Introduction
Signaling between the ER and mitochondria regulates a variety of fundamental cellular processes. These include energy metabolism, Ca
2+ homeostasis, phospholipid synthesis, mitochondrial biogenesis and trafficking, ER stress responses, autophagy and inflammation [
7,
34,
37,
39]. This signaling is facilitated by close physical contacts between the two organelles such that up to approximately 20% of the mitochondrial surface is closely apposed (10–30 nm distances) to ER membranes. These regions of ER are termed mitochondria associated ER membranes (MAM) [
7,
34,
37,
39].
The mechanisms by which ER membranes are recruited to the mitochondrial surface are not fully understood but it is widely agreed that the process involves “tethering proteins” which act to scaffold and anchor the two organelles in close proximity. One such tether involves an interaction between the integral ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the outer mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51) [
9,
45]. The VAPB-PTPIP51 tethers are now known to facilitate inositol 1,4,5-trisphosphate (IP3) receptor mediated delivery of Ca
2+ from ER stores to mitochondria, mitochondrial ATP production and autophagy, all of which are known to be regulated by ER-mitochondria crosstalk [
9,
13,
33,
45,
46].
The pivotal roles that ER-mitochondria signaling plays in so many important physiological functions suggest that damage to this signaling will have detrimental effects on cellular homeostasis. Indeed, perturbation of ER-mitochondria contacts and signaling is associated with the major human neurodegenerative diseases Alzheimer’s disease, Parkinson’s disease and FTD/ALS [
1,
23,
24,
34]. Notably, disruption to the VAPB-PTPIP51 tethers has been linked to Parkinson’s disease and FTD/ALS [
9,
33,
45,
46]. FTD is the second most common form of presenile dementia after Alzheimer’s disease and is now known to be clinically, genetically and pathologically linked to ALS, the most common form of motor neuron disease [
24,
28].
Loss of synaptic function is a key pathogenic feature of Parkinson’s disease and FTD/ALS. Indeed, synaptic loss underlies the cognitive and motor dysfunctions that characterise these disorders [
3,
17,
43]. Both ER and mitochondria are known to be present in synaptic regions [
16,
18,
31,
50]. However, any role of the VAPB-PTPIP51 tethers and ER-mitochondria signaling in synaptic function is currently unknown. Such knowledge is essential not only for comprehending the normal roles of ER-mitochondria signaling in synaptic function, but also for determining any pathological role that disruption to the VAPB-PTPIP51 tethers might play in Parkinson’s disease and FTD/ALS. Here, we show that VAPB and PTPIP51 are present and interact in synaptic regions, that their interactions are stimulated by neuronal activity, and that loss of VAPB and PTPIP51 disrupts synaptic function.
Materials and methods
Plasmids and siRNAs
SPLICS
s and SypHy-RGECO reporter plasmids were as described [
6,
20]; pEGFPC1 was from Clontech. Verified non-targeting control and rat VAPB and PTPIP51 siRNAs were purchased from GE Healthcare Dharmacon (Accell range). Sequences were: VAPB A-091473-17# 5′-GUGCUGUUCUUUAUUGUUG-3′, A-091473-18# 5′- CUUAUGGAUUCAAAACUUA-3′, A-091473-19# 5′-GGUUCAGUCUAUGUUUGCU-3′, A-091473-20# 5′-GUUACAGCCUUUCGAUUAU-3′; PTPIP51 (Fam82a2) A-092062-13# 5′-CCUUUAAUGUCAUACCUUA-3′, A-092062-14# 5′-GCUUUAGCUUCAAGGAACA-3′, A-092062-15# 5′- GCUACAGCCUUGUUUGAAA-3′, A-092062-16# 5′- CUCUGGACCUUGAUAUGGA-3′.
