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Aggregation of αSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons.

Taschenberger G, Garrido M, Tereshchenko Y, Bähr M, Zweckstetter M, Kügler S - Acta Neuropathol. (2011)

Bottom Line: To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors.Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation.These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.

View Article: PubMed Central - PubMed

Affiliation: Center of Molecular Physiology of the Brain, Department of Neurology, University Medicine Göttingen, Germany. gtasche@gwdg.de

ABSTRACT
Fibrillar αSynuclein is the major constituent of Lewy bodies and Lewy neurites, the protein deposits characteristic for Parkinson's disease (PD). Multiplications of the αSynuclein gene, as well as point mutations cause familial PD. However, the exact role of αSynuclein in neurodegeneration remains uncertain. Recent research in invertebrates has suggested that oligomeric rather than fibrillizing αSynuclein mediates neurotoxicity. To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors. The formation of proteinase K-resistant αSynuclein aggregates was correlated to the loss of nigral dopaminergic (DA) neurons and striatal fibers. Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation. However, only the αSynuclein variants capable of forming fibrils (WT/A30P), but not the oligomeric αSynuclein species induced a sustained progressive loss of adult nigral DA neurons. These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.

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PK-resistant aggregates of αSynuclein and ThioS-positive structures represent different entities in adult rat DA neurons. To further characterize PK-resistant aggregates, digested sections were immunofluorescently stained with a αSynuclein antibody, followed by a ThioS stain. We detected abundant ThioS-positive structures in DA neurons independent from αSynuclein expression, i.e., also in DA neurons of the contralateral nigra. However, these structures were in no case immunoreactive for aggregated αSynuclein, although we exploited several different αSynuclein antibodies (Syn211, aa121–135 of human αSynuclein; LB509, aa115–122, generated against Lewy bodies as immunogen; clone 42, aa 91–99 of rat and human αSynuclein; and 5C2, directed against the NAC domain). The figure shows ThioS/αSynuclein (Syn211 antibody, confocal detection) double staining after PK digestion (10 min, 55°C, 10 μg/ml) in SNpc 4 weeks after virus injection. DA neurons in the SN showed several small ThioS-positive inclusions (d–f) and many αSynuclein-positive aggregates (a), however, both structures did not colocalize (g). As shown before, neurons infected with WT αSynuclein (a) showed considerably more αSynuclein aggregates than neurons expressing A56P (b) or TP αSynuclein (c)
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Fig3: PK-resistant aggregates of αSynuclein and ThioS-positive structures represent different entities in adult rat DA neurons. To further characterize PK-resistant aggregates, digested sections were immunofluorescently stained with a αSynuclein antibody, followed by a ThioS stain. We detected abundant ThioS-positive structures in DA neurons independent from αSynuclein expression, i.e., also in DA neurons of the contralateral nigra. However, these structures were in no case immunoreactive for aggregated αSynuclein, although we exploited several different αSynuclein antibodies (Syn211, aa121–135 of human αSynuclein; LB509, aa115–122, generated against Lewy bodies as immunogen; clone 42, aa 91–99 of rat and human αSynuclein; and 5C2, directed against the NAC domain). The figure shows ThioS/αSynuclein (Syn211 antibody, confocal detection) double staining after PK digestion (10 min, 55°C, 10 μg/ml) in SNpc 4 weeks after virus injection. DA neurons in the SN showed several small ThioS-positive inclusions (d–f) and many αSynuclein-positive aggregates (a), however, both structures did not colocalize (g). As shown before, neurons infected with WT αSynuclein (a) showed considerably more αSynuclein aggregates than neurons expressing A56P (b) or TP αSynuclein (c)

