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169 Protein phosphatase calcineurin and dementia
FIFTH INTERNATIONAL
CONFERENCE
ON ALZHEIMER'S
167 Neuronal Death and Immunosuppressant-Immunophilin-Transcription Factor Systems N. Ogawa* Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700, Japan Possible involvement of the immune response and oxidative stress in the brain have been revealed not only in several animal models of pathological states, but also in neurodegenerative diseases including Alzheimer-type dementia. As a useful model of neurodegenerative diseases, We previously reported that a late onset reduction of muscarinic acetylcholine receptors (LORMAR) occurs in the gerbil hippocampus after transient ischemia, accompanying the accumulation of glia. Concomitant with the progressive reduction in muscarinic receptors, there is an increase in lipid peroxides which may reflect the activity of reactive microglia. This suggests that the LORMAR may occur as a result of an immune response and oxidative stress. We also showed that this LORMAR was prevented by daily post-ischemic administration of the immunosuppressant cyclosporin A (CsA). The effectiveness of CsA against the LORMAR indicates that an immune mechanism may be involved in progressive brain damage, and that immunosuppressants may be efficacious in preventing the occurence and inhibiting the progressive neurodegeneration. Oxidative stress exhibited the opposite effects on DNA-binding activities of AP-1 and CREBin cultured neuronal and glial cells in vitro, and increased DNA-binding activity of AP-1 in vivo. Furthermore, administration of CsA can mimic increased DNA-binding activity of AP-I, and the oxidativestress may be related to the immune response to neural damage. CsA exerts its effect by inhibiting the production of the T-lymphocyte activating factor interleukin-2 (IL-2) in helper T-lymphocytes. CsA binds to its cytosolic receptor protein, cyclophilin (CyP), which is a peptidyl-prolyl cistrans isomerase. The CsA-CyP complex interacts with the Ca2+/calmodulin regulated phosphatase, calcineurin, and inhibits the activation of a specific transcription factor NF-AT by calcineurin, and as a result suppresses IL-2 gene expression. Transcriptional factor AP- 1 interacts with NF-AT, both of which are involved in T-lymphocyte activation via binding to their responsive elements, the TRE and NF-AT sites located in the enhanser region of the IL-2 gene. These findings suggest that an immune response via activation of transcriptional factors in the brain is involved in oxidative stress-induced neurodegeneration such as that seen in Alzheimer-type dementia. Thus, immunosuppressants may be useful in preventing progressive brain damage.
DISEASE
$43
Himeji 670, Japan, and 2Kinsmen Laboratory of Neurological Research, University of British Columbia, VancouverV6T1Z3, Canada Protein phosphorylation and dephosphorylation, and Ca++ concentrations have all been recognized to play key roles in a wide range of intracellular signal transductions. Ca~/calmodulin-dependentprotein phosphatase, calcineurin(CN), is a serine/threonine-specific protein phosphatase as highly enriched as protein kinase C in the central nervous and immune systems. There are several reasons why CN might be implicated in Alzheimer disease (AD) pathology. (1) It might be involved in the abnormal phosphorylation of tan protein, leading to neurofibrillary tangles, or in the abnormal processing of amyloid precursor protein (APP). The regulation of APP processing is not yet fully understood, but it is known to be affected by modulating the activities of protein kinases and phosphatases, as well as by the level of Ca++. (2) CN might also play a role in the chronic inflammatory process in AD brain - a process evidenced by the many immune system proteins and activated microgiia found in association with AD lesions. The inhibition of CN by the immunosuppressants, cyclosporin A and FK506, when bound to the immunophilins, has been thought to account for their pharmacological activities. (3) Some have suggested that some neuronal death in AD may be by an excitotoxic mechanism and CN has recently been found to desensitize NMDA receptors and to be associated with IP3 receptor-FKBP12 complex, thus affecting both long term potentiation and the excitotoxicity caused by a dynamic regulationof Ca"" flux through such receptor channels. The isoforms of CN (CN-Act and CN-A~) have been shown to be differently distributed to rat brain. Hence, we used isoform-specific antibodies in an immunohistochemical study of their localization in AD and control brains. In all cases, some neurons and gila-like cells were stained, exhibiting granular or homogenous patterns in their processes and perikarya. Some AD pathology was also positive. The two isoforms showed distinct distributions of positive cells, processes and AD lesions, indicating they may also differ in both their normal physiological roles and their involvement in AD pathology.
