- Email: [email protected]
Melatonin ameliorates Alzheimer-like pathological changes and spatial memory retention impairment induced by calyculin A
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Poster Presentations P1 APOLIPOPROTEIN E-ISOFORM DEPENDENT HIPPOCAMPAL NEURODEGENERATION TO KAINIC ACID-INDUCED EXCITOTOXICITY
Xing-Mei Zhang1, Xi-Jing Mao1, Hong-Liang Zhang1, Therese Pham1, Abdu Adem2, Bengt Winblad1, Jie Zhu1, 1Karolinska Institute, Stockholm, Sweden; 2United Arab Emirates University, Al Ain, United Arab Emirates. Contact e-mail: [email protected] Background: The diverse effects of apolipoprotein E (apoE) isoforms in the neurodegenerative disorders have been evidenced. In addition to its physiological role in cholesterol transport, apoE has an intricate biological function in modulating immune responses. In the present study, we aimed to investigate the individual roles of apoE isoforms in the kainic acid (KA)-induced hippocampal neurodegeneration with consideration the immune responses. Methods: ApoE2, apoE3 and apoE4 transgenic mice as well as wild-type male mice (7-weeks-old) were treated with KA (40mg/kg bodyweight) intranasally. The KA-induced seizure activity and pathological changes were recorded. Additionally, before and after KA administration, all groups of mice were performed a series of behavioral tests, including elevated plus-maze, open-field test, novel objects exploration test and the spontaneous alteration behavior in a Y-maze. Microglia activation and astrocyte proliferation were evaluated by immunohistochemistry. The inflammatory molecules secreted by microglia such as MHC-II, CD86, F4/80, CD95, inducible nitric oxide synthase (iNOS), TNF-a, IL-6, IL-12 and IL-17 were detected by flow cytometry. Results: We found that the same amount of KA induced severer seizure activity and pathological changes in apoE4 transgenic mice compared to other groups of mice. In open-field test, KA-treated apoE4 mice showed lower locomotion and rearing activity than KA-treated other groups of mice. In elevated plus-maze test, KA administration resulted in higher counts of head drops in the cross-area in apoE4 transgenic mice, indicating the changed risk assessment. Detected by flow cytometry, microglia-secreted CD86 and MHCII elevated in KA-treated apoE4 transgenic mice. Additionally, there was also an apoE-isoform dependent elevation of iNOS expression 1 day after KA insult. Conclusions: In conclusion, overexpression of apoE4 deteriorated KA-induced hippocampal neurodegeneration, which might be resulted from the up-regulated microglia activation and followed more secretion of the inflammatory molecules. P1-326
ROLE OF NEUROINFLAMMATION IN HYPERTENSION-INDUCED BRAIN AMYLOID PATHOLOGY
Daniela Carnevale1, Giada Mascio1, Germana Cocozza1, Giuseppe Cifelli1, Luisa Minghetti2, Giuseppe Lembo1, 1IRCCS Neuromed, Pozzilli, Italy; 2 Istituto Superiore di Sanita`, Roma, Italy. Contact e-mail: daniela. [email protected] Background: Associations between hypertension and sporadic Alzheimer’s Disease (AD) have been identified but clear links between their pathophysiology have not yet been demonstrated. Inflammation is recognized as a pathophysiological trait of both hypertension and AD. Hypertension-triggered hemodynamic changes can be expected to activate neurovascular unit signaling, eventually leading to activation of microglia, the brain resident macrophages, and to neuroinflammation. Thus, we explored if hypertension could influence b-amyloid (Ab) deposition by promoting neuroinflammation. Methods: The possible dynamic interaction among hypertension, inflammation and Ab-deposition was studied in a hypertensive mouse model obtained by transverse aortic coarctation (TAC), selectively imposing cerebral circulation pressure overload. In this model Ab deposits are detectable in brain parenchyma and around vasculature as early as four weeks after TAC. Thus, brain pathology was analyzed 3 weeks after TAC, an early time preceding Ab deposition, and at 8 weeks after TAC, a later time with over Ab. Results: TAC mice displayed alterations of brain immune surveillance, as revealed by microglial morphological changes at the time preceding overt Ab deposition, thus demonstrating that the neuroinflammatory reaction is most likely the consequence of hypertension. Microglia reaction was further supported by changes in mRNA levels of both pro- and anti-inflammatory cytokines. This response was even more pronounced at the later time
