ABSTRACT
Neurons display extreme degrees of polarization, including compartment-specific organelle morphology. In cortical pyramidal neurons, dendritic mitochondria are long and tubular whereas axonal mitochondria display uniformly short length. Here, we explored the functional significance of maintaining small mitochondria for axonal development in vitro and in vivo. We report that the Drp1 ‘receptor’ Mitochondrial fission factor (MFF) is required for determining the size of mitochondria entering the axon and then for maintenance of their size along the distal portions of the axon without affecting their trafficking properties, presynaptic capture, membrane potential or capacity for ATP production. Strikingly, this increase in presynaptic mitochondrial size upon MFF downregulation augments their capacity for Ca2+ ([Ca2+]m) uptake during neurotransmission, leading to reduced presynaptic [Ca2+]c accumulation, decreased presynaptic release and terminal axon branching. Our results uncover a novel mechanism controlling neurotransmitter release and axon branching through fission-dependent regulation of presynaptic mitochondrial size.
INTRODUCTION
Neurons are among the most highly polarized cells found in nature. This high level of polarization underlies the formation of structural compartments such as the axon, dendrites and spines which in turn dictates information processing and transfer in neural circuits. In order to set up this extreme degree of compartmentalization and maintain it throughout the life of the organism, critical cellular and molecular effectors of polarization, including mRNAs, proteins, and organelles, must be either differentially trafficked or functionally regulated in a spatially precise way 1.
Mitochondria are one of the most abundant organelles found in neurons and are localized throughout the axonal and somatodendritic domains 2–6. Several studies have observed that dendritic mitochondria have a long and tubular shape, while axonal mitochondria appear to be short and punctate 2–10. However, the functional implications of this structural difference have not been addressed. In general, mitochondria play many important physiological functions such as ATP production via oxidative phosphorylation, Ca2+ uptake, lipid biogenesis, and can trigger apoptosis through cytochrome-c release 11. Dendritic mitochondrial morphology and function have mainly been studied in the context of neurodegenerative diseases and their contribution to activity-dependent homeostasis 4,6,12. In axons, the presynaptic capture of mitochondria is both necessary and sufficient for terminal axon branching in cortical neurons 13 and these presynaptically captured mitochondria generate ATP and buffer Ca2+ during synaptic transmission 14–16. Interestingly, presynaptic Ca2+ clearance by individual mitochondria underlies bouton-specific regulation of presynaptic vesicle release along cortical and hippocampal axons 15,16. Mitochondrial function has been suggested to differ in specific neuronal compartments 4,17, but the outcome of disrupting mitochondrial size in a compartment-specific manner remains to be determined.
Mitochondrial size is regulated through the competing processes of fission and fusion 18. Mitochondrial fusion is regulated by two distinct molecular effectors: Mfn1/2 mediates outer membrane fusion 19,20 while Opa1 regulates inner membrane fusion 21,22. Fission occurs by the ‘pinching’ of a mitochondrion into two new mitochondria via oligomerization of Drp1, a dynamin-like GTPase, at the outer membrane 23–25. Because Drp1 is a cytoplasmic protein, it must be recruited to the outer mitochondrial membrane via Drp1 ‘receptors’. There are currently four known Drp1 receptors: MFF, FIS1, MiD49 (Scmr7) and MiD51 (Scmr7L), whose relative contribution to Drp1-dependent fission is still debated 26–36.
Our understanding of the relative contribution of fission and fusion for neuronal morphogenesis or synaptic function remains fragmented. Loss of Drp1 is embryonic lethal and leads to defects in brain development, neuronal process outgrowth and synapse formation. However, Drp1 affects many cellular processes other than mitochondrial fission and also severely affects neuronal viability, so this observation does not unequivocally implicate mitochondrial fission as the cause of the defects in neuronal development 37–42. Mitochondrial fusion is also required for proper development as each of the Mfn1, Mfn2 and Opa1 knockouts are lethal20,43,44, however their role in cortical neuron development is less well studied.
We establish that the small mitochondrial size characterizing the axon of cortical pyramidal neurons is dependent on mitochondrial fission factor (MFF) but not FIS1, the other abundant Drp1 receptor in these neurons. We find that downregulation of Mff results in a striking increase of the size of mitochondria entering the axon, and an inversion of the fission/fusion ratio along the axon. This elongation of axonal mitochondria does not affect dendritic mitochondria, presynaptic capture, trafficking along axons, mitochondrial membrane potential or the capacity to generate ATP. However, this Mff-dependent increase in presynaptic mitochondrial length significantly increased total Ca2+ uptake capacity, resulting in decreased evoked neurotransmitter release and decreased terminal axon branching in vivo. Our results identify a novel molecular mechanism limiting presynaptic mitochondrial size and demonstrate its functional importance for neurotransmitter release and axon branching via the limitation of mitochondrial Ca2+ buffering capacity.
RESULTS
Mitochondrial Morphology is Strikingly Different in Axons and Dendrites
In order to visualize mitochondrial morphology and function in developing and adult cortical neurons in vitro and in vivo, we implemented a strategy for sparse labeling via ex utero or in utero electroporation (EUE and IUE respectively) 45,46. We developed Flp recombinase-dependent plasmids expressing either a cytoplasmic fluorescent protein (tdTomato (red), EGFP (green) or mTAGBFP2 (blue)), or a mitochondrial matrix-targeted fluorescent protein (mt-YFP, mt-DsRED), based on a previous report 47. When co-electroporated with a low concentration of Flp-e plasmid at E15.5, this strategy resulted in sparse labeling of layer 2/3 cortical pyramidal neurons 13,48,49, and allowed us to visualize mitochondrial morphology at high resolution in single, optically-isolated neurons both in vitro(with EUE) and in vivo (with IUE). Following EUE at E15.5, we acutely dissociated and cultured cortical pyramidal neurons at high density for three weeks (21 Days In Vitro, (DIV)) and observed striking differences in mitochondrial morphology and occupancy between axons and dendrites (Fig. 1a-c). In dendrites, mitochondria were elongated (0.52 to 8.88 μm (10-90th percentile)) and occupied the majority of proximal and distal dendritic processes (69.6 ± 2.54%), while throughout the axon, mitochondria are short, relatively standard in length (0.3 to 1.08 μm (10-90th percentile)) and occupied only an average of 4.95 (± 0.4%) of the axonal length. To confirm this was not an in vitro artifact, we also performed IUE at E15.5 and visualized sparsely labeled layer 2/3 cortical neurons at (P)ostnatal day 21. Remarkably, quantitative measurement of mitochondrial size and occupancy were highly conserved in vivo (Fig. 1d-f) where dendritic mitochondria measured 1.31 to 13.28 μm (10-90th percentile) in length and occupied 69.58 ± 2.23% of dendritic processes, while axonal mitochondria measured 0.45 to 1.13 μm (10-90th percentile) and occupied 8.41 ± 0.75% of axon length (Fig. 1g-h). These observations confirm that mitochondrial morphology is strikingly different in the axonal and dendritic compartments of cortical layer 2/3 pyramidal neurons both in vitro and in vivo.
