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. 2007 Oct;14(10):1721-32.
doi: 10.1038/sj.cdd.4402180. Epub 2007 Jun 15.

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration

Z Tan  1 , X Sun, F-S Hou, H-W Oh, L G W Hilgenberg, E M Hol, F W van Leeuwen, M A Smith, D K O'Dowd, S S Schreiber
Affiliations

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration

Z Tan et al. Cell Death Differ. 2007 Oct.

Abstract

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.

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Figures

Figure 1
Figure 1. UbB + 1 causes neuritic beading and mitochondrial buildup in neuronal processes
(a) Fluorescence images of rat cortical neurons 16 h after transfection with pEGFP–UbB + 1, pEGFP-Ub or pEGFP alone. Quantification shows a significant increase in the percentage of beading cells and the number of beads per cell following transfection with pEGFP-UbB + 1. * and Δ indicate significant differences (P’s * <0.05, ** <0.01, N = 4; Δ<0.05, N = 20, as per ANOVA and Fisher’s PLSD). Bars depict mean±S.E. (b) Neurons were co-transfected with pEGFP–UbB + 1 and pDsRed2-Mito. Confocal microscopy shows colocalization of the mitochondrial marker in EGFP– UbB + 1-associated neuritic beads. (c) A transmission electron micrograph reveals beading structures in an axon from a pEGFP–UbB + 1-transfected cell. (d and e) At higher magnification, the boxed areas in (c) show the presence of microtubules (MT), mitochondria or mitochondria-like structures (Mi), spheroids (broad arrows in e) and smaller vesicular bodies. (f) A neuritic bead containing mitochondria in clumps, MT and vesicular bodies. An adjacent process is demarcated by delimiting membranes (DM, arrowheads). Scale bar = 50 µm (a), 20 µm (insets a), 150 µm (inset b) shown at higher magnification in other panels (30 µm), 1 µm (c) and 0.4 µm in (d–f)
Figure 2
Figure 2. Time-lapse analysis of mitochondrial movements
(a) Comparison of photomicrographs at two different times after transfection demonstrates retrograde (arrowheads shifted up) and anterograde (arrows shifted down) motions of mitochondria-containing aggregates. Scale bar = 10 µm. (b) Quantification of mitochondrial aggregate motions in neuronal processes. The number of labeled mitochondrial aggregates that moved retrogradely was significantly reduced in EGFP–UbB + 1-transfected cells (P’s * <0.01, ** <0.005, N = 25, as per ANOVA and Fisher’s PLSD). No significant difference was detected between the number of mitochondrial aggregates that moved retrogradely in cells expressing EGFP-Ub and Ub alone (P’s Δ> 0.45, N = 25, as per ANOVA); no statistically significant change was detected in the number of mitochondrial aggregates that moved anterogradely in EGFP–UbB + 1-expressing cells or in cells expressing either EGFP–Ub or EGFP alone (P’s ∇ > 0.50, N = 25, as per ANOVA). Bars depict mean±S.E.
Figure 3
Figure 3. Biochemical analyses of mitochondrial motors
(a) Whole-cell lysates, mitochondrial (mito) and cytosolic (cyto) fractions were prepared from primary neurons transfected with either pEGFP, pEGFP–UbB + 1 or pEGFP–Ub. Western blotting was performed with a panel of antibodies at the indicated times. (b) The relative changes in dynein P74 abundance in both mitochondrial and cytosol fractions are shown based on the initial levels in EGFP-transfected cells. Bars represent the mean±S.E. from four independent experiments (*P<0.05, as per Student’s t-test). (c) Fluorescence microscopy revealed neuritic beading of mitochondria (red) in primary neurons co-transfected with pDsRed2-Mito and UbB + 1 (green) in pcDNA3.1. (d). Whole–cell lysates (whole cell), mitochondrial (mito) and cytosolic (cyto) fractions were prepared from neurons 24 h after transfection with empty pcDNA3.1 (ctl) or with Ub or UbB + 1 cDNA cassette. Western blotting was performed with indicated antibodies. (e) Cell-free reconstitution assay. Isolated mitochondria were incubated with increasing amounts of Ub species or bovine serum album (ctl), followed by P74 Western blotting. * indicates a significant difference of the group incubated with either 2.0 µg UbB + 1 or Ub5 + 1 relative to the control (P’s<0.05, N = 4, as per ANOVA and Fisher’s PLSD)
Figure 4
