Eyal A, Szargel R, Avraham E, Liani E, Haskin J, Rott R, Engelender S. Synphilin-1A: an aggregation-prone isoform of synphilin-1 that causes neuronal death and is present in aggregates from alpha-synucleinopathy patients. Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5917-22. PubMed.
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This very interesting report by Simone Engelender and colleagues identifies a previously unrecognized isoform of synphilin-1, which is the product of alternative splicing of the same gene. Designated as synphilin-1A, this new protein has a different initiation codon, lacks exons 3 and 4 leading to a frame shift, and inserts a new exon 9A in the C-terminus, forming a smaller protein of 75 kDa. The tendency of this new protein to cause cytotoxicity, which is attenuated by inclusion formation, and its ability to recruit other proteins implicated in α-synucleinopathies, provides yet another dimension in the role of proteins and inclusions in the genesis of these neurodegenerative disorders. This elegant study also adds to the growing body of published reports suggesting that inclusions, including those formed by synphilin-1 and α-synuclein in our hands (Tanaka et al., 2004), can be cytoprotective. As is often the case with the initial stages of a novel finding, this report raises several questions and begs for more investigations.
Synphilin-1A interacts with both the longer synphilin-1 and with α-synuclein using overexpression systems in transfected cells. Whether these interactions also occur in vivo at physiologic concentrations of these proteins remains to be demonstrated. The authors conclude that synphilin-1A recruits synphilin-1 into the insoluble fraction of cells based on studies in transfected HEK293 cells. However, no synphilin-1 is found in the insoluble fraction of DLB brains, even though synphilin-1A is detected. Lack of synphilin-1 in insoluble DLB brain extracts is particularly intriguing in view of prior data indicating that synphilin-1 is a component of Lewy bodies. Further, the presence of the 75 kDa synphilin-1A band in the triton X-100-insoluble fraction may not necessarily indicate aggregation. In addition, the authors find that synphilin-1A increases the concentration of its longer isoform synphilin-1 in the presence of the E3 ubiquitin ligase SIAH-1. It would be of interest to find out the mechanism of synphilin-1 stabilization and whether competition for SIAH-1 plays a role. Another curious observation from the data in this publication is what appears to be nuclear localization of synphilin-1A in a healthy looking cell without inclusions. If this is a reproducible observation rather than an artifact of overexpression, the functional consequences of nuclear synphilin-1A, and whether this relates to its putative role in cell death, need further investigation.
The bigger unanswered question that arises from this important report is what is the mechanism of cytotoxicity induced by synphilin-1A overexpression. Can the suggestion from cultured cells that overexpression of this protein is deleterious but that its ability to aggregate somehow rescues cells applicable to the brain of patients? The next phase of studies made possible by this exciting paper should shed light on these questions.
References:
Tanaka M, Kim YM, Lee G, Junn E, Iwatsubo T, Mouradian MM. Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J Biol Chem. 2004 Feb 6;279(6):4625-31. PubMed.
View all comments by Eunsung JunnUniversity College London
Eyal et al. describe the cloning and characterization of a new isoform of synphilin-1, synphilin-1A (Sph-1A), which is prone to aggregation and demonstrates marked cellular toxicity. The two transcripts originate from the same gene (SNCAIP) but utilize distinct initiation codons. In addition, exons 3 and 4 are spliced out, while an extra exon, 9A, is incorporated. Using a specific polyclonal antibody, Eyal et al. demonstrate that Sph-IA is a 75 kDa protein expressed in rat and human brain in significant amounts, as well as liver, lung, and spleen. However, by comparison with synphilin-1, Sph-1A expression is lower (~15 percent of synphilin-1 levels).
Overexpression of Sph-1A leads to large perinuclear aggregates in two heterologous cell lines (HEK293 and SH-SY5Y) and primary neuronal cultures, even without treatment with proteosome inhibitors. This property contrasted with that of synphilin-1, which was reported to form aggregates only upon proteasomal inhibition. Our group has recently shown that endogenous synphilin-1 forms aggresomes in SH-SY5Y cells upon proteasomal inhibition (Bandopadhyay et al., 2005). Sph-1A was shown to interact with synphilin-1 and α-synuclein (two proteins implicated in Parkinson disease that are components of Lewy bodies) by coimmunoprecipitation from cells overexpressing these proteins. However, it remains to be determined whether these interactions are also relevant to Parkinson disease.
Overexpression of Sph-1A appears to have a detrimental effect on neurons, causing them to retract their processes. This implies that excessive Sph-1A levels compromise normal neuronal function. Markers of neuronal toxicity were also observed in these neurons, including nuclear fragmentation and cell death. Sph-1A overexpression also leads to large protein aggregates in around 8 percent of Sph-1A transfected neurons, while treatment with lactacystin led to formation of “organized” inclusions in about 40 percent of these neurons. The authors also noted an inverse relationship between inclusion formation and cell death, that is, cells containing inclusions were more likely to survive. This observation lends further fuel to the debate concerning whether inclusion formation is beneficial or detrimental to normal neuronal function.
Data from human brain homogenates showed an accumulation of detergent-insoluble Sph-1A in postmortem tissue from patients with dementia with Lewy bodies (DLB) compared to controls. However, interestingly this was not observed for synphilin-1. Sph-1A also localized to Lewy bodies in brain sections from Parkinson nigra and DLB tissue, strongly suggesting a role for Sph-1A in the pathogenesis of inclusion formation. No doubt the prevalence of Sph-1A positive inclusion bodies in these diseases will be investigated in the near future. It would also be interesting to see if Sph-1A is present in glial cytoplasmic inclusions in multiple system atrophy (MSA).
References:
Bandopadhyay R, Kingsbury AE, Cookson MR, Reid AR, Evans IM, Hope AD, Pittman AM, Lashley T, Canet-Aviles R, Miller DW, McLendon C, Strand C, Leonard AJ, Abou-Sleiman PM, Healy DG, Ariga H, Wood NW, de Silva R, Revesz T, Hardy JA, Lees AJ. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson's disease. Brain. 2004 Feb;127(Pt 2):420-30. PubMed.
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