Abstract
Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening.
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CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
Parkinson disease (PD) is a progressive neurodegenerative disease that affects both subcortical and cortical brain regions, leading to deficits in motor control and cognitive decline. Although most cases of PD are sporadic, familial mutations account for nearly 10% of patients with PD.1 Interestingly, mutations in the leucine-rich repeat kinase 2 (LRRK2) have been identified in approximately 5% of familial PD cases and 1% of sporadic PD cases.2 Although the underlying pathogenesis remains poorly understood, changes in autophagy regulation and mitochondrial homeostasis are emerging as common pathways in toxin and genetic models of PD.3,4
Previous studies have highlighted the importance of mitochondrial quality control in PD models using mitochondrial neurotoxins or proteins encoded by recessive PD-associated genes (Parkin, DJ-1, and PINK1) that traffic to the mitochondria.5–8 Although these mitochondrial toxins and mutant proteins perturb mitochondrial respiration, degradation, and calcium buffering, less is known about how nonmitochondrially targeted PD gene products, such as LRRK2 or α-synuclein, affect mitochondrial homeostasis.9–11 Recent studies suggest that autophagy regulation may be a point of convergence among the Parkin, PINK1, DJ-1, α-synuclein, and LRRK2 pathways.6,12–15 Autophagy is a highly conserved, cytoplasmic recycling pathway that engulfs proteins, aggregates, and organelles in autophagosomes and traffics them to the lysosome for degradation.16 Autophagic turnover of mitochondria and aggregate-prone proteins is one way to maintain healthy, functioning neurons; however, excessive degradation may have deleterious effects leading to neurodegeneration.17 Therefore, PD-associated genes may modulate mitochondrial homeostasis by directly regulating mitochondrial function or by affecting mitochondrial degradation.4
Although LRRK2 function remains unknown, previous studies indicate a role in regulating neurite morphological characteristics, cytoskeletal dynamics, or the endosomal-autophagic system.12,18–25 PD-associated mutations in the GTPase domain (R1441C) and the kinase domain (G2019S) have previously been shown to cause neurite shortening and autophagy induction.12,19,22,23,25 LRRK2 may also play roles in synaptic transmission26–28 and protein translation.29,30 A recent study of skin fibroblasts from patients bearing the G2019S mutation showed altered mitochondrial function.31 Because LRRK2 partially associates with mitochondria18,32 and interacts with PD-associated proteins that regulate mitochondrial stress,33 we investigated mechanisms by which mutations in LRRK2 affect mitochondrial homeostasis in neurons.
Materials and Methods
All experiments were performed in accordance with research protocols approved by the University of Pittsburgh (Pittsburgh, PA) Institutional Animal Care and Use Committee.
Mitochondrial Content and Dendrite Length Measurements
Mouse E15 cortical neurons were maintained in Neurobasal, supplemented with 2% B27 and 2 mmol/L Glutamax (all from Invitrogen, Carlsbad, CA). The cortical neurons analyzed were pyramidal neurons based on soma size (212 ± 6 μm2) and dendrite morphological characteristics.34,35 For mitochondrial content and dendrite length measurements, neurons were transfected with the indicated plasmids at 7 days in vitro (DIV). Neurons were then fixed with 4% paraformaldehyde 5 days after transfection (12 DIV) for analysis of mitochondrial content and autophagy and 14 days after transfection (21 DIV) for dendrite length. Neurons were stained with antibodies to microtubule associated protein-2 (MAP2) (Millipore, Billerica, MA) to identify dendrites and with anti-green fluorescent protein (GFP; Invitrogen) to identify transfected neurons for dendrite length analysis. Axons were defined as MAP2-negative processes that exhibited known morphological characteristics (thin and uniform diameter and length of more than two high-power fields) that distinguish axons from dendrites.36 ImageJ software version 1.42q (NIH) was used to measure the mitochondrial content in primary dendrites, axons, and soma; the primary dendrite area/neuron; and the summated dendrite length/neuron, as illustrated in Supplemental Figure S1.
Mitochondrial content was calculated from raw images as follows: area of cytochrome c oxidase subunit 8 targeting sequence (COX8)–GFP–labeled mitochondria/area for each compartment. For mitochondrial content and autophagy analysis, neurons were treated with 1 nmol/L bafilomycin A (Merck KGaA, Darmstadt, Germany), 2 μmol/L 1,2-bis (aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Invitrogen), 2 mmol/L EGTA (Sigma-Aldrich Corp, St Louis, MO), 1 μmol/L nitrendipine (Tocris Bioscience, Ellisville, MO), 1 μmol/L NiCl2 (Sigma-Aldrich Corp), 50 nmol/L ω-agatoxin (Tocris Bioscience), or 100 nmol/L w-conotoxin (Tocris Bioscience) 3 days after transfection and analyzed 2 days later. For dendrite length analysis, neurons were treated as indicated at 10 days after transfection and analyzed 4 days later.
