PINK1 Regulates Mitochondrial Trafficking in Dendrites of Cortical Neurons through Mitochondrial PKA
Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson\xe2\x80\x99s disease, are two neuroprotective serine/threonine kinases that regulate dendrite remodeling, and mitochondrial function. We have previously shown that PINK1 regulates dendrite morphology by enhancing PKA activity. Here, we show the molecular mechanisms by which PINK1 and PKA in the mitochondrion interact to regulate dendrite remodeling, mitochondrial morphology, content, and trafficking in dendrites. PINK1-deficient cortical neurons exhibit impaired mitochondrial trafficking, reduced mitochondrial content, fragmented mitochondria, and a reduction in dendrite outgrowth compared to wild-type neurons. Transient expression of wild-type, but not a PKA-binding deficient mutant of the PKA-mitochondrial scaffold dual-specificity A Kinase Anchoring Protein 1 (D-AKAP1), restores mitochondrial trafficking, morphology, and content in dendrites of PINK1-deficient cortical neurons suggesting that recruiting PKA to the mitochondrion reverses mitochondrial pathology in dendrites induced by loss of PINK1. Mechanistically, full-length and cleaved forms of PINK1 increase the binding of the regulatory subunit \xce\xb2 of PKA (PKA/RII\xce\xb2) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RII\xce\xb2 and PKA activity. D-AKAP1/PKA governs mitochondrial trafficking in dendrites via the Miro-2/TRAK2 complex and by increasing the phosphorylation of Miro-2. Our study identifies a new role of D-AKAP1 in regulating mitochondrial trafficking through Miro-2, and supports a model in which PINK1 and mitochondrial PKA participate in a similar neuroprotective signaling pathway to maintain dendrite connectivity.
INTRODUCTION<!>Cell Culture<!>Immunofluorescence and live cell imaging<!>PKA Activity Assay<!>Western Blot and Immunoprecipitation<!>Phos-tag and LC-MS/MS<!>Confocal Imaging<!>Immunohistochemistry and Image Analyses<!>Cytotoxicity Assays<!>Statistical Analysis<!>Power analyses<!>Loss of PINK1 alters dendrite outgrowth and mitochondrial movement and content in dendrites<!>Loss of PINK1 adversely affects PKA signaling in cortical neurons<!>D-AKAP1/PKA reverses PINK1 deficiency-induced loss of dendrite length and mitochondrial trafficking in cortical neurons<!>Cytosolic PINK1 enhances mitochondrial transport in dendrites through activation of mitochondrial PKA signaling<!>PINK1 and mitochondrial PKA synergize mitochondrial interconnectivity in dendrites, but not mitochondrial trafficking or dendrite outgrowth<!>PINK1 enhances the interaction of D-AKAP1 with PKA/RII\xce\xb2<!>Effects of PKA/D-AKAP1 on mitochondrial trafficking: possible mechanisms<!>1) Drp1<!>2) Mfn2<!>3) Miro-2/TRAK2<!>Miro-2 enhances neurite length and promotes mitochondrial transport in dendrites via activating mitochondrial PKA<!>D-AKAP1/PKA mediates phosphorylation of Miro2<!>DISCUSSION
<p>Mitochondrial dysfunction underlies the pathogenesis of both sporadic and genetic forms of Parkinson's disease (PD). Increased oxidative stress and abnormalities in mitochondrial respiration have been documented in cells and tissues from PD patients (Hoepken et al. 2007, Exner et al. 2007). The involvement of PINK1, an autosomal recessive PD gene product localized to mitochondria, has further strengthened the link between PD and mitochondrial biology (Dodson & Guo 2007).</p><p>PINK1 is a neuroprotective serine/threonine (ser/thr) kinase localized to both the mitochondria and the cytosol (Zhou et al. 2008). At the mitochondrion, PINK1 is critical for maintaining mitochondrial function and structure as loss of endogenous PINK1 is associated with mitochondrial fragmentation, overt macroautophagy/mitophagy, and elevated production of mitochondria-derived reactive oxygen species (ROS) (Deng et al. 2005, Mills et al. 2008, Dagda & Chu 2009). Cytosolic PINK1, triggers pro-survival and trophic signaling pathways (Dagda et al. 2014, Murata et al. 2011) to promote dendritic extension, a physiological effect that requires PKA signaling in mitochondria (Dagda et al. 2014).</p><p>Mitochondrial PKA (referred to as D-AKAP1/PKA) is also a regulator of neuronal differentiation, mitochondrial structure and function, autophagy/mitophagy, and dendrite homeostasis (Dickey & Strack 2011, Dagda et al. 2011, Merrill et al. 2011). The PKA holoenzyme is composed of two catalytic subunits bound to two regulatory subunits of two different isoforms. Type II regulatory (RII) subunits anchor PKA holoenzyme to distinct dual-specificity A-kinase anchoring proteins (D-AKAPs), which restrict PKA signaling to distinct subcellular sites (Feliciello et al. 2001). The RIIβ subunit of PKA (PKA/RIIβ) is predominantly enriched in neurons, specifically in postsynaptic compartments (Glantz et al. 1992). In cardiac myocytes, PKA-mediated autophosphorylation of RIIβ increases its affinity for AKAP14/15, lowers the activation threshold of PKA, and enhances localized PKA signaling (Manni et al. 2008, Zakhary et al. 2000). The upstream signaling pathways that enhance RIIβ phosphorylation and localization of type II PKA holoenzymes in dendrites of neurons during development are, however, not well understood. PKA is targeted to the mitochondrion by D-AKAP1, a neuroprotective mitochondria-directed scaffold of PKA (also termed as AKAP140/149, AKAP121, and sAKAP84). Although mutations in specific components of the regulatory machinery of PKA or AKAPs have not been associated with familial PD, deregulated PKA signaling has been reported in postmortem PD brain tissue and chemical and genetic models of PD (Howells et al. 2000, Sandebring et al. 2009, Parisiadou et al. 2014, Chalovich et al. 2006, Dagda et al. 2014).