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710 Abnormality of neurofilament protein amount in Alzheimer brains
S176
FIFTH INTERNATIONAL
CONFERENCE
ON ALZHEIMER'S
DISEASE
707
709
Cyclin-dependent Kinase 5 (cdk5) and p35 in Lewy Bodies (LB)
Adenylyl Cyclase Signal Transduetion in Alzheimer's Disease Lymphocytes.
S. Nakamura, Y. Kawamoto, S. Nakano, 1. Akiguchi and J. Kimura Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan Objective. Accumulated evidence has suggested that neurofilament proteins (NFs) are integral components of LB fibrils. High molecular weight (NF-H), middle molecular weight (NF-M), and low molecular weight protein subunits have been identified in LB fibrils. Among these, NF-H and NF-M in LB are abnormally phosphorylated. It has been hypothesized that the phosphorylation of NF proteins is a crucial step for the conversion of NF proteins to the insoluble fibrils of the LB. Recently cdk5, a kinase that is capable of phosphorylating the KSP motif in NF-H and NF-M, was found in LB of PD brains. However, cdk5 needs p35 as a regulatory subunit to be an activative form. If cdk5 is involved in the phosphorylation of NF in LB fibrils, p35 should be associated with LB. Thus, in the present study, we examined the immunohistochemical localization of p35 in PD brains. Methods. Paraffin-embedded sections of postmortem brains from 7 patients with PD and 6 controls were examined, lmmunohistochemical staining of p35 were performed using two polyclonal antibodies raised against synthetic peptides corresponding to N-terminal and C-terminal regions of the human p35. Anti-cdk5 antibodies were purchased from Santa-Cruz Biotechnology, Inc, USA. Results. In controls, p35 was immunostained in some neurons and varicose fibers. In PD brains, in addition to neurons and fibers, we found p35immunoreactive LB in the substantia nigra and locus ceruleus, p35immunoreactivity was mainly observed in the halo of LB. Cdk5 was also immunosatined in LB in accordance with the previous study. Discussion and Conclusion. Cdks are essential molecules for cell-cycle control and 7 types ofcdk have been identified. Among ".hese kinases, cdk5 is unique in that mRNA for this kinase has been shown to be relatively abundant in neurons which are post-mitotic. Another unique characteristic is that the protein kinase activity of cdk5 in brain is regulated by a regulatory subunit, p35, which is distinct from cyclins. Members of the cdk family need to associate with cyclins to assume an active form. However, p35 displays no sequence homology to cyclins, and mRNA for p35 is expressed exclusively in the brain. Thus cdk5 has been suggested to be associated with neuronal functions unrelated to the cell cycle in neurons. One of such functions is the ability to phosphorylate KSP motif of Cterminal tail region of NF-M and NF-H, which are major comonents of LB fibrils. In this context, the present results strongly support the hypothesis that cdkS-p35 complex is involved in the formation of LB fibrils in PD.
7O8 Loss of Ryanodine Receptor Calcium Release Channels in Alzheimer's Disease Brain C. O' Neill 1", M. Kelliher 1 and R. Ravid 2. 1Department of Biochemistry, University College, Lee Maltings, Cork, Ireland. 2 The Netherlands Brain Bank, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands. Alzheimer's disease (AD) has been hypothesised to be characterised by deregulated calcium homeostasis. The ryanodine receptor (RYR) and the inositol 1,4,5 receptor (IP3) channel proteins play crucial roles in the regulation of neuronal intracellular calcium signalling, being responsible for the stimulus induced release of stored calcium. Although levels of the IP3 receptor have been found to be severely compromised in the AD brain, nothing is known about RYR function. The objective of this study was to examine the functional integrity of the RYR in control and AD post mortem brain. In the present study we compared high affinity 3H-Ryanodine binding and its modulation by calcium and magnesium in the temporal cortex from a series of Alzheimer's disease (n = 8) and matched control (n=7) subjects. At these concentrations ryanodine is known to bind to the open conformation of the channel protein. Results show 3H- Ryanodine to be reduced by 44% in AD samples when compared to controls. Kinetic analysis of RYR binding revealed a similar meaa Kd of 5ram for control a~d AD samples. However the maximum number of binding sites Bmax was significantly decreased from 461 to 287 fmol/mg of protein in the AD brain. Examination of the calcium dependence of 3H ryanodine binding is a reflection of the ability of calcium to open or close the channel. Despite the reduced number of ryanodine binding sites in AD the kinetics for opening and closing the channel in response to calcium was the same in control and AD samples. Magnesium is a known inhibitor of RYR channel opening. Comparative inhibition curves for magnesium revealed that the AD samples had a slightly higher IC50 value (10mM) than the control samples (7.5raM), implicating some deficiency in channel closing in response to this cation. We conclude that levels of the RYR receptor are severely reduced in Alzheimer's disease temporal cortex. This reduction in association with reduced levels of the IP3 receptor is bound to have severe repercussions in the regulation of intracellular calcium levels in the Alzheimer's disease brain and its role in such diverse processes as neuronal activation, secretion and cell death. Supported by The HealthResearchBoard and The AI zheimerSocietyof Ireland