SH-SY5Y and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker, Walkersville, MD) with 10% fetal bovine serum (BioWhittaker), 2 mmol/L l-glutamine (BioWhittaker), and 10 μmol/L retinoic acid (Sigma-Aldrich Corp) for 3 days before transfection. For neurite length, autophagosome quantification, and mitochondrial content in SH-SY5Y cells, the cells were cotransfected with GFP and LRRK2 [wild type (WT), mutants, or vector] at 2 days before imaging. For BAPTA-AM treatments, cells were incubated in BAPTA-AM supplemented media for 1 day before imaging. The mitochondria were identified in GFP-positive cells by staining for endogenous TOM20 (Santa Cruz Biotechnology, Santa Cruz, CA). Autophagy was inhibited using RNA interference (RNAi) against human ATG7 (Thermo Scientific, Rockford, IL), as previously described.12
Mitochondrial Trafficking
Cortical neurons were transfected at 7 DIV and imaged at 2, 3, 4, or 5 days after transfection. Neurons were placed in warm Dulbecco’s PBS, and mitochondrial movement in the proximal dendrites (up to 75 μm from the soma) was imaged every 5 seconds for a total of 5 minutes37 on a 37°C microscope stage using an inverted epifluorescence microscope (Olympus IX71) and Microsuite Basic Edition version 2.3 (build 1121) imaging software (Olympus America, Center Valley, PA). The net mitochondrial movement was tracked and quantified using ImageJ software version 1.46r (NIH, Bethesda, MD), with the MTrackJ plug-in. Mitochondrial movement was defined as a >2-μm change in position within the imaging period. The percentages of mitochondria moving in the anterograde and retrograde directions were quantified for each neuron and expressed as a ratio. The velocities of individual mitochondria were quantified at 3 and 5 days after transfection.
Autophagosome Quantification
GFP–light chain 3 (LC3)–labeled autophagosomes were identified as bright puncta (>1.5 SDs higher than the mean cytoplasmic fluorescence), ranging from 0.5 to 1.0 μm in diameter. The puncta were counted in each cellular compartment for primary neurons.
Mitochondrial Polarization
Cortical neurons were transfected at 7 DIV. Neurons were stained with 50 nmol/L tetramethylrhodamine methyl ester (TMRM; Invitrogen) and imaged in Krebs-Ringer buffer with 12.5 nmol/L TMRM on day 3 after transfection, using a Nikon A1 confocal microscope (Melville, NY) with <1% laser power at 37°C. TMRM equilibrates across the plasma and mitochondrial membranes in a nernstian manner; thus, the resulting whole cell fluorescence after TMRM loading reflects the potentials of both membranes.38 Mitochondrial membrane potential (Ψm) was calculated as the ratio of mitochondrial/cytosolic TMRM staining intensity, as previously described.39 This ratio accounts for variations in cellular polarization that affect the uptake of TMRM, thus allowing for the comparison of ratios between mitochondrial and cytosolic TMRM staining across multiple cells. The staining intensity of proximal dendrites (<75 μm from the soma) was measured in the cytosol and mitochondria using ImageJ software. Several mitochondrial regions of interest were selected in the dendrites of each cell and divided by adjacent cytosolic control regions to obtain the nernstian quotient. The average was taken as the relative mitochondrial polarization of dendritic mitochondria.
SDS-PAGE and Immunoblotting
Cortical neurons or SH-SY5Y cells were lysed in 25 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, and 1% Triton X-100. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Blots were probed with antibodies against ATG7 (Rockland Inc., Gilbersville, PA), glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, MA), Lamin A/C (Cell Signaling Technology, Danvers, MA), LC3 (Nanotools, Teningen, Germany), and LRRK2 (C41-2; Michael J. Fox Foundation, New York City, NY).
Calcium Imaging
Cortical neurons were incubated with 5 mmol/L Fura2-AM (Invitrogen) in normal buffer: 10 mmol/L HEPES (pH 7.6), 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 10 mmol/L glucose at 37°C for 1 hour. Cells were washed in normal buffer for 10 minutes, and then imaged at 25°C; 50 mmol/L KCl in normal buffer was added to determine the peak amplitude of calcium influx. Changes in cytoplasmic calcium were followed by measuring the Fura2 340nm/380nm fluorescence ratio in regions of interest containing one cell. The signal deflection from the baseline (ΔF) was normalized to the mean baseline value (F), which was established prior to stimulating the cells. Calcium buffering capacity was defined as the difference in Fura2-AM intensity between peak amplitude and the plateau phase during KCl stimulation. To study calcium clearance, intracellular calcium was allowed to reach the plateau phase in the presence of 50 mmol/L KCl in normal buffer; Fura2-AM intensity was then measured on washout with normal buffer. The curve between the Fura2-AM staining at the plateau phase with 50 mmol/L KCl and at baseline after KCl washout was fit by a mono-exponential equation, which was used to calculate the time constant (τ). In some experiments, the calcium clearance was measured in the presence of 10 μmol/L carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich Corp).
