- Email: [email protected]
PL-02-02: Amyloid precursor sorting and processing: Regulation by transmitters, hormones, and protein phosphorylation mechanisms
T122
Plenary PL-02
toskeleton integrity was assessed by immunohistochemistry and western blotting against tubulin, MAP2, neurofilament, and Tau protein. The presence of Caspase-6-cleaved proteins in AD was assessed by immunohistochemistry with neoepitope antisera. Results: Tubulin, MAP2, and Tau, but not Neurofilament proteins, are cleaved by Caspase-6. Whereas tubulin and MAP2 are homogenously distributed in the neurites of primary human neurons in normal culture conditions, they take a beaded appearance in serum-deprived conditions. Tau protein also appears beaded in serum deprivation and additionally redistributes around the nuclei. In contrast, the neurofilaments remain intact with serum deprivation. Caspase-6 inhibitors prevent the cytoskeleton beading and Tau redistribution. Tubulin and Tau cleaved by Caspase-6 are present in AD brains. Conclusions: These results show that the activation of Caspase-6 in human neurons disrupts the integrity of the cytoskeleton. Because of the importance of the cytoskeleton in proper vesicular and protein trafficking, it is likely that the activation of Caspase-6 not only disrupts the architecture but the function of neurons. Together, these results suggest that the activation of Caspase-6 disrupts cytoskeletal integrity and neuronal function in AD. MONDAY, JULY 28, 2008 PLENARY PL-02 PL-02-01
TAU MODIFICATIONS, AGGREGATION AND TOXICITY
Lester I. Binder, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Tau proteins normally interact with microtubules in the nervous system. During the course of neurodegenerative diseases such as Alzheimer’s disease (AD) and tauopathies, these proteins dissociate from the microtubule and begin to aggregate. The resultant tau filaments are thought by many to be toxic although the nature of this toxicity is, as yet, unknown. The tau molecule aggregates by association with its repeat region; this area of the molecule that normally mediates tau’s association with microtubules. We have discovered that the carboxy and amino regions of the molecule constitute control elements in tau aggregation with the carboxy tail region being inhibitory and the amino region being facilitative of the filament assembly process. Methods: For the past several years, we have been studying mechanisms by which the cell modulates these regions of the molecule in disease. Using molecular biological, biochemical and immunological procedures, we have discovered that these regions can be nitrated on tyrosines or truncated by enzymes such as caspases. Results: The result of changes such as these are modeled in vitro to assess their affect on tau filament formation and assayed in situ in AD and tauopathies using appropriately targeted monoclonal antibodies. Additionally, we have used different tau isoforms and modified polymers to assess their affect on axonal transport in isolated axoplasm from the squid giant axon. Tau filaments and certain non-canonical isoforms of tau that contain only the amino region of the molecule inhibit anterograde but not retrograde transport through a signal transduction cascade involving specific phosphatases and GSK3. Conclusions: Taken together, our results indicate that the amino terminal region of tau may be a primary site of toxicity in neurodegenerative disease. Supported by Grants AG14453, AG09466, AG021184, and AG021661. PL-02-02
AMYLOID PRECURSOR SORTING AND PROCESSING: REGULATION BY TRANSMITTERS, HORMONES, AND PROTEIN PHOSPHORYLATION MECHANISMS
Sam Gandy, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: The identification of potential phospho-acceptor sites in the cytoplasmic tail of the Alzheimer’s amyloid precursor (APP), reported by our group in 1988, led to the discovery of a robust mechanism for direct and indirect regulation of APP sorting and processing by secretases. Over the ensuing twenty years, many labs have confirmed and extended the key initial
observation: i.e., that protein phosphatases 1 and 2A (PP1, PP2A), protein kinase C (PKC), extracellular signaled regulated protein kinase (ERK), cyclindependent protein kinase 5 (cdk5), rho kinase (ROCK), and the transmitters and hormones linked to these enzymes (acetylcholine, interleukins, estradiol, testosterone, insulin, isoprenoids) control formation of sAPP␣ (via ectodomain shedding) and 〈, usually in reciprocal fashion. Methods: Studies in continuous cultures, primary cultures, brain slices, and experimental animals have confirmed the physiological relevance of “regulated cleavage” of APP. Work from our own lab will be reviewed as well as collaborative and/or independent work from over 50 labs. Results: Activation of biogenesis of APP-bearing transport vesicles at the trans Golgi network is one subcellular mechanism underlying “regulated cleavage”, but indirect evidence also implicates plasma membrane localized mechanisms. The physiological impact of direct phosphorylation of APP (especially by cdk5) remains an area of ongoing interest and evolution of our understanding. Conclusions: Regulated cleavage of APP is one of the most studied and most robust mechanisms for dynamic modulation of APP ectodomain shedding and A generation via integration, on a moment to moment basis, of multiple intracellular signals, including those transduced from the cell surface. Human studies and drug trials suggest that direct and indirect protein phosphorylation signaling mechanisms may contribute to the predisposition to the cerebral amyloidosis of Alzheimer’s disease and potentially to its pharmacotherapy. Supported by NIA, the Atkins Foundation, and Cure Alzheimer’s Fund. PL-02-03
