Systemic DNA-damage responses in development and aging

BN Seminar
 
Speaker: Björn Schumacher (University of Cologne)

Subject: Systemic DNA Damage responses in development and aging

Location: TU Delft

Date: Thursday, 29.10.2015, 16:00-17:00

Author: Edgar Schönfeld

It is estimated that each DNA molecule experiences tens of thousands lesions daily. While unrepaired damages foster ageing, erroneous repair promotes cancer. Mutations in nucleotide excision repair (NER) pathways, however, result in impaired development. Mutations in specific proteins involved in global-genome NER (GG-NER) result in the syndrome Xeroderma Pigmentosum, which is characterized by an enormously increased risk of UV-light-induced skin cancer. Cockayne Syndrome, on the other hand, is attributed to defects in transcription-coupled repair (TCR) and entails premature aging.

schumacher_03_2015
Simplified working principle of GG-NER and TCR (source: http://cecad.uni-koeln.de/Dr-Bjoern-Schumacher.107.0.html)

Prof. Schumacher is interested in understanding the processes that are responsible for somatic maintenance and thereby ageing. To address this question, the worm C.elegans was chosen as an animal model, as the GG-NER and TCR pathways are evolutionarily conserved in this animal. Xeroderma Pigmentosum is caused among others by point mutations in the gene XPC, while Cockayne Syndrome develops as a result of mutations in the genes CSA or CSB. Prof. Schumacher’s team radiated C. elegans with UV-light. Worms with mutations in XPC developed normally, despite being sterile. Worms with defective CSB, on the other hand, developed a healthy germ line, but mitosis in somatic cells was impaired. This suggested that GG-NER is of vital significance for germ cells, while TC-NER is essential for somatic tissues. Previously it was thought that these two branches of NER function during different stages of development. The next step was the identification of genes responding to UV-induced DNA damage. In order to do so the transcriptome of the treated worms was analyzed and a lot of genes proved to be differentially expressed. Parallely the program “Wormpath” was written, which uses statistical methods to identify the network of genetic interactions that correlates most with the observed gene expression pattern. Such a network was found and the genes DAF-16 and DAF-2 played a pivotal role in it. These genes are also known for being involved in aging. DAF-2 mutant worms live twice as long as wild-type C.elegans. It became evident that DAF-2 activates DAF-16 if persistent DNA-damage is present. The researchers also demonstrated that DAF-16 helps to overcome damage-induced developmental arrest through enhancement of tissue functionality. DAF-16 activation alone is sufficient to promote developmental growth after UV-induced damage. In this way, DAF-16 ensures that cells can develop normally even in the presence of persistent DNA-damage, even in the absence of DNA-repair. This damage tolerance ensures tissue functionality amidst the accumulating number of mutations in the process of aging.
What I find particularly interesting is that there seem to be mechanisms that counteract cell cycle arrest, which is meant to give the cell time to repair its DNA. In my eyes, this implies that cells are willing to sacrifice a bit of their genomic integrity to guarantee the functionality of the whole tissue. Apart from that, the “Wormpath” programme impresses me.

Material-cell interactions

Speaker: Prof. Christine Payne
Department: Bionanoscience
Subject: Material-cell interactions
Location: Delft University of Technology
Date: 2015-10-15
Author: Romano van Genderen

This talk by professor Payne was divided into two parts, the first about nanoparticle-cell interactions and the other one about the synthesis of conducting polymers by cells.

She started by explaining the use of nanoparticles in current research. These particles are often used for tracking and sensing molecules in biological research. They also have a hypothetical use as delivery agents for drugs and nucleic acids into cells. But if they want to use this second application of nanoparticles in humans instead of in controlled testing environments, they first must investigate a specific point, the interaction between serum (blood without any living cells or coagulation factors present) and these particles; this is the case because the drugs must obviously be injected into the blood stream. This is why they investigated the nanoparticles in FBS (foetal bovine serum) and MEM (minimal essential medium). They observed the presence of a so-called corona, which is a coating of proteins on the surface of the nanoparticle. This corona consists mostly of albumin, the most common protein in the blood which plays a very important role in regulating the osmotic pressure of the blood. This detection was done using epi-fluorescence.

