- Email: [email protected]
P1-155 ER stress is entangled with Aβ pathology
Poster Session P I : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis
~
CIRCADIAN C L O C K G E N E S O S C I L L A T E IN T H E HUMAN PINEAL GLAND AND A R E DESYNCHRONIZED IN A L Z H E I M E R D I S E A S E
YingHui Wu* 1, David F. Fischer 1, Jiang-Nlng Zhou2, Dick F. S waab 1. t Netherlands Institute for Brain research, Amsterdam, Netherlands; 2University of Science and Technology of China, Hefei, Anhui, China. Contact e-mail: [email protected]
Background: Circadian disturbances, such as nightly restlessness, are major clinical problems in Alzheimer's disease (AD). The deterioration of the neurons in the biological clock, the suprachiasmatic nucleus (SCN), and a dysfunction of the pineal gland in AD are supposed to be responsible for the circadian disorders. Circadian clock genes and melatonin synthesis oscillate in the rodent pineal gland under the control of the sympathetic iunervation originating from the SCN. Objective: To investigate circadian clock genes (hBmall, hCryl, hPerl, hClock), betal-adi'energic receptor mRNA levels, and melatonin levels in relation to the AD process in the human pineal gland. Methods: Gene expression levels of clock genes and betal-adrenergic receptor were measured with quantitative PCR and melatonin levels with radioimmunoassay in postmortem pineal glands of 24 aged controls (Braak stage 0), 22 cognitively intact subjects with the earliest AD changes (Braak stage I-II), and 22 late stage AD patients (Braak stage VI) (obtained from the Netherlands Brain Bank). Conclusion: In controls, hBmalI was rhythmically expressed, with a trough during the light-phase, while hCryl displayed a peak at night, hPerl and betaladrenergic receptor gene expression were positively correlated with two peaks at the light-dark transition periods, hCIock was not rhythmically expressed in controls. In both the preclinical and clinical stages of AD, clock gene expression was arrhythmic, as were the betal-adrenergic receptor mRNA and melatonin levels. Moreover, the positive correlation between hPerl and betal-adrenergic receptor mRNA had disappeared. We conclude that circadian clock genes oscillate in the human pineal gland, but are desynchronized already in the earliest, preclinical stages of AD. Our data also suggest that a dysregulation of the SCN is most probably responsible for the pineal changes in AD.
•
NARROWING THE VISUAL SEARCH FIELD FACILITATES P E R F O R M A N C E IN PATIENTS T H O U G H T T O BE IN T H E P R O D R O M A L STAGE OF
ALZHE1MER'S DISEASE (AD) Matthew Smith .1 , K. Thurairajah 1, M. Borrie 1, S. Murtha 2. 1Dept of Geriatric Medicine, Parkwood Hospital, London, ON, Canada; 2york University, Toronto, ON, Canada. Contact e-mail: matthew.smith @sjhc.london, on. ca
Background: The ability to focus attention is slower in the elderly than the young, particularly when there are a greater number of distractors present. Impairment of speed of visual processing and selective attention have also been documented in AD. However, reaction time improves if the distractors are clumped around the target. Therefore, moving the stimuli into the center of the attentional spotlight facilitates performance in the normal elderly. Objective: It is not known whether patients thought to be in the prodromal phase of Alzheimer disease would show a similar facilitation of performance. Methods: Fifteen Normal Elderly Control (NEC, 4m, llf) and thirteen patients diagnosed with Mild Cognitive Impairment (MCI; 8m, 5f) were tested on the visual search task. The within group variables were target presence, number of distractors, type of distractor, and most importantly, distractor location. Results: Initial analysis indicated no significant differences between Age, Education, and simple reaction time. When the target was absent, the only significant interactions with group were group x location x distractor type and group x location x distractor #. In addition, although the people with MCI were generally slower, the between groups effect was not significant. For the MCI group, clumping improved performance when there were fewer (6) distractors (220 ms faster) but had no effect on performance when there were 12 distractors, whereas the NEC group were fairly efficient at the task with 6 distractors and performance was facilitated (270 ms faster) when there were 12 distractors. Analysis
S 139
of the target present data revealed two interactions with group (group x location x distractor # and group x location) as well as a significant between groups effect (MCI slower). In the MCI group clumping the distractors around the target facilitated performance mainly when there were a greater number of distractors but they were still significantly slower than the NEC. Conclusion: It appears possible to improve visual attention in MCI participants particularly if the target is actually present but never to the point that they perform as well as the elderly control subjects. This has implications for cognitive rehabilitations strategies in this group of patients.