THE NEUROPATHOLOGY OF THE NONALZHEIMER’S DISEASE DEMENTIAS
Dennis W. Dickson, Neuropathology Laboratory, Mayo Clinic College of Medicine, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Fundamental to all neurodegenerative disorders is selective loss of neurons. The anatomical distribution of neuronal loss correlates with the clinical syndrome, but a number of different disorders can have the same clinical syndrome. Modern classifications of neurodegenerative disorders are based on biological attributes: amyloidoses (A - Alzheimer’s disease (AD); PrP - Creutzfeldt-Jakob disease (CJD)); tauopathies (Pick’s disease (PiD); corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD)); ␣-synucleinopathies (dementia with Lewy bodies (DLB), Parkinson disease dementia (PDD), atypical multiple system atrophy (MSA)); and TDP-43 proteinopathies (frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U); frontotemporal lobar degeneration with motor neuron degeneration (FTLD-MND)). Methods: Diagnostic neuropathology employs histology and immunohistochemistry. The latter provides specificity, information about molecular pathology and can be standardized in multi-center studies. Results: AD is characterized by amyloid plaques composed of A and neurofibrillary tangles (NFT) composed of tau. Other disorders (e.g., CJD) may have non-A amyloid (e.g., PrP). Tau-immunoreactive neuronal inclusions characterize PiD, PSP, CBD and AGD. Unlike AD, most tauopathies have glial lesions - “astrocytic plaques” in CBD, “tufted astrocytes” in PSP and oligodendroglial “coiled bodies” in AGD. Tauopathies are subdivided according to the tau isoform that accumulates. PSP, CBD and AGD are 4R tauopathies and PiD is a 3R tauopathy. Lewy bodies, neuronal cytoplasmic inclusions composed of ␣-synuclein, are the histopathologic hallmark of Parkinson’s disease (PD). They are more numerous in PDD and DLB. In MSA ␣-synuclein accumulates in oligodendroglia. Until recently, ubiquitin immunohistochemistry was used to diagnose FTLD-U and FTLD-MND, but TDP-43 is now known to be a more specific marker. These disorders are referred to as TDP-43 proteinopathies. A few cases of FTLD-U are negative for TDP-43. In some of these disorders other proteins (e.g., ␣-internexin) make up the inclusions. Conclusions: Given the frequency of AD, it is important that all neurodegenerative disorders be screened for AD pathology. Subsequently, the diagnostic algorithm should exclude ␣-synucleinopathies, tauopathies and TDP-43 proteinopathies. Less common disorders may require additional methods to determine the specific type of amyloid that accumulates or the specific protein composition of intraneuronal inclusions.
Plenary PL-02
toskeleton integrity was assessed by immunohistochemistry and western blotting against tubulin, MAP2, neurofilament, and Tau protein. The presence of Caspase-6-cleaved proteins in AD was assessed by immunohistochemistry with neoepitope antisera. Results: Tubulin, MAP2, and Tau, but not Neurofilament proteins, are cleaved by Caspase-6. Whereas tubulin and MAP2 are homogenously distributed in the neurites of primary human neurons in normal culture conditions, they take a beaded appearance in serum-deprived conditions. Tau protein also appears beaded in serum deprivation and additionally redistributes around the nuclei. In contrast, the neurofilaments remain intact with serum deprivation. Caspase-6 inhibitors prevent the cytoskeleton beading and Tau redistribution. Tubulin and Tau cleaved by Caspase-6 are present in AD brains. Conclusions: These results show that the activation of Caspase-6 in human neurons disrupts the integrity of the cytoskeleton. Because of the importance of the cytoskeleton in proper vesicular and protein trafficking, it is likely that the activation of Caspase-6 not only disrupts the architecture but the function of neurons. Together, these results suggest that the activation of Caspase-6 disrupts cytoskeletal integrity and neuronal function in AD. MONDAY, JULY 28, 2008 PLENARY PL-02 PL-02-01
TAU MODIFICATIONS, AGGREGATION AND TOXICITY
Lester I. Binder, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Tau proteins normally interact with microtubules in the nervous system. During the course of neurodegenerative diseases such as Alzheimer’s disease (AD) and tauopathies, these proteins dissociate from the microtubule and begin to aggregate. The resultant tau filaments are thought by many to be toxic although the nature of this toxicity is, as yet, unknown. The tau molecule aggregates by association with its repeat region; this area of the molecule that normally mediates tau’s association with microtubules. We have discovered that the carboxy and amino regions of the molecule constitute control elements in tau aggregation with the carboxy tail region being inhibitory and the amino region being facilitative of the filament assembly process. Methods: For the past several years, we have been studying mechanisms by which the cell modulates these regions of the molecule in disease. Using molecular biological, biochemical and immunological procedures, we have discovered that these regions can be nitrated on tyrosines or truncated by enzymes such as caspases. Results: The result of changes such as these are modeled in vitro to assess their affect on tau filament formation and assayed in situ in AD and tauopathies using appropriately targeted monoclonal antibodies. Additionally, we have used different tau isoforms and modified polymers to assess their affect on axonal transport in isolated axoplasm from the squid giant axon. Tau filaments and certain non-canonical isoforms of tau that contain only the amino region of the molecule inhibit anterograde but not retrograde transport through a signal transduction cascade involving specific phosphatases and GSK3. Conclusions: Taken together, our results indicate that the amino terminal region of tau may be a primary site of toxicity in neurodegenerative disease. Supported by Grants AG14453, AG09466, AG021184, and AG021661. PL-02-02
