The Study Of Quality Control Mechanism Of Synaptic Mitochondria In Alzheimer’s Disease

Research BackgroundAlzheimer’s disease is one of the most common neurodegenerative diseases in the senile population. Most AD patients are sporadic cases, with insidious onset and slow progressive clinical course. As an aging-related disease, the prevalence of AD is increasing as the worldwide problem of ageing population is becoming more and more serious. Mitochondrial dysfunction is one of the most important mechanisms in the development of AD, mainly including several aspects such as abnormal mitochondrial dynamics, mitochondrial respiratory chain dysfunction, mitochondrial oxidative stress and DNA damage, and abnormal mitophagy. All these changings are closely associated with Aβ deposition. Synaptic mitochondria dysfunction is one of the early sign of AD and has a strong association with the mechanism of synaptic mitochondrial damage in AD. The findings of the accumulation of damaged synaptic mitochondria in AD brains and the critical role of mitophagy in removing mitochondria with poor quality seem to lead the credibility to the hypothesis that impaired mitophagic pathway underlies AD-relevant mitochondrial abnormalities.Mitophagy is a kind of macrophagy which can selectively remove damaged mitochondria and keep the mitochondria group healthy. It is a very conservative part of autophagy in evolution, which only focus on unhealthy mitochondria and remove them so that mitochondrial quality control can be regulated. While, the mechanism of mitophagy is not clear yet. Wildly accepted pathway of mitophagy in neural cells is PINK1/Parkin pathway. Studies have shown that, whether in maintaining normal function of neurons, or in the development of nervous system disease, mitochondrial autophagy mediated by PINK1/Parkin plays a very important role in mitochondrial quality regulation. We have enough supports to suppose that PINK1/Parkin mediated mitochondrial autophagy in AD is also very important in the mechanism of regulation of damaged synaptic mitochondria clearance and much damaged mitochondria aggregation.Changes of mitochondrial DNA copy number in AD brain aroused our interest in DNA quality control related protein. A-SUCL-β is a mitochondrial DNA quality control associated protein. Lack of gene SUCLA2, which encodes A-SUCL-P causes mitochondrial DNA depletion and kinds of syndromes of mitochondrial encephalomyopathy. SUCLA2 encode the β subunit of SUCL, which is also called protein A-SUCL-β. SUCL is an enzyme in the Krebs cycle, exists in the mitochondrial matrix, and participates in the reversible reaction which can catalyze succinyl-CoA and ADP or GDP into succinate, coenzyme A, and ATP or GTP. SUCL is a heterogeneous dimer, composed by a common alpha subunit SUCL-a, and a substrate specific beta subunits:A-SUCL-β (encoded by SUCLA2) or G-SUCL-β (encoded by SUCLG2). So, SUCL-a with A-SUCL-β can catalyze the synthesis of ATP, and similarly SUCL-a with G-SUCL-β can catalyze the synthesis of GTP. A-SUCL-β and G-SUCL-β are expressed tissue specifically. In human, A-SUCL-β is highly expressed in heart, brain and kidney, while G-SUCL-β is abundant in heart, kidney and liver, with very low level in brain, however. And in the brain tissue, A-SUCL-β specifically highly expressed in neurons, almost no expression in other cell types. This also support the specific clinical manifestation of encephalopathy caused by SUCLA2 mutations. All these details suggests that A-SUCL-p plays an important role in the development and maintaining of normal function of the neuron. Based on the mitochondrial DNA damage in AD and the important role of A-SUCL-β in neuron development and mitochondria DNA quality control, we supposed that A-SUCL-β might also attend the pathogenesis of AD, while data for this area remains blank.Research Goals1. To study the synaptic mitochondrial dysfunction and damaged mitochondrial quality control pathways in AD.2. To find more evidence of A-SUCL-β involving in AD synaptic mitochondrial quality control.Methodology1. AD mouse model.We used 5XFAD transgenic mouse as AD mouse model, and the non-transgenic littermate with same genic background as the control. For each cage, we had one 5XFAD transgenic male mouse and 1 or 2 non-transgenic SJL female mouse/mice for the first generation. Mice