Afterwards they added cells to the mixture and discovered that cationic nanoparticles only bind the cells if free protein is present. But if the nanoparticle is anionic, it only binds when protein bound to the nanoparticles is present. This is specific for the protein albumin. This shows that the protein albumin changes shape in the presence of a cationic or an anionic nanoparticle. These two different structures of albumin have different receptors on the cell surface. To be sure this did not depend on the material of the nanoparticles (polystyrene), they repeated the experiment using quantum dots, and the same result occurred.

To investigate if the albumin really does have two different structures, they used circular dichroism (CD) spectroscopy, where circularly polarised light is used to detect differences in the secondary structure of proteins. This showed that the protein keeps the same shape when bound to the anionic nanoparticle, but that the cationic particle causes it to change shape to a form with far less α-helices.

Afterwards she mentioned another kind of nanoparticle, which is not really a real nanoparticle but a nanoparticle aggregate. This is titanium dioxide (TiO2), also known as an additive in food called E171. Because this molecule also enters the blood stream, it must be known how it reacts to albumin and if it changes its structure to make sure it is not harmful. In small doses it is not, but it has been shown that it can change peroxiredoxin genes which influence the breakdown of hydrogen peroxide in the cell.

The second and shorter part of the talk was about the synthesis of conducting polymers by cells. This is the polymerisation of EDOT and PSS to a conducting compound called PEDOT:PSS. This was usually done using the peroxidase proteins of plants as catalysts, but now they can also do it using animal peroxidase. But the peculiar thing here is that the protein does not even need to be active, because even after boiling the protein, the reaction still occurred. They thought that perhaps the iron inside the heme bound to the peroxidase plays a role, so they used an iron importer molecule; this did also cause the reaction to occur. So only iron must be present.

http://pfam.xfam.org/structure/getimage?id=2gj1

Image 1: The structure of a peroxidase, namely peroxidase from a P. chrysosporium. Note the heme molecule bound in the middle and especially the iron-II bound (gray atom surrounded by 4 blue atoms) http://pfam.xfam.org/structure/getimage?id=2gj1

But the kind of protein used does actually matter in the form of the polymer used. Using a protein that binds heme causes a so-called bipolaron state, which leads to a very high conductivity of around 20 S/cm. But a protein that binds bare iron-II causes a polaron state, which has a far lower conductivity. This conductivity is still low compared to metal wires, but it definitely makes up for this lower conductivity in flexibility.
This talk was really fascinating because it showed how theoretical physics eventually got a practical use, the nanoparticles for drug delivery and the wires for in electronics. Especially the conducting polymers were completely new for me; I never expected that cells could make such practical structures.

Material-Cell interactions

Speaker:         Prof. Christine Payne

Department:    Bionanoscience

Subject:          Material-Cell interactions

Location:         Delft

Date:               15-10- 2015

Author: Carolien Bastiaanssen

The lab of Christine Payne at Georgia Tech does research to understand how cells interact with materials. Two main focusses of their research are nanoparticle-cell interactions and conducting polymer-cell interactions. Some of the techniques they use are spectroscopy, calorimetry and fluorescence microscopy.

Nanoparticles (NPs) have several important cellular applications. They can be used for imaging, sensing, and nucleic acid or drug delivery. Payne wants to understand how the adsorption of proteins onto the surface of NPs influences their interactions with cells. The layer of proteins around a NP is called the corona. To study the influence of the charge of the NP, cationic and anionic NPs made from polystyrene were mixed with serum (FBS) where the main protein is BSA (net negative charge). The corona proteins were isolated using centrifugation and resuspension in water. After which the supernatant was loaded onto a gel and analysed using electrophoresis. The experiment showed that both cationic and anionic NPs bind BSA.

cationic and anionic nanoparticles binding

The images above were obtained with a fluorescence microscope. They show the cells in blue and the NPs in green. The upper two images show the cationic NPs and the lower two images show the anionic NPs. On the left the cells are surrounded by medium (MEM) only and in the right two images the medium also contains BSA. Source: Fleischer and Payne, Nanoparticle-cell interactions: molecular structure of the protein corona and cellular outcomes, Accounts of Chemical Research, 2014 Jul 11

