Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction

Abstract

Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus with recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 hr before irradiation, and compared with a control adenovirus. The control-treated glands have ∼50% reduction in salivary flow 60 days post-irradiation, whereas neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation, but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

Keywords: adenovirus; gene therapy; irradiation; neurturin; salivary gland; xerostomia.

Figure 1

An Adenovirus Expressing Neurturin Improves Ex Vivo SMG Growth after Irradiation Similar to Recombinant NRTN (A) E13.5 SMGs were treated for 24 hr with increasing doses of AdNRTN, then IR (5 Gy) and cultured for a further 72 hr. The endbuds were counted and normalized to baseline. AdNRTN 10⁴ vp/c was used for subsequent experiments. N = 12 SMG/group. Error bars represent SEM. ANOVA with post hoc Dunnett’s test, **p < 0.001 when compared with IR control without AdNRTN treatment. (B) Bright-field images of E14 SMGs at T0 and again at 72 hr showing the increase in size after treatment with AdNRTN at 10⁴ vp/c and NRTN before IR. SMGs were treated for 24 hr with a control vector (AdC), AdNRTN (10⁴ vp/c), NRTN (1 ng/mL), then IR (5 Gy) and cultured for a further 72 hr. Scale bars: 200 μm. (C) The number of endbuds from (B) were counted and normalized to the number of endbuds at time 0. N = 4. Error bars represent the SEM. ANOVA with post hoc Dunnett’s test, *p < 0.01 when compared with IR control. (D) There is an increase in Nrtn expression with AdNRTN treatment, no change in Ret, and increased Tubb3 expression with IR. Gene expression relative to non-IR control and normalized to ribosomal protein S29 (Rps29) shows N = 3. Error bars represent the SEM. ANOVA compared with non-IR control, *p < 0.05. vp/c, viral particles per cell.

Figure 2

AdNRTN Treatment Increases Parasympathetic Innervation of Ex Vivo SMG Culture after Radiation (A) Whole-mount image of a SMGs cultured for 72 hr after IR. The SMGs were immunostaining for nerves (Tubb3), NRTN, and perlecan, which stain the basement membrane. The area in the white box is shown in (B). (B) Higher magnification of terminal endbuds and nerves. Individual endbud cells with increased NRTN expression are apparent with AdNRTN treatment (white arrows). Scale bars: 250 μm (A); 50 μm (B).

Figure 3

AdNRTN Treatment Reduces Apoptosis of Parasympathetic Ganglia in Irradiated E14 SMG (A) SMG immunostained to visualize the PSG cell bodies (Tubb3, red), apoptotic cells (cleaved caspase-3, green), and nuclei (Hoechst, blue). White arrows show cells in the IR group that express both caspase-3 and Tubb3. Scale bar: 20 μm. (B) There is a reduction in apoptosis 48 hr after IR with AdNRTN and recombinant NRTN. Scale bar: 20 μm. N = 3. Error bars represent SEM. ANOVA compared with IR, *p < 0.05.

Figure 4

AdNRTN Treatment of SMGs before IR Increases Salivary Flow Rates Similar to the Non-IR Group Pilocarpine-stimulated whole saliva (in μL/15 min) was measured 60 days after IR. There was ∼60% reduction in saliva flow with IR compared with the non-IR group, and the control vector (AdC) did not increase saliva after IR. Non-IR SMGs were also treated with AdNRTN (10¹⁰ vp/g), and the AdNRTN (10⁸, 10⁹, and 10¹⁰ vp/g) treatment of IR SMGs improved saliva volume similar to the non-IR group. N = 8 mice, except non-IR AdNRTN vp/g, which had N = 4. Error bars represent the SEM. ANOVA with post hoc Dunnett’s test, ***p < 0.001 compared with non-IR.

Figure 5

Treatment of SMGs In Vivo with Increasing Doses of AdNRTN Increases Nrtn Transcripts within the Gland and Improves Some Neuronal Markers after IR (A) qPCR analysis of Nrtn transcripts in SMGs 60 days after IR and treatment with increasing doses of AdNRTN. (B) Additional qPCR of genes related to NRTN signaling in IR SMGs treated with AdNRTN 10⁹ compared with IR treatment alone. The expression of the NRTN receptors Gfra2 and Ret kinase, neuronal markers Tubb3, and the parasympathetic markers Chat and Vacht and the sympathetic markers Th and Adra2b. Gene expression was normalized to non-IR SMGs and the ribosomal housekeeping gene Rps29 (data not shown). qPCRs were done in triplicate, and N = 11–12 SMGs analyzed. ^#Vacht expression was not detected in 5 of 11 SMGs. Error bars represent the SEM. ANOVA with post hoc Dunnett’s test compared with non-IR, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

AdNRTN Treatment of Adult SMG In Vivo Also Increases Staining of the Neuronal Markers VAChT and TH (A) H&E staining of adult SMG appears similar among all treatment groups after IR of mouse SMGs. (B) Sections were also immunostained for nerves (Tubb3), VAChT, a marker of parasympathetic function, TH, a sympathetic marker, and nuclei (Hoechst). (C) Staining was quantified (five sections/gland) and normalized to nuclear staining. N = 3–4 SMGs/group. Error bars represent SEM. ANOVA compared with non-IR, *p < 0.05; **p < 0.01. Scale bars: 20 μm.