Angiogenin in Parkinson disease models: role of Akt phosphorylation and evaluation of AAV-mediated angiogenin expression in MPTP treated mice.

The angiogenic factor, angiogenin, has been recently linked to both Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). We have recently shown that endogenous angiogenin levels are dramatically reduced in an alpha-synuclein mouse model of PD and that exogenous angiogenin protects against cell loss in neurotoxin-based cellular models of PD. Here, we extend our studies to examine whether activation of the prosurvival Akt pathway is required for angiogenin's neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+), as observed in ALS models, and to test the effect of virally-mediated overexpression of angiogenin in an in vivo PD model. Using a dominant negative Akt construct, we demonstrate that inhibition of the Akt pathway does not reduce the protective effect of angiogenin against MPP+ toxicity in the dopaminergic SH-SY5Y cell line. Furthermore, an ALS-associated mutant of angiogenin, K40I, which fails to induce Akt phosphorylation, was similar to wildtype angiogenin in protection against MPP+. These results confirm previous work showing neuroprotective effects of angiogenin against MPP+, and indicate that Akt is not required for this protective effect. We also investigated whether adeno-associated viral serotype 2 (AAV2)-mediated overexpression of angiogenin protects against dopaminergic neuron loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. We found that angiogenin overexpression using this approach does not reduce the MPTP-induced degeneration of dopaminergic cells in the substantia nigra, nor limit the depletion of dopamine and its metabolites in the striatum. Together, these findings extend the evidence for protective effects of angiogenin in vitro, but also suggest that further study of in vivo models is required to translate these effects into meaningful therapies

Angiogenin overexpression does not reduce MPTP-induced dopaminergic neuronal loss in the substantia nigra.

<p>Four weeks following AAV2 injection, MPTP was administered at 30 mg/kg/day for five days. Mice were sacrificed three weeks following the last MPTP injection and brain sections stained for tyrosine hydroxylase to identify DA neurons in the substantia nigra. Stereology counts of TH-positive nigral neurons in the AAV-injected side are shown for each of the four conditions: GFP-saline (n = 7), Ang-saline (n = 4), GFP-MPTP (n = 17), and Ang-MPTP (n = 16). MPTP induced a 28% reduction in TH-stained neurons in the substantia nigra of GFP-injected mice, and angiogenin did not ameliorate dopaminergic neuron loss induced by MPTP. *p<0.05, **p<0.01 (One-way ANOVA with Tukey's post-hoc test). Error bars reflect SEM.</p

Angiogenin is expressed in TH-positive neurons in the substantia nigra after viral injection.

<p>A. An AAV2-Ang injected mouse was sacrificed eight weeks after injection and stained with a primary antibody against the V5 tag fused to angiogenin and a CY3-conjugated secondary antibody, demonstrating overexpression of angiogenin in the substantia nigra. Scale bar = 100 µm. B. Same section of tissue stained with a primary antibody against tyrosine hydroxylase (TH) and an Alexa488-conjugated secondary antibody to identify dopaminergic nigral neurons. Scale bar = 100 µm. C. A merge of V5-angiogenin staining and TH staining demonstrating colocalization in dopaminergic cells in the substantia nigra. Scale bar = 100 µm. D. Higher magnification image demonstrating V5-angiogenin localized perinuclearly and accumulated in vesicular-like puncta within the cell. Scale bar = 10 µm. E. Control immunostaining of the substantia nigra from an AAV2-GFP injected mouse. No V5-angiogenin staining was observed. Scale bar = 100 µm. F. Same section stained for TH to identify dopaminergic neurons in the substantia nigra of an AAV2-GFP-injected mouse. Scale bar = 100 µm. G. GFP immunostaining of the substantia nigra from another section from the same AAV-GFP injected mouse, demonstrating GFP expression in the substantia nigra. H. Same section stained for TH to identify dopaminergic neurons in the substantia nigra of an AAV-GFP injected mouse. Scale bar = 100 µm.</p

Angiogenin does not prevent MPTP-induced depletion of dopamine and its metabolites in the striatum.