for research were all from the second generation in which half were 5XFAD heterozygous and half were non-transgenic control mice theoretically. Pups were weaned out to new cages when they were older than 3 weeks and then tagged by ear tag and identified by PCR with their tale samples.2. Synaptic mitochondria purification.All steps of synaptic mitochondria preparation have to be operated on ice or 4℃. Briefly, brain cortices were placed in a 5× volume of ice-cold isolation buffer. The tissue was homogenized with a Dounce homogenizer. The resultant homogenate was centrifuged at 1,300 x g for 5 min, and the supernatant was layered on a 3×2-mL discontinuous gradient of 15%,23%, and 40%(vol/vol) Percoll and centrifuged at 34,000 x g for 8 min. After centrifugation, band 2 (the interface between 15% and 23% containing synaptosomes) and band 3 (the interface between 23% and 40% containing non-synaptic mitochondria) were removed from the density gradient. The fractions were then resuspended in 20 mL of isolation buffer containing 0.02% digitonin and incubated on ice for 10 min. The suspensions were then centrifuged at 16,500 x g for 15 min. The resulting loose pellets were washed for a second time by centrifugation at 8,000 x g for 10 min. Pellets were collected and resuspended in isolation buffer. A discontinuous Percoll density gradient centrifugation was performed as described above for a second time. Band 3 was obtained and resuspended in isolation buffer to centrifuge at 16,500 x g for 15 min. The resultant pellet was washed in isolation buffer at 8,000 x g for 10 min. The final synaptic mitochondrial pellet was resuspended in isolation buffer and stored on ice. Protein concentration was determined using the Bio-Rad DC protein assay (BioRad Laboratories). Or mitochondria should be stored in 80℃ for later use.3. Westernblot analysis.Samples were lysed in 4X LDS loading buffer containing β-ME, separated by SDS/PAGE, and then transferred to a PVDF membrane. After blocking in TBST buffer (20 mM Tris-HCl,150 mM sodium chloride,0.1% Tween-20) containing 5% (wt/vol) nonfat dry milk for 1 h at room temperature, the membrane was then incubated and gently shaken overnight (at 4℃) with primary antibodies. This was followed by incubation with the corresponding secondary antibody for 1h at room temperature. After another 3 times of wash in TBS and then ECL incubation, blot was imaged in imaging system from BioRad. Image J software (National Institutes of Health) was used to analyze the scanned blots and to quantify protein signal intensity.4. Primary neurons cultureBriefly, brain cortexes were dissected from day 0-1 pups in cold HBBS (without Ca2+ and Mg2+), cut into tiny pieces, dissociated with 0.05% trypsin at 37℃ for 15 min, and then triturated with a pipette in NeuroNeurobasal A medium (Invitrogen). After nondispersed tissue settled for 3 min, the supernatant was transferred to a 15-mL tube and centrifuged for 5 min at 200 x g. The pellet was gently resuspended in neuron culture medium (Neurobasal A medium containing 2% B27 and 0.5 mM L-glutamine) and plated onto poly-D-lysine-coated culture plate. Half of the culture medium was replaced by fresh medium every 3 days.5.Mitochondiral DNA PCR amplificationEach PCR amplification reaction was set in a volume of 20μl with 0.4μg of purified synaptic mitochondria,200mM each of forward/reverse primers,0.2μl of Taq DNA polymerase and 2μl of 10X PCR buffer. Conditions on a master cycler gradient thermocycler were as follows:23 cycles of amplification (0.5min at 94℃,2 min at 55℃ and 40s at 72℃) and final extension for 10 min at 72℃. Amplified products were analyzed by electrophoresis in 2% agarose gel with ethidium bromide staining.6. Data analysisImages from westernblot and PCR were all captured by imaging system from BioRad and analyzed by ImageJ software from NIH by gray values. We used the gray values of mitochondrial loading control or whole cell loading control from same band as 1 and then acceded statistic analysis. One-way ANOVA analysis and Student T-Test were used for repeated measure analysis on SPSS. P< 0.05 was considered significant. All data were expressed as the mean±SEM.Results1. Synaptic mitochondrial dysfunction in 5xFAD transgenic miceMore Aβ deposition in AD brain from both 4-month-old and 8-month-old mice compared with control