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. Author manuscript; available in PMC: 2019 Aug 16.
Published in final edited form as: J Neurochem. 2018 Aug 16;146(4):403–415. doi: 10.1111/jnc.14484

Cyclophilin D regulates neuronal activity-induced filopodiagenesis by fine-tuning dendritic mitochondrial calcium dynamics

Shaomei Sui 1,2, Jing Tian 2, Esha Gauba 2, Qi Wang 1,2, Lan Guo 2, Heng Du 1,2,*
PMCID: PMC6107423  NIHMSID: NIHMS975503  PMID: 29900530

Abstract

Recent studies have highlighted the role of mitochondria in dendritic protrusion growth and plasticity. However, the detailed mechanisms that mitochondria regulate dendritic filopodia morphogenesis remain elusive. Cyclophilin D (CypD, gene name: Ppif) controls the opening of mitochondrial permeability transition pore (mPTP). Although the pathological relevance of CypD has been intensively investigated, little is known about its physiological function in neurons. Here, we have found that genetic depletion of or pharmaceutical inhibiton of CypD blunts the outgrowth of dendritic filopodia in response to KCl-stimulated neuronal depolarization. Further cell biological studies suggest that such inhibitory effect of CypD loss-of-function is closely associated with compromised flexibility of dendritic mitochondrial calcium regulation during neuronal depolarization, as well as the resultant changes in intra-dendritic calcium homeostasis, calcium signaling activation, dendritic mitochondrial motility and redistribution. Interestingly, loss of CypD attenuates oxidative stress-induced mitochondrial calcium perturbations and dendritic protrusion injury. Therefore, our study has revealed the physiological function of CypD in dendritic plasticity by acting as a fine-tuner of mitochondrial calcium homeostasis. Moreover, CypD plays distinct roles in neuronal physiology and pathology.

Keywords: mitochondria, mitochondrial permeability transition, cyclophilin D, dendritic spine morphogenesis, depolarization

Summarizing schematic

Dendritic spinogenesis are energy demanding and regulated by local calcium transients. Cyclophilin D (CypD)-mediated mitochondrial permeability transition (mPT) is a critical pathway for mitochondrial release. But whether CypD-medaited mPTP contributes to neuronal activity-induced dendritic spinogenesis is unknown. Here, we have found that transient opening of CypD-mediated (mPTP) regulates intra-dendritic calcium dynamics by mediating mitochondrial calcium release and the downstream signaling, eventually promoting activity-induced dendritic protrusion outgrowth. This phenomenon highlights the role of CypD in neuronal physiology and implicates the limitation of CypD inhibition as a preventive strategy for diseases.

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Introduction

Mitochondria are vital organelles for neuronal physiology. In recent years, emerging evidence has implicated the role of mitochondria in the regulation of dendritic protrusion plasticity in response to neuronal activity (Li et al. 2004, Sung et al. 2008). Dendritic protrusions are critical for synaptic plasticity and transmission in neurons. Heterogeneous in shape, size and function, dendritic protrusions are recognized as dendritic spines and filopodia (Hering & Sheng 2001). Generally accepted as the precursors of spines, filopodia demonstrate apparent differences from spines in several aspects such as the shape and capacity to form synapses, as well as the content of organelles and some key molecules (Ziv & Smith 1996, Hering & Sheng 2001). It has been repeatedly identified that the emergence and remodeling of dendritic filopodia are energy demanding and regulated by local calcium transients in a sophisticated manner (Hu & Hsueh 2014, Lohmann et al. 2005, Sala & Segal 2014). With little dispute that mitochondria are the major energy provider in dendrites, but whether and how mitochondria regulate dendritic filopodiagenesis through its function in modulating local calcium homeostasis is still unclear.

Cyclophilin D (CypD, gene name: Ppif−/−) is a mitochondrial peptidyl-prolyl cis-trans isomerase (PPIase) that locates in mitochondrial matrix. Albeit its function has not been fully elucidated, CypD is a genetically determined regulator that promotes mitochondrial permeability transition (mPT) (Baines et al. 2005, Nakagawa et al. 2005). mPT through the formation of mPT pore (mPTP) refers to increased permeability of inner mitochondrial membrane (IMM) to ions and small solutes (Baines et al. 2005, Nakagawa et al. 2005). A pronounced consequence of mPTP opening is mitochondrial calcium release. Although mPTP has been recognized for decades, its role in cell biology still remains puzzling. In addition to its pathological relevance to diseases (Du et al. 2008, Gauba et al. 2017, Schinzel et al. 2005, Thomas et al. 2012, Kim et al. 2012), increasing evidence has suggested the physiological function of mPTP (Kwong & Molkentin 2015, Agarwal et al. 2017, Hausenloy et al. 2004, Saotome et al. 2009). Such conflicting roles of mPTP are reconciled by the concept that transient mPTP opening is a physiological event for the maintenance of normal mitochondrial calcium homeostsis; while prolonged mPTP is detrimental (Kwong & Molkentin 2015). Of note, mPTP is an identified mitochondrial calcium release pathway and blockade of mPTP increases mitochondrial calcium retension. If it holds true that the transient opening of CypD-mediated mPTP regulates mitochondrial calcium dynamics under physiological conditions, it would be hypothesized that the loss of CypD could potentially affect dendritic filopodia growth in response to neuronal activity.