along with more evident Ab deposition. Thus, the hypertension-driven neuroinflammation could exert an important pathophysiological role in the onset of AD. Therefore, we explored whether a strategy aimed at immune system stimulation and known to modulate neuroinflammation, could modify the main traits of the disease. Remarkably, we found that a challenge of peripheral LPS, given during the progression of the pathology, induced a strong reduction of Ab deposits in the brain. Conclusions: Our results indicate that hypertension per se triggers a neuroinflammatory reaction, which precede evident Ab deposition. The reduced amyloid burden following mild stimulation of peripheral immune system suggests that targeting inflammation in the appropriate time window may be successful in limiting vascular triggered AD neurodegenerative processes. P1-327
ANALYSIS OF CHLAMYDIA PNEUMONIAEINFECTED MONOCYTES FOLLOWING INCUBATION WITH A NOVEL PEPTIDE, ACALY18: A POTENTIAL TREATMENT FOR INFECTION IN ALZHEIMER’S DISEASE
Brian J. Balin1, James D. Thacker2, Corey M. Caruthers1, Susan T. Hingley1, Elizabeth K. Ruszak1, Juliana D. Zoga1, Denah M. Appelt1, 1Philadelphia College of Osteopathic Medicine, Philadelphia, PA, USA; 2Institute for Hepatitis and Virus Research, Doylestown, PA, USA. Contact e-mail: [email protected] Background: Our laboratory has been studying the role of infection with the obligate intracellular bacterium, Chlamydia pneumoniae, in sporadic late-onset Alzheimer disease (LOAD). This infection may be a trigger for the pathology observed in LOAD as a function of initiating neuroinflammation following entry of the organism into the brain. We have hypothesized that one entry mechanism may be by blood-borne infected monocytes trafficking the infection into the brain. Methods: Our current studies focus on infection of monocytes in vitro followed by analysis of infection using immunofluorescence labeling and RTPCR-microarray techniques. In addition, we are studying a novel approach utilizing a unique peptide, acALY18, derived from the endogenously expressed endoplasmic reticulum protein TRPC1, to eradicate the organism at 24-48 hr post-infection, thereby limiting its capacity to develop into a chronic/persistent infection. The peptide appears to stimulate the innate immune system through activation of the inflammasome. Results: C. pneumoniae prominently and stably infected THP1 monocytes at 24-48hr. Numerous large inclusions were labeled using specific anti-chlamydial monoclonal antibodies. Monocyte gene expression, both for markers of innate and adaptive immunity as well as for Alzheimer disease, was significantly altered. Three genes were up-regulated as compared to 45 genes down-regulated in the immunity array at 48hr post-infection, whereas in the Alzheimer array, 4 genes including those for cathepsins B and D were up-regulated as compared to 5 down-regulated. Intriguingly, following incubation of C. pneumoniae-infected cells with acALY18 peptide (25-50nM) at 24hr post-infection, there was a dramatic clearance of the organism from the monocytes (80% infected and untreated to 13-15% infected after treatment). Furthermore, gene regulation was altered following peptide treatment as there were 39 up-regulated including those for CASP1 and IL-1 at least 4 fold, and no genes down-regulated in the immunity array. Conclusions: Our data suggest that C. pneumoniae-infected monocytes are altered significantly to promote a chronic/persistent infection that may account for the presence of C. pneumoniae in LOAD. Furthermore, stimulating the innate immune response using the novel peptide, acALY18, promotes clearance of C. pneumoniae from infected monocytes; this peptide may be a viable candidate for treating C. pneumoniae infections in Alzheimer disease. P1-328
MELATONIN AMELIORATES ALZHEIMER-LIKE PATHOLOGICAL CHANGES AND SPATIAL MEMORY RETENTION IMPAIRMENT INDUCED BY CALYCULIN A
