Final Report Summary - CHAPERONES IN ND (The Role of Molecular Chaperones in Parkinson' s Disease)
Parkinson's disease is a chronic and progressive neurodegenerative disorder that affects mainly the aging population, and is caused by the degeneration of dopaminergic neurons from the substantia nigra in the brain. The pathology of dopaminergic neurons is characterised by the formation of intra-cellular protein inclusions called Lewy Bodies, primarily composed of aggregated alpha-synuclein (a-syn). a-Syn aggregation is a highly specific amyloid formation reaction that proceeds through oligomeric intermediates, referred to as protofibrils that disappear upon elongation of amyloid fibrils.
The aggregation of a-syn and the linked pathogenesis are very complex processes which are not yet fully understood. On-pathway intermediates of the amyloid formation are considered as the toxic species and transmission between neuronal cells may take place similar to the transmission of prion particles. Recent advances showed how molecular chaperones are linked to these processes. The molecular chaperone Hsp104 from yeast was suggested to act predominantly on stable a-syn aggregates and thereby serves as a protein disaggregation machine. Hsp104 is an ATP-fulled molecular machine which is active in extracting and re-naturing proteins from stable aggregates. Over-expression of Hsp104 is protective against a-syn toxicity in a rat model of Parkinson's disease. However, it is not clear how Hsp104 can confer protection against a-syn-induced toxicity in neuronal cells and how it interacts with a-syn on a molecular level.
Our study aims to elucidate the mechanism how molecular chaperones interfere with a-syn related toxicity and Parkinson's disease pathology. In particular, we aimed to determine the ability of molecular chaperones to prevent or revert a-syn amyloid formation and to study the molecular mechanisms by which molecular chaperones interact with a-syn and modulate its structural features and aggregation properties.
In order to study the interaction of a-syn with Hsp104, recombinant proteins were cloned, expressed and characterised. Amyloid formation assays were developed to study the influence of Hsp104 on a-syn aggregation in vitro and enzymatic ATPase assays were performed to assess the effect of substrate binding on the ATPase activity of Hsp104. For biophysical interaction studies protein derivatives were designed which were either carrying a Histidine tag, a tryptophane residue or a fluorescent dye.
Following key results were found:
(i) Hsp104 affects the aggregation of a-syn in a sub-stoichiometrical range.
(ii) The hexameric form of Hsp104 is the active species in inhibiting amyloid formation; however, nucleotide presence is not needed for this inhibitory effect in amyloid formation assays.
(iii) Hsp104 is interacting with monomeric a-syn as found by fluorescence anisotropy and ATPase activity assays. Whereas both, the N-terminal domain and NAC region of a-syn are sufficient for binding to Hsp104 which was studied by ATPase activity and fluorescence binding assays.
(iv) Further, Hsp104 specifically interacts with intermediates of a-syn on the pathway of amyloid formation, e.g. oligomers and protofibrils as found in seeded aggregation assays.
We conclude that Hsp104 inhibits amyloid formation of a-syn in a specific, dosage-dependent manner by blocking the seeding capacity of a-syn oligomers in the lag phase of amyloid formation. This effect is independent from ATP presence or the protein disaggregation activity of Hsp104. Hence, Hsp104 is protecting against a-syn toxicity by inhibiting the on-pathway of amyloid formation. Our finding suggests that this process is specific and not related to the ATPase activity or protein unfolding activity of Hsp104.
The aggregation of a-syn and the linked pathogenesis are very complex processes which are not yet fully understood. On-pathway intermediates of the amyloid formation are considered as the toxic species and transmission between neuronal cells may take place similar to the transmission of prion particles. Recent advances showed how molecular chaperones are linked to these processes. The molecular chaperone Hsp104 from yeast was suggested to act predominantly on stable a-syn aggregates and thereby serves as a protein disaggregation machine. Hsp104 is an ATP-fulled molecular machine which is active in extracting and re-naturing proteins from stable aggregates. Over-expression of Hsp104 is protective against a-syn toxicity in a rat model of Parkinson's disease. However, it is not clear how Hsp104 can confer protection against a-syn-induced toxicity in neuronal cells and how it interacts with a-syn on a molecular level.
Our study aims to elucidate the mechanism how molecular chaperones interfere with a-syn related toxicity and Parkinson's disease pathology. In particular, we aimed to determine the ability of molecular chaperones to prevent or revert a-syn amyloid formation and to study the molecular mechanisms by which molecular chaperones interact with a-syn and modulate its structural features and aggregation properties.
In order to study the interaction of a-syn with Hsp104, recombinant proteins were cloned, expressed and characterised. Amyloid formation assays were developed to study the influence of Hsp104 on a-syn aggregation in vitro and enzymatic ATPase assays were performed to assess the effect of substrate binding on the ATPase activity of Hsp104. For biophysical interaction studies protein derivatives were designed which were either carrying a Histidine tag, a tryptophane residue or a fluorescent dye.
Following key results were found:
(i) Hsp104 affects the aggregation of a-syn in a sub-stoichiometrical range.
(ii) The hexameric form of Hsp104 is the active species in inhibiting amyloid formation; however, nucleotide presence is not needed for this inhibitory effect in amyloid formation assays.
(iii) Hsp104 is interacting with monomeric a-syn as found by fluorescence anisotropy and ATPase activity assays. Whereas both, the N-terminal domain and NAC region of a-syn are sufficient for binding to Hsp104 which was studied by ATPase activity and fluorescence binding assays.
(iv) Further, Hsp104 specifically interacts with intermediates of a-syn on the pathway of amyloid formation, e.g. oligomers and protofibrils as found in seeded aggregation assays.
We conclude that Hsp104 inhibits amyloid formation of a-syn in a specific, dosage-dependent manner by blocking the seeding capacity of a-syn oligomers in the lag phase of amyloid formation. This effect is independent from ATP presence or the protein disaggregation activity of Hsp104. Hence, Hsp104 is protecting against a-syn toxicity by inhibiting the on-pathway of amyloid formation. Our finding suggests that this process is specific and not related to the ATPase activity or protein unfolding activity of Hsp104.