Final Report Summary - LEARNING AND MEMORY (The zebrafish as a new vertebrate model for molecular and cellular mechanisms of learning and memory, including synaptic dysfunction in Alzheimer's disease)
A majority of our most common neurological diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, age-related dementia and multiple sclerosis, are manifested by memory loss and a reduced potential for learning. Additionally, a substantial portion of our population suffers from various forms of learning difficulties. Despite all the efforts to solve the mystery of learning and memory, there has not been any complete cellular model of learning in a vertebrate model available.
The zebrafish (Danio rerio) is one of the newer model systems that has become a strong candidate for biomedical research. The strengths of the zebrafish are the developed transgenic methods and its sequenced genome. We have used this model to study how molecular, cellular and network modifications underlie learning and memory. In parallel, we have investigated the usefulness of the model to study memory deficiencies induced by Alzheimer-associated proteins, such as Amyloid beta.
The goal has been to establish the links between genes, molecular mechanisms, network function, and behavior of learning and memory, to help us understand both the normal and diseased brain. The project nicely combines basic and clinical research, as well as uses a multidisciplinary approach (the implementation of the latest developed methods in chemistry and physics to neuropsychological memory testing) that is still a unique feature of the zebrafish as a model system.
We have chosen to study the learning of a fairly simple behavior, i.e. the startle response to able to localize synaptic changes in the network underlying the behavior. Pathological processes affecting the learning of this behavior will be easier to track since we know the underlying network and its structures. In this project, we have developed new behavioral protocols to study learning of this behavior. The transmitter systems involved in mediating the learning has also been investigated and methods of in vivo recordings of cellular activity during the learning were developed. A review of calcium imaging recordings in the zebrafish including the experiences from these studies was published in: Kettunen, P. (2012) Calcium imaging in the zebrafish. In: Calcium Signaling. Advances in Experimental Medicine and Biology, Vol. 740,p. 1039-1072, ed. S. Islam.
The next step was to develop a new model for memory impairments related to AD, based on injections of the AD protein Abeta into the larval zebrafish. We were able to show that learning is indeed blocked by Abeta, and that commonly used AD medications are protecting against this memory impairments. This opens up the possibility to use the zebrafish as a future tool for drug discovery. These findings are described in an unpublished manuscript.
We have also studied the function of another AD-related protein, amyloid precursor protein (APP), from where Abeta is being cleaved. To this end, we knocked-down one homologue of APP in the zebrafish, app-b, using morpholinos and studied axonal outgrowth, behavioral phenotypes and synaptic formation. Interestingly, we were able to show that app-b is involved in axonal outgrowth, synaptic formation and regulation of the startle response. The next step is to investigate the function of APP in learning and synaptic plasticity using our zebrafish learning model. The experiments are described in: Abramsson, A., Kettunen, P., Lott, E., Li, M., Arnér A., and Zetterberg, H. (2013) The zebrafish amyloid precursor protein-b is required for motor neuron guidance and synapse formation. Developmental Biology 381:377–388.
The zebrafish has a great potential to serve as a means to study the function of disease genes found in patient materials. We have access of large collections of biological samples from patients suffering from AD and other neurodegenerative diseases that can serve as a means to understand associations between gene variants and the risk of developing neurodegenerative diseases. Then, the functional implications of these gene variants can be tested in the zebrafish. One important gene for learning processes is ARC and therefore we performed single nucleotide (SNP) analysis of this gene in cerebrospinal fluid (CSF) from patients diagnosed with AD. The result showed that one gene variant of ARC, including a SNP in a region regulating its professing, is reducing the risk of developing AD. Unfortunately, a homologue of ARC has not yet been reported in the zebrafish, but transgenic methods will still allow for further analysis of the gene function in this model. This paper was the first one reporting a genetic association of ARC and the risk of developing a disease. (Landgren, S., von Otter, M., Nilsson S., Seibt Palmér, M., Lundvall, C., Lennart Minthon, L., Bogdanovic, N., Andreasen, N., Gustafson, D.R. Skoog, I., Wallin, A., Blennow, K., Zetterberg, H. and Kettunen P. (2012) A novel ARC gene polymorphism is associated with reduced risk of Alzheimer's disease. Journal of Neural Transmission 119(7):833-42.)
This project shows the potential of using the zebrafish to study gene function, synaptic plasticity and behavior related to normal function and disease states. We are now developing our preparations to serve as tools for developing new therapies for CNS diseases, particularly neurodegenerative diseases. We are convinced that the zebrafish will mean a more efficient and highly cost-effective way to develop novel and better treatments for AD. This will mean that we can replace initial studies on rodents and reduce the experiments in higher vertebrates.
The zebrafish (Danio rerio) is one of the newer model systems that has become a strong candidate for biomedical research. The strengths of the zebrafish are the developed transgenic methods and its sequenced genome. We have used this model to study how molecular, cellular and network modifications underlie learning and memory. In parallel, we have investigated the usefulness of the model to study memory deficiencies induced by Alzheimer-associated proteins, such as Amyloid beta.
The goal has been to establish the links between genes, molecular mechanisms, network function, and behavior of learning and memory, to help us understand both the normal and diseased brain. The project nicely combines basic and clinical research, as well as uses a multidisciplinary approach (the implementation of the latest developed methods in chemistry and physics to neuropsychological memory testing) that is still a unique feature of the zebrafish as a model system.
We have chosen to study the learning of a fairly simple behavior, i.e. the startle response to able to localize synaptic changes in the network underlying the behavior. Pathological processes affecting the learning of this behavior will be easier to track since we know the underlying network and its structures. In this project, we have developed new behavioral protocols to study learning of this behavior. The transmitter systems involved in mediating the learning has also been investigated and methods of in vivo recordings of cellular activity during the learning were developed. A review of calcium imaging recordings in the zebrafish including the experiences from these studies was published in: Kettunen, P. (2012) Calcium imaging in the zebrafish. In: Calcium Signaling. Advances in Experimental Medicine and Biology, Vol. 740,p. 1039-1072, ed. S. Islam.
The next step was to develop a new model for memory impairments related to AD, based on injections of the AD protein Abeta into the larval zebrafish. We were able to show that learning is indeed blocked by Abeta, and that commonly used AD medications are protecting against this memory impairments. This opens up the possibility to use the zebrafish as a future tool for drug discovery. These findings are described in an unpublished manuscript.
We have also studied the function of another AD-related protein, amyloid precursor protein (APP), from where Abeta is being cleaved. To this end, we knocked-down one homologue of APP in the zebrafish, app-b, using morpholinos and studied axonal outgrowth, behavioral phenotypes and synaptic formation. Interestingly, we were able to show that app-b is involved in axonal outgrowth, synaptic formation and regulation of the startle response. The next step is to investigate the function of APP in learning and synaptic plasticity using our zebrafish learning model. The experiments are described in: Abramsson, A., Kettunen, P., Lott, E., Li, M., Arnér A., and Zetterberg, H. (2013) The zebrafish amyloid precursor protein-b is required for motor neuron guidance and synapse formation. Developmental Biology 381:377–388.
The zebrafish has a great potential to serve as a means to study the function of disease genes found in patient materials. We have access of large collections of biological samples from patients suffering from AD and other neurodegenerative diseases that can serve as a means to understand associations between gene variants and the risk of developing neurodegenerative diseases. Then, the functional implications of these gene variants can be tested in the zebrafish. One important gene for learning processes is ARC and therefore we performed single nucleotide (SNP) analysis of this gene in cerebrospinal fluid (CSF) from patients diagnosed with AD. The result showed that one gene variant of ARC, including a SNP in a region regulating its professing, is reducing the risk of developing AD. Unfortunately, a homologue of ARC has not yet been reported in the zebrafish, but transgenic methods will still allow for further analysis of the gene function in this model. This paper was the first one reporting a genetic association of ARC and the risk of developing a disease. (Landgren, S., von Otter, M., Nilsson S., Seibt Palmér, M., Lundvall, C., Lennart Minthon, L., Bogdanovic, N., Andreasen, N., Gustafson, D.R. Skoog, I., Wallin, A., Blennow, K., Zetterberg, H. and Kettunen P. (2012) A novel ARC gene polymorphism is associated with reduced risk of Alzheimer's disease. Journal of Neural Transmission 119(7):833-42.)
This project shows the potential of using the zebrafish to study gene function, synaptic plasticity and behavior related to normal function and disease states. We are now developing our preparations to serve as tools for developing new therapies for CNS diseases, particularly neurodegenerative diseases. We are convinced that the zebrafish will mean a more efficient and highly cost-effective way to develop novel and better treatments for AD. This will mean that we can replace initial studies on rodents and reduce the experiments in higher vertebrates.