- Email: [email protected]
S2-03-01: Tau-associated neurodegeneration in Alzheimer's Disease and tauopathy
T126
Symposia S2-03: Disease Mechanisms: Tau and Tauopathies
Background: Age-related neurodegenerative diseases share a number of important pathological features, such as accumulation of misfolded proteins as amyloid oligomers and fibrils and dysregulation of cellular function. Recent evidence suggests that soluble amyloid oligomers may represent the primary pathological species of protein aggregates and not the mature amyloid fibrils. Methods: We have produced two specific conformation-dependent antisera and a number of monoclonal antibodies that recognize mutually exclusive, generic epitopes associated with prefibrillar oligomers and amyloid fibrils. Results: These antibodies specifically recognize these assembly states from many different types of amyloids, indicating that the assembly states share a generic structural motif that may mediate toxicity by common mechanisms. Analysis of synthetic A oligomers by western blotting indicates that the antisera specifically and mutually exclusively stain homogeneous preparations of prefibrillar oligomers and fibrils, but the sizes of bands recognized by the different antibodies are broadly overlapping. These results indicate that size is not a reliable indicator of oligomer conformation. Both antisera detect naturally occurring amyloid oligomers in human AD brain, but the soluble fibrillar oligomers correlate best with disease. Monoclonal antibodies specific for prefibrillar oligomers indicate that structural polymorphisms exist in A prefibrillar oligomers that vary in their reactivity with monoclonal antibodies. Conclusions: These results suggest that distinct structural variants of soluble A oligomers exist, analogous to different strains of prions. Conformation dependent monoclonal antibodies are facile tools for distinguishing different types of soluble oligomers, both in vitro and in vivo and for elucidating the structure of amyloid oligomer variants. MONDAY, JULY 28, 2008 SYMPOSIA S2-03 DISEASE MECHANISMS: TAU AND TAUOPATHIES S2-03-01
TAU-ASSOCIATED NEURODEGENERATION IN ALZHEIMER’S DISEASE AND TAUOPATHY
Maria Grazia Spillantini, Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: Tau is a microtubule-associated protein involved in microtubule assembly and stabilization. Filamentous deposits made of the tau protein constitute a major defining characteristic of several neurodegenerative diseases known as tauopathies including Alzheimer’s disease. The involvement of tau in neurodegeneration has been clarified by the identification of genetic mutations in the Tau gene in cases with familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP17). It is known that these mutations act by destabilizing the binding of tau to microtubules or by altering the ratio of tau isoforms with 3 and 4 repeats that is about 1 in normal human adult brain. Whether mutations in the Tau gene act through a loss or gain of function is not yet clear. The aim of our study is to understand the mechanisms linking tau function/ dysfunction to neurodegeneration and the significance of the tau aggregates that invariably can be found in tauopathies. Tau consists of a microtubule-binding domain in the carboxy-terminal part of the protein and a projection domain in the amino-terminal part. The function of the projection domain remains unclear and mutations in the Tau gene in this region have been reported to affect tau microtubule binding. Methods: Using a bacterial two hybrid system, in vitro assays, immunoprecipitation, immunohistochemistry and electron microscopy we have investigated whether and how any protein was interacting with the amino-terminal part of tau. Results: We found that the projection domain of tau binds to the carboxy-terminal part of dynactin p150 that is part of the motor complex dynein. Furthermore,mutations of a conserved arginine in the projection domain of tau found in FTDP-17 patients affect the binding of tau to dynactin and the distribution of dynactin appears to be altered in brain of patients with some Tau gene mutations. Conclusions: These results suggest a direct involvement of tau in axonal transport and have important implications for understanding the
pathogenesis of tauopathies. This work was supported by the UK Alzheimer’s Research Trust and MRC. S2-03-02
THE TAU TOP 10: 10 THINGS YOU NEED TO KNOW ABOUT THE GENETICS OF MAPT AND COGNITIVE DECLINE
Amanda J. Myers, NIH/NIA, Bethesda, MD, USA. Contact e-mail: [email protected] Background: While paired helical filaments of hyperphosphorylated Tau have been implicated in tauopathy neuropathology for many years, it is only recently that genetic studies have mapped specific variants to disease risk. Methods: Many different approaches have been taken to understand the genetics of the MAPT locus. MAPT was first studied genetically using linkage analysis in families with a rare form of tauopathy (FTDP-17). Current studies have employed both SNP analysis as well as more complex genomics analysis to piece apart the genetic structure of this complex locus. Recent studies have employed different techniques to examine the functional effects of many of the risk variants of MAPT. Results: It is now clear that MAPT variation is strongly associated with risk for progressive supranuclear palsy, corticobasal degeneration and certain forms of FTDP17. Specific sub-variation at the MAPT locus appears to also be implicated in risk for both Alzheimer’s and Parkinson’s