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Regulation of phosphorylation of tau by protein kinases in rat brain
Poster Presentation:
S150
REGULATION OF PHOSPHORYLATION KINASES IN RAT BRAIN Amitabhu
Sengupta,
in Developmental
lnp
Grundke-Iqbal,
Disabilities.
Stafen
Khalid Island,
Iqbol,
OF TAU BY PROTEIN
NYS Instfor
Basic
Research
NY
Abnormally hyperphosphorylated tau is the major protein component of neurofibrillary tangles in Alzheimer’s disease brain. In order to investigate the role of protein kinases in the abnormal phosphorylation of tau, metabolically active chopped slices made from brains of adult rats were incubated with or without various specific kinase activators in oxygenated artificial cerebrospinal fluid. Detectable activities of A-kinase. CaM kinase 11 and GSK-3 were found in rat brain. Using inhibitors of the above kmases, HA-100 (an inhibitor of A-kinase). KN-62 (a specific inhibitor of CaM kinase II) and LiCl (an inhibitor of GSK-3) the basal kinaw activities were inhibited to the extent of 750/c, 70% and 60% respectively. These basal kinase activities were stimulated more than 2.fold by adding isoproterenol, bradykinin and wortmannin respectively. Since hyperphosphorylation of tau is believed to be the result of the concerted action of several kinases rather than a single one, we examined whether two or more kinases can be stimulated step hy Etep by external activator?. We found that A-kinase, CaM kinase I1 and GSK-3 can be activated sequentially in the rat brain slices by external activators (isoproterenol, bradykinin and wortmannin) without interfering with one another. Tao from the tiswe where both CaM kinase I1 and GSK-3 activities were &mlated was found to bind microtubules to a significantly lesser extent compared to control tissue (where the kinases were not stimulated). Under these conditions, the phosphorylation of tau at Thr-231, Ser.396/404 and Ser.422 sites were significantly enhanced. These data suggest that CaM kinase II and GSK-3 are involved in the regulation of phorphorylation of tau and hyperphosphorylation of tau i\ a synergistic action of more than one kinase.
ABNORMAL HYPERPHOSPHORYLATION 16781TEINS IN ALZHEIMER’S DISEASE BRAIN Shrijay
Vljaynn,
Inge
Grundke-Iqhal,
D~~~~eloppment~lDi.whilities,
Staten
Khalid island,
lqhal,
OF NON-TAU
NYS Inst for
Basic
PRO-
Research
in
NY
We undertook a study to investigate whether proteins other than tau are also hyperphorphorylated in Alzheimer disease (AD) brain. Frontal gray matter from AD (n=7) and age-matched control(n=fi) case? were homogenized and centrifuged at 100,000 x g for 30 min. While the 100,000 x g supematant had no significant changes in phosphate levels between the two groups, the pellet showed an increase of -1 I pmoles phosphate per microgram protein in AD over the control cases. Hyperphosphorylation of proteins was examined by employing antibodies to phosphoserine (P-Ser) and phosphothreonine (P-Thr) on Western blots. Interestingly, a -54kD non-&u protein band, which did not label with anti-tau antibodies, also showed a -36% increase in phosphoaerine immunoreactivity in the AD brain pellet compared to age-matched controls. The -54kD protein was purified by preparative SDS-PAGE and identified as @tubalin, both by masr spectrometry and specific antibody to p-tubulin. Since microtubules are rarely seen in tangle-bearing neurons in AD brain, hyperphosphorylated P-tub& could also have been contributing to the breakdown of the microtubule network, apart from tau. In an attempt to look for other proteins that may also be hyperphosphorylated in AD brain, preparative SDS-PAGE purified protein fractions were examined for hyperphosphorylation by employing P-Ser and P-Thr antibodies on Western blots. A -YOkD protein was observed to be significantly hyperphosphorylated at threonine residues. The increase in hyperphosphorylation was 200% more than in age-matched controls. There was also a significant increave in phosphorylation at wine residues. These findings support the hypothesis that there 1s an imbalance in the phosphorylation-dephosphorylation system m AD brain, resulting in the hyperphosphorylation of several neuronal phospho-proteins, which in turn may reult in the alteration of their biological activity, as has been demonstrated for tau.
STRUCTURAL MATURATION OF PRESENILIN PROTEIN COM)6791 PLEXES IS INHIBITED BY MUTATION OF THE CONSERVED INTRAMEMBRANE Gung Yu. Fusheng George-Hyslop,
Chen,
ASPARATE
Masuki Nishimum,
Univ of Toronto.
