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Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.

Koprich JB, Johnston TH, Reyes MG, Sun X, Brotchie JM - Mol Neurodegener (2010)

Bottom Line: The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01).At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Toronto Western Research Institute, Toronto Western Hospital, University Health Network, 399 Bathurst Street, Toronto, ON, M5T 2S8, Canada. jkoprich@uhnres.utoronto.ca.

ABSTRACT

Background: The pathological hallmarks of Parkinson's disease (PD) include the presence of alpha-synuclein (α-syn) rich Lewy bodies and neurites and the loss of dopaminergic (DA) neurons of the substantia nigra (SN). Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV), or green fluorescent protein (GFP), the latter to control for the possibility that high levels of protein in themselves might contribute to damage.

Results: We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01). At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.

Conclusions: In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model. The dynamics of the evolution of the pathology however, provide advantages over current models with respect to providing an initial screen to assess efficacy of novel treatments that might prevent/reverse α-syn aggregation.

No MeSH data available.


Related in: MedlinePlus

Alpha-synuclein deposits in nigral neurons are insoluble aggregates. Midbrain sections from rats injected with AAV1/2 A53T α-syn were treated with proteinase K (B, PK+) or the buffer used to dissolve PK (A, PK-) and subsequently immunostained for human α-syn to determine its solubility. Panel A (PK-) shows that α-syn expression fills the substantia nigra expressing in both nigral neurons and neurites. In panel B, following PK digestion, many neurons maintain expression of α-syn aggregates (insoluble α-syn), while the neurites appear to have been largely cleared (soluble α-syn). Arrows point to examples of α-syn filled neurons. Scale bar in panel A is 200 μm.
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Figure 2: Alpha-synuclein deposits in nigral neurons are insoluble aggregates. Midbrain sections from rats injected with AAV1/2 A53T α-syn were treated with proteinase K (B, PK+) or the buffer used to dissolve PK (A, PK-) and subsequently immunostained for human α-syn to determine its solubility. Panel A (PK-) shows that α-syn expression fills the substantia nigra expressing in both nigral neurons and neurites. In panel B, following PK digestion, many neurons maintain expression of α-syn aggregates (insoluble α-syn), while the neurites appear to have been largely cleared (soluble α-syn). Arrows point to examples of α-syn filled neurons. Scale bar in panel A is 200 μm.

Mentions: A pathological feature of PD is that a significant proportion of α-syn inclusions in nigral neurons are considered to be aggregated (insoluble inclusions) [5,7,28]. In order to evaluate the solubility of the α-syn deposits seen in this model we conducted a proteinase K (PK) digestion on midbrain sections from AAV1/2 A53T α-syn rats. We found that the majority α-syn inclusions in our model were resistant to PK digestion and could thus be considered insoluble aggregates (Figure 2).


Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.

Koprich JB, Johnston TH, Reyes MG, Sun X, Brotchie JM - Mol Neurodegener (2010)

Alpha-synuclein deposits in nigral neurons are insoluble aggregates. Midbrain sections from rats injected with AAV1/2 A53T α-syn were treated with proteinase K (B, PK+) or the buffer used to dissolve PK (A, PK-) and subsequently immunostained for human α-syn to determine its solubility. Panel A (PK-) shows that α-syn expression fills the substantia nigra expressing in both nigral neurons and neurites. In panel B, following PK digestion, many neurons maintain expression of α-syn aggregates (insoluble α-syn), while the neurites appear to have been largely cleared (soluble α-syn). Arrows point to examples of α-syn filled neurons. Scale bar in panel A is 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984491&req=5

Figure 2: Alpha-synuclein deposits in nigral neurons are insoluble aggregates. Midbrain sections from rats injected with AAV1/2 A53T α-syn were treated with proteinase K (B, PK+) or the buffer used to dissolve PK (A, PK-) and subsequently immunostained for human α-syn to determine its solubility. Panel A (PK-) shows that α-syn expression fills the substantia nigra expressing in both nigral neurons and neurites. In panel B, following PK digestion, many neurons maintain expression of α-syn aggregates (insoluble α-syn), while the neurites appear to have been largely cleared (soluble α-syn). Arrows point to examples of α-syn filled neurons. Scale bar in panel A is 200 μm.
Mentions: A pathological feature of PD is that a significant proportion of α-syn inclusions in nigral neurons are considered to be aggregated (insoluble inclusions) [5,7,28]. In order to evaluate the solubility of the α-syn deposits seen in this model we conducted a proteinase K (PK) digestion on midbrain sections from AAV1/2 A53T α-syn rats. We found that the majority α-syn inclusions in our model were resistant to PK digestion and could thus be considered insoluble aggregates (Figure 2).

Bottom Line: The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01).At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Toronto Western Research Institute, Toronto Western Hospital, University Health Network, 399 Bathurst Street, Toronto, ON, M5T 2S8, Canada. jkoprich@uhnres.utoronto.ca.

ABSTRACT

Background: The pathological hallmarks of Parkinson's disease (PD) include the presence of alpha-synuclein (α-syn) rich Lewy bodies and neurites and the loss of dopaminergic (DA) neurons of the substantia nigra (SN). Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV), or green fluorescent protein (GFP), the latter to control for the possibility that high levels of protein in themselves might contribute to damage.

Results: We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01). At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.

Conclusions: In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model. The dynamics of the evolution of the pathology however, provide advantages over current models with respect to providing an initial screen to assess efficacy of novel treatments that might prevent/reverse α-syn aggregation.

No MeSH data available.


Related in: MedlinePlus