Mitochondria modulate programmed neuritic retraction

Abstract

Neuritic retraction in the absence of overt neuronal death is a shared feature of normal aging and neurodegenerative disorders, but the intracellular mechanisms modulating this process are not understood. We propose that cumulative distal mitochondrial protein damage results in impaired protein import, leading to mitochondrial dysfunction and focal activation of the canonical apoptosis pathway in neurites. This is a controlled process that may not lead to neuronal death and, thus, we term this phenomenon "neuritosis." Consistent with our hypothesis, we show that in primary cerebrocortical neurons, mitochondrial distance from the soma correlates with increased mitochondrial protein damage, PINK1 accumulation, reactive oxygen species production, and decreased mitochondrial membrane potential and depolarization threshold. Furthermore, we demonstrate that the distance-dependent mitochondrial membrane potential gradient exists in vivo in mice. We demonstrate that impaired distal mitochondria have a lower threshold for focal/nonlethal neuritic caspase-3 activation in normal neurons that is exacerbated in aging, stress, and neurodegenerative conditions, thus delineating a fundamental mechanistic underpinning for synaptic vulnerability.

Keywords: caspase-3; mitochondrial membrane potential; mutant huntingtin; neurite retraction; neurodegeneration.

Fig. 1.

Elevation of focal axodendritic caspase-3 activity is associated with decreased neuritic mitochondrial cyt c content. (A) Synaptosomes isolated from mouse brains are characterized by elevated caspase-like (DEVD-ase) activity (*P < 0.001, two-tailed t test) compared with whole-forebrain lysates. Data are shown as a box chart; n = 6. (B) Immunoblotting shows a reduction of apoptosis-inhibiting protein (XIAP), reduction in pro-caspase-3 (p32), increase of the cleaved caspase-3 precursor (p29), and appearance of cleaved caspase-3 in PCNs upon aging in culture. Each lane represents one culture; this is representative of three wells. (C) Elevated DEVD-ase activity in neurites is more robust in mature (DIV14) compared with young (DIV8) PCNs and is decreased in the presence of a pan-caspase inhibitor (zVAD-fmk; 5 μM). Each condition was repeated five times and the images are representative. Boxed regions are enlarged in the corresponding images of the bottom row. (D) Immunoblotting quantification shows a neurite-specific decrease of XIAP in PCNs of DIV10. Each lane represents cells or neurites collected from one well of a multiwell cell-culture plate as described in Methods; representative image of n = 3 (*P = 0.012, paired t test). (E) Representative immunoblot and quantitation for PINK1 and parkin in PCNs, neurites, and isolated mitochondria. Immunoblotting shows that isolated neuritic mitochondria accumulate more full-length PINK1 and less parkin compared with PCN mitochondria (DIV10). Mitochondrial cyt c oxidase (respiratory complex IV) subunit 1 (COX-I) was used as a loading control (*P = 0.03, t test; n = 3; data are shown as mean +/− SD). (F) Immunostaining of PCNs cultured in a microfluidic chamber to compartmentalize neurites and the soma. PCNs (DIV14) were fixed and stained against TOM20 and cyt c. (G) Close-up of the soma and axon up to 75 μm (Top) and >150 μm (Bottom) from the nuclear compartments demonstrates reduced cyt c signal. Red mitochondria have reduced cyt c (green) content. (H) Analysis of TOM20 and cyt c signal shows a distance-dependent decrease of the ratio of colocalized red (TOM20) and green (cyt c) signals. Each dot represents one mitochondrial volume (total 716 mitochondrial volumes per neuron). (I) Decreased colocalization [presented as Pearson’s colocalization coefficient (51)] of TOM20 and cyt c in distal (>150 μm from nucleus) compared with perinuclear mitochondrial volumes (*P < 0.0001, paired t test; n = 9 neurons, total 4,894 mitochondrial volumes; data are shown as mean +/− SD). (J) Content of mitochondrial proteins CI (subunit NDUFB8), CII (subunit SDHB), CIII (core protein 2), COX-I, CV (alpha subunit), and VDAC (voltage-dependent anion-selective channel protein 1, a porin of the outer mitochondrial membrane) of the cytosolic fraction of PCNs and of neurites (2 μg total protein) (DIV14) as well as mitochondria isolated from PCNs and neurites (10 μg total protein). Tubulin was used as loading control for cytosolic fractions and an impurity indicator for isolated mitochondria; representative image of n = 3.

Fig. 2.