168 Differential Pbospborylation of Three-Repeat Human Tau Isoforms by Several Protein Kinases T. Singh*, I. Grundke-Iqbal, and IC Iqbal New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA The paired helical filaments (PHF) found in the brain of patients with Alzheimer disease (AD) are composed primarily of the microtubuleassociated protein tau. The six isoforms of tau are present in a hyperphosphorylated state in PHF. It is not known whether all tau isoforms serve equally well as substrates for various kinases. In this study we have compared the phosphorylation of human tan isoforms containing three microtubule-binding repeats and zero (r3), one (r3S) or two (r3L) Nterminal inserts. Four kinases (A-kinase, CK-1, CaM kinase II, GSK-3) were used for this purpose. With A-kinase, CK-1 and CaM kinase II the extent of phosphorylation was r 3 L > r3S> r3. With GSK-3 it was r3L r3S> r3. Tau 3 was a poor substrate for either CaM kinase II or CK-1, 32p incorporation being only 5 % and 11%, respectively, of that observed by these kinases when r 3 L w a s the substrate. After prephosphorylation of the three tau isoforms by A-kinase, a subsequent phosphorylation by GSK-3 was stimulated several fold over tau that was not prephnsphorylated. Under these conditions the extent of 32p incorporation was r 3 L > r3S> r3. Both CK-1 and GSK-3 phosphorylated ser 396 and ser 404 more rapidly in r3L compared to r3 or r3S. Our results suggest that I) the presence of Nterminal inserts in tan isoforms are important structural determinants that modulate the specificity of several kinases; 2) the different tau isoforms may be present at different states of phosphorylation in PHF. (Supported in part by NIH grants AG08076, AG05892, NS18105, AGl1932 and Zenith Award (ICI.) from the Alzheimer Disease Assoc.).
169 Protein Phosphatase Calcineurin and Dementia T. Kawamata t*, T. Hashimoto l, P. L. McGeer 2, and C. Tanaka I IHyogn Institute for Aging Brain and Cognitive Disorders, 520 Saisho-Koh,
170 APP Metabolism is regulated in vivo by Protein Kinase C in Rat Brain M. Di Luca Institute of Pharmacological Sciences, School of Pharmacy, University of Milano, via Balzaretti 9, 20123 Milano, Italy The A~ protein is derived from a longer precursor: the arnyloid precursor protein (APP). The mature precursor can be processed in a secretory pathway with a proteolitic cleavage within the ~A4 region occurring at or near the cell surface and with an ectodomain secreted in extracellular fluids (APPs). An alternative pathway is represented by the endosomaUlysosornal cleavage which produces a complex set of carboxy-terminal derivatives that include potentially amyloidogenic forms. It is well known that the metabolism of APP is a highly regulated event profoundly influenced by either extracellular signals or intracelhilar messengers. In particular it has been long demonstrated that Protein Kinase C (PKC) activation stimulates the secretion of APPs in a number of cell lines. To ascertain the possible role of PKC in regulating APP metabolism in vivo, we have used an animal model in which the PKC system is permanently hyperactivated. These animals are obtained by in utero exposure to a potent neuroselective antiproliferative agent: Methylazoxymethanol (MAM). As a result of the treatment, the animals show marked hypoplasia of cortical intermediate layers and hippocampal CA 1 region. In these regions a strong alteration of synaptic plasticity processes has been detected as a result of electrophysiological (LTP is not inducible in hypoplastic areas), behavioural (selective alteration of cognitive behaviour) and biochemical investigations. Interestingly, PKC phosphorylation processes are permanently increased in the presynaptic compartement of MAM-rat cortex and hippocumpus. This characteristic renders the MAM-rat an intriguing experimental model to test PKC involvement in modulating APPs production in vivo. APPs has beeen detected by Western blot analysis in soluble fractions obtained from hypoplastic areas and correlated to normal rats: the results show a clear increase in APPs production in cortex and hippocampus, while the total amount of APP does not show any modification between controls and MAM-rats, as revealed by immunostaining with an antibody raised against the C-terminal sequence. In addition, in a brain area not affected by MAM treatment (i.e. cerebellum), both PKC basal activation and secretion of APP are similar in control and MAM-rats, thus indicating that the altered metabolism of APP is restricted only to those areas where PKC system is upregulated. Moreover modulators of PKC activation, such as phorbol esters and H-7, induced expected changes in APP secretion in hippocampal slices from both control and MAM-treated rats, further suggesting an in vivo role for this enzyme in regulating the metabolism of mature APP. Finally, these data suggest that pharmacological treatments inducing an increase of PKC activity can indeed modulate APP secretion in vivo.