Ying Yang, Jing Feng, Yin Li, Yun Cao, Jie Deng, Qi-Miao Feng, Qing Tian, Jian-Zhi Wang, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: yangying3636@ yahoo.com
Poster Presentations P1 Background: Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of intracellular neurofibrillary tangles (NFTs). It is reported that hyperphosphorylated tau and neurofilaments (NFs) which are the major protein components of NFTs might be the result of an imbalanced regulation in protein kinases and protein phosphatases in affected neurons. Accumulating data has demonstrated that PP-2A plays key role in Alzheimer-like hyperphosphorylation of cytosketal proteins and spatial memory retention. However, the mechanisms of spatial memory impairment are not clear, and there is no effective measure to prevent both AD-like pathological changes and spatial memory retention impairment. Methods: In this study, we tested the in vivo effects of melatonin (MT) on these AD-like pathological changes and spatial memory retention deficit in rats by bilateral injection of calyculin A (CA), a potent and specific inhibitor of protein phosphatase-2A (PP-2A) and protein phosphatase-1 (PP-1) into hippocampus. We employed Morris Water Maze test to examine spatial memory of adult rats, used western blot and immunofluorescence to detect alterations of cytosketal proteins phosphorylation, and pre- and post-synaptic molecules. Results: In this study, we found bilateral hippocampal injection of 2 ml of 16mM CA induced approximately 40% inhibition of PP-2A but no inhibition of PP-1, whereas 32mM CA induced 65% inhibition of PP-2A and 35% inhibition of PP-1. Administration of MT intraperitoneally for 9 consecutive days before bilateral hippocampal injection of 2 ml of 16mM CA could prevent CA-induced synaptophysin loss, memory retention deficits, as well as hyperphosphorylation of tau and neurofilaments (NFs). Furthermore, MT partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307 (Y307), a crucial site negatively regulating the activity of PP-2A. Conclusions: These results suggest that MT could serve as a potential therapeutic agent for preventing AD-like pathological changes and behavioral abnormality via modulating the activity of PP-2A. P1-329
CHRONIC TYPE II DIABETES DIFFERENTIALLY CHANGE THE EXPRESSION PATTERN OF TAU ISOFORMS AND HYPERPHOSPHORYLATION DEPENDING ON TAU SOLUBILITY
Sun Ah Park, Hyun Jung Jung, Soonchunhyang University Bucheon Hospital, Bucheon, Gyeonggi-do, Republic of Korea. Contact e-mail: [email protected] Background: Type II diabetes (T2DM) has been suggested to be a strong risk factor for Alzheimer’s disease (AD). Its pathomechanism has not been fully identified, however increased tau phosphorylation has been consistently reported in animal model of T2DM. To further understand the role of tau in T2DM in relation with AD, we started this experiment. Methods: The pattern of expression of tau isoforms and phosphorylation were explored after serial extraction of brain from old diabetic Otsuka Long Evans Tokushima Fatty (OLETF) rats (13 months old) and their control, agematched nondiabetic Long Evans Tokushima Otsuka (LETO). TBS and subsequent 2% SDS extraction were performed. And tau phoshorylation at Ser199, Ser396, Thr 212, and Thr231 was investigated using specific antibody. Results: The expression pattern of tau isoforms in TBS extracts was not different between OLETF and LETO on western blot, however the SDS extractable tau showed different pattern of isoforms in OLETF, increased at the size of 48kD (ON) and 52kD (1N), while decreased at 62kD (2N). For the tau phoshorylation we found the increased phosphorylation at Thr212 and Thr231 but no change at Ser199 and Ser396 in TBS extracts of OLETF rats in comparison with LETO. On the other hand, no detectable level of phosphorylation except at Ser199 was noted in SDS extracts both in OLETF and LETO. Even at this site the extent of tau phosphorylation was minimal and not different between OLETF and LETO. Conclusions: In summary, the increased phosphorylation of tau in T2DM was only present in the soluble fraction of tau not in insoluble fraction. Additionally, change in pattern of tau isoforms showing the increase in lighter isoforms, while decrease in heavier form was noted in insoluble fraction in T2DM. The different expression pattern of tau isoforms and increased hyperphosphorylation