Statistical Analysis
All graphical data are compiled from multiple independent experiments. One-way analysis of variance was used to compare the treatment groups, followed by post hoc t-tests with Bonferroni corrections for multiple comparisons. The sample size for all graphical data was calculated to provide a statistical power of 0.75. A corrected P value of <0.05 was considered statistically significant.
Results
To determine whether LRRK2 mutants affected mitochondrial homeostasis, we measured the percent mitochondrial content [100 × (mitochondrial area/cytoplasmic area)] in the axons, dendrites, and soma of cortical neurons co-expressing COX8-GFP with LRRK2-WT, LRRK2-G2019S, or LRRK2-R1441C (Figure 1A and Supplemental Figure S1). Neurons were counterstained for MAP2 to distinguish dendrites from axons (Figure 1A and Supplemental Figure S2A). We found that LRRK2 PD-associated mutants, but not WT LRRK2, caused significant reductions in mitochondrial content in dendrites, but not axons, at 5 days after transfection (Figure 1, B and E, and Supplemental Figure S2A). The LRRK2-G2019S mutant also caused a significant decrease in somatic mitochondrial content that was not observed with the LRRK2-R1441C mutant (Figure 1D and Supplemental Figure S2B), although the LRRK2 plasmids resulted in equivalent protein expression (Supplemental Figure S2C). There were no morphological changes to dendrites at this time point (Figure 1C), indicating that the decrease in mitochondria density preceded subsequent reductions in dendritic area observed at 14 days after transfection.23 The kinase-deficient LRRK2 K1906M mutant had no effect on dendritic mitochondrial content (Supplemental Figure S3A). Because decreased dendritic mitochondrial content could underlie the dendrite/neurite retraction observed in culture and mouse models of LRRK2-related PD,19,22,23,25 we further investigated causes of this decrease in mitochondrial content.
Figure 1.
Mutant LRRK2 reduces mitochondrial content in dendrites. A: Representative micrographs of cortical neurons co-expressing the indicated form of LRRK2, or vector, and COX8-GFP (green). Dendrites were identified by MAP2 staining (red). For single-channel figure panels, the green channel was extracted to gray scale. Proximal dendrites from boxes in A are presented below at a five-fold higher magnification. The COX8-GFP fluorescence channel is shown above the corresponding MAP2 red channel. B: Quantification of means ± SEM dendritic mitochondrial content [100 × (mitochondrial pixels/dendrite pixels)] (n = 19 to 32 neurons per group compiled from three independent experiments). C: Quantification of means ± SEM dendritic area (n = 19 to 32 neurons per group compiled from three independent experiments). D and E: Quantification of the means ± SEM percentage mitochondrial content in the soma and axons of neurons (n = 14 to 32 neurons per group compiled from three independent experiments). *P < 0.05 versus vector. Scale bars: 50 μm (A, upper panels); 10 μm (A, lower panels).
Mitochondrial trafficking is one process that could regulate the quantity of mitochondria by delivering or removing mitochondria from the dendrites. To determine whether LRRK2 mutants affected mitochondrial trafficking, we measured anterograde (away from the soma) and retrograde (toward the soma) movement of COX8-GFP–labeled mitochondria in dendrites at 2 to 5 days after transfection. The percentage of mobile mitochondria was not significantly changed between treatment conditions (eg, at 5 days, vector = 15.64 ± 0.42, WT = 15.71 ± 0.11, G2019S = 17.52 ± 1.41, and R1441C = 16.74 ± 1.64; P = 0.454). The ratio of anterograde/retrograde moving mitochondria per cell was likewise not significantly altered (Figure 2A). There was a trend toward a greater spread of faster-moving anterograde mitochondria in LRRK2-transfected neurons, but this did not result in significant alterations in the average anterograde (Figure 2B) or retrograde (Figure 2C) velocities in vector versus LRRK2 (WT or mutant) expressing neurons. Because significant changes in mitochondrial trafficking were not observed, we investigated the potential role of mitochondrial degradation.
Figure 2.
Mutant LRRK2 does not elicit defects in mitochondrial trafficking, but it induces autophagy to degrade mitochondria. A: Net mitochondrial movement was quantified per neuron as the ratios of mitochondria moving in the anterograde/retrograde (Ant:Ret) directions (n = 4 to 17 neurons per group compiled from three independent experiments). B: The anterograde velocities of individual mitochondria moving away from the soma are plotted, along with bars showing the means ± SEM (n = 32 to 43 mitochondria per group compiled from day 3 and day 5 measurements). C: The retrograde velocities of individual mitochondria moving toward the soma are plotted, along with bars showing the means ± SEM (n = 33 to 45 mitochondria per group compiled from day 3 and day 5 measurements). D: Quantification of means ± SEM GFP-LC3–labeled autophagosomes per neuron (n = 20 to 30 neurons per group compiled from three independent experiments). E: Quantification of means ± SEM dendritic mitochondrial content in the presence and absence of bafilomycin (n = 5 to 32 neurons per group compiled from three independent experiments). F: Quantification of means ± SEM mitochondrial content in the neurites of SH-SY5Y cells in the presence and absence of ATG7 RNAi (n = 26 to 58 cells per group compiled from three independent experiments). Mitochondria were stained for endogenous TOM20. Inset: Efficacy of ATG7 knockdown. *P < 0.05 versus vector.
