Protection of nigral dopaminergic neurons by AAV1 transduction with Rheb(S16H) against neurotoxic inflammation in vivo

Abstract

We recently reported that adeno-associated virus serotype 1 (AAV1) transduction of murine nigral dopaminergic (DA) neurons with constitutively active ras homolog enriched in brain with a mutation of serine to histidine at position 16 [Rheb(S16H)] induced the production of neurotrophic factors, resulting in neuroprotective effects on the nigrostriatal DA system in animal models of Parkinson's disease (PD). To further investigate whether AAV1-Rheb(S16H) transduction has neuroprotective potential against neurotoxic inflammation, which is known to be a potential event related to PD pathogenesis, we examined the effects of Rheb(S16H) expression in nigral DA neurons under a neurotoxic inflammatory environment induced by the endogenous microglial activator prothrombin kringle-2 (pKr-2). Our observations showed that Rheb(S16H) transduction played a role in the neuroprotection of the nigrostriatal DA system against pKr-2-induced neurotoxic inflammation, even though there were similar levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β), in the AAV1-Rheb(S16H)-treated substantia nigra (SN) compared to the SN treated with pKr-2 alone; the neuroprotective effects may be mediated by the activation of neurotrophic signaling pathways following Rheb(S16H) transduction of nigral DA neurons. We conclude that AAV1-Rheb(S16H) transduction of neuronal populations to activate the production of neurotrophic factors and intracellular neurotrophic signaling pathways may offer promise for protecting adult neurons from extracellular neurotoxic inflammation.

Figure 1

Transduction of nigral DA neurons with AAV1-GFP and AAV1-Rheb(S16H) in normal adult mice. (a) The expression of GFP and FLAG (brown reaction product) in the SN at 3 weeks after AAV1-GFP and AAV1-Rheb(S16H) administration for each viral injection. No expression of FLAG was observed in the non-injected control side (CON). The area inside the dotted lines is the substantia nigra pars compacta. Scale bar, 200 μm. (b) Double-immunofluorescence staining for TH (red) and GFP (green) and TH and FLAG (green) in the SN. Arrow heads indicate increased neuronal size induced by Rheb(S16H) expression in the nigral DA neurons. Scale bar, 20 μm. (c) Double-immunofluorescence staining for GFAP/Iba1 (red) and GFP (green), and GFAP/Iba1 and FLAG (green) in the SN. Scale bar, 20 μm.

Figure 2

Rheb(S16H) protects the nigrostriatal DA pathway from pKr-2-induced neurotoxicity in vivo. (a) Representative coronal sections of the SN and striatum following TH immunostaining. CON, contralateral control side. Scale bars, 200 and 500 μm in the SN and striatum, respectively. The small panels show higher magnifications of the SN. Scale bar, 50 μm. (b) Quantification of TH-positive neurons at 7 days after pKr-2 treatment. EXP, ipsilateral injected side. The histogram quantitatively shows TH-positive neurons in the counting area of the ipsilateral SN as a percentage of those in the contralateral controls. *P<0.001 vs contralateral controls, ^#P<0.01 vs pKr-2 alone (one-way ANOVA and Tukey’s post hoc analysis; n=4, each experimental group). All values are expressed as the mean±standard error of the mean (s.e.m.). (c) Quantitative determination of striatal TH immunostaining. The histogram shows the optical density of TH-positive fibers at 7 days after pKr-2 treatment. *P<0.001 vs contralateral controls, ^#P<0.001 vs pKr-2 alone (one-way ANOVA and Tukey’s post hoc analysis; n=4, each experimental group). All values are expressed as the mean±s.e.m. (d, e) Western blot analysis of TH expression at 7 days after pKr-2 treatment in the SN and striatum. The density of TH bands was normalized to the density of the β-actin band for each sample. *P<0.01 and ^**P<0.05 vs contralateral controls, ^#P<0.01 and ^##P<0.05 vs pKr-2 alone (one-way ANOVA and Tukey’s post hoc analysis; n=3, each experimental group). All values are expressed as the mean±s.e.m. (f) Representative horizontal sections of the medial forebrain bundle (MFB) following TH immunofluorescence staining. Scale bar, 10 μm. (g) Quantification of TH-positive axons at 7 days after injection of pKr-2. The histogram quantitatively shows TH-positive axons in the counting area of the ipsilateral MFB as a percentage of those in the contralateral controls. *P<0.001 and ^**P<0.01 vs contralateral controls, ^#P<0.001 vs pKr-2 alone (one-way ANOVA and Tukey’s post hoc analysis; n=4, between groups). All values are expressed as the mean±standard error of the mean (s.e.m.).

