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. 2018 Aug 22;38(34):7505-7515.
doi: 10.1523/JNEUROSCI.0541-18.2018. Epub 2018 Jul 20.

Evidence for Compartmentalized Axonal Mitochondrial Biogenesis: Mitochondrial DNA Replication Increases in Distal Axons As an Early Response to Parkinson's Disease-Relevant Stress

Victor S Van Laar  1   2 , Beth Arnold  1   2 , Evan H Howlett  1   2 , Michael J Calderon  3   4 , Claudette M St Croix  3   4 , J Timothy Greenamyre  1   2 , Laurie H Sanders  1   2   5 , Sarah B Berman  6   2
Affiliations

Evidence for Compartmentalized Axonal Mitochondrial Biogenesis: Mitochondrial DNA Replication Increases in Distal Axons As an Early Response to Parkinson's Disease-Relevant Stress

Victor S Van Laar et al. J Neurosci. .

Abstract

Dysregulation of mitochondrial biogenesis is implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). However, it is not clear how mitochondrial biogenesis is regulated in neurons, with their unique compartmentalized anatomy and energetic demands. This is particularly relevant in PD because selectively vulnerable neurons feature long, highly arborized axons where degeneration initiates. We previously found that exposure of neurons to chronic, sublethal doses of rotenone, a complex I inhibitor linked to PD, causes early increases in mitochondrial density specifically in distal axons, suggesting possible upregulation of mitochondrial biogenesis within axons. Here, we directly evaluated for evidence of mitochondrial biogenesis in distal axons and examined whether PD-relevant stress causes compartmentalized alterations. Using BrdU labeling and imaging to quantify replicating mitochondrial DNA (mtDNA) in primary rat neurons (pooled from both sexes), we provide evidence of mtDNA replication in axons along with cell bodies and proximal dendrites. We found that exposure to chronic, sublethal rotenone increases mtDNA replication first in neurites and later extending to cell bodies, complementing our mitochondrial density data. Further, isolating axons from cell bodies and dendrites, we discovered that rotenone exposure upregulates mtDNA replication in distal axons. Utilizing superresolution stimulated emission depletion (STED) imaging, we identified mtDNA replication at sites of mitochondrial-endoplasmic reticulum contacts in axons. Our evidence suggests that mitochondrial biogenesis occurs not only in cell bodies, but also in distal axons, and is altered under PD-relevant stress conditions in an anatomically compartmentalized manner. We hypothesize that this contributes to vulnerability in neurodegenerative diseases.SIGNIFICANCE STATEMENT Mitochondrial biogenesis is crucial for maintaining mitochondrial and cellular health and has been linked to neurodegenerative disease pathogenesis. However, regulation of this process is poorly understood in CNS neurons, which rely on mitochondrial function for survival. Our findings offer fundamental insight into these regulatory mechanisms by demonstrating that replication of mitochondrial DNA, an essential precursor for biogenesis, can occur in distal regions of CNS neuron axons independent of the soma. Further, this process is upregulated specifically in axons as an early response to neurodegeneration-relevant stress. This is the first demonstration of the compartmentalized regulation of CNS neuronal mitochondrial biogenesis in response to stress and may prove a useful target in development of therapeutic strategies for neurodegenerative disease.

Keywords: Parkinson's disease; axonal; biogenesis; mitochondrial; neurodegeneration; rotenone.

