Mitochondrial angiotensin receptors in dopaminergic neurons. Role in cell protection and aging-related vulnerability to neurodegeneration

Abstract

The renin-angiotensin system (RAS) was initially considered as a circulating humoral system controlling blood pressure, being kidney the key control organ. In addition to the 'classical' humoral RAS, a second level in RAS, local or tissular RAS, has been identified in a variety of tissues, in which local RAS play a key role in degenerative and aging-related diseases. The local brain RAS plays a major role in brain function and neurodegeneration. It is normally assumed that the effects are mediated by the cell-surface-specific G-protein-coupled angiotensin type 1 and 2 receptors (AT1 and AT2). A combination of in vivo (rats, wild-type mice and knockout mice) and in vitro (primary mesencephalic cultures, dopaminergic neuron cell line cultures) experimental approaches (confocal microscopy, electron microscopy, laser capture microdissection, transfection of fluorescent-tagged receptors, treatments with fluorescent angiotensin, western blot, polymerase chain reaction, HPLC, mitochondrial respirometry and other functional assays) were used in the present study. We report the discovery of AT1 and AT2 receptors in brain mitochondria, particularly mitochondria of dopaminergic neurons. Activation of AT1 receptors in mitochondria regulates superoxide production, via Nox4, and increases respiration. Mitochondrial AT2 receptors are much more abundant and increase after treatment of cells with oxidative stress inducers, and produce, via nitric oxide, a decrease in mitochondrial respiration. Mitochondria from the nigral region of aged rats displayed altered expression of AT1 and AT2 receptors. AT2-mediated regulation of mitochondrial respiration represents an unrecognized primary line of defence against oxidative stress, which may be particularly important in neurons with increased levels of oxidative stress such as dopaminergic neurons. Altered expression of AT1 and AT2 receptors with aging may induce mitochondrial dysfunction, the main risk factor for neurodegeneration.

Figure 1

Specificity of angiotensin receptor antibodies and localization of angiotensin receptors in mitochondria of dopaminergic neurons. (a) The expression of major RAS components in dopaminergic neurons was confirmed by RT-PCR and laser microdissection of dopaminergic neurons retrogradly labeled by intrastriatal injection of fluorescent red retrobeads (RRB). SN section showing labeled dopaminergic neurons before and after laser microdissection for RT-PCR. Expression of TH, AGT, AT1, AT2 and β-actin mRNA in laser-microdissected dopaminergic neurons (right) and homogenates of SN used as a positive control (left) are also shown. (b) Western blot densitometric bands corresponding to 10 μg of AT1 or AT2 receptor overexpression lysate containing a C-terminal DDK epitope tag (DYKDDDDK) fused (left), and 10 μg of empty vector transfected control cell lysate HEK293 (EV, right). A band of 45 kDa was detected with the AT1 antibody, while a band of 50 kDa was detected with the AT2 antibody. A monoclonal antibody against DDK detected the corresponding band in the protein lysates. Colocalization of mitochondria and AT1 and AT2 receptors in primary cultures of the nigral region (c) and neurons from the MES 23.5 dopaminergic cell line (d). Electron microscopy of AT1 and AT2 labeling in a neuronal process (e) and cytoplasm (f) of a dopaminergic neuron. Immunolabeling for AT1 was observed in the outer membrane and cristae (white arrows) of mitochondria. In addition, strong AT1 labeling was also seen in clusters of free ribosomes (empty arrowheads). Immunolabeling for AT2 was present in mitochondrial membranes (white arrows), in the rough endoplasmic reticulum (black arrowheads) and in small clusters of free ribosomes, some of which were in close proximity to labeled mitochondria (empty arrowheads). AGT, angiotensinogen; DA, dopaminergic; MTDR, MitoTracker Deep Red; SN, substantia nigra; TH, tyrosine hydroxylase. Scale bars: (a) 50 and 200 μm (SN section); (c) 5 μm; (d) 10 μm; (e and f) 500 nm

Figure 2

Presence of fluorescence-tagged angiotensin receptors and fluorescent angiotensin II in mitochondria. Colocalization (c, f and i) of the fluorescent mitochondrial marker MTDR (a, d and g) with AT2-YFP (b), AT1-EGFP (e) or AII-488 (h). Fluorescent AII-488 colocalized with mitochondria 8 h after treatment of cultures (g–i). Cells transfected with AT1-EGFP required simultaneous treatment with the AT1 receptor antagonist losartan, in order to minimize AT1-induced superoxide toxicity and cell death. AT2-YFP, angiotensin receptor type 2 tagged to yellow fluorescent protein; AT1-EGFP, angiotensin receptor type 1 tagged to enhanced green fluorescent protein; AII-488, Alexa Fluor 488-fluorescent angiotensin II; MTDR, MitoTracker Deep Red. Scale bar: 5 μm