Axonal Mitochondria Size is Regulated by Mitochondrial Fission Factor (MFF)
Based on publicly available RNA-seq data from the cerebral cortex and hippocampus 50,51 only two of the four known mediators of mitochondrial fission (so called Drp1 ‘receptors’) are highly expressed in excitatory pyramidal neurons: Mitochondrial Fission Factor (Mff) and Mitochondrial Fission 1 (Fis1). To determine if MFF-and/or FIS1-mediated fission are required for the regulation of mitochondrial size in the axon, we first validated knockdown constructs for both mouse Mff and Fis1 via western blot and immunocytochemistry (Fig. S1). Following ex utero or in utero electroporation of the validated shRNA constructs for Mff along with plasmid DNA encoding cytoplasmic filler (tdTomato) and a mitochondria-targeted fluorescent protein (mt-YFP) at E15.5 and visualizatison of neurons at 21DIV or P21, mitochondrial length and occupancy along the axon were dramatically increased compared to control both in vitro and in vivo (Fig. 2a-d and Fig. 2i-n). This increase in mitochondrial length and occupancy is completely rescued by the co-expression of a cDNA plasmid encoding human MFF, which is impervious to the shRNA directed against mouse Mff (Fig. 2e-h), demonstrating that this effect is not the result of an ‘off-target’ effect of the shRNA. In contrast, Fis1 knockdown had no effect on axonal mitochondrial length but did show a small but significant increase in mitochondrial occupancy which could be consistent with its recently proposed role in mitophagy 31 (Fig. 2o-s). Interestingly, Mff knockdown had no effect on mitochondrial length or occupancy in dendrites in vivo suggesting a low level of basal MFF activity in this compartment (Fig. S2). These results establish that MFF, but not FIS1, is required for the maintenance of small mitochondrial size in the axon. Interestingly, MFF is present on both somatodendritic and axonal mitochondria (Fig. S1c-n) suggesting that fission is regulated in a manner other than localization of MFF to a particular set of mitochondria (See Discussion).
Reduced MFF Activity Produces Elongated Axonal Mitochondria Via Increased Mitochondrial Length Upon Axonal Entry as well as Decreased Fission Along the Axon Shaft
To better characterize the role of MFF in regulating axonal mitochondrial size, we used the mitochondrial matrix-targeted, photo-convertible fluorescent protein mEos2 52,53 which allowed us to quantify mitochondrial fission and fusion. We employed two distinct imaging strategies to determine where MFF activity is required for axonal mitochondrial size maintenance; (1) photo-conversion of mitochondria located in the cell body followed by time-lapse imaging of mitochondrial entry into the axon, or (2) photo-conversion of a small fraction of mitochondria located along the axon shaft followed by time-lapse imaging and probing of fusion and fission events (Fig. 3a, Fig. S3a). Time-lapse imaging of mitochondrial entry into the axon following photo-conversion in the cell body revealed that loss of MFF affects both the frequency of axonal entry as well as their size. On average ~8 mitochondria (8.1 ± 1.7) entered the axon from the cell body per hour in control neurons, but upon Mff knockdown this decreased by more than half (2.8 ± 0.78) (Fig. S3b-d). Concurrently, the length of mitochondria entering the axon increased four-fold upon Mff knockdown compared to control neurons (Fig. S3e, Movie S1), establishing that MFF activity in the cell body is required for proper axonal mitochondria size.
To determine if MFF-dependent fission is also required along the axon for the maintenance of mitochondrial size, we performed time-lapse imaging following photo-conversion of a small subset of mitochondria along the axon shaft. In control axons, the hourly rate of fission (4.2 ± 0.53) and fusion (3.1 ± 0.42) slightly favors fission. However, upon loss of MFF activity, axonal fission is much less likely (1.8 ± 0.30) while fusion isslightly more likely (4.2 ± 0.52) (Fig. 3b-d) leading to a dramatic reversal in the fission/fusion ratio (1.5 013 (control shRNA) vs. 0.46 ± 0.08 (Mff shRNA) Fig. 3d, Movie S2). These results demonstrate that MFF is required both for regulation of mitochondrial size during axonal entry as well as along the axon shaft for maintenance of small mitochondrial size.
Mff Knockdown Disrupts Terminal Axon Branching
To determine the consequence of decreased MFF expression on axonal development, we performed unilateral cortical in utero electroporation (IUE) of the validated Mff shRNA constructs along with a plasmid encoding cytoplasmic tdTomato at E15.5, and collected coronal sections at P21 to visualize axonal growth and branching. In control brains, axons grew across the corpus callosum reaching the contralateral hemisphere where they invaded the upper layers forming a stereotyped branching pattern in layers 2/3 and 5 (Fig. 4a-c) 13,48,49. Knockdown of Mff did not affect neuronal migration, axon formation and axon growth across the midline. Axons from Mff knockdown neurons reached their target territory on the contralateral cortex all the way to superficial layers 2/3. However, terminal axon branching was dramatically reduced, especially in layers 2/3 (Fig. 4d-f). This striking reduction of cortical axon branching was rescued upon reintroduction of shRNA-impervious human MFF validating the specificity of our shRNA directed against Mff (Fig. 4g-k). These results emphasize the importance of MFF-dependent fission for the control of axonal mitochondria size and axonal branching in vivo.