We first measured the levels of steady-state autophagy in cortical neurons expressing mutant LRRK2. The microtubule-associated protein LC3 is a key autophagy mediator that covalently attaches to autophagosome membranes.40 By using GFP fused to LC3 (GFP-LC3), we investigated whether mutant LRRK2 altered the number of autophagosomes. Mutant LRRK2 caused an increase in autophagosomes compared with vector control (Figure 2D). To determine whether autolysosomal degradation contributed to the decrease in dendritic mitochondria, we inhibited lysosomal degradation with bafilomycin A using nontoxic doses previously shown to stabilize LC3-II from degradation,41,42 and confirmed using Western blot analysis and the tandem tfLC3 flux reporter42 (Supplemental Figure S3, B–D). The inhibition of autophagic degradation prevented the loss of dendritic mitochondria in neurons expressing mutant LRRK2 (Figure 2E). To further assess the involvement of autophagy, SH-SY5Y cells were used because they recapitulated the mutant LRRK2 effects observed in neurons (ie, increased autophagosomes,12,19 neurite shortening,12,19 and the selective reduction in the mitochondrial content of neurites) (Figure 2F), with no changes observed in the cell body for percentage of cytoplasmic area occupied by mitochondria (vector = 12.50 ± 0.54, WT = 14.56 ± 0.64, G2019S = 11.31 ± 0.64, and R1441C = 13.45 ± 0.83; P = 0.10). The inhibition of autophagy in SH-SY5Y cells using RNAi against ATG7, a protein essential for autophagy induction, also reversed the effects of LRRK2 mutants on neuritic mitochondrial density (Figure 2F).
Because mitochondrial depolarization triggers mitochondrial degradation through autophagy (mitophagy) under multiple conditions,6,43,44 we assessed whether LRRK2 mutants affected mitochondrial inner membrane polarization. LRRK2 mutants, but not WT LRRK2, caused a significant decrease in dendritic mitochondrial polarization measured by TMRM staining 3 days after transfection (Figure 3, A and B). At this early time point, there was no change in mitochondrial content (Figure 3C), indicating that decreases in mitochondrial polarization preceded the dendritic mitochondrial loss seen after 5 days.
Figure 3.
Mutant LRRK2 induces mitochondrial depolarization. A: Representative micrographs of TMRM staining in the proximal dendrites of neurons co-expressing the indicated form of LRRK2, or vector, and GFP. Scale bar = 20 μm. B: The means ± SEM mitochondrial membrane potential was calculated as the mean mitochondrial intensity/cytoplasmic background in the dendritic segments analyzed (n = 16 to 29 neurons per group compiled from three independent experiments). C: The means ± SEM mitochondrial fraction of proximal dendrites was calculated as the area occupied by TMRM-stained mitochondria/GFP-stained area of the proximal dendrites (n = 16 to 29 neurons per group compiled from three independent experiments). *P < 0.05 versus vector.
Calcium homeostasis is essential for both neuronal and mitochondrial function, and aberrant elevations in intracellular calcium could lead to mitochondrial and cellular dysfunction.45 In addition to the endoplasmic reticulum, mitochondria contribute to the maintenance of neuronal calcium homeostasis.45,46 Because calcium metabolism and mitochondrial function are intrinsically linked,47 we investigated whether calcium metabolism was perturbed in neurons expressing mutant LRRK2. First, we measured the levels of intracellular calcium with Fura2-AM in transfected neurons after KCl stimulation. There was no difference in the peak intracellular calcium level after stimulation; however, there was a significant decrease in calcium signal recovery in neurons expressing mutant LRRK2 (Figure 4, A–C). The calcium signal recovery is affected by calcium channel inactivation and calcium efflux due to sequestration into organelles, such as endoplasmic reticulum or mitochondria, and pumping across the plasma membrane. To eliminate the channel inactivation component, we directly measured total calcium efflux from the cytoplasm as the rate of calcium signal decay after removal of KCl. The time constant (τ) of calcium efflux was used as a measure of its efficacy. We found that calcium efflux from the cytoplasm was significantly less efficient in mutant LRRK2-expressing neurons (Figure 4, D and E).
Figure 4.
