The Study Of HUMMR On Hypoxia Exposure Induced Neuronal Mitochondria Injury

Owing to low atmospheric pressure caused by high altitude, the oxygen content in Tibetan Plateau area is only ~13%, which is far less than that in flat area(~21%). Researches have reported that long-term exposure to high altitude environment will affect the development and function of nervous system, leading to learning and memory impairment.Generally thinking, the function of hippocampus is responsible for learning and memory construction, and hippocampal neurons are critical for the process of learning and memory.The Hypoxia up-regulated mitochondrial movement regulator, HUMMER, is a mitochondrial protein, which locates on the mitochondrial outer membrane. Previous studies have found that HUMMR is a hypoxic stress protein and plays a regulatory role in mitochondria axonal transport. However, it had not been reported that whether HUMMER is involved in hypoxia-induced mitochondrial damage. MEF2s(myocyte enhancer factor 2 family), initially identified as nuclear factors for muscular cell differentiation, have been revealed to play a crucial role in the activation of the genetic programs that regulate cell proliferation, differentiation, and apoptosis in multiple cells. Recent studies have demonstrated that MEF2 D exists in rodent neuronal mitochondria and can regulate the expression of mitochondrial DNA(mt DNA). Given to the essential role of MEF2 D and HUMMER in regulating mitochondrial function, our study will focus on their relationship in hypoxia-induced mitochondrial damage and possible molecular mechanisms.Objective Through the establishment of in vitro model of hypoxia-induced mitochondrial damage, our objectives are to determine the effect of hypoxia on neuronal mitochondrial damage and clarify the role of HUMMR protein in hypoxia-induced mitochondrial damage. Next, we try to explore the role of MEF2 D and HUMMER in regulating mitochondrial function. The results of the study will provide guidelines for the prevention and treatment of the mitochondria injury related diseases induced by hypobaric hypoxia exposure.Methods 1. In vitro hypoxia exposure model: primary cultured rat hippocampal neurons and SH-SY5 Y cells were treatment with 1% O2 environment. The 1% O2 environment is set by the hypoxic workstation produced by DWS Company. 2. Plasmids Construction: HUMMR-GFP over-expression plasmids and HUMMR-sh RNA lentivirus plasmids. 3. Lentivirus plasmid Construction: using ps-PAX2, p MD2.G, HUMMR-sh RNA-PLKO recombinant lentiviral vector co-transfected with 293 T cells, packed into a complete virus. 4. Immunofluorescence staining: The Mito-Tracker Red mitochondrial fluorescent probes and p Ds Red2-Mito plasmids were used to mitochondrial staining and observe mitochondrial distribution and morphology by confocal microscope.5. Mitochondrial membrane potential detection: By JC-1 fluorescence probe staining and observed by fluorescence microscopy. 6. Ultrastructure of mitochondria observation by electron microscope. 7. Related protein expression levels were detected by Western blot methods.Results. 1. The effect of hypobaric hypoxia exposure on the structure and function of mitochondria: Both SH-SY5 Y cells and primary hippocampal neurons showed fragmentations in mitochondria after exposure to 1% oxygen concentration for 24 h and 48 h compared to control. What’s more, the results of mitochondrial membrane potential detection showed a significant decrease in experiment group compared with that in control group. 2. The expression of related proteins under hypoxia conditions: Western blot results showed that the protein levels of HUMMER, MEF2 D and mitochondrial related proteins(MFN2)in hypoxia exposure group were far more lower than those in control group. 3. The expression of HUMMER protein on hypoxia-induced mitochondrial fragmentation: Owing to result of the down-expression of HUMMER protein in neurons under hypoxia conditions, the HUMMR-GFP expression plasmids were constructed and transfected into SH-SY5 Y cells and primary cultured hippocampus neurons. The immunofluorescence staining results showed reduced mitochondrial fragmentation and western blot results showed a recovery of MFN2 protein expression in high-expression group comparing to control group under 1% hypoxia conditions. 4. The effect of interference with HUMMER protein expressions on hypoxia-induced mitochondrial fragmentation: Small hairpin RNA(sh RNA) was designed against HUMMR protein and delivered by adenovirus to SH-SY5 Y cells and primary cultured hippocampus neurons cultured under 1% hypoxia and normal condition. Under 1% hypoxia conditions, the fragmented mitochondria in neurons interfered with sh RNA were much larger than that of neurons treated with sham vectors, and reduced HUMMR expression aggravated the down-expression of MFN2 protein. 5. The regulation of MEF2 D on the expression of HUMMER protein and mitochondrial fragmentation. 1) Compared with the control group, the protein expression of MEF2 D in mice hippocampus tissues and SH-SY5 Y cells in hypoxia exposure group were reduced. 2) Transfection with MEF2 D plasmids helped reduce the percentage of fragmented mitochondria in neurons exposed to hypoxia and attenuate the down expression of HUMMER proteins in some degree.Conclusion. In this study, we established the in vitro model to confirm that hypoxia led to neural mitochondria fragmentation and reduction of mitochondrial membrane potentials. Our results also showed that HUMMR protein was involved in the process of mitochondria fragmentation, up-regulation of HUMMER protein reduced the percentage of mitochondrial fragmentation and down-regulation of HUMMER increased the percentage. What’s more, the nuclear transcription factor MEF2 D influenced the expression of the mitochondria fusion protein MFN2 levels through increasing the expression of HUMMER protein, eventually leading to the mitochondria fragmentation. The results will provide guidelines for the prevention and treatment of the mitochondria injury related diseases induced by hypoxia exposure.