Antibodies and chemicals
Rabbit and rat antibodies to VAPB and PTPIP51 were as described [
9]. Rabbit anti-PTPIP51 and chicken anti-MAP 2 were from Gentex. Rabbit anti-translocase of the outer mitochondrial membrane protein-20 (TOM20), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and goat anti-synaptophysin were from Santa Cruz Biotechnology. Mouse anti-β-Tubulin Isotype III antibody and mouse anti-β-Actin were from Sigma. Mouse anti-post synaptic density protein-95 (PSD95) was from Millipore, mouse anti-protein disulphide isomerase (PDI) was from Thermo Fisher Scientific and mouse anti-phosphorylated neurofilament heavy chain (NFH) (antibody SMI31) was from Sternberger Monoclonals Inc. Species specific goat and donkey anti-mouse, −rabbit and -chicken Igs coupled to AlexaFluor-488, − 594 or − 647 were from Invitrogen, Jackson ImmunoResearch, ThermoFisher or Abcam. FM 4–64 was from Invitrogen, DL-2-amino-5-phosphonovaleric acid (AP5) was from Cayman chemical company and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was from Santa Cruz.
Hippocampal neuronal culture and transfection
Hippocampal neurons were obtained from embryonic day 18 rat embryos and cultured in Neurobasal medium containing B27 supplement, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Invitrogen). Neurons were cultured on poly-d-lysine-coated glass cover slips in 12-well plates and analysed at DIV20–23. For siRNA studies, neurons were untreated or treated with 1 μM of each siRNA for 72 h prior to analyses. For transfection studies, neurons were transfected at DIV5 using Lipofectamine 2000 (Invitrogen) (0.5 μg plasmid DNA, 1 μl Lipofectamine 2000 per well) according to the manufacturer’s instructions.
Protein fractionation, SDS–PAGE and immunoblotting
Cells were harvested for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) by washing with calcium-free phosphate buffered saline (PBS) pre-warmed at 37 °C and scraping into SDS-PAGE sample buffer containing protease inhibitors (Complete Roche), 1 mM Na
3VO
4 and 5 mM NaF. Samples were then heated for 10 min at 100 °C, sonicated and centrifuged at 10000 g (av) for 10 min. Total, cytosolic and synaptoneurosome proteins were prepared from rat brains essentially as described [
35,
47]. Protein concentrations were determined using a commercial BCA assay (Pierce). Samples were prepared for SDS-PAGE by addition of sample buffer and then resolved on 10 or 15% SDS-PAGE gels and transferred to nitrocellulose membranes (Schleicher & Schuell Bioscence) by wet electroblotting (BioRad). Membranes were blocked with Tris-HCl-buffered saline (TBS) containing 5% dried milk and 0.1% Tween-20 for 1 h at 20 °C, and then incubated with primary antibodies in blocking buffer for 16 h at 4 °C. Following washing in TBS containing 0.1% Tween-20, the blots were incubated with horseradish peroxidase conjugated secondary antibodies and developed using chemiluminescence with a Luminata Forte Western HRP substrate system according to the manufacturer’s instructions (Millipore). Chemiluminescence signals were detected using a BioRad ChemiDoc MP Imaging system.
Immunofluorescence staining and proximity ligation assays
Neurons grown on coverslips were fixed for 15 min at 20 °C with 4% (
w/
v) paraformaldehyde in PBS and then permeabilized with PBS containing 0.5% Triton X-100 for 15 min. Samples were then preincubated with blocking buffer (PBS containing 10% goat or 2% donkey serum and 0.5% Triton X-100) for 1 h and incubated with primary antibodies diluted in blocking buffer for 16 h at 4 °C. Following washing in PBS containing 0.5% Triton X-100, the samples were incubated with goat/donkey anti-rabbit, mouse, rat or chicken Igs coupled to AlexaFluor − 488, − 594 or 647 in PBS for 1 h, washed in PBS and then mounted in Vectashield mounting medium (Vector Laboratories). Proximity ligation assays (PLAs) to identify the VAPB-PTPIP51 interaction were performed essentially as described previously using Duolink reagents (Sigma-Aldrich) [
13]. Briefly, neurons were fixed in 4% paraformaldehyde in PBS and probed with rat anti-PTPIP51 and rabbit anti-VAPB antibodies, and signals developed using a Duolink In Situ Orange kit (Sigma-Aldrich). Following PLAs, neurons were immunolabeled for synaptophysin and PSD95.