</p><p>Mitochondria are highly dynamic organelles that undergo constant fission/fusion, trafficking, and turnover (mitophagy). The mitochondrial trafficking machinery in neurons consists of the microtubule motors kinesin and dynein, and the mitochondrial adaptor proteins TRAK1/TRAK2 and Miro1/Miro2. The mitochondrial Rho GTPases Miro1 and Miro2 are outer mitochondrial membrane (OMM)-anchored proteins that govern bidirectional trafficking of mitochondria in neurites (Russo et al. 2009, Birsa et al. 2013) by interacting with kinesin and dynein motors and the TRAK-family of adaptor proteins. Disruption of mitochondrial trafficking in neurites induced by oxidative stress or dysfunction in the mitochondrial transport machinery, can lead to severe defects in synaptic function and plasticity, and aberrations in neuronal morphology that eventually lead to the demise of neurons (MacAskill et al. 2010, Sheng & Cai 2012).</p><p>PINK1 is a critical regulator of mitochondrial trafficking and quality control (Weihofen et al. 2009). In mouse hippocampal axons, PINK1 phosphorylates Miro1 at the OMM to promote its degradation via Parkin, in turn stalling mitochondria and facilitating their autophagic/lysosome-mediated sequestration (Wang et al. 2011). In dendrites, the cytosolic pool of PINK1 upregulates mitochondrial content in dendrites and elicits downstream PKA signaling to induce dendrite outgrowth (Dagda et al. 2014). We have previously reported that redirecting endogenous PKA to the mitochondrion can reverse mitochondrial dysfunction in undifferentiated PINK1-deficient neuroblastoma cells (Dagda et al. 2011). However, the specific underlying mechanisms of how PINK1 and PKA modulate dendritic homeostasis and mitochondrial trafficking in dendrites of primary neurons are not clear.</p><p>We hypothesized that PINK1 regulates mitochondrial trafficking and content in dendrites to synergize dendrite homeostasis in an AKAP- and mitochondrial PKA-dependent manner. Here we show that increasing PKA signaling at the mitochondrion reverses impaired mitochondrial trafficking and loss of mitochondrial content in dendrites of PINK1-deficient primary neurons. In addition, upregulating PKA activity in the cytosol restored dendrite length in PINK1-deficient neurons. Mechanistically, D-AKAP1/PKA governs mitochondrial trafficking via the Miro2/TRAK2 complex and by phosphorylating Miro-2. PINK1 enhances the D-AKAP1-RIIβ association, and augments the autocatalysis-mediated phosphorylation of RIIβ. Overall, our data suggests that PINK1 modulates two pools of PKA, (1) cytosolic PKA, to regulate dendrite outgrowth, and (2) mitochondrial PKA, via D-AKAP1, to regulate mitochondrial trafficking/distribution in dendrites. By being able to regulate both pools of PKA, PINK1 plays an important role in both development and protection of dendritic arbors in neurons to foster dendrite homeostasis.</p><!><p>COS-7 cells (cultured in DMEM containing 4.5 g/L D-glucose, 2mM sodium pyruvate, 2mM glutamine, 10% fetal bovine serum) are more amenable than neuroblastoma cells for biochemical analyses with their higher transfection efficiency (>95% C0S7 vs. 40% SH-SY5Y). Hence, COS-7 cells were used for immunoprecipitation experiments to analyze phosphorylation of Miro2-Myc and processing of PINK1-3XFlag. All experiments requiring the use of ATTC-approved cell lines and treatment/transfection of cells with pharmacological compounds, reagents and recombinant DNA was performed via a Memorandum of Understanding and Agreement on the Use of Biological Agents and Recombinant DNA (MOUA #B2016-06, expiration date: 03-2019) that was approved by the University of Nevada, Reno Institutional Board Committee.</p><p>Primary cortical neurons were prepared as previously described (Dagda et al. 2014, Dagda et al. 2011), and used to investigate the effects of PINK1 and PKA on neuron-specific processes. All experiments involving mice were performed in accordance with ARRIVE guidelines and were approved by the University of Nevada, Reno's Institutional Animal Care and Use Committee (IACUC, Protocol # 00572). In brief, primary cortical neurons were prepared from timed-pregnant female E17 wild type C57BL/6 (referred to as PINK1+/+ or wild-type, RRID:IMSR_JAX:000664) or timed-pregnant female PINK1 knockout mice (Catalog#: B6.129S4-Pink1tm1Shn/J, RRID:IMSR_JAX:017946, Jackson Laboratories; referred to as PINK−/−). The PINK1 genotype of each pup was confirmed by PCR. At 5 days in vitro (DIV), primary cortical neurons were transiently transfected with the different plasmids described above. All analyses of mitochondrial trafficking and dendrite length were conducted 48hrs. post-transfection.