In the current study we test the hypothesis by employing primary cultured neurons from CypD-deficient (Ppif−/−) pups and their nontransgenic (Ppif+/+) littermates. Although showing similar baseline density of dendritic protrusions, Ppif−/− neurons exhibit deterred filopodia growth in response to neuronal activity. Moreover, such effect is replicated by pharmaceutical CypD inhibition. Further analysis shows that such inhibitory effect of CypD deficiency on filopodia emergence is closely associated with decreased flexibility of dendritic mitochondrial calcium dynamics, which alters the patterns of the intra-dendritic calcium homeostasis, the activation of calcium signaling as well as dendritic mitochondrial redistribution. Notably, loss of CypD attenuates dendritic mitochondrial calcium perturbations and dendritic protrusion injury in the oxidative-stress environment. The most parsimonious interpretation of the results is that CypD regulates the flexibility of dendritic filopodia development by acting as a regulatory factor for the fine-tuning of mitochondrial calcium dynamics. Furthermore, CypD seems to be a double-edged sword for neurons in physiology and pathology.

Materials and Methods

Primary neurons culture and treatment

The animals and procedures used were in accordance with the guidelines and approval of Shandong University and the University of Texas at Dallas Institutional Animal Care and Use Committees (IACUC). Male and female Ppif−/− mice (B6;129-Ppiftm1Jmol/J, RRID:IMSR_JAX:009071) and B6;129 (RRID:IMSR_TAC:b6129) mice were purchased from Jackson Lab. Ppif−/− mice were backcrossed with C57BL/6J (RRID:IMSR_JAX:000664) mice for 10 generations to obtain Ppif+/+ and Ppif−/− littermates at the C57BL/6J background. All the adult mice were identified by genotyping and ear tag. The breeding cages (1male mouse and 2–3 female mice) were housed in a temperature-controlled (23°C) facility with a 12 hr light/dark cycle and were given free access to breeding diet and water. The plastic cages were 46 cm in length by 25 cm in width by 15 cm in height. The study was not pre-registered. The investigators who analyzed the data were blind to the genotype and treatment group. Primary neurons were cultured as we previously described (Beck et al. 2016). Briefly, Cortices or hippocampi were dissected from day 0 pups and immediately put in cold Hank’s balanced salt solution (HBSS, Sigma-Aldrich), dissociated with 0.05% trypsin (Sigma-Aldrich) at 37 ºC for 15 min followed by 10–15 times trituration. Cells were then passed through 100 μm mesh cell strainer (Fisher brand) and centrifuged for 5min at 200g. The pellet was gently resuspended in neuron culture medium (Neurobasal A with 2% B27 supplement and 0.5mM L-glutamine, Gibco) and plated on poly-D-lysine (Sigma-Aldrich) coated culture plates (Corning) or chamber slides (Nunc) with an appropriate density(Beck et al. 2016). 5-fluoro-2′-deoxyuridine (FDU, Sigma-Aldrich) was added to the neuron cultures to inhibit non-neuronal cell proliferation. Neurons were cultured to 14–21 days in vitro (DIV) for experiments. For KCl treatment, cultured neurons were incubated with culture medium containing 90mM KCl (final concentration) for 3 min followed by a wash with neuron culture medium for 10 minutes. This is considered to be 1 cycle. To induce oxidative stress, 1mM H2O2 (Sigma-Aldrich) was added to neurons 90 min before KCl treatment. For the treatment of cyclosporine A (CsA), neurons were pre-incubated with 1 μM CsA (Sigma-Aldrich 30024) for 2 hours. CsA was mantained throughout the experiments. For the treatment of KB-R7943, CGP37157 and BAPTA, KB-R7943 (1μM, Sigma-Aldrich, K4144), CGP37157 (1μM,Sigma-Aldrich, C8874) or BAPTA (20 μM, Sigma-Aldrich, A4926) was added to neuronal cultures for 5 minutes proceeding to the KCl stimulation. The agents were maintained throughout the experiments.

Synaptic mitochondria isolation

Synaptic mitochondria were isolated from tissue as previously described (Beck et al. 2016), brain tissues were homogenized in ice cold isolation buffer (225 mM mannitol, 75 mM sucrose, 2 mM K2PO4, 0.1% BSA, 5 mM Hepes, 1 mM EGTA (pH 7.2)) followed by a centrifugation at 1,300g for 3 min. The resultant supernatant was layered on a 3 × 2-ml discontinuous gradient of 15, 23 and 40% (vol/vol) Percoll and centrifuged at 34,000g for 8 min on a Beckman Coulter ultracentrifuge (Optima XPN-90 Ultracentrifuge). The interface between 15 and 23% (Band containing synaptosomes) was collected and synaptosomes were permeabilized by 0.02% digitonin. Percoll density gradient centrifugation was performed as described above for a second time. The interface between 23 and 40% (mitochondria released from synaptosomes) was collected and wahsed by centrifugation. Protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories).

ATP measurement

ATP content levels of neurons were analyzed by using ATP Luminescent assay kit (Abcam) following the manufacturer’s instructions.

Fluoresence dyes

For Calcein AM-cobalt chloride quenching assay, neurons were subjected to the staining of 1mM Calcein AM (Life Technologies) for 30min and then incubated with 1mM cobalt chloride (Sigma-Aldrich) for 30min to remove cytosolic Calcein staining (Beck et al. 2016) before imaging. For dendritic calcium measurement, 1μM Fluo-4 AM (Life Technologies) was loaded on neurons for 30min, followed by a wash with neuron culture medium before imaging. For mitochondrial calcium measurement, 1μM Rhod-2 AM (Life Technologies) was used for 30min followed by wash-out before imaging.