S269
depending on tau solubility are expected to play a cooperative role in chronic T2DM contributing AD pathology. P1-330
CHRONIC SODIUM NITRITE INTAKE INDUCED SPATIAL MEMORY DEFICIT IN RATS AND THE UNDERLYING MECHANISMS
Qing Tian, Institute of Neuroscience, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: Sodium nitrite is widely used to fix color in preserved food. It was reported that certain dose of sodium nitrite was used to induce acute experimental amnesia. The effect of chronic intake of sodium nitrite on learning and memory is not established. Methods: To explore the effect of chronic intake of sodium nitrite on spatial learning and memory in rats and the underlying mechanisms. Results: Rats drank water containing 667mg/L sodium nitrite for 60 days, then, the spatial learning and memory were detected by Morris water maze. Phosphorylation of tau and neurofilament (NF), expression of protein phosphatase 2A catalytic subunit (PP2Ac) were detected with certain antibodies by immunocytochemistry. The hippocampal acetylcholine (Ach) level was assayed by microdialysis and high-performance liquid chromatography. Conclusions: Down-regulation of PP2A may underlie both abnormal hyperphosphorylation of cytoskeletal proteins and cholinergic deficiency in sodium nitrite drinking rats with spatial learning and memory impairment. P1-331
ROLE OF GSK3b IN BEHAVIORAL ABNORMALITIES AND TAU HYPERPHOSPHORYLATION IN OLFACTORY BULBECTOMIZED RATS
Juan Hu, Lingqiang Zhu, Jianzhi Wang, Institute of Neuroscience, Tongji Medical College, Huazhong University, Wuhan, China. Contact e-mail: [email protected] Background: Bilateral olfactory bulbectomy (OBX) initiates in the brain a pathological process similar to human Alzheimer’s disease in location, biochemistry, and behavioral manifestations. Such animals could be considered as a model of sporadic form of Alzheimer’s disease (AD) rather than of a depression model. Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles (NFTs) in Alzheimer’s disease. Glycogen Synthase Kinase-3b (GSK3b) play a key role in tau phosphorylation at most AD-related epitopes in vivo. There is no effective cure to arrest its hyperphosphorylation until now. Methods: In this study we used Sprague Dawley rats with bilateral olfactory bulbectomy (OBX) as one of the AD models and measured their learning/memory abilities and levels of tau phosphorylation. The rats were assigned to two groups: sham-operated rats, SO, and olfactory bulbectomized rats, OBX and tested with the Morris water paradigm 2 weeks after OBX. A selective GSK3b inhibitor, SB216763 was injected in tail vein every day after OBX to reverse the tau hyperphosphorylation. Results: Here, we found SB216763 (0.6mg/kg/day), a selective GSK3b inhibitor, significantly improved memory deficits as assessed by Morris water maze in OBX rats. The results by Western blot showed that the immunoreaction of tau at Ser9 epitopes of GSK-3b was weakened significantly in temporal lobe cortex when comparing the OBX group to sham-operated controls, while no obvious change was observed in the total GSK-3b. Immuoreaction at pT205, pT231 epitopes were enhanced, suggesting the activity of GSK-3b was elevated. Similar results were observed by immunohistochemistry staining, in which hyperphosphorylated tau was mainly detected in cortex and mossy fibers of hippocampal CA3 region. SB-216763 can reverse these changes. Morris water maze test showed that the latency to find the hidden platform and the distance to reach the platform were shorter in SB team compared with the DMSO team after OBX. Conclusions: Taken together, tail vein injection with SB216763 alleviated behavioral deficits in OBX rats. We also demonstrated the the preventive effect of SB216763 in Alzheimer-like tau hyperphosphorylation. Our findings suggest GSK3b play a critical role in behavioral deficits and tau hyperphosphorylation in OBX rats.