Distal neuritic mitochondria have higher levels of oxidized proteins, increased ROS production, and slower protein import compared with perinuclear mitochondria. (A) Representative dual-channel image of a DIV5 neuron using a 40×, N.A. 0.95 objective taken 24 h after transfection with MitoTimer and shown as a maximum-intensity profile for a series of z stacks. (Scale bars, 20 μm.) (B) Quantification of the protein content quality of proximal (≤10 µm from nucleus) and distal (≥70 µm from nucleus) mitochondria (*P < 0.0001, paired-sample t test; n = 21 neurons, total 3,590 mitochondrial volumes). (C) DIV7 neurons were imaged 24 h after transfection with mtGFP (green) and 1 h after addition of MitoSOX (violet) and MitoView633 (red) dyes. The MitoSOX/MitoView633 ratio represents Δψ_m-independent MitoSOX fluorescence intensity (shown as a heatmap in which blue is low and red is high) affected by mitochondrial O₂^•−. (Scale bars, 20 μm.) (D) Quantification of the MitoSOX/MitoView633 ratio of proximal (≤10 µm from nucleus) and distal (≥70 µm from nucleus) mitochondria (*P = 0.001, paired t test; n = 11 PCNs, total 2,750 mitochondrial volumes). (E) Induction of mitochondrial protein import occurs earliest in the perinuclear area. DIV5 PCNs transfected with mtGFP were imaged over the indicated time period. Representative image from three independent experiments. (Scale bar, 10 μm.) (F) Time-dependent changes in the mtGFP signal intensities of distal (≥50 µm from nucleus) mitochondria (d) in comparison with perinuclear (≤10 µm from nucleus) mitochondria (p). All images (z stacks) were collected with a confocal fluorescence microscope using a 40×, N.A. 0.95 objective. Data are shown for 5 neurons imaged 3 d posttransfection and 12 neurons imaged 6, 9, and 12 d posttransfection. The asterisk indicates statistically significant differences between the mitochondrial populations on days 3 and 12 posttransfection (*P < 0.001, Mann–Whitney U test). All data are shown as mean +/− SD.

Fig. 3.

Δψ_m is negatively correlated with distance from the nucleus. (A) Representative image of a PCN transfected with mtGFP and loaded with 50 nM TMRM. (A, Left) TMRM signal, GFP signal, and merged. (A, Right) TMRM fluorescence intensity heatmap demonstrating a gradual proximal-to-distal gradient of loss of Δψ_m. (Scale bars, 20 µm.) (B) Representative analysis of the TMRM and mtGFP fluorescence intensities’ dependence on the distance between mitochondria and nuclei for DIV5 neurons (Left) and DIV14 neurons (Right) transfected 2 and 9 d before imaging. TMRM and GFP fluorescence are presented as mean values of the voxel intensities for a given mitochondrial volume defined via mtGFP signal after 3D reconstruction of z-stack images collected using a 40×, N.A. 0.95 objective. Symbols represent individual mitochondrial volumes. Apparent linear regression with the slope indicated is shown as a line plot. (C) Comparison of TMRM fluorescence of anterograde and retrograde mitochondria normalized by TMRM signal intensity of stationary mitochondria. Data are for 25 mitochondria (14 anterograde and 11 retrograde) observed in 12 neurons in five independent experiments (*P = 0.015, Wilcoxon signed-rank test; data are shown as mean +/− SD). (D) Comparison of the MitoTimer fluorescence of anterograde and retrograde mitochondria (9 anterograde and 10 retrograde observed in six neurons in three independent experiments) (*P = 0.006, Mann–Whitney U test; data are shown as mean +/− SD). Ant, anterograde; Ret, retrograde.

Fig. 4.

In vivo dual-channel two-photon images of mouse spinal cord neuronal mitochondria. (A) Representative images of a Thy1-CFP mouse spinal cord exposed to TMRM. (A, Left) TMRM signal, CFP signal, and merged images. (A, Right) TMRM fluorescence intensity heatmap demonstrating a gradual proximal-to-distal gradient of Δψ_m. (B) Representative analysis of the TMRM and mtCFP fluorescence intensity, and their ratio along the imaged spinal cord. TMRM and CFP fluorescence intensities are presented as mean values of the pixel intensities for a given mitochondrial volume defined as mtCFP signal after 3D reconstruction of z-stack images collected using a 20×, N.A. 1 objective. Each dot represents a mitochondrial volume. Plotted linear regression line of best fit shows the slope of the TMRM signal (normalized to mtCFP) to be significantly different from zero (R² = 0.06, P = 5 × 10⁻⁶).

Fig. 5.

