Final Report Summary - RNANEUROTOX (Small RNA toxicity and DNA damage response in Huntington´s disease)
INTRODUCTION
Huntington’s disease (HD) is a fatal disease of the neuronal system that affects muscle coordination, causes mental and behavioral problems and ultimately death. HD is caused by a mutation in the DNA of the so-called Huntingtin gene. This results in the production of an incorrect protein that is toxic to neuronal cells, inducing protein aggregation and an abnormal DNA damage response amongst others. As a result, the cells die and the brain mass declines, affecting proper function of the neuronal system.
Recently, the group of Prof. Estivill has shown that not only the mutated Huntingtin DNA but also the RNA molecule is toxic to neuronal cells. RNA is an intermediate step in the transition from DNA to protein: DNA is first copied to an RNA transcript and subsequently this RNA is used as a translator to produce the final protein product. While RNA toxicity in HD has recently been proven, the mechanism of this toxic effect is currently not known.
Research in RNA biology has shown that certain small RNA products can induce a DNA damage response. Since HD is characterized by small RNA toxicity in addition to an abnormal DNA damage response, we hypothesize that these two processes are linked: the mutated Huntingtin RNA transcripts induce a DNA damage response and thereby cause toxicity.
PROJECT OBJECTIVES
The aim of this project is to study the mechanism underlying RNA toxicity in HD. This will be addressed by the following objectives:
Objective 1: Provide evidence that mutant Huntingtin RNA products induce a DNA damage response in cultured cells
Objective 2: Test whether RNA toxicity can be modulated by inhibition of the DNA damage response
Objective 3: Validation of the obtained results in HD affected mouse and human samples
PROGRESS SUMMARY
The first objective was addressed by a microarray analysis, a technique to study changes in gene expression. For this experiment we have used a system of differentiated human neuroblastoma cells, the main cell type that is affected in HD patients. By treating these cells with the mutant Huntingtin RNA, we were able to study the effect of RNA toxicity on gene expression. Untreated cells served as a control. While several genes were deregulated, we have focused specifically on genes that are implicated in the DNA damage response pathway. We selected 10 genes as interesting targets:
1) ADORA2A and MEIS2; genes that were previously shown to be downregulated in affected brain areas of HD patients
2) GTF2H5, RAD50, TP53, XAB2; genes involved in DNA damage
3) ATM, ATR, MRE11A, NBN; DNA damage genes previously shown to be implicated in HD
These three sets of genes were selected for a validation assay using qPCR, an alternative technique to study changes in gene expression. Unfortunately, we could not verify the microarray results, neither deregulation of the genes from set 1 nor set 2 was confirmed by qPCR. We found only two genes from set 3 that were deregulated: ATM and ATR. However, these results were not convincing, as they were not verified by a second qPCR analysis using a different control gene.
In conclusion, we were not able to show a convincing link between RNA toxicity and the DNA damage response pathway. At this point, the next step would be to try another approach to proof our hypothesis. We propose to use cell biology experiments like western blot and immunofluorescence as described in the fellowship application. However, we were not able to continue with these experiments as the fellowship was prematurely terminated, due to a unique new career opportunity for the fellow.
EXPECTED RESULTS AND IMPACT
The long-term goal of this research project is to contribute to a better understanding of the molecular mechanisms in Huntington’s disease. This knowledge might be translated as well to several other disorders of the neuronal system. These results should open new avenues in the therapeutic perspectives of these disorders. Next, our results will provide insights into non-coding RNA biology, a field of which knowledge is currently far from complete. Third, our studies will broaden our knowledge about the DNA damage response pathway, with a broad spectrum of implications in several diseases ranging from neurological disorders to cancer. Therefore, our results will contribute to the understanding of several research fields and the biological mechanisms underlying a broad spectrum of diseases. This knowledge will help society in general to increase their knowledge of biology, it will help assist the medical and pharmaceutical system to explore new therapeutic targets for neurological disorders and hopefully it might contribute to a cure for Huntington’s disease.