Toronto,
RESIDUES
Erin Holmes,
Paul E Fraser,
Peter
H St
ON Canada
The pre$enilin proteins (PSI and PS2) are components of heteropolymeric high molecular weight protein complexes. Inclusion of the presenilins in these complexes follows a complex and tightly regulated pathway in which the PS holoprotein is first incorporated into a lower molecular weight immature complex in the rough ER. Maturation of thi? complex in the smooth ER and Golgi is associated with an increase in apparent molecular weight and with endoproteolysis. Mutation of either of two conserved aspartate residues in PSI or PS2 inhibits both endoproteolysis and proteolytic processing of the P-amyloid precursor protein and Notch. This has implied that these aspartate residues may he part of a catalytic Eite and that the presenilins may
Alzheimer’s
Disease and Related Dementias 111
be novel auto-catalytic aspattyl proteases. We used glycerol velocity fractionation to examine the native states of wild type and aspartate-mutant presenilins. We show that two aspartate PSl mutants (D257A and D385A) are in a smaller polymeric complex, as compared to wild type PSI NTF/CTF. Moreover, an Alzheimer-associated PSI AElO variant that does not undergo proteolysis was detected in gradient fractions overlapping that of the endogenous mature PSl NTF/CTF. Mutation of aspartate 385 in PSlAEIO(PSlAF.l0-D385A) causes the holoprotein to mainly distribute in lower molecular weight fractions, which overlap that of PSID257A and D38SA. We also used sucrose gradient fractionation to examine the subcellular distribution pattern of the aspaxtate mutant presemlins and found that they are predominantly located in the rER, while the wild type presenilin NTF/CTF fragments are predominantly m the smooth ER and Golgi. Furthermore, when over-expressed, the aspartate mutant PSI or PS2 proteins displace/replace the endogenous wild type presenilin proteins from both mature and immature complexe,, thereby causing the observed losq-of-function effect. Taken together. these data suggest that mutation of the conserved aspartates affects structural maturation of preaenilin protein complexes in distmct subcellular compartmenta.
16801THE Cews
Eloah
HOME VISIT: AN ALTERNATIVE
Frrrrtti,
Pm/o
Henriqur
Bertolucci.
OF CARE IN DEMENTIA
Univ Fed de Sao Paula,
Sue Puulo
Brazd Introduction:
Alzheimer’s disease, as well as other types of dementia, reqwes an effective support. Several doubts are brought to the consultant by the caregiver, always concerning the management of their patients. Homecare is a program included on the nursing assessment at the Behavioral Neurology Unit - UNIFESP-EPM. It has become an effective alternative to keep biopsychosocial maintenance to both patient and caregiver, specially when it refers to the dependence in ADLsllADLs execution, changes in mood and behavior, which are so common along the dlaease evolution. Objectives: Provide resources to patient/caregivers at home, through educational actions at first, and giving assistance afterwards. Nursing intervention must be adapted to the family financial conditions and needs, and also according to the dependence level of the patient. Comparative studies between assessment scales, when at home and in the hospital, were done. Method: 59 patientslcaregivers were enrolled in this study. The dependence degree varied between I to 4, according to the CDR. Patients classified as CDR 3 and 4 were visited monthly, CDR I and 2 each 3 and 2 months respectively. The Katz Index was used to measure the dependence to the ADLsflADLs, and the NPI - adapted - to assess the behavioral changes. The nursmg interventions followed the Nanda and Carpenito’s criteria. Rrsulrs: There has been an improvement in 17 (81%) of the 21 items which concerned the dependence of ADLsllADLs. A reduction of 72.5% on the total of the behavioral disturbance wa\ shown after the nursing intervention. Conclusion: Specific nursing strategies may provide a better quality of hfe to the demented patients and their family/caregiver!, at any stage of dementia evolution.
MINIMAL 1681) EASE ON DEFICITS:
EFFECTS OF PRECLINICAL ALZHEIMER’S DISCROSS-SECTIONAL AGE-RELATED COGNITIVE EVIDENCE FROM THE KUNGSHOLMEN STUDY.
We used data from a population-based sample of very old adults to investigate the impact of preclinical Abheimer’s disease (AD) on cross-sectional age-related differences in cognitive performance. Contrary to expectations, eliminating those who developed AD across a three-year interval did not alter the magnitude of baseline age differences in memory, verbal, and vwospatlal performance. This pattern of reultc was obtained although preclinical AD was associated with both lower cognitive performance and higher age. These findings suggest that age-related cognitive deficits in nondemented populations are little affected by persons who are in a preclinical phase of AD.