Mutant LRRK2 causes an imbalance in calcium homeostasis. A: A representative calcium trace illustrates the experimental paradigm and the measures of peak amplitude and buffering capacity during KCl stimulation. B: The means ± SEM peak amplitude of calcium influx measured by Fura2-AM intensity after the addition of 50 mmol/L KCl (n = 6 to 10 neurons per group compiled from three independent experiments). C: The means ± SEM buffering capacity (peak amplitude − plateau intensity) measured by Fura2-AM in the presence of 50 mmol/L KCl (n = 6 to 10 neurons per group compiled from three independent experiments). D: Representative calcium traces illustrating calcium clearance after KCl withdrawal. Inset: Mono-exponential curve fit to the respective groups. E: Quantification of means ± SEM calcium decay time constant (τ) after KCl withdrawal (n = 11 to 17 neurons per group compiled from three independent experiments). Max, maximum. *P < 0.05 versus vector.
To determine whether the mitochondrial status was responsible for the delayed calcium signal recovery, we measured the τ of calcium efflux in mutant LRRK2-expressing neurons treated with the mitochondrial proton uncoupler, CCCP (Figure 5, A and B). Mitochondrial depolarization by CCCP eliminated the difference in the τ of calcium efflux between neurons expressing mutant LRRK2 (relative) and either vector or WT LRRK2 (Figure 5B), implicating a role for altered mitochondrial handling of calcium in the pathophysiological characteristics of mutant LRRK2.
Figure 5.
Perturbed mitochondrial calcium handling is upstream of mitochondrial depolarization, degradation, and dendrite shortening elicited by mutant LRRK2. A: Representative calcium traces illustrating the experimental paradigm for studying the effects of 10 μmol/L CCCP on calcium efflux. B: Quantification of the means ± SEM calcium decay time constant (τ) in the presence of 10 μmol/L CCCP in normal buffer (n = 8 to 10 neurons per group compiled from three independent experiments). C: Quantification of means ± SEM dendritic mitochondrial membrane potential in the presence or absence of BAPTA-AM (n = 15 to 24 neurons per group compiled from three independent experiments). D: Quantification of means ± SEM GFP-LC3–labeled autophagosomes per neuron in the presence or absence of BAPTA-AM (n = 13 to 30 neurons per group compiled from three independent experiments). E: Quantification of means ± SEM dendritic mitochondrial content in the presence or absence of BAPTA-AM (n = 19 to 34 neurons per group compiled from three independent experiments). F: Quantification of means ± SEM summated dendrite length in the presence and absence of BAPTA-AM (n = 10 to 35 neurons per group compiled from three independent experiments). G: Quantification of means ± SEM neurite autophagosomes in SH-SY5Y cells as the number of autophagosomes per 100 μm of neurite length (n = 30 to 75 cells per group compiled from three independent experiments). H: Quantification of means ± SEM neurite length in SH-SY5Y cells (n = 30 to 75 cells per group compiled from three independent experiments). *P < 0.05 versus vector. DMSO, dimethyl sulfoxide.
To determine whether calcium dysregulation contributed to the increased turnover of mitochondria elicited by mutant LRRK2, we measured the mitochondrial membrane potential in neurons treated with the calcium chelator, BAPTA-AM. Treatment with BAPTA-AM prevented mitochondrial depolarization caused by mutant LRRK2 (Figure 5C). Furthermore, BAPTA-AM prevented elevations in autophagy and mitochondrial degradation in neurons and SH-SY5Y cells (Figure 5, D, E, and G), suggesting that mutant LRRK2-elicited deficits in calcium homeostasis led to mitochondrial depolarization and mitophagy. Calcium chelation was also sufficient to reverse the subsequent neurite shortening elicited by mutant LRRK2 in both neurons and SH-SY5Y cells (Figure 5, F and H).
To identify the calcium pools involved in mutant LRRK2-induced mitochondrial degradation, we treated neurons with EGTA to chelate extracellular calcium. Similar to BAPTA-AM, EGTA prevented the reduction in the percentage of dendritic area occupied by mitochondria (vector = 32.87 ± 4.57, WT = 35.18 ± 2.79, G2019S = 30.55 ± 3.12, and R1441C = 32.14 ± 3.35; P = 0.78), suggesting that extracellular calcium influx was required for mutant LRRK2-induced mitophagy in unstimulated cultures. Because primary cortical neurons exhibited spontaneous synaptic activity,48 we studied whether voltage-gated calcium channel inhibition would prevent the calcium influx–induced mutant LRRK2 phenotype. There are three families of voltage-gated calcium channels: the Cav1 (L-type), the Cav2 (P/Q-, N-, and R-types), and the Cav3 (T-type). The L- and T-types are primarily located in the somatodendritic compartment, whereas the P/Q-, N-, and R-type channels are found, to some extent, in both axonal and somatodendritic compartments.49 T-type channels primarily facilitate small, transient currents, whereas the other channels underlie larger calcium fluxes. Therefore, we assessed whether inhibiting L-type or P/Q-, N-, and R-type channels would modulate mutant LRRK2-induced mitochondrial degradation.