Image acquisition, processing and quantifications for fluorescence dyes

The images were obtained using a Nikon Eclipse Ti-E inverted microscope with on-stage incubator (37°C, 5% CO2), perfect focus and time-lapse system. The images of Calcein AM were taken every minute. The images of Fluo-4 AM andRhod-2 AM were acquired every 5 sec or 1 sec. Nikon NIS Advanced Research software was used to analyze all images for fluorescence intensities. The change in Calcein AM intensity was expressed as a relative fold change in the mean fluorescence intensity, and the fluorescence intensity at time 1 of baseline was expressed as 100. Estimation of fluorescence intensity of Fluo-4 AM and Rhod-2 AM was presented as ΔF/Fo, which was calculated using the following formula: ΔF/Fo = (F-Fbase)/Fbase, where F is the measured fluorescence intensity of the indicator, Fbase is the average fluorescence intensity before KCl treatment. Before calculating the intensity using above formula, the fluorescence intensity values were subtracted by the background signal determined from the average of areas adjacent to the object.

Mitochondria motility measurement

To measure dendritic mitochondria movement, imaging of mitochondrial targeted DsRed was performed every 5 sec on Nikon inverted fluorescent microscope with on-stage incubator (37ºC, 5% CO2). The same neuron was imaged before (resting condition) and during the 90mM KCl treatment (depolarization) using the same microscopic and camera settings. A mitochondrion was considered to be nonmobile if it remained stationary for the entire recording period; movement was counted only if the displacement was more than the length of the mitochondrion (Du et al. 2010). The direction of movement was recorded as stationary, anterograde and retrograde, and it was determined by comparing the displacement between the initial and final positions relative to the cell body. Mitochondrial movement toward the distal end of a dendrite is considered to be anterograde, whereas that toward the proximal end is considered to be retrograde. Kymographs were generated using maximum intensity projection integrated in Nikon NIS Advanced Research software to demonstrate the overall movement traces of mitochondria during the recording period.

Dendritic mitochondrial length, volume and dendritic protrusion measurements

Hippocampal neurons were infected by lentivirus expressing mitochondrial targeted DsRed at 7 DIV. At 14 DIV the neurons were treated with 90mM KCl and then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking in 5% BSA with 0.2% Triton X-100 (Sigma-Aldrich) for 1hr at room temperature, the neurons were stained with rabbit anti-Microtubule associated protein 2 (MAP2, Cell Signaling Technology, #8707, 1:200, RRID:AB_2722660) to determine dendrites followed by Alexa Fluor 405 conjugated goat anti-rabbit IgG(H+L) secondary antibody (Invitrogen, A31556, 1:400, RRID:AB_221605). ActinGreen 488 ReadyProbes Reagent (Life technologies, R37110) was used to stain F-actin to visualize dendritic protrusions. Dendritic segments between 70 and 100μm from the soma were used for the analysis. Images were collected under a Nikon confocal microscope using 40× Oil Immersion Objective Lens at 0.2μm thickness of each step size. 3D reconstruction was generated using Nikon-Elements advanced Research software from confocal image stacks. The measurements of dendritic mitochondrial length, volume and dendritic protrusion were conducted by using Nikon NIS Advanced Research software. A DsRed labeled particle with clear boundary was considered to be a mitochondrion. The appendages stemming from the dendrites shorter than 7μm and longer than 1/3 of the dendritic diameter were considered as a dendritic protrusion. Filopodia were identified as thin (head: neck diameter ratio less than 1.2) and long (the length is more than 3 times of the neck diameter) dendritic protrusions. The rest dendritic protrusions were considered to be dendritic spines.

Lentivirus production

Mitochondria targeted DsRed (Clontech) was inserted into lentivirus vector with human polyubiquitin promoter-C (Addgene). HEK293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transiently cotransfected with lentivirus vector carrying mitochondria targeted DsRed, packaging vector psPAX2 (Addgene) and envelope vector pMD2.G (Addgene) by using standard calcium phosphate precipitation method. At 24 hr post transfection, the medium was replaced with fresh medium, and lentivirus-containing medium was harvested 24hr later. The virus supernatant was filtered using a 0.45 μm filter (Millipore) to remove cell debris, concentrated by ultracentrifugation (82,700g at 4°C for 2 h), resuspended in neuron culture medium, and stored at −80°C until use.

Immunoblotting analysis

Neurons were collected in sample loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA and 0.02% bromophenol blue) with Protease Inhibitor Cocktail Set V (Millipore), Sodium Flouride (Sigma-Aldrich) and Sodium Orthovanadate (Sigma-Aldrich). Proteins were separated by NuPAGE™ Novex™ 12% Bis-Tris Protein Gels (Invitrogen) and then transferred onto PVDF membranes (Bio-Rad Laboratories). After blocking in 5% w/v nonfat dry milk in TBS buffer (20 mM Tris-HCl, 150mM sodium chloride) for 1hr at room temperature, the membrane was probed overnight at 4 °C with diluted primary antibodies against phospho-GluR1 Ser831 (Millipore, 04-823, 1:5,000, RRID:AB_1977218), GluR1 (Cell Signaling Technology, 13185S, 1:4,000), phospho-CaMK II (alpha subunit: Thr286, beta subunit: Thr287) (Cell Signaling Technology, 12716S, 1:10,000, RRID:AB_2713889), CaMK II (Santa Cruz, sc-5306, 1:2,000, RRID:AB_626788), CaMK II β (Invitrogen, 139800, 1:1,000, RRID:AB_2533045), and β actin (Sigma-Aldrich, A5441, 1:10,000), MCU (Cell Signaling Technology, 14997, 1:5,000), mNCX (Novus Biologicals, C2C12, 1:1,000) followed by incubation with the appropriate secondary antibodies, HRP conjugated goat anti-mouse IgG (H+L) (Invitrogen, 626520, 1:5,000, RRID:AB_2533947) or HRP conjugated goat anti-rabbit IgG (H+L) (Invitrogen, 656120, 1:5,000, RRID:AB_2533967) for 1 hr at room temperature. Images were collected on a Bio-Rad Chemidoc Imaging System. Image J (National Institutes of Health) was used to analyze the blots and to quantify protein signal intensity.