Microscopy
Super resolution structured illumination microscopy (SIM) was performed using Nikon Eclipse Ti-E Inverted microscopes with 100× 1.49 NA CFI objectives and equipped with Nikon N-SIM or Visitech iSIM Super Resolution Systems. Images were captured using an Andor iXon EMCCD camera and reconstructed using Nikon imaging software Elements Advanced Research with N-SIM module or Nikon deconvolution software for iSIM.
Live cell imaging was performed by time-lapse microscopy using a Nikon Eclipse Ti microscope equipped with an Intenslight C-HGFI light source, CFI Apo Lambda S 60x/1.40 objective, TiND6 PFS-S Perfect Focus Unit and EGFP, DsRed and EGFP/DsRed dual filter sets (Chroma Technology). Images were captured using an Andor Neo sCMOD camera. For dual imaging, EGFP and DsRed signals were captured simultaneously using an EGFP/DsRed dual filter set, an Andor TuCam camera adapter system equipped with an emission GFP/RFP dichroic filter set and two Andor Neo sCMOD cameras. Movements were recorded using Nikon NIS-Elements AR software at 2 or 3 s time-lapse intervals. Temperature was maintained at 37 °C using a microscope incubation chamber (Solent Scientific). During recordings, neurons were kept under constant perfusion (0.5 ml/min) with external solution using a Bio-Logic MSC200 fast perfusion system. External solution comprised 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM CaCl2, 1.2 mM Na2HPO4, 10 mM glucose in 20 mM HEPES buffer pH 7.4.
Electrical field stimulations were performed in a Chamlide EC-B18 field stimulation chamber and field stimulation (25 mA pulses of 1 ms duration) delivered by an Isolated Stimulator DS3 controlled by a Train/Delay Generator DG2A (Digitimer). Analyses involving FM 4–64 were performed essentially as described [
19]. Briefly, FM 4–64 (2.5 μM) was added in external solution and loaded into neurons with electrical field stimulation (20 Hz for 60 s) which was applied with an insert adaptor in the culture plates (RC-37FS, Warner instruments). FM 4–64 dye was removed from the surface membranes by incubation in external solution containing NMDA and AMPA receptor antagonists (50 μM AP5, 10 μM CNQX) followed by incubation in Ca
2+-free external solution. Neurons were then transferred to the microscope field stimulation chamber and analysed in time-lapse. For analyses of SPLICS
s, field stimulations were delivered at frequency of 30 Hz for 10 s. For analyses of FM 4–64, 3 field stimulations of 60 s at 20 Hz were applied at 60 s intervals as described [
19]. For analyses of SypHy-RGECO signals, 3 field stimulations of 10 s at 30 Hz were applied at 60 s intervals essentially as described [
20]. VAPB-PTPIP51 PLA field stimulations were conducted in the culture plates using the insert adaptor delivering stimulations of 30 Hz for 10 s. Neurons were then fixed and processed for PLAs and immunostaining.
Confocal microscopy images were acquired using a Leica TCS-SP5 confocal microscope using a 63x HCX PL APO lambda blue CS 1.4 oil UV objective. Z-stack images were analysed and processed using Leica Applied Systems (LAS AF6000) image acquisition software.
PLA signals were quantified in close proximity (less than 1 μm) to synaptic contacts identified by synaptophysin/PSD95 apposition using NIH ImageJ in 20 μm segments of dendrites after the first dendritic branchpoint. ER-mitochondria contacts were quantified by analyses of PDI/TOM20 colocalization with Pearson’s coefficient using Nikon Imaging Software Elements AR. Dendritic spine densities were quantified using NeuronStudio software (CNIC). Active spines involving apposition of spines to synaptophysin signals were quantified using ImageJ in 20 μm segments of dendrites located after the first branch. Time-lapse movies were processed offline using the NIS-Elements AR software and ImageJ. FM 4–64 and SypHy-RGECO signals were quantified as described [
19,
20].
