Mitochondrial Structural And Functional Analysis In Forebrain Specific Presenilin-1 And Presenilin-2 Double Knockout Mice

The huge amount of evidence has proved the importance of mitochondrial abnormalities and energy dysfunction in pathphysiological development of several neurodegenerative diseases, including Parkinson's disease (PD),Alzheimer's disease (AD), Amyotrophic lateralizing sclerosis (ALS) and Huntington disease (HD). Mitochondrial dysfunction has also been implicated in many age-dependent diseases, and the incidences of these diseases are increasing as the increase of old-age population. Despite of these, however, the accurate mechanisms of these diseases are not fully understood. The mitochondrial abnormalities and energy dysfunction are considered as early pathology phenomena. Mitochondria are the places that not only produce energy, but also produce ROS.Mitochondrial dysfunction would cause energy deficiency and oxidative stress, which would eventually lead to apoptosis. Mitochondria are crucial in neuronal apoptosis, participating not exclusively in the deficiency of membrane potential, the activation of caspase enzymes, and releasing of cytochrome c. Exploring mitochondrial functions and mechanisms under pathological settings shall help us to gain further insight into the underlying patholodical mechanisms as well as the pharmacological modifications of these neurodegenerative diseases.Mice lacking both presenilin 2 and presenilin 1 in the forebrain showed progressive neurodegenerative phenotypes including age-dependent memory decline, forebrain degeneration and hyperphosphorylated Tau. There is no Aβdeposit in the brain of this transgenic model, and the reason and mechanism of the pathology phenomena are not clarified yet. In this study, we investigated mitochondrial structural and functional abnormalities responsible for the presenilins deficiencies and the role of mitochondria in the pathology process in cDKO mice.Furthermore, as cDKO mice displayed AD-like symptoms without Aβdeposit, dose it mean that Aβdeposit is not necessary for AD development? We employed SHSY5Y cell to test the toxicity of Aβon mitochondria. The results demonstrated that Aβcaused mitochondrial dysfunction in SHSY5Y cell.In summary, given the fact that there are multifactorial effects that cause the loss of neurons, ventricular enlargement and memory decline, our data indicate that while the Aβ-induced functional changes in neuronal mitochondria is at least a very important element for accelerating neurodegenerative process, in particular in cDKO mice the PS deficiency-related mitochondria dysfunction may represent an important machnistic way involved in neurodegeneration.1.Investigating the mitochondrial structure and function in cDKO miceCortexs were taken from 3,6,9 and 12-month-old cDKO and WT mice and mitochondria were isolated from cortex via differential centrifugation. We observed the mitochondrial architecture, swelling, and vacuolation with electron microscope technique. The results showed no significant difference in the morphology of mitochondria from cDKO and WT mice until 12-month old. In accordance with the structural changes, the assessment of mitochondrial electron transfer chain in terms of the activities of complexesⅠ,Ⅱ,ⅢandⅣshowed, while no significant difference exhibited in the early stage, the cDKO mice of 9-and 12-month old showed a significant decline in complexⅠactivity. The analysis ofα-CaMKⅡand P-AMPK expression also proved abnormal metabolism in the older cDKO mice.Meanwhile, we analyzed several genes modulatingmitochondrial fusion and fission. We found Mfn2, Drpl and OPA1 mRNA expressions were decreased in 12-month old cDKO mice compared with WT mice, and OPA1 gene expression declined at early of 3-month old. These results indicated that the balance of fusion and fission of mitochondria was disrupted in cDKO mice. In addition to its modulating role in mitochondrial fusion process, OPA1 is also considered to be an antipoptosis protein. The loss of neurons and ventricular enlargement we observed in cDKO mice would be possibly related with the quick releasing of cytochrome C involved with OPA1. 2. Effects of Aβ1-42 on the structure and function of mitochondria in SHSH5Y cellWe treated the SHSY5Y cells with 10μM Aβ1-42 for 24h, followed by the measurement of mitochondrial potential, expression level of genes regulating the dynamic balance of mitochondria, the activities of enzymes of electron transfer chain, and the expression of cytochrome C oxidase and AMPK. These results revealed that Aβ1-42 could down-regulate mitochondrial potential, inhibit the activity of the complexes, destroy the dynamic balance between mitochondria fission and fusion, and disturb energy metabolism. However, the pattern for energy deficiency as well as the complex enzyme activeities inhibited and the dependence of AMPK were different from what we observed in cDKO mice independent of Aβdeposit, indicating a potential critical role of PS,not merely for y-secretase, in mitochondrial structure and function.