Statistical analysis

Cells were examined randomly for imaging and analysis. No randomization was performed. But data collection and analysis were conducted blindly to the conditions of the experiments by researchers blinded to the genotype/group performed. For animal experiments, mice of both genders were selected randomly according to the simple randomization method. No samples were excluded from the study. Unpaired Student’s t-tests or One-way ANOVA followed by Bonferroni post hoc analysis wherever appropriate were used for repeated measure analysis on SPSS software (IBM, version 21). Shapiro–Wilk tests were performed to examine normality of each dataset. Significance was set at P<0.05. All data were expressed as mean ± SEM.

Results

Loss of CypD deters activity-dependent dendritic filopodia outgrowth

To determine the role of CypD in neuronal activity-induced dendritic protrusion plasticity, we exposed Ppif+/+ and Ppif−/− neurons to repetitive KCl pulses, which is a common practice to mimic physiological neuronal depolarization in tissues and cell cultures. There was no significant difference in the baseline density of dendritic protrusions between Ppif+/+ and Ppif−/− neurons (Fig. 1a&d). Moreover, the baseline density of dendritic spines or filopodia was comparable between the two types of neurons (Fig. 1b, c&d), suggesting CypD deficiency has little effect on dendritic protrusion architecture in quiescent neurons. Repetitive KCl challenge induced substantially increased dendritic protrusion density in Ppif+/+ neurons (Fig. 1a&d); while in sharp contrast, CypD-deficient neurons only exhibited marginal changes (Fig. 1a&d). Further analysis showed that neurons had small changes in the density of dendritic spines in response to KCl treatment regardless of the presence or absence of CypD (Fig. 1b&d). However, depolarization aroused a dramatic filopodia outgrowth in Ppif+/+ neurons (Fig. 1c&d); while Ppif−/− neurons only showed a small response (Fig. 1c&d). Therefore, the results seem to suggest that CypD promotes the emergence of dendritic filopodia in response to neuronal activity. Of note, CypD is a deeply conservative protein. Yet the physiological function of CypD has not been fully depicted. Although studies have suggested its regulatory role in metabolism and protein acetylation (Nguyen et al. 2013, Elrod et al. 2010), the most well-documented notion is that CypD is a critical regulator of mPTP, the opening of which affects mitochondrial calcium homeostasis (Baines et al. 2005, Elrod & Molkentin 2013). To examine whether such effects of CypD loss on dendritic filopodiagenesis is associated with CypD-mediated mPTP, we exposed Ppif+/+ neurons to acute treatment of cyclosporine A (CsA), which is a potent inhibitor of CypD to blunt mitochondrial permeability transition. Our results showed that the pharmaceutical blockade of CypD-mediated mPTP replicated the above phenotypes of genetic CypD depletion (Figs. 1a–d). Therefore, the results seem to link the deleterious influence of CypD loss on dendritic protrusion outgrowth during neuronal depolarization to CypD-mediated mPTP activation.

Fig. 1. Loss of CypD blunts depolarization-induced dendritic filopodiagenesis.

Fig. 1

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(a) The density of dendritic protrusions. (b) The density of dendritic spines. (c) The density of filopodia. *P<0.05 vs other groups in the same condition (One-way ANOVA followed by Bonferroni post hoc analysis, n=45–63 segments). (d) Representative images of dendritic protrusions. White arrows indicate dendritic spines and white arrow heads indicate filopodia. Scale bar=5μm. Data were collected from 3–5 independent batches of culture.

Loss of CypD compromises the flexibility of dendritic mitochondrial calcium dynamics during depolarization

Neuronal activity induces dendritic calcium elevation (Lohmann et al. 2005), which is the driving factor for dendritic protrusion growth and remodeling. Such link between activity-induced intra-dendritic calcium transient and dendritic spinogenesis was also confirmed in our system as evidenced by the observations with an intracellular calcium chelator, BATPA (Supplementary Fig. 1a–c). Mitochondria involve in buffering intra-dendritic calcium in response to synaptic activity-induced calcium influx (Pivovarova et al. 2002). To determine whether loss of CypD alters intra-dendritic calcium homeostasis during neuronal depolarization, we examined intra-dendritic calcium levels in repetitive KCl-treated Ppif+/+, Ppif−/− as well as CsA-exposed Ppif+/+ neurons. Kinetic calcium level changes in dendritic segments were observed, by proxy, using the staining of Fluo-4 AM, a sensitive and specific fluorescent indictor of calcium (Sergeant et al. 2006). In all the three groups of neurons, KCl induced a significant rise in Fluo-4 AM intensity followed by a quick drop approaching the baseline levels (Fig. 2a). However, the return of intra-dendritic calcium to baseline levels was significantly faster in Ppif−/− and CsA-exposed Ppif+/+ neurons in comparison to their Ppif+/+ counterparts (Fig. 2a). The results indicate that loss of CypD promotes intra-dendritic calcium clearance to reach homeostasis during neuronal depolarization.