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Figures

Figure 1.
Figure 1.
Incorporation of BrdU and EdU into neuronal mitochondria in cell bodies and axons within 1–3 h of exposure. A–C, Primary neurons (DIV14) expressing mitochondrially targeted photoactivatible GFP (Mitochondria) were treated with 10 μm BrdU for 1 h (A) or 3 h (B). Control cells were incubated with 100 μm ddC, an inhibitor of mitochondrial DNA polymerase gamma, for 6 h before and then during 3 h of BrdU exposure (C). Cells were immunofluorescently stained for BrdU and confocal imaging revealed BrdU puncta associated with mitochondria in cell bodies (A, B), but minimal, if any, incorporation when ddC was present (C), confirming specificity for mtDNA replication. D, BrdU puncta were also found in mitochondria of distal axons after 1 h of BrdU exposure. E, Primary neurons (DIV14) expressing mitochondrially targeted DsRed2 (Mitochondria) were treated with EdU for 3 h and then stained using the EdU Click-iT system. EdU-positive puncta were again observed in mitochondria of the cell body and axons (E, arrow).
Figure 2.
Figure 2.
Quantifying localized mtDNA replication in soma and neurites of primary neurons in response to chronic exposure to rotenone. A, B, Representative confocal z-stack images of neurons exposed to 10 μm BrdU for 1 h following exposure to 1 week of DMSO vehicle control (A) or 1 nm rotenone (B). C, Quantitative analysis of BrdU puncta revealed no difference in number of puncta per cell body between control and rotenone at 1 week (t(78) = 0.012; p = 0.99; two-tailed unpaired t test; puncta/cell body ± SEM). D, In neurites, we observed a significantly increased number of BrdU puncta of rotenone-exposed neurons compared with vehicle control at 1 week (difference in means = +13.2; t(78) = 2.02; *p = 0.047; two-tailed unpaired t test; puncta in processes/field ± SEM; n = 38 control and n = 42 rotenone image fields representing three independent neuronal preps). E, F, Representative confocal z-stack images of neurons exposed to 10 μm BrdU for 1 h following exposure to 2 weeks of DMSO vehicle control (E) or 1 nm rotenone (F). G, H, Quantitative analysis of BrdU puncta revealed significant rotenone-associated increases in both cell bodies (G; difference in means = +1.2; t(81) = 2.34; *p = 0.022; two-tailed unpaired t test; puncta/cell body ± SEM) and neurites (H; difference in means = +11.3; t(81) = 2.32; *p = 0.023; two-tailed unpaired t test; puncta in processes/field ± SEM) compared with vehicle control at 2 weeks (n = 40 control and n = 43 rotenone image fields representing three independent neuron preps ± SEM).
Figure 3.
Figure 3.
Distal axonal mtDNA replication is increased in response to chronic rotenone. A, Primary neurons were seeded into one side of microfluidic devices (Xona Microfluidics) to environmentally separate cell bodies and dendrites from axons for BrdU incorporation assessments. B, Neurons were transfected to express mitochondrially targeted mtDsRed2 (Mitochondria) and at DIV14 were immunofluorescently stained to detect ENT-1 to ensure that axons were capable of importing BrdU on their own. TLI, Transmitted light image. C, Following 1 week of DMSO vehicle (shown) or 1 nm rotenone, only the axons were exposed to 10 μm BrdU for 3 h. Confocal imaging of immunofluorescence for BrdU incorporation show that axon-localized BrdU puncta can be found distally from the microfluidic grooves (arrows), with no BrdU incorporation on the cell chamber side of the device. This suggests that mtDNA replication does occur locally in distal axons. D, Quantification of BrdU puncta demonstrated that mtDNA replication in distal axons was significantly increased following 1 week of chronic 1 nm rotenone exposure compared with DMSO vehicle control (n = 123 image fields per condition representing six independent neuron preps; difference in means = +3.9; t(244) = 4; *p = 0.0001; two-tailed unpaired t test; puncta in processes/field ± SEM).
Figure 4.
Figure 4.
