PINK1 regulates mitochondrial trafficking in dendrites of cortical neurons through mitochondrial PKA

Abstract

Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's 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 β of PKA (PKA/RIIβ) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RIIβ 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.

Keywords: Miro-2; PTEN-induced kinase 1; Parkinson's disease; Protein Kinase A; dual specificity A-kinase anchoring protein 1; mitochondrial trafficking.

Figure 1. Loss of endogenous PINK1 reduces dendrite outgrowth and is associated with mitochondrial pathology in dendrites

A) Representative epifluorescence images of midbrain dopamine neurons, identified by tyrosine hydroxylase (TH) immunoreactivity, in brain slices of 10-month-old wild-type and PINK1−/− mice. “S” indicates soma, arrows point to dendrites. Quantification of dendrite length (bottom) revealed that dopamine neurons in PINK1 −/− mice show a significant reduction in dendrite length compared to wild-type (^*:p<0.05 for PINK1+/+ vs. PINK1−/−, n=30–40 neurons analyzed per genotype, N=3 PINK1+/+, N=4 PINK1−/−). B) Representative fluorescence images of wild-type and PINK1−/− primary cortical neurons cultured for 2-8DIVs and immunostained for dendrites using MAP2B antibody and with DAPI. Arrows point to MAP2-positive neurons. Right: Compilation of mean dendrite length per neuron at the indicated time points in wild-type vs. PINK1−/− cortical neurons. The average growth rate of dendrites is shown for each time point per genotype (red and blue). The average neurite length is significantly reduced in PINK1−/− neurons for all time points analyzed (N=3, ^*:p<0.05 vs. wild-type, n=65–70 neurons analyzed per experiment). C) 6DIV primary cortical neurons transiently expressing Mito-RFP were analyzed for mitochondrial movement in dendrites by time lapse epifluorescence microscopy. The bar graph shows the mean average distance traveled per mitochondrion in the anterograde (away from cell body) or retrograde direction (towards cell body), in wild-type or PINK1−/− neurons. Loss of PINK1 resulted in a significant reduction in anterograde mitochondrial displacement (N=3, ^*:p<0.05 vs. wild-type). D) Representative fluorescence micrographs of dendrites from 6DIV primary cortical neurons transiently expressing Mito-RFP to analyze for mitochondrial length and distribution in dendrites. Bottom: Quantification of average length per mitochondrion (left) and mitochondrial density within dendrites (right) indicated a significant reduction in both parameters in PINK1−/− neurons compared to wild-type (^*:p<0.05 vs. wild-type, N=3).

Figure 2. PINK1 deficiency disrupts PKA signaling

A) Total intracellular PKA activity measured in midbrain of wild-type and PINK1−/− mice. Loss of PINK1 reduces total PKA activity in PINK1−/− mice (N=4 wild-type vs. 3 for PINK1−/−, ^*:p<0.05 vs. wild-type). B) Compilation of number of substantia nigra/ventral tegmental (SN-VTA) neurons per epifluorescence field that stained for total or phosphorylated PKA/RIIβ. Brain sections obtained from wild-type and PINK1−/− mice were immunostained with total or phospho-PKA/RIIβ antibody. Loss of PINK1 reduced levels of phospho-PKA/RIIβ without affecting total levels of PKA/RIIβ (^*:p<0.05 vs. wild-type, N= 3 wild-type vs 4 for PINK1−/−, 18–20 brain sections analyzed per genotype). C) Representative western blot showing steady-state levels of endogenous total PKA/RIIβ, and phosphorylated PKA/RIIβ in whole brain lysates of wild type and PINK1−/− mice. The bar graph shows mean integrated density of phosphorylated or total PKA/RIIβ. (^*:p<0.05 vs. wild-type, N= 3 for each genotype). D) Cell lysates derived from vector and stable PINK1 overexpressing SHSY5Y cell lines were immunoprobed for total and phosphorylated PKA/RIIβ, and for β-actin as loading control (representative western blot; N=3). Overexpression of PINK1 increased levels of phosphorylated PKA/RIIβ without affecting the levels of total PKA/RIIβ. E) Compilation of dendrite length in 7DIV wild-type and PINK1−/− primary cortical neurons treated with vehicle control (water) or dbt-cAMP (250μM, 24 hrs.). cAMP treatment significantly increased the dendrite length of both wild-type and PINK1−/− neurons (^*:p<0.05 vs. vehicle in PINK1+/+, #:p < 0.05 vs. vehicle in PINK1−/−, N=3). F) Representative epifluorescence micrographs of primary cortical neurons transiently expressing GFP or PKA/RIIβ-GFP and immunostained for MAP2B to identify dendrites and with DAPI. Individual channels for GFP and MAP2B were separated to appreciate differences in dendrite lengths between transfection conditions. PKA/RIIβ increases dendrite length in PINK1−/− cortical neurons compared to Mito-GFP (^*:p<0.05 vs. Mito-GFP, N=3).