Disruption of mitochondrial import causes early mitochondrial depolarization of distal mitochondria followed by caspase-associated neuronal cell death. (A) TOM40 knockdown (TOM40KD) induced mitochondrial depolarization followed by segmentation in a distal-to-proximal direction. Live-cell imaging of TOM40KD neurons in the presence of TMRM and cell-impermeable RedDot2 nuclear (stains nucleus blue in dead cells) dyes. DIV5 cortical neurons were transfected with a U6-TOM40/CMV-GFP plasmid. At DIV8, loss of mitochondrial membrane potential and cell death were assessed using TMRM and RedDot2 using live confocal imaging. Time 0 indicates the beginning of imaging, and timestamps on images are h:min:s. To better appreciate the temporal and site-specific pattern of Δψ_m loss,_, Bottom panels demonstrate TMRM staining only of the green transfected neuron. (Scale bars in all panels, 20 μm.) (B) Survival plot of neurons transfected with U6-TOM40/CMV-GFP (TOM40KD) (n = 49 neurons, three independent experiments) and U6-Scr/CMV-GFP (Scr, scrambled) (n = 47 neurons, three independent experiments). Equality of survival functions tested with log rank is shown in the graph. (C) Prolonged (72-h) incubation of PCNs with 2 µM MitoBloCK-6 (MB-6; Δψ_m-independent mitochondrial protein import blocker) resulted in lowering of the Δψ_m of distal (>50 μm from nucleus) (*P = 0.005, t test) compared with somal (≤10 μm from nucleus) mitochondria. (D) Prolonged (72-h) incubation of PCNs with MitoBloCK-6 induced caspase-3 activation (*P = 0.027, paired-sample t test). Data are shown as a box chart; n = 3. (E) Neuritic caspase-3 activation in PCNs (DIV21) after 72 h of incubation with 2 µM MitoBloCK-6 visualized as CFP/FRET signal ratio in neurons transfected with genetically encoded caspase-3 FRET substrate. Image color ranged from blue to red, representing minimum and maximum DEVD-ase activity, respectively. A representative image of five pyramidal cortical neurons obtained from four independent experiments is shown. (F) Lower protein import activity as measured by pOTC cleavage and generation of mOTC of synaptosomal (Syn) mitochondria compared with nonsynaptosomal (NS) mitochondria at equal Δψ_m. Protein import activity was quantified in synaptosomal mitochondria and in isolated nonsynaptosomal mitochondria supplied with 20 to 40 nM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

Fig. 6.

mHTT expression is associated with elevated synaptosomal caspase-3 activity in R6/2 mice compared with WT, elevated caspase-3 activity in neurites, and lower Δψ_m in distal mitochondria. (A) Synaptosomes isolated from 6-wk-old (early symptomatic) R6/2 mouse forebrains have elevated DEVD-ase activity (*P = 0.008, t test) compared with WT samples, while DEVD-ase activity in WT and R6/2 forebrains is similar. Data are shown as a box chart; n = 6. (B) Elevated caspase-3 activity in neurites is more robust in R6/2 PCNs. The image is representative of 13 WT and 9 R6/2 PCNs obtained from three independent experiments. (C) Content of mitochondria of oxidized proteins is increased in R6/2 synaptosomal mitochondria associated with disease progression. Mitochondria were isolated from brains of R6/2 and WT mice at 3, 6, and 12 wk of age (n = 3 independent isolations of five brains per isolation). Mitochondrial protein quality was assessed by using a Protein Carbonyl Colorimetric Assay Kit (Cayman Chemical). The asterisk indicates statistically significant differences between the WT and R6/2 at 6 wk of age (*P = 0.0014, t test). The double asterisks indicate statistically significant differences between the WT and R6/2 at 12 wk of age (**P = 0.007, t test). (D) Distal mitochondria in R6/2 neurons have lower Δψ_m. DIV5 PCNs from WT or R6/2 were transfected with mtGFP and loaded with 50 nM TMRM to probe Δψ_m. Images were analyzed to obtain a mean value of TMRM fluorescence per mitochondrial volume defined by mtGFP signal. The image is representative of 17 WT and 17 R6/2 PCNs obtained from five independent experiments. (E) Representative analysis of the TMRM fluorescence intensity changes is presented as a gradient of the Δψ_m decrease with distance from the nucleus (slope). (F) Each symbol represents the TMRM fluorescence slope of transfected neurons; n = 17 obtained from five independent experiments. The asterisk indicates statistically significant differences between WT and R6/2 (*P = 0.003, t test).