disease to a lesser extent. The region encompassing the MAPT locus has a complex genomic architecture, caused by several low copy tandem repeats which created an inversion occurring ⬃ 3 million years ago. Experiments assessing the downstream effects of risk variation in MAPT suggest that both splicing and expression are important factors in disease risk. Transgenic animal models have demonstrated a direct causative relationship between changes in MAPT expression and downstream behavioral effects. Conclusions: MAPT is one of the most widely studied loci for neurodegenerative diseases. Recent data have now mapped genetic variation, genomic variation and the downstream effects in MAPT that are causative for disease. These pathways implicate expression differences as a major player in Tau pathology which might have implications beyond Tau to the pathology of neurodegenerative diseases in general. S2-03-03
TAU DEGRADATION AND THERAPY
Leonard Petrucelli, Mayo Clinic, Jacksonville, FL, USA. Contact email: [email protected] Background: Intracellular aggregation of abnormal phosphorylated species of the microtubule-associated protein tau (p-tau) is a major pathological feature of a family of neurodegenerative disorders, collectively referred to as the tauopathies. The most common tauopathy is Alzheimer’s disease (AD), where p-tau aggregates in neurofibrillary tangles, dystrophic neurites in senile plaques and in cell processes in the neuropil. While these lesions represent visible evidence of p-tau aggregation, the formation of soluble toxic tau species may be more important mediators of tau-associated neurodegeneration. If so, then decreasing p-tau through refolding or degradation may be a plausible therapeutic strategy. Chaperones are molecular machines designed to maintain proteins in a properly folded state. Misfolded or misassembled proteins that cannot be corrected by chaperones are ubiquitinated and targeted for degradation by the proteasome. We and others proposed that the chaperone and proteasome systems may act in concert in clearing the toxic forms of phosphorylated tau (p-tau) by pharmacologically manipulating the activity of Hsp90. When Hsp90 encounters a damaged protein, it can send it down one of two pathways, either refolding the protein, or targeting it for degradation with the help of one of its co-factors, CHIP. Small molecules that block the Hsp90 ATPase activity, and thus its folding capacity, enhance the degradation of substrates. Methods: Biochemical, immunohistochemical and neuropathological approaches were employed for these studies. Results: We report that peripheral administration of a novel Hsp90 inhibitor (EC102) promotes selective decreases in p-tau species in a mouse model of tauopathy. Moreover, Hsp90 derived from affected tissue of human AD brain binds to EC102
Symposia S2-03: Disease Mechanisms: Tau and Tauopathies
Background: Age-related neurodegenerative diseases share a number of important pathological features, such as accumulation of misfolded proteins as amyloid oligomers and fibrils and dysregulation of cellular function. Recent evidence suggests that soluble amyloid oligomers may represent the primary pathological species of protein aggregates and not the mature amyloid fibrils. Methods: We have produced two specific conformation-dependent antisera and a number of monoclonal antibodies that recognize mutually exclusive, generic epitopes associated with prefibrillar oligomers and amyloid fibrils. Results: These antibodies specifically recognize these assembly states from many different types of amyloids, indicating that the assembly states share a generic structural motif that may mediate toxicity by common mechanisms. Analysis of synthetic A oligomers by western blotting indicates that the antisera specifically and mutually exclusively stain homogeneous preparations of prefibrillar oligomers and fibrils, but the sizes of bands recognized by the different antibodies are broadly overlapping. These results indicate that size is not a reliable indicator of oligomer conformation. Both antisera detect naturally occurring amyloid oligomers in human AD brain, but the soluble fibrillar oligomers correlate best with disease. Monoclonal antibodies specific for prefibrillar oligomers indicate that structural polymorphisms exist in A prefibrillar oligomers that vary in their reactivity with monoclonal antibodies. Conclusions: These results suggest that distinct structural variants of soluble A oligomers exist, analogous to different strains of prions. Conformation dependent monoclonal antibodies are facile tools for distinguishing different types of soluble oligomers, both in vitro and in vivo and for elucidating the structure of amyloid oligomer variants. MONDAY, JULY 28, 2008 SYMPOSIA S2-03 DISEASE MECHANISMS: TAU AND TAUOPATHIES S2-03-01
TAU-ASSOCIATED NEURODEGENERATION IN ALZHEIMER’S DISEASE AND TAUOPATHY
