized here. Peroxisomes contain a catalyst, called catalase. By depositing monomers in these peroxisomes, it has been shown that PEDOT:PSS is formed. By firstly boiling catalase (so that it loses its enzymatic activities), the experiment has been repeated several times. Even though the enzyme was denatured, still PEDOT:PSS was produced. This indicates that enzyme activity is not required. This  experiment has been done with other proteins as well, like transferrin. Transferrin is associated with transport of iron in the blood. The results were exactly the same, PEDOT:PSS was produced. This shows that enzymatic activity is not needed for PEDOT:PSS formation, however an iron containing protein in necessary for the reaction to work.  In contract to nowadays techniques to form PEDOT:PSS, this method allows formation in a single step.

I liked this seminar very much. All kinds of research techniques were discussed, like spectrometry, quantum dots and fluorescence microscopy. It is very nice to recognize those techniques and know their basics work.

 

 

 

T-cell mediated neurodegeneration and repair

Speaker: Frauke Zipp

Location: Erasmus MC

Date: 6 February 2017

Author: Carolien Bastiaanssen

 Frauke Zipp conducts research to learn more about the crosstalk of the immune system and the nervous system in neurology. In this field Multiple Sclerosis (MS) is studied as a model disease. In her talk she first explained about the pathology of MS and the current view on the mechanisms behind this disease. Next she showed some of the results of her own experimental work. Finally she highlighted several novel concepts that followed from recent experimental work.

mri-scan-ms

Figure 1: The white marks indicated by the red arrows are referred to as plaques. They are the result of damaged myelin. Adapted from http://www.radiologyassistant.nl/en/p4556dea65db62/multiple-sclerosis.html

MS is an immune-mediated disease. It is a chronic inflammation of the central nervous system (CNS), where the patient’s immune system attacks cells in the CNS by mistake. Especially the coating that protects nerve fibers, called myelin, is attacked by T-cells that entered the CNS. The damaged myelin causes scar tissue and as a consequence nerve impulses are distorted or interrupted. The demyelination can be visualized using an MRI scan. The scar tissue will show as white plaques (Figure 1). The current view is that due to the damaged myelin, there is less nutrition and support for the axons. Thus leading to axonal damage (Figure 2).  However there is also evidence that suggests demyelination does not have to occur before axon degeneration (Tomassy et al. 2014, Science). Both CD4 and CD8 cells can interact directly with axons, making it possible to attack the axon without demyelination. MS patients show a wide variety of symptoms including walking difficulties, numbness or spasticity. It is a very heterogenic disease with periodic relapses. It is unknown what triggers the disease, although it is thought that a combination of genetic susceptibility and environmental factors are involved.

demyelination

Figure 2: At the top of the image healthy neurons and axons are shown. The axons are protected by a myelin sheath. When the protective layer of myelin is broken down, a process called demyelination, the axons are exposed, leading to axonal damage. Adapted from: Susuki, K. (2010), Myelin: A specialized membrane for cell communication, Nature Education, 3(9):59

Zipp and her colleagues monitored in vivo what happens during MS in the brain stem. For this purpose they used an experimental autoimmune encephalomyelitis (EAE) mouse model. This enabled the live imaging of T-cells in the CNS. The experiments showed that T helper 17 (Th17) cells are attracted by CD11c+ cells. Depletion of the CD11c+ cells caused a significant reduction in the number of Th17 cells that entered the CNS. Another result that followed from their experiments was that Th17 cells are able to interact directly with demyelinating axons on their own without the recruitment of other cells.

In her talk, Frauke Zipp presented three novel concepts. First of all she described counterbalancing inflammatory processes. During their experiments they observed macrophage cells in the CNS called microglia. These cells can transport Th17 cells within the CNS. The microglia engulfs a living Th17 cell and then two different outcomes are possible. Either the Th17 cell escapes and lives on or the it goes into apoptosis. This work shows one of the mechanisms the CNS employs in an attempt to defend itself.

The second novel concept that was presented concerns the characteristic relapses in MS. Using the animal model, Zipp and her colleagues mimicked the relapses. They found that during a relapse there was no demyelination.

The final novel concept concerned neuroprotection, regeneration, plasticity and homeostasis. There are patients that have little lesions growing into large damage sites. Some damaged sites even disappear and are thus in some way repaired. In the animal model the same phenomenon is observed. It was for example shown that T helper 2 cells promote axonal outgrowth and that they can promote axonal regeneration after spinal cord injury in vivo.

The research of Frauke Zipp, has led to a different view in the field of neuroimmunology. Her interdisciplinary approach has provided new insights into the mechanisms of MS. By gaining a better understanding of these mechanisms, she hopes to contribute to the development of therapeutic strategies and the improvement of diagnostics.

 

 

Crosstalk of immune and nervous system

The second speaker of this two-part seminar series by the Department of Neurology was by professor Zipp. She immediately started talking about the model disease in her field, namely multiple sclerosis. She showed that on an MRI image there are two differently colored parts associated with MS, one showing the inflammatory pathology of MS because of a too permissive blood-brain barrier, the other showing the degenerative pathology of the shrinking parenchyma.

What happens in MS is the following. Firstly a specific kind of T-cell that specifically targets the central nervous system (CNS) targets an antigen in the CNS. Then they transmigrate the blood-brain barrier. When they enter the CNS they attack the myelin of the nerves’ axons. But there is not only a process of demyelination. Bare neurons are also attacked. This was shown by showing that in the cortex you get swollen meninges. Because the cortex is unmyelinated, the bare neurons can also be attacked. In this process the T-17 helper cells play a very large role.

FIGURE_01_metabolism

Figure 1: Overview of the inflammation process in MS

Over time MS happens in a peculiar way. It has periodic relapses and remissions. In 40% of the cases it eventually loses this profile, leading to a continuous increase of disabilities. But in the other 60% the relapses eventually stop. It is not yet sure if this is the case because of neuron repair or because the neurons of this population is for some reason more durable than the other 40%.

Next she showed some of her experimental data. First she showed that the processes of dendritic cells play a role in the blood-brain barrier, acting like a kind of gatekeeper specifically keeping the T-17 helper cells out of the CNS. Also, through a transgenic cell staining it was seen that the T-17 cells directly attack the myelin without recruiting other cells.

Next she introduced three novel concepts in her field.

Firstly, she explained the process called counterbalancing. The microglia, a type of cells in the CNS, can carry and transport the T-17 helper cells inside of their protrusions. This is done to migrate the cells. It can end in 2 ways, either the engulfed cells die due to apoptosis or they escape. This process can be used by the T-17 cells to move through the CNS to harder to reach places. A specific compound called wortmannin has been found that blocks this process. This process is not specific to T-17 cells. In essence, this process is the microglia trying to phagocytize the cells, but it is doing more harm than good.

Secondly, it was seen that in mice there is no demyelination during a relapse, maybe explaining the two different populations.

Finally, the process of neuroprotection was explained. It has been seen that repair of damage was seen in patients. Another kind of T-cell, the Th-2 cells, is able to increase the axonal regeneration. The neurons of MS patients possess Interleukin 4 receptors. This compound reduces the severity of the MS by inducing axonic growth.

I found this last fact to be especially amazing, since an interleukin, a compound exclusively related to the immune system, is apparently able to interact with the nervous system. This suggests that the usual view of the human body as a set of different systems can be very wrong. Because of this, interdisciplinary work such as the work of professor Zipp should be appreciated way more, because these innovations will really improve health care a whole lot.