Effect of chronic rotenone on neuronal expression of PGC1α, COXIV, and ATP5G1. Primary neurons were treated with DMSO vehicle control or 1 nm rotenone for 1 week (A–E) or 2 weeks (F–J) and collected for Western blot and immunochemical detection analyses of PGC1α and COXIV (A, F), and of ATP5G1 (B, G). C, We observed that 1 week of chronic rotenone exposure did not alter PGC1α levels (t(21) = 0.17; p = 0.87; two-tailed unpaired t test). D, Levels of COXIV were significantly increased after 1 week of rotenone exposure compared with vehicle control (difference in means = +51.4; t(21) = 2.5; *p = 0.020; two-tailed unpaired t test). E, Levels of ATP5G1 were also significantly increased after 1 week of rotenone exposure compared with vehicle control difference in means = +43.2; t(24) = 2.3; *p = 0.028; two-tailed unpaired t test). H, After 2 weeks of chronic rotenone exposure, PGC1α levels were significantly increased compared with vehicle control (difference in means = +11.7; t(22) = 3.7; *p = 0.0013; two-tailed unpaired t test). I, Levels of COXIV remained significantly elevated after 2 weeks of rotenone exposure compared with vehicle control (difference in means = +33.2; t(22) = 2.14; p = 0.044; two-tailed unpaired t test). J, Levels of ATP5G1 also remained significantly increased after 2 weeks of rotenone exposure compared with vehicle control (difference in means = +23.3; t(24) = 2.66; p = 0.014; two-tailed unpaired t test; n = 11–13; percentage of control ± SEM). To assess neuroanatomical localization of changes in COXIV protein, primary neurons were grown in microfluidic devices (Xona Microfluidics) to environmentally separate cell bodies and dendrites from axons. K, M, Neurons were treated with DMSO vehicle control or 1 nm rotenone for 1 week and then fixed for fluorescent immunochemical detection of COXIV and detection of actin via phalloidin. L, Quantitative fluorescence analyses demonstrated that, after 1 week of 1 nm rotenone, COXIV protein levels in axons significantly increased compared with vehicle control control (difference in means = +31.8; t(47) = 3.72; p = 0.0005; two-tailed unpaired t test; n = 26 control and n = 23 rotenone image fields representing three independent neuron preps ± SEM). N, COXIV levels in cell bodies, however, were unchanged following 1 week of rotenone compared with control (t(151) = 0.17; p = 0.87; two-tailed unpaired t test; n = 91 control and n = 62 rotenone cells representing three independent neuron preps ± SEM).
Figure 5.
Figure 5.
STED superresolution microscopy reveals mitochondrial–ER interaction at axonal mtDNA replication sites, and mtSSBP localization suggests active mtDNA replication in axons. A–C, Primary cortical neurons were cotransfected with mtDsRed2 (Mitochondria, Mitochondrion) and GFP-tagged endoplasmic reticulum protein Sec61β (ER). At DIV14, cells were exposed to EdU (10 μm) for 3 h and then fixed and stained for EdU using the Click-iT Alexa Fluor 647 kit and for GFP via immunofluorescence. STED superresolution microscopy was used to examine EdU staining relative to both mitochondria and ER in the soma (A) and in axons (B, C). We observed ER–mitochondrial interaction in axons (B, green arrowheads) and specifically at sites of mtDNA EdU incorporation (C, white arrow). D, Primary neurons were transfected with PA-mtGFP (Mitochondria) and at DIV14 exposed to BrdU (10 μm) for 3 h. Cells were immunofluorescently stained for BrdU and mtSSBP. We observed mtSSBP colocalized with BrdU puncta within distal axonal mitochondria, suggesting active mtDNA replication.
Figure 6.
Figure 6.
Proposed model of compartmentalized mitochondrial biogenesis response to stress in neurons. Early response to chronic stress: As an initial response to low, chronic mitochondrial stress, high-energy demanding arborized distal axons upregulate mitochondrial biogenesis locally, increasing mtDNA replication, mRNA translation, and mitochondrial density to preserve axonal health and function. This happens independently of the soma, where no significant changes in mitochondrial biogenesis have yet occurred. Later response to chronic stress: As prolonged stress continues to tax mitochondrial function, nuclear upregulation of the “master mitochondrial biogenesis regulator” transcription coactivator PGC1α increases activation of mitochondrial biogenesis transcription factors (TFs), including NRF-1 and NRF-2. Mitochondrial biogenesis increases in the soma, leading to increased somal mitochondrial density and increased resources (such as nuclear-expressed mitochondrially targeted transcription factors, proteins, and mRNAs) are available for transport down the axon to maintain localized mitochondrial biogenesis distally. Pathogenic conditions in vulnerable neurons: Neurons vulnerable to mitochondrial stressors may lack the ability to quickly upregulate local mitochondrial biogenesis in distal axons in response to stress. The poor early response to mitochondrial distress in the distal axon may lead to loss of the axonal projection and subsequent death of the neuron.
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