Discussion
Loss of synapses and synaptic dysfunction are principal features of the major human neurodegenerative diseases Alzheimer’s disease, Parkinson’s disease and FTD/ALS [
3,
17,
43]. Damage to ER-mitochondria contacts and signaling is increasingly linked to these diseases and for Parkinson’s disease and FTD/ALS this includes disruption to the VAPB-PTPIP51 ER-mitochondria tethering proteins [
1,
23,
33,
34,
45,
46]. However, whilst ER and mitochondria are both present in synaptic regions [
16,
18,
31,
50] any role that the VAPB-PTPIP51 tethers play in synaptic function is not currently known. Such knowledge is essential for determining whether disruption to ER-mitochondria signaling and the VAPB-PTPIP51 tethers contributes to synaptic dysfunction in Parkinson’s disease and FTD/ALS.
Here we show that the VAPB-PTPIP51 ER-mitochondria tethers are present and interact at synapses. We also demonstrate that stimulating synaptic activity increases ER-mitochondria contacts in synaptic regions and that this involves increased interactions between VAPB and PTPIP51. Finally, we show that siRNA loss of VAPB or PTPIP51 to reduce ER-mitochondria contacts inhibits synaptic activity including alterations to synaptic vesicle release and dendritic spine numbers. Together, our results demonstrate that ER-mitochondria contacts mediated by the VAPB-PTPIP51 tethers regulate synaptic function.
To determine how VAPB and PTPIP51 siRNA loss affects presynaptic function, we utilised two experimental approaches. The first involved the synaptic vesicle recycling dye FM 4–64 which is loaded into synaptic vesicles as they form via endocytosis and then released following induction of synaptic activity [
19]. Loss of VAPB and PTPIP51 both reduced FM 4–64 release consistent with inhibition of presynaptic activity. The initial loading of FM 4–64 requires stimulation of synaptic activity and so it would be interesting in future studies to determine whether loss of VAPB and PTPIP51 affects this process. Such studies would assist in determining whether the VAPB-PTPIP51 tethers affect synaptic vesicle endocytosis and recycling.
The second approach utilised a genetic indicator SypHy-RGECO that involves fusion of the synaptic vesicle protein synaptophysin to both a red shifted Ca2+ indicator (R-GECO1) and a GFP-based pH sensor (pHluorin). Synaptic activity induces increased Ca2+ levels and also changes in pH which are the result of release of neurotransmitter from the acidic synaptic vesicle into the more basic synaptic cleft. Since the pHluorin sensor is more active in basic conditions, stimulation of synaptic activity generates increases in both R-GECO1 and pHluorin signals. The SypHy-RGECO indicator therefore enables optical correlates of Ca2+ and pH changes to be simultaneously monitored in synaptic vesicles. As was the case with FM 4–64 experiments, loss of VAPB and PTPIP51 both inhibited presynaptic activity in assays involving SypHy-RGECO.
As detailed above, following their release, synaptic vesicles are endocytosed for re-cycling and this involves their re-acidification. Following stimulation of synaptic activity, pHluorin signals initially increase with synaptic vesicle release but then decrease as the vesicles are endocytosed. Over the times analysed in our experiments, we observed the expected increase in pHluorin signals following induction of synaptic activity but no marked decreases. However, the times taken for these decreases are variable and dependent firstly upon endocytosis rates and then the times taken for re-acidification of vesicles by vATPase proton pumps. The kinetics are also dependent upon experimental conditions such as the strength of electrical stimulation used to induce synaptic activity [
38] and the type and strength of buffer used to bathe the neurons; stronger buffers require longer to acidify [
2]. Finally, different indicator plasmids (e.g. fusion of pHluorin to synaptophysin, synaptobrevin and VGLUT1) can provide differences in rates of endocytosis. Interestingly, like us others have shown that the SypHy-RGECO indicator plasmid we use generates relatively stable high signals following electrical field stimulation [
20]. Future studies that involve analyses of SypHy-RGECO signals at later time points will help determine how the SypHy-RGECO indicator responds to vesicle recycling.
