An ominous double may be at work in Parkinson disease (PD) and other neurodegenerative diseases. In this week’s PNAS online, researchers report that a heretofore undetected and unusual isoform of synphilin-1 causes cellular toxicity. The doppelgänger also turns up in Lewy bodies, the dense, α-synuclein-containing protein aggregates that are pathological hallmarks of disease. Dubbed synphilin-1A, the toxic protein may lead to new insights into α-synucleinopathies, including PD, multiple system atrophy, and diffuse Lewy body disease (DLBD).

The shady synphilin-1A was exposed when Simone Engelender and colleagues at Technion-Israel Institute of Technology were sifting through the expressed sequence tag (EST) databases at the National Center for Biotechnology Information website. EST sequences are usually deposited by scientists who have analyzed complementary DNA or messenger RNA, and as such, ESTs reflect RNA sequences that are transcribed, spliced, and presumably functionally important. But first authors Allon Eyal and colleagues noticed something odd about several synphilin ESTs. Three of them contained a 71-base sequence predicted to lie in an intron. In other words, that sequence should never end up in mRNA—in theory. In practice, however, it turns out that between exons 9 and 10 of synphilin lies another small exon that everyone else had missed. Eyal and colleagues coined it exon 9A.

But the mystery does not stop there. When Eyal and colleagues made an antibody to the peptide encoded by exon 9A, then used it to detect synphilin-1A in the brain, they found that the protein that bound to the antibody was only 75 kDa, about 25 kDa smaller than the predicted sequence.

Realizing that the 75 kDa protein is consistent with the loss of exons 3 and 4 through alternative splicing, the authors faced another enigma. Splicing out exons 3 and 4 would cause a frame shift that introduces a stop codon in exon 5, leading to a protein of less than 10 kDa. So how could synphilin-1A turn out to be 75 kDa? The answer lies in an apparently unique type of frame shift. Synphilin-1A uses a different start codon to synphilin-1, giving the A isoform a different N-terminus. But the splicing out of exons 3 and 4 resets the reading frame so that the C-terminal end of the two synphilin-1s are the same. “To our knowledge, two totally functional alternatively spliced transcripts originating from the same gene have never before been translated by the use of two different initial reading frames,” write the authors. If this turns out to be a more general phenomenon, then the number of proteins encoded by the genome could be markedly increased, the authors suggest.

Having figured out the size and nature of synphilin-1A, the authors then went on to characterize it more fully. They found that it is widely expressed in the brain, including in the substantia nigra, the region most affected in PD, and in the cerebral cortex, where synuclein inclusions are also found. Quantitatively, synphilin-1A represents slightly less than 15 percent of total synphilin-1. The A isoform is also found in synaptic vesicles and colocalizes with synaptophysin, suggesting that even with an alternative N-terminus, the protein may play an important role in the synapse.

But the protein may also be detrimental to neurons. When the authors overexpressed it in primary cortical cultures, they found that the cells retracted their processes. Markers of apoptosis also indicated that about 20 percent of the neurons had entered a cell death pathway. A smaller number of neurons also formed large aggregates, which, oddly enough, do not form when synphilin-1 is overexpressed in neurons, suggesting that the A isoform has a greater propensity to aggregate. The authors suggest that the shorter N-terminus on the 1A form might expose ankyrin-like domains on the protein, which could then interact with each other.

The researchers also observed an inverse relationship between the formation of inclusions and cell death. When they challenged neurons overexpressing synphilin-1A with MG132, the proteasomal inhibitor, about 40 percent of neurons formed organized inclusions. MG132 also increased cell death, but only in those neurons that had no inclusions. This finding speaks to a raging debate about whether inclusions are protective or detrimental. “Our findings clearly suggest a cytoprotective role for inclusion bodies,” the researchers write. Indeed, recent evidence suggests that inclusions may protect neurons expressing mutant huntingtin, the protein that causes Huntington disease (see ARF related news story).

Whether synphilin-1A plays any role in PD or other α-synucleinopathies has yet to be determined, but Eyal and colleagues did find α-synuclein in the synphilin-1A inclusions. They also found that the A isoform traps synphilin-1 in inclusions and prevents its degradation through the ubiquitin proteasome pathway. The finding suggests that even though it is not expressed as much as synphilin-1, the A isoform might pack a double punch, by trapping an equal amount of its more abundant twin, assuming a 1:1 binding.

As for patient data, Eyal and colleagues report that insoluble synphilin-1A is present in brain samples from DLBD patients, and it is also present in Lewy bodies from both PD and DLBD patients. Curiously though, in one out of three DLBD patient samples, the authors found that detergent-soluble synphilin-1A was totally missing, but as they point out, because of small sample numbers, no correlation can be made between soluble synphilin-1A and disease at this time.—Tom Fagan

Comments

Make a Comment

To make a comment you must login or register.

Comments on this content

  1. 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:

    . Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J Biol Chem. 2004 Feb 6;279(6):4625-31. PubMed.

  2. 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:

    . The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson's disease. Brain. 2004 Feb;127(Pt 2):420-30. PubMed.

References

News Citations

  1. New Microscope Resolves Role of Huntington Inclusions—Neuroprotection

Further Reading

Primary Papers

  1. . 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.