S150
REGULATION OF PHOSPHORYLATION KINASES IN RAT BRAIN Amitabhu
Sengupta,
in Developmental
lnp
Grundke-Iqbal,
Disabilities.
Stafen
Khalid Island,
Iqbol,
OF TAU BY PROTEIN
NYS Instfor
Basic
Research
NY
Abnormally hyperphosphorylated tau is the major protein component of neurofibrillary tangles in Alzheimer’s disease brain. In order to investigate the role of protein kinases in the abnormal phosphorylation of tau, metabolically active chopped slices made from brains of adult rats were incubated with or without various specific kinase activators in oxygenated artificial cerebrospinal fluid. Detectable activities of A-kinase. CaM kinase 11 and GSK-3 were found in rat brain. Using inhibitors of the above kmases, HA-100 (an inhibitor of A-kinase). KN-62 (a specific inhibitor of CaM kinase II) and LiCl (an inhibitor of GSK-3) the basal kinaw activities were inhibited to the extent of 750/c, 70% and 60% respectively. These basal kinase activities were stimulated more than 2.fold by adding isoproterenol, bradykinin and wortmannin respectively. Since hyperphosphorylation of tau is believed to be the result of the concerted action of several kinases rather than a single one, we examined whether two or more kinases can be stimulated step hy Etep by external activator?. We found that A-kinase, CaM kinase I1 and GSK-3 can be activated sequentially in the rat brain slices by external activators (isoproterenol, bradykinin and wortmannin) without interfering with one another. Tao from the tiswe where both CaM kinase I1 and GSK-3 activities were &mlated was found to bind microtubules to a significantly lesser extent compared to control tissue (where the kinases were not stimulated). Under these conditions, the phosphorylation of tau at Thr-231, Ser.396/404 and Ser.422 sites were significantly enhanced. These data suggest that CaM kinase II and GSK-3 are involved in the regulation of phorphorylation of tau and hyperphosphorylation of tau i\ a synergistic action of more than one kinase.
ABNORMAL HYPERPHOSPHORYLATION 16781TEINS IN ALZHEIMER’S DISEASE BRAIN Shrijay
Vljaynn,
Inge
Grundke-Iqhal,
D~~~~eloppment~lDi.whilities,
Staten
Khalid island,
lqhal,
OF NON-TAU
NYS Inst for
Basic
PRO-
Research
in
NY
We undertook a study to investigate whether proteins other than tau are also hyperphorphorylated in Alzheimer disease (AD) brain. Frontal gray matter from AD (n=7) and age-matched control(n=fi) case? were homogenized and centrifuged at 100,000 x g for 30 min. While the 100,000 x g supematant had no significant changes in phosphate levels between the two groups, the pellet showed an increase of -1 I pmoles phosphate per microgram protein in AD over the control cases. Hyperphosphorylation of proteins was examined by employing antibodies to phosphoserine (P-Ser) and phosphothreonine (P-Thr) on Western blots. Interestingly, a -54kD non-&u protein band, which did not label with anti-tau antibodies, also showed a -36% increase in phosphoaerine immunoreactivity in the AD brain pellet compared to age-matched controls. The -54kD protein was purified by preparative SDS-PAGE and identified as @tubalin, both by masr spectrometry and specific antibody to p-tubulin. Since microtubules are rarely seen in tangle-bearing neurons in AD brain, hyperphosphorylated P-tub& could also have been contributing to the breakdown of the microtubule network, apart from tau. In an attempt to look for other proteins that may also be hyperphosphorylated in AD brain, preparative SDS-PAGE purified protein fractions were examined for hyperphosphorylation by employing P-Ser and P-Thr antibodies on Western blots. A -YOkD protein was observed to be significantly hyperphosphorylated at threonine residues. The increase in hyperphosphorylation was 200% more than in age-matched controls. There was also a significant increave in phosphorylation at wine residues. These findings support the hypothesis that there 1s an imbalance in the phosphorylation-dephosphorylation system m AD brain, resulting in the hyperphosphorylation of several neuronal phospho-proteins, which in turn may reult in the alteration of their biological activity, as has been demonstrated for tau.
STRUCTURAL MATURATION OF PRESENILIN PROTEIN COM)6791 PLEXES IS INHIBITED BY MUTATION OF THE CONSERVED INTRAMEMBRANE Gung Yu. Fusheng George-Hyslop,
Chen,
ASPARATE
Masuki Nishimum,
Univ of Toronto.
