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Proteoglycans and regenerative sprouting of neurites
NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE MECHANISMS OF NEURONAL DEGENERATION
115 MODIFICATIONS OF MEMBRANE B-AMYLOID PRECURSOR PROTEIN ASSEMBLIES IN ALZHEIMER'S DISEASE AND IN SQUID GIANT NERVE FIBERS. *J. Metuzals I, H. M. Fishman 2, V. Montpetit I, Y. Robitaille 3, S. Gauthier 3. IDepartment of Pathology. University of Ottawa, Ottawa, Ontario, Canada; 2Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas, USA; 3McGill Centre for the Study of Aging, Montreal General Hospital, Montreal, Quebec, Canada. The membrane - cytoskeleton complex is of importance for the normal functioning of the neuron as well as for Alzheimer's disease (AD) and Creutzfeldt-Jacob disease (CJD). The modifications of the 6-amyloid precursor protein (B-APP) in association with the corresponding changes of the membranes and the attached cytoskeleton are crucial. We have investigated 15 frontal lobe biopsies from AD patients by immuno-electron microscopy using antisera against A4 and C-terminal of the B-APP. We have also investigated the spongiform vacuolation induced experimentally in the squid giant nerve fiber. Membrane profiles display a wide variety of modifications in all parts of the neurons in AD brains. Membranous aggregates, vesicles of all sizes, virus-like particles and vacuoles of different outlines can be regularly observed. In dendrites the vesicles are in spongiform arrays similar to those observed in CJD brains. These dendrites show randomly arranged filaments and paired helical filaments (PHFs) in association with the membranes. The membranes display different forms of endocytotic buds similar to viruses and coated vesicles. We conclude that the spongiform dendritic modifications in AD represent the initial phase of the neuron response to different types of stress - temperature, injury, toxic agents and viral infection. The experiments with squid giant nerve fibers prove that the spongiform vacuolation of the neuroplasm is not an artifact induced by the treatment of the specimen. Peptides of the transmembrane B-APP and of the cytoskeleton, modified by Ca*+-activated proteases, reassemble into insoluble PHFs. Retroviruses may be involved in the pathogenesis of AD. Overlapping of the pathogenic processes between AD and CJD may exist. Supported by MRC of Canada and AHAF.
116 ABNORMAL PHOSPHORYLATION OF TAU PRECEDES UBIQUlTINATION IN NEUROFIBRILLARY PATHOLOGY OF ALZHEIMER DISEASE. *C. Bancher', I. Grundke-lqbai', K. Iqbal', V.A. Fried=, H.T. Smith =, H.M. Wisniewski.' 'NYS Inst. for Basic Res. Staten Island, NY and =St. Jude Children's Ras. Hosp. Memphis, TN, USA. Neurofibrillary tangles (NFT) of paired helical filaments (PHF) are a hallmark of the neuropathology characteristic of Alzheimer disease. Based on a morphological classification of NFT by different stages of maturation, we have shown previously that the intraneuronai accumulation of abnormally phophorylated form of microtubule associated protein tau precedes the formation of compact NFT. The expression of an epitope of ubiquitin recognized by an antibody raised against PHF appeared to occur later in the process of tangle formation (Brain Research, 477:90-99, 1989). In the present study we report further immunocytochemical characterization ofthe pretangle neurons. Formalin fixed, paraffin embedded tissue blocks of hippocampus and adjacent temporal cortex of histopathologically confirmed Alzheimer disease patients and of an aged individual with profound mental retardation have been cut into 5- to 6- um thick gapless serial sections and used for immunohistochemical staining with antibodies to different antigenic sites of tau and ubiquitin. On tissue sections of Alzheimer brain, four antibodies to tau stain not only neurofibrillary tangles and dystrophic plaque and neuropil neurites but also a subset of hippocampal and cortical neuronal cytoplasms devoid of a tangle we believe to be at a stage of neuronal alteration preceding the formation of paired helical filaments (PHF). Pratreatment of tissue sections with alkaline phosphatase leads to an increase in staining intensity and in number of immunoreactive lesions with antibodies directed to an amino terminal and to a mid-region of the tau molecule. The diffuse neuronal staining could not be observed with any of seven monoclonal antibodies recognizing ubiquitin. We conclude
281
(1) that abnormal phosphorylation of tau occurs prior to its incorporation into PHF and leads to its accumulation in the nerve cell body and (2) that ubiquitin is seen associated only when a neurofibrillary tangle of PHF is already formed.