Fig. 2. Transient mPTP opening regulates dendritic mitochondrial calcium transients.

Fig. 2

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(a) Intra-dendritic calcium level measurement. P<0.05 vs other groups (One-way ANOVA followed by Bonferroni post hoc analysis, n=6–9 neurons). (b) Intra-dendritic mitochondrial calcium level measurement. P<0.01 vs other groups (One-way ANOVA followed by Bonferroni post hoc analysis, n=125–159 mitochondria). (c) Calcium levels in the “net gain”, “flickering” and “net loss” types of mitochondria in Ppif+/+, Ppif−/− and CsA-treated Ppif+/+ neurons (n=125–159 mitochondria). (d) Percentages of the “net gain”, “flickering” and “net loss” types of mitochondria in the indicated types of neurons. *P<0.05 vs other groups in the same condition (One-way ANOVA followed by Bonferroni post hoc analysis, n=125–159 mitochondria). Data were collected from 3–5 independent batches of culture.

Next, we examined the impact of CypD functional loss on intra-dendritic mitochondrial calcium dynamics. Intra-mitochondrial calcium was monitored by the staining of Rhod-2 AM (Kovacs et al. 2005). Within the observation window, Ppif+/+, Ppif−/− and CsA-exposed Ppif+/+ neurons had a substantial elevation in Rhod-2AM intensity from their baseline levels; while the increase was more rigorous in Ppif−/− and CsA-exposed Ppif+/+ dendritic mitochondria (Fig. 2b). The results suggest that CypD loss-of-function fortifies mitochondrial calcium uptake in response to depolarization-induced intra-dendritic calcium elevation, which is consistent with a previous observation (Barsukova et al. 2011).

In view of the heterogeneity of mitochondrial responses to neuronal depolarization (Kovacs et al. 2005), we then performed a single-mitochondrion based analysis of intra-mitochondrial calcium dynamics. Interestingly, in both types of neurons three distinct patterns of dendritic mitochondrial calcium changes during neuronal depolarization were identified. To be more specific, we found that some dendritic mitochondria maintained constantly higher Rhod-2AM intensity than their baseline levels (that is, “net gain” of calcium), and some showed constantly lowered Rhod-2AM levels than their baseline levels (that is, “net loss” of calcium); while the rest mitochondria underwent repetitive transition between the “net gain” and “net loss” states (that is, “flickering” in calcium levels) (Fig. 2c&d). Of note, the percentages of the three types of Ppif+/+ mitochondria are almost comparable; whereas most of the Ppif−/− dendritic mitochondria were at the “net gain” state during neuronal depolarization (Fig. 2d). Importantly, CsA-exposed Ppif+/+ mitochondria represented a similar change as seen with the genetic CypD-deficient ones (Fig. 2c&d). Given that mitochondrial calcium flashes indicate transient mitochondrial calcium release (Agarwal et al. 2017), the results seem to suggest that CypD mediates dendritic mitochondrial calcium extrusion during neuronal depolarization through transient mPTP opening. Indeed, in a parallel experiment we have observed increased transient mPTP opening in KCl-challenged Ppif+/+ neurons, which was prohibited by the loss of CypD (Supplementary Fig. 2a–i).

However, given the complexity of the regulation of mitochondrial calcium homeostasis, we sought to determine whether CypD also affects mitochondrial calcium uptake. To this end, we examined and compared the function of mitochondrial calcium uniporter (MCU), a major mitochondrial calcium influx pathway (Kwong et al. 2015, Baughman et al. 2011) in neurons with or without CypD expression. We have found that the genetic depletion of CypD has little influence on the expression levels of MCU both in neuron cultures (Supplementary Fig. 3a) and synaptic mitochondria (that is, specific neuronal mitochondria (Du et al. 2010, Dunkley et al. 1988)) isolated from adult mice (Supplementary Fig. 3b). Further blockade of MCU by using its specific inhibitor, KB-R7943 induced significant suppression on intra-dendritic calcium clearance (Supplementary Fig. 3c) and dendritic mitochondrial calcium uptake (Supplementary Fig. 3d) in both types of neurons without genotypic difference. The little impact of CypD deficiency on MCU-mediated mitochondrial calcium uptake seems to implicate that the elevated mitochondrial calcium levels in KCl-stimulated CypD-deficient neurons is not likely to be associated with enhanced mitochondrial calcium uptake but rather a result of increased mitochondrial calcium retention due to lessened calcium efflux.

Indeed, CypD-mediated transient mPTP is not the only pathway for mitochondrial calcium release. Mitochondrial sodium-calcium exchanger (mNCX) also mediates mitochondrial calcium efflux (Rizzuto et al. 2012, Palty et al. 2010). However, we have found similar expression levels of mNCX (Supplementary Fig. 4a) as well as comparable response to mNCX inhibitor, CGP37157-mediated mitochondrial calcium retention (Supplementary Fig. 4b) in both types of neurons. These findings seem to rule out the contribution of mNCX to the phenotypic effect of CypD depletion.

Importantly, both KB-R7943 and CGP37157 mediated remarkable defects in dendritic protrusion outgrowth in depolarized neurons (Supplementary Figs. 3e&f, 4c&d). These results along with the impacts of CypD depletion highlight the essential role of normal mitochondrial calcium dynamics in sustaining dendritic spinogenesis.