Figure 4. Ultrastructural analysis of the spatial relationships between mitochondria and MTs
(a) Electron micrographs taken from transfected neurons. The first two panels show mitochondria flanked by MTs (arrows) on two sides (i.e., MT-associated), whereas the third panel shows a mitochondrion with MTs on only one side. (b) There was a significant decrease in MT-associated mitochondria relative to the total number of mitochondria-like structures in EGFP–UbB + 1-transfected cells. (*P’s<0.01, N = 4, as per ANOVA and Fisher’s PLSD). Bars depict mean±S.E.
Figure 5
Figure 5. Neuritic beading is associated with mitochondrial stress
(a) Neurons were stained with TMRM after either EGFP–UbB + 1 or EGFP–Ub transfection. TMRM fluorescence (red) is present in both neurites (arrows) and cell soma (arrowheads) after EGFP–Ub transfection, but only in the cell soma after EGFP–UbB + 1 transfection. (b) The number of cells with reduced TMRM signal in neurites (left) or whole cell (right) relative to EGFP-positive cells following transfection. P’s<0.01 for each time point, N = 4. (c) Mitochondrial superoxides were stained by MSR in EGFP–UbB + 1-associated neuritic beads (arrows), but not in EGFP–Ub-transfected cells. (d) The number of MSR-relative to EGFP-positive cells following plasmid transfection. * Indicates significant differences (P’s * <0.05, ** <0.01, N = 4, as per ANOVA and Fisher’s PLSD). Bars depict mean±S.E. (e) Cytochrome c abundance in cytosol following plasmid transfection. GAPDH was used as loading control. (f) Caspase-3 activation (green) and UbB + 1-positive beads (red) are colocalized in some processes (merged, arrow). Scale bar = 40 µm (inset f), 10 µm in other panels of (f), and 15 µm in (a and c)
Figure 6
Figure 6. Accumulation of polyUb conjugates, p53 activation and neuronal cell death
(a) Whole-cell lysates were prepared from primary neurons transfected with EGFP or EGFP fusion constructs 24 h after transfection. Western blotting using a Ub antibody showed increased abundance of polyUb conjugates in UbB + 1-transfected cells. Lysates from neurons treated with MG115 (5 µM, 24 h) was used a positive control. Bottom: changes in abundance of total polyUb conjugate ladders are expressed relative to those in EGFP control cells. The data depict the mean number of pixels±S.E.M from four independent experiments. (b) p53 and free Ub abundance at indicated times following plasmid transfection. All the lanes shown in each panel were from the same gel. β-Actin was used as loading control. (c) Representative examples in EGFP–UbB + 1-transfected neurons of: nuclear p53 immunoreactivity (arrow, top row); activated p53 following co-transfection with pp53-TA-DsRed2 (second row); positive nuclear TUNEL staining (red, third row) with neuritic beads; positive MSR staining (red, fourth row) in p53-null neurons. Scale bar = 60 µm in second row and 30 µm in other panels. (d) The number of TUNEL- or p53 reporter-positive cells relative to EGFP-positive cells after EGFP–UbB + 1 transfection. Bars are mean±S.E. TUNEL studies were performed in the presence or absence of DEVD-CHO. (e) p53-null neurons were transfected with EGFP–UbB + 1, and the number of MSR- or TUNEL-positive cells relative to EGFP-positive cells were counted. Bars are mean±S.E.
Figure 7
Figure 7. shRNA-mediated suppression of UbB + 1
(a) Schematic of RFP/DsRed2.U6.silencer vector encoding a specific shRNA directed by murine U6 promoter and a CMV-directed fluorescent reporter. UbB + 1 shRNA targets UbB + 1 mRNA, which differs by only two nucleotides (highlighted in red) from UbB mRNA. (b) Mouse cortical neurons were co-transfected with indicated plasmids and visualized by dual fluorescence. Specific suppression of EGFP–UbB + 1 by RFP.UbB + 1sh was detected in the cells 24 h after transfection (right column). Note: one EGFP–UbB + 1-positive cell with neuritic beading in the right lower two panels is RFP.UbB + 1sh-negative. Scale bar represents 30 µm for all the panels. (c) The number of EGFP-positive cells (left panel) or cells with neuritic beads (right panel) relative to RFP-positive cells following co-transfection of EGFP-UbB + 1 and either RFP.Controlsh or RFP.UbB + 1sh. The bars represent mean±S.E. **P’s<0.001
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