Poster Session PI: Molecular Mechanisms of Neurodegeneration- fl-Amyloidosis
•
ASSEMBLY-DEPENDENT ABETA CONFORMATIONAL TRANSITIONS REVEALED
USING I N T R I N S I C F L U O R E S C E N C E Samir Maji, Margaret M. Condron, David B. Teplow*. Brigham and Women's Hospital, Boston, MA, USA. Contact e-mail: teplow @cnd. bwh.harvard, edu
Background: Alzheimer's disease (AD) is characterized by the presence of insoluble, fibrous aggregates of the 40-42 residue amyloid beta-protein (Abeta). A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. We report the use of intrinsic Tyr fluorescence to study the conformational changes occurring in Abeta during its oligomerization and fibril assembly. Objective(s): To use the fluorescence of Tyr residues introduced at specific sites along the entire length of the Abeta peptide as a probe of local environmental changes occurring during oligomerization and fibril formation. Methods: Abeta(1-40) and Abeta(1-42) were synthesized chemically. Single Tyr substitutions were introduced at positions 1, 10 (native Tyr), 20, 30, and 40 or 42 (for Abeta(1-40) or Abeta(1-42), respectively). For all but the native sequences, a fluorornetrically "silent" Phe residue was substituted for the native Tyr 1°. Fluorescence was then monitored periodically during peptide assembly. Solvent accessibility of the Tyr ring was studied using a mixed solvent (DMSO-water mixture) system. Results: Abeta(1-40) and Abeta(1-42) existed initially in a predominantly disordered state, however significant difference in solvent accessibility were observed between the two peptides. In Abeta(1-40), Vat4° is shielded from solvent. In Abeta(142), residues 30-42 are solvent shielded. Assembly-dependent changes in fluorescence during aggregation of native Abeta(1-40) and Abeta(1-42), and their Tyr-substituted analogues, showed that the conformation of the CHC differed between Abeta(1-40) and Abeta(1-42). Freshly solubilized Abeta( 140) displayed structural organization within the CHC, which quenched Tyr fluorescence. During aggregation, disruption of this structured occurred, resulting in a significant increase in fluorescence. This increase occurred most notably during the conversion of a helix-containing intermediate into the beta-sheet-rich conformer composing protofibrils and fibrils. Conclusion: The data suggest that CHC experiences major conformational changes during the helix/coil-to-beta-sheet conversion of Abeta(1-40) during assembly. In contrast, during Abeta(1-42) assembly, its more ordered C-terminus may be most significant.