In a next experiment Payne and her colleagues studied the effect of FBS on the biding of NPs to cells. Again they used the cationic and anionic polystyrene NPs. Epi-fluorescence microscopy was used to generate the images. This experiment showed that cationic NPs bind significantly more often to cells when FBS is present than when it is not present. Anionic NPs on the other hand bind significantly more often to cells when FBS is not present. This indicates that cationic NPs with BSA use a different cellular receptor than free BSA, while the anionic NPs with BSA use the same cellular receptor as free BSA. They had a suspicion to which receptors the different NPs bind. To determine whether this is really the case, they looked at competition effects between the NPs and a protein that is known to bind to this specific receptor. When NPs bind to different receptors it suggests that they have different conformations. CD spectroscopy was used to analyse small changes in the secondary structure of the proteins absorbed on the NPs. All these experiments lead to the conclusion that the protein corona structure determines which cell surface receptors are used to bind NPs.

The second part of the talk by Christine Payne was focussed on conducting polymer-cell interactions. The lab of Payne wants to synthesize PEDOT:PSS in cells. PEDOT:PSS are conducting polymers made from PDOT and PSS monomers. Peroxisomes are organelles and they are present in most eukaryotic cells. They contain catalase, a catalyst which is similar to plant enzymes used for the synthesis of PDOT:PSS. Payne and her colleagues deposited the monomers in the peroxisomes and saw that PEDOT:PSS was generated. They repeated this experiment, but now they first boiled the catalase. This caused the enzyme to become denatured and it lost all its enzymatic activity. Yet they still saw that PDOT:PSS was produced. The experiment was done with different proteins as well. One of the proteins they tried was transferrin which transports iron in your blood. This protein was used successful to produce PDOT:PSS. It seems that enzymatic activity is not necessary for the synthesis of PEDOT:PSS, but an iron containing protein is needed. In contrast to the current way of synthesising PEDOT:PSS, this procedure allows you to synthesise this conducting biopolymer in a single step.

I really liked the subject of the seminar by Christine Payne. There were several moments of recognition when something I recently learned in a course was mentioned. Yet there were also parts I could not follow completely.

the many layers of the neocortex

[Cleo Bagchus]
[4386736]

Speaker:      Randy Bruno
Department: Department of neuroscience
Subject:        the many layers of the neocortex
Location:     Rotterdam, Erasmus MC
Date:               05-10-15

The neocortex is a part of the brain, made up of six layers. The neocortex has a severe influence on cognition. It is involved in functions like conscious thought, recognition, problem solving, motor commands and sensory reception.

Randy Bruno focuses on sensory reception. Sensory information first travels centrally to the thalamus. The thalamus relays the information to other structures in the brain, including the neocortex. The axons from the thalamus innervate the neocortex and especially layer four (L4). Many people believe that the information follows the pathway according to part A of the image. The information first enters L4, than continues to L 2/3 that dispatches it to L5. There was a small part of the information that directly entered L5, but this was always believed to be an unimportant shortcut. After this there is sensory transformation.

Randy Bruno has done research to disprove this theory. To do this he used the whisker system of rodents. This is a system a bit more sensitive than human skin. The axons from the thalamus are projected into every layer of the cortex. The axons are bimodal, with a peak in L5/6 and L4, but L4 is most densely innervated. Axons spike depolarization in each layer. You can look at the time of depolarization. L4 has an early onset, while L2/3 has a late onset. If the other system was true, L5/6 should have a later onset than L2/3, but this is not correct. Half of the cells in L5/6 have an onset as early as L4. So the shortcut may give much information.

Bruno performed an experiment in which a drug (lidocain) was pipetted into L4. This drugs blocks sodium channels, so prevents a response. You can look at the response evoked in L5 with sensory stimulants. According to the old theory there should be no response. But there was almost no difference with a functioning L4. The amplitude of the peak and the time of depolarization were very similar in both cases.

These experiments would disprove the old theory. They give prove for the theory of the system portrayed in part B of the image. Almost all input of L5 comes from the primary pathway and not via L2/3. This is sensory information.