</p><!><p>For neurite length measurements, cortical neurons were fixed with 4% paraformaldehyde (PFA), permeabilized in PBS containing 0.1% Triton X-100 (PBST), immunolabeled for GFP and MAP2B to identify dendrites in transfected cells, and counterstained with 1.25μg/ml DAPI to visualize nuclei. Immunolabeled cells were imaged by using an EVOS-FL Cell Imaging System, equipped with EVOS Light cubes specific for GFP (Ex/Em of 470/510) and RFP (Ex/Em of 531/593), at a magnification of 20× (0.45NA). Neurite lengths were analyzed by using the NIH Image J plug-in program 'NeuronJ' (Erik Meijering, Biomedical Imaging Group Rotterdam, Netherlands) as previously described (Dagda et al. 2014). Neurite length was assessed for at least 25–30 neurons per experiment.</p><p>For live cell imaging, primary cortical neurons were co-transfected with PKA- modulating or PINK1 plasmids, and with Mito-RFP. The movement of mitochondria was monitored by time-lapse microscopy every 10s for 4 min. at a magnification of 100× by using the EVOS-FL Cell Imaging System as previously described (Dagda et al. 2014). Kymographs were analyzed using Image J v 1.44 (Bethesda, MD, USA) and compatible plug-in 'Multiple Kymograph' (J. Rietdorf, A. Seitz, EMBL, Heidelberg). Movement was characterized as "anterograde" if mitochondria moved away from cell body, and "retrograde" for movement towards cell body. Hence, to assign the direction of moving mitochondria for each neuron of interest in a consistent manner, the "movement tracks" of mitochondria were always traced from left to right of the cell body. Mitochondrial velocity and distance were assessed for at least 50–70 mitochondria per cell, for 6–7 transfected primary cortical neurons per condition. Mitochondrial content in dendrites was assessed using the line tool of Image J to calculate the percentage of dendrite length occupied by mitochondria as previously described (Dagda et al. 2014). All neurite length and mitochondrial movement quantitations were scored blindly by an independent participant who was not directly involved with the experiment.</p><!><p>PKA activity was measured in midbrain of wild-type and PINK−/− mice by determining the level of PKA-mediated substrate phosphorylation by ELISA using a colorimetric PKA activity kit (Enzo Life Sciences; ADI-EKS-390A) as previously published (Kamga Pride et al. 2014) with minor modifications. To determine PKA-specific activity, midbrain lysates were treated with H89, a pharmacological inhibitor of PKA (10μM, 5 min), and the H89-recalcitrant kinase activity was subtracted from total kinase activity of untreated tissue lysates.</p><!><p>Proteins (1% TritonX-100 with protease/phosphatase inhibitors) were resolved on 10% Tris-HCl polyacrylamide gels as previously described (Dagda et al. 2009). Miro-2, FLAG or GFP immunoreactivity from PINK1 and D-AKAP1 constructs were determined in cell lysates with rabbit anti-FLAG, goat anti-GFP, rabbit anti-Miro2, or with rabbit anti-β-tubulin antibodies. Mouse brain lysates were obtained from 10-month-old mice (wild-type or PINK1−/−) to determine the expression levels of phospho- and total PKA/RIIβ.</p><p>For analyzing the ability of D-AKAP1-GFP to immunoprecipitate (IP) with p-RIIβ, cell lysates from COS7 cells transiently co-expressing full-length PINK1-FLAG and D-AKAP1-GFP were incubated with anti-GFP mb-Agarose beads, as per manufacturer's instructions. Proteins were resolved on 10% Tris-HCl polyacrylamide gels, and transferred to PVDF membranes. The membrane was blocked in 5% BSA and incubated with GFP and p-RIIβ antibodies at 4°C. Following overnight incubation, PVDF membranes were incubated with the appropriate secondary antibodies conjugated to HRP and analyzed by film detection of chemiluminescence. Densitometric analyses were done by measuring the integrated density of each protein of interest, corrected for background, and normalized to β-tubulin or β-actin by using Image J as previously published (Dagda et al. 2008, Dagda et al. 2009).</p><!><p>Phos-tag and LC-MS/MS analyses were performed to analyze the phosphorylation status of two species (68 and 75kDa) of endogenous and overexpressed Miro-2 under PKA-enhancing conditions in COS7 cells. COS-7 cells were transiently transfected with Miro-2-Myc alone or cotransfected with GFP-tagged D-AKAP1, D-AKAP1-∆PKA, or full-length PINK-3X Flag for two days, in the presence or absence of cAMP (db-cAMP, 250μM, 2 hrs.). Cell lysates (RIPA buffer containing protease/phosphatase inhibitors) were resolved on a 7.5% Tris-HCl polyacrylamide gel containing 10μM Phos-tag, transferred onto a PVDF membrane, and immunoblotted for total and phosphorylated Miro-2. For every amino acid phosphorylated in Miro2, an upward mobility shift of 5–10 kDa above endogenous Miro-2 is predicted to occur. The mean intensities for phosphorylated Miro-2 (75kDa) were quantified using Image J and normalized to total Miro-2 levels. Additional details are available in the Supplemental Materials.