Figure 3

The functional preservation of Rheb(S16H) against the pKr-2-induced neurotoxic events in vivo. (a–c) The amounts of striatal dopamine and its metabolites, such as DOPAC and HVA, were measured by HPLC, and the level was quantitatively expressed as a percentage of the level in contralateral controls. *P<0.001 and ^**P<0.01 vs contralateral controls, ^#P<0.001 vs pKr-2 alone (one-way ANOVA and Tukey’s post hoc analysis; n=5, each experimental group). All values are expressed as the mean±standard error of the mean (s.e.m.). (d) Locomotor activity was measured with the rota-rod test. *P<0.05 vs control mice, ^#P<0.05 vs pKr-2 alone (t-test analysis; n=8, each experimental group). All values are expressed as the mean±s.e.m.

Figure 4

Increase in neuroinflammatory responses induced by pKr-2 treatment in the SN. (a) Double-immunofluorescence staining for TNF-α and IL-1β within Iba1-positive microglia in the SN at 1 day after pKr-2 treatment. CON, contralateral control side. Scale bars, 20 μm. (b, c) Western blot analysis of Iba1, TNF-α and IL-1β at 1 day after pKr-2 treatment in the SN. The density of Iba1, TNF-α, and IL-1β bands was normalized to the density of the β-actin band for each sample. *P<0.01 and ^**P<0.05 vs contralateral controls (one-way ANOVA and Tukey’s post hoc analysis; n=4, each experimental group). All values are expressed as the mean±s.e.m.

Figure 5

Preservation of mTORC1 activation and neurotrophic factors induced by Rheb(S16H) in neuroinflammatory conditions. (a) Double-immunofluorescence staining for TH (red) and p-4E-BP1 (green), TH and GDNF (green), and TH and BDNF (green) in the SN at 3 weeks after AAV1-Rheb(S16H) administration. CON, contralateral control side. Scale bars, 20 μm. (b) Western blot analysis of 4E-BP1, p-4E-BP1, GDNF and BDNF in the SN at 1 day after pKr-2 treatment. The density of p-4E-BP1, GDNF, and BDNF bands was normalized to the density of the β-actin band for each sample. *P<0.001 and ^**P<0.01 vs contralateral controls, pKr-2 alone and pKr-2 in the presence of GFP (one-way ANOVA and Tukey’s post hoc analysis; n=4, each experimental group). All values are expressed as the mean±s.e.m. (c) Western blot analysis of 4E-BP1, p-4E-BP1, GDNF, and BDNF in the SN at 7 days after pKr-2 treatment. The density of p-4E-BP1, GDNF, and BDNF bands was normalized to the density of the β-actin band for each sample. *P<0.001, ^**P<0.01 and ^***P<0.05 vs contralateral controls, ^##P<0.01 and ^###P<0.05 vs pKr-2 alone (one-way ANOVA and Tukey’s post hoc analysis; n=4, each experimental group). All values are expressed as the mean±s.e.m.

Figure 6

Upregulation of Rheb(S16H)-induced GDNF and BDNF contributes to protection of the nigrostriatal DA projection against pKr-2-induced neurotoxicity in vivo. (a) Representative coronal sections of the SN and striatum following TH immunostaining. Scale bars, 200 and 500 μm in the SN and striatum, respectively. The small panels show higher magnifications of the SN. Scale bar, 50 μm. Quantification of TH-positive neurons at 7 days after injection of pKr-2 and neutralizing antibodies against GDNF, BDNF, and Mix (BDNF+GDNF) in the presence of Rheb(S16H) and quantitative determination of striatal TH immunostaining. (b) TH-positive neurons in the counting area of the ipsilateral SN expressed as a percentage of those in the contralateral controls. CON, contralateral control side; EXP, ipsilateral injected side. *P<0.001 vs contralateral controls, ^##P<0.01 vs pKr-2 alone in the presence of Rheb(S16H), ^$P<0.01 vs pKr-2 and GDNF neutralizing antibody in the presence of Rheb(S16H) (one-way ANOVA and Tukey’s post hoc analysis; n=5, each experimental group). (c) Optical density of TH-positive fibers at 7 days after injection of pKr-2 and neutralizing antibodies against GDNF, BDNF and Mix in the presence of Rheb(S16H). ^**P<0.01 vs contralateral controls, ^##P<0.01 vs pKr-2 alone in the presence of Rheb(S16H) (one-way ANOVA and Tukey’s post hoc analysis; n=5, each experimental group). All values are expressed as the mean±s.e.m.