Reduced MFF Activity Does Not Affect Presynaptic Mitochondrial Capture
Based on our previous work demonstrating the importance of presynaptic mitochondrial capture for terminal axonal branching 13, we tested whether altered mitochondrial size had any effect on axonal mitochondrial transport and/or their capture at presynaptic sites. As previously shown mitochondrial motility in vitro and in vivo progressively decreases along the axons of pyramidal neurons in vitro and in vivo 53–55, so we quantified mitochondrial motility at both 7DIV and 21DIV (Fig. S4a-b, Movie S3). We observed a modest but significant increase in the number of stationary mitochondria at 7DIV in MFF-deficient axons compared to control. Strikingly as seen in Movie S3, these elongated axonal mitochondria are clearly capable of sustained trafficking along the axon (Fig. S4c-d). At 21DIV, the vast majority of these elongated mitochondria become immobilized at specific points along the axon as observed in control axons (94.8 ± 3.53% versus 91.3 ± 5% in control). To determine if altering mitochondrial size along the axon affected their capture at presynaptic boutons in vivo, we performed unilateral cortical in utero electroporation of the validated shRNA constructs for Mff along with plasmid DNA encoding cytoplasmic tdTomato (cell filler), mt-YFP (mitochondrial marker) and VGLUT1-HA (presynaptic marker) at E15.5 and collected coronal sections at P21 to visualize co-localization of mitochondria and VGLUT1 positive presynaptic sites. Interestingly, elongation of axonal mitochondrial size had no significant effect on localization to presynaptic sites or the density of VGLUT1 presynaptic boutons along layer 2/3 cortical axons (Fig. S4e-h). These results demonstrate that elongation of axonal mitochondria size does not impact their capture at presynaptic boutons and suggested that we should instead focus on potential changes in mitochondrial function that might accompany the observed increase in mitochondrial size upon knockdown of Mff.
Reduced MFF Activity and Elongation of Axonal Mitochondria Does Not Decrease Mitochondrial Membrane Potential, Mitochondrial ATP Levels or Redox Potential
We first tested if either mitochondrial membrane potential or mitochondrial ATP levels were affected following a reduction in MFF levels and increased mitochondrial size along cortical axons. To determine if these elongated axonal mitochondria maintain their membrane potential, we used Tetramethylrhodamine, Methyl Ester (TMRM, 20nM). We electroporated cortical neurons with either control shRNA or Mff shRNA and unique combinations of mitochondrial and cytoplasmic fluorescent proteins (mt-BFP and Venus for control vs. mt-YFP and mTAGBFP2 for Mff knockdown), and mixed control and knockdown neurons at a 1:1 ratio (Fig. S5). This approach allowed us to image mitochondrial membrane potential in control and Mff knockdown neurons under the exact same culture conditions. Upon quantification of the Fm/Fc ratio for individual mitochondria along the axon 56, the longer axonal mitochondria upon Mff knockdown actually have an increased ratio (data not shown) but after accounting for mitochondrial area there is no change in the Fm/Fc ratio versus the control mitochondria (15.0 ± 2.63 in Mff knockown vs 13.8 ± 1.08 for control, Fig. 5a-b).
Taking a similar approach, we measured intra-mitochondrial ATP levels using a genetically-encoded probe, ATEAM1.03, targeted to the mitochondrial matrix 57,58 (mt-ATEAM and mCardinal for control vs. mt-ATEAM and mScarlet for Mff knockdown). Again, we found no significant change in the YFP/CFP ratio for mitochondria upon Mff knockdown (2.55 ± 0.07) versus control (2.38 ± 0.08) imaged with the same settings and culture conditions at 21DIV (Fig. 5c-d).
Finally, we tested if there was a change in the redox potential of elongated, Mff-deficient mitochondria using a genetically-encoded redox-sensitive probe targeted to the mitochondrial matrix (mt-roGFP2; 59,60). Upon Mff knockdown, there was no significant change in the ratio of 405nm/488nm excitation (0.77 ± 0.04) compared to neurons expressing control shRNA (0.70 ± 0.04) at 21DIV (Fig. 5e-f). These results strongly argue that reducing MFF-dependent mitochondrial fission in axons does not affect their ability to perform oxidative phosphorylation and therefore their ability to generate ATP or their redox state.
Axonal Mitochondrial Elongation upon Mff Knockdown Increases Mitochondrial Calcium Uptake at Presynaptic Sites
Based on previous results from our lab and others demonstrating that mitochondrial Ca2+ uptake plays a critical role in regulating presynaptic release at en passant boutons of pyramidal neurons 15,16, we tested if mitochondrial Ca2+ uptake was affected upon Mff knockdown. Our hypothesis was that the total amount of Ca2+ uptake capacity of mitochondria is in part dependent on their total matrix volume. To monitor presynaptic mitochondrial Ca2+ dynamics ([Ca2+]m), we used a mitochondrial matrix targeted genetically-encoded Ca2+ sensor, mt-GCaMP5G 15,61, and introduced this with VGLUT1-mCherry and mt-mTagBFP2 to cortical neurons using ex utero electroporation at E15.5. At 17-23DIV, we imaged intra-mitochondrial Ca2+ dynamics before and following stimulation of presynaptic release with 20 action potentials (AP) at 10Hz using a concentric bipolar electrode 15,62. During stimulation, Ca2+ import occurs at points of these long mitochondria in direct contact with presynaptic sites, followed by diffusion of mitochondrial Ca2+ along the mitochondria in Mff knockdown neurons (Fig. 6b, Fig. S6 and Movie S4). In addition, long mitochondria show significantly faster extrusion of mitochondrial Ca2+ after stimulation compared to control mitochondria (t=13.2 ± 1.5s of versus 8.2 ± 1.1s in control, Fig. 6a-b and e). To determine if these elongated mitochondria accumulate more Ca2+, we measured the integrated intensity of mt-GCaMP5G over the entire length of individual mitochondria associated with single presynaptic VGLUT1+ boutons. Interestingly, elongated presynaptic mitochondria import significantly higher amounts of Ca2+ than punctate mitochondria (as measured by area under the curve or total charge transfer: 2.3×105 ± 0.41×105 versus 1.4×105 ± 0.19×105 in control, Fig. 6c-d).