117 ABNORMALLY PHOSPHORYLATED TAU ISOLATED FROM ALZHEIMER DISEASE BRAIN CYTOSOL IS NOT UBIQUITINATED * E. KoepkeSecundo, I. Grundke-lqbal and K. Iqbal. Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A. Polypeptide composition of PHF isolated from Alzheimer disease (AD) brain has revealed that micro-tubule associated protein tau is a major component of these aberrant fibrils and tau in PHF has been shown to be abnormally phosphorylated. A second protein which has been identified in PHF is ubiquitin. In the present study we present evidence showing that tau in AD brain is abnormally phosphorylated prior to its ;ncorporation into tangles. Abnormally phosphorylated tau from AD brain was isolated from the 27,000 x g to 103,000 x g fraction of brain homogenats prepared in the presence of a cocktail of phosphatase and protease inhibitors. Unlike normal tau, the abnormally phosphorylated tau was found to sediment at 103,000 x g, and was found to be non-congophilic. The pellet was extracted in 8 M urea and then renatured from it by dialysis against 50 mM Tris, pH 7.2. After this treatment the abnormal tau stayed in solution and could not be sedimanted even at 333,850 x g for 15 minutes. Further purification of the abnormal tau was carried out by phosphocellulose chromatography where it eluted ahead of the normal tau. The abnormal phosphorylation of the isolated tau was confirmed by Western blots developed with monoclonal antibody Tau-1. However, unlike PHF tau, this unpolymerized abnormally phosphorylated tau was not labeled on Western blots with monoclonal antibodies 3-39 and 5-25 which recognize the amino acid residues 50-65 and 64-76 of ubiquitin respectively. These studies show a method for the isolation of abnormally phosphorylated tau in unpolymerized form and suggest (1) that accumulation of abnormally phosphorylated tau precedes its polymerization into PHF and (2) that the incorporation of ubiquitin in AIzheimer neurofibrillary tangles is a later event which most likely is initiated after the formation of PHF. (Supported in part by AHAF and NIH grants NS 18105, AGO5892, AGO4220).
118 PROTEOGLYCAN$AND REGENERATIVESPROUTING OF NEURITES. *R.J. RlopeUe,fEE. Dow, P. Johnson-Green,P. Merchar¢ Apps Medical ResearchCentre, Kingston General Hospital and Queen's University, Kingston, Ontario, K7L 2V7 Canada. Proteoglycansof neuronaland astrocytlc origin are endowed with neurite growthpromoting activity that differs quantitativelyand qualitativelyfrom neurite extension on other substrstes. Primaryastrocyteain vitro produce four proteoglycans- hyaluronate, heparan sulphate, chondroltln sulphate,and dermatan sulphate, while regenerstlng neuronsin vitro produce only haparan sulphate and chondroitin sulphate. In both cases, haparansulphate proteoglynans(HSPG's) from neurons and astrocytes are endowed with neurite growth-promoting activity when complexed to laminin. During regenerative growth of CNS axons in vivo, biosynthesis and orthograde axonal transport of heparan sulphate and chrondroitln sulphate proteoglycans are Increased, and In NGF-responslveneurons this trophlc factor Induces HSPG's with neurite-promotingactivity. These observations bear upon the findings of altered neurites, expression of microtubule-associated proteins, and accumulation of proteoglycans in Alzheimer's disease, and may suggest that soma pathological changes in the disease reflect an abortiveregenerativeresponseby neurons to an unknowninsult. Supportedby MRC Canada,NIH USA. 119 UBIQUITIN CARBOXYL-TERMINAL HYDROLASE (PGP9.5] IS SELECTIVELY PRESENT IN UBIQUITINATED INCLUSION BODIES CHARACTERISTIC OF HUMAN NEURODEGENERATIVE DISEASE. *R. John Mayer', Helen McDermott', Michael Landont , James Lowe'- Keith D. Wilkinson'. Departments of Biochemistry', Pathology2 , Queen's Medical Centre, Nottingham, U.K. and Department of Biochemistry', Emory University School of Medicine, Atlanta, U.S.A.