A. G-arlind*, R. F. Cowburn and B. Winblad Karolinska Institute Department of Clinical Neuroseience and Family Medicine Division of Geriatric Medicine, B 84, Huddinge University Hospital S-141 86 Huddinge, Sweden The adenylyl cyclase signal transduction system represents an important mechanism through which many neurotransmitter substances mediate neural activity. The integrity of this system in Alzheimer's disease brain is essential for the success of a number of neurotransmitter based therapies. However, over the last few years evidence has accumulated of dysfunctions in the adenylyl cyclase system in Alzheimer's disease postmortem brain, including a widespread Gs protein dysfunction. Moreover, these changes seem not to be restricted merely to neural tissue or to late stages of the disease. In the present study we have investigated basal, forsknlin- and isoprenaline-stimulated adenylyl eyclase activity in Alzheimer's disease lymphocytes. Blood samples were collected from 12 healthy control subjects and 12 Alzheimer's disease patients and adanylyl cyclase assays performed on lymphocyte fractions using a commercial kit (Amersham International, UK). In accordance with studies on post-mortem brain, the receptormediated isoprenaline-stimulated adenylyl cyclase activity in Alzheimer's disease lymphoeytes was significantly lower as compared to control lymphocytes. There was no significant difference in basal or forskolinstimulated enzyme activity between the groups. These results indicate that there is a disturbance in the receptoradenylyl eyclase coupling in Alzheimer's disease lymphocytas, possibly at the Gs protein level, whilst the catalytic subunit of the enzyme seems to be intact. Furthermore, dysfunction of the adenylyl cyclase system in Alzheimer's disease is not restricted to brain regions with histopathological changes. These findings has important consequences for the development of therapeutic strategies aimed at enhancing the adenylyl cyclase system.
710 Abnormality of Neurofilament Protein Amount in Alzheimer B r a i n s Y. Nakamura*, M. Takeda and T. Nishimura Department of Neuropsychiatry, Osaka University Medical S c h o o l ; " 2-2 Yamadaoka, Suita-shi, Osaka, 565, Japan We have reported abnormality of distribution, composition and phosphorylation level of neurofilament proteins in Alzheimer brains. The protein amounts of neurofilamant L (NF-L) and H (NF-H) in Alzheimer and control brains were measured by western blotting to investigate the pathologic changes of neumfilament proteins in Alzheimer brains in detail. Frozen blocks of frontal lobe were homogenized with 2 volumes (v/w) of 10 M urea, and centrifuged at 100,000 x g. The supematants were analyzed by western blotting using four monocinnal antibodies bound to NF-H (NE-!4, SMI34, SMI-32, N52) and a polyclonal antibody (anti-TL) bound to tail region of NF-L. The protein amounts recognized by the antibodies were densito-metrically analyzed by Image Master (Pharmacia). The standard curves were prepared by the data of experiments of purified NF-H and NF-L, and the protein amounts recognized by the antibodies were calculated. NE14 and SMI-34 bound to phosphorylated epitope of NF-H showed that NF-H content of Alzbeimer brains was approximately 1 ,ug/mg protein, which was almost identical to that of control brains. SMI-32 bound to nonphosphorylated epitope of NF-H showed that NF-H content of Alzheimer brains was approximately 60 % of that of control brains (0.74 ,ug/mg protein). N52 phosphorylation-independently bound to NF-H showed NF-H content of Alzheimer brains was approximately 1.7 ,ug/mg protein, which was almost identical to that of control brains. These results indicate the decrease of nonphosphorylated form of NF-H in Alzheimer brains despite of constant amount of NF-H. On the other hand, anti-TL bound to NF-L showed that NF-L content of Alzheimer brains was approximately 60 % of that of control brains (4.23 pg/mg protein), indicating the decrease of NF-L in Aizhelmer brains. These findings showed abnormal composition of neurofilament subunit proteins and the significant decrease of total NF-L compared with total NF-H in Alz.heimer brains. The decrease of non-phosphorylated form of NF-H imply acceleration of phosphorylation or reduction of de-phnsphorylation in Aizheimer brains. These abnormalities of neurofilament may induce the disorder of axonal transport, resulting the deterioration of neuronal functions.