AMYLOID PRECURSOR SORTING AND PROCESSING: REGULATION BY TRANSMITTERS, HORMONES, AND PROTEIN PHOSPHORYLATION MECHANISMS
Sam Gandy, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: The identification of potential phospho-acceptor sites in the cytoplasmic tail of the Alzheimer’s amyloid precursor (APP), reported by our group in 1988, led to the discovery of a robust mechanism for direct and indirect regulation of APP sorting and processing by secretases. Over the ensuing twenty years, many labs have confirmed and extended the key initial
observation: i.e., that protein phosphatases 1 and 2A (PP1, PP2A), protein kinase C (PKC), extracellular signaled regulated protein kinase (ERK), cyclindependent protein kinase 5 (cdk5), rho kinase (ROCK), and the transmitters and hormones linked to these enzymes (acetylcholine, interleukins, estradiol, testosterone, insulin, isoprenoids) control formation of sAPP␣ (via ectodomain shedding) and 〈, usually in reciprocal fashion. Methods: Studies in continuous cultures, primary cultures, brain slices, and experimental animals have confirmed the physiological relevance of “regulated cleavage” of APP. Work from our own lab will be reviewed as well as collaborative and/or independent work from over 50 labs. Results: Activation of biogenesis of APP-bearing transport vesicles at the trans Golgi network is one subcellular mechanism underlying “regulated cleavage”, but indirect evidence also implicates plasma membrane localized mechanisms. The physiological impact of direct phosphorylation of APP (especially by cdk5) remains an area of ongoing interest and evolution of our understanding. Conclusions: Regulated cleavage of APP is one of the most studied and most robust mechanisms for dynamic modulation of APP ectodomain shedding and A generation via integration, on a moment to moment basis, of multiple intracellular signals, including those transduced from the cell surface. Human studies and drug trials suggest that direct and indirect protein phosphorylation signaling mechanisms may contribute to the predisposition to the cerebral amyloidosis of Alzheimer’s disease and potentially to its pharmacotherapy. Supported by NIA, the Atkins Foundation, and Cure Alzheimer’s Fund. PL-02-03
THE NEUROPATHOLOGY OF THE NONALZHEIMER’S DISEASE DEMENTIAS
Dennis W. Dickson, Neuropathology Laboratory, Mayo Clinic College of Medicine, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Fundamental to all neurodegenerative disorders is selective loss of neurons. The anatomical distribution of neuronal loss correlates with the clinical syndrome, but a number of different disorders can have the same clinical syndrome. Modern classifications of neurodegenerative disorders are based on biological attributes: amyloidoses (A - Alzheimer’s disease (AD); PrP - Creutzfeldt-Jakob disease (CJD)); tauopathies (Pick’s disease (PiD); corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD)); ␣-synucleinopathies (dementia with Lewy bodies (DLB), Parkinson disease dementia (PDD), atypical multiple system atrophy (MSA)); and TDP-43 proteinopathies (frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U); frontotemporal lobar degeneration with motor neuron degeneration (FTLD-MND)). Methods: Diagnostic neuropathology employs histology and immunohistochemistry. The latter provides specificity, information about molecular pathology and can be standardized in multi-center studies. Results: AD is characterized by amyloid plaques composed of A and neurofibrillary tangles (NFT) composed of tau. Other disorders (e.g., CJD) may have non-A amyloid (e.g., PrP). Tau-immunoreactive neuronal inclusions characterize PiD, PSP, CBD and AGD. Unlike AD, most tauopathies have glial lesions - “astrocytic plaques” in CBD, “tufted astrocytes” in PSP and oligodendroglial “coiled bodies” in AGD. Tauopathies are subdivided according to the tau isoform that accumulates. PSP, CBD and AGD are 4R tauopathies and PiD is a 3R tauopathy. Lewy bodies, neuronal cytoplasmic inclusions composed of ␣-synuclein, are the histopathologic hallmark of Parkinson’s disease (PD). They are more numerous in PDD and DLB. In MSA ␣-synuclein accumulates in oligodendroglia. Until recently, ubiquitin immunohistochemistry was used to diagnose FTLD-U and FTLD-MND, but TDP-43 is now known to be a more specific marker. These disorders are referred to as TDP-43 proteinopathies. A few cases of FTLD-U are negative for TDP-43. In some of these disorders other proteins (e.g., ␣-internexin) make up the inclusions. Conclusions: Given the frequency of AD, it is important that all neurodegenerative disorders be screened for AD pathology. Subsequently, the diagnostic algorithm should exclude ␣-synucleinopathies, tauopathies and TDP-43 proteinopathies. Less common disorders may require additional methods to determine the specific type of amyloid that accumulates or the specific protein composition of intraneuronal inclusions.