</p><!><p>Confocal imaging analysis was performed to ascertain whether co-expressing D-AKAP1 and PINK1 promotes mitophagy. Additional details are available in the Supplemental Material.</p><!><p>Immunohistochemical analyses was performed in midbrain tissue obtained from WT and PINK1−/− mice (male and female, 1:1 ratio) to determine abundance of total and phosphorylated PKA/RII β and to determine neurite lengths of midbrain neurons. Additional details are available in the Supplemental Materials and Methods.</p><!><p>Primary cortical neurons (wild-type and PINK1−/−) were transfected with Mito-GFP or D-AKAP1-GFP at 4DIV. At 5DIV, wild-type cultures were treated with rotenone (60nM, 24 h.) to induce cytotoxicity. 24hrs. post-treatment, wild-type and PINK1−/− neurons were fixed with 4% PFA and immunostained for GFP and for cleaved caspase-3, a hallmark of late-stage apoptosis. Active cell death was calculated as percentage of GFP-positive neurons labeled for active caspase-3. All assays were performed blind to experimental conditions.</p><!><p>Unless indicated otherwise, results are expressed as mean ± S.E.M. from three independent experiments. Data was analyzed by Student's t test (two-tailed) for pairwise comparisons. Multiple group comparisons were done by performing one-way ANOVA followed by Bonferroni-corrected Tukey's test. P values less than 0.05 were considered statistically significant.</p><!><p>For both immunohistochemistry and in vivo protein expression analyses using western blot, a total sample size of 8 (4 mice per group) was required based on preliminary data which yielded a large effect size (Cohen's d= 1.99, β: 0.80 and α=0.05).</p><!><p>Cytosolic PINK1 remodels dendrites and mitochondrial content in primary cortical neurons (Dagda et al. 2014). Consistent with a role of PINK1 in maintaining dendrite homeostasis, we observed a significant decrease in dendrite length and complexity (# of intersections per neuron) per midbrain dopamine neuron of 10-month-old PINK1 −/− mice compared to wild-type mice (Fig. 1A, S1A). This result suggests that PINK1 regulates either dendrite outgrowth and/or dendrite maintenance by enhancing the stability of dendritic arbors. To determine the role of PINK1 in dendrite outgrowth or maintenance, we performed a time-course analysis of dendrite length in wild-type and PINK1-deficient primary cortical neurons. PINK1-deficient neurons showed significantly decreased rates of dendrite outgrowth compared to wild-type neurons at all time points examined. Dendrite length plateaued at 5 DIV for both wild-type and PINK1−/− neurons, with non-significant changes at 8 DIV, indicating that PINK1 plays a critical role in dendrite outgrowth rather than maintenance during neuronal development (Fig. 1B).</p><p>PINK1 appears to have different roles depending on its localization. In axons, PINK1 phosphorylates Miro1 at the OMM to promote its degradation via Parkin, in turn stalling mitochondria (Wang et al. 2011). In dendrites, PINK1 regulates mitochondrial transport, morphology and content (Dagda et al. 2009, Dagda et al. 2011). Consistent with this concept, we observed that loss of endogenous PINK1 impaired anterograde trafficking of dendritic mitochondria (Fig. 1C), fragmented mitochondria, and decreased mitochondrial content within dendrites (Fig. 1D). Overall, these results indicate that endogenous PINK1 is a critical regulator of dendrite outgrowth, and proper mitochondrial distribution in dendrites. The decreased dendrite outgrowth in PINK1-deficient neurons is not due to cell death, as PINK1deficiency does not significantly increase basal apoptosis of primary cortical neurons (Fig. S1B).</p><!><p>PINK1 modulates PKA signaling as transient or stable expression of full-length PINK1 in primary cortical neurons or SH-SY5Y cells increases intracellular PKA signaling (Dagda et al. 2014). Conversely, here we show that midbrains from 10-month-old PINK1 −/− mice exhibited significantly reduced global PKA activity compared to wild-type mice (Fig. 2A). PKA-mediated autophosphorylation of RIIβ increases its affinity for AKAPs, and lowers the activation threshold of PKA to enhance localized PKA signaling (Manni et al. 2008, Zakhary et al. 2000). Hence, a high level of phosphorylated of PKA/RIIβ is a surrogate marker for the presence of active compartmentalized PKA in neurons. The number of neurons expressing phosphorylated PKA/RIIβ (phospho-RIIβ) within dendrites was significantly reduced in the substantia nigra of PINK1−/− mice compared to wild-type, while expression of total PKA/RIIβ was not affected (Fig. 2B, S2A). Consistent with this observation, we observed a significant reduction in expression of phospho-PKA/RIIβ, but no change in the expression levels of total PKA/RIIβ, in whole brain lysates from 10-month-old PINK1−/− mice compared to wild-type mice (Fig. 2C). We