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Research article
Björn Falkenburger
Dresden University of Technology: Technische Universitat Dresden
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
During the pathogenesis of Parkinson’s disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo.
Methods
The disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or its dominant-negative T22N mutant as a control. The cellular integrity was quantified by morphological and biochemical analyses.
Results
In primary neurons, WT Rab7 rescued the αSyn -induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn -induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain.
Conclusion
Rab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.
Keywords
alpha-synuclein, Rab7, protein aggregation, Parkinson’s disease, DNA damage, oxidative stress, autophagy, neuroprotection
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- SzegoRab7aSynFigureSI1.jpg
A) αSyn-induced activation of caspase-3 in the presence of 100 µM NMDA (n=3, mean ± SD, ANOVA followed by Bonferroni posthoc test; **p=0,00421). (B) Quantification of the amount of human αSyn (normalized to β-tubulin signal) in the Triton X-100 soluble fraction of lysates from three independent preparations (n=3, mean ± SD, ANOVA followed by Bonferroni posthoc test). Representative western blot is shown on Figure 1D. (C) Uncropped membranes from Figure 1D showing human aSyn, rodent aSyn, PSD95, HA-tag and β-tubulin signal.
- SzegoRab7aSynFigureSI2.jpg
(A) Representative images showing HA-tag staining in the substantia nigra, contralateral to the rAAV injection. Images showing HA-tag staining in the ipsilateral hemisphere are presented on Figure 2C. Scale bar: 200 µm (B) Linear regression of the numbers of dopaminergic neurons in the substantia nigra (from Figure 2D, E) and densities of dopaminergic axon terminals in the striatum (from Figure 3B, C; p=0,8546; r2=0,001917). (C) Representative images showing colocalization of phospho-αSyn (red) and TH (green) in the striatum of αSyn + DN Rab7 (left panel) or WT Rab7 (right panel) rAAV-injected animals. Scale bar: 20 µm (D) Quantification of total area fraction of the striatal DAT signal (n=10, mean ± SD, t-test; p=0,0534). Representative images showing DAT (green) and VMAT2 (red) staining are on Figure 3K, thresholded images used for total DAT signal measurement are on Figure 3K2 and K2.
- SzegoRab7aSynFigureSI3.jpg
(A) Representative images showing the astroglia marker GFAP (green) and the microglia marker Iba1 (red) in the contralateral (left panel) and ipsilateral (right panel) striatum of αSyn + DN Rab7 (upper panel) and αSyn + WT Rab7 (lower panel) rAAV-injected animals. Scale bar: 100 µm (B) Quantification of the area fraction of GFAP signal (normalized to the contralateral hemisphere; n=10, mean ± SD, t-test; *p=0,020). (C) Quantification of the area fraction of Iba1 signal (normalized to the contralateral hemisphere, n=10, mean ± SD, t-test). (D) Linear regression of the GFAP ratio (from supplemental Figure 3B) and the density of phospho-αSyn-positive structures in the striatum (from Figure 3E, p=0,0119, r2=0,3028). (E) Linear regression of the GFAP ratio (from supplemental Figure 3B) and the density of dopaminergic terminals in the striatum (from Figure 3C; p=0,0663, r2=0,1752)..
- SzegoRab7aSynFigureSI4.jpg
(A) Representative, stiched images showing tubulin-stained axons grown in microfluidic chambers (axonal compartment), 5 days after mechanical lesion. Primary cultures were infected with (GFP (left) or αSyn (right)) and with (GFP (upper), DN Rab7 (middle) or WT Rab7 (lower) rAAV. Scale bar: 100 µm (B) Quantification of the length of tubulin-stained axons in the axonal compartment of microfluidic chambers (n=3, mean ± SD, ANOVA followed by Bonferroni posthoc test; p=0,0468).
- SzegoRab7aSynSupplementalTableS1.docx
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This is a preprint. It has not completed peer review.
Research article
Björn Falkenburger
Dresden University of Technology: Technische Universitat Dresden
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 11 Mar, 2021
No community comments so far
Subject Areas
Neurology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
During the pathogenesis of Parkinson’s disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo.
Methods
The disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or its dominant-negative T22N mutant as a control. The cellular integrity was quantified by morphological and biochemical analyses.
Results
In primary neurons, WT Rab7 rescued the αSyn -induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn -induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain.
Conclusion
Rab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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