Huntington’s disease (HD) is a fatal disease of the neuronal system that affects muscle coordination, causes mental and behavioral problems and ultimately death. HD is caused by a mutation in the DNA of the so-called Huntingtin gene. This results in the production of an incorrect protein that is toxic to neuronal cells, inducing protein aggregation and an abnormal DNA damage response amongst others. As a result, the cells die and the brain mass declines, affecting proper function of the neuronal system.
Recently, the group of Prof. Estivill has shown that not only the mutated Huntingtin DNA but also the RNA molecule is toxic to neuronal cells. RNA is an intermediate step in the transition from DNA to protein: DNA is first copied to an RNA transcript and subsequently this RNA is used as a translator to produce the final protein product. While RNA toxicity in HD has recently been proven, the mechanism of this toxic effect is currently not known.
Research in RNA biology has shown that certain small RNA products can induce a DNA damage response. Since HD is characterized by small RNA toxicity in addition to an abnormal DNA damage response, we hypothesize that these two processes are linked: the mutated Huntingtin RNA transcripts induce a DNA damage response and thereby cause toxicity.
PROJECT OBJECTIVES
The aim of this project is to study the mechanism underlying RNA toxicity in HD. This will be addressed by the following objectives:
Objective 1: Provide evidence that mutant Huntingtin RNA products induce a DNA damage response in cultured cells
Objective 2: Test whether RNA toxicity can be modulated by inhibition of the DNA damage response
Objective 3: Validation of the obtained results in HD affected mouse and human samples
PROGRESS SUMMARY
The first objective was addressed by a microarray analysis, a technique to study changes in gene expression. For this experiment we have used a system of differentiated human neuroblastoma cells, the main cell type that is affected in HD patients. By treating these cells with the mutant Huntingtin RNA, we were able to study the effect of RNA toxicity on gene expression. Untreated cells served as a control. While several genes were deregulated, we have focused specifically on genes that are implicated in the DNA damage response pathway. We selected 10 genes as interesting targets:
1) ADORA2A and MEIS2; genes that were previously shown to be downregulated in affected brain areas of HD patients
2) GTF2H5, RAD50, TP53, XAB2; genes involved in DNA damage
3) ATM, ATR, MRE11A, NBN; DNA damage genes previously shown to be implicated in HD
These three sets of genes were selected for a validation assay using qPCR, an alternative technique to study changes in gene expression. Unfortunately, we could not verify the microarray results, neither deregulation of the genes from set 1 nor set 2 was confirmed by qPCR. We found only two genes from set 3 that were deregulated: ATM and ATR. However, these results were not convincing, as they were not verified by a second qPCR analysis using a different control gene.
In conclusion, we were not able to show a convincing link between RNA toxicity and the DNA damage response pathway. At this point, the next step would be to try another approach to proof our hypothesis. We propose to use cell biology experiments like western blot and immunofluorescence as described in the fellowship application. However, we were not able to continue with these experiments as the fellowship was prematurely terminated, due to a unique new career opportunity for the fellow.
EXPECTED RESULTS AND IMPACT
The long-term goal of this research project is to contribute to a better understanding of the molecular mechanisms in Huntington’s disease. This knowledge might be translated as well to several other disorders of the neuronal system. These results should open new avenues in the therapeutic perspectives of these disorders. Next, our results will provide insights into non-coding RNA biology, a field of which knowledge is currently far from complete. Third, our studies will broaden our knowledge about the DNA damage response pathway, with a broad spectrum of implications in several diseases ranging from neurological disorders to cancer. Therefore, our results will contribute to the understanding of several research fields and the biological mechanisms underlying a broad spectrum of diseases. This knowledge will help society in general to increase their knowledge of biology, it will help assist the medical and pharmaceutical system to explore new therapeutic targets for neurological disorders and hopefully it might contribute to a cure for Huntington’s disease.