Figure 3. Overexpression of D-AKAP1 enhances mitochondrial trafficking in dendrites of primary neurons

A) Left: Representative kymographs depicting mitochondrial movement in wild-type and PINK1−/− primary cortical neurons transfected with Mito-RFP and other plasmids as indicated. The x-axis represents the position of mitochondria (μm) and the y-axis denotes time (seconds, from top to bottom). Right: Quantification of average anterograde velocity (top), and anterograde displacement (bottom) of dendritic mitochondria in 6DIV wild-type and PINK1−/− primary cortical neurons. D-AKAP1, but not D-AKAP1-ΔPKA, significantly increases mitochondrial movement in the anterograde direction (^*:p < 0.05 vs. MitoGFP-PINK1+/+; ^#:p < 0.05 vs. MitoGFP-PINK1−/−, n=75–128 mitochondria from 10 dendrites and three separate transfections per genotype). B) Top: Representative fluorescence micrographs of dendrites from 6DIV primary cortical neurons transiently expressing Mito-RFP and the indicated plasmids. Bottom: Quantification of average mitochondrial length within dendrites of wild-type and PINK1−/− cortical neurons. D-AKAP1, but not D-AKAP1-ΔPKA, increased mitochondrial length of PINK1−/− neurons (^*:p < 0.05 vs. Mito-GFP in PINK1+/+; ^#:p < 0.05 vs. Mito-GFP in PINK1−/−, n=65–100 mitochondria from 10 dendrites and three separate transfections per genotype). C) Left: Representative kymographs depicting mitochondrial movement in wild-type primary cortical neurons transfected with Mito-RFP and other plasmids as indicated. Quantification of average mitochondrial velocity (middle) and total average distance per mitochondrion (right) in wild-type primary neurons transiently co-transfected with the indicated plasmids showed that cytosolic PINK1 (ΔN111-PINK1) significantly increased anterograde mitochondrial movement, which was blocked by co-expression of Mito-PKI (^*:p < 0.05 vs. Mito-GFP; ^#:p < 0.05 vs. ΔN111-PINK1, 50–70 mitochondria analyzed per condition, N=3).

Figure 4. PINK1 enhances the association of PKA/RIIβ to D-AKAP1

A) Representative western blot showing that transient expression of PINK1 increases the amount of PKA/RIIβ that IPs with D-AKAP1-GFP. COS7 cells were transiently transfected with an empty vector or full-length PINK1-FLAG and the indicated plasmids for 2 days. D-AKAP1-GFP or D-AKAP1-ΔPKA-GFP were IPed using an anti-GFP antibody, and immunoblotted with an PKA/RIIβ or a PINK1 antibody (N=3). B) Representative western blot showing that PINK1 activity enhances the interaction of PKA/RIIβ with D-AKAP1. COS7 cells were transiently transfected with GFP or D-AKAP1-GFP and co-transfected with the indicated plasmids for 2 days. D-AKAP1-GFP was IPed with an anti-GFP antibody, and immunoblotted with a PKA/RIIβ or a PINK1 antibody. Black arrowhead points to full-length PINK1, white arrowheads point to processed forms of PINK1. Wild-type PINK1 overexpression significantly increased the amount of PKA/RIIβ that IPed with D-AKAP1, an effect that was reduced by PINK1 K219M (N=3). Discontinuity in the western blot images (shown as a break) indicate that certain lanes have been cropped to maintain visual clarity. All samples were run on the same gel and immunoblotted at the same time. Bottom graph shows the densitometry quantification of the ratio of the amount p-RIIβ that pulled down with D-AKAP1-GFP relative to total PINK1 levels (endogenous and PINK1-Flag) and normalized relative to empty vector control (^*:p<0.05 vs. Vector, N=3). C) Representative western blot showing that transient co-expression of PINK1 with D-AKAP1 significantly enhances phosphorylation of Drp1, suggesting increased PKA activity. COS7 cells were transiently transfected with the indicated plasmids. Cell lysates were analyzed for expression of phosphorylated- and total Drp1 using specific antibodies. The double arrow points to the location of p-Drp1 immunoreactive bands. Numbers indicated in red, below the blots, indicate mean integrated density of phospho-Drp1 for the respective transfection conditions shown for the representative blot. The bar graph below the blot shows fold change ±SEM of the mean integrated densities of the immunoreactive bands specific for phosphorylated Drp1 (p-Drp1) divided by total Drp1, and normalized to the D-AKAP1 transfection condition, compiled from three experiments (^*:p<0.05 vs. D-AKAP1, N=3, t-test). Discontinuity in the western blot images (shown as a break, thick white line) indicate that certain lanes have been cropped to show relevant lanes and maintain visual clarity. All samples were run on the same gel and immunoblotted at the same time.