~
E
R
S T R E S S IS E N T A N G L E D W I T H A[3 P A T H O L O G Y
Takashi Kudo *l , Dalsnke Kanayama I , Kazunori Imaizumi 2, Taichi Katayama 3 , Masayasu Oknchi 1, Takeshi Tabira 4, Masaya Tohyama 3, Masatoshi Takeda 1. IDept " Neuropsychiat., Osaka Univ. Med. Sch., Osaka, Japan; 2Div. of Structual Cell Biol., Nara Inst. Of Sci. and Technol., Nara, Japan; 3Dept. Anat. and Neurosci., Osaka Univ. Meal. Sch., Osaka, Japan; 4Natl. Inst. for Longevity Sci., Aichi, Japan. Contact e-mail: [email protected]
Background: We previously reported that presenilinl (PS1) mutations deteriorates endoplasmic reticulum (ER) stress response due to downregnlated ER stress transducers. Therefore, neurons with mutant PS1 is vulnerable
S 140
Poster Session P1." Molecular Mechanisms ofNeurodegeneration - ~-Amyloidosis
to ER stress (Katayama et al., Nat Cell Biol. 1999; 1(8):479-85, J Biol Chem. 2001 27; 276(17):13935-40 and Yasuda et al., Biochem Biophys Res Commun. 2002 16; 296(2):313-8). Objective(s): Elevated levels of [3-amyloid (A[~) are present in the brains of individuals with either the sporadic or familial form of Alzheimer's disease (AD). However, the relationship between A{3 production and the deterioration of ER stress is yet to be elucidated. Methods a n d Results: In this study, we show, by Western blotting and Northern blotting, that ER stress response: the phosphorylation of eIF2 and PERK, and the induction of Bip/GRF78, are activated in neurons exposed to A~. It is also reported that these activations stimulate the cleavage of caspase-12 to induce neuronal apoptosis. We characterized that caspase-4, which is also activated by A[3, is an ER stress-specific caspase in humans, corresponding to caspase-12 in rodents. Under ER stress, protein degradation by proteasome plays an important role. ER-associated protein degradation (ERAD) transports malfolded proteins in ER to proteasome to degrade them. Our study shows that AI3 alters the proteasome activity. Thus it is possible that AI3 may affect ERAD to cause ER stress and ER stress-induced apoptosis. These findings suggest that the attenuation of ERAD by accumulated A[3 may contribute to A~ -dependent death in AD patients. Another our study shows the possibility that ER stress attenuate Al3production or A1340/42 ratio. Conclusions: Taken together, ER stress is entangled with Al3pathology. From this point of view, we also investigate whether ER-chaperone inducers, that unload ER stress, are strong therapeutic strategy for AD.
We and others previously showed that [3-amyloid precursor protein ([3APP) is clipped by gamma-secretase at two sites, gamma- and epsilon-cleavage sites, within the transmembrane domain. This dual cleavage is observed on some other substrates of gamma-secretase, but its mechanism is totally unknown. When we think about the gamma-secretase inhibitor as a therapeutic approach, elucidation of its mechanism and/or distinction between the two cleavages would be important to find a way for eliminating its adverse effects. Objective(s): To learn more about the mechanism, we sought to find intermediate products by the gamma-secretase cleavage. Methods: We have developed 1P/Western blotting system, which makes possible to clearly distinguish each A[~ from A131-37 through A131-49. Using this protocol, we analysed intracellular A[3 and secreted A[3 in various cell lines to see if A[3 species longer than AI~42/AI~43 are present. Results: While only a trace amount of A~43 was detected in the culture media, a substantial amount of A1343 was found in the cell lysate. Most importantly, several longer A[3 species, such as A[345, AIM6, and AIM8, were indeed detectable within the cell, but virtually not in the culture media. Familial Alzheimer's disease-associated mutations of PS1/2 affected differentially the cellular levels of longer A[3 species as well as those of A1340 and A1342. The production of long AI3 was suppressed by dominant-negative Asp mutant of PS and gamma-secretase inhibitors, indicating that the generation of long A[3 is gamma-secretase-dependent. Conclusions: These results indicate that multiple cleavage sites by gamma-secretase exist between gamma- and epsilon-cleavage sites.
