Downregulation of tyrosine hydroxylase phenotype after AAV injection above substantia nigra: Caution in experimental models of Parkinson's disease

Abstract

Adeno-associated virus (AAV) vector-mediated delivery of human α-synuclein (α-syn) gene in rat substantia nigra (SN) results in increased expression of α-syn protein in the SN and striatum which can progressively degenerate dopaminergic neurons. Therefore, this model is thought to recapitulate the neurodegeneration in Parkinson's disease. Here, using AAV to deliver α-syn above the SN in male and female rats resulted in clear expression of human α-syn in the SN and striatum. The protein was associated with moderate behavioral deficits and some loss of tyrosine hydroxylase (TH) in the nigrostriatal areas. However, the immunohistochemistry results were highly variable and showed little to no correlation with behavior and the amount of α-syn present. Expression of green fluorescent protein (GFP) was used as a control to monitor gene delivery and expression efficacy. AAV-GFP resulted in a similar or greater TH loss compared to AAV-α-syn and therefore an additional vector that does not express a protein was tested. Vectors with double-floxed inverse open reading frame (DIO ORF) encoding fluorescent proteins that generate RNA that is not translated also resulted in TH downregulation in the SN but showed no significant behavioral deficits. These results demonstrate that although expression of wild-type human α-syn can cause neurodegeneration, the variability and lack of correlation with outcome measures are drawbacks with the model. Furthermore, design and control selection should be considered carefully because of conflicting conclusions due to AAV downregulation of TH, and we recommend caution with having highly regulated TH as the only marker for the dopamine system.

Keywords: DIO; GFP; Parkinson’s disease; adeno-associated virus; alpha-synuclein; dopamine; substantia nigra.

Figure 1.. Demonstrating different injection paradigms tested in these experiments and resulting outcomes.

A. Green fluorescent protein (GFP) expression in substantia nigra and striatum using AAV5-eGFP injected above the substantia nigra into one site for male Wistar rats used in the experiments. B. GFP expression in the substantia nigra and striatum using AAV5-eGFP injected above the substantia nigra into two sites for male Wistar rats used in the experiments. C. Human α-synuclein expression in substantia nigra and striatum using AAV-α-synuclein (mixture of AAV2 and AAV5-α-synuclein) injected above the substantia nigra into two sites using male Wistar rats. 40x images of substantia nigra for the injected and uninjected sides are also shown. ±SD is used for the error bar of graphs. D. Number of rearings for Wistar rats (n=79) and Sprague Dawley (n=79) rats used in the experiments. E. Representative photomicrograph of a male Wistar rat injected with AAV5-eGFP showing tyrosine hydroxylase (TH) immunostaining in the striatum and substantia nigra. F. Total rotations (ipsilateral - contralateral) on the amphetamine-induced rotational assay (120 mins) for AAV5-eGFP (n=5) injected rats and AAV5-α-synuclein (n=7) injected male Wistar rats.

Figure 2.. There is a wide variation in nigrostriatal TH for AAV-α-synuclein injected rats that does not correlate with outcome measures.

A. Relative tyrosine hydroxylase (TH) optical density (percentage of the uninjected side) for AAV-α-synuclein (mixture of AAV2-α-synuclein and AAV5-α-synuclein, referred to as AAV-α-synuclein) injected rats 8 weeks after injection (n=43). B. Photomicrographs of two example brains showing the striatum and substantia nigra injected with AAV-α-synuclein and one with 6-OHDA. C. Correlation between relative TH optical density and α-synuclein integrated optical density for AAV-α-synuclein injected rats. D. TH+ cells in the substantia nigra as a percentage of the uninjected side for AAV-α-syn rats (n=25). E. Cylinder test results for AAV-α-synuclein injected rats at 8 weeks after injection (n=43). Results are presented as ipsilateral, contralateral, and both paw touches as a percentage of total paw touches. F. Correlation between ipsilateral paw use on the cylinder test and TH optical density for AAV-α-synuclein injected rats. G. Correlation between ipsilateral paw use on the cylinder test and TH optical density for 6-OHDA injected rats. H. Correlation between ipsilateral paw use on the cylinder test and dopamine content (as a percentage of the uninjected side) for AAV-α-synuclein injected rats. I. Correlation between total rotations (ipsilateral - contralateral rotations) on the amphetamine-induced rotation assay (120 minutes) and TH optical density for AAV-α-synuclein injected rats. ±SD is used for the error bar of graphs. All results are from 8 weeks post-AAV injection, female Sprague-Dawley rats used.

Figure 3.. Comparing AAV-α-synuclein, AAV-eGFP and AAV-DIO-mCherry in a single experiment.

A. Cylinder test results for AAV2 and AAV5 mixture vector injected rats at 8 weeks after injection. Results are represented as ipsilateral, contralateral, and both paw touches as a percentage of total paw touches. Both paw touches represents the number of times the animal placed both paws on the cylinder wall simultaneously. N=10/group. B. Total rotations (ipsilateral - contralateral) on the amphetamine-induced rotation assay (120 minutes) for all three AAV vector injected rats at 8 weeks. C. Relative tyrosine hydroxylase (TH) optical density (percentage of the uninjected side) for AAV vector injected rats at 8 weeks post-injection. D. Dopamine concentration (ng/g) for AAV vector injected rats at 8 weeks after AAV injection. Results are shown as both the total amount of dopamine measured for both the injected and uninjected side. N=5/group. E) Dopamine turnover for the injected side striatum. Calculated by summing the concentrations (ng/g) of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and dividing that by the concentration of dopamine (DA). F) Percentage of TH+ cells in the substantia nigra pars compacta (percent of the uninjected side) for AAV vector injected rats at 8 weeks, analyzed using MATLAB. G) Estimated number of TH+ cells in the substantia nigra pars compacta at 8 weeks post-AAV injection, analyzed using Aiforia^®. H) Representative images of TH staining in the substantia nigra at 8 weeks post-injection. I) Percentage of Nissl+ cells in the substantia nigra pars compacta area (percent of the control side) for AAV vector rats at 8 weeks. J) Representative image of Nissl-stained substantia nigra at 8 weeks post-injection. ±SD is used for the error bar of graphs. Female Sprague-Dawley rats used.

Figure 4.. Testing AAV1-DIO-iRFP and AAV1-lacZ-DIO as additional negative controls.

A. Schematic figure of AAV1-lacZ-DIO (pAAV-lacZ-DIO-iRFP). B. Expression patterns of AAV1-DIO-iRFP and AAV1-lacZ-DIO vectors in cells without Cre (Cre-) and with Cre (Cre+). C. Cylinder test results for AAV1-DIO-iRFP and AAV1-lacZ-DIO injected rats 8 weeks after injection (n=10/group). Results are represented as ipsilateral, contralateral, and both paw touches as a percentage of total paw touches. D. Total rotations (ipsilateral - contralateral) on the amphetamine-induced rotation assay (120 minutes) for AAV1-DIO-iRFP and AAV1-lacZ-DIO rats 8 weeks after injection. ±SD is used for the error bar of graphs. E. Relative tyrosine hydroxylase (TH) optical density (percentage of the uninjected side) for AAV1-DIO-iRFP and AAV1-lacZ-DIO injected rats 8 weeks after injection. F. Correlation between ipsilateral paw use and TH optical density for AAV1-DIO-iRFP injected rats. G. Correlation between ipsilateral paw use and TH optical density for AAV1-lacZ-DIO injected rats. Female Sprague-Dawley rats used.