We found that nitrendipine, an inhibitor of L-type calcium channels, prevented mitochondrial degradation in neurons expressing mutant LRRK2 (Figure 6A). However, a mixture of NiCl2, ω-agatoxin, and ω-conotoxin that inhibit N-, P/Q-, and R-type calcium channels, respectively, was effective only in the R1441C model (Figure 6A). Furthermore, inhibition of L-type calcium channels and, to a lesser extent, N-, P/Q-, and R-type calcium channels prevented dendritic shortening caused by mutant LRRK2 (Figure 6B). Together, these results suggested that altered calcium homeostasis in mutant LRRK2-expressing neurons contributed to mitochondrial depolarization, mitophagy, and dendritic shortening. By normalizing the intracellular levels of calcium via voltage-gated calcium channel inhibitors, we prevented the mitophagy and dendritic shortening caused by mutant LRRK2.
Figure 6.
Inhibition of L-type calcium channels prevents mutant LRRK2-induced mitophagy and dendritic shortening. A: Quantification of dendritic mitochondrial content in the presence or absence of voltage-gated calcium channel inhibitors: nitrendipine (Nit) or a mixture of NiCl2, ω-agatoxin, and ω-conotoxin (Mix) (n = 14 to 31 neurons per group compiled from three independent experiments). B: Quantification of total dendrite length in the presence or absence of voltage-gated calcium channel inhibitors (n = 10 to 35 neurons per group compiled from three independent experiments). Data are given as means ± SEM. *P < 0.05 versus vector. DMSO, dimethyl sulfoxide.
Discussion
Mitochondrial function is important to maintain healthy, functioning neurons; furthermore, mitochondrial dysfunction has been implicated in PD.3,10,50 Although data from toxin and several recessive genetic models support a key pathogenic role for mitochondrial dysfunction, the relationship of these models to sporadic PD is complicated by differences in pathological and clinical features. This study identifies a mechanistic link between LRRK2 mutations, which cause late-onset PD more closely resembling sporadic disease,51 and mitochondrial homeostasis. Herein, we show that mutant LRRK2 causes a deficit in calcium recovery after chemical depolarization of neurons, which would be predicted to result in relatively sustained exposure to elevated cytosolic calcium, contributing to mitochondrial depolarization and, ultimately, mitochondrial degradation (Figures 2 and 3). Similar to our findings, transgenic mice expressing the LRRK2-G2019S mutation also show mitochondrial alterations associated with increased autophagy.25 Furthermore, decreased mitochondrial function has been observed in cells derived from patients carrying the LRRK2-G2019S mutation,31 and mitophagy is observed in sporadic patients with PD.52 Because LRRK2 mutations are implicated in both sporadic and familial PD, and are usually associated with classic pathological and clinical features of PD, these data further strengthen the notion of mitochondrial pathobiological features as an important mechanism in PD.
In this model, mitochondrial loss, which occurs 5 days after transfection (Figure 1), precedes dendritic shortening, which begins 9 days after transfection23 and continues at least another 5 days (Figures 5F and 6B). These findings are supported by previous research showing that sufficient dendritic mitochondrial content is required for proper dendritic morphological characteristics, neurotransmission, and synaptic plasticity.50 A long-term reduction in mitochondrial content, such as in neurons expressing mutant LRRK2, may further exacerbate deficiencies in calcium buffering capacity, decreasing the ability to generate proper levels of ATP and biosynthetic precursors within dendrites. Li and colleagues53 have shown that acute decreases in dendritic mitochondrial content rapidly lead to synapse and spine loss after 4 days. The current study extends these findings to show that chronic genetic stress leading to mitochondrial degradation can also elicit more extensive dendrite shortening after 14 days.
Both the reduction in mitochondrial content and the subsequent reduction in dendrite length are mediated by autophagy. We have previously shown that inhibition of autophagy through RNAi or through phosphoregulation of LC3 suppresses neurite shortening caused by mutant LRRK2.12,19 However, the substrate(s) removed by autophagy remained undefined. The current data implicate autophagic degradation of mitochondria as a proximal pathogenic mechanism that underlies neurite degeneration. Autophagy-mediated neurite shortening has also been reported during axotomy, growth factor withdrawal, and neurotoxin treatment in other primary neuron and in vivo systems.54,55 Part of the autophagy-mediated neurite shortening in these other models of neurodegeneration may also be due to a mitophagy-dependent reduction in mitochondrial support of dendrites or axons.
