Progranulin gene delivery protects dopaminergic neurons in a mouse model of Parkinson's disease

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by tremor, rigidity and akinesia/bradykinesia resulting from the progressive loss of nigrostriatal dopaminergic neurons. To date, only symptomatic treatment is available for PD patients, with no effective means of slowing or stopping the progression of the disease. Progranulin (PGRN) is a 593 amino acid multifunction protein that is widely distributed throughout the CNS, localized primarily in neurons and microglia. PGRN has been demonstrated to be a potent regulator of neuroinflammation and also acts as an autocrine neurotrophic factor, important for long-term neuronal survival. Thus, enhancing PGRN expression may strengthen the cells resistance to disease. In the present study, we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of PGRN gene delivery as a therapy for the prevention or treatment of PD. Viral vector delivery of the PGRN gene was an effective means of elevating PGRN expression in nigrostriatal neurons. When PGRN expression was elevated in the SNC, nigrostriatal neurons were protected from MPTP toxicity in mice, along with a preservation of striatal dopamine content and turnover. Further, protection of nigrostriatal neurons by PGRN gene therapy was accompanied by reductions in markers of MPTP-induced inflammation and apoptosis as well as a complete preservation of locomotor function. We conclude that PGRN gene therapy may have beneficial effects in the treatment of PD.

Figure 1. Immunohistochemical analysis of enhanced PGRN expression in the SN_C following ND-602.

A) Fluorescent photomicrographs depicting GFP expression in TH⁺ neurons of the ipsilateral SN_C 4 weeks following unilateral intranigral ND-602 infusion. B) Fluorescent photomicrographs depicting PGRN immunolabeling in the ipsilateral SN_C 4 weeks following unilateral intranigral infusion of either ND-602 or GFP control. C) In GFP controls, MPTP produced a significant reduction in PGRN immunolabeling within the substantia nigra of both hemispheres. Unilateral intranigral delivery of ND-602 significantly elevated PGRN immunolabeling in the ipsilateral SN_C of both vehicle- and MPTP-treated animals. No effect of ND-602 was seen in the contralateral nigra. Each bar represents the mean (±S.E.M., n = 5–9) optical density (arbitrary units) averaged across 4 coronal sections through the SN_C. D) The density of PGRN immunolabeling within TH⁺ neurons of the ipsilateral SN_C was significantly elevated in those animals treated with ND-602. Inset fluorescent photomicrograph depicts PGRN immunolabeling in dopaminergic neurons of the ipsilateral SN_C following ND-602 treatment. Each bar represents the mean (±S.E.M., n = 5–9) optical density (arbitrary units) measured in 10 cells in each of 4 coronal sections through the ipsilateral SN_C. **sig. diff. from vehicle control, p<0.001. ++sig. diff. from GFP control, p<0.001.

Figure 2. Locomotor deficits in MPTP-intoxicated mice treated with ND-602.

A) Following MPTP intoxication, animals displayed significantly reduced locomotor activity, as assessed by total distance traveled in one hour. However, MPTP failed to reduce locomotor activity in those animals treated with ND-602. Each bar represents the mean (±S.E.M., n = 5–6) distance traveled (cm) in one hour. B) The average velocity of locomotion was significantly reduced following MPTP intoxication. However, in those animals treated with viral ND-602, no such reduction was observed. Velocity in those animals was significantly higher than in their GFP-treated counterparts. Each bar represents the mean (±S.E.M., n = 5–6) velocity (cm/s) observed over a one hour period. C) When traversing the horizontal ladder, MPTP-treated animals displayed reduced locomotor coordination as evidenced by significantly more foot slips. However, in those animals treated with ND-602, no such elevation in foot slips was observed. Locomotor coordination in those animals was significantly better than in their GFP-treated counterparts. Each bar represents the mean (±S.E.M., n = 5–6) number of foot slips recorded. D) Following MPTP intoxication, animals took significantly longer to traverse a horizontal ladder. However, those animals treated with ND-602 showed no increase in travel time and were significantly faster than their GFP-treated counterparts. Each bar represents the mean (±S.E.M., n = 5–6) time(s) to traverse a horizontal ladder. **sig. diff. from vehicle control, p<0.001; *p<0.05. ++sig. diff. from GFP control, p<0.001; +p<0.05.

Figure 3. Nigrostriatal integrity of MPTP-intoxicated mice treated with ND-602.

A) Representative fluorescent photomicrographs depicting TH immunolabeling in the ipsilateral SN_C following MPTP intoxication in ND-602- and GFP-treated animals. Unbiased stereological counts of B) TH⁺ and C) Nissl⁺ cells in the SN_C were significantly reduced following MPTP intoxication. However, no significant cell loss was observed ipsilateral to viral vector delivery of PGRN by ND-602. Significantly more TH⁺ and Nissl⁺ cells were noted ipsilateral to ND-602 infusion relative to GFP controls. Each bar represents the mean (±S.E.M., n = 5–9) number of TH or Nissl immunopositive cells counted in the SN_C. D) Representative fluorescent photomicrographs depicting DAT immunolabeling in the ipsilateral striatum following MPTP intoxication in ND-602- and GFP-treated animals. E) MPTP intoxication resulted in a significant loss of DAT immunolabeling in the striatum. In those animals treated with ND-602, the loss of striatal DAT was significantly attenuated in the ipsilateral hemisphere. Each bar represents the mean (±S.E.M., n = 5–9) optical density (arbitrary units) measured in three striatal regions across 4 coronal sections through the striatum. **sig. diff. from vehicle control, p<0.001; *p<0.05. ++sig. diff. from GFP control, p<0.001.

Figure 4. Striatal dopamine and turnover in MPTP-intoxicated mice following ND-602.

A) Levels of striatal dopamine were significantly reduced following MPTP intoxication. However, in those animals treated with viral vector delivery of PGRN by ND-602, no decline in dopamine was observed ipsilateral to vector delivery. Levels were reduced, however, in the contralateral, untreated, hemisphere. The dopamine metabolites, B) DOPAC and C) HVA, were similarly reduced by MPTP, with no reductions observed ipsilateral to ND-602 delivery. D) Striatal dopamine turnover was significantly elevated by MPTP intoxication. However, no change in turnover was observed ipsilateral to ND-602 treatment. Each bar represents the mean (±S.E.M., n = 5–8) µg of dopamine, DOPAC or HVA detected per gram of wet tissue weight. For dopamine turnover, the calculation, DOPAC + HVA/dopamine was applied. **sig. diff. from vehicle control, p<0.001. ++sig. diff. from GFP control, p<0.001.

Figure 5. Nigrostriatal inflammation in MPTP-intoxicated mice treated with ND-602.

Representative fluorescent photomicrographs of isolectin (ILB4) labeling in the A) SN and C) striatum two weeks following the last MPTP injection. MPTP intoxication resulted in a significant elevation in the number of activated microglia in both the B) SN_C and D) striatum. In both regions, this activation was significantly reduced in those animals treated with ND-602. Each bar represents the mean (±S.E.M., n = 5–6) number of ILB4-positive cells estimated per mm³. **sig. diff. from vehicle control, p<0.001; *p<0.05. ++sig. diff. from GFP control, p<0.001.