115 MODIFICATIONS OF MEMBRANE B-AMYLOID PRECURSOR PROTEIN ASSEMBLIES IN ALZHEIMER'S DISEASE AND IN SQUID GIANT NERVE FIBERS. *J. Metuzals I, H. M. Fishman 2, V. Montpetit I, Y. Robitaille 3, S. Gauthier 3. IDepartment of Pathology. University of Ottawa, Ottawa, Ontario, Canada; 2Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas, USA; 3McGill Centre for the Study of Aging, Montreal General Hospital, Montreal, Quebec, Canada. The membrane - cytoskeleton complex is of importance for the normal functioning of the neuron as well as for Alzheimer's disease (AD) and Creutzfeldt-Jacob disease (CJD). The modifications of the 6-amyloid precursor protein (B-APP) in association with the corresponding changes of the membranes and the attached cytoskeleton are crucial. We have investigated 15 frontal lobe biopsies from AD patients by immuno-electron microscopy using antisera against A4 and C-terminal of the B-APP. We have also investigated the spongiform vacuolation induced experimentally in the squid giant nerve fiber. Membrane profiles display a wide variety of modifications in all parts of the neurons in AD brains. Membranous aggregates, vesicles of all sizes, virus-like particles and vacuoles of different outlines can be regularly observed. In dendrites the vesicles are in spongiform arrays similar to those observed in CJD brains. These dendrites show randomly arranged filaments and paired helical filaments (PHFs) in association with the membranes. The membranes display different forms of endocytotic buds similar to viruses and coated vesicles. We conclude that the spongiform dendritic modifications in AD represent the initial phase of the neuron response to different types of stress - temperature, injury, toxic agents and viral infection. The experiments with squid giant nerve fibers prove that the spongiform vacuolation of the neuroplasm is not an artifact induced by the treatment of the specimen. Peptides of the transmembrane B-APP and of the cytoskeleton, modified by Ca*+-activated proteases, reassemble into insoluble PHFs. Retroviruses may be involved in the pathogenesis of AD. Overlapping of the pathogenic processes between AD and CJD may exist. Supported by MRC of Canada and AHAF.
116 ABNORMAL PHOSPHORYLATION OF TAU PRECEDES UBIQUlTINATION IN NEUROFIBRILLARY PATHOLOGY OF ALZHEIMER DISEASE. *C. Bancher', I. Grundke-lqbai', K. Iqbal', V.A. Fried=, H.T. Smith =, H.M. Wisniewski.' 'NYS Inst. for Basic Res. Staten Island, NY and =St. Jude Children's Ras. Hosp. Memphis, TN, USA. Neurofibrillary tangles (NFT) of paired helical filaments (PHF) are a hallmark of the neuropathology characteristic of Alzheimer disease. Based on a morphological classification of NFT by different stages of maturation, we have shown previously that the intraneuronai accumulation of abnormally phophorylated form of microtubule associated protein tau precedes the formation of compact NFT. The expression of an epitope of ubiquitin recognized by an antibody raised against PHF appeared to occur later in the process of tangle formation (Brain Research, 477:90-99, 1989). In the present study we report further immunocytochemical characterization ofthe pretangle neurons. Formalin fixed, paraffin embedded tissue blocks of hippocampus and adjacent temporal cortex of histopathologically confirmed Alzheimer disease patients and of an aged individual with profound mental retardation have been cut into 5- to 6- um thick gapless serial sections and used for immunohistochemical staining with antibodies to different antigenic sites of tau and ubiquitin. On tissue sections of Alzheimer brain, four antibodies to tau stain not only neurofibrillary tangles and dystrophic plaque and neuropil neurites but also a subset of hippocampal and cortical neuronal cytoplasms devoid of a tangle we believe to be at a stage of neuronal alteration preceding the formation of paired helical filaments (PHF). Pratreatment of tissue sections with alkaline phosphatase leads to an increase in staining intensity and in number of immunoreactive lesions with antibodies directed to an amino terminal and to a mid-region of the tau molecule. The diffuse neuronal staining could not be observed with any of seven monoclonal antibodies recognizing ubiquitin. We conclude
281
(1) that abnormal phosphorylation of tau occurs prior to its incorporation into PHF and leads to its accumulation in the nerve cell body and (2) that ubiquitin is seen associated only when a neurofibrillary tangle of PHF is already formed.
