Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Bandopadhyay, Rina

doi:10.3791/53415

Cited by 29 publications

(28 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Then, by increasing the detergent strength of the extraction buffer (2% SDS) to solubilize the residual pellet obtained from the first extraction, it is possible to isolate and detect the monomeric and oligomeric αSyn forms. Finally, the insoluble pellet from both the 1% Triton X-100 and 2% SDS extraction contains high molecular weight (HMW) oligomers and aggregates of αSyn [ 33 , 34 , 35 , 36 ]. Likewise, in the 1% Triton X-100 soluble fraction, we observed two bands of about 14 and 16 kDa, corresponding to endogenous monomeric αSyn (Syn-EN) and ectopic monomeric αSyn (Syn-OE).…”

Section: Resultsmentioning

confidence: 99%

The Down-Regulation of Clusterin Expression Enhances the αSynuclein Aggregation Process

Lenzi

Ramazzina

Russo

et al. 2020

IJMS

View full text Add to dashboard Cite

Parkinson’s Disease (PD) is a progressive neurodegenerative disease characterized by the presence of proteinaceous aggregates of αSynuclein (αSyn) in the dopaminergic neurons. Chaperones are key components of the proteostasis network that are able to counteract αSyn’s aggregation, as well as its toxic effects. Clusterin (CLU), a molecular chaperone, was consistently found to interfere with Aβ aggregation in Alzheimer’s Disease (AD). However, its role in PD pathogenesis has yet to be extensively investigated. In this study, we assessed the involvement of CLU in the αSyn aggregation process by using SH-SY5Y cells stably overexpressing αSyn (SH-Syn). First, we showed that αSyn overexpression caused a strong increase in CLU expression without affecting levels of Hsp27, Hsp70, and Hsp90, which are the chaperones widely recognized to counteract αSyn burden. Then, we demonstrated that αSyn aggregation, induced by proteasome inhibition, determines a strong increase of CLU in insoluble aggregates. Remarkably, we revealed that CLU down-regulation results in an increase of αSyn aggregates in SH-Syn without significantly affecting cell viability and the Unfolded Protein Response (UPR). Furthermore, we demonstrated the direct molecular interaction between CLU and αSyn via a co-immunoprecipitation (co-IP) assay. All together, these findings provide incontrovertible evidence that CLU is an important player in the response orchestrated by the cell to cope with αSyn burden.

show abstract

Section: Resultsmentioning

confidence: 99%

The Down-Regulation of Clusterin Expression Enhances the αSynuclein Aggregation Process

Lenzi

Ramazzina

Russo

et al. 2020

IJMS

View full text Add to dashboard Cite

show abstract

“…For a-syn immunoblotting, brain homogenates were processed in order to collect the TBS-and SDS-soluble fraction of a-syn as described. 54 Briefly, brains were homogenated with TBS (pH 7.4), clarified from non-homogenated residue and submitted to 100,000 Â g centrifugation at 4 C for 1 hr. The resulting supernatant represents the TBS-soluble fraction.…”

Section: Immunoblottingmentioning

confidence: 99%

AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy

et al. 2017

View full text Add to dashboard Cite

The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.

show abstract

“…The supernatants containing RIPA-soluble a-Syn aggregates were used for optical assessment of Venus fluorescent signal intensities. The pellets containing RIPA-insoluble a-Syn aggregates were dissolved in urea/thiourea buffer, sonicated, and subjected to FRA, a method commonly used to visualize and quantify insoluble protein aggregates (43)(44)(45)(46). This approach allowed us to detect changes in abundance of soluble and insoluble a-Syn aggregates in flies.…”

Section: Quantification Of Soluble and Insoluble A-syn Aggregates Fromentioning

confidence: 99%

Monitoring α‐synuclein multimerization in vivo

Prasad¹,

Wasser²,

Hans

et al. 2018

FASEB j.

View full text Add to dashboard Cite

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α‐synuclein (α‐Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α‐Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α‐Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α‐Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α‐Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α‐Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α‐Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α‐Syn at serine 129 enhances the accumulation and toxicity of α‐Syn. Altogether, our study establishes a novel model to study α‐Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.—Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α‐synuclein multimerization in vivo. FASEB J. 33, 2116–2131 (2019). http://www.fasebj.org

show abstract

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Cited by 29 publications

References 34 publications

The Down-Regulation of Clusterin Expression Enhances the αSynuclein Aggregation Process

The Down-Regulation of Clusterin Expression Enhances the αSynuclein Aggregation Process

AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy

Monitoring α‐synuclein multimerization in vivo

Contact Info

Product

Resources

About