There are axons passing from L2/3 to L5, but they do not give sensory information. Researchers are now trying to determine what kind of information is relayed. The other layers are probably not redundant. They have different biological structures and relay information to different places. Researches do not know what information is dispatched in these layers.

Screen-Shot-2013-07-23-at-5.31.43-PM

http://www.neuwritewest.org/blog/4167 11-10-15 (Deep cortical layers are activated directly by thalamus by C.M. Constantinople and R.M. Bruno in Science 28-06-2013)

The lecturer spoke clearly, but he still used very technical terms, especially on the slides. This sometimes made the lecture difficult to follow without a purely biological background. The lack of biological information made it easier to hear that the old theory was incorrect. Other people had difficulty with this.

I believe this is a very important topic. The neocortex so heavily influences our behavior and without it we would not be human. So I believe it is very important to know which kind of information is relayed in the neocortex.

The many layers of the Neocortex

Speaker:                     Randy M. Bruno

Department:             Neuroscience

Subject:                      The many layers of the Neocortex

Location:                    Erasmus MC Rotterdam

Date:                           05-10-2015

 Author: Katja Slangewal

Sensation, perception, finding patterns, conscious movement, reasoning, abstract thinking and solving problems, in all these processes the Neocortex is involved (see figure 1). Randy Bruno does research to tis part of the brain and he has found some very interesting processes. As he spoilers in the beginning of his seminar ‘the books were wrong’. The rest of the talk told us why.

Figure 2

Figure 1: The neocortex (from: http://www.lookfordiagnosis.com/mesh_info.php?term=Neocortex&lang=1)

The Neocortex is the upper part of the brain; it covers the inside and is made up of six layers. The most outer layer is layer I, going in it ends in layer VI. Information from the outside world is processed in the cortex as signals. These signals travel from the senses via neurons into the neocortex. Long time it was believed this signals arrive in the cortex in layer IV and are distributed over the rest of the layers afterwards. This was found by using the ‘follow the wires’ tactic. While looking to the images of neurons going into the neocortex it seems true, most dendrites end in Layer IV, this region is really dense. Though there are small (though obvious) dendrites in Layers V and VI as well. These dendrites were mostly ignored so far, but why are they there?

Different tests were done to find out. At first the reaction time of the different dendrites were measured. It was clear that layer IV responded a lot faster than Layer II and III. Though layer V and VI gave weird results, their reaction times were spread out. Some were faster than every single dendrite in layer IV and some were really slow. Also the connection between the different layers was tested. The connection probability gave a linear output depending on the depth, the deeper in the brain, the less the chance of finding a connection. Important is the number of known cells in the different layers, this also decreases when you get deeper. Combining this gives a maximum of connection just where axons enter layer V and VI. So now the question is how much of layer V and VI input derives from a direct pathway and how much from an indirect pathway? This was tested by deactivating layer IV and surprisingly 100% of the input goes via a direct pathway. This and some other tests conclude that every axon goes to layer IV as well as the border of layer V and VI, without the so called ‘sensory talk’ between the layers (See figure 2).

Figure 2:

Figure 2A: the old understanding of the signalling pathway. Figure 2B: the new understanding of the signalling pathway. (from: Christine M. Constantinople, Randy M. Bruno, Deep cortical layers are activated directly by Thalamus, Science: Volume 340, no. 6140, 28 June 2013, pages 1591-1594 http://www.sciencemag.org/content/340/6140/1591.full)

These results give raise to some questions. The upper layers and lower layers both get direct input; within milliseconds a signal is distributed all over the brain. This information hasn’t gone through the whole cortex before arriving in certain layers, so what does the cortex do? How can parts of the brain not communicate while being next to each other?  Some people believed the two layers are comparable with a hard-drive and its copy, though this is hard to believe. Maybe the upper layers and lower layers are just sensitive to different patterns.

This was tested as well. When measuring the signal, not only high spikes were seen, but also noise. Most of the experiments ignore the noise and focus on the high spikes, this time it was done the other way around and indeed some signals give more spikes in the lower layers than in the top layers and some reactions spikes are only seen in certain layers. So the layers are indeed sensitive to different patterns.