Poster Presentations P1 APOLIPOPROTEIN E-ISOFORM DEPENDENT HIPPOCAMPAL NEURODEGENERATION TO KAINIC ACID-INDUCED EXCITOTOXICITY
Xing-Mei Zhang1, Xi-Jing Mao1, Hong-Liang Zhang1, Therese Pham1, Abdu Adem2, Bengt Winblad1, Jie Zhu1, 1Karolinska Institute, Stockholm, Sweden; 2United Arab Emirates University, Al Ain, United Arab Emirates. Contact e-mail: [email protected] Background: The diverse effects of apolipoprotein E (apoE) isoforms in the neurodegenerative disorders have been evidenced. In addition to its physiological role in cholesterol transport, apoE has an intricate biological function in modulating immune responses. In the present study, we aimed to investigate the individual roles of apoE isoforms in the kainic acid (KA)-induced hippocampal neurodegeneration with consideration the immune responses. Methods: ApoE2, apoE3 and apoE4 transgenic mice as well as wild-type male mice (7-weeks-old) were treated with KA (40mg/kg bodyweight) intranasally. The KA-induced seizure activity and pathological changes were recorded. Additionally, before and after KA administration, all groups of mice were performed a series of behavioral tests, including elevated plus-maze, open-field test, novel objects exploration test and the spontaneous alteration behavior in a Y-maze. Microglia activation and astrocyte proliferation were evaluated by immunohistochemistry. The inflammatory molecules secreted by microglia such as MHC-II, CD86, F4/80, CD95, inducible nitric oxide synthase (iNOS), TNF-a, IL-6, IL-12 and IL-17 were detected by flow cytometry. Results: We found that the same amount of KA induced severer seizure activity and pathological changes in apoE4 transgenic mice compared to other groups of mice. In open-field test, KA-treated apoE4 mice showed lower locomotion and rearing activity than KA-treated other groups of mice. In elevated plus-maze test, KA administration resulted in higher counts of head drops in the cross-area in apoE4 transgenic mice, indicating the changed risk assessment. Detected by flow cytometry, microglia-secreted CD86 and MHCII elevated in KA-treated apoE4 transgenic mice. Additionally, there was also an apoE-isoform dependent elevation of iNOS expression 1 day after KA insult. Conclusions: In conclusion, overexpression of apoE4 deteriorated KA-induced hippocampal neurodegeneration, which might be resulted from the up-regulated microglia activation and followed more secretion of the inflammatory molecules. P1-326
ROLE OF NEUROINFLAMMATION IN HYPERTENSION-INDUCED BRAIN AMYLOID PATHOLOGY
Daniela Carnevale1, Giada Mascio1, Germana Cocozza1, Giuseppe Cifelli1, Luisa Minghetti2, Giuseppe Lembo1, 1IRCCS Neuromed, Pozzilli, Italy; 2 Istituto Superiore di Sanita`, Roma, Italy. Contact e-mail: daniela. [email protected] Background: Associations between hypertension and sporadic Alzheimer’s Disease (AD) have been identified but clear links between their pathophysiology have not yet been demonstrated. Inflammation is recognized as a pathophysiological trait of both hypertension and AD. Hypertension-triggered hemodynamic changes can be expected to activate neurovascular unit signaling, eventually leading to activation of microglia, the brain resident macrophages, and to neuroinflammation. Thus, we explored if hypertension could influence b-amyloid (Ab) deposition by promoting neuroinflammation. Methods: The possible dynamic interaction among hypertension, inflammation and Ab-deposition was studied in a hypertensive mouse model obtained by transverse aortic coarctation (TAC), selectively imposing cerebral circulation pressure overload. In this model Ab deposits are detectable in brain parenchyma and around vasculature as early as four weeks after TAC. Thus, brain pathology was analyzed 3 weeks after TAC, an early time preceding Ab deposition, and at 8 weeks after TAC, a later time with over Ab. Results: TAC mice displayed alterations of brain immune surveillance, as revealed by microglial morphological changes at the time preceding overt Ab deposition, thus demonstrating that the neuroinflammatory reaction is most likely the consequence of hypertension. Microglia reaction was further supported by changes in mRNA levels of both pro- and anti-inflammatory cytokines. This response was even more pronounced at the later time