then examined the extent by which overexpressing PINK1 increases the levels of phospho-PKA/RIIβ. Indeed, stable overexpression of PINK1-3X Flag resulted in a robust increase in the ratio of phospho-PKA/RIIβ to total PKA/RIIβ compared to a control cell line (Fig. 2D). Next, we investigated whether restoring intracellular PKA activity reverses the decreased mean dendrite length caused by loss of endogenous PINK1. Indeed, we observed that treating PINK1-deficient neurons with dibutyryl cAMP (250μM, 24hrs.) or transiently transfecting them with PKA/RIIβ alone, restored the mean dendrite length as wild-type neurons (Fig. 2E, F, S2B). These observations suggest that deregulated PKA signaling contributes to the loss of dendrite length/complexity in PINK1-deficient neurons, and that direct restoration of PKA activity or PKA/RIIβ levels in dendrites can completely, or partially, rescue loss of dendrite length due to PINK1-deficiency.</p><!><p>The ability of cAMP to reverse the loss of dendrite length in PINK1-deficient neurons suggests that distinct subcellular pools of PKA can restore dendrite connectivity. PKA is targeted to mitochondria by associating with D-AKAP1, a mitochondria-directed scaffold of PKA (Dagda & Das Banerjee 2015). As D-AKAP1/PKA has been shown to reverse mitochondrial pathology in undifferentiated PINK1-deficient neuronal cells (Dagda et al. 2011), we surmised that D-AKAP1/PKA can also ameliorate loss of dendrites and dendritic mitochondrial pathology. To redirect PKA to the mitochondrion, wild-type and PINK1-deficient cortical neurons were transfected with the common PKA-binding region shared among splice variants of D-AKAP1 (Carnegie & Scott 2003), or with a PKA-binding deficient mutant of D-AKAP1 (D-AKAP1-ΔPKA). Transient expression of D-AKAP1 restored anterograde mitochondrial movement in PINK1-deficient neurons to a similar extent as wild-type neurons (Fig. 3A). Transient expression of D-AKAP1-ΔPKA was unable to restore mitochondrial trafficking in cortical neurons suggesting that binding of PKA to D-AKAP1 is essential for stimulating mitochondrial movement in PINK1-deficient neurons (Fig. 3A). Consistent with the ability of D-AKAP1/PKA to promote mitochondrial interconnectivity (fusion) and neuroprotection (Merrill et al. 2011, Dickey & Strack 2011), we observed that transient expression of D-AKAP1, but not D-AKAP1-ΔPKA, partially reversed mitochondrial fragmentation in PINK1-deficient neurons compared to wild-type primary cortical neurons expressing Mito-RFP (Fig. 3B). D-AKAP1-GFP, partially but significantly, also increased dendrite length in PINK1-deficient neurons (Fig S3A) suggesting that D-AKAP1 can ameliorate mitochondrial dysfunction in dendrites in a genetic PD model of PINK1 deficiency. In a chemical model of PD, transient expression of D-AKAP1-GFP robustly protected primary cortical neurons against cell death induced by the complex I inhibitor rotenone (Fig. S3B), but only partially increased dendrite length of rotenone-treated cells (Fig. S3C).</p><!><p>Cytosolic PINK1 requires mitochondrial PKA signaling to remodel dendrites in primary cortical neurons (Dagda et al. 2014). However, the downstream signaling players involved in regulating PINK1-mediated mitochondrial trafficking are not known. We hypothesize that PINK1 requires mitochondrial PKA signaling to stimulate mitochondrial trafficking. We observed that transient expression of cytosolic PINK1 (ΔN111-PINK1) significantly increased the mean anterograde mitochondrial velocity and distance traveled per mitochondrion, with no effect on retrograde movement (Fig. 3C). Transient co-expression of an inhibitor of PKA targeted to the mitochondrion (Mito-PKI) – which reduces endogenous PKA activity in mitochondria by at least 40% in neuronal cells (Dagda et al. 2014) (Merrill et al. 2011)- completely blocked the ability of ΔN111-PINK1 to enhance mitochondrial trafficking in dendrites suggesting that PINK1 requires PKA to stimulate mitochondrial trafficking. Mito-PKI alone had no effect on basal mitochondrial trafficking suggesting that mitochondrial PKA is not sufficient to modulate mitochondrial trafficking in dendrites.</p><!><p>Next, we investigated the consequences of up-regulating both ser/thr kinases on mitochondrial trafficking, content and dendrite length in primary cortical neurons. As expected, we observed that transiently expressing full-length PINK1 or D-AKAP1 significantly increased anterograde mitochondrial velocity and distance traveled per mitochondrion (Fig. S4A, B). Co-expressing full-length PINK1 and D-AKAP1 also significantly increased the anterograde mitochondrial velocity and distance, but to a lesser extent compared to neurons transiently expressing full-length PINK1 alone. Similar effects were observed for dendrite length (Fig. S4C). Co-expression with D-AKAP1-ΔPKA, on the other hand, negated the ability of PINK1 to induce mitochondrial trafficking suggesting that PINK1 requires binding of PKA to D-AKAP1 to govern mitochondrial trafficking. Interestingly, we observed an additive effect of co-expressing full-length PINK1 and D-AKAP1 on mitochondrial interconnectivity in dendrites compared to transient expression of either FL-PINK1 or D-AKAP1-GFP (Fig. S4D). The data suggests that both ser/thr kinases interact in parallel pathways or in a linear signaling pathway to regulate different aspects of mitochondrial and dendrite homeostasis.