The Effect Of Hemojuvelin Gene On Learning And Memory And Exercise Tolerance In Mice

Hemojuvelin was found when studying the juvenile hemochromatosis.So far,there is no effective treatment for juvenile hemochromatosis,largely due to our poor understanding of its underlying mechanisms.With the improvement of people’s living,the high level of iron is closely related to the occurrence of many metabolic syndromes.The disorders of iron metabolism have serious effects on relevant organs,and the imbalance of iron homeostasis induces many disease which threatening to people’s health.Although some researchers has found close relationship between brain iron deposition and cognitive dysfunction,the deficit of learning and memory and exercise endurance in hemojuvelin knockout mice has not been reported.And the underlying mechanisms are unclear.In present study,we’ investigated the learning and memory and exercise endurance in adult male wild type and knockout mice using behavioral experiments,combined with western blot,real-time PCR and immunohistochemistry.The purpose of this study is to discover characteristics and possible molecular mechanisms of hemojuvelin knockout mice,and provide new drug candidates and molecular targets for the treatment of iron accumulation and muscle-related diseases.We first observed that the deficit of learning and memory in hemojuvelin knockout mice,and apoptosis in hippocampus and prefrontal cortex.And,we also found that exercise endurance was reduced in hemojuvelin knockout mice,and the TGF-β1/Smad3 pathway was actived in gastrocnemius.These results are as follows:1.The deficit of recognition memory and patial reference memory in hemojuvelin knockout miceIn novel object recognition task experiments,we found a significant different in test phase performance in wild type mice between novel object preference index and familiar object preference index,but not in hemojuvelin knockout mice.In Morris water maze test,we found that with the increase of training days the latency decrease gradually in knockout mice and wild type mice,and in the fourth and fifth day the knockout mice have a longer latency compared to wild type mice.Knockout mice had decreased numbers of crossing the platform and spent less time in target quadrant compared to wild type mice.The results showed that recognition memory and spatial reference memory were damaged in hemojuvelin knockout mice.2.The apoptosis was triggered in hippocampus and prefrontal cortex of hemojuvelin knockout miceWestern blot showed that the ratio of Bax/Bcl-2 was significantly increased in hippocampus and prefrontal cortex,compared with wild type mice.Real-time PCR showed that in hippocampus and prefrontal cortex,the level of Bax mRNA was increased,compared with wild type mice.And,the level of Bcl-2 mRNA was decreased,compared with wild type mice.3.The deficit of exercise endurance in hemojuvelin knockout miceThe result of exercise endurance showed that,it was significantly increased in running distance and time,compared with hemojuvelin knockout mice.This result indicated that the exercise endurance was reduced in hemojuvelin knockout mice.4.Hemojuvelin led to decrease of Troponin-I SS and increase of Troponin-I FS in gastrocnemius and soleusWestern blot results showed that the expression of Troponin-I SS was significantly decrease,and Troponin-I FS have an increasing trend in gasttrocnemius,compared to wild type mice.The expression of Troponin-I SS was decreased,and Troponin-I FS have an increasing trend in soleus,compared to wild type mice.5.Hemojuvelin led to the decrease of slow-twitch muscle fiber and increase of fast-twitch muscle fiber in gastrocnemius and soleusReal-time PCR result showed that the mRNA levels of MHC-I,MHC-IIa and MHC-Ⅱd were also significantly decreased,but MHC-IIb mRNA was significantly increased compared with the gastrocnemius of wild type mice.The mRNA levels of MHC-I,MHC-Ⅱa and MHC-IId were also decreased,but MHC-IIb mRNA was significantly increased,compared with the soleus of wild type mice.The result of immunofluorescence staining showed that the content of MHC-IIa muscle fibers decreased significantly,and MHC-IIb muscle fibers increased significantly in hemojuvelin knockout mice,compared with the gastrocnemius of wild type mice.6.TGF-β1/Smad3 pathway was activated after hemojuvelin knockout in gastrocnemiusThe results of western blot showed that the protein expression of TGF-β1,TβRⅡ(P<0.05),p-Smad3 and p-Smad2 were significantly increased,but not p-Smad1/5/8 and Smad1/5/8,compared with the gastrocnemius of wild type mice.This result indicated that TGF-β1/Smad3 pathway was activated after hemojuvelin knockout in gastrocnemius.7.TGF-β1/Smad3 pathway was activated after interference hemojuvelin in myoblast cell C2C12The results of western blot showed that the protein expression of hemojuvelin was significantly decreased,while TGF-β1、TβRⅡ、p-Smad3 and p-Smad2 were also significantly increased,but not p-Smad1/5/8 and Smad1/5/8,compared with the ConsiRNA group.This result indicated that TGF-βl/Smad3 pathway was activated after interference hemojuvelin in myoblast cell C2C12.Conclusion:Recognition memory and spatial reference memory were damaged in hemojuvelin knockout mice,and trigged excessive apoptosis in hippocampus and prefrontal cortex,which may be one of the molecular mechanisms for learning and memory damaging.The exercise endurance was decreased in hemojuvelin knockout mice,and slow-to-fast muscle fibers transition was the direct reason for the decline of aerobic exercise endurance.TGF-β1/Smad3 signaling pathway was activated in gastrocnemius,which was one of molecular mechanisms for the reduction in aerobic exercise endurance in hemojuvelin knockout mice.