along with more evident Ab deposition. Thus, the hypertension-driven neuroinflammation could exert an important pathophysiological role in the onset of AD. Therefore, we explored whether a strategy aimed at immune system stimulation and known to modulate neuroinflammation, could modify the main traits of the disease. Remarkably, we found that a challenge of peripheral LPS, given during the progression of the pathology, induced a strong reduction of Ab deposits in the brain. Conclusions: Our results indicate that hypertension per se triggers a neuroinflammatory reaction, which precede evident Ab deposition. The reduced amyloid burden following mild stimulation of peripheral immune system suggests that targeting inflammation in the appropriate time window may be successful in limiting vascular triggered AD neurodegenerative processes. P1-327
ANALYSIS OF CHLAMYDIA PNEUMONIAEINFECTED MONOCYTES FOLLOWING INCUBATION WITH A NOVEL PEPTIDE, ACALY18: A POTENTIAL TREATMENT FOR INFECTION IN ALZHEIMER’S DISEASE
Brian J. Balin1, James D. Thacker2, Corey M. Caruthers1, Susan T. Hingley1, Elizabeth K. Ruszak1, Juliana D. Zoga1, Denah M. Appelt1, 1Philadelphia College of Osteopathic Medicine, Philadelphia, PA, USA; 2Institute for Hepatitis and Virus Research, Doylestown, PA, USA. Contact e-mail: [email protected] Background: Our laboratory has been studying the role of infection with the obligate intracellular bacterium, Chlamydia pneumoniae, in sporadic late-onset Alzheimer disease (LOAD). This infection may be a trigger for the pathology observed in LOAD as a function of initiating neuroinflammation following entry of the organism into the brain. We have hypothesized that one entry mechanism may be by blood-borne infected monocytes trafficking the infection into the brain. Methods: Our current studies focus on infection of monocytes in vitro followed by analysis of infection using immunofluorescence labeling and RTPCR-microarray techniques. In addition, we are studying a novel approach utilizing a unique peptide, acALY18, derived from the endogenously expressed endoplasmic reticulum protein TRPC1, to eradicate the organism at 24-48 hr post-infection, thereby limiting its capacity to develop into a chronic/persistent infection. The peptide appears to stimulate the innate immune system through activation of the inflammasome. Results: C. pneumoniae prominently and stably infected THP1 monocytes at 24-48hr. Numerous large inclusions were labeled using specific anti-chlamydial monoclonal antibodies. Monocyte gene expression, both for markers of innate and adaptive immunity as well as for Alzheimer disease, was significantly altered. Three genes were up-regulated as compared to 45 genes down-regulated in the immunity array at 48hr post-infection, whereas in the Alzheimer array, 4 genes including those for cathepsins B and D were up-regulated as compared to 5 down-regulated. Intriguingly, following incubation of C. pneumoniae-infected cells with acALY18 peptide (25-50nM) at 24hr post-infection, there was a dramatic clearance of the organism from the monocytes (80% infected and untreated to 13-15% infected after treatment). Furthermore, gene regulation was altered following peptide treatment as there were 39 up-regulated including those for CASP1 and IL-1 at least 4 fold, and no genes down-regulated in the immunity array. Conclusions: Our data suggest that C. pneumoniae-infected monocytes are altered significantly to promote a chronic/persistent infection that may account for the presence of C. pneumoniae in LOAD. Furthermore, stimulating the innate immune response using the novel peptide, acALY18, promotes clearance of C. pneumoniae from infected monocytes; this peptide may be a viable candidate for treating C. pneumoniae infections in Alzheimer disease. P1-328
MELATONIN AMELIORATES ALZHEIMER-LIKE PATHOLOGICAL CHANGES AND SPATIAL MEMORY RETENTION IMPAIRMENT INDUCED BY CALYCULIN A