<p>Three weeks after MPTP or saline injections, AAV2-GFP and AAV2-Ang-injected mice were sacrificed and striatal tissue immediately removed and frozen for HPLC. DA and its metabolites, DOPAC and HVA, were analyzed by HPLC. GFP-Saline n = 10, Ang-Saline n = 10, GFP-MPTP n = 9, Ang-MPTP n = 6. Error bars reflect SEM. A. Striatal dopamine levels as determined by HPLC. AAV2-Ang mice treated with MPTP showed a comparable loss in striatal DA. ***p<0.001 (One-way ANOVA with Tukey's post-hoc test). B. Striatal DOPAC levels as determined by HPLC. A similar reduction in DOPAC was seen in MPTP-treated AAV2-Ang injected mice as compared to AAV2-Ang MPTP. ***p<0.001 (One-way ANOVA with Tukey's post-hoc test). C. Striatal HVA levels as determined by HPLC. AAV2-Ang-injected mice treated with MPTP had a similar decrease in HVA as compared to AAV2-GFP mice treated with MPTP. ***p<0.001 (One-way ANOVA with Tukey's post-hoc test).</p

Dominant negative Akt does not inhibit angiogenin's protective effect against MPP+.

<p>A. Western blot demonstrating reduced Akt phosphorylation in SH-SY5Y cells transfected with the K179M DN-Akt construct. Twenty-four hours following transfection of empty pCMV5 vector (pC) or DN-Akt vector (DN), SH-SY5Y cells were treated with Insulin Growth Factor (50 ng/mL). Thirty seconds after Insulin Growth Factor treatment, cell lysates were collected for Western blotting. Actin was used as a loading control. B. Representative Western blot demonstrating reduced caspase-3 cleavage following angiogenin treatment in SH-SY5Y cells transfected with either empty vector or HA-tagged DN-Akt using Amaxa nucleoporation. Twenty-four hours after transfection, cells were pretreated with angiogenin (100 nM). MPP+ (0.75 mM) was then applied with fresh angiogenin for an additional 24 hours prior to collection. HA-tagged DN-Akt expression was comparable among all DN-Akt-transfected conditions. Actin was used as loading control. C. Densitometric quantification of caspase-3 cleavage bands normalized to actin. Results reflect three independent experiments. *p<0.05, ***p<0.001 (One-way ANOVA with Tukey's post-hoc test). Error bars reflect SEM.</p

K40I fails to activate Akt phosphorylation but protects against MPP+.

<p>A. Immunostaining of SH-SY5Y cells treated with exogenous angiogenin (100 nM) or K40I (100 nM) for 12 hours. Control is immunostaining of SH-SY5Y cells not treated with recombinant angiogenin and reflects low endogenous angiogenin levels in SH-SY5Y cells. Both wildtype and K40I recombinant angiogenin are taken up by SH-SY5Y cells. As the intensity in staining is stronger for wildtype angiogenin compared to K40I, either less K40I is taken up compared to wildtype or the antibody used better recognizes the wildtype form. Scale bar = 25 µm. B. Quantification of angiogenin immunostaining of SH-SY5Y cells treated with exogenous wildtype or K40I angiogenin. 10 random fields per well were imaged by confocal microscopy, and intensity was quantified using Image J. Average of 30 cells was analyzed per field, and two separate immunostaining runs were quantified. ***p<0.001 (students' t-test). C. Western blot demonstrating increased Akt phosphorylation after 30 sec treatment with recombinant angiogenin (100 nM) but not with the K40I angiogenin mutant (100 nM) in SH-SY5Y cells. Actin was used as the loading control. D. LDH assay of SH-SY5Y cells treated with MPP+ with and without wildtype or K40I angiogenin. Cells were pretreated with wildtype or K40I angiogenin (50–200 nM) for 12 hrs and then co-incubated with MPP+ (0.75 µM) and fresh angiogenin or K40I for another twenty-four hours. Angiogenin demonstrated a protective effect that was not significantly different from K40I. n = 4–6, **p<0.01, ***p<0.001 compared to MPP+ treatment (One-way ANOVA with Tukey's post-hoc test). There is no significant difference between wildtype and K40I-treated conditions. Error bars reflect SEM.</p