annual report

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I’d like to start with some general information about my project. First of all – why we are interested in DNA damage. As you see it plays a role in some disorders like Alzheimer’s disease, as well as healthy ageing or even learning.

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This is why we are curious to find cellular consequences of this damage - this is a general aim of the project. But the most important questions I’ve asked this year were:

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Is there a link between DNA damage and senescence in neurons, and the second one: can a long-term neuronal culture be a model of aging brain.

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To answer these questions I use neuronal primary culture obtained from rat brains by papain dissociation

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As you see on this staining It’s not a pure neuronal culture, there are different types of glia and progenitors, which proliferate, so in fact the percentage of neurons falls down in time.

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And the first result I want to share with you is that the level of senescence associated beta galactosidase increases in time up to almost 100% after 4 weeks.

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This process was shown before, however, we are the first who distinguished between senescent neurons and glia by adding immunofluorescence after this biochemical step.

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Moreover, to find out if this process is caused by DNA damage, we detected and counted 53BP1 foci in neurons nuclei. 53BP1 binds to DNA breaks, so what we see here is a spontaneous DNA damage, which fluctuates at a low level, but goes up after 3 weeks.

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And this is my first proof that senescence here is not caused by DNA damage, since the damage rises after, not before, the onset of senescence.

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To be sure I induced more DNA damage in neurons with doxorubicin... and this slide is just to show you that doxorubicin provokes DNA damage in dose-depended manner without massive apoptosis in neurons (you see a lack of caspase 3).

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And all these biochemical tests indicated 10 nanomolar dose as the best for this study.

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And now having these two cultures – one with induced DNA damage and the untreated one I performed the same steps to check if there are more senescent cells here.

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And this result was a surprise for me as the red bars not only weren’t higher, but they were even lower than the blue bars from untreated cells. So it looks like doxorubicin even prevented the process of senescence.

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That was the second proof, now let’s turn to the third one. Once again we have this model of untreated and treated neurons, again with biochemical test for beta galactosidase and fluorescent detection of neuronal markers and DNA damage protein.

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However, this time I have categorized the DNA damage foci between positive, negative cells and those with mild activity of beta galactosidase. And take a look at those bars - they are all almost the same.

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So senescent and non-senescent cells have statistically the same level of DNA damage And this is my third proof that there is no cause an effect relation between DNA damage and senescence in this particular model.

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Now the next part of my results concerning the second aim – the utility of primary neuronal culture as a model of aging brain. I’ve already shown you the most common symptom of senescence (beta galactosidase),

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but to have more markers I checked for the level of proinflammatory cytokines, commonly released by senescent cells. So far I have detected two of them - interleukin 6 rises almost linear after two weeks and TNF alpha

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quite similar, however, with this unexpectedly high level at the first stage of the culture. And this is my current hypothesis for the reason of this premature senescence in a culture – I guess it might be caused by the initially high level of TNFalpha.

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Another important process linked to senescence is autophagy. In my last year’s attempt I used the lysotracker dye which showed an increased number of lysosomes so I assumed that the level of autophagy will be increased as well.

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But this year I checked a more specific marker – the LC3 protein level and the result was quite opposite. LC3 has two forms – the first is cytoplasmic, the second is observed on autophagic vesicles and as you see they both go down in time.

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The most important thing is the ratio of vesicular to cytoplasmic form – here - it also drops. And this result is consistent with many observations in other cell types showing that autophagy usually precedes senescence in order to rebuild the cell.

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I wanted to have a second proof and also to distinguish between autophagy in neurons and glia. In order to do that I used this construct with LC3, GFP and RFP cloned into one plasmid giving both green and red light in autophagic vesicles in proper pH.

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Here are the results of live observations in different days of culture. A typical neuron in early stage had a few autophagic vesicles. Two weeks later another typical cell with almost none of them.

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But still with very high level of acidic vesicles. I also observed, that this process is even more visible in glia.

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in conclusion: I have two main findings

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I have very specific plans for starting this summer: I was granted an internship in National... where I will obtain IPS cells from patients without ATM protein (crucial for DNA damage response) I will differentiate them into neurons and perform a set of