FIFTH INTERNATIONAL
CONFERENCE
ON ALZHEIMER'S
DISEASE
707
709
Cyclin-dependent Kinase 5 (cdk5) and p35 in Lewy Bodies (LB)
Adenylyl Cyclase Signal Transduetion in Alzheimer's Disease Lymphocytes.
S. Nakamura, Y. Kawamoto, S. Nakano, 1. Akiguchi and J. Kimura Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan Objective. Accumulated evidence has suggested that neurofilament proteins (NFs) are integral components of LB fibrils. High molecular weight (NF-H), middle molecular weight (NF-M), and low molecular weight protein subunits have been identified in LB fibrils. Among these, NF-H and NF-M in LB are abnormally phosphorylated. It has been hypothesized that the phosphorylation of NF proteins is a crucial step for the conversion of NF proteins to the insoluble fibrils of the LB. Recently cdk5, a kinase that is capable of phosphorylating the KSP motif in NF-H and NF-M, was found in LB of PD brains. However, cdk5 needs p35 as a regulatory subunit to be an activative form. If cdk5 is involved in the phosphorylation of NF in LB fibrils, p35 should be associated with LB. Thus, in the present study, we examined the immunohistochemical localization of p35 in PD brains. Methods. Paraffin-embedded sections of postmortem brains from 7 patients with PD and 6 controls were examined, lmmunohistochemical staining of p35 were performed using two polyclonal antibodies raised against synthetic peptides corresponding to N-terminal and C-terminal regions of the human p35. Anti-cdk5 antibodies were purchased from Santa-Cruz Biotechnology, Inc, USA. Results. In controls, p35 was immunostained in some neurons and varicose fibers. In PD brains, in addition to neurons and fibers, we found p35immunoreactive LB in the substantia nigra and locus ceruleus, p35immunoreactivity was mainly observed in the halo of LB. Cdk5 was also immunosatined in LB in accordance with the previous study. Discussion and Conclusion. Cdks are essential molecules for cell-cycle control and 7 types ofcdk have been identified. Among ".hese kinases, cdk5 is unique in that mRNA for this kinase has been shown to be relatively abundant in neurons which are post-mitotic. Another unique characteristic is that the protein kinase activity of cdk5 in brain is regulated by a regulatory subunit, p35, which is distinct from cyclins. Members of the cdk family need to associate with cyclins to assume an active form. However, p35 displays no sequence homology to cyclins, and mRNA for p35 is expressed exclusively in the brain. Thus cdk5 has been suggested to be associated with neuronal functions unrelated to the cell cycle in neurons. One of such functions is the ability to phosphorylate KSP motif of Cterminal tail region of NF-M and NF-H, which are major comonents of LB fibrils. In this context, the present results strongly support the hypothesis that cdkS-p35 complex is involved in the formation of LB fibrils in PD.
7O8 Loss of Ryanodine Receptor Calcium Release Channels in Alzheimer's Disease Brain C. O' Neill 1", M. Kelliher 1 and R. Ravid 2. 1Department of Biochemistry, University College, Lee Maltings, Cork, Ireland. 2 The Netherlands Brain Bank, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands. Alzheimer's disease (AD) has been hypothesised to be characterised by deregulated calcium homeostasis. The ryanodine receptor (RYR) and the inositol 1,4,5 receptor (IP3) channel proteins play crucial roles in the regulation of neuronal intracellular calcium signalling, being responsible for the stimulus induced release of stored calcium. Although levels of the IP3 receptor have been found to be severely compromised in the AD brain, nothing is known about RYR function. The objective of this study was to examine the functional integrity of the RYR in control and AD post mortem brain. In the present study we compared high affinity 3H-Ryanodine binding and its modulation by calcium and magnesium in the temporal cortex from a series of Alzheimer's disease (n = 8) and matched control (n=7) subjects. At these concentrations ryanodine is known to bind to the open conformation of the channel protein. Results show 3H- Ryanodine to be reduced by 44% in AD samples when compared to controls. Kinetic analysis of RYR binding revealed a similar meaa Kd of 5ram for control a~d AD samples. However the maximum number of binding sites Bmax was significantly decreased from 461 to 287 fmol/mg of protein in the AD brain. Examination of the calcium dependence of 3H ryanodine binding is a reflection of the ability of calcium to open or close the channel. Despite the reduced number of ryanodine binding sites in AD the kinetics for opening and closing the channel in response to calcium was the same in control and AD samples. Magnesium is a known inhibitor of RYR channel opening. Comparative inhibition curves for magnesium revealed that the AD samples had a slightly higher IC50 value (10mM) than the control samples (7.5raM), implicating some deficiency in channel closing in response to this cation. We conclude that levels of the RYR receptor are severely reduced in Alzheimer's disease temporal cortex. This reduction in association with reduced levels of the IP3 receptor is bound to have severe repercussions in the regulation of intracellular calcium levels in the Alzheimer's disease brain and its role in such diverse processes as neuronal activation, secretion and cell death. Supported by The HealthResearchBoard and The AI zheimerSocietyof Ireland