•
•
INTRANEURONAL AMYLOID-fll-42 PRODUCTION T R I G G E R E D BY S U S T A I N E D I N C R E A S E O F CYTOSOLIC CALCIUM CONCENTRATION I N D U C E D N E U R O N A L DEATH
ALTERATIONS IN BACE1 AND BACE2 EXPRESSION AND IN THE KINETICS OF ~ - S E C R E T A S E A C T I V I T Y IN T H E A L Z H E I M E R ' S D I S E A S E T E M P O R A L CORTEX
Nathaiie Pierrot*, J.N. Octave. Universit~ catholique de Louvain,
John Stockiey* i, Rivka Ravid 2, Cora O'Neill 1. 1Department of
Brurelles, Belgium. Contact e-mail: [email protected]
Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland; 2The Netherland's Brain Bank, Meibergdreef 33, 1105 AZ, Amsterdam, Netherlands. Contact e-mail: [email protected]
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques, which contain an amyloid core made of [3-amyloid peptide (A[3). A[3 is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signaling has been causally implicated in aging and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurons. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the c~-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the [3-cleavage of the protein (CTF[3). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal A[~1-42, which is inhibited by nimodipine, a specific antagonist of L-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal A[~, which requires influx of extraceflular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolie calcium is needed to induce the production of intraneuronal A[31-42 from human APP. Our results show that this accumulation of intraneuronal AIM-42 induces neuronal death, which is prevented by a functional y-secretase inhibitor.
•
P R E S E N C E O F L O N G Aft IN T H E LYSATE: AN I M P L I C A T I O N F O R T H E M E C H A N I S M OF G A M M A - S E C R E T A S E CLEAVAGE
Maho Morishima*, Yue Qi, Yasuo Ihara. Department of Neuropathology,
School of Medicine, University of Tokyo, Tokyo, Japan. Contact e-mail: maho @m. u-tokyo.ac.jp Background: Accumulating evidence suggests that various type I membrane proteins is degraded through regulated intramembrane proteolysis (RIP) by gamma-secretase. Gamma-secretase is a high molecular weight complex with characteristics of aspartic protease. Presenilin (PS) is essential for its activity and believed to compose the catalytic core of gamma-secretase. Nicastrin, Aph-1, and Pen-2 are also identified as its required components.
Background: [3-secretase is associated with two type-I aspartic proteases BACE1 and BACE2. BACE1 is believed to be the rate-limiting step for A[3 production in Alzheimer's disease (AD) brain and previous studies have detected increased expression and activity of BACE1 in AD brain. However, kinetics of [~-secretase activity has not been examined in AD, nor has BACE2 protein expression analysed. The present study compared BACE1/2 expression and [3-secretase kinetics in particulate fractions from AD (u = 9) and control (n = 7) temporal cortex. Results: BACE1 immunoreactivity was detected at 75 kDa, 70 kDa and 50 kDa in AD and control cases. Levels of the major BACE1 immunoreactive band (75 kDa), believed to represent mature BACE1, were significantly decreased (37%, p < 0.001) in AD cases compared to controls, as was the 70 kDa band (30%, p < 0.005). In contrast, levels of the minor 50 kDa band, believed to represent a less glycosylated BACE1 form, were significantly increased (84%, p < 0.001) in AD samples compared to controls. This increase showed strong correlation with the severity of neurofibrillary pathology. BACE2 was also detected in all cases, with major immunoreactive bands at 75 kDa and 70 kDa and a minor band at 50 kDa. Reduced levels of the higher molecular weight bands (35%, p < 0.001) and increased levels of the 50 kDa band were detected in AD cases (56%, p < 0.005) compared to controls. Examination of [3-secretase activity in control brain particulate fractions, using the wild type APP peptide substrate MCA-EVKMDAEFK(DNP)-NH2 showed activity to be potently inhibited (ICs0 = 60 nM) by a selective [3-secretase inhibitor [KTEEISEVN(STAT)VAEF]. Comparative analysis of [%secretase enzyme kinetics showed enzyme activity to be saturable, mean Km = 3.43 tzM and Vmax of 3.65rim/rain in control cases and in AD cases where increased mean K m = 5.3 tzM AD; Vmax = 4.89nndmin. values were detected. Conclusion: Decreased levels of mature forms of BACE1 and BACE2 were detected in AD temporal cortex with increases in lower M W less mature forms. These alterations are associated with changes in Vmax and Km of [3-secretase activity in AD brain.
