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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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Loss of TH-positive innervation in the striatum after expression of a fibrillar αSynuclein variant (WT) or the prefibrillar variant TP. Representative images illustrating the axonal fiber innervation in the striatum 14 weeks after AAV-mediated gene transfer into the SN. Overview of coronal sections showing transgene expression of EGFP (a), WT αSynuclein (b), and TP αSynuclein (c). Nigrostriatal fiber terminals in the striatum were filled with the transgenic protein. Immunohistochemical staining for TH (d–f) revealed that in contrast to the AAV-EGFP-injected animals (d), the expression of αSynuclein led to the appearance of degenerative changes and a loss of striatal fibers, which was more pronounced for WT (e). Scale bar 500 μm. g–o Higher magnification images covering the dorsal, medial and ventral area of a representative striatal section of an intact non-transduced control side (g–i), WT-transduced (j–l), and TP-transduced (m–o) brain. TH-immunostained sections showing the extent of loss of striatal innervation. Remaining fibers in the WT αSynuclein-treated animals were sparse. Scale bar 100 μm. p Quantification of fiber density revealed a clear reduction in TH-positive striatal innervation in the αSynuclein transduced animals at 14 weeks after injection (significant difference from the EGFP-transduced control group at p < 0.001). The difference between the WT and TP variant was also significant (p = 0.048), whereas the difference between A30P and TP was not
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Fig6: Loss of TH-positive innervation in the striatum after expression of a fibrillar αSynuclein variant (WT) or the prefibrillar variant TP. Representative images illustrating the axonal fiber innervation in the striatum 14 weeks after AAV-mediated gene transfer into the SN. Overview of coronal sections showing transgene expression of EGFP (a), WT αSynuclein (b), and TP αSynuclein (c). Nigrostriatal fiber terminals in the striatum were filled with the transgenic protein. Immunohistochemical staining for TH (d–f) revealed that in contrast to the AAV-EGFP-injected animals (d), the expression of αSynuclein led to the appearance of degenerative changes and a loss of striatal fibers, which was more pronounced for WT (e). Scale bar 500 μm. g–o Higher magnification images covering the dorsal, medial and ventral area of a representative striatal section of an intact non-transduced control side (g–i), WT-transduced (j–l), and TP-transduced (m–o) brain. TH-immunostained sections showing the extent of loss of striatal innervation. Remaining fibers in the WT αSynuclein-treated animals were sparse. Scale bar 100 μm. p Quantification of fiber density revealed a clear reduction in TH-positive striatal innervation in the αSynuclein transduced animals at 14 weeks after injection (significant difference from the EGFP-transduced control group at p < 0.001). The difference between the WT and TP variant was also significant (p = 0.048), whereas the difference between A30P and TP was not

Mentions: Nigral αSynuclein overexpression resulted in αSynuclein immunoreactivity in striatal projections of nigral DA neurons. The measured density of DA projections in the striatum correlated with the extent of nigral neurodegeneration, i.e., αSynuclein WT overexpression induced significantly more fiber loss as compared to TP and A30P overexpression (Fig. 6). αSynuclein immunoreactive striatal fibers displayed large numbers of dystrophic swollen profiles, which colocalized with the DA marker TH and morphologically differed considerably between WT, TP and A30P αSynuclein-expressing specimen (Fig. 7). Following WT overexpression, extensive beading of neuronal processes could be detected (Fig. 7a, d). In contrast, TP overexpression resulted in the appearance of large axonal retraction bulb-like structures, while the axon proximal to the retraction bulb showed fewer varicosities (Fig. 7b, e). A30P overexpression, which in nigral DA neurons resulted in fewer PK-resistant aggregates than WT but more such aggregates than TP, displayed an intermediate axonopathy. It induced fewer small, bead-like varicosities but more large retraction bulb-like profiles than WT αSynuclein (Fig. 7c, f). Thus, it appeared that aggregating αSynuclein WT resulted in extensive beading of neuronal processes, while less- and non-aggregating αSynuclein variants demonstrated a preference for generating large axonal retraction bulb-like structures. These results suggest that fibrillar and prefibrillar variants of αSynuclein induce different modes of axonopathy in nigral DA neurons.Fig. 6


