Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates
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eLife assessment
The authors attempted to show that syntaxin 6 (Stx6) delays PrP fibril formation and in presence of Stx6, PrP forms amorphous aggregates which are more toxic to neuronal cells, indicative of Stx6's anti-chaperone activity. This useful study has potential to provide important understanding of the molecular mechanism of PrP aggregation and neurotoxicity. However, the evidence supporting the physiological relevance and robustness of the assays is incomplete.
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Abstract
Prions replicate via the autocatalytic conversion of cellular prion protein (PrP C ) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt–Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an ‘anti-chaperone’, which promotes toxic aggregation intermediates by inhibiting fibril formation.
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Author Response
Reviewer #2 (Public Review):
The work reports a minor modification in the protocol for Prp formation in vitro. Using this the authors evaluate the role of Syntaxin 6 in modulating prion formation in vitro and the toxicity of the amyloid fibrils in cell culture models. The authors show that while prions/amyloids formed by PrP are non-toxic, mixed aggregates formed by Stx6/PrP are toxic; they claim that this is due to the toxic aggregation intermediates that accumulate more in the presence of Stx6. However, the basis of enhanced toxicity of Stx6/PrP mixed aggregates is not clear and doesn't seem to be physiologically relevant; there is no evidence that Stx6 and PrP forms mixed aggregates in vivo. Which is the toxic component of the Stx6/Prp co-aggregate? Is it the Stx6 component or the Prp component? Additionally, the …
Author Response
Reviewer #2 (Public Review):
The work reports a minor modification in the protocol for Prp formation in vitro. Using this the authors evaluate the role of Syntaxin 6 in modulating prion formation in vitro and the toxicity of the amyloid fibrils in cell culture models. The authors show that while prions/amyloids formed by PrP are non-toxic, mixed aggregates formed by Stx6/PrP are toxic; they claim that this is due to the toxic aggregation intermediates that accumulate more in the presence of Stx6. However, the basis of enhanced toxicity of Stx6/PrP mixed aggregates is not clear and doesn't seem to be physiologically relevant; there is no evidence that Stx6 and PrP forms mixed aggregates in vivo. Which is the toxic component of the Stx6/Prp co-aggregate? Is it the Stx6 component or the Prp component? Additionally, the authors do not have mechanistic explanation for the effect of Stx6 on PrP prion formation
We thank the reviewer for his assessment and we agree that more in vivo data was needed to support the physiological relevance of the effect of syntaxin-6 on PrP. We now provide two new key experiments demonstrating interaction of STX6 with PrP in a cell model of prion disease and testing the effect of Stx6 knockout on the replication of infectious RML prions in PMCA assays (Figures 4, 4S1, 4S2). Please refer to our response to reviewer 1, point 1 for more details. We respectfully disagree that the native aggregation assay represents a minor modification of PrP fibril formation protocols. While this statement may be true in the narrow technical sense, it is striking that in more than 25 years of prion research, no aggregation assay under near-native assay conditions had been developed. The conditions of previous assays, which relied on thermal or chemical denaturation to facilitate PrP misfolding, were inherently incapable of assessing the effect of protein modifiers of PrP fibril formation. Therefore, the NAA opens a wide field of new experiments to mechanistically probe modulators of PrP aggregation and toxicity under physiologically relevant conditions. The protein syntaxin-6 proves a test case for this new capability.
The reviewer may have misunderstood the mechanistic hypothesis for neurotoxicicty that is supported by our data. We are not claiming that the co-aggregates between PrP and syntaxin-6 are toxic. As our data demonstrate, aggregation endpoints in the presence of STX6 have little neurotoxicity, as do fibrillar aggregation endpoints without the presence of STX6 (Figures 5 and 5S1). Rather, based on the well-established oligomer toxicity hypothesis, we are concluding that STX6, by delaying or preventing formation of mature amyloid fibrils, caused toxic aggregation intermediates to persist. Our new data from secondary seeding assays (Figure 5S2) demonstrate that at the aggregation time points when the maximum amounts of neurotoxic species are present (20 h), no seeding competent fibrils have yet been formed. The presence of STX6 prolongs this period and therefore increases toxicity (Figures 5 and 5S1). These data directly support the established theories for the basis of amyloid toxicity and, additionally, caution that an intervention to delay amyloid formation can have deleterious effects on toxicity. We have now made this point more clearly in our discussion. Of course, we, like many other protein misfolding laboratories in the world, are also working hard on isolating and characterizing the toxic species in prion and other protein misfolding diseases, which, as the reviewer suggests, will be a very important milestone in understanding these diseases.