We next tested if the increased mitochondrial Ca2+ uptake we observed in elongated mitochondria upon Mff knockdown was impacted by changes in mitochondrial Ca2+ extrusion by applying a mitochondrial Na+/Ca2+ exchanger (NCLX) antagonist, CGP 37157, as NCLX is one of the main mitochondrial Ca2+ extrusion mechanisms. Interestingly, this inhibitor effectively delays Ca2+ extrusion from mitochondria and actually increases the difference of total Ca2+ charge transfer between Mff knockdown and control (5.9×105 ± 7.7×104 versus 2.2×105 ± 2.1×104 in control, Fig. 6f-g). These results demonstrate that the longer presynaptic mitochondria induced by Mff knockdown underlies a significant increase in their Ca2+ buffering capacity.
Increased Uptake of Ca2+ by Elongated Mitochondria in Mff Knockdown Neurons Decreases Presynaptic Ca2+ Levels
To understand the impact of this increased mitochondrial Ca2+ uptake, we tested if presynaptic cytoplasmic Ca2+ ([Ca2+]cyto) is affected by the increased Ca2+ uptake from mitochondria in Mff knockdown axons. Combining GCaMP5G targeted to presynaptic boutons (VGLUT1-GCaMP5G) with mt-mTagBFP2 and VGLUT1-mCherry allowed us to measure presynaptic Ca2+ dynamics at presynaptic boutons associated with mitochondria15. All three plasmids with either control or Mff shRNA were introduced by ex utero cortical electroporation at E15.5 and imaged in axons of pyramidal neurons at 17-23DIV using the stimulation protocol described above (20APs at 10Hz). Interestingly, the peak of [Ca2+]cyto signals from long mitochondria-associated presynaptic boutons was 20% lower (0.46 ± 0.025 versus 0.56 ± 0.02 in control, Fig. 7a-b). In addition, both the peak values of presynaptic [Ca2+]cyto and the total charge transfer (area under the curve) were significantly reduced in Mff knockdown neurons (Fig. 7c-d). These results show that the increased [Ca2+]m characterizing MFF-deficient mitochondria significantly impacts presynaptic [Ca2+]cyto clearance.
Presynaptic Release at Sites of Elongated Mitochondria is Impaired in Mff Knockdown Neurons
Presynaptic Ca2+ influx through voltage-gated Ca2+ channels (VGCC) triggers synaptic vesicle exocytosis, therefore the amount of presynaptic Ca2+ quantitatively regulates neurotransmitter release at individual boutons 15,63. To monitor presynaptic release at individual boutons, we employed pHluorin-tagged synaptophysin (syp-pHluorin) 64. In short, pH-sensitive GFP, pHluorin, is fused to the luminal domain of synaptophysin, and syp-pHluorin fluorescence is quenched because of the low/acidic luminal pH of neurotransmitter vesicles 65. When exposed to extracellular pH following vesicle exocytosis, the luminal pH equilibrates closer to pH 7 and pHluorin emits fluorescence. We introduced the syp-pHluorin, synaptophysin-mCherry (constitutive presynaptic marker), and mt-mTagBFP2 with either control or Mff shRNA using ex utero electroporation at E15.5 and imaged at 20-23DIV. Consistent with decreased presynaptic [Ca2+]cyto levels, presynaptic boutons associated with long mitochondria in Mff knockdown axons displayed significantly decreased evoked neurotransmitter vesicle release (0.074 ± 0.012 versus 0.13 ± 0.014 in control, Fig. 7e-g). These results demonstrate that, in cortical axons, regulation of mitochondrial size by MFF-dependent fission is critical for proper presynaptic Ca2+ homeostasis, neurotransmitter release and results in decreased terminal axon branching in vivo.
Discussion
In the present study, we uncovered a novel mechanism by which neurons regulate presynaptic release and axonal development. Our results demonstrate that loss of MFF activity increased mitochondria length by reducing fission for both mitochondria entering the axon as well as along the axonal shaft. Strikingly, mitochondrial elongation did not affect mitochondrial trafficking, presynaptic positioning, membrane potential, or their ability to produce ATP; however, it dramatically increased their Ca2+ buffering capacity. This size-dependent increase in mitochondrial Ca2+ uptake lessened presynaptic cytoplasmic Ca2+ levels causing reduced neurotransmitter release upon evoked activity and resulted in decreased terminal axon branching in vivo (Fig. S7).
These results suggest a two-step model for MFF-dependent regulation of axonal mitochondrial size where MFF activity is required both at the time of axonal entry as well as for maintenance along the length of the axon. Our data reveals that in control cortical axons, mitochondria enter with an average size of ~1µm, and maintain this short length along the axon by coupling almost every fusion event to a fission event. Our results represent the first evidence that axonal mitochondrial size is regulated before or upon entry into the axon, and raises intriguing possibilities regarding the underlying mechanism(s). Two non-mutually exclusive scenarios exist: (1) mitochondria are “tagged” as axonal (vs. dendritic) mitochondria soon after biogenesis in the cell body; and/or (2) mitochondria allowed to enter the axon are ‘filtered’ based upon size. The most compelling evidence supporting scenario one may be that in hippocampal and cortical neurons axonal and dendritic mitochondria interact with the distinct motor adaptor proteins TRAK1 or TRAK2 respectively 66,67. However, the mechanism and site of first interaction between TRAK1/2 and mitochondria remain unknown i.e. do they interact in the cell body to determine specificity or are TRAK1/2 themselves targeted to the axonal or dendritic compartments and engaged locally? Evidence for scenario two and the existence of a size filter only allowing mitochondria of a short length to enter the axon is currently unexplored. However, recent work demonstrating the presence of an actin based filter at the axon initial segment (AIS) 68,69 and evidence of myosin motors opposing kinesin-directed mitochondrial movement 70 could represent such a mitochondria-size selection filter.
While not the focus of this manuscript, the question of how MFF-dependent fission is regulated differentially in the axon remains poorly understood and should be the focus of further investigations. In fact, it is further complicated by the finding that MFF is present on the outer membrane of both axonal and dendritic mitochondria (Fig. S1). Coupled with the results showing no significant change in dendritic mitochondria length upon Mff knockdown (Fig. S2), this implies that MFF recruitment, activation or interaction with Drp1 must be differentially regulated in the axonal and somatodendritic compartments. Cortical pyramidal neurons could accomplish this via: (1) subcellular targeting of different isoforms of MFF which can be generated through alternative splicing and have different affinities for Drp1 32, (2) MFF can be post-translationally modified and ‘activated’ to increase its ability to recruit Drp1 71, (3) although MiD51 and MiD49 are expressed at extremely low levels in neurons, they may potentially alter the MFF/Drp1 interaction from promoting fission to inhibiting fission 72,73, and finally (4) axonal or dendritic Drp1 may be post-translationally modified to increase or reduce its ability to interact with MFF 10,74–76.