ones was observed by immunofluorescent staining. Data from Western blot and ELISA also showed that there was abundant Aβ in synaptic mitochondria in AD brain. At the same time, mtDNA PCR data suggested less mtDNA copy number in synaptic mitochondria of 8-month-old 5xFAD transgenic mice (p=0.02), while no significant change for 4-month-old ones.We also found dynamic imbalance of synaptic mitochondrial in 5xFAD transgenic mice. In 8-month-old mice, data showed less Mfn2 expressed in synaptic mitochondria of 5xFAD transgenic mice compared with control (p<0.01). Similar, the expression level of OPA1 was also lower significantly in 5xFAD transgenic mice (p=0.005). While more Dlp1 in AD synaptic mitochondria (p=0.001) compared with that in the control group.2. Dysfunction of mitophagy pathway in AD synaptic mitochondriaCompared with control group, more Parkin was trans-located to synaptic mitochondria in 5xFAD transgenic mice (p<0.001), while no difference of Parkin expression level in whole brain homogenate was found between two groups. There was no significant difference of PINK1 level between two groups, either in synaptic mitochondria or in whole brain homogenate.At the same time, higher P62 level was found in 8-month-old synaptic mitochondria and no change in whole brain homogenate. Similarly, more LC3 was found in AD synaptic mitochondria, suggesting that more synaptic mitochondria were tagged by LC3 and more mitophagosome formation in 5xFAD transgenic mice.From electron microscopy scanning images of brain section from AD and control mice, we observed a lot of accumulation of autophagosomes containing mitochondria at synapses in 5xFAD transgenic mice, which were mitophagosomes.3. Less A-SUCL-β in synaptic mitochondria of 5xFAD transgenic miceCompared with control mice with the same age, less A-SUCL-P was detected in synaptic mitochondria both in 4-month-old (p=0.03) and 8-month-old (p=0.02) 5xFAD transgenic mice. Between two age groups of non-transgenic control mice, there was also less A-SUCL-β in synaptic mitochondria of 8-month-old than those of 4-month-old mice, however, with no significant difference, suggesting that reduced A-SUCL-β was not caused only by aging.At the same time, we detected the expression level of A-SUCL-β in the brain homogenate, and didn’t find significant difference between 5xFAD transgenic mice and control group, supporting that this kind of down-regulation of A-SUCL-β lever is neuronal specific.4. The association between less A-SUCL-β and Aβ toxicity.We treated primary cultured neurons from non-transgenic C57 mice with Aβ oligomers and collected cells for Western blot. Data showed that less A-SUCL-β was found in neurons treated with 1μM Aβ oligomer compared with non-treated control groups. While in neurons only treated with H2O2, we didn’t find any change in A-SUCL-β level.5. Dysfunction of mitochondria quality control in SUCLA2-KO/KI cell linesWe did two kinds of genetic modifications to 293T cells, SUCLA2 knock-out and knock-in. Data from Western blot showed that OPA1 levels were higher both in SUCLA2 KO/KI cell lines compared with control (p<0.05), while no difference for Mfn2. Interestingly, we found also higher expression level of D1p1 both in SUCLA2 KO/KI cell lines compared with control group (p<0.05).We detected also mitophagy related proteins in cell lines, and data showed that more Parkin and LC3, while less PINK 1 was detected both in SUCLA2 KO and KI cell lines compared with control group (p<0.05).Conclusion1. Our data demonstrated that the induction of Parkin-mediated mitiophagy on synaptosomal mitochondria was strongly activated in 5xFAD transgenic mice; furthermore, the failure to remove damaged synaptic mitochondria in AD-relevant pathological state is more likely to be associated with deregulation of the late stages s of pathway or more specifically, the clearance of mitophagosomes.2. The level of A-SUCL-β is reduced in the synaptic mitochondria in AD, which is associated with the Aβ deposition in the synaptic mitochondria. So we can conclude that A-SUCL-β plays an important role in mitochondrial quality control in AD synaptic mitochondria.3. Our study has improved our understanding of synaptic mitochondrial pathology in AD and has implications for mitophagy regulation as a potential therapeutic strategy for the treatment of AD.