Figure 3

AT1 and AT2 receptors in isolated mitochondria (IM) from ventral mesencephalon and the dopaminergic neuron cell line MES 23.5. Effect of oxidative stress and aging. (a–c) Western blot (WB) of whole homogenate (Wh; n=4) and pure IM (Mt; n=4) from the nigral region (a) and MES 23.5 cells (c) showing different compartment markers used to assess the purity of the IM: voltage-dependent anion channel (VDAC) as a mitochondrial marker, tubulin as a cytosol marker and histone deacetylase 2 (HDAC2) as a nuclear marker. Note the higher expression of mitochondrial AT2 compared with AT1 receptors (b). (d)The levels of lactate dehydrogenase specific activity (LDH; micromol of substrate/min/mg; a marker for cytosolic and synaptosomal contamination) of the nigral region and MES 23.5 pure IM (Mt; n=6) were negligible relative to those in Wh. (e and f) WB of IM from the MES 23.5 cells transfected with AT2-YFP (yellow fluorescent protein; n=4) or AT1-EGFP (enhanced green fluorescent protein; n=3) showing the presence of fluorescent-tagged angiotensin receptors in IM in comparison with non-transfected cells (Co, control); 24 h treatment of cells, low doses of MPP⁺ (n=4) or N-acetyl-l-cysteine (NAC; n=3) induced increased expression of mitochondrial AT2 and AT1 receptors, respectively (AT2-YFP+MPP⁺; AT1-EGFP+NAC). (g–i) In young (n=3) and aged (n=4) rats, the expression of AT1 and AT2 receptors was analyzed by RT-PCR in nigral dopaminergic neurons labeled with red retrobeads (RRB) and isolated by laser microdissection (g–h), and by WB of IM from the nigral region of young (n=5–8) and aged rats (n=5-6) (i). Aging induced a significant increase in AT1 expression and a significant decrease in AT2 expression in both dopaminergic neurons and in IM. The results were normalized to the values of young animals. Data are means±S.E.M. *P<0.05 relative to the corresponding controls (Student's t-test). Scale bars: 50 μm

Figure 4

Effect of mitochondrial angiotensin receptors on mitochondrial respiration. (a and b) Activation of mitochondrial AT1 receptors with AII (i.e. AII+ PD123,319) induces an increase in both oxidative phosphorylation (P) and maximum respiratory rate (E), which was inhibited by pre-treatment of isolated mitochondria with the NOX4 inhibitor thioridazine (n=3–8). (c and d) Knockout mice for AT1 receptors (KO AT1; n=5) show lower mitochondrial respiration rates compared with wild-type littermate controls (WT). (e and f) Activation of mitochondrial AT2 receptors by AII (i.e. AII+losartan) produces a significant decrease in activated respiration (OXPHOS, P) and maximum respiration rate (maximum electron transport system, ETS, E) associated with complex I, which was blocked by pre-incubation of isolated mitochondria with the nitric oxide synthase (NOS) inhibitor, N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME; n=5–8). (g and h) Mice lacking AT2 receptors (KO AT2; n=5) show an increased respiratory activity compared with wild-type mice (WT). Data are mean±S.E.M. *P<0.05 compared with control. ^#P<0.05 compared with the group treated with AII. One-way analysis of variance (ANOVA) and Bonferroni post hoc test (a, b, e, f) and Student's t-test(c, d, g, h)

Figure 5

Effect of mitochondrial angiotensin receptors on mitochondrial transmembrane potential, nitric oxide and superoxide production. (a) In isolated mitochondria, activation of AT1 and AT2 receptors (AII+ the AT2 blocker PD123,319, and AII+ the AT1 blocker losartan, respectively) did not induce any significant change in mitochondrial membrane potential maintenance; however, the potassium ionophore valinomycin led to loss of approximately 40% of transmembrane potential relative to non-treated mitochondria (n=5). (b) Activation of AT2 receptors (AII+ the AT1 blocker losartan) induced an increase in levels of NO that was inhibited by pre-treatment with the NOS inhibitor l-NAME (n=5–8). (c) The presence of Nox4 in isolated mitochondria was also shown by western blot assay with a rabbit monoclonal anti-Nox4 antibody, which detected a band of 60  kDa, and an antibody against the mitochondrial marker VDAC. The Nox4 signal increased with mitochondrial content (5, 10, 50 μg of mitochondrial sample loaded in the acrylamide gel). (d) In isolated mitochondria, activation of AT1 receptors with AII (AII+the AT2 receptor antagonist PD123,319) resulted in increased levels of superoxide, and simultaneous treatment with the NOX4 inhibitor thioridazine led to the inhibition of total superoxide to about a 40% of untreated controls (n=4–8). Data are mean±S.E.M. *P<0.05 compared with control. ^#P<0.05 compared with the group treated with AII (one-way analysis of variance (ANOVA) and Bonferroni post hoc test). l-NAME, l-arginine methyl ester hydrochloride; NOS, nitric oxide synthase

Figure 6

Model of the role that AT1 and AT2 receptors play in modulating oxidative phosphorylation in brain mitochondria. Activation of AT1 receptors in mitochondria regulates superoxide production, via Nox4, and increases respiration. Mitochondrial AT2 receptors are much more abundant and induce, via nitric oxide, a decrease in mitochondrial respiration, modulating oxidative phosphorylation without significant alteration in mitochondrial membrane potential, which indicates that the bioenergetic properties of the mitochondria are not affected. Mitochondrial AT2 receptor expression increased after treatment of cells with oxidative stress (OS) inducers (such as low doses of MPP⁺) and decreased with aging. Mitochondrial AT1 expression increased with aging and after treatment of cells with antioxidants (such as N-acetyl-cysteine, NAC). At mitochondrial level, AT2 receptors may act as respiratory modulators and counteract low levels of OS, which may be particularly important in cells with an increase in levels of OS such as dopaminergic neurons. Aging induces altered expression of mitochondrial AT1 and AT2 receptors that may induce mitochondrial dysfunction, the main risk factor for neurodegeneration