~
CIRCADIAN C L O C K G E N E S O S C I L L A T E IN T H E HUMAN PINEAL GLAND AND A R E DESYNCHRONIZED IN A L Z H E I M E R D I S E A S E
YingHui Wu* 1, David F. Fischer 1, Jiang-Nlng Zhou2, Dick F. S waab 1. t Netherlands Institute for Brain research, Amsterdam, Netherlands; 2University of Science and Technology of China, Hefei, Anhui, China. Contact e-mail: [email protected]
Background: Circadian disturbances, such as nightly restlessness, are major clinical problems in Alzheimer's disease (AD). The deterioration of the neurons in the biological clock, the suprachiasmatic nucleus (SCN), and a dysfunction of the pineal gland in AD are supposed to be responsible for the circadian disorders. Circadian clock genes and melatonin synthesis oscillate in the rodent pineal gland under the control of the sympathetic iunervation originating from the SCN. Objective: To investigate circadian clock genes (hBmall, hCryl, hPerl, hClock), betal-adi'energic receptor mRNA levels, and melatonin levels in relation to the AD process in the human pineal gland. Methods: Gene expression levels of clock genes and betal-adrenergic receptor were measured with quantitative PCR and melatonin levels with radioimmunoassay in postmortem pineal glands of 24 aged controls (Braak stage 0), 22 cognitively intact subjects with the earliest AD changes (Braak stage I-II), and 22 late stage AD patients (Braak stage VI) (obtained from the Netherlands Brain Bank). Conclusion: In controls, hBmalI was rhythmically expressed, with a trough during the light-phase, while hCryl displayed a peak at night, hPerl and betaladrenergic receptor gene expression were positively correlated with two peaks at the light-dark transition periods, hCIock was not rhythmically expressed in controls. In both the preclinical and clinical stages of AD, clock gene expression was arrhythmic, as were the betal-adrenergic receptor mRNA and melatonin levels. Moreover, the positive correlation between hPerl and betal-adrenergic receptor mRNA had disappeared. We conclude that circadian clock genes oscillate in the human pineal gland, but are desynchronized already in the earliest, preclinical stages of AD. Our data also suggest that a dysregulation of the SCN is most probably responsible for the pineal changes in AD.
•
NARROWING THE VISUAL SEARCH FIELD FACILITATES P E R F O R M A N C E IN PATIENTS T H O U G H T T O BE IN T H E P R O D R O M A L STAGE OF
ALZHE1MER'S DISEASE (AD) Matthew Smith .1 , K. Thurairajah 1, M. Borrie 1, S. Murtha 2. 1Dept of Geriatric Medicine, Parkwood Hospital, London, ON, Canada; 2york University, Toronto, ON, Canada. Contact e-mail: matthew.smith @sjhc.london, on. ca
Background: The ability to focus attention is slower in the elderly than the young, particularly when there are a greater number of distractors present. Impairment of speed of visual processing and selective attention have also been documented in AD. However, reaction time improves if the distractors are clumped around the target. Therefore, moving the stimuli into the center of the attentional spotlight facilitates performance in the normal elderly. Objective: It is not known whether patients thought to be in the prodromal phase of Alzheimer disease would show a similar facilitation of performance. Methods: Fifteen Normal Elderly Control (NEC, 4m, llf) and thirteen patients diagnosed with Mild Cognitive Impairment (MCI; 8m, 5f) were tested on the visual search task. The within group variables were target presence, number of distractors, type of distractor, and most importantly, distractor location. Results: Initial analysis indicated no significant differences between Age, Education, and simple reaction time. When the target was absent, the only significant interactions with group were group x location x distractor type and group x location x distractor #. In addition, although the people with MCI were generally slower, the between groups effect was not significant. For the MCI group, clumping improved performance when there were fewer (6) distractors (220 ms faster) but had no effect on performance when there were 12 distractors, whereas the NEC group were fairly efficient at the task with 6 distractors and performance was facilitated (270 ms faster) when there were 12 distractors. Analysis
S 139
of the target present data revealed two interactions with group (group x location x distractor # and group x location) as well as a significant between groups effect (MCI slower). In the MCI group clumping the distractors around the target facilitated performance mainly when there were a greater number of distractors but they were still significantly slower than the NEC. Conclusion: It appears possible to improve visual attention in MCI participants particularly if the target is actually present but never to the point that they perform as well as the elderly control subjects. This has implications for cognitive rehabilitations strategies in this group of patients.
