Figure 1.
TOM40 protein levels are decreased in PD brains and in α-Syn transgenic mice.
(A) Western blot analysis showing levels of mitochondrial proteins TOM40 and TOM20 as well as of α-Syn on postmortem human brain samples from control subjects and PD patients (β-Actin signal is shown as normalization control). (B) Densitometric analysis of TOM40 and TOM20 immunoreactivity. (C) Immunohistochemical detection of TOM40 and TOM20 in control and PD brains. (D) Optical density analysis of TOM40 and TOM20 immunoreactivity. (E) Western blot analysis of Tom40, Tom20 and human-α-Syn levels on whole brain homogenates from α-Syn-tg mice and non-transgenic littermate controls. (F) Densitometric analysis of Tom40 and Tom20 on mice brains. (G) Immunohistochemical detection of Tom40 and Tom20 in α-Syn-tg and non-transgenic mice. (H) Optical density analysis of Tom40 and Tom20 on mice brains. *p<0.05 per Student’s t test. Scale bar represents 25 µm.
Figure 2.
Tom40 is reduced in B103 cells overexpressing wild type and A53T-α-Syn.
B103 neuroblastoma rat cells were infected with lentivirus encoding human α-Syn wild type (LV- α-Syn) or its mutated isoforms A53T (LV-α-Syn-A53T) and A30P (LV-α-Syn-A30P); Tom40 (LV-Tom40) or a combination of these vectors. (A) Western blot detection of α-Syn, Tom40 and Tom20 on whole cell lysates from infected B103 cells. (B) Densitometric analysis of Tom40 and Tom20 levels. (C) Efficient overexpression of Tom40 is achieved in B103 cells by infection with LV-Tom40. (D) Immunohistochemical detection of α-Syn and Tom40 in B103 cells infected with the corresponding lentiviral constructs. (E) Pixel intensity analysis of α-Syn levels, showing reduction of α-Syn in Tom40-overexpressing cells. *p<0.05 per Student’s t test. Scale bar represents 10 µm.
Figure 3.
Mitochondrial impairment and oxidative stress induced by α-Syn-accumulation is reduced by overexpression of Tom40.
B103 neuroblastoma rat cells were infected with lentivirus encoding human α-Syn wild type (LV- α-Syn) or its mutated isoforms A53T (LV-α-Syn-A53T) and A30P (LV-α-Syn-A30P); Tom40 (LV-Tom40) or a combination of these vectors. (A) Immunohistochemical detection of mitochondria by in vivo labeling with MitoTracker. (C) In vivo detection of mitochondria-derived reactive oxygen species (ROS) in cytoplasm by CellROX fluorogenic probes. (E) Fluorescent immunolabeling of oxidative DNA lesions (anti-8-OHdG). (B, D and F) Pixel intensity analysis of the corresponding immunosignals. Scale bar represents 10 µm. *p<0.05 per Student’s t test.
Figure 4.
Alpha-synuclein overexpression results in oxidative DNA damage, alterations in respiratory chain complex I and mitochondrial DNA deletions in transgenic mice brains.
(A) Immuno-histochemical detection of 8-OHdG suggesting increased oxidative DNA damage in α-Syn tg mice brains. (B) 8-OHdG levels were quantified on whole brain in α-Syn tg and non-transgenic control mice by ELISA. Scale bar represents 25 µm. (C) Long extension PCR showing large-scale mtDNA deletions in α-Syn tg mice. (D) Quantitative determination of mtDNA deletions by real-time PCR showing increased mtDNA deletion levels in the brain of α-Syn tg mice compared to control animals. (E) Immunostaining for α-Syn (green signal) and NeuN (red signal; arrow marks the identical neuron in double labeling) used for Laser Capture Microdissection (LCM). Scale bar represents 250 µm in the upper panel and 25 µm in the close-up lower panel. (F) Quantification of mtDNA deletion levels by real-time PCR on individual neurons with either intense or negative α-Syn immunoreactivity after LCM. (G) Western blot analysis of mitochondrial OXPHOS in brain homogenates from α-Syn tg and wt mice. Lane 1 shows mitochondrial proteins standard marker. (H) Densitometric analysis of the levels of Complexes I, II, III and IV. *p<0.05 per Student’s t test.
Figure 5.
Lentiviral delivery of Tom40 ameliorates α-Syn-induced mitochondrial alterations in transgenic mice.
(A) Tom40 immunostaining in sagittal brain sections at the level of the hippocampus of non-transgenic (non-tg) controls and α-Syn-tg mice injected with control lentivirus (LV-control) or with lentiviral delivery of Tom40. (B) Stereological analysis of Tom40 immunoreactivity (optical density) showing increased signal after LV-Tom40 treatment. (C) Fluorescent immunolabeling of oxidative DNA lesions (anti-8-OHdG) in control and α-Syn tg mice treated with LV-control or LV-Tom40. Scale bar represents 100 µm in the upper panel and 15 µm in the lower panel. (D) Stereological analysis of anti-8-OHdG staining intensity reveals normal oxidative lesion load in tg animals overexpressing Tom40. (E) Quantification of ATP levels on brain homogenates from mice treated with LV-control or LV-Tom40. *p<0.05 per Student’s t test.
Figure 6.
Tom40 overexpression reduces accumulation of α-Syn in neuronal cells.
(A and B) Immunostaining of neuronal (anti-Neu N, upper panel) and glial (anti-GFAP, lower panel) cells in non-transgenic control animals and α-Syn tg mice that received LV-control or LV-Tom40 gene therapy. Scale bar represents 250 µm. (C and D) Stereological analysis of anti-NeuN and anti-GFAP-labeling, respectively (number of positive cells). (E) Effect of Tom40 overexpression on α-Syn levels: immunostaining of α-Syn on sagittal brain sections from non-transgenic (non-tg) controls, α-Syn-tg mice that received control lentivirus and α-Syn-tg mice injected with Tom40-lentivirus. Scale bar represents 250 µm in the upper panel and 50 µm in the lower panel. (F) Stereological analysis of α-Syn immunosignal (number of positive cells) showing reduced α-Syn levels on Tom40-overexpressing animals. *p<0.05 per Student’s t test.