</p><!><p>The data thus far suggests two plausible models by which D-AKAP1/PKA can reverse dendrite pathology: (1) both ser/thr kinases converge at the mitochondrion to modulate mitochondrial and dendrite homeostasis, or (2) PINK1 and D-AKAP1/PKA participate in parallel neuroprotective signaling pathways to phosphorylate common substrates. Based on the converging effects of PINK1 and D-AKAP1/PKA on mitochondrial trafficking and dendrite morphology, we hypothesized that PINK1 may increase the binding of D-AKAP1 with PKA. Indeed, we observed that transient co-expression of PINK1 with D-AKAP1 increased the amount of PKA/RIIβ that IPs with wild-type D-AKAP1 (Fig. 4A). Transient co-expression with mutant PINK1-K219M, on the other hand, reduced the amount of PKA/RIIβ that IPed with D-AKAP1 compared to wild-type PINK1 (Fig. 4B). Next, we surmised that the increased phosphorylation of PKA/RIIβ by PINK1 leads to increased activity of mitochondrial PKA (D-AKAP1/PKA). To address this hypothesis, we analyzed the intensity of PKA-mediated phosphorylation of Drp1, which is substrate of D-AKAP1/PKA (Cribbs & Strack 2007, Merrill et al. 2011), by Western blot. COS-7 cells transiently expressing Mito-GFP, Mito-GFP and PINK1-FLAG, D-AKAP1-GFP, or D-AKAP1-GFP and PINK1-FLAG were compared to PKA targeted to the mitochondrion (Mito-PKA) as positive control for elevating p-Drp1 levels. By using an antibody that recognizes the PKA phosphorylation site in Drp1, we observed that transient expression of D-AKAP1 alone modestly increases p-Drp1 levels compared to cells expressing Mito-GFP, consistent with a previous study (Dagda et al. 2011). However, co-expressing of PINK1 with D-AKAP1 significantly enhanced the level of Drp1 phosphorylation compared to D-AKAP1 expression alone (~two fold increase), albeit not to the same level as in cells expressing Mito-PKA (Fig. 4C). Overall, our data suggests that PINK1 enhances the association of PKA/RIIβ with D-AKAP1 to elevate PKA activity in the mitochondrion, presumably by enhancing PKA-mediated autocatalytic phosphorylation of RIIβ.</p><p>Unexpectedly, our IP/Western blot data showed that co-expressing D-AKAP1 with PINK1 reduces the amount of processed forms of exogenous PINK1 (55 and 44kDa bands) relative to full-length PINK1 (Fig. S5A). An increase in OMM-localized PINK1 (~66kDa) is associated with increased mitophagy of damaged mitochondria (Narendra et al. 2010). We, however, failed to see induction of mitophagy in COS7 cells co-expressing full-length PINK1 and D-AKAP1 compared to empty-vector transfected cells (number of lysosomes localized to mitochondria; Empty vector: 4.1 ±1.1; PINK1: 4.1±1.8; D-AKAP1: 8.5 ±2.1; PINK1/D-AKAP1: 8.7±2.1, p=0.115, PINK1/D-AKAP1 vs. empty vector; p=0.108, PINK1/D-AKAP1 vs. PINK1; One-Way ANOVA, Tukey's test), ruling out activation of mitophagy as the potential cause for reduced processing of PINK1 (Fig. S5B).</p><!><p>In an effort to identify potential mechanisms by which D-AKAP1/PKA regulates mitochondrial trafficking, we examined the role of known modulators of mitochondrial transport on PKA-mediated mitochondrial movement in dendrites.</p><!><p>Drp1 is a GTPase protein that regulates mitochondrial fission. D-AKAP1/PKA phosphorylates human Drp1 at serine 637 to promote mitochondrial fusion and enhance mitochondrial content in dendrites of hippocampal neurons (Merrill et al. 2011, Dickey & Strack 2011). However, we did not observe any significant effects of transiently expressing wild-type Drp1, or the PKA phospho-mimetic mutant of Drp1 (S656D), on mitochondrial movement or density in dendrites. (Fig. S6A–C).</p><!><p>The mitochondrial fusion modulator mitofusin-2 (Mfn2) is a bona fide substrate of PINK1 and PKA (Chen & Dorn 2013, Zhou et al. 2010), and regulates mitochondrial trafficking and their distribution in axons of neurons by binding to the Miro/Milton complex (Misko et al. 2010). Although D-AKAP1 IPed endogenous Mfn2 in a PKA-independent manner (Fig. S7A), transient expression of wild-type Mfn2 alone was not sufficient to increase dendrite length (Fig. S7B) or mitochondrial trafficking in primary cortical neurons (Fig. S7C–D).</p><!