Poster Session PI: Molecular Mechanisms of Neurodegeneration- fl-Amyloidosis
•
ASSEMBLY-DEPENDENT ABETA CONFORMATIONAL TRANSITIONS REVEALED
USING I N T R I N S I C F L U O R E S C E N C E Samir Maji, Margaret M. Condron, David B. Teplow*. Brigham and Women's Hospital, Boston, MA, USA. Contact e-mail: teplow @cnd. bwh.harvard, edu
Background: Alzheimer's disease (AD) is characterized by the presence of insoluble, fibrous aggregates of the 40-42 residue amyloid beta-protein (Abeta). A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. We report the use of intrinsic Tyr fluorescence to study the conformational changes occurring in Abeta during its oligomerization and fibril assembly. Objective(s): To use the fluorescence of Tyr residues introduced at specific sites along the entire length of the Abeta peptide as a probe of local environmental changes occurring during oligomerization and fibril formation. Methods: Abeta(1-40) and Abeta(1-42) were synthesized chemically. Single Tyr substitutions were introduced at positions 1, 10 (native Tyr), 20, 30, and 40 or 42 (for Abeta(1-40) or Abeta(1-42), respectively). For all but the native sequences, a fluorornetrically "silent" Phe residue was substituted for the native Tyr 1°. Fluorescence was then monitored periodically during peptide assembly. Solvent accessibility of the Tyr ring was studied using a mixed solvent (DMSO-water mixture) system. Results: Abeta(1-40) and Abeta(1-42) existed initially in a predominantly disordered state, however significant difference in solvent accessibility were observed between the two peptides. In Abeta(1-40), Vat4° is shielded from solvent. In Abeta(142), residues 30-42 are solvent shielded. Assembly-dependent changes in fluorescence during aggregation of native Abeta(1-40) and Abeta(1-42), and their Tyr-substituted analogues, showed that the conformation of the CHC differed between Abeta(1-40) and Abeta(1-42). Freshly solubilized Abeta( 140) displayed structural organization within the CHC, which quenched Tyr fluorescence. During aggregation, disruption of this structured occurred, resulting in a significant increase in fluorescence. This increase occurred most notably during the conversion of a helix-containing intermediate into the beta-sheet-rich conformer composing protofibrils and fibrils. Conclusion: The data suggest that CHC experiences major conformational changes during the helix/coil-to-beta-sheet conversion of Abeta(1-40) during assembly. In contrast, during Abeta(1-42) assembly, its more ordered C-terminus may be most significant.