Reviewer #3 (Public Review):
The autocatalytic replication mechanism of misfolded Prion-like proteins (PrP) into amyloid aggregates is associated with a plethora of deleterious neurodegenerative diseases. Despite of the huge amount of research, the underlying molecular events of self-replication and identification of the toxic species are not fully understood. Many recent studies have indicated that non-fibrillar oligomeric intermediates could be more neurotoxic compared to the Prion fibrils. Various cellular factors, like the participation of other proteins and chaperone activity, also play an important role in PrP misfolding, aggregation, and neurotoxicity. The present work focuses on understanding the PrP aggregation mechanism with the identification of the associated toxic species and cellular factors. One of the significant strengths of the work is performing the aggregation assay in near-native conditions. In contrast, most in vitro studies use harsh conditions (such as high temperature, denaturant, detergent, low pH, etc.) to promote protein aggregation. The authors successfully observed the well-known seeding property of the PrP in this aggregation assay that bypasses the primary nucleation during aggregation. Moreover, the authors have shown that syntaxin 6 (Stx6), a known risk factor in prion-mediated Creutzfeldt-Jakob disease, delays fibril formation and prolongs the persistence of toxic intermediates, thus playing an anti-chaperone activity. This study will contribute to understanding the molecular mechanism of PrP aggregation and neurotoxicity. However, further studies are required to identify and characterize the toxic intermediate in the near future precisely.
We thank the reviewer for his thoughtful and accurate summary. We fully agree that the nature of the toxic species in protein misfolding diseases is a key challenge of the field and we hope that our study contributes to solving this puzzle.
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eLife assessment
The authors attempted to show that syntaxin 6 (Stx6) delays PrP fibril formation and in presence of Stx6, PrP forms amorphous aggregates which are more toxic to neuronal cells, indicative of Stx6's anti-chaperone activity. This useful study has potential to provide important understanding of the molecular mechanism of PrP aggregation and neurotoxicity. However, the evidence supporting the physiological relevance and robustness of the assays is incomplete.
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Reviewer #1 (Public Review):
Sangar and co-workers have shown the role of Syntaxin 6 on prion (PrP) protein aggregation and have claimed a possible molecular mechanism behind its role in pathogenesis of Prion diseases like CJD. Authors have elaborately shown the aggregation kinetics of PrP in absence and presence of Stx6 by their in-house developed NAA assay which allowed them to catch the interaction of PrP with other cellular proteins like Stx6 or Hspa1a. By in vitro aggregation kinetics assays, authors show that Stx6 delays the aggregation kinetics of PrP and leads to amorphous aggregates rather than well-formed fibrils. By a neurite length detection assay with treatment of preformed PrP-fibrils (with or without Stx6 interaction), they show enhanced toxicity of Prp-Stx6 aggregates compared to PrP fibrils. However, there are a number …
Reviewer #1 (Public Review):
Sangar and co-workers have shown the role of Syntaxin 6 on prion (PrP) protein aggregation and have claimed a possible molecular mechanism behind its role in pathogenesis of Prion diseases like CJD. Authors have elaborately shown the aggregation kinetics of PrP in absence and presence of Stx6 by their in-house developed NAA assay which allowed them to catch the interaction of PrP with other cellular proteins like Stx6 or Hspa1a. By in vitro aggregation kinetics assays, authors show that Stx6 delays the aggregation kinetics of PrP and leads to amorphous aggregates rather than well-formed fibrils. By a neurite length detection assay with treatment of preformed PrP-fibrils (with or without Stx6 interaction), they show enhanced toxicity of Prp-Stx6 aggregates compared to PrP fibrils. However, there are a number of concerns related to physiological relevance that need to be addressed by the authors.