Aside from these presynaptic effects, we also found that siRNA loss of VAPB and PTPIP51 decreased total dendritic spine numbers and also the numbers of active spines as determined by their apposition to presynaptic synaptophysin. The VAPB-PTPIP51 ER-mitochondria tethers therefore have roles in both pre- and postsynaptic function. Others have recently shown that ER-mitochondria signaling can affect postsynaptic function although whether this involved the VAPB-PTPIP51 tethers was not reported [
18]. Since synapse function is intimately related to both pre- and postsynaptic changes, these reductions in dendritic spine number induced by loss of VAPB and PTPIP51 may therefore influence some of the presynaptic changes we observe.
The precise mechanisms by which the VAPB-PTPIP51 ER-mitochondria tethers affect synaptic function are not fully clear. However, dynamic changes in Ca
2+ signaling are fundamental to synaptic transmission. Presynaptic Ca
2+ levels regulate neurotransmitter release and dendritic Ca
2+ alterations control synaptic plasticity [
10,
40,
41,
44]. Synaptic transmission is also metabolically expensive and ATP production to drive this transmission is associated with mitochondrial Ca
2+ levels since several dehydrogenases in the tricarboxylic acid cycle are Ca
2+ regulated [
12,
14,
15,
25,
34]. A primary function of ER-mitochondria contacts including those mediated by the VAPB-PTPIP51 tethering proteins is to facilitate delivery of Ca
2+ to mitochondria from ER stores [
7,
9,
12,
15,
34,
45]. Indeed, the VAPB-PTPIP51 tethers have been linked to mitochondrial ATP production [
13,
33,
46]. Our findings that the VAPB-PTPIP51 tethers regulate synaptic function are therefore consistent with the known roles of these proteins in Ca
2+ homeostasis and the generation of mitochondrial ATP.
Aside from these roles, ER-mitochondria contacts also control a number of other fundamental cellular functions. For example, the contacts regulate lipid metabolism since the synthesis of some phospholipids involves precursor exchange between the two organelles and the tight contacts facilitate this exchange [
7,
12,
34]. ER-mitochondria signaling involving the VAPB-PTPIP51 tethers also affects autophagosome formation [
13]. Changes in lipid metabolism and autophagy are both known to influence synaptic function [
26,
32]. Thus, the VAPB-PTPIP51 tethers may also modulate synaptic function via their roles in these other processes.
Finally, PTPIP51 has been associated with changes in the activities of a number of signaling molecules and in particular, mitogen-activated protein kinase (MAP kinase) [
4]. MAP kinase plays major roles in synaptic function [
29]. In addition, and based upon its expression in different brain regions, PTPIP51 has been linked to learning and memory [
5]. It will be interesting to determine whether the role of PTPIP51 and VAPB in regulating ER-mitochondria Ca
2+ leads to downstream changes in MAP kinase and other signaling molecules.
As detailed above, damage to ER-mitochondria signaling has been associated with Alzheimer’s disease, Parkinson’s disease and FTD/ALS and for Parkinson’s disease and FTD/ALS this can involves disruption of the VAPB-PTPIP51 tethers [
1,
23,
24,
33,
34,
45,
46]. Synaptic damage is a key feature of all these diseases but the mechanisms underlying this damage are not fully understood. Our findings that the VAPB-PTPIP51 tethers regulate synaptic activity therefore provide a novel route linking neurodegenerative disease insults with synaptic dysfunction. Future studies to determine whether such insults affect ER-mitochondria contacts and the VAPB-PTPIP51 tethers in synaptic regions would provide further insight into this topic. In addition, it will also be informative to determine whether correction of damaged VAPB-PTPIP51 tethers rescues synaptic damage. Interestingly, increasing ER-mitochondria contacts via overexpression of VAPB rescues α-synuclein induced damage to mitochondrial Ca
2+ levels [
33]. Also, exogenous viral delivery of VAPB is protective in ALS mutant superoxide dismutase-1 transgenic mice [
22]. Thus, the findings we report here pave the way for future studies that address whether synaptic damage in neurodegenerative diseases is linked to changes in VAPB-PTPIP51 interactions and ER-mitochondria signaling.