Our data illustrates that the long mitochondria produced upon Mff knockdown possessed a comparable membrane potential and maintained similar levels of matrix ATP (Fig. 5); in fact, based on their increased size, we might expect that they actually have an increased capacity to produce ATP. A previous report observed a requirement for activity-driven ATP synthesis in synaptic function 14; however, ATP production could only be seen upon a prolonged stimulation condition (600APs at 10Hz). It is now clear that mitochondrial Ca2+ buffering is important for synaptic function in more physiological conditions 15,16 and we observed significant increases in presynaptic mitochondrial Ca2+ uptake upon Mff-knockdown leading to reduced synaptic vesicle release with physiological stimulation regimes (20APs @ 10Hz). In addition, blocking glycolysis or ATP production mainly affected endocytosis of synaptic vesicles (SVs), but not exocytosis 14. Layer 2/3 cortical neurons are well known to have less spontaneous activity than other pyramidal neurons 77,78; therefore, our results in Mff-deficient axons is more likely to be a result of abnormal SV release due to increased mitochondrial Ca2+ uptake than altered ATP production.
A recent study showed that the Bcl-xL-Drp1 complex regulates SV endocytosis, and MFF may form a complex with these proteins 79. Consistent with our data, Mff knockdown in hippocampal neurons showed reduced exocytosis of SVs. While the authors of this study normalized the pHluorin signal with F0 value from base line signals, we used the Fmax value obtained by NH4Cl incubation in order to normalize for any potential changes in total vesicle pool size. Even with this more optimal normalization method, MFF-deficient neurons still have reduced SV release. Future experiments will be required to determine the importance of both MFF-mediated decreases on synaptic vesicle recycling and increased mitochondrial Ca2+ uptake on neurotransmitter release.
In addition to mitochondria, axonal endoplasmic reticulum (ER) was recently shown to be involved in presynaptic Ca2+ clearance upon stimulation of neurotransmitter release 80. Although inhibition of ER Ca2+ uptake can change plasma membrane Ca2+ entry through STIM1, a recent 3D-EM study revealed the presence of ER and mitochondria contacts along the axon and specifically at presynaptic sites 81. Future investigation is needed to characterize the relative contribution of axonal ER and mitochondria, and ER-mitochondria coupling for presynaptic Ca2+ homeostasis.
The role of neuronal activity in axonal development and branching has been well established over the last twenty years. Early work demonstrated that developing neurons require spontaneous activity and spontaneous neurotransmitter release for axonal development 82–86 while more recent work has revealed the requirement for activity on both the presynaptic and postsynaptic sides 48,49,87–90. Finally, axonal branches which make presynaptic boutons and are synchronized with postsynaptic neuronal activity are more likely to be stabilized when compared to axons with desynchronized activity 91–93. Based on these previous results and the observation that layer 2/3 terminal branching is the most affected, it seems likely that the loss of terminal axon branching we observe upon Mff knockdown is a result of the decreased neurotransmitter release we observed along these axons.
Taken together, our data demonstrate for the first time that maintenance of small mitochondrial size in CNS axons through MFF-dependent fission is critical to limit presynaptic Ca2+ dynamics, neurotransmitter release, terminal axonal branching and therefore proper development of circuit connectivity.
Author Contributions
Conceptualization, T.L.L., R.S., F.P.; Methodology, T.L.L., S.K., F.P.; Investigation, T.L.L., S.K., A.L.; Writing, T.L.L., S.K., F.P.; Funding Acquisition, T.L.L. and F.P.; Resources, R.S.; Supervision, T.L.L., and F.P.
METHODS
Mice for in utero electroporation
All animals were handled according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University. Time-pregnant CD1 females were purchased from Charles Rivers. Timed-pregnant hybrid F1 females were obtained by mating inbred 129/SvJ females (Charles Rivers), and C57Bl/6J males (Charles Rivers) in house. At the time of in utero electroporation (Embryonic Day 15.5), littermates were randomly assigned to experimental groups without regard to their sex.
Mice for ex utero electroporation and primary culture
All animals were handled according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University. Time-pregnant CD1 females were purchased from Charles Rivers. At the time of ex utero electroporation (E15.5), littermates were randomly assigned to experimental groups without regard to their sex.
HEK 293T Cells
Human Embryonic Kidney (HEK) 293T cells were purchased from ATCC.
Plasmids
pCAG mt-ATEAM was made by placing the DNA encoding mt-ATEAM (gift from Hiromi Imamura) 3’to the CAG promoter via PCR and Infusion cloning (Clonetech). pCAG mt-Grx1-roGFP2 was made by placing the DNA encoding mt-Grx1-roGFP2 (Addgene plasmid #64977) 3’ to the CAG promoter as above. pCAG vGLUT1-HA was created by replacing the mCherry in pCAG vGLUT1-mCherry with an HA tag via restriction digest and PCR. pCAG mTAGBFP2 was created by removing the mitochondrial targeting sequence in pCAG mt-mTAGBFP2 via restriction digest and re-ligation. pCAFNF mTAGBFP2 and mt-YFP were made by PCR of the DNA encoding mTAGBFP2 or mt-YFP and placement 3’ to the CAG FNF cassette (Addgene plasmid #13772) via PCR and Infusion cloning (Clonetech). pCAG HA-mMff and pCAG HA-mFis1 were created via PCR of the DNA encoding mouse Mff or mouse Fis1 from a neuronal mouse cDNA library, and sub-cloned 3’ to the CAG promoter and a HA tag.
In utero electroporation
A mix of endotoxin-free plasmid preparation (2mg/mL total concentration) and 0.5% Fast Green (Sigma) was injected into one lateral hemisphere of E15.5 embryos using a Picospritzer III (Parker). Electroporation (ECM 830, BTX) was performed with gold paddles to target cortical progenitors in E15.5 embryos by placing the anode (positively charged electrode) on the side of DNA injection and the cathode on the other side of the head. Five pulses of 45 V for 50 ms with 500 ms interval were used for electroporation. Animals were sacrificed 21 days after birth (P21) by terminal perfusion of 4% para-formaldehyde (PFA, Electron Microscopy Sciences) followed by overnight postfixation in 4% PFA. For sparse labeling via in utero electroporation, Flp dependent plasmids (pCAFNF, Addgene) were used along with 640 pg/µl of pCAG Flp-e (Addgene).