Ying Yang, Jing Feng, Yin Li, Yun Cao, Jie Deng, Qi-Miao Feng, Qing Tian, Jian-Zhi Wang, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: yangying3636@ yahoo.com
Poster Presentations P1 Background: Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of intracellular neurofibrillary tangles (NFTs). It is reported that hyperphosphorylated tau and neurofilaments (NFs) which are the major protein components of NFTs might be the result of an imbalanced regulation in protein kinases and protein phosphatases in affected neurons. Accumulating data has demonstrated that PP-2A plays key role in Alzheimer-like hyperphosphorylation of cytosketal proteins and spatial memory retention. However, the mechanisms of spatial memory impairment are not clear, and there is no effective measure to prevent both AD-like pathological changes and spatial memory retention impairment. Methods: In this study, we tested the in vivo effects of melatonin (MT) on these AD-like pathological changes and spatial memory retention deficit in rats by bilateral injection of calyculin A (CA), a potent and specific inhibitor of protein phosphatase-2A (PP-2A) and protein phosphatase-1 (PP-1) into hippocampus. We employed Morris Water Maze test to examine spatial memory of adult rats, used western blot and immunofluorescence to detect alterations of cytosketal proteins phosphorylation, and pre- and post-synaptic molecules. Results: In this study, we found bilateral hippocampal injection of 2 ml of 16mM CA induced approximately 40% inhibition of PP-2A but no inhibition of PP-1, whereas 32mM CA induced 65% inhibition of PP-2A and 35% inhibition of PP-1. Administration of MT intraperitoneally for 9 consecutive days before bilateral hippocampal injection of 2 ml of 16mM CA could prevent CA-induced synaptophysin loss, memory retention deficits, as well as hyperphosphorylation of tau and neurofilaments (NFs). Furthermore, MT partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307 (Y307), a crucial site negatively regulating the activity of PP-2A. Conclusions: These results suggest that MT could serve as a potential therapeutic agent for preventing AD-like pathological changes and behavioral abnormality via modulating the activity of PP-2A. P1-329
CHRONIC TYPE II DIABETES DIFFERENTIALLY CHANGE THE EXPRESSION PATTERN OF TAU ISOFORMS AND HYPERPHOSPHORYLATION DEPENDING ON TAU SOLUBILITY
Sun Ah Park, Hyun Jung Jung, Soonchunhyang University Bucheon Hospital, Bucheon, Gyeonggi-do, Republic of Korea. Contact e-mail: [email protected] Background: Type II diabetes (T2DM) has been suggested to be a strong risk factor for Alzheimer’s disease (AD). Its pathomechanism has not been fully identified, however increased tau phosphorylation has been consistently reported in animal model of T2DM. To further understand the role of tau in T2DM in relation with AD, we started this experiment. Methods: The pattern of expression of tau isoforms and phosphorylation were explored after serial extraction of brain from old diabetic Otsuka Long Evans Tokushima Fatty (OLETF) rats (13 months old) and their control, agematched nondiabetic Long Evans Tokushima Otsuka (LETO). TBS and subsequent 2% SDS extraction were performed. And tau phoshorylation at Ser199, Ser396, Thr 212, and Thr231 was investigated using specific antibody. Results: The expression pattern of tau isoforms in TBS extracts was not different between OLETF and LETO on western blot, however the SDS extractable tau showed different pattern of isoforms in OLETF, increased at the size of 48kD (ON) and 52kD (1N), while decreased at 62kD (2N). For the tau phoshorylation we found the increased phosphorylation at Thr212 and Thr231 but no change at Ser199 and Ser396 in TBS extracts of OLETF rats in comparison with LETO. On the other hand, no detectable level of phosphorylation except at Ser199 was noted in SDS extracts both in OLETF and LETO. Even at this site the extent of tau phosphorylation was minimal and not different between OLETF and LETO. Conclusions: In summary, the increased phosphorylation of tau in T2DM was only present in the soluble fraction of tau not in insoluble fraction. Additionally, change in pattern of tau isoforms showing the increase in lighter isoforms, while decrease in heavier form was noted in insoluble fraction in T2DM. The different expression pattern of tau isoforms and increased hyperphosphorylation
S269
depending on tau solubility are expected to play a cooperative role in chronic T2DM contributing AD pathology. P1-330
CHRONIC SODIUM NITRITE INTAKE INDUCED SPATIAL MEMORY DEFICIT IN RATS AND THE UNDERLYING MECHANISMS
Qing Tian, Institute of Neuroscience, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: Sodium nitrite is widely used to fix color in preserved food. It was reported that certain dose of sodium nitrite was used to induce acute experimental amnesia. The effect of chronic intake of sodium nitrite on learning and memory is not established. Methods: To explore the effect of chronic intake of sodium nitrite on spatial learning and memory in rats and the underlying mechanisms. Results: Rats drank water containing 667mg/L sodium nitrite for 60 days, then, the spatial learning and memory were detected by Morris water maze. Phosphorylation of tau and neurofilament (NF), expression of protein phosphatase 2A catalytic subunit (PP2Ac) were detected with certain antibodies by immunocytochemistry. The hippocampal acetylcholine (Ach) level was assayed by microdialysis and high-performance liquid chromatography. Conclusions: Down-regulation of PP2A may underlie both abnormal hyperphosphorylation of cytoskeletal proteins and cholinergic deficiency in sodium nitrite drinking rats with spatial learning and memory impairment. P1-331
ROLE OF GSK3b IN BEHAVIORAL ABNORMALITIES AND TAU HYPERPHOSPHORYLATION IN OLFACTORY BULBECTOMIZED RATS
Juan Hu, Lingqiang Zhu, Jianzhi Wang, Institute of Neuroscience, Tongji Medical College, Huazhong University, Wuhan, China. Contact e-mail: [email protected] Background: Bilateral olfactory bulbectomy (OBX) initiates in the brain a pathological process similar to human Alzheimer’s disease in location, biochemistry, and behavioral manifestations. Such animals could be considered as a model of sporadic form of Alzheimer’s disease (AD) rather than of a depression model. Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles (NFTs) in Alzheimer’s disease. Glycogen Synthase Kinase-3b (GSK3b) play a key role in tau phosphorylation at most AD-related epitopes in vivo. There is no effective cure to arrest its hyperphosphorylation until now. Methods: In this study we used Sprague Dawley rats with bilateral olfactory bulbectomy (OBX) as one of the AD models and measured their learning/memory abilities and levels of tau phosphorylation. The rats were assigned to two groups: sham-operated rats, SO, and olfactory bulbectomized rats, OBX and tested with the Morris water paradigm 2 weeks after OBX. A selective GSK3b inhibitor, SB216763 was injected in tail vein every day after OBX to reverse the tau hyperphosphorylation. Results: Here, we found SB216763 (0.6mg/kg/day), a selective GSK3b inhibitor, significantly improved memory deficits as assessed by Morris water maze in OBX rats. The results by Western blot showed that the immunoreaction of tau at Ser9 epitopes of GSK-3b was weakened significantly in temporal lobe cortex when comparing the OBX group to sham-operated controls, while no obvious change was observed in the total GSK-3b. Immuoreaction at pT205, pT231 epitopes were enhanced, suggesting the activity of GSK-3b was elevated. Similar results were observed by immunohistochemistry staining, in which hyperphosphorylated tau was mainly detected in cortex and mossy fibers of hippocampal CA3 region. SB-216763 can reverse these changes. Morris water maze test showed that the latency to find the hidden platform and the distance to reach the platform were shorter in SB team compared with the DMSO team after OBX. Conclusions: Taken together, tail vein injection with SB216763 alleviated behavioral deficits in OBX rats. We also demonstrated the the preventive effect of SB216763 in Alzheimer-like tau hyperphosphorylation. Our findings suggest GSK3b play a critical role in behavioral deficits and tau hyperphosphorylation in OBX rats.