Mentions: We then assessed the formation of aggregates in midbrain tissue sections derived from adult rats by quantifying the amount of PK-resistant αSynuclein. These animals were injected into the SNpc with AAV vectors expressing WT, A30P, A56P or TP αSynuclein 4 weeks before tissue sampling. Control rats received identical injections of a AAV vector encoding EGFP. Tissue samples were subjected to the maximum possible dose and incubation time of PK, which was just below the threshold of completely digesting the tissue specimen. Untreated sections adjacent to the treated ones served as controls. This method has been shown to be the most sensitive procedure to detect αSynuclein aggregates in tissue specimen including post-mortem patient’s brain [4, 32]. After immunohistochemistry with the human αSynuclein specific antibody syn211, PK-resistant aggregates were identified as small intensely stained inclusions of about 0.5 μm in diameter in cell bodies of DA neurons. The neuropil of untreated slices showed relatively large, focal bead-like swellings immunoreactive for αSynuclein. With PK treatment, we detected smaller profiles but with similar spatial arrangement (Fig. 2a–o). PK-resistant aggregates in the neuropil were quantified by calculating the fraction of αSynuclein-positive material using the ImageJ software. PK-resistant aggregates in neuronal cell bodies were quantified by counting of individual aggregates. Without PK treatment, αSynuclein immunoreactivity in the neuropil was equal for all αSynuclein variants 4 weeks post transduction. Following PK treatment, WT and A30P expressing specimen demonstrated significantly higher levels of αSynuclein immunoreactivity as compared to A56P and TP (Fig. 2p). The number of PK-resistant intraneuronal αSynuclein aggregates was significantly higher in WT- and A30P-expressing neurons (99.1 ± 12 and 53.6 ± 20.6, respectively) compared to A56P- and TP-expressing neurons (5.9 ± 3.3 and 3.4 ± 3.8, respectively) (Fig. 2q). These PK-resistant aggregates did not co-stain with ThioS (Fig. 3). No aggregates were detectable in neurons of undigested sections, in PK-digested sections after EGFP overexpression or in the contralateral nigra (not shown). In contrast to the biochemical PK-resistance assay conducted with cell lysates from cultured non-DA cortical neurons, we found that expression of WT αSynuclein in adult rat DA neurons produced significantly more aggregates than expression of A30P, suggesting that the duration of the expression, the age of the neurons or the DA environment may differentially impact on aggregate formation.Fig. 2


Aggregation of αSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons.

Taschenberger G, Garrido M, Tereshchenko Y, Bähr M, Zweckstetter M, Kügler S - Acta Neuropathol. (2011)

PK-resistant aggregates of αSynuclein and ThioS-positive structures represent different entities in adult rat DA neurons. To further characterize PK-resistant aggregates, digested sections were immunofluorescently stained with a αSynuclein antibody, followed by a ThioS stain. We detected abundant ThioS-positive structures in DA neurons independent from αSynuclein expression, i.e., also in DA neurons of the contralateral nigra. However, these structures were in no case immunoreactive for aggregated αSynuclein, although we exploited several different αSynuclein antibodies (Syn211, aa121–135 of human αSynuclein; LB509, aa115–122, generated against Lewy bodies as immunogen; clone 42, aa 91–99 of rat and human αSynuclein; and 5C2, directed against the NAC domain). The figure shows ThioS/αSynuclein (Syn211 antibody, confocal detection) double staining after PK digestion (10 min, 55°C, 10 μg/ml) in SNpc 4 weeks after virus injection. DA neurons in the SN showed several small ThioS-positive inclusions (d–f) and many αSynuclein-positive aggregates (a), however, both structures did not colocalize (g). As shown before, neurons infected with WT αSynuclein (a) showed considerably more αSynuclein aggregates than neurons expressing A56P (b) or TP αSynuclein (c)
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Related In: Results  -  Collection

