A30P mutant α-synuclein impairs autophagic flux by inactivating JNK signaling to enhance ZKSCAN3 activity in midbrain dopaminergic neurons

Lei, Zhinian; Cao, Guangliang; Wei, Gang

doi:10.1038/s41419-019-1364-0

Cited by 42 publications

(35 citation statements)

References 43 publications

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“…Although several a-syn PDassociated mutants have been shown to promote a-syn oligomerization and/or fibril formation in vitro (de Oliveira & Silva, 2019;Ruf et al, 2019), in cells (M. B. Fares et al, 2014;Khalaf et al, 2014;Lei, et al, 2019) and in vivo (Mbefo et al, 2015;Paumier et al, 2013), Lázaro et al reported identical Venus BiFC signals from WT a-syn and the PD-linked mutants A30P, E46K, H50Q, G51D and A53T (Lázaro et al, 2014). Similar observation were made by Outeiro et al using a GFP-BiFC cellular assay (Outeiro et al, 2008).…”

Section: Discussionsupporting

confidence: 58%

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Frey

AlOkda

Lashuel

2020

Preprint

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Abbreviations:a-syn: a-synuclein; a-syn-V: a-synuclein-full length Venus; Vn-a-syn: n-terminal fragment of Venus fused to the amino-terminus of a-syn; a-syn-Vc: c-terminal fragment of Venus fused to the carboxy-terminus of a-syn; BiFC: bimolecular fluorescent complementation; PD: Parkinson´s disease; SEC: size exclusion chromatography Abstract Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed, including mouse, rat and Drosophila models, based on the assumption that an increase in the fluorescence signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that 1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; 2) provide a structural characterization of the oligomers and aggregates formed by the BiFC fragments; or 3) investigate the extent to which the oligomers by the fluorescent complex resemble oligomers that form on the pathway to α-syn fibrillization. To address this knowledge gap, we first analyzed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 cells using multiple approaches, including size exclusion chromatography, semiquantitative Western blot analysis, in-cell crosslinking, immunocytochemistry and sedimentation assays. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that 1) the two BiFC fragments are not expressed at the same level since C-terminal-Venus fused to α-syn (a-syn-Vc) was present in very low abundance; 2) the fragment with Nterminal-Venus fused to α-syn (Vn-a-syn) exhibited a high propensity to form oligomers and higher-order aggregates; 3) the expression of either or both fragments does not result in the formation of α-syn fibrils, cellular inclusions or the accumulation of pS129 immunoreactive αsyn aggregates. Furthermore, our results suggest that only a small fraction of Vn-a-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of a-syn-Vc in Vn-a-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that oligomerization, aggregation and cell-tocell transmission assays and model systems based on a-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

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Section: Discussionsupporting

confidence: 58%

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Frey

AlOkda

Lashuel

2020

Preprint

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“…In primary VM neurons transfected with A30P aSyn AAV vectors, aggregated p62 increased in a manner similar to that of the E46K mutant aSyn (Yan et al, 2014;Lei et al, 2019). A30P did not alter p62 mRNA levels, and 3-MA treatment increased p62 in both the mutant and empty AAV vector cells, supporting the conclusion that the insoluble aggregates were due to A30P-mediated autophagy inhibition (Lei et al, 2019). Like E46K, A30P also reduced LC3-II synthesis (but not degradation), suggesting a similar mechanism of impaired autophagosome formation (Yan et al, 2014;Lei et al, 2019).…”

Section: A30pmentioning

confidence: 82%

Current Evidence for a Bidirectional Loop Between the Lysosome and Alpha-Synuclein Proteoforms

Wildburger

Hartke

Schidlitzki

et al. 2020

Front. Cell Dev. Biol.

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Cumulative evidence collected in recent decades suggests that lysosomal dysfunction contributes to neurodegenerative diseases, especially if amyloid proteins are involved. Among these, alpha-synuclein (aSyn) that progressively accumulates and aggregates in Lewy bodies is undisputedly a main culprit in Parkinson disease (PD) pathogenesis. Lysosomal dysfunction is evident in brains of PD patients, and mutations in lysosomal enzymes are a major risk factor of PD. At first glance, the role of protein-degrading lysosomes in a disease with pathological protein accumulation seems obvious and should guide the development of straightforward and rational therapeutic targets. However, our review demonstrates that the story is more complicated for aSyn. The protein can possess diverse posttranslational modifications, aggregate formations, and truncations, all of which contribute to a growing known set of proteoforms. These interfere directly or indirectly with lysosome function, reducing their own degradation, and thereby accelerating the protein aggregation and disease process. Conversely, unbalanced lysosomal enzymatic processes can produce truncated aSyn proteoforms that may be more toxic and prone to aggregation. This highlights the possibility of enhancing lysosomal function as a treatment for PD, if it can be confirmed that this approach effectively reduces harmful aSyn proteoforms and does not produce novel, toxic proteoforms.

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“…During the symptomatic stage of PD, when most dopaminergic neurons in the midbrain are already lost, the TFEB-mediated transcription of Beclin-1, CTSD, and LAMP1 is decreased compared to the pre-symptomatic stage [151]. Moreover, the expression of human A30P α-synuclein increased the nuclear translocation of the transcriptional repressor zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3), which regulates the transcription of LC3 and p62 [152]. As expected, the pathological features of PD, including neurodegeneration in the midbrain, motor dysfunction, and α-synuclein accumulation, were improved by TFEB overexpression [153,154].…”

Section: Parkinson's Disease (Pd)mentioning

confidence: 99%

Autophagy in Neurodegenerative Diseases: A Hunter for Aggregates

Park

Kang

Lee

2020

IJMS

138

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Cells have developed elaborate quality-control mechanisms for proteins and organelles to maintain cellular homeostasis. Such quality-control mechanisms are maintained by conformational folding via molecular chaperones and by degradation through the ubiquitin-proteasome or autophagy-lysosome system. Accumulating evidence suggests that impaired autophagy contributes to the accumulation of intracellular inclusion bodies consisting of misfolded proteins, which is a hallmark of most neurodegenerative diseases. In addition, genetic mutations in core autophagy-related genes have been reported to be linked to neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Conversely, the pathogenic proteins, such as amyloid β and α-synuclein, are detrimental to the autophagy pathway. Here, we review the recent advances in understanding the relationship between autophagic defects and the pathogenesis of neurodegenerative diseases and suggest autophagy induction as a promising strategy for the treatment of these conditions.

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A30P mutant α-synuclein impairs autophagic flux by inactivating JNK signaling to enhance ZKSCAN3 activity in midbrain dopaminergic neurons

Cited by 42 publications

References 43 publications

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Current Evidence for a Bidirectional Loop Between the Lysosome and Alpha-Synuclein Proteoforms

Autophagy in Neurodegenerative Diseases: A Hunter for Aggregates

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