><p>Miro2 interacts with kinesin and/or dynein motors through the TRAK1/2 proteins to govern bidirectional trafficking of mitochondria in neurites (Russo et al. 2009, Birsa et al. 2013). While TRAK1 is localized to axons and dendrites, TRAK2 is predominantly localized in dendrites (Loss & Stephenson 2015, van Spronsen et al. 2013). Hence, we examined the extent to which TRAK2 and Miro-2 are required for the effects of D-AKAP1/PKA on mitochondrial trafficking in dendrites. PINK1−/− primary cortical neurons were transfected with either Mito-GFP or D-AKAP1, and co-transfected with an empty vector or a (TRAK2-DN) that acts in a dominant negative fashion to block basal mitochondrial trafficking and mitochondrial content within dendrites (Kimura & Murakami 2014). While transient expression of D-AKAP1 was able to significantly elevate anterograde and retrograde velocities in PINK1-deficient neurons, transiently co-expressing TRAK2-DN significantly blocked the ability of D-AKAP1 to enhance mitochondrial velocity and displacement (Fig. 5A–C). These data indicate that D-AKAP1/PKA requires the TRAK2/Miro complex to stimulate mitochondrial movement in dendrites.</p><!><p>Given that TRAK2/Miro is required for the ability of D-AKAP1/PKA to modulate mitochondrial trafficking (Fig. 5), we then examined the extent by which Miro-2 phenocopies the ability of D-AKAP1/PKA to stimulate mitochondrial movement. Indeed, transient expression of Miro-2 alone was sufficient to increase dendrite outgrowth and mitochondrial anterograde transport in dendrites of wild-type and PINK1-deficient primary cortical neurons (Fig. 6 A–C, E–H). Transient expression of Miro-2, however, had no significant effect on retrograde velocity or distance traveled per mitochondrion (Fig. 6–B, E). The ability of Miro-2 to enhance anterograde mitochondrial trafficking and dendrite length requires PKA as co-expressing Mito-PKI, or co-treating primary cortical neurons with H89, blocked the ability of Miro-2 to increase mitochondrial trafficking and dendrite length (Fig. 6B–C. E–H).</p><!><p>So far our data suggests that Miro2 may be a potential substrate of D-AKAP1/PKA. Indeed, algorithmic analyses of potential phosphosites predict that Miro2 is phosphorylated by PKA at 13 amino acid ser/thr residues (ExPasy NetPhosK 1.0 Tool) (Blom et al. 2004). To ascertain whether Miro2 is phosphorylated by mitochondrial PKA, exogenous Miro2-Myc was IPed from COS7 cells transiently co-expressing Miro2-Myc and D-AKAP1-GFP, D-AKAP1-ΔPKA-GFP, or full-length PINK1-3X-Flag. IPs were analyzed for total phosphorylation of ser/thr residues by performing Phostag assays and by immunoblotting with an anti-phospho-ser/thr antibody. We observed that Miro2-Myc is highly phosphorylated at basal conditions, as evidenced by an immunoreactive band that electrophoretically migrates 7kDa higher (~75k Da) compared to its predicted molecular weight (68kDa) (Fig. 7A) in Phostag gels, and that can be abolished by incubating cell lysates with alkaline phosphatase (Fig. S8A). Transiently co-expressing Miro2 with D-AKAP1-GFP, but not D-AKAP1-ΔPKA, significantly elevated the basal phosphorylation levels of Miro2 (band at 75 kDa and a doublet slightly above 68kDa). Co-expression of PINK1 or treatment of Miro2-Myc and PINK1 co-expressing cells with db-cAMP (250μM, 2hrs) also modestly increased the phosphorylation of Miro2 (~69 and 75kDa bands) (Fig. 7B). LC-MS/MS analyses confirmed that both the 68kDa and 75kDa immunoreactive bands are specific for Miro2 (~30% coverage, 8 unique peptides identified to be specific for Miro2 with >90% probability per Miro2 species). In addition, the 75kDa band of Miro2, which is enhanced by PKA-modulating conditions, was significantly phosphorylated at threonine 47 (100% peptide probability, 100% phosphorylation site probability, Ascore: 38.52, ~36% protein coverage, 756.89 m/z, 1,511.77 Da) compared to the 68kDa band at basal conditions. This site is predicted to be phosphorylated with high confidence (GPS 3.0, ExPASy). Furthermore, LC MS/MS analysis of the 68kDa band of Miro2 did not identify any phosphopeptides (Fig. S8B). The above results suggest that Miro2 phosphorylation is modulated by PINK1 and PKA alike, and that both ser/thr kinases may act synergistically to phosphorylate Miro2 at similar and/or distinct ser/thr amino acid residues.</p><!><p>We have previously shown that loss of mitochondrial homeostasis caused by loss of PINK1 function is associated with reduced dendrite connectivity and neurodegeneration (Dagda et al. 2011, Dagda et al. 2014), suggesting that PINK1 is a regulator of dendrite homeostasis and development. Here, we show that midbrain dopamine neurons in PINK1 −/− mice and PINK1-deficient primary cortical neurons exhibit significantly decreased dendrite length in the absence of significant cell death, but accompanied by a significant reduction in global PKA activity and increased mitochondrial pathology in dendrites. The loss of dendrites is causally linked to reduced PKA signaling as pharmacologically activating PKA can restore dendrite length in vitro. Interestingly, while mitochondrially- targeted PKA can compensate for defects in mitochondrial trafficking and density in PINK1-deficient neurons, it only partly reverses the decrease in dendrite length compared to db-cAMP treatment. These observations suggest that non-mitochondrial pools of PKA are required to stimulate dendrite outgrowth, presumably by eliciting dendrite biogenesis (dendritogenesis). How PINK1 elicits PKA signaling in the cytosolic and nuclear compartments to promote dendritogenesis requires further study.