Loss of CypD blunts CaMK IIactivation during neuronal depolarization

CaMK II (calmodulin-dependent kinase II) is a critical downstream calcium signaling following calcium transients and plays a critical role in activity-induced dendritic protrusion growth (Shi & Ethell 2006, Lisman et al. 2002). In this regard, we examined the influence of CypD depletion on the activation of CaMK II. Ppif+/+ and Ppif−/− neurons were exposed to KCl followed by a wash to terminate KCl effect. Although KCl treatment induced the activation of CaMK II α and β in both types of neurons, Ppif+/+ neurons demonstrated a greater response to KCl-induced CaMK II α and β phosphorylation (Fig. 3a, b &d). Given that AMPA receptor subunit GluR1 is activated by CaMK II and GluR1 phosphorylation has been shown to potentiate dendritic spine morphogenesis(Inglis et al. 2002), we next examined the phosphorylation levels of GluR1 at Ser831, which is CaMK II-sensitive. After KCl stimulation, GluR1 activation in Ppif+/+ neurons was greater than that in their Ppif−/− counterparts without any effect on total GluR1 levels (Fig. 3c&d). The results suggest that CypD potentiates dendritic protrusion plasticity-related calcium signaling activation during neuronal depolarization.

Fig. 3. CypD deficiency suppresses CaMK IIand GluR1 phosphoryaltion in response to depolarization.

Fig. 3

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(a) CaMK II α phosphorylation. (b) CaMK II β phosphorylation. (c) GluR1 Ser831 phosphorylation (Unpaired Student’s t-tests, n=4). (d) Representative Western-blot bands of the indicated proteins.

Loss of CypD alters the patterns of dendritic mitochondrial motility and distribution during neuronal depolarization

Mitochondria are dynamic organelles. Previous studies have shown mitochondrial trafficking to dendritic spines in response to neuronal activity (Li et al. 2004). Such mitochondrial relocation is believed to be vital for depolarization-induced dendritic spine plasticity (Li et al. 2004). Of note, CaMK II plays a critical role in potentiating synaptic cargo (including mitochondria) trafficking (Schlager & Hoogenraad 2009). It is therefore necessary to examine the impact of transient mPTP on dendritic mitochondrial motility. In this regard, we measured dendritic mitochondrial transport. Although at the baseline condition only a small percentage of dendritic mitochondria was movable in both Ppif+/+ and Ppif−/− neurons, Ppif+/+ dendritic mitochondria underwent more active movement (Fig. 4a–e). After depolarization, Ppif−/− dendritic mitochondria quickly stopped moving; while Ppif+/+ dendritic mitochondria were still actively transported in both anterograde and retrograde directions for a relatively longer time before they reached stabilization in their physical position (Fig. 4c, d&e).

Fig. 4. CypD deficiency inhibits dendritic mitochondrial movement and redistribution in response to depolarization.

Fig. 4

Fig. 4

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(a) The percentage of movable mitochondria in Ppif+/+ and Ppif−/− neurons (Unpaired Student’s t-tests, n=11, 9 neurons, respectively). (b) The percentage of stationary mitochondria in Ppif+/+ and Ppif−/− neurons (Unpaired Student’s t-tests, n=11, 9 neurons, respectively). (c) The percentage of anterograde mitochondria in Ppif+/+ and Ppif−/− neurons (Unpaired Student’s t-tests, n=11, 9 neurons, respectively). (d) The percentage of retrograde mitochondria in Ppif+/+ and Ppif−/− neurons (Unpaired Student’s t-tests, n=11, 9 neurons, respectively). (e) Kymograph data of mitochondrial movement. Scale bar=10μm. (f) The percentage of mitochondria-containing dendritic protrusions (n=29–58 dendritic segments). (g) Representative images of dendritic protrusions (green) and mitochondria (red). Scale bar=5μm. Data were collected from 3–5 independent batches of culture.

Altered mitochondrial motility may affect mitochondrial distribution. To address this question, we examined and compared the distribution patterns of dendritic mitochondria between the two types of neurons. At the resting condition, 6.32 ± 2.76% of Ppif+/+ dendritic protrusions contained mitochondrial fraction. The percentage of mitochondria-occupying dendritic protrusions was significantly elevated to 16.52 ± 3.39% and 23.52 ± 3.82% post 1x and 4x KCl-induced depolarization, respectively (Fig. 4f&g). In sharp contrast, approximately a quarter of Ppif−/− dendritic protrusions were occupied with mitochondrial content at the resting condition, which is significantly higher than that of their Ppif+/+ counterparts (Fig. 4f&g). However, Ppif−/− neurons exhibited little change in the percentage of mitochondrial-containing dendritic protrusions on exposure to either one time or repetitive KCl treatment (Fig. 4f&g). Therefore, CypD seems to confer flexibility to dendritic mitochondrial motility and redistribution, which are critical for the outgrowth of dendritic filopodia in response to neuronal activity.

CypD deficiency attenuates dendritic mitochondrial calcium perturbations and dendritic protrusion injury in oxidative-stress environment