~
E
R
S T R E S S IS E N T A N G L E D W I T H A[3 P A T H O L O G Y
Takashi Kudo *l , Dalsnke Kanayama I , Kazunori Imaizumi 2, Taichi Katayama 3 , Masayasu Oknchi 1, Takeshi Tabira 4, Masaya Tohyama 3, Masatoshi Takeda 1. IDept " Neuropsychiat., Osaka Univ. Med. Sch., Osaka, Japan; 2Div. of Structual Cell Biol., Nara Inst. Of Sci. and Technol., Nara, Japan; 3Dept. Anat. and Neurosci., Osaka Univ. Meal. Sch., Osaka, Japan; 4Natl. Inst. for Longevity Sci., Aichi, Japan. Contact e-mail: [email protected]
Background: We previously reported that presenilinl (PS1) mutations deteriorates endoplasmic reticulum (ER) stress response due to downregnlated ER stress transducers. Therefore, neurons with mutant PS1 is vulnerable
S 140
Poster Session P1." Molecular Mechanisms ofNeurodegeneration - ~-Amyloidosis
to ER stress (Katayama et al., Nat Cell Biol. 1999; 1(8):479-85, J Biol Chem. 2001 27; 276(17):13935-40 and Yasuda et al., Biochem Biophys Res Commun. 2002 16; 296(2):313-8). Objective(s): Elevated levels of [3-amyloid (A[~) are present in the brains of individuals with either the sporadic or familial form of Alzheimer's disease (AD). However, the relationship between A{3 production and the deterioration of ER stress is yet to be elucidated. Methods a n d Results: In this study, we show, by Western blotting and Northern blotting, that ER stress response: the phosphorylation of eIF2 and PERK, and the induction of Bip/GRF78, are activated in neurons exposed to A~. It is also reported that these activations stimulate the cleavage of caspase-12 to induce neuronal apoptosis. We characterized that caspase-4, which is also activated by A[3, is an ER stress-specific caspase in humans, corresponding to caspase-12 in rodents. Under ER stress, protein degradation by proteasome plays an important role. ER-associated protein degradation (ERAD) transports malfolded proteins in ER to proteasome to degrade them. Our study shows that AI3 alters the proteasome activity. Thus it is possible that AI3 may affect ERAD to cause ER stress and ER stress-induced apoptosis. These findings suggest that the attenuation of ERAD by accumulated A[3 may contribute to A~ -dependent death in AD patients. Another our study shows the possibility that ER stress attenuate Al3production or A1340/42 ratio. Conclusions: Taken together, ER stress is entangled with Al3pathology. From this point of view, we also investigate whether ER-chaperone inducers, that unload ER stress, are strong therapeutic strategy for AD.
We and others previously showed that [3-amyloid precursor protein ([3APP) is clipped by gamma-secretase at two sites, gamma- and epsilon-cleavage sites, within the transmembrane domain. This dual cleavage is observed on some other substrates of gamma-secretase, but its mechanism is totally unknown. When we think about the gamma-secretase inhibitor as a therapeutic approach, elucidation of its mechanism and/or distinction between the two cleavages would be important to find a way for eliminating its adverse effects. Objective(s): To learn more about the mechanism, we sought to find intermediate products by the gamma-secretase cleavage. Methods: We have developed 1P/Western blotting system, which makes possible to clearly distinguish each A[~ from A131-37 through A131-49. Using this protocol, we analysed intracellular A[3 and secreted A[3 in various cell lines to see if A[3 species longer than AI~42/AI~43 are present. Results: While only a trace amount of A~43 was detected in the culture media, a substantial amount of A1343 was found in the cell lysate. Most importantly, several longer A[3 species, such as A[345, AIM6, and AIM8, were indeed detectable within the cell, but virtually not in the culture media. Familial Alzheimer's disease-associated mutations of PS1/2 affected differentially the cellular levels of longer A[3 species as well as those of A1340 and A1342. The production of long AI3 was suppressed by dominant-negative Asp mutant of PS and gamma-secretase inhibitors, indicating that the generation of long A[3 is gamma-secretase-dependent. Conclusions: These results indicate that multiple cleavage sites by gamma-secretase exist between gamma- and epsilon-cleavage sites.