</p><p>There is some evidence that decreased PKA signaling in neurons contributes to the etiology of PD. For instance, non-degenerating midbrain dopamine neurons from postmortem PD brain tissue show reduced mRNA levels of PKA-regulated genes (Howells et al. 2000). In this study, we provide compelling evidence that dendrites of PINK1-deficient cortical neurons exhibit decreased PKA signaling, robust mitochondrial pathology, and reduced growth rates. The ability of endogenous D-AKAP1 to associate with phosphorylated PKA/RIIβ is compromised in the kinase-deficient mutant of PINK1 (K219M). Likewise, loss of PINK1 is associated with decreased levels of phosphorylated RIIβ in vivo, whereas total RIIβ or D-AKAP1 levels are not significantly altered. In summary, our data suggest that PINK1 modulates PKA-mediated autocatalytic phosphorylation.</p><p>The neuroprotective role of D-AKAP1 has traditionally been thought to involve phosphorylation of the pro-apoptotic protein BAD and the fission modulator Drp1 to prevent neuronal apoptosis and elicit mitochondrial fusion, respectively (Affaitati et al. 2003, Carlucci et al. 2008, Merrill et al. 2011). Our study extends the neuroprotective effects of D-AKAP1 to include enhancing mitochondrial trafficking and interconnectivity in dendrites via PKA-meditated phosphorylation of Miro2. We show that D-AKAP1 can reverse mitochondrial dysfunction in dendrites by redirecting the endogenous pool of PKA to the mitochondrion. D-AKAP1/PKA acts as a downstream "effector" kinase that mediates PINK1-regulated anterograde mitochondrial trafficking in dendrites, but can also act independently of PINK1. This ability of D-AKAP1 to elicit anterograde mitochondrial trafficking in dendrites does not involve PKA-mediated phosphorylation of Drp1, but PKA-mediated phosphorylation of Miro2. Interestingly, D-AKAP1/PKA can also partially ameliorate the deficiencies in dendrite outgrowth in PINK1-deficient neurons, and in neurons exposed to a sublethal dose of the complex I inhibitor rotenone. These observations suggest that while D-AKAP1 plays a major role in mitochondrial homeostasis, other factors likely interplay to mediate the dendrite outgrowth effects of PINK1.</p><p>Our data raises the possibility that PINK1 plays dual roles in regulating mitochondrial trafficking in neurons based on its localization in neuronal compartments and levels of oxidative stress. In axons, PINK1 phosphorylates Miro1 on serine 156 to promote its degradation via Parkin under oxidative stress (Wang et al. 2011). However, under physiological conditions, PINK1 stimulates mitochondrial trafficking and content in dendrites. Given that the effects of D-AKAP1 on dendritic mitochondria depends on its ability to associate with PKA-RIIβ, the disparate effects of PINK1 on mitochondrial trafficking and distribution in axons vs. dendrites can be partly explained by the selective distribution of type II PKA holoenzyme to dendrites (Harada et al. 2002). In the absence of oxidative stress, binding of PINK1 to D-AKAP1 enhances the association of PKA/RIIβ with D-AKAP1 and increases the autocatalysis-mediated phosphorylation of PKA/RIIβ, presumably to "prime" PKA holoenzymes for activation by cAMP at the mitochondrion. Mechanistically, increased PKA signaling leads to enhanced mitochondrial interconnectivity and trafficking in dendrites by phosphorylating Drp1 and Miro2, respectively. Our data appears to contradict a recent study that suggests that PINK1 uncouples the PKA holoenzyme from D-AKAP1 to initiate Drp1-mediated fission and autophagic sequestration of damaged/depolarized mitochondria (Pryde et al. 2016). However, when considered in the context of our data, the aforementioned study suggests that PINK1 plays a role in maintenance of dendrite and mitochondrial homeostasis under baseline conditions, but facilitates mitophagy of damaged mitochondria in the presence of oxidative stress by uncoupling mitochondrial PKA signaling, both with neuroprotective end-results (Fig. 7C).</p><p>Our PINK1/D-AKAP1 co-expression studies further suggest PINK1 and D-AKAP1 interact to sense mitochondrial health and maintain mitochondrial distribution in dendrites, which may also facilitate the ability of processed PINK1 to interact with Miro. Our data is in agreement with the recent reports of Akabane et al. and Pryde et al. that suggest that PKA and PINK1 negatively regulate each other- where PKA indirectly stimulates the degradation of PINK1 by phosphorylating the adaptor protein MIC60 (Akabane et al. 2016), or whereby OMM-localized PINK1 can induce the dissociation of PKA from D-AKAP1 to initiate mitophagy of damaged mitochondria (Pryde et al. 2016). This negative feedback loop involving PINK1 and D-AKAP1/PKA, may explain the lack of synergism between the two kinases for certain aspects of mitochondrial function, and merits further exploration.</p><p>In summary, D-AKAP1/PKA and PINK1 show neuroprotective convergence at the OMM of healthy mitochondria to modulate mitochondrial trafficking, content, interconnectivity and dendrite morphogenesis.</p>