- Email: [email protected]
P4-265: Changes of synaptic ultrastructure, synaptophysin and choline acetyltransferase in hippocampus of age-increasing rats
Poster Presentations P4:
T749
P4-266
MODIFICATION OF SYNAPTIC PLASTICITY BY TNF AND SPHINGOMYELINASE: IMPLICATIONS FOR COGNITIVE IMPAIRMENT IN ALZHEIMER’S DISEASE
Creutzfeldt-Jakob’s disease. Recent evidence suggests that rather than amyloid plaques, pre-fibrillar small oligomeric amyloid intermediates may be the most neurotoxic species. Notably oligomers from diverse diseaserelated amyloids share a remarkably similar quaternary structure and are equally cytotoxic, suggesting common mechanisms of action. With this in mind, we investigated the role of calcineurin (CaN), a protein phosphatase abundant in neurons and involved in synaptic plasticity and neuronal death, in mediating the cytotoxic and cognitive effects of various amyloid oligomers. Methods: The effect of monomeric, oligomeric and fibrillar A, ␣-synuclein, prion, Huntington’s polyQ and amylin (IAPP) were assessed on 1) CaN activity, CREB-mediated transcription and CaN-dependent cell death in human SY5Y neuroblastoma cells; 2) CaN-dependent long term potentiation (LTP) in hippocampal slices and 3) CaN-dependent cognitive function after icv injection in mice. Results: We found that all oligomeric amyloids tested induced CaN and evoked CaN-dependent cell death, whereas their monomeric and fibrillar counterparts were ineffective. We also found that oligomeric A, ␣-synuclein and prion decreased CREBdriven transcription, an event important for LTP and negatively regulated by CaN. Indeed, we found CaN activity to be increased and LTP to be depressed by oligomeric A and ␣-synuclein and this effect was prevented by inhibition of CaN. Lastly, oligomeric A and ␣-synuclein similarly induced CaN and CaN-dependent memory deficits when acutely injected icv in mice. Conclusions: Collectively, these results suggest that there may be a dysfunctional signaling pathway commonly elicited by oligomeric amyloid proteins that involves CaN hyper-activity and produces decreased LTP, memory impairments and eventually cell death, all events playing a central role in such amyloid diseases as AD, PD and Creutzfeldt-Jacob disease. These results further suggest that inhibition of CNS CaN shall be considered as a possible pharmacological target for the treatment of cognitive deficits and neurodegeneration in amyloid diseases. P4-265
CHANGES OF SYNAPTIC ULTRASTRUCTURE, SYNAPTOPHYSIN AND CHOLINE ACETYLTRANSFERASE IN HIPPOCAMPUS OF AGE-INCREASING RATS
Rong Wang, Lin Li, Yu Tang, Lan Zhang, Beijing Xuan-Wu Hospital, Capital Medical University, Beijing, China. Contact e-mail: [email protected] Background: The aim of the present study is to investigate the changes in hippocampal synapses and their relation with learning-memory abilities at different ages, and evaluate the effects of 2,3,5,4’-tetrahydroxystilbene-2O--D-glucoside (TSG), which is one of the major components of a traditional Chinese herb Polygonum multiflorum, on brain aging. Methods: SD rats at the age of 1, 3, 6, 18 and 24 months were used. TSG at doses of 30 and 60mg/kg/day was intragastrically administered to 21-month-old rats for 3 months, respectively. Learning-memory abilities were determined by Morris water maze (MWM) and passageway water maze test. The ultra-structure of synapses in hippocampal CA1 region was observed by electronic microscopy. The expression of synaptophysin (SYP) in whole hippocampus was measured by using immunohistochemistry. Results: Compared with rats at 6 months of age, both the 1-month-old rats and 24-month-old rats showed longer escape latency and swimming distance in MWM test, while more errors are detected in the passageway water maze test, with smaller number of synapses and synaptic vesicles and less expression of SYP in the hippocampus. Treatment with high-dose TSG in rats at 24 months of age had significant improvement in the learningmemory abilities in the water maze tests associated with an increase in the number of synapses and synaptic vesicles, and an elevation of expression of SYP in the hippocampus. Conclusions: In conclusion, hippocampal synapses count and SYP expression decreased in aged rats, which may be one of the mechanisms involved in learning-memory deficit. TSG reversed the above changes in aged rats, suggesting that TSG may be beneficial for the treatment of Alzheimer disease or cognitive impairment in old people.
Yue Wang, NIA/NIH, Baltimore, MD, USA. Contact e-mail: [email protected] Background: Levels of the pro-inflammatory cytokine tumor necrosis factor-␣ (TNF), and the sphingomyelin metabolite ceramide, are increased in association with the A and neurofibrillary pathology of AD (Exp Neurol 1999;160:289-99. PNAS 2004; 101:2070-5). In response to TNF and oxidative stress, sphingomyelin can be rapidly cleaved by neutral sphingomyelinase (nSmase) to produce ceramide. Because recent findings suggest that TNