AQP9 in Parkinson's disease and diabetes
1 Formål:
Aquaporin-9 is an aquaglyceroporin expressed in liver and to a lesser extent in the brain. Data from our laboratory have shown that genetic deletion of AQP9 (AQP9-KO) is protective in models of Parkinson's disease. Previous reports have shown that diabetic mice are more vulnerable to toxin-induced PD. To investigate whether AQP9 expression is increased in the brain of diabetic mice. We will analyze the expression levels of AQP9 (mRNA and protein) in different brain regions of mice with Type 1 diabetes mellitus (DM1) and type 2 diabetes mellitus (DM2). DM1 will be induced by injection of Streptozotocin, while we will purchase mouse models of DM2 (LPR db/db from Jacksons).
2 Skadevirkninger
We will use well-established models. There will not be any additional damage to the animals or the environment. Mice injected with Streptozotocin will develop diabetes, which is the purpose of the project. There are reports of other long-term effects such as diabetic neuropathy and hyperalgesia (REF: Cunha, J.M., Funez, M.I., Cunha, F.Q., Parada, C.A., & Ferreira, S.H.. (2009). Brazilian Journal of Medical and Biological Research, 42(2), 197-206. https://doi.org/10.1590/S0100-879X2009000200008), but these effects appear at a later time point than the scope of our experiments. So, since we are going to sacrifice the animals within two weeks after the injections these effects are not considered as relevant for our experiments.
3 Forventet nytteverdi
If successful, this project will give valuable knowledge about the link between diabetes mellitus and Parkinson’s disease which may be used for diagnosis or development of new diagnostic and/or therapeutical strategies.
4 Antall dyr og art
30 C57BL6 WT mice (male, from our internal AQP9- breeding colony) (half will be treated with Streptazotocin to induce diabetes type 1)
10 AQP9-KO from the same breeding colony as the mice above
10 Leptin receptor KO mice (LPR db/db; a model for type 2 diabetes mellitus) and 10 sex-and age-matched WT mice fra Jacksons laboratories
5 Hvordan etterleve 3R
Since we need to study effect of a systemic disease on brain, we can not use cell lines or other reduced models and will have to use animals. We have tried to use the same animal for several techniques- by dividing the brain into two hemispheres (where the same animal can be used for protein and mRNA measurements) or using a perfusion method which enables use to use the tissue for light and electron microscopy).
We are going for the minimum number of animals we can use to get a quantitative result. We will be analyzing the brains with four different techniques. We will divide the brains into two hemispheres and use each hemisphere for each technique. It will give 5 samples from each experimental setting (diabetes type 1 and diabetes type 2) for each technique. Based on the previous experiments analyzing AQP9 levels, n=5 is the minimum we can use. For the DM1 mice, we will only be using mice that develop DM1 and there is a possibility that not all the mice will develop DM1.
Aquaporin-9 is an aquaglyceroporin expressed in liver and to a lesser extent in the brain. Data from our laboratory have shown that genetic deletion of AQP9 (AQP9-KO) is protective in models of Parkinson's disease. Previous reports have shown that diabetic mice are more vulnerable to toxin-induced PD. To investigate whether AQP9 expression is increased in the brain of diabetic mice. We will analyze the expression levels of AQP9 (mRNA and protein) in different brain regions of mice with Type 1 diabetes mellitus (DM1) and type 2 diabetes mellitus (DM2). DM1 will be induced by injection of Streptozotocin, while we will purchase mouse models of DM2 (LPR db/db from Jacksons).
2 Skadevirkninger
We will use well-established models. There will not be any additional damage to the animals or the environment. Mice injected with Streptozotocin will develop diabetes, which is the purpose of the project. There are reports of other long-term effects such as diabetic neuropathy and hyperalgesia (REF: Cunha, J.M., Funez, M.I., Cunha, F.Q., Parada, C.A., & Ferreira, S.H.. (2009). Brazilian Journal of Medical and Biological Research, 42(2), 197-206. https://doi.org/10.1590/S0100-879X2009000200008), but these effects appear at a later time point than the scope of our experiments. So, since we are going to sacrifice the animals within two weeks after the injections these effects are not considered as relevant for our experiments.
3 Forventet nytteverdi
If successful, this project will give valuable knowledge about the link between diabetes mellitus and Parkinson’s disease which may be used for diagnosis or development of new diagnostic and/or therapeutical strategies.
4 Antall dyr og art
30 C57BL6 WT mice (male, from our internal AQP9- breeding colony) (half will be treated with Streptazotocin to induce diabetes type 1)
10 AQP9-KO from the same breeding colony as the mice above
10 Leptin receptor KO mice (LPR db/db; a model for type 2 diabetes mellitus) and 10 sex-and age-matched WT mice fra Jacksons laboratories
5 Hvordan etterleve 3R
Since we need to study effect of a systemic disease on brain, we can not use cell lines or other reduced models and will have to use animals. We have tried to use the same animal for several techniques- by dividing the brain into two hemispheres (where the same animal can be used for protein and mRNA measurements) or using a perfusion method which enables use to use the tissue for light and electron microscopy).
We are going for the minimum number of animals we can use to get a quantitative result. We will be analyzing the brains with four different techniques. We will divide the brains into two hemispheres and use each hemisphere for each technique. It will give 5 samples from each experimental setting (diabetes type 1 and diabetes type 2) for each technique. Based on the previous experiments analyzing AQP9 levels, n=5 is the minimum we can use. For the DM1 mice, we will only be using mice that develop DM1 and there is a possibility that not all the mice will develop DM1.