•
•
INTRANEURONAL AMYLOID-fll-42 PRODUCTION T R I G G E R E D BY S U S T A I N E D I N C R E A S E O F CYTOSOLIC CALCIUM CONCENTRATION I N D U C E D N E U R O N A L DEATH
ALTERATIONS IN BACE1 AND BACE2 EXPRESSION AND IN THE KINETICS OF ~ - S E C R E T A S E A C T I V I T Y IN T H E A L Z H E I M E R ' S D I S E A S E T E M P O R A L CORTEX
Nathaiie Pierrot*, J.N. Octave. Universit~ catholique de Louvain,
John Stockiey* i, Rivka Ravid 2, Cora O'Neill 1. 1Department of
Brurelles, Belgium. Contact e-mail: [email protected]
Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland; 2The Netherland's Brain Bank, Meibergdreef 33, 1105 AZ, Amsterdam, Netherlands. Contact e-mail: [email protected]
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques, which contain an amyloid core made of [3-amyloid peptide (A[3). A[3 is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signaling has been causally implicated in aging and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurons. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the c~-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the [3-cleavage of the protein (CTF[3). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal A[~1-42, which is inhibited by nimodipine, a specific antagonist of L-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal A[~, which requires influx of extraceflular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolie calcium is needed to induce the production of intraneuronal A[31-42 from human APP. Our results show that this accumulation of intraneuronal AIM-42 induces neuronal death, which is prevented by a functional y-secretase inhibitor.
•
P R E S E N C E O F L O N G Aft IN T H E LYSATE: AN I M P L I C A T I O N F O R T H E M E C H A N I S M OF G A M M A - S E C R E T A S E CLEAVAGE
Maho Morishima*, Yue Qi, Yasuo Ihara. Department of Neuropathology,
School of Medicine, University of Tokyo, Tokyo, Japan. Contact e-mail: maho @m. u-tokyo.ac.jp Background: Accumulating evidence suggests that various type I membrane proteins is degraded through regulated intramembrane proteolysis (RIP) by gamma-secretase. Gamma-secretase is a high molecular weight complex with characteristics of aspartic protease. Presenilin (PS) is essential for its activity and believed to compose the catalytic core of gamma-secretase. Nicastrin, Aph-1, and Pen-2 are also identified as its required components.
Background: [3-secretase is associated with two type-I aspartic proteases BACE1 and BACE2. BACE1 is believed to be the rate-limiting step for A[3 production in Alzheimer's disease (AD) brain and previous studies have detected increased expression and activity of BACE1 in AD brain. However, kinetics of [~-secretase activity has not been examined in AD, nor has BACE2 protein expression analysed. The present study compared BACE1/2 expression and [3-secretase kinetics in particulate fractions from AD (u = 9) and control (n = 7) temporal cortex. Results: BACE1 immunoreactivity was detected at 75 kDa, 70 kDa and 50 kDa in AD and control cases. Levels of the major BACE1 immunoreactive band (75 kDa), believed to represent mature BACE1, were significantly decreased (37%, p < 0.001) in AD cases compared to controls, as was the 70 kDa band (30%, p < 0.005). In contrast, levels of the minor 50 kDa band, believed to represent a less glycosylated BACE1 form, were significantly increased (84%, p < 0.001) in AD samples compared to controls. This increase showed strong correlation with the severity of neurofibrillary pathology. BACE2 was also detected in all cases, with major immunoreactive bands at 75 kDa and 70 kDa and a minor band at 50 kDa. Reduced levels of the higher molecular weight bands (35%, p < 0.001) and increased levels of the 50 kDa band were detected in AD cases (56%, p < 0.005) compared to controls. Examination of [3-secretase activity in control brain particulate fractions, using the wild type APP peptide substrate MCA-EVKMDAEFK(DNP)-NH2 showed activity to be potently inhibited (ICs0 = 60 nM) by a selective [3-secretase inhibitor [KTEEISEVN(STAT)VAEF]. Comparative analysis of [%secretase enzyme kinetics showed enzyme activity to be saturable, mean Km = 3.43 tzM and Vmax of 3.65rim/rain in control cases and in AD cases where increased mean K m = 5.3 tzM AD; Vmax = 4.89nndmin. values were detected. Conclusion: Decreased levels of mature forms of BACE1 and BACE2 were detected in AD temporal cortex with increases in lower M W less mature forms. These alterations are associated with changes in Vmax and Km of [3-secretase activity in AD brain.