Previous studies have suggested the protection of CypD deficiency on dendritic spine architecture at pathological states (Du et al. 2014), which seems to contradict the aforementioned inhibitory effect of CypD loss on dendritic spinogenesis. We hypothesized that this discrepancy is the result of prolonged mPTP opening in pathological conditions. To address this question, we exposed Ppif+/+ and Ppif−/− neurons to H2O2-induced pathological oxidative insults on neurons. We first examined dendritic mitochondrial calcium dynamics as increased “net loss” type of mitochondria is an indicator of lasting mitochondrial calcium release. Dendritic mitochondria in H2O2-insulted Ppif+/+ neurons demonstrated decreased mitochondrial calcium levels in response to KCl treatment (Fig. 5a), implicating lasting calcium release in these mitochondria. Indeed, further analysis showed that most of Ppif+/+ dendritic mitochondria were of “net loss” type, which serves as a strong indicator of the opening of prolonged mPTP (Fig. 5b). By contrast, the majority of CypD-deficient dendritic mitochondria still had the ability to detain calcium, although their calcium retention capacity was also impaired (Fig. 5b). In parallel with the changes in mitochondrial calcium transients, H2O2-insulted Ppif+/+ neurons had a significantly lowered dendritic protrusion density in comparison to that of the Ppif−/− neurons at either resting or KCl-treated conditions (Fig. 5c&d).The results suggest that at pathological state the protective effect of CypD deficiency on dendritic protrusions is at least in part associated with the blockade of prolonged mPTP opening-induced mitochondrial calcium perturbations.

Fig. 5. CypD deficiency protects mitochondrial calcium dynamics and dendritic protrusions from oxidative stress.

Fig. 5

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(a) Intra-dendritic mitochondrial calcium level measurement in oxidative-stress environment (Unpaired Student’s t-tests, n=125, 112 mitochondria, respectively). (b) Percentages of the “net gain”, “flickering” and “net loss” types of mitochondria in Ppif+/+ and Ppif−/− neurons (Unpaired Student’s t-tests, n=125, 112 mitochondria, respectively). (c) The measurement of dendritic protrusion density (Unpaired Student’s t-tests, n=62, 57, 60 and 53 dendritic segments, respectively). (d) Representative images of dendritic protrusion staining. White arrows indicate dendritic protrusions. Scale bar = 5 μm. Data were collected from 3–4 independent batches of culture.

Discussion

In this study, we have shown that loss of CypD compromises the flexibility of mitochondrial calcium dynamics during neuronal activity, which is closely associated with the blockade of transient mPTP activation. mPTP is proposed to be a non-selective mitochondrial pore, the opening of which promotes IMM permeability. Current studies overwhelmingly focused on the role of mPTP in diseases. Indeed, over-activation of mPTP has been associated with mitochondrial abnormalities in Alzheimer’s disease (AD) (Du et al. 2008), Parkinson’s disease (PD) (Thomas et al. 2012), and brain ischemia (Schinzel et al. 2005) as well as in many other pathological scenarios (Gauba et al. 2017, Perrucci et al. 2016, Kim et al. 2012). It is therefore proposed that the inhibition of mPTP opening is a promising strategy for the treatment and prevention of neurological disorders. While the pathological role of mPTP has been intensively investigated, recent studies have implicated the physiological function of CypD and transient mPTP. Elrod and the colleagues reported that CypD deficiency increases the propensity of heart failure in aging mice. Such deleterious effect of CypD depletion is at least in part due to its influence on mitochondrial calcium homeostasis (Elrod et al. 2010). Moreover, several in vitro and in vivo studies have suggested the physiological role of CypD and transient mPTP in synaptic plasticity, synaptic transmission and cognition (Weeber et al. 2002, Levy et al. 2003) as well as in the regulation of astrocyte microdomain calcium transients (Agarwal et al. 2017). These studies have suggested the critical role of CypD and the transient opening of CypD-mediated mPTP in the cells whose function is significantly reliant on calcium transients. Here, we have found that blockade of transient mPTP by CypD depletion or pharmaceutical approach deters dendritic filopodia growth in response to neuronal activity. Such inhibitory effect correlates to increased dendritic mitochondrial calcium retention, which blunts activity-induced calcium signaling activation, as well as dendritic mitochondrial motility and redistribution. Therefore, CypD through transient opening of mPTP seems to act as a critical regulator of the fine-tuning of intra-dendritic calcium dynamics during neuronal activity. Interestingly, we have also found that CypD-deficiency confers resistance to oxidative stress induced calcium perturbation in dendritic mitochondria, which results in preserved dendritic protrusions. Such observations further confirm the connection between mitochondrial calcium regulation and dendritic protrusion plasticity, as well as reconcile the conflicting roles of CypD in physiological and pathological conditions.

Notably, although increasing evidence has implicated the importance of dendritic mitochondria in neuronal activity-induced dendritic protrusion plasticity (Li et al. 2004, Sung et al. 2008, Cheng et al. 2012, Yu et al. 2011), the role of mitochondria in dendritic protrusion remodeling and structure is still under debate. A critical issue is that whether mitochondrial distribute to dendritic protrusions. Previous electro-microscopic studies suggest that mitochondrial content is rarely found in dendritic spines and filopodia (Sorra & Harris 2000, Chicurel & Harris 1992). Indeed, we have noticed that only ~5% of dendritic protrusions were occupied by mitochondrial content at resting conditions, which is similar to the electro-microscopic observations. However, during depolarization the percentage of mitochondrial containing dendritic protrusions is remarkably elevated, which conforms to the findings from several other groups (Li et al. 2004, Sung et al. 2008). The results implicate that dendritic mitochondrial insertion into dendritic protrusions is highly related to neuronal activity and is probably transient. It is true that observations on in vitro cultured neurons may not fully represent the in vivo changes. Therefore, future studies on this question in a more physiological in vivo setting are necessary. Nevertheless, in this study we focused on the growth of filopodia, which are the nascent appendages of dendritic spines. It is suggested that neuronal activity-induced calcium transients promote the growth and motility of filopodia; while high levels of intra-dendritic calcium facilitates filopodia stabilization and maturation (Lohmann et al. 2005, Hu & Hsueh 2014). In this case, our results suggest that the impact of dendritic mitochondria on the regulation of intra-dendritic calcium transients is critical for the outgrowth of filopodia from dendritic shafts, which is indirectly evidenced by the concurrence of refrained filopodia emergence and the rigidity of dendritic mitochondrial calcium regulation in CypD-deficient neurons.

