BDNF Induced Mitochondrial Stationary And Docking At Presynaptic Site Contribute To BDNF Enhanced Synaptic Transmission

BackgroundMitochondrion as an important organelle executes multiple functions including ATP production, maintaining intracellular Ca2+homeostasis and regulation of apoptosis. Particularly in neurons proper mitochondrial trafficking and distribution along neurites are involved in synaptic plasticity modulation such as presynaptically neurotransmitter release, postsynaptically membrane trafficking of ion channels and dendritic spine maturation.The mechanisms underlying mitochondrial motility is now revealing. In vivo and in vitro studies have identified several kinesin family members including KIF5A/B/C, KIF1Ba and KLP6involved in mitochondrial anterograde transport. In addition to the motor proteins, intracellular signaling pathways that regulate mitochondrial movement have also been investigated. It has been reported nerve growth factor (NGF) affects mitochondrial motility by its downstream phosphoinositide3-kinase (PI3K) signaling pathway. David Edelman’s group has demonstrated Akt-glycogen synthase kinase3β (GSK3β) is associated with axonal mitochondrial transport stimulated by serotonin and dopamine. Calcium has been demonstrated to be one of the critical factors that regulate mitochondrial transport. Recently, a complex composed of KIF5, Milton or TRAK and Miro1was reported to mediate mitochondrial transport along microtubules, and Miro1as the Ca2+sensor could regulate mitochondrial anterograde and retrograde movement in both axon and dendrite. Brain-derived neurotrophic factor (BDNF) is broadly expressed in central nervous system and plays critical roles in regulating neuronal survival, development and synaptic plasticity. The actions of BDNF are dictated by two classes of receptors on the cell surface:the TrkB and the p75receptors. Upon activation of TrkB receptor, several signaling pathways including MAP kinase (MAPK), PI3K and phospholipase-Cy (PLCy) are activated. It has been reported that in addition to regulation of synaptic plasticity, BDNF could stimulate brain mitochondrial metabolism by increasing the efficiency of respiratory coupling and ATP synthesis. However, through which mechanism BDNF regulates mitochondrial function is still unclear. In particular, it’s still unknown whether BDNF could regulate mitochondrial transport and distribution in neurons.In this study we investigated the effects of BDNF on mitochondrial motility in primary hippocampal-cortical neurons. We report that BDNF treatment could induce mitochondrial docking and accumulation at presynaptic site, which effect is mediated by elevated intracellular Ca2+through PI3K, PLCy signaling pathways and TRPC channels in a Mirol-dependent manner.Results1. BDNF treatment induces reduced mitochondrial motility in neuritesTo measure mitochondrial movement, we carried out live-cell microscopy of rat neurons.We discoverd that BDNF treated neurons for15min significantly reduced mitochondrial motility. To determine whether BDNF could affect other organelles movement, we labeled Rab5with GFP-tag to indicate the motility of endosome. No changes in the percentage of moving endosomes were observed upon BDNF treatment, indicating that the effect of BDNF on mitochondria motility is specific.We further investigated whether BDNF induced mitochondrial stopping was associated with mitochondrial membrane potential and ATP production. After BDNF treatment for15min, we observed neither membrane potential nor ATP production was affected.2. The activation of PI3K and PLCy signaling pathways are required for BDNF induced mitochondrial stoppingSince BDNF treatment triggered more mitochondria into the stationary pool, we next investigated the mechanisms underlying this phenomenon. We applied inhibitors to block the signaling pathway and then demonstrate thet BDNF induced mitochondrial stationary depends on TrkB-PLCy-IP3R and TrkB-PI3K signaling pathway.3. Elevation of intracellular Ca2+is necessary for BDNF induced mitochondrial stoppingIt has been known that BDNF induces the elevation of intracellular Ca2+, and Ca2+is an important regulator of mitochondrial movement. We hypothesize that Ca2+plays an important role in BDNF regulated mitochondrial motility. So we used permeable Ca2+buffer BAPTA-AM and extracellular Ca2+depletion reagent EGTA to block Ca2+elevation. We investigated that pre-incubation with BAPTA-AM or EGTA, which blocked BDNF-induced intracellular Ca2+elevation, could abolish the effect of BDNF on mitochondrial mobility.Multiple evidences have demonstrated TRPC channels are Ca2+permeant channels activated by BDNF. We next investigated whether they are involved in BDNF evoked stopping of mitochondria. When primary hippocampal-cortical neurons were bath incubated with SKF-96365, a known nonspecific TRP channels inhibitor, BDNF induced mitochondrial stopping was completely abolished.4. Mirol mediates BDNF induced mitochondrial stationaryWe next investigated how the intracellular Ca2+regulated mitochondrial motility in response to BDNF. Recently, several studies have demonstrated that Miro1, a mitochondrial outer-membrane protein, acts as a Ca2+sensor to regulate mitochondrial motility. We questioned whether Miro1also plays a role in BDNF regulated mitochondrial movement. To address this question, we transfected wild type (wt) or mutant Miro1(Miro1KK, EF hand mutation resulting in disability of binding with Ca2+) into cultured hippocampal-cortical neurons, and found in neurons expressing Miro1KK, BDNF lost the ability to cause mitochondria stationary, which suggests that Mirol mediates BDNF induced mitochondria stopping by binding with Ca2+. 5. BDNF induced mitochondria into the stationary pool contributes to the effect of BDNF on synaptic transmissionSince mitochondria provides energy and calcium buffering which are important for neuronal function especially for synaptic plasticity, we asked whether BDNF induced arrested mitochondrial moving played a role in the BDNF mediated synaptic plasticity modulation. After investigating BDNF treated neurons which were transtected with Miro1and Miro1KK, we found the percentage of mitochondria colocalized with SV2punctas significantly increased. These data suggested BDNF treatment induces more mitochondria accumulation at presynaptic site and Mirol is necessary.We next demonstrated whether BDNF induced Miro1-dependent mitochondrial presynaptic accumulation is associated with BDNF modulated synaptic plasticity. FM1-43was used to label the presynaptic sites. The neurons expressing wtMiro1or Miro1KK had been investigated. Consistent with previous reported, BDNF could increase K+evoked neurotransmitter release in control neurons. However, this effect was blocked by Miro1KK mutant but not wtMiro1, suggesting Miro1as Ca2+sensor mediates BDNF enhanced neurotransmitter release.Conclusion1. BDNF treatment induces reduced mitochondrial motility in neurites.2. Elevation of intracellular Ca2+is necessary for BDNF induced mitochondrial stopping.3. Miro1mediates BDNF induced mitochondrial stationary.4. BDNF induced mitochondria into the stationary pool contributes to the effect of BDNF on synaptic transmission.