117 ABNORMALLY PHOSPHORYLATED TAU ISOLATED FROM ALZHEIMER DISEASE BRAIN CYTOSOL IS NOT UBIQUITINATED * E. KoepkeSecundo, I. Grundke-lqbal and K. Iqbal. Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A. Polypeptide composition of PHF isolated from Alzheimer disease (AD) brain has revealed that micro-tubule associated protein tau is a major component of these aberrant fibrils and tau in PHF has been shown to be abnormally phosphorylated. A second protein which has been identified in PHF is ubiquitin. In the present study we present evidence showing that tau in AD brain is abnormally phosphorylated prior to its ;ncorporation into tangles. Abnormally phosphorylated tau from AD brain was isolated from the 27,000 x g to 103,000 x g fraction of brain homogenats prepared in the presence of a cocktail of phosphatase and protease inhibitors. Unlike normal tau, the abnormally phosphorylated tau was found to sediment at 103,000 x g, and was found to be non-congophilic. The pellet was extracted in 8 M urea and then renatured from it by dialysis against 50 mM Tris, pH 7.2. After this treatment the abnormal tau stayed in solution and could not be sedimanted even at 333,850 x g for 15 minutes. Further purification of the abnormal tau was carried out by phosphocellulose chromatography where it eluted ahead of the normal tau. The abnormal phosphorylation of the isolated tau was confirmed by Western blots developed with monoclonal antibody Tau-1. However, unlike PHF tau, this unpolymerized abnormally phosphorylated tau was not labeled on Western blots with monoclonal antibodies 3-39 and 5-25 which recognize the amino acid residues 50-65 and 64-76 of ubiquitin respectively. These studies show a method for the isolation of abnormally phosphorylated tau in unpolymerized form and suggest (1) that accumulation of abnormally phosphorylated tau precedes its polymerization into PHF and (2) that the incorporation of ubiquitin in AIzheimer neurofibrillary tangles is a later event which most likely is initiated after the formation of PHF. (Supported in part by AHAF and NIH grants NS 18105, AGO5892, AGO4220).
118 PROTEOGLYCAN$AND REGENERATIVESPROUTING OF NEURITES. *R.J. RlopeUe,fEE. Dow, P. Johnson-Green,P. Merchar¢ Apps Medical ResearchCentre, Kingston General Hospital and Queen's University, Kingston, Ontario, K7L 2V7 Canada. Proteoglycansof neuronaland astrocytlc origin are endowed with neurite growthpromoting activity that differs quantitativelyand qualitativelyfrom neurite extension on other substrstes. Primaryastrocyteain vitro produce four proteoglycans- hyaluronate, heparan sulphate, chondroltln sulphate,and dermatan sulphate, while regenerstlng neuronsin vitro produce only haparan sulphate and chondroitin sulphate. In both cases, haparansulphate proteoglynans(HSPG's) from neurons and astrocytes are endowed with neurite growth-promoting activity when complexed to laminin. During regenerative growth of CNS axons in vivo, biosynthesis and orthograde axonal transport of heparan sulphate and chrondroitln sulphate proteoglycans are Increased, and In NGF-responslveneurons this trophlc factor Induces HSPG's with neurite-promotingactivity. These observations bear upon the findings of altered neurites, expression of microtubule-associated proteins, and accumulation of proteoglycans in Alzheimer's disease, and may suggest that soma pathological changes in the disease reflect an abortiveregenerativeresponseby neurons to an unknowninsult. Supportedby MRC Canada,NIH USA. 119 UBIQUITIN CARBOXYL-TERMINAL HYDROLASE (PGP9.5] IS SELECTIVELY PRESENT IN UBIQUITINATED INCLUSION BODIES CHARACTERISTIC OF HUMAN NEURODEGENERATIVE DISEASE. *R. John Mayer', Helen McDermott', Michael Landont , James Lowe'- Keith D. Wilkinson'. Departments of Biochemistry', Pathology2 , Queen's Medical Centre, Nottingham, U.K. and Department of Biochemistry', Emory University School of Medicine, Atlanta, U.S.A.