Silencing of tuberin enhances photoreceptor survival and function in a preclinical model of retinitis pigmentosa (an american ophthalmological society thesis)

Abstract

Purpose: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis.

Methods: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin's role in regulating survival.

Results: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration.

Conclusions: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP.

FIGURE 1

PDE6B alleles. Most PDE6β missense mutations in retinitis pigmentosa occur in the catalytic domain. PDE6β contains two GAF domains (green) and a catalytic domain (yellow). Black arrowheads indicate the position of missense mutations in retinitis pigmentosa. Mouse Pde6b^rd1 nonsense and Pde6b^H620Q missense mutations are indicated on top.

FIGURE 2

Low levels of PDE6 result in high concentrations of cGMP. PDE6 is a heterotetrameric enzyme consisting of a catalytic α-subunit, a catalytic β-subunit, and two inhibitory γ-subunits in the rod photoreceptors. PDE6 is highly important in the phototransduction cascade, as it is the primary regulator of cytoplasmic cGMP concentration in the photoreceptor cells. In the dark, PDE6 is in an inactive form, with the γ inhibitory subunits bound (PDE6αβγ), and cGMP levels within the photoreceptor cell outer segment are high (several micromolars). The main molecular components of phototransduction are known; however, the links between abnormally high cGMP and intracellular Ca²⁺ and cell death are unclear.

FIGURE 3

AGC kinases are activated before the onset of degeneration. Pde6b^H620Q/Pde6b^H620Q retinal lysates exhibit increased PKG, PKA, PKC, and PKB activity compared to wild-type (WT) lysates at P14. WT and H620Q P14 and P18 retinal lysates were tested in different in vitro enzyme immunometric kinase assays for PKA, PKC, PKG, and PKB activity. Differences between WT and H620Q P14 or P18, respectively, are calculated with the Student t test P values at P14 (WT vs H620Q): PKA, P<.001, n=3; PKC, P<.001, n=3; PKG, P<.001, n=3; and PKB, P<.05, n=3. Levels of significance: *significant, P<.05; **highly significant, P< .01; ***very highly significant, P<.001.

FIGURE 4

Kinase substrate array and kinases in the mTOR survival pathway. Representative kinase substrates (oligopeptides) were immobilized on arrays and hybridized with outer segment (OS) extract labeled with ³³P-ATP to detect kinase activities in P14 (left panel) and P18 Pde6b^H620Q/Pde6b^H620Q OS (right panel). Lysates were analyzed using 1,164 different synthetic peptide substrates and 12 controls; substrates were spotted twice., Dots represent phosphorylated peptide substrates (based on incorporation of ³³P). Circled dots represent preferentially phosphorylated substrates (left panel, P14; right panel, P18).

FIGURE 5

Increased activity of the mTOR survival pathway at the onset of degeneration. Left, Schematized version of the mTOR survival pathway when stress is absent and during the stress response when the pathway is activated. Right, Phosphosubstrate immunostaining in Pde6b^H620Q/Pde6b^H620Q mutant P14 (before degeneration) and P18 (onset of degeneration) retinas: PDK1 p(S241), left column; tuberin p(S939), middle column; RPS6 p(S236), right column. Yellow and red arrowheads indicate regions of increased staining using anti-PDK1 pS241, anti-tuberin pS939, and anti-RPS6 pS236 antibodies. OS, photoreceptor outer segments; INL, inner nuclear layer.

FIGURE 6

Monotherapy with shRNA_tuberin can decrease tuberin levels in a dose-dependent manner. Top, Schematic representation of the shRNA lentiviral vector. This self-inactivating (SIN) vector consists of a 5′ long terminal repeat (LTR), a packaging signal ψ, a tRNA primer binding site, a lentiviral reverse response element (RRE), the shRNA driven by U6 promoter, and a 3′LTR. This pLKO.1 viral vector also contains a central polypurine tract/DNA flap (cPPT) and a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The Simian virus 40 (SV40) polyadenylation signal is located at the 3′ end of the cDNA. Arrows indicate the direction of transcription. AmpR, ampicillin resistance gene; ori, origin of replication. Bottom, Immunoblot showing that phosphotuberin levels are inversely proportional to the amount of tuberin shRNA. Phosphorylated residue detected by the phosphotuberin antibody (phospho-Ser939).

FIGURE 7

Monotherapy with shRNA_tuberin improved photoreceptor survival and electroretinogram (ERG) function in 6-week-old mouse Pde6b^H620Q/ Pde6b^H620Q retinas. Upper left, Survival of photoreceptors. Retinal sections were stained to assess changes in the outer nuclear layer (ONL), which contains photoreceptor nuclei. Treated retinas showed a 26% average increase in ONL thickness compared to control eyes, suggesting that tuberin knockdown slows or prevents photoreceptor loss. Vertical yellow bars represent the ONL thickness. GC, ganglion cells; INL, inner nuclear layer. Upper right, ONL enhancement in mice (n=4) 8 weeks post tuberin shRNA transduction as indicated by the solid bar. Lower panel, Representative maximum ERG responses (in μV) of shRNA-transduced eyes (blue) compared to saline-injected control eyes (red). ERGs were performed on both eyes (injected and control) simultaneously.

FIGURE 8

Improved photoreceptor survival of PDE mutant photoreceptors after tripartite AAV8 transduction. Top, Schematic representation of the AAV2/8(Y733F)-Rho::Pde6α tripartite vector. pZac2.1 plasmid vector displaying the Pde6α complementary DNA (cDNA) fragment driven by 1.1 kb of the murine rhodopsin promoter. The Simian virus 40 (SV40) polyadenylation signal is located at the 3’ end of the cDNA. The H1 promoter drives expression of the tuberin_shRNA. The U6 promoter drives expression of the Cnga1_shRNA. Arrows indicate the direction of transcription. 5′- and 3′-ITR, inverted terminal repeats of AAV; AmpR, ampicillin resistance gene; F1 ori, origin of replication. Left, Hematoxylin-eosin stained retinal section of a 1-month-old C57BL/6J control. Middle, Pde6a^D670G/Pde6a^D670G eye transduced by Rho::Pde6a-shRNA_tuberin-shRNA_Cnga1 at 1 month. Right, Untreated Pde6a^D670G/Pde6a^D670 at 1 month. Vertical yellow bars represent the thickness of the outer nuclear layer. Images were obtained at 40× magnification at the same location of the retina, 0.3 mm distal from the optic nerve head.