Ex utero cortical electroporation
A mix of endotoxin-free plasmid preparation (2-5mg/mL) and 0.5% Fast Green (Sigma) mixture was injected using a Picospritzer III (Parker) into the lateral ventricles of isolated head of E15.5 mouse embryo, and electoporated using an electroporator (ECM 830, BTX) with four pulses of 20V for 100ms with a 500ms interval. Following ex utero electroporation we performed dissociated neuronal culture as described below.
Primary neuronal culture
Embyonic mouse cortices (E15.5) were dissected in Hank’s Balanced Salt Solution (HBSS) supplemented with HEPES (10mM, pH7.4), and incubated in HBSS containing papain (Worthington; 14U/ml) and DNase I (100 μg/ml) for 20min at 37°C. Then, samples were washed with HBSS, and dissociated by pipetting. Cell suspension was plated on poly-D-lysine (1mg/ml, Sigma)-coated glass bottom dishes (MatTek) or coverslips (BD bioscience) in Neurobasal media (Invitrogen) containing B27 (1x), Glutamax (1x), FBS (2.5%) and penicillin/streptomycin (0.5x, all supplements were from Invitrogen). After 5 to 7 days, media was changed with supplemented Neurobasal media without FBS.
Immunocytochemistry
Primary culture-Cells were fixed for 10 minutes at room temperature in 4% (w/v) paraformaldehyde (PFA, EMS) in PBS (Sigma), then incubated for 30 minutes in 0.1% Triton X-100 (Sigma), 1% BSA (Sigma), 5% Normal Goat Serum (Invitrogen) in PBS to permeabilize and block nonspecific staining, after washing with PBS. Primary and secondary antibodies were diluted in the buffer described above. Primary antibodies were incubated at room temperature for 1 hour and secondary antibodies were incubated for 30 minutes at room temperature. Coverslips were mounted on slides with Fluoromount G (EMS). Primary antibodies used for immunocytochemistry in this study are chicken anti-GFP (5 μg/ml, Aves Lab – recognizes GFP and YFP), mouse anti-HA (1:500, Covance), rabbit anti-RFP (1:1,000, Abcam – recognizes mTagBFP2, DsRED and tdTomato). All secondary antibodies were Alexa-conjugated (Invitrogen) and used at a 1:2000 dilution. Nuclear DNA was stained using Hoechst 33258 (1:10,000, Pierce)
Endogenous MFF staining-Cells were fixed for 10 minutes at room temperature in 4% PFA in PBS, followed directly with −20°C, 100% methanol at −20°C for 6 minutes. After washing with room temperature PBS, cells were incubated for 30 minutes in 1% BSA (Sigma), 5% Normal Goat Serum in PBS to block nonspecific staining. Primary and secondary antibodies were diluted in the buffer above. Primary antibodies were incubated at room temperature for 1 hour and secondary antibodies were incubated for 30 minutes at room temperature. Coverslips were mounted on slides with Fluoromount G (EMS). Primary antibodies used for this section are chicken anti-GFP (5 μg/ml, Aves Lab – recognizes GFP and YFP), rabbit anti-MFF (1:200, Protein Tech). All secondary antibodies were Alexa-conjugated (Invitrogen) and used at a 1:2000 dilution. Brain sections-Post fixed brains were sectioned via vibratome (Leica VT1200) at 100 µm. Floating sections were then incubated for 2 hours in 0.4% Triton X-100, 1% BSA, 5% Normal Goat Serum in PBS to block nonspecific staining. Primary and secondary antibodies were diluted in the buffer described above. Primary and secondary antibodies were incubated at 4°C overnight. Sections were mounted on slides and coversliped with Aqua PolyMount (Polymount Sicences, Inc). Primary and secondary antibodies are the same as above.
Imaging
Fixed samples were imaged on a Nikon Ti-E microscope with an A1 confocal. All equipment and solid state lasers (Coherent, 405nm, 488nm, 561nm, and 647nm) were controlled via Nikon Elements software. Nikon objectives used include 20x (0.75NA), 40x (0.95NA) or 60x oil (1.4NA). Optical sectioning was performed at Nyquist for the longest wavelength. Analysis of mitochondrial length and occupancy were performed in Nikon Elements.
Live imaging-Electroporated cortical neurons were imaged at 15-21DIV with EMCCD camera (Andor, iXon3-897) on an inverted Nikon Ti-E microscope (40x objective NA0.95 with 1.5x digital zoom or 60x objective NA1.4) with Nikon Elements. 488nm and 561nm lasers shuttered by Acousto-Optic Tunable Filters (AOTF) or 395nm, 470nm, and 555nm Spectra X LED lights (Lumencor) were used for the light source, and a custom quad-band excitation/dichroic/emission cube (based off Chroma, 89400) followed by clean up filters (Chroma, ET435/26, ET525/50, ET600/50) were applied for excitation and emission. We used modified normal tyrode solution as a bath solution at 37°C (Tokai Hit Chamber), which contained (in mM): 145 NaCl, 2.5 KCl, 10 HEPES pH7.4, 2 CaCl2, 1 MgCl2, 10 glucose.
For ATEAM (gift from Hiromi Imamura) imaging a CFP/YFP cube (Chroma, 59217) followed by clean up filters (Chroma, ET475/20, ET540/21) was used, while for roGFP2 (Addgene) imaging, the custom cube above was used followed by clean up with ET525/50.
For Tetramethylrhodamine (Sigma, TMRM) imaging, cells were incubated in the solution above and 20nM TMRM for 20 minutes at 37°C, and imaged in the solution with 5nM TMRM.