Another interesting finding is that we have noticed that depolarization causes dendritic mitochondrial calcium flashing, which is significantly blocked by the depletion of CypD. Flashing mitochondrial calcium is a strong indicator of the fast transition between mitochondrial net calcium uptake and release during synaptic activation (Kovacs et al. 2005). Indeed, we cannot exclude the involvement of mNCX and/or MCU to mitochondrial calcium flickering. But we have observed the little difference in the expression levels of MCU and mNCX as well as the similar response to MCU and mNCX inhibitors between Ppif+/+ and Ppif−/− neurons, suggesting the genotypic effects of CypD loss on the patterns of dendritic mitochondrial calcium dynamics are primarily associated with the deactivation of transient mPTP. Therefore, these findings serve as strong evidence supporting the involvement of transient opening of CypD-mediated mPTP in calcium-induced mitochondrial calcium release during neuronal activity. In this context, transient mPTP activation via CypD seems to be an active actor in physiological calcium oscillations (Dupont et al. 2011). Moreover, CypD-deficient neurons also had “flickering” type of dendritic mitochondria. This could be a result of CypD-independent mitochondrial calcium release in depolarization-challenged neurons. Based on our observations of the prompt response of CypD-deficient neurons to mNCX inhibitor, the opening of mNCX-related calcium release channels may constitute a critical mechanism for mitochondrial calcium release in Ppif−/− neurons. And this non-CypD-related mitochondrial calcium release pathway seems to at least in part explain that CypD-deficient neurons still maintain baseline levels of dendritic protrusions and have response to neuronal depolarization. Therefore, we propose a model in which synergistic mitochondrial calcium regulation is critical for dendritic spinogenesis and CypD-mediated transient mPTP is a significant contributor.

Lastly, the molecular identity of mPTP still remains enigmatic. Emerging evidence has suggested that mitochondrial F1Fo ATP synthase forms the structure of mPTP (Giorgio et al. 2017, Bonora et al. 2017, Alavian et al. 2014), the opening of which is probably through the interaction of CypD with F1Fo ATP synthase oligomycin sensitivity-conferring protein (OSCP) (Beck et al. 2016, Giorgio et al. 2013, Giorgio et al. 2009, Alavian et al. 2014, Bonora et al. 2017), although a recent study suggests that calcium may also directly trigger mPTP opening through the interaction with F1Fo ATP synthase β subunit(Giorgio et al. 2017). Accordingly, transient mPTP formation may exert dual impacts in regulating the status of mitochondrial calcium buffering and ATP production to prevent excess calcium overloading and ROS accumulation during neuronal depolarization. In this regard, transient opening of CypD-mediated mPTP may also serve as a mitochondrial adaptive strategy in response to extensive workload during synaptic activity to release mitochondrial stress i.e. calcium overloading and ROS (Bernardi & von Stockum 2012).

In summary, we have observed the physiological function of CypD in the regulation of activity-driven filopodiagenesis. Indeed, the development of dendritic spine and filopodia is the result of complicated mechanistic pathways (Woolfrey & Srivastava 2016, Hering & Sheng 2001). Furthermore, the function of CypD in cell biology has not yet been fully depicted. Thus, studies to fully explore the impact of CypD on dendritic protrusion growth and regulation may yield critical findings in the future. The simplest interpretation of the results is that CypD has its physiological function in neurophysiology, which is at least in part associated with the influence of transient opening of CypD-medaited mPTP on the regulation of mitochondrial calcium homeostasis. Moreover, the protective effect of CypD deficiency on dendritic protrusions against oxidative stress further endorses the therapeutic value of CypD inhibition for the treatment of neurological diseases. However, in view of the physiological function of CypD, it should be with caution to use CypD inhibitors as preventive strategies for neurological disorders.

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Acknowledgments

This study is supported by research funding from National Natural Science Foundation of China (31271145, 81200847), and Natural Science Foundation of Shandong Province (JQ201318), National Institutes of Health (R00AG037716, R01AG053588), Alzheimer’s Association (NIRG-12-242803, AARG-16-442863), and China Scholarship Council (201606220203).

Abbreviations used

CypD

Cyclophilin D

PPIase

peptidyl-prolyl cis-trans isomerase

mPT

mitochondrial permeability transition

mPTP

mitochondrial permeability transition pore

IMM

inner mitochondrial membrane

HBSS

Hank’s balanced salt solution

FDU

5-fluoro-2′-deoxyuridine

MAP2

Microtubule associated protein 2

CaMK II

calmodulin-dependent kinase II

AD

Alzheimer’s disease

PD

Parkinson’s disease

RRID

Research resource Identifier

OSCP

oligomycin sensitivity-conferring protein

MCU

mitochondrial calcium uniporter

mNCX

mitochondrial sodium-calcium exchanger

BAPTA

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

CGP37157

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one

KB-R7943

2-[4-[(4-nitrophenyl)methoxy]phenyl]ethyl ester, methanesulfonate (1:1), Carbamimidothioic acid.

Footnotes

Conflict of Interest:

The authors have no conflict of interest to claim.

Author contributions:

S.S., J.T., E.G., Q.W. performed experiments and analyzed data. L.G. designed the experiments and analyzed the data. H.D. designed the experiments and composed the manuscript.

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