I really liked the seminar of Randy Bruno, it was relatively easy to follow and he had a good kind of humor included in his talk. He did use a lot of technical terms which I didn’t understand at the moment, though the core of his talk was clear and interesting. It taught me a lot about the working of the brain and I am looking forward to learn more about this process.

The many layers of the neocortex

Speaker: Randy M. Bruno

Department: Dept. of Neuroscience

Subject: The many layers of the neocortex

Location: EMC Rotterdam

Date: 5 October 2015

Author: Carolien Bastiaanssen

The neocortex is deeply involved in any human and animal cognition. Sensation, problem solving, executing motor problems, you name it, the neocortex plays an influential role in it. This six layer structure in the brain is the focus of Randy Bruno and his team, they want to know how the neocortex does all this. In this seminar mr Bruno presented their latest results about the different layers in the neocortex and how they are (not) dependent.

Our sensory systems transduce information in the periphery. Next it is propagated centrally to the thalamus, which relays this information to primary areas of the cortex. The conventional model is that axons from the thalamus go to layer four of the neocortex. The neocortex’s cells transduce the signal to layers two and three, that activate layers five and six. These last layers give feedback to “the outer world”.

Mr Bruno works on the rodent whisker system. This system is very similar to our sensory system of the skin. They observed that most branches of the axons from the thalamus end in layer four, but there are also many branches ending in the higher layers five and six. He asked himself why this would be. Could it be some sort of shortcut?

To answer this question they used rats and recorded the reaction of neurons from many layers after a stimulus was applied. The results showed that cells in layer four responded before cells in layers two and three responded, this was expected and it is in line with the theory mentioned before. Yet they also saw that half of the layer five cells depolarised at the same time or even before the layer four cells. And the other half of the layer five cells reacted later than layer four but earlier than the cells from layer two and three.  Another experiment to study the connection between the different layers of the cortex was done in mice. A pipe filled with artificial cerebrospinal fluid (ACSF) was inserted in layer four of the neocortex and a patch was inserted a few micrometres away in the same layer. This allowed them to observe the action potential and random activity. When the ACSF was swapped for lidocaine, a local anaesthetic, there was no field potential anymore and just a flat line was observed. Changing back to ACSF allowed for a full recovery of the signal. Next the patch was moved to layer five. Lidocaine was used to deactivate 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.

Next mr Bruno wanted to know what the difference is in the function of cells in different layers of the neocortex. He and his team looked at the response of mice neurons to many different kinds of stimuli. They could move the whiskers of the mice separately and in any direction. The neurons of interest do not fire so often therefore they looked at the voltage and not at peaks above the threshold. This enabled them to find the pattern of stimuli needed to get the maximum response. For layer four and layer five and six cells the optimized stimulus gave better response than a random stimulus. Yet for layer two and three cells there was no difference. A second experiment again used mice, these mice were trained to identify a pole in the dark. The animal self-initiates the test by using a lever. Then a pole will sometimes come down and the animal has to move its whiskers to detect it. When it correctly identifies a pole and lets go of the lever it is rewarded with a drop of water. If the make a mistake they are punished with a time out. When the trial starts a peak in activity is observed at the tops of apical dendrites that rise from layer five to layer one. There is a second peak around the time the animal expects or gets its reward. Layer four cells however do not have these apical dendrites and therefor they do not have reward-related activity. So the reward-related signal in layer five is independent of the signal in layer 4.

LayersOfTheNeocortex.ai

Figure 1: The model on the left is the conventional model, in which a chain of events transforms the sensory information of the thalamus. The model on the right is the model mr Bruno proposed, with two strata. Source: Constantinople, CM. Bruno, RM. Deep cortical layers are activated directly by thalamus, Science, 2013 Jan 28

Mr Bruno’s findings indicate that instead of a chain of events, there are two independent strata and layer four is not the first stage of the cortical processing. This contradicts with the way people in the field of neurology have thought about the way the neocortex works for decades. It was no surprise that several individuals in the audience asked sceptical questions. The response from the audience shows that these ideas are revolutionary. It was interesting to see how mr Bruno did not just accept the common theory but that he looked at some small details which were ignored before.