When amyloid-β (Aβ) peptides get together, the trouble starts. A conformational change in Aβ, followed by its aggregation into toxic clumps, is a central event in the development of Alzheimer disease. Three papers this week look at chaperone proteins that can prevent that aggregation, either of Aβ or of another protein implicated in neurodegeneration, α-synuclein. All three papers suggest strategies to keep these proteins well-folded and well clear of clinging companions.
The first paper describes an Aβ-targeted chaperone with the cumbersome name of lipocalin-type prostaglandin D synthase/β-trace (L-PGDS), which just happens to be the most abundant protein in human cerebral spinal fluid. The work, published in last week’s early edition of PNAS from Yoshihiro Urade and colleagues of the Osaka Bioscience Institute in Japan shows that the protein is an endogenous inhibitor of Aβ aggregation, and can prevent deposition of Aβ in mouse brain.
The L-PGDS story starts with an observation that the protein is present in amyloid plaques in Tg2576 mice, and in plaques in the postmortem brain of a person with AD. First author Takahisa Kanekiyo and colleagues followed up this observation by examining in detail the binding of L-PGDS to Aβ peptides. Using surface plasmon resonance methods, they determined that L-PGDS purified from human CSF bound to immobilized Aβ40) peptide or fibrils at low concentrations (Kd = 50 nM). Likewise, monomeric Aβ(1-40) bound to immobilized L-PGDS.
Using shorter peptides, the researchers localized the recognition sequence on Aβ to residues 25-28, a key region involved in the conformational transition from random coil to β-sheet (see ARF related news story). This led them to think that L-PGDS might affect Aβ aggregation, and they showed this was the case in vitro. Addition of 1 uM L-PGDS to 50 uM Aβ(1-40 or 1-42) inhibited either spontaneous or seeded aggregation, as measured by thioflavin T fluorescence. Aggregation was completely blocked by 5 uM L-PGDS, a concentration comparable to that found in the CSF. They used circular dichroism measurements to show that the synthase prevented Aβ from adopting a β-sheet structure. They also showed that neither heat-inactivated nor a catalytically inactive point mutant prevented aggregation, suggesting that an intact structure was important for the activity.
Further experiments indicate that L-PGDS provides the major Aβ anti-aggregation activity in CSF and in brain. Human CSF was able to prevent aggregation of Aβ in vitro, and depleting the CSF of the L-PGDS with a specific antibody removed the anti-aggregation activity. They also showed the activity in vivo, by injecting Aβ(1-42) into the brains of L-PGDS knockout mice. The mice showed accelerated deposition of the Aβ after 3 hours compared to normal mice. In contrast, L-PGDS-overexpressing mice showed reduced deposition.
“These data indicate that L-PGDS/β-trace is a major endogenous Aβ chaperone in the brain and suggest that disturbance of this function may be involved in the onset and progression of AD,” the authors conclude. They point out that L-PGDS binds Aβ with affinity similar to ApoE, but in contrast to ApoE, this protein is produced in the brain and highly abundant in the CSF. “L-PGDS/β-trace could be considered more essential than ApoE for the prevention of Aβ aggregation in the brain,” they write.
Could changes in L-PGDS levels contribute to AD? Proteomic analysis of CSF identified the protein as one that is decreased in AD patients (Puchades et al., 2002). Conversely, environmental enrichment, which reduced Aβ deposition, increased L-PGDS gene transcription (see ARF related news story).
α-synuclein to the Rescue?
A second paper considers the actions of another, perhaps more familiar protein, also found in amyloid plaques. The protein, α-synuclein, is itself prone to aggregation, forming intracellular Lewy bodies in Parkinson disease and in a subset of AD cases. More than 10 years ago, researchers identified a fragment of α-synuclein in Alzheimer plaques (Iwai et al., 1995). Because this piece was very hydrophobic, some proposed that it aided plaque formation.
To test this idea, Verena Kallhoff, Erica Peethumnongsin, and Hui Zheng at the Baylor College of Medicine in Houston, Texas, crossed transgenic APP mice (Tg2576) with α-synuclein knockouts. They hypothesized that getting rid of synuclein might diminish plaques, but instead they saw the opposite: mice without α-synuclein developed much higher plaque loads as they aged. The researchers showed that lack of α-synuclein did not alter Aβ levels in 6-month-old animals, nor change the age of onset of plaque deposition, but in 18-month-old knockout mice they saw a three to four times higher plaque load compared to intact APP mice. They conclude that α-synuclein is not involved in the seeding of plaques, but may suppress plaque pathology at later stages.
“Our data that the amyloid plaques are increased in the absence of α-synuclein at old age suggests that α-synuclein may indeed serve as a chaperone, helping the cells to clear protein deposits,” they write. The data bolster the emerging view of α-synuclein as a neuroprotector (see ARF related news story and Quilty et al., 2006). The paper appeared in the March 16 issue of Molecular Neurodegeneration.
β-synuclein Changes the Channel
Neuroprotective or not, α-synuclein eventually goes the way of many proteins implicated in neurodegenerative disease, aggregating into fibrillar structures that eventually form large inclusions in cells. In a paper appearing in the April issue of the FEBS Journal, Eliezer Masliah and colleagues at the University of California at San Diego show the results of computer modeling aimed at understanding α-synuclein aggregation. They note that while the structure of α-synuclein before and after aggregating has been the subject of intense study, little is known of the early steps in the process. First author Igor Tsigelny and colleagues employed a supercomputer to perform molecular modeling and molecular dynamics simulation of synuclein aggregation on membranes, where it is thought to occur. Their results suggest that the early steps of aggregation involve formation of α-synuclein homodimers on the membrane. Some homodimer structures are dead ends, which do not grow further, but others can accommodate additional α-synuclein molecules and progress to higher oligomers including hexamers with a pore-like structure.
Structural and biochemical studies agreed with this model. To see if α-synuclein formed a functional pore, the researchers overexpressed the protein in cells. The result was an increase in plasma membrane cation channel activity. Addition of β-synuclein, a non-aggregating protein which may actually protect against α-synuclein toxicity, stopped the recruitment of additional α-synuclein units to growing multimers and prevented channel formation. Their results suggest that the protective effects of β-synuclein may stem from its ability to block aggregation and channel formation.—Pat McCaffrey
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