CONFERENCE
ON ALZHEIMER'S
167 Neuronal Death and Immunosuppressant-Immunophilin-Transcription Factor Systems N. Ogawa* Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700, Japan Possible involvement of the immune response and oxidative stress in the brain have been revealed not only in several animal models of pathological states, but also in neurodegenerative diseases including Alzheimer-type dementia. As a useful model of neurodegenerative diseases, We previously reported that a late onset reduction of muscarinic acetylcholine receptors (LORMAR) occurs in the gerbil hippocampus after transient ischemia, accompanying the accumulation of glia. Concomitant with the progressive reduction in muscarinic receptors, there is an increase in lipid peroxides which may reflect the activity of reactive microglia. This suggests that the LORMAR may occur as a result of an immune response and oxidative stress. We also showed that this LORMAR was prevented by daily post-ischemic administration of the immunosuppressant cyclosporin A (CsA). The effectiveness of CsA against the LORMAR indicates that an immune mechanism may be involved in progressive brain damage, and that immunosuppressants may be efficacious in preventing the occurence and inhibiting the progressive neurodegeneration. Oxidative stress exhibited the opposite effects on DNA-binding activities of AP-1 and CREBin cultured neuronal and glial cells in vitro, and increased DNA-binding activity of AP-1 in vivo. Furthermore, administration of CsA can mimic increased DNA-binding activity of AP-I, and the oxidativestress may be related to the immune response to neural damage. CsA exerts its effect by inhibiting the production of the T-lymphocyte activating factor interleukin-2 (IL-2) in helper T-lymphocytes. CsA binds to its cytosolic receptor protein, cyclophilin (CyP), which is a peptidyl-prolyl cistrans isomerase. The CsA-CyP complex interacts with the Ca2+/calmodulin regulated phosphatase, calcineurin, and inhibits the activation of a specific transcription factor NF-AT by calcineurin, and as a result suppresses IL-2 gene expression. Transcriptional factor AP- 1 interacts with NF-AT, both of which are involved in T-lymphocyte activation via binding to their responsive elements, the TRE and NF-AT sites located in the enhanser region of the IL-2 gene. These findings suggest that an immune response via activation of transcriptional factors in the brain is involved in oxidative stress-induced neurodegeneration such as that seen in Alzheimer-type dementia. Thus, immunosuppressants may be useful in preventing progressive brain damage.
DISEASE
$43
Himeji 670, Japan, and 2Kinsmen Laboratory of Neurological Research, University of British Columbia, VancouverV6T1Z3, Canada Protein phosphorylation and dephosphorylation, and Ca++ concentrations have all been recognized to play key roles in a wide range of intracellular signal transductions. Ca~/calmodulin-dependentprotein phosphatase, calcineurin(CN), is a serine/threonine-specific protein phosphatase as highly enriched as protein kinase C in the central nervous and immune systems. There are several reasons why CN might be implicated in Alzheimer disease (AD) pathology. (1) It might be involved in the abnormal phosphorylation of tan protein, leading to neurofibrillary tangles, or in the abnormal processing of amyloid precursor protein (APP). The regulation of APP processing is not yet fully understood, but it is known to be affected by modulating the activities of protein kinases and phosphatases, as well as by the level of Ca++. (2) CN might also play a role in the chronic inflammatory process in AD brain - a process evidenced by the many immune system proteins and activated microgiia found in association with AD lesions. The inhibition of CN by the immunosuppressants, cyclosporin A and FK506, when bound to the immunophilins, has been thought to account for their pharmacological activities. (3) Some have suggested that some neuronal death in AD may be by an excitotoxic mechanism and CN has recently been found to desensitize NMDA receptors and to be associated with IP3 receptor-FKBP12 complex, thus affecting both long term potentiation and the excitotoxicity caused by a dynamic regulationof Ca"" flux through such receptor channels. The isoforms of CN (CN-Act and CN-A~) have been shown to be differently distributed to rat brain. Hence, we used isoform-specific antibodies in an immunohistochemical study of their localization in AD and control brains. In all cases, some neurons and gila-like cells were stained, exhibiting granular or homogenous patterns in their processes and perikarya. Some AD pathology was also positive. The two isoforms showed distinct distributions of positive cells, processes and AD lesions, indicating they may also differ in both their normal physiological roles and their involvement in AD pathology.