For calcium imaging on evoked release, we added APV (50μM, Tocris) and CNQX (20μM, Tocris) in bath solution. Evoked releases were triggered by 1ms current injections with a concentric bipolar electrode (FHC) placed 20μm away from transfected axons. We applied 20APs at 10Hz with 30V using the stimulator (Model 2100, A-M systems) and imaged with 500ms or 100ms interval (2Hz or 10Hz) during 90sec for mt-GCaMP5G signals and 100ms interval (10Hz) for 15sec for VGLUT1-GCaMP5G signals. At the end of acquiring, we added the calcium ionophore ionomycin (5μM, EMD Millipore) and continued imaging with 1sec interval to obtain Fmax value. For blocking mitochondrial Na+/Ca2+ exchanger, we added CGP 37157 (10μM, Tocris) following mt-GCaMP5G imaging with 500ms interval. After 3min, we imaged the same area with the identical condition (20APs at 10Hz).
For syp-pHluorin imaging, 20APs were applied at 10Hz and neurons were imaged with 100ms interval (10Hz) during 60sec, then the bath solution was changed with tyrode solution containing 55mM NH4Cl for Fmax value.
Images were analyzed using a Fiji (Image J) plug-in, Time Series Analyzer (v3.0). Each vGLUT1-GCaMP5G or syp-pHluorin puncta and nearby backgrounds were selected by circular ROIs and intensities were measured by plug-in. For mt-GCaMP5G measurement, single synapse-associated mitochondria were pooled for comparing uptake of presynaptic Ca2+ from a single presynaptic bouton. Full-length mitochondria were marked by a freehand selection tool and total intensities were measured. After intensities were corrected for background subtraction, ΔF values were calculated from (F-F0). F0 values were defined by averaging 10 frames before stimulation, and Fmax values were determined by averaging 10 frames of maximum plateau values following ionomycin or NH4Cl application, then, used for normalization. Long mitochondria in Mff knockdown axons for Ca2+ and pHluorin imaging were selected by the length over 3µm. Diffusion of long mitochondrial Ca2+ from a single presynapse is also analyzed by Fiji. Line ROIs (1µm, width 3) were placed on a presynapse-overlapped site and every 2µm distance from the site. Then, Plot Z-axis Profile function was performed for obtaining intensities from each line, and time to peak was defined from the plot.
Cell Line Transfection and Western Blot
HEK cells were transfected via jetPrime (Polyplus) by following the manufacture‘s instructions. Cells were harvested in cold DPBS and lysed in ice-cold lysis buffer containing 25 mM Tris (pH7.5), 2 mM MgCl2, 600 mM NaCl, 2 mM EDTA, 0.5% NP-40, 1X protease and phosphatase cocktail inhibitors (Sigma) and Benzonase (0.25 U/μl of lysis buffer; Novagen). Aliquots of the proteins were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham). After transfer, the membrane was washed 3X in Tris Buffer Saline (10 mM Tris-HCl pH 7.4, 150 mM NaCl) with 0.1% of Tween 20 (T-TBS), blocked for 1 hr at room temperature in Odyssey Blocking Buffer (TBS, LI-COR), followed by 4°C overnight incubation with the appropriate primary antibody in the above buffer. The following day, the membrane was washed 3X in T-TBS, incubated at room temperature for 1 hr with IRDye secondary antibodies (LI-COR) at 1:10,000 dilution in Odyssey Blocking Buffer (TBS), followed by 3X T-TBS washes. Visualization was performed by quantitative fluorescence using an Odyssey CLx imager (LI-COR). Signal intensity was quantified using Image Studio software (LI-COR). Primary antibodies used for Western-blotting are mouse anti-HA (1:2,000, Covance), rabbit anti-ERp72 (1:1000, Cell Signaling Technologies).
Quantification and Statistical Analysis
All statistical analysis and graphs were performed/created in Graphpad’s Prism 6. Statistical tests, p values, and (n) numbers are presented in the figure legends. Gaussian distribution was tested using D’Agostino & Pearson’s omnibus normality test. We applied non-parametric tests when data from groups tested deviated significantly from normality. All analysis were performed on raw imaging data without any adjustments. Images in figures have been adjusted for brightness and contrast (identical for control and experimental conditions in groups compared).
RESOURCES
Supplemental Figure 1. Validation of Mff shRNA knockdown constructs
(a) Western blot of shRNA knockdown efficiency via overexpression of HA-tagged mouse MFF (top) in HEK cells. Antibody against an endogenous ER protein (ERp72; bottom) was used as the loading control. (b) Western blot of shRNA knockdown efficiency via overexpression of HA-tagged mouse Fis1 (top) in HEK cells. Endogenous ERp72 (bottom) was used as the loading control. (c-e) Representative images of a neuron electroporated with mt-YFP and control shRNA via ex utero electroporation at E15.5, and stained at 7DIV with antibodies for GFP and Mff. (f-h) Representative images of neurons electroporated with mt-YFP and a (1:1) mixture of Mff shRNAs via ex utero electroporation at E15.5, and stained at 7DIV with antibodies for GFP and Mff. (i-k) Representative images of an axon from a neuron electroporated with mt-YFP and control shRNA via ex utero electroporation at E15.5, and stained at 7DIV with antibodies for GFP and MFF. (l-n) Representative images of an axon from a neuron electroporated with mt-YFP and a (1:1) mixture of Mff shRNAs via ex utero electroporation at E15.5, and stained at 7DIV with antibodies for GFP and Mff. Orange arrows point to the same mitochondria in each set of images. Related to Figure 1.
Supplemental Figure 2. Decreased MFF activity does not increase dendritic mitochondrial length
(a-c) Representative images of a dendrite from a neuron electroporated with mt-YFP, tdTomato and control shRNA via in utero electroporation at E15.5, and stained at P21 with antibodies for GFP and tdTomato. (d-f) Representative images of a dendrite from a neuron electroporated with mt-YFP, tdTomato and a mixture of Mff shRNAs via in utero electroporation at E15.5, and stained at P21 with antibodies for GFP and tdTomato. (g) Quantification of mitochondrial length in the dendrites. Data is represented at minimum to maximum box plots, with the box denoting 25th, 50th and 75th percentile. (h) Quantification of the percent of the dendrite occupied by mitochondria. Data is represented at minimum to maximum box plots, with the box denoting 25th, 50th and 75th percentile. ncontrol shRNA = 26 dendrites, 267 mitochondria; nMFF shRNA = 19 dendrites, 146 mitochondria. n.s., p>0.05 according to Mann-Whitney test. Related to Figure 2.