Toronto,
RESIDUES
Erin Holmes,
Paul E Fraser,
Peter
H St
ON Canada
The pre$enilin proteins (PSI and PS2) are components of heteropolymeric high molecular weight protein complexes. Inclusion of the presenilins in these complexes follows a complex and tightly regulated pathway in which the PS holoprotein is first incorporated into a lower molecular weight immature complex in the rough ER. Maturation of thi? complex in the smooth ER and Golgi is associated with an increase in apparent molecular weight and with endoproteolysis. Mutation of either of two conserved aspartate residues in PSI or PS2 inhibits both endoproteolysis and proteolytic processing of the P-amyloid precursor protein and Notch. This has implied that these aspartate residues may he part of a catalytic Eite and that the presenilins may
Alzheimer’s
Disease and Related Dementias 111
be novel auto-catalytic aspattyl proteases. We used glycerol velocity fractionation to examine the native states of wild type and aspartate-mutant presenilins. We show that two aspartate PSl mutants (D257A and D385A) are in a smaller polymeric complex, as compared to wild type PSI NTF/CTF. Moreover, an Alzheimer-associated PSI AElO variant that does not undergo proteolysis was detected in gradient fractions overlapping that of the endogenous mature PSl NTF/CTF. Mutation of aspartate 385 in PSlAEIO(PSlAF.l0-D385A) causes the holoprotein to mainly distribute in lower molecular weight fractions, which overlap that of PSID257A and D38SA. We also used sucrose gradient fractionation to examine the subcellular distribution pattern of the aspaxtate mutant presemlins and found that they are predominantly located in the rER, while the wild type presenilin NTF/CTF fragments are predominantly m the smooth ER and Golgi. Furthermore, when over-expressed, the aspartate mutant PSI or PS2 proteins displace/replace the endogenous wild type presenilin proteins from both mature and immature complexe,, thereby causing the observed losq-of-function effect. Taken together. these data suggest that mutation of the conserved aspartates affects structural maturation of preaenilin protein complexes in distmct subcellular compartmenta.
16801THE Cews
Eloah
HOME VISIT: AN ALTERNATIVE
Frrrrtti,
Pm/o
Henriqur
Bertolucci.
OF CARE IN DEMENTIA
Univ Fed de Sao Paula,
Sue Puulo
Brazd Introduction:
Alzheimer’s disease, as well as other types of dementia, reqwes an effective support. Several doubts are brought to the consultant by the caregiver, always concerning the management of their patients. Homecare is a program included on the nursing assessment at the Behavioral Neurology Unit - UNIFESP-EPM. It has become an effective alternative to keep biopsychosocial maintenance to both patient and caregiver, specially when it refers to the dependence in ADLsllADLs execution, changes in mood and behavior, which are so common along the dlaease evolution. Objectives: Provide resources to patient/caregivers at home, through educational actions at first, and giving assistance afterwards. Nursing intervention must be adapted to the family financial conditions and needs, and also according to the dependence level of the patient. Comparative studies between assessment scales, when at home and in the hospital, were done. Method: 59 patientslcaregivers were enrolled in this study. The dependence degree varied between I to 4, according to the CDR. Patients classified as CDR 3 and 4 were visited monthly, CDR I and 2 each 3 and 2 months respectively. The Katz Index was used to measure the dependence to the ADLsflADLs, and the NPI - adapted - to assess the behavioral changes. The nursmg interventions followed the Nanda and Carpenito’s criteria. Rrsulrs: There has been an improvement in 17 (81%) of the 21 items which concerned the dependence of ADLsllADLs. A reduction of 72.5% on the total of the behavioral disturbance wa\ shown after the nursing intervention. Conclusion: Specific nursing strategies may provide a better quality of hfe to the demented patients and their family/caregiver!, at any stage of dementia evolution.
MINIMAL 1681) EASE ON DEFICITS:
EFFECTS OF PRECLINICAL ALZHEIMER’S DISCROSS-SECTIONAL AGE-RELATED COGNITIVE EVIDENCE FROM THE KUNGSHOLMEN STUDY.
We used data from a population-based sample of very old adults to investigate the impact of preclinical Abheimer’s disease (AD) on cross-sectional age-related differences in cognitive performance. Contrary to expectations, eliminating those who developed AD across a three-year interval did not alter the magnitude of baseline age differences in memory, verbal, and vwospatlal performance. This pattern of reultc was obtained although preclinical AD was associated with both lower cognitive performance and higher age. These findings suggest that age-related cognitive deficits in nondemented populations are little affected by persons who are in a preclinical phase of AD.