A. G-arlind*, R. F. Cowburn and B. Winblad Karolinska Institute Department of Clinical Neuroseience and Family Medicine Division of Geriatric Medicine, B 84, Huddinge University Hospital S-141 86 Huddinge, Sweden The adenylyl cyclase signal transduction system represents an important mechanism through which many neurotransmitter substances mediate neural activity. The integrity of this system in Alzheimer's disease brain is essential for the success of a number of neurotransmitter based therapies. However, over the last few years evidence has accumulated of dysfunctions in the adenylyl cyclase system in Alzheimer's disease postmortem brain, including a widespread Gs protein dysfunction. Moreover, these changes seem not to be restricted merely to neural tissue or to late stages of the disease. In the present study we have investigated basal, forsknlin- and isoprenaline-stimulated adenylyl eyclase activity in Alzheimer's disease lymphocytes. Blood samples were collected from 12 healthy control subjects and 12 Alzheimer's disease patients and adanylyl cyclase assays performed on lymphocyte fractions using a commercial kit (Amersham International, UK). In accordance with studies on post-mortem brain, the receptormediated isoprenaline-stimulated adenylyl cyclase activity in Alzheimer's disease lymphoeytes was significantly lower as compared to control lymphocytes. There was no significant difference in basal or forskolinstimulated enzyme activity between the groups. These results indicate that there is a disturbance in the receptoradenylyl eyclase coupling in Alzheimer's disease lymphocytas, possibly at the Gs protein level, whilst the catalytic subunit of the enzyme seems to be intact. Furthermore, dysfunction of the adenylyl cyclase system in Alzheimer's disease is not restricted to brain regions with histopathological changes. These findings has important consequences for the development of therapeutic strategies aimed at enhancing the adenylyl cyclase system.
710 Abnormality of Neurofilament Protein Amount in Alzheimer B r a i n s Y. Nakamura*, M. Takeda and T. Nishimura Department of Neuropsychiatry, Osaka University Medical S c h o o l ; " 2-2 Yamadaoka, Suita-shi, Osaka, 565, Japan We have reported abnormality of distribution, composition and phosphorylation level of neurofilament proteins in Alzheimer brains. The protein amounts of neurofilamant L (NF-L) and H (NF-H) in Alzheimer and control brains were measured by western blotting to investigate the pathologic changes of neumfilament proteins in Alzheimer brains in detail. Frozen blocks of frontal lobe were homogenized with 2 volumes (v/w) of 10 M urea, and centrifuged at 100,000 x g. The supematants were analyzed by western blotting using four monocinnal antibodies bound to NF-H (NE-!4, SMI34, SMI-32, N52) and a polyclonal antibody (anti-TL) bound to tail region of NF-L. The protein amounts recognized by the antibodies were densito-metrically analyzed by Image Master (Pharmacia). The standard curves were prepared by the data of experiments of purified NF-H and NF-L, and the protein amounts recognized by the antibodies were calculated. NE14 and SMI-34 bound to phosphorylated epitope of NF-H showed that NF-H content of Alzbeimer brains was approximately 1 ,ug/mg protein, which was almost identical to that of control brains. SMI-32 bound to nonphosphorylated epitope of NF-H showed that NF-H content of Alzheimer brains was approximately 60 % of that of control brains (0.74 ,ug/mg protein). N52 phosphorylation-independently bound to NF-H showed NF-H content of Alzheimer brains was approximately 1.7 ,ug/mg protein, which was almost identical to that of control brains. These results indicate the decrease of nonphosphorylated form of NF-H in Alzheimer brains despite of constant amount of NF-H. On the other hand, anti-TL bound to NF-L showed that NF-L content of Alzheimer brains was approximately 60 % of that of control brains (4.23 pg/mg protein), indicating the decrease of NF-L in Aizhelmer brains. These findings showed abnormal composition of neurofilament subunit proteins and the significant decrease of total NF-L compared with total NF-H in Alz.heimer brains. The decrease of non-phosphorylated form of NF-H imply acceleration of phosphorylation or reduction of de-phnsphorylation in Aizheimer brains. These abnormalities of neurofilament may induce the disorder of axonal transport, resulting the deterioration of neuronal functions.