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)

Loss of TH-positive innervation in the striatum after expression of a fibrillar αSynuclein variant (WT) or the prefibrillar variant TP. Representative images illustrating the axonal fiber innervation in the striatum 14 weeks after AAV-mediated gene transfer into the SN. Overview of coronal sections showing transgene expression of EGFP (a), WT αSynuclein (b), and TP αSynuclein (c). Nigrostriatal fiber terminals in the striatum were filled with the transgenic protein. Immunohistochemical staining for TH (d–f) revealed that in contrast to the AAV-EGFP-injected animals (d), the expression of αSynuclein led to the appearance of degenerative changes and a loss of striatal fibers, which was more pronounced for WT (e). Scale bar 500 μm. g–o Higher magnification images covering the dorsal, medial and ventral area of a representative striatal section of an intact non-transduced control side (g–i), WT-transduced (j–l), and TP-transduced (m–o) brain. TH-immunostained sections showing the extent of loss of striatal innervation. Remaining fibers in the WT αSynuclein-treated animals were sparse. Scale bar 100 μm. p Quantification of fiber density revealed a clear reduction in TH-positive striatal innervation in the αSynuclein transduced animals at 14 weeks after injection (significant difference from the EGFP-transduced control group at p < 0.001). The difference between the WT and TP variant was also significant (p = 0.048), whereas the difference between A30P and TP was not
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Fig6: Loss of TH-positive innervation in the striatum after expression of a fibrillar αSynuclein variant (WT) or the prefibrillar variant TP. Representative images illustrating the axonal fiber innervation in the striatum 14 weeks after AAV-mediated gene transfer into the SN. Overview of coronal sections showing transgene expression of EGFP (a), WT αSynuclein (b), and TP αSynuclein (c). Nigrostriatal fiber terminals in the striatum were filled with the transgenic protein. Immunohistochemical staining for TH (d–f) revealed that in contrast to the AAV-EGFP-injected animals (d), the expression of αSynuclein led to the appearance of degenerative changes and a loss of striatal fibers, which was more pronounced for WT (e). Scale bar 500 μm. g–o Higher magnification images covering the dorsal, medial and ventral area of a representative striatal section of an intact non-transduced control side (g–i), WT-transduced (j–l), and TP-transduced (m–o) brain. TH-immunostained sections showing the extent of loss of striatal innervation. Remaining fibers in the WT αSynuclein-treated animals were sparse. Scale bar 100 μm. p Quantification of fiber density revealed a clear reduction in TH-positive striatal innervation in the αSynuclein transduced animals at 14 weeks after injection (significant difference from the EGFP-transduced control group at p < 0.001). The difference between the WT and TP variant was also significant (p = 0.048), whereas the difference between A30P and TP was not
Mentions: Nigral αSynuclein overexpression resulted in αSynuclein immunoreactivity in striatal projections of nigral DA neurons. The measured density of DA projections in the striatum correlated with the extent of nigral neurodegeneration, i.e., αSynuclein WT overexpression induced significantly more fiber loss as compared to TP and A30P overexpression (Fig. 6). αSynuclein immunoreactive striatal fibers displayed large numbers of dystrophic swollen profiles, which colocalized with the DA marker TH and morphologically differed considerably between WT, TP and A30P αSynuclein-expressing specimen (Fig. 7). Following WT overexpression, extensive beading of neuronal processes could be detected (Fig. 7a, d). In contrast, TP overexpression resulted in the appearance of large axonal retraction bulb-like structures, while the axon proximal to the retraction bulb showed fewer varicosities (Fig. 7b, e). A30P overexpression, which in nigral DA neurons resulted in fewer PK-resistant aggregates than WT but more such aggregates than TP, displayed an intermediate axonopathy. It induced fewer small, bead-like varicosities but more large retraction bulb-like profiles than WT αSynuclein (Fig. 7c, f). Thus, it appeared that aggregating αSynuclein WT resulted in extensive beading of neuronal processes, while less- and non-aggregating αSynuclein variants demonstrated a preference for generating large axonal retraction bulb-like structures. These results suggest that fibrillar and prefibrillar variants of αSynuclein induce different modes of axonopathy in nigral DA neurons.Fig. 6

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