Supplemental Figure 3. Fewer mitochondria enter the axon but have increased length upon loss of MFF expression
(a) Schematic of the imaging paradigm used for measuring axonal entry of mitochondria via mitochondrial-targeted mEos2. (b) Selected timeframes of mitochondria entering the axon of a 10DIV neuron ex utero electroporated with control shRNA and mt-mEos2. (c) Selected timeframes of mitochondria entering the axon of a 10DIV neuron ex utero electroporated with Mff shRNA and mt-mEos2. Fewer mitochondria enter the axon upon Mff knockdown, but are much longer. See supplemental video 2. (d) Quantification of the number of axonal entry events per hour in 7-11DIV axons. Data is represented as a scatter plot with mean ± sem. ***p<0.001 for control vs. Mff shRNA. Mann-Whitney test. (e) Cumulative frequency of mitochondrial length upon axonal entry for control and Mff shRNA mediated knockdown in 7-11DIV axons. **** p<0.0001 for control vs. Mff shRNA fission. Mann-Whitney test. ncontrol shRNA = 12 axons, 63 mitochondria; nMFF shRNA = 19 axons, 48 mitochondria. Related to Figure 3.
Supplemental Figure 4. Loss of MFF activity does not affect the final localization of axonal mitochondria at presynaptic sites
(a) Quantification of the percent of stationary mitochondria for 15min at 7DIV. Data is represented as a scatter plot with mean ± sd. ncontrol shRNA = 14 axons; nMFF shRNA = 16 axons. See supplemental video 3. (b) Quantification of the percent of stationary mitochondria for 15min at 21DIV. Data is represented as a scatter plot with mean ± sd. ncontrol shRNA = 10 axons; nMFF shRNA = 9 axons. (c) Quantification of average velocity formotile mitochondria in axons from (A) Data is represented as a scatter plot with mean ± sd. (d) Quantification of run length for motile mitochondria in axons from (A). Data is represented as a scatter plot with mean ± sd. (e) Representative images of an axon from a neuron electroporated with mt-YFP, VGLUT1-HA, tdTomato and control shRNA (left panels) or a mixture of Mff shRNAs (right panels) via in utero electroporation at E15.5, and stained at P21 with antibodies for GFP, HA and tdTomato. (f) Quantification of the percent of mitochondria at presynaptic sites. (g) Quantification of the percent of presynaptic sites with mitochondria. (h) Quantification of the number of VGLUT1-HA puncta per 100 microns of axon. Data is represented as scatter plots with mean ± sd. ncontrol shRNA = 16 axons; nMFF shRNA = 26 axons. Mann-Whitney test; p values in figure. Related to Figures 5 and 6.
Supplemental Figure 5. Paradigm for the measure of mitochondrial membrane potential in both control and Mff-deficient dissociated neuronal co-culture
(a) Schematic representation of how neurons were labeled with color-swapped fluorescent proteins to identify axons from control and Mff shRNA-expressing neurons before TMRM labeling. Following ex utero electroporation with DNA plasmids encoding control shRNA, mt-mTAGBFP2 and YFP or Mff shRNA, mt-YFP and mTAGBFP2, dissociated neurons were mixed at a 1:1 ratio and plated on coverslips for co-cultures. This strategy allows for the simultaneous imaging of mitochondrial membrane potential (TMRM-see Fig. 5a-b) in both control and Mff knockdown neurons under the exact same culture conditions. (b) Representative field of view for mTAGBFP2/mt-mTAGBFP2. (c) Representative field of view for YFP/mt-YFP. (d) Merged channels shown in B-C where control (sky blue arrowheads) and Mff knockdown (orange arrows) axons can be visualized in the same field of view. Related to Figure 5.
Supplemental Figure 6. Long mitochondria in Mff knockdown neurons show diffusion of imported Ca2+
(a-b) Cropped mitochondrial Ca2+ time-lapse images of control and Mff knockdown axons. Presynaptic mitochondrial Ca2+ signals were captured with 100ms interval for monitoring diffusion of imported Ca2+ through mitochondrial matrix. Ca2+ propagation in long mitochondria of Mff knockdown axons occurs from presynaptic sites. (c) For quantification of diffusion time, analysis was performed with single presynapse-overlapped mitochondria from Mff knockdown neurons. (d-e) Graphs display the latency of time to peak depending on distance from presynaptic sites. nMFF shRNA = 8 dishes, 8 mitochondria. p=0.0012 for 0 vs. 4, 0.0067 for 0 vs. 6. One-way ANOVA, Bonferroni‘s multiple comparison test. Related to Figure 6.
Supplemental Figure 7. MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size
(a) In control neurons, MFF activity is required for the small size of axonal mitochondria both upon axonal entry, as well as for maintenance along the axon. Mitochondrial size is maintained by coupling the majority of mitochondrial fusion events to a fission event. (b) Loss of MFF activity increases mitochondrial entry size and decreases the ratio of fission to fusion along the axon. This leads to increased mitochondrial size along the axon and reduced terminal branching. (c) In control axons, ~50% of presynaptic boutons are occupied by mitochondria. Upon neuronal activity, the mitochondria buffer a significant amount of Ca2+ influx, in an MCU dependent manner, thereby reducing presynaptic vesicle release as compared to presynaptic sites without a mitochondria. (d) Upon MFF knockdown, the increased mitochondrial matrix volume allows for increased Ca2+ uptake after neuronal activity, and strongly reduces presynaptic vesicle release at these sites. The reduction in terminal axon branching observed following MFF knockdown is likely due to this reduction in presynaptic release as presynaptic release is required for the stabilization of axonal branches.
Supplemental Movie 1: Mitochondrial fission and fusion dynamics along the axon upon Mff knockdown. Related to Figures 2 & 3.
Supplemental Movie 2: Dynamics of mitochondrial entry into the axon upon Mff knockdown. Related to Figures 2 and 3.
Supplemental Movie 3: Axonal mitochondrial motility upon Mff knockdown. Related to Figures 2 & 3.
Supplemental Movie 4: Presynaptic mitochondrial Ca2+ uptake upon evoked activity in Mff knockdown axons, Related to Figure 6.
Acknowledgements
The authors would like to thank members of the F.P. and R.S. labs for helpful suggestions, technical advice, and critical reading of the manuscript. Funding sources include NIH-R01NS089456 (F.P.), NIH-K99NS091526 (T.L.L.), Human Frontiers Science Program Long-term Fellowship (S.K.), and an Award from the Fondation Roger De Spoelberch (F.P.).