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Fig3: PK-resistant aggregates of αSynuclein and ThioS-positive structures represent different entities in adult rat DA neurons. To further characterize PK-resistant aggregates, digested sections were immunofluorescently stained with a αSynuclein antibody, followed by a ThioS stain. We detected abundant ThioS-positive structures in DA neurons independent from αSynuclein expression, i.e., also in DA neurons of the contralateral nigra. However, these structures were in no case immunoreactive for aggregated αSynuclein, although we exploited several different αSynuclein antibodies (Syn211, aa121–135 of human αSynuclein; LB509, aa115–122, generated against Lewy bodies as immunogen; clone 42, aa 91–99 of rat and human αSynuclein; and 5C2, directed against the NAC domain). The figure shows ThioS/αSynuclein (Syn211 antibody, confocal detection) double staining after PK digestion (10 min, 55°C, 10 μg/ml) in SNpc 4 weeks after virus injection. DA neurons in the SN showed several small ThioS-positive inclusions (d–f) and many αSynuclein-positive aggregates (a), however, both structures did not colocalize (g). As shown before, neurons infected with WT αSynuclein (a) showed considerably more αSynuclein aggregates than neurons expressing A56P (b) or TP αSynuclein (c)
Mentions: We then assessed the formation of aggregates in midbrain tissue sections derived from adult rats by quantifying the amount of PK-resistant αSynuclein. These animals were injected into the SNpc with AAV vectors expressing WT, A30P, A56P or TP αSynuclein 4 weeks before tissue sampling. Control rats received identical injections of a AAV vector encoding EGFP. Tissue samples were subjected to the maximum possible dose and incubation time of PK, which was just below the threshold of completely digesting the tissue specimen. Untreated sections adjacent to the treated ones served as controls. This method has been shown to be the most sensitive procedure to detect αSynuclein aggregates in tissue specimen including post-mortem patient’s brain [4, 32]. After immunohistochemistry with the human αSynuclein specific antibody syn211, PK-resistant aggregates were identified as small intensely stained inclusions of about 0.5 μm in diameter in cell bodies of DA neurons. The neuropil of untreated slices showed relatively large, focal bead-like swellings immunoreactive for αSynuclein. With PK treatment, we detected smaller profiles but with similar spatial arrangement (Fig. 2a–o). PK-resistant aggregates in the neuropil were quantified by calculating the fraction of αSynuclein-positive material using the ImageJ software. PK-resistant aggregates in neuronal cell bodies were quantified by counting of individual aggregates. Without PK treatment, αSynuclein immunoreactivity in the neuropil was equal for all αSynuclein variants 4 weeks post transduction. Following PK treatment, WT and A30P expressing specimen demonstrated significantly higher levels of αSynuclein immunoreactivity as compared to A56P and TP (Fig. 2p). The number of PK-resistant intraneuronal αSynuclein aggregates was significantly higher in WT- and A30P-expressing neurons (99.1 ± 12 and 53.6 ± 20.6, respectively) compared to A56P- and TP-expressing neurons (5.9 ± 3.3 and 3.4 ± 3.8, respectively) (Fig. 2q). These PK-resistant aggregates did not co-stain with ThioS (Fig. 3). No aggregates were detectable in neurons of undigested sections, in PK-digested sections after EGFP overexpression or in the contralateral nigra (not shown). In contrast to the biochemical PK-resistance assay conducted with cell lysates from cultured non-DA cortical neurons, we found that expression of WT αSynuclein in adult rat DA neurons produced significantly more aggregates than expression of A30P, suggesting that the duration of the expression, the age of the neurons or the DA environment may differentially impact on aggregate formation.Fig. 2

Bottom Line: To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors.Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation.These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.

View Article: PubMed Central - PubMed

Affiliation: Center of Molecular Physiology of the Brain, Department of Neurology, University Medicine Göttingen, Germany. gtasche@gwdg.de

ABSTRACT
Fibrillar αSynuclein is the major constituent of Lewy bodies and Lewy neurites, the protein deposits characteristic for Parkinson's disease (PD). Multiplications of the αSynuclein gene, as well as point mutations cause familial PD. However, the exact role of αSynuclein in neurodegeneration remains uncertain. Recent research in invertebrates has suggested that oligomeric rather than fibrillizing αSynuclein mediates neurotoxicity. To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors. The formation of proteinase K-resistant αSynuclein aggregates was correlated to the loss of nigral dopaminergic (DA) neurons and striatal fibers. Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation. However, only the αSynuclein variants capable of forming fibrils (WT/A30P), but not the oligomeric αSynuclein species induced a sustained progressive loss of adult nigral DA neurons. These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.

Show MeSH
Related in: MedlinePlus