- Email: [email protected]
S3-02-01 Principles of assembly of tau protein into Alzheimer paired helical filaments
Symposium $3-02: Conformational Disorders ratio of A[3Dutch40 : 42 towards A~Dutch42 is sufficient to redistribute the amyloid pathology from the vasculature to the parenchyma. Thus far, we have not detected arnyloid deposits in L68Q mutated cystatin tg mice. Crosses of APP and cystatin C tg mice await further analysis. Conclusion: These mouse models are unique tools with which to further study the pathogenic mechanism by which CAA directly affects cognition and neurodegeneration in AD. They will also prove useful in the development of therapeutic strategies.
$47
Our hypothesis is that these processes will share properties that suggest possible mechanisms of neurotoxicity. Results: We have characterized an aggregate that resembles known protein pores and that is an intermediate in or side-product of amyloid fibril formation. Amyloid pores comprising many amyloid proteins have been characterized, by our lab and by others. Conclusion: the amyloid pore may be the/one pathogenic protein aggregate. Our current efforts are focussed on disproving this hypothesis.
I$3-02-03 I PRION PROTEIN CONVERSIONS AND TSE S y m p o s i u m $3-02: C o n f o r m a t i o n a l Disorders
•
P R I N C I P L E S OF ASSEMBLY O F TAU PROTEIN
INTO ALZHEIMER PAIRED HELICAL FILAMENTS Eckhard Mandelkow*, Martin von Bergen, Stefan Barghom, Li Li, Jacek Biernat, Eva-Maria Mandelkow. Max-PlanckInstitute, Hamburg,
Germany. Contact e-mail: [email protected] Several neurudegenerative diseases, including Alzheimer's disease, are "tauopathies" characterized by the abnormal aggregation of tan protein into paired helical filaments (PHF) and related forms of assembly in a highly phosphorylated state, The selective aggregation of this protein is enigmatic because tau is, in principle, one of the most soluble proteins known, owing to its hydrophilic character. For the same reason, tan is not folded in a compact fashion, but rather behaves like a Gaussian polymer in solution without a defined shape; in fact tan is a prototypic "natively unfolded" protein. Nevertheless, abnormal aggregation can be induced in vitro, using recombinant tan protein and constructs derived from them. Some principles have emerged, for example: (1) Dimerization of tan by oxidative crosslinking of cysteines helps to overcome the barrier of filament initiation. (2) The kinetics of aggregation can be accelerated by anionic cofactors (heparin, poly-Glu, RNA, acidic peptides, or fatty acid micelles), presumably because these compensate the cationic nature of tau. (3) Tan aggregates by a nucleation-elongation mechanism where the rate limiting step is the formation of a nucleus involving oligomers of 40-7 tau dimers. (4) Phosphorylation at certain sites, particularly in the repeat domain, tends to disrupt tau-microtubule interactions and therefore increases the pool of soluble tan molecules. Likewise, phosphorylation disrupts the tan-tau interaction leading to the aggregation into PHFs. Thus, the abnormal phosphorylation of tan in Alzheimer's disease could be regarded as a protective mechanism against PHF aggregation. (5) Certain motifs of tan are prone to induce beta-sheet formation and aggregation, such as the hexapepfide motifs 306-VQIVYK-311 and 275-VQIINK-280 in the repeat domain. Thus only a small fraction of tau is responsible for its "amyloid" behavior with a cross-beta s~ucture. (6) Most tau missense mutations occurring in frontotemporal dementias (FFDP-17) are located in the microtubule-binding domain. Some of them strongly enhance PHF aggregation (notably P301L and delta-K280) because they enhance the tendency for beta-structure formation at the two hexapepfide motifs. These features allow one to distinguish pathological from normal conformations of tau, and to develop strategies for preventing unwanted aggregation of tan by inhibitory compounds. - Supported by DFG.
$ 3 - 0 2 - 0 2 ] S T R U C T U R A L B I O L O G Y O F SYNUCLEIN Peter Lansbury*. Brigham and Women's Hospital, Cambridge, MA, USA.
Contact e-mail: [email protected]
Background: Amyloid fibril formation is characteristic of most neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. The relationship between this process and neurodegeneration has not been unequivocally determined, despite compelling circumstantial evidence that it is pathogenic. Objective: To determine if a discrete protein aggregate is initiating a chain of events that culminate in neuronal death. Method: We study the in vitro aggregation properties of a-synuclein, which is linked to Parkinson's disease, and superoxide dismutase, which is linked to ALS.
INFECTIONS Byron Caughey* 1, Gerald S. Baron 2, Ana Cristina Magalhaes 2, Marco Antonio Prado 3, David A. Kocisko 2 . INIH/NIAID Rocky Mountain Labs,
Hamilton, MT, USA; 2NIH~IAID Rocky Mt LABS, Hamilton, MZ USA; 3Federal University of Minas Gerais, Belt Horizonte, Brazil. Contact e-mail: [email protected]
Background: The conformational conversion of the normal, protease-
sensitive prion protein ( P r Pc) to the protease-resistant prion protein (PrP-res or PrP sc) is critical in transmissible spongiform encephalopathies (TSE) or priori diseases. Our studies of PrP conversion and the effects of cofactors have provided insight into the molecular bases for PrP-res formation, TSE species barriers, and agent strains. Objectives: 1) To determine how membrane associations of PrP isoforms affect PrP conversion and the infection of cells. 2) To follow the trafficking of PrP-res within acutely infected cells. 3) To identify inhibitors of PrP-res propagation that might serve as anti-TSE drugs. Methods: Cell-free reactions between membrane-botmd PrP isoforms were used to assess the influence of membranes on PrP conversion. The influence of membranes on PrP-res-associated scrapie infectivity was monitored by long-term PrP-res production in cultured murine neuro-2a and SN56 cells. Chronically scrapie-infected cells were used to assay for PrP-res inhibitors. Results: Efficient cell-free conversion reactions between membrane-bound, GPI-anchored PrP C and PrP-res molecules required the molecules to be incorporated into the same membrane. Membrane-associated PrP-res was several fold more efficient than membrane-free PrP-res at inducing longterm PrP-res propagation in cultured cells. High throughput screening of compounds on scrapie-infected tissue culture cells identified many new inhibitors of PrP-res formation. Conclusions: Cell-free studies of the interactions between membrane-bound PrP isoforms suggest that the propagation of infection between cells might require transfer of PrP-res into the membranes of recipient cells. Membranous PrP-res preparations are more efficient at inducing PrP-res formation in cultured cells than membrane-free preparations. SN56 (septal neuronal) cells are valuable as a new experimental model for investigating acute TSE infection of cells, the subeellular trafficking of PrP-res, and the effects of neuronal differentiation. Recently identified new inhibitors of PrP-res accumulation may serve both as potential therapeutic agents and probes of the molecular mechanism of pathological PrP accumulation.
$3-02-04 ] NEUROSERPIN PROVIDES A STRUCTURAL MODEL FOR THE CAUSATION AND RATIONAL THERAPY OF DEMENTIA Robin W. Carrell* 1, Aiwu Zhou 2, David A. Lomas 1.1Dept of Medicine,
University of Cambridge, CIMR, Hills Road, Cambridge, UnitedKingdom; 2Dept of Haematology, University of Cambridge, UnitedKingdom. Contact e-mail: [email protected]
Background: The mutations of neuruserpin provide a s~ucturally defined model of the changes underlying the conformational dementias in general. Neuroserpin is a brain-specific member of the serpin family and shares the tightly conserved structure that typifies this family of protease inhibitors. Mutations affecting the structural stability of neuroserpin result in familial nenrodegeneration and dementia (Nature 1999, 401: 376--9). Four such mutations have now been identified, each being homologously identical to mutations that have been well-studied in other serpins where they result in a range of conformational diseases - notably so with the mutations of alpha-l-antitrypsin that result in a variably slow onset of cirrhosis.
$47
Our hypothesis is that these processes will share properties that suggest possible mechanisms of neurotoxicity. Results: We have characterized an aggregate that resembles known protein pores and that is an intermediate in or side-product of amyloid fibril formation. Amyloid pores comprising many amyloid proteins have been characterized, by our lab and by others. Conclusion: the amyloid pore may be the/one pathogenic protein aggregate. Our current efforts are focussed on disproving this hypothesis.
I$3-02-03 I PRION PROTEIN CONVERSIONS AND TSE S y m p o s i u m $3-02: C o n f o r m a t i o n a l Disorders
•
P R I N C I P L E S OF ASSEMBLY O F TAU PROTEIN
INTO ALZHEIMER PAIRED HELICAL FILAMENTS Eckhard Mandelkow*, Martin von Bergen, Stefan Barghom, Li Li, Jacek Biernat, Eva-Maria Mandelkow. Max-PlanckInstitute, Hamburg,
Germany. Contact e-mail: [email protected] Several neurudegenerative diseases, including Alzheimer's disease, are "tauopathies" characterized by the abnormal aggregation of tan protein into paired helical filaments (PHF) and related forms of assembly in a highly phosphorylated state, The selective aggregation of this protein is enigmatic because tau is, in principle, one of the most soluble proteins known, owing to its hydrophilic character. For the same reason, tan is not folded in a compact fashion, but rather behaves like a Gaussian polymer in solution without a defined shape; in fact tan is a prototypic "natively unfolded" protein. Nevertheless, abnormal aggregation can be induced in vitro, using recombinant tan protein and constructs derived from them. Some principles have emerged, for example: (1) Dimerization of tan by oxidative crosslinking of cysteines helps to overcome the barrier of filament initiation. (2) The kinetics of aggregation can be accelerated by anionic cofactors (heparin, poly-Glu, RNA, acidic peptides, or fatty acid micelles), presumably because these compensate the cationic nature of tau. (3) Tan aggregates by a nucleation-elongation mechanism where the rate limiting step is the formation of a nucleus involving oligomers of 40-7 tau dimers. (4) Phosphorylation at certain sites, particularly in the repeat domain, tends to disrupt tau-microtubule interactions and therefore increases the pool of soluble tan molecules. Likewise, phosphorylation disrupts the tan-tau interaction leading to the aggregation into PHFs. Thus, the abnormal phosphorylation of tan in Alzheimer's disease could be regarded as a protective mechanism against PHF aggregation. (5) Certain motifs of tan are prone to induce beta-sheet formation and aggregation, such as the hexapepfide motifs 306-VQIVYK-311 and 275-VQIINK-280 in the repeat domain. Thus only a small fraction of tau is responsible for its "amyloid" behavior with a cross-beta s~ucture. (6) Most tau missense mutations occurring in frontotemporal dementias (FFDP-17) are located in the microtubule-binding domain. Some of them strongly enhance PHF aggregation (notably P301L and delta-K280) because they enhance the tendency for beta-structure formation at the two hexapepfide motifs. These features allow one to distinguish pathological from normal conformations of tau, and to develop strategies for preventing unwanted aggregation of tan by inhibitory compounds. - Supported by DFG.
$ 3 - 0 2 - 0 2 ] S T R U C T U R A L B I O L O G Y O F SYNUCLEIN Peter Lansbury*. Brigham and Women's Hospital, Cambridge, MA, USA.
Contact e-mail: [email protected]
Background: Amyloid fibril formation is characteristic of most neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. The relationship between this process and neurodegeneration has not been unequivocally determined, despite compelling circumstantial evidence that it is pathogenic. Objective: To determine if a discrete protein aggregate is initiating a chain of events that culminate in neuronal death. Method: We study the in vitro aggregation properties of a-synuclein, which is linked to Parkinson's disease, and superoxide dismutase, which is linked to ALS.
INFECTIONS Byron Caughey* 1, Gerald S. Baron 2, Ana Cristina Magalhaes 2, Marco Antonio Prado 3, David A. Kocisko 2 . INIH/NIAID Rocky Mountain Labs,
Hamilton, MT, USA; 2NIH~IAID Rocky Mt LABS, Hamilton, MZ USA; 3Federal University of Minas Gerais, Belt Horizonte, Brazil. Contact e-mail: [email protected]
Background: The conformational conversion of the normal, protease-
sensitive prion protein ( P r Pc) to the protease-resistant prion protein (PrP-res or PrP sc) is critical in transmissible spongiform encephalopathies (TSE) or priori diseases. Our studies of PrP conversion and the effects of cofactors have provided insight into the molecular bases for PrP-res formation, TSE species barriers, and agent strains. Objectives: 1) To determine how membrane associations of PrP isoforms affect PrP conversion and the infection of cells. 2) To follow the trafficking of PrP-res within acutely infected cells. 3) To identify inhibitors of PrP-res propagation that might serve as anti-TSE drugs. Methods: Cell-free reactions between membrane-botmd PrP isoforms were used to assess the influence of membranes on PrP conversion. The influence of membranes on PrP-res-associated scrapie infectivity was monitored by long-term PrP-res production in cultured murine neuro-2a and SN56 cells. Chronically scrapie-infected cells were used to assay for PrP-res inhibitors. Results: Efficient cell-free conversion reactions between membrane-bound, GPI-anchored PrP C and PrP-res molecules required the molecules to be incorporated into the same membrane. Membrane-associated PrP-res was several fold more efficient than membrane-free PrP-res at inducing longterm PrP-res propagation in cultured cells. High throughput screening of compounds on scrapie-infected tissue culture cells identified many new inhibitors of PrP-res formation. Conclusions: Cell-free studies of the interactions between membrane-bound PrP isoforms suggest that the propagation of infection between cells might require transfer of PrP-res into the membranes of recipient cells. Membranous PrP-res preparations are more efficient at inducing PrP-res formation in cultured cells than membrane-free preparations. SN56 (septal neuronal) cells are valuable as a new experimental model for investigating acute TSE infection of cells, the subeellular trafficking of PrP-res, and the effects of neuronal differentiation. Recently identified new inhibitors of PrP-res accumulation may serve both as potential therapeutic agents and probes of the molecular mechanism of pathological PrP accumulation.
$3-02-04 ] NEUROSERPIN PROVIDES A STRUCTURAL MODEL FOR THE CAUSATION AND RATIONAL THERAPY OF DEMENTIA Robin W. Carrell* 1, Aiwu Zhou 2, David A. Lomas 1.1Dept of Medicine,
University of Cambridge, CIMR, Hills Road, Cambridge, UnitedKingdom; 2Dept of Haematology, University of Cambridge, UnitedKingdom. Contact e-mail: [email protected]
Background: The mutations of neuruserpin provide a s~ucturally defined model of the changes underlying the conformational dementias in general. Neuroserpin is a brain-specific member of the serpin family and shares the tightly conserved structure that typifies this family of protease inhibitors. Mutations affecting the structural stability of neuroserpin result in familial nenrodegeneration and dementia (Nature 1999, 401: 376--9). Four such mutations have now been identified, each being homologously identical to mutations that have been well-studied in other serpins where they result in a range of conformational diseases - notably so with the mutations of alpha-l-antitrypsin that result in a variably slow onset of cirrhosis.