Because dendrites undergo large fluxes in calcium, the deficiencies in calcium handling could directly increase mitochondrial stress because of their important role in buffering excess intracellular calcium.45 Indeed, chelation of either intracellular or extracellular calcium or inhibition of voltage-gated calcium channels prevented mitochondrial degradation and dendrite shortening elicited by the G2019S or R1441C mutations in LRRK2. The observation of mitochondrial stress caused by LRRK2 mutants supports the previous findings that animals expressing mutations in LRRK2 are hypersensitive to mitochondrial toxins.56,57 This calcium-induced mitochondrial stress could also explain why Parkin, which plays roles in mitochondrial homeostasis and synaptic pruning, prevents degeneration caused by mutant LRRK2.56,58
Although this study cannot exclude a primary mitochondrial insult upstream of the calcium imbalance, WT LRRK2 overexpression may regulate lysosomal calcium mobilization in kidney cells.59 In our system, either calcium chelation or inhibition of voltage-gated calcium channels prevented mitochondrial degradation and neurite shortening. Together, these data support a hypothesis by which mitochondrial dysfunction, elicited by mutant LRRK2, results from calcium buffering deficiencies. Restoration of calcium homeostasis using voltage-gated calcium channel inhibitors raises an interesting therapeutic possibility, because L-type calcium channel inhibitors also protect in toxin models of PD.60–63 Furthermore, a tight link between voltage-gated calcium channels and mitochondrial function has recently been uncovered.47 The current data lend further support to the concept that inhibition of voltage-gated calcium channels may provide a therapeutic target for familial and sporadic PD.
Our experiments with voltage-gated calcium channel inhibitors uncovered an interesting aspect of LRRK2 biological features. Although both the G2019S and R1441C mutations cause calcium imbalance, mitochondrial degradation, and dendritic shortening, they have subtle differences in their responses to calcium channel inhibitors. Inhibiting the primarily somatodendritic L-type channels prevented mitochondrial degradation and neurite shortening elicited by both mutations (Figure 6). However, inhibition of the P/Q-, N-, and R-type channels only suppressed the effects of the R1441C mutation (Figure 6). One possible explanation for this discrepancy could be the degree of calcium buffering deficiency in neurons expressing these mutations. The G2019S mutation trended toward a greater decrease in calcium signal recovery, a greater delay in calcium clearance, and significantly depleted mitochondria in the soma, whereas the R1441C mutations had milder effects on these parameters (Figures 1D and 4, C–E). This suggests that the degree of the calcium imbalance may govern the levels and extent of mitochondrial injury and mitophagy.
Another explanation could be the localization of the injury versus the localization of the calcium channels. The L-type channels are primarily somatodendritic, but the P/Q-, N-, and R-type channels are primarily axonal, with minor somatodendritic localization.49 It would follow that NiCl2, ω-agatoxin, and ω-conotoxin could suppress a minor deficit in calcium clearance in the somatodendritic compartment, whereas nitrendipine is needed to suppress the greater defect elicited by LRRK2-G2019S.
Differences in the molecular functions of LRRK2 containing these mutations could also contribute to these observations. Similar to the current study, the G2019S mutation produces a more severe locomotion defect in worms than the R1441C mutation.64 Furthermore, the R1441C mutation has less affinity for binding to LRRK2-interacting proteins when compared with the G2019S mutation,65–67 suggesting possible signaling pathway differences between the two mutants. One underlying mechanism for these differences in signaling could be the level of kinase activity, which is consistently higher in the G2019S versus the R1441C.21,23,68–75 The degree of injury and efficacy of inhibitors in this study correlates with previously observed levels of kinase activity68 and the extent of calcium imbalance. These findings indicate that, although different mutations in LRRK2 converge in causing parkinsonian neurodegeneration, they may do so via distinct, as well as converging, pathways to produce the mutant LRRK2 phenotypes of mitochondrial loss, autophagy dysregulation, and neurite shortening.
In conclusion, we found that mutations in LRRK2 cause calcium imbalance and mitochondrial degradation preceding dendritic shortening. These data suggest that excess cytosolic or mitochondrial calcium perturb mitochondrial homeostasis, thus playing a pathogenic role in the mutant LRRK2 model of PD. Restoring calcium homeostasis through calcium chelation or inhibition of voltage-gated calcium channels was sufficient to prevent the mutant LRRK2 phenotype. Disruption of calcium homeostasis is an early, reversible event, linking mutations in LRRK2 to mitochondrial dysfunction and neurodegeneration. Restoration of calcium homeostasis may provide a new therapeutic strategy for familial and sporadic PD.
Acknowledgments
We thank Jeff Lee for technical assistance with calcium imaging studies, Jason Callio for preparation of primary neuron cultures, and Vivek Patel for assistance with optimization of live cell imaging parameters.
Footnotes
Supported by NIH grants AG026389 and NS065789 (C.T.C.). S.J.C. and E.S. were supported in part by NIH training grants F31NS064728 and T32EB001026, respectively.
Current address of S.J.C., Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, California.
Supplemental Data
Image-based mitochondrial content measurements. A: Mitochondrial content was measured in the soma, dendrites, and axons of mouse cortical neurons co-expressing mitochondrially targeted COX8-GFP (green) and human LRRK2, and immunostained for MAP2 (red). B: Dendrites of transfected neurons were manually traced, and the dendrite area was automatically quantified using ImageJ software and outlined for mitochondrial measurements. C: The green channel was extracted to gray scale. A uniform background subtraction was applied to the gray scale image, which was then thresholded and converted to a binary image (D). The dendritic outlines were applied to the binary image, and the mitochondrial area was quantified as green pixels encompassed by the dendritic outlines. E: The mitochondrial area was divided by the corresponding dendrite area to calculate the mitochondrial content for each dendrite, which was then averaged among all dendrites per neuron. Similar analytical procedures were used for the quantification of somatic and axonal mitochondrial content. ROI, region of interest.