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Reviewer #2 (Public Review):
The work reports a minor modification in the protocol for Prp formation in vitro. Using this the authors evaluate the role of Syntaxin 6 in modulating prion formation in vitro and the toxicity of the amyloid fibrils in cell culture models. The authors show that while prions/amyloids formed by PrP are non-toxic, mixed aggregates formed by Stx6/PrP are toxic; they claim that this is due to the toxic aggregation intermediates that accumulate more in the presence of Stx6. However, the basis of enhanced toxicity of Stx6/PrP mixed aggregates is not clear and doesn't seem to be physiologically relevant; there is no evidence that Stx6 and PrP forms mixed aggregates in vivo. Which is the toxic component of the Stx6/Prp co-aggregate? Is it the Stx6 component or the Prp component? Additionally, the authors do not have …
Reviewer #2 (Public Review):
The work reports a minor modification in the protocol for Prp formation in vitro. Using this the authors evaluate the role of Syntaxin 6 in modulating prion formation in vitro and the toxicity of the amyloid fibrils in cell culture models. The authors show that while prions/amyloids formed by PrP are non-toxic, mixed aggregates formed by Stx6/PrP are toxic; they claim that this is due to the toxic aggregation intermediates that accumulate more in the presence of Stx6. However, the basis of enhanced toxicity of Stx6/PrP mixed aggregates is not clear and doesn't seem to be physiologically relevant; there is no evidence that Stx6 and PrP forms mixed aggregates in vivo. Which is the toxic component of the Stx6/Prp co-aggregate? Is it the Stx6 component or the Prp component? Additionally, the authors do not have mechanistic explanation for the effect of Stx6 on PrP prion formation
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Reviewer #3 (Public Review):
The autocatalytic replication mechanism of misfolded Prion-like proteins (PrP) into amyloid aggregates is associated with a plethora of deleterious neurodegenerative diseases. Despite of the huge amount of research, the underlying molecular events of self-replication and identification of the toxic species are not fully understood. Many recent studies have indicated that non-fibrillar oligomeric intermediates could be more neurotoxic compared to the Prion fibrils. Various cellular factors, like the participation of other proteins and chaperone activity, also play an important role in PrP misfolding, aggregation, and neurotoxicity. The present work focuses on understanding the PrP aggregation mechanism with the identification of the associated toxic species and cellular factors. One of the significant …
Reviewer #3 (Public Review):
The autocatalytic replication mechanism of misfolded Prion-like proteins (PrP) into amyloid aggregates is associated with a plethora of deleterious neurodegenerative diseases. Despite of the huge amount of research, the underlying molecular events of self-replication and identification of the toxic species are not fully understood. Many recent studies have indicated that non-fibrillar oligomeric intermediates could be more neurotoxic compared to the Prion fibrils. Various cellular factors, like the participation of other proteins and chaperone activity, also play an important role in PrP misfolding, aggregation, and neurotoxicity. The present work focuses on understanding the PrP aggregation mechanism with the identification of the associated toxic species and cellular factors. One of the significant strengths of the work is performing the aggregation assay in near-native conditions. In contrast, most in vitro studies use harsh conditions (such as high temperature, denaturant, detergent, low pH, etc.) to promote protein aggregation. The authors successfully observed the well-known seeding property of the PrP in this aggregation assay that bypasses the primary nucleation during aggregation. Moreover, the authors have shown that syntaxin 6 (Stx6), a known risk factor in prion-mediated Creutzfeldt-Jakob disease, delays fibril formation and prolongs the persistence of toxic intermediates, thus playing an anti-chaperone activity. This study will contribute to understanding the molecular mechanism of PrP aggregation and neurotoxicity. However, further studies are required to identify and characterize the toxic intermediate in the near future precisely.
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