168 Differential Pbospborylation of Three-Repeat Human Tau Isoforms by Several Protein Kinases T. Singh*, I. Grundke-Iqbal, and IC Iqbal New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA The paired helical filaments (PHF) found in the brain of patients with Alzheimer disease (AD) are composed primarily of the microtubuleassociated protein tau. The six isoforms of tau are present in a hyperphosphorylated state in PHF. It is not known whether all tau isoforms serve equally well as substrates for various kinases. In this study we have compared the phosphorylation of human tan isoforms containing three microtubule-binding repeats and zero (r3), one (r3S) or two (r3L) Nterminal inserts. Four kinases (A-kinase, CK-1, CaM kinase II, GSK-3) were used for this purpose. With A-kinase, CK-1 and CaM kinase II the extent of phosphorylation was r 3 L > r3S> r3. With GSK-3 it was r3L r3S> r3. Tau 3 was a poor substrate for either CaM kinase II or CK-1, 32p incorporation being only 5 % and 11%, respectively, of that observed by these kinases when r 3 L w a s the substrate. After prephosphorylation of the three tau isoforms by A-kinase, a subsequent phosphorylation by GSK-3 was stimulated several fold over tau that was not prephnsphorylated. Under these conditions the extent of 32p incorporation was r 3 L > r3S> r3. Both CK-1 and GSK-3 phosphorylated ser 396 and ser 404 more rapidly in r3L compared to r3 or r3S. Our results suggest that I) the presence of Nterminal inserts in tan isoforms are important structural determinants that modulate the specificity of several kinases; 2) the different tau isoforms may be present at different states of phosphorylation in PHF. (Supported in part by NIH grants AG08076, AG05892, NS18105, AGl1932 and Zenith Award (ICI.) from the Alzheimer Disease Assoc.).
169 Protein Phosphatase Calcineurin and Dementia T. Kawamata t*, T. Hashimoto l, P. L. McGeer 2, and C. Tanaka I IHyogn Institute for Aging Brain and Cognitive Disorders, 520 Saisho-Koh,
170 APP Metabolism is regulated in vivo by Protein Kinase C in Rat Brain M. Di Luca Institute of Pharmacological Sciences, School of Pharmacy, University of Milano, via Balzaretti 9, 20123 Milano, Italy The A~ protein is derived from a longer precursor: the arnyloid precursor protein (APP). The mature precursor can be processed in a secretory pathway with a proteolitic cleavage within the ~A4 region occurring at or near the cell surface and with an ectodomain secreted in extracellular fluids (APPs). An alternative pathway is represented by the endosomaUlysosornal cleavage which produces a complex set of carboxy-terminal derivatives that include potentially amyloidogenic forms. It is well known that the metabolism of APP is a highly regulated event profoundly influenced by either extracellular signals or intracelhilar messengers. In particular it has been long demonstrated that Protein Kinase C (PKC) activation stimulates the secretion of APPs in a number of cell lines. To ascertain the possible role of PKC in regulating APP metabolism in vivo, we have used an animal model in which the PKC system is permanently hyperactivated. These animals are obtained by in utero exposure to a potent neuroselective antiproliferative agent: Methylazoxymethanol (MAM). As a result of the treatment, the animals show marked hypoplasia of cortical intermediate layers and hippocampal CA 1 region. In these regions a strong alteration of synaptic plasticity processes has been detected as a result of electrophysiological (LTP is not inducible in hypoplastic areas), behavioural (selective alteration of cognitive behaviour) and biochemical investigations. Interestingly, PKC phosphorylation processes are permanently increased in the presynaptic compartement of MAM-rat cortex and hippocumpus. This characteristic renders the MAM-rat an intriguing experimental model to test PKC involvement in modulating APPs production in vivo. APPs has beeen detected by Western blot analysis in soluble fractions obtained from hypoplastic areas and correlated to normal rats: the results show a clear increase in APPs production in cortex and hippocampus, while the total amount of APP does not show any modification between controls and MAM-rats, as revealed by immunostaining with an antibody raised against the C-terminal sequence. In addition, in a brain area not affected by MAM treatment (i.e. cerebellum), both PKC basal activation and secretion of APP are similar in control and MAM-rats, thus indicating that the altered metabolism of APP is restricted only to those areas where PKC system is upregulated. Moreover modulators of PKC activation, such as phorbol esters and H-7, induced expected changes in APP secretion in hippocampal slices from both control and MAM-treated rats, further suggesting an in vivo role for this enzyme in regulating the metabolism of mature APP. Finally, these data suggest that pharmacological treatments inducing an increase of PKC activity can indeed modulate APP secretion in vivo.