Mitochondrial content in axonal and somatic compartments. COX8-GFP was co-expressed with the indicated LRRK2 plasmid or with vector for 5 days. A: Axons were identified as MAP2-negative processes exhibiting a thin, uniform diameter, and a length of more than two high-power fields. Images show proximal axonal segments (arrowheads). B: Somatic mitochondria were defined by a smooth arc that excluded dendritic and axonal projections. Scale bar = 50 μm. C: Immunoblot showing expression of LRRK2 constructs in HEK293 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Effects of kinase-deficient LRRK2 K1906M and bafilomycin flux assays. Primary cortical neurons were transfected with COX8-GFP and the indicated LRRK2 construct or vector at 7 DIV. Cells were fixed 5 days after transfection. The mean ± SEM mitochondrial content of proximal dendrites [100 × (mitochondrial area/cytoplasmic area)] was quantified (n = 19 to 25 neurons per group compiled from two independent experiments). B and C: SH-SY5Y cells transfected with tfLC3, a tandem mRFP-EGFP-LC3 flux reporter, were treated with 1 nmol/L bafilomycin A1 for 24 hours and then imaged using a Zeiss Meta 510 microscope (Thornwood, NY). B: Early autophagic vacuoles (AVs) were defined as puncta exhibiting both green and red fluorescence, whereas late AVs (red only) were quantified by subtracting the number of green puncta from the number of red puncta. C: Representative images show a predominant increase in early AVs (arrows), with no significant change in late AVs (arrowheads), consistent with a maturation defect. D: Mouse primary cortical neurons were treated with 1 nmol/L bafilomycin A1 for 48 hours after 10 DIV. An immunoblot from these lysates shows increased levels of LC3-II, indicating inhibition of LC3-II turnover. *P < 0.05 versus vector. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRFP, monomeric red fluorescent protein.
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Supplementary Materials
Image-based mitochondrial content measurements. A: Mitochondrial content was measured in the soma, dendrites, and axons of mouse cortical neurons co-expressing mitochondrially targeted COX8-GFP (green) and human LRRK2, and immunostained for MAP2 (red). B: Dendrites of transfected neurons were manually traced, and the dendrite area was automatically quantified using ImageJ software and outlined for mitochondrial measurements. C: The green channel was extracted to gray scale. A uniform background subtraction was applied to the gray scale image, which was then thresholded and converted to a binary image (D). The dendritic outlines were applied to the binary image, and the mitochondrial area was quantified as green pixels encompassed by the dendritic outlines. E: The mitochondrial area was divided by the corresponding dendrite area to calculate the mitochondrial content for each dendrite, which was then averaged among all dendrites per neuron. Similar analytical procedures were used for the quantification of somatic and axonal mitochondrial content. ROI, region of interest.
Mitochondrial content in axonal and somatic compartments. COX8-GFP was co-expressed with the indicated LRRK2 plasmid or with vector for 5 days. A: Axons were identified as MAP2-negative processes exhibiting a thin, uniform diameter, and a length of more than two high-power fields. Images show proximal axonal segments (arrowheads). B: Somatic mitochondria were defined by a smooth arc that excluded dendritic and axonal projections. Scale bar = 50 μm. C: Immunoblot showing expression of LRRK2 constructs in HEK293 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Effects of kinase-deficient LRRK2 K1906M and bafilomycin flux assays. Primary cortical neurons were transfected with COX8-GFP and the indicated LRRK2 construct or vector at 7 DIV. Cells were fixed 5 days after transfection. The mean ± SEM mitochondrial content of proximal dendrites [100 × (mitochondrial area/cytoplasmic area)] was quantified (n = 19 to 25 neurons per group compiled from two independent experiments). B and C: SH-SY5Y cells transfected with tfLC3, a tandem mRFP-EGFP-LC3 flux reporter, were treated with 1 nmol/L bafilomycin A1 for 24 hours and then imaged using a Zeiss Meta 510 microscope (Thornwood, NY). B: Early autophagic vacuoles (AVs) were defined as puncta exhibiting both green and red fluorescence, whereas late AVs (red only) were quantified by subtracting the number of green puncta from the number of red puncta. C: Representative images show a predominant increase in early AVs (arrows), with no significant change in late AVs (arrowheads), consistent with a maturation defect. D: Mouse primary cortical neurons were treated with 1 nmol/L bafilomycin A1 for 48 hours after 10 DIV. An immunoblot from these lysates shows increased levels of LC3-II, indicating inhibition of LC3-II turnover. *P < 0.05 versus vector. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRFP, monomeric red fluorescent protein.