Figure 5. D-AKAP1/PKA requires the TRAK-2/Miro2 complex to regulate mitochondrial trafficking in neurons

A) Transfection with D-AKAP1-GFP significantly increased mitochondrial velocity in the anterograde direction in PINK1 −/− primary cortical neurons compared to Mito-GFP. Co-transfection of D-AKAP1 with TRAK2-DN, a Miro2-binding deficient mutant of TRAK2, blocked the ability of D-AKAP1-GFP to increase anterograde mitochondrial movement (^*:p < 0.05 vs Mito-GFP-Vector, compiled from N=3). B) The average mitochondrial retrograde velocity was significantly increased in primary neurons transiently expressing D-AKAP1-GFP compared to vector-transfected neurons, but blocked in PINK1−/− cortical neurons expressing D-AKAP1-GFP and TRAK2-DN (^*:p < 0.05 vs Mito-GFP-Vector, compiled from N=3). C) Quantitation of total distance moved per mitochondrion shows that co-expressing TRAK2-DN significantly reduced the effect of D-AKAP1-GFP on mitochondrial movement (^*:p < 0.05 vs. Mito-GFP-Vector; ^#:p < 0.05 vs. D-AKAP1-Vector, compiled from N=3).

Figure 6. Miro2 enhances neurite length and mitochondrial transport in dendrites via mitochondrial PKA

A) Left: Representative kymographs depicting mitochondrial movement in wild-type primary cortical neurons transiently transfected with Mito-RFP, co-transfected with the indicated plasmids, and treated with H89 (0.5μM, 2hrs) or vehicle control. Middle and right: Compilation of mitochondrial velocities and displacement in the above dendrites shows that Miro2 expression significantly increased overall mitochondrial velocity and displacement, which was blocked by inhibiting PKA with H89 treatment or transient expression of Mito-PKI (^*:p<0.05 vs. Mito-GFP, ^#:p<0.05 vs. Miro2-Myc, n=72–118 mitochondria per condition, N=3). B) Left: Representative kymographs depicting mitochondrial movement in PINK1−/−primary cortical neurons transiently transfected with Mito-RFP and co-transfected with the indicated plasmids and treated with H89 (0.5μM, 2hrs) or vehicle control. Middle and right: Compilation of mitochondrial velocities and displacement in the above dendrites shows that Miro2 expression significantly increased overall mitochondrial velocity and displacement, which was blocked by inhibiting PKA with H89 treatment or transient expression of Mito-PKI (^*:p<0.05 vs. Mito-GFP, ^#:p<0.05 vs. Miro2-Myc, N=3). C, D) Top: Representative epifluorescence images of 6DIV PINK1−/− and wild-type primary cortical neurons expressing Mito-GFP, Miro-2-Myc or Mito-PKI and/or treated with H89. Bottom: Compilation of dendrite length analyses showed that transient expression of Miro-2 significantly increased dendrite length per neuron in both wild-type and PINK1−/− neurons. This effect was inhibited by both H89 and Mito-PKI (^*:p<0.05 vs. Mito-GFP, :^#:p<0.05 vs. Miro2-Myc, N=3).

Figure 7. D-AKAP1/PKA promotes phosphorylation of Miro2

A) Analyses of phosphorylation of Miro2 as determined by Phostag/Western blot assay of Miro-2-Myc IPed from COS-7 cells expressing the indicated plasmids in the presence or absence of dbt-cAMP (250μM, 2hrs) treatment. Following electrophoresis on a Phostag gel, the membrane was immunoprobed with an anti-Myc, an anti-phospho-Ser/Thr, and an anti-Miro-2 antibody. The anti-phospho-ser/thr antibody recognized a 75kDa band and a doublet that migrated at approximately 68kDa suggesting that Miro-2 is phosphorylated at multiple amino acids by PKA. Discontinuity in the western blot images (shown by a break) indicate that certain lanes have been cropped to maintain visual clarity. All samples were run on the same gel and immunoblotted at the same time. B) Quantitation of phosphorylation levels of Miro-2 as assessed by measuring the mean intensity of immunoreactive bands for phosphorylated Miro-2 normalized to total exogenous Miro-2-Myc shows that D-AKAP1 expression results in a two-fold increase in phosphorylation of Miro-2-Myc (N=3, ^*:p<0.05 vs. Miro-2-Myc). C) Conceptual model: During homeostasis, PINK1 enhances the autocatalysis-mediated phosphorylation and binding of PKA-RIIβ to D-AKAP1, thereby priming PKA for activation by cAMP at the mitochondrion leading to increased PKA signaling in dendrites. The functional consequences of PINK1-mediated increase in association of PKA/RIIβ to D-AKAP1 includes enhanced anterograde mitochondrial trafficking in dendrites by employing the TRAK2/Miro-2 complex and via PKA-mediated phosphorylation of Miro-2.