Maria Grazia Spillantini, Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: Tau is a microtubule-associated protein involved in microtubule assembly and stabilization. Filamentous deposits made of the tau protein constitute a major defining characteristic of several neurodegenerative diseases known as tauopathies including Alzheimer’s disease. The involvement of tau in neurodegeneration has been clarified by the identification of genetic mutations in the Tau gene in cases with familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP17). It is known that these mutations act by destabilizing the binding of tau to microtubules or by altering the ratio of tau isoforms with 3 and 4 repeats that is about 1 in normal human adult brain. Whether mutations in the Tau gene act through a loss or gain of function is not yet clear. The aim of our study is to understand the mechanisms linking tau function/ dysfunction to neurodegeneration and the significance of the tau aggregates that invariably can be found in tauopathies. Tau consists of a microtubule-binding domain in the carboxy-terminal part of the protein and a projection domain in the amino-terminal part. The function of the projection domain remains unclear and mutations in the Tau gene in this region have been reported to affect tau microtubule binding. Methods: Using a bacterial two hybrid system, in vitro assays, immunoprecipitation, immunohistochemistry and electron microscopy we have investigated whether and how any protein was interacting with the amino-terminal part of tau. Results: We found that the projection domain of tau binds to the carboxy-terminal part of dynactin p150 that is part of the motor complex dynein. Furthermore,mutations of a conserved arginine in the projection domain of tau found in FTDP-17 patients affect the binding of tau to dynactin and the distribution of dynactin appears to be altered in brain of patients with some Tau gene mutations. Conclusions: These results suggest a direct involvement of tau in axonal transport and have important implications for understanding the
pathogenesis of tauopathies. This work was supported by the UK Alzheimer’s Research Trust and MRC. S2-03-02
THE TAU TOP 10: 10 THINGS YOU NEED TO KNOW ABOUT THE GENETICS OF MAPT AND COGNITIVE DECLINE
Amanda J. Myers, NIH/NIA, Bethesda, MD, USA. Contact e-mail: [email protected] Background: While paired helical filaments of hyperphosphorylated Tau have been implicated in tauopathy neuropathology for many years, it is only recently that genetic studies have mapped specific variants to disease risk. Methods: Many different approaches have been taken to understand the genetics of the MAPT locus. MAPT was first studied genetically using linkage analysis in families with a rare form of tauopathy (FTDP-17). Current studies have employed both SNP analysis as well as more complex genomics analysis to piece apart the genetic structure of this complex locus. Recent studies have employed different techniques to examine the functional effects of many of the risk variants of MAPT. Results: It is now clear that MAPT variation is strongly associated with risk for progressive supranuclear palsy, corticobasal degeneration and certain forms of FTDP17. Specific sub-variation at the MAPT locus appears to also be implicated in risk for both Alzheimer’s and Parkinson’s disease to a lesser extent. The region encompassing the MAPT locus has a complex genomic architecture, caused by several low copy tandem repeats which created an inversion occurring ⬃ 3 million years ago. Experiments assessing the downstream effects of risk variation in MAPT suggest that both splicing and expression are important factors in disease risk. Transgenic animal models have demonstrated a direct causative relationship between changes in MAPT expression and downstream behavioral effects. Conclusions: MAPT is one of the most widely studied loci for neurodegenerative diseases. Recent data have now mapped genetic variation, genomic variation and the downstream effects in MAPT that are causative for disease. These pathways implicate expression differences as a major player in Tau pathology which might have implications beyond Tau to the pathology of neurodegenerative diseases in general. S2-03-03
TAU DEGRADATION AND THERAPY
Leonard Petrucelli, Mayo Clinic, Jacksonville, FL, USA. Contact email: [email protected] Background: Intracellular aggregation of abnormal phosphorylated species of the microtubule-associated protein tau (p-tau) is a major pathological feature of a family of neurodegenerative disorders, collectively referred to as the tauopathies. The most common tauopathy is Alzheimer’s disease (AD), where p-tau aggregates in neurofibrillary tangles, dystrophic neurites in senile plaques and in cell processes in the neuropil. While these lesions represent visible evidence of p-tau aggregation, the formation of soluble toxic tau species may be more important mediators of tau-associated neurodegeneration. If so, then decreasing p-tau through refolding or degradation may be a plausible therapeutic strategy. Chaperones are molecular machines designed to maintain proteins in a properly folded state. Misfolded or misassembled proteins that cannot be corrected by chaperones are ubiquitinated and targeted for degradation by the proteasome. We and others proposed that the chaperone and proteasome systems may act in concert in clearing the toxic forms of phosphorylated tau (p-tau) by pharmacologically manipulating the activity of Hsp90. When Hsp90 encounters a damaged protein, it can send it down one of two pathways, either refolding the protein, or targeting it for degradation with the help of one of its co-factors, CHIP. Small molecules that block the Hsp90 ATPase activity, and thus its folding capacity, enhance the degradation of substrates. Methods: Biochemical, immunohistochemical and neuropathological approaches were employed for these studies. Results: We report that peripheral administration of a novel Hsp90 inhibitor (EC102) promotes selective decreases in p-tau species in a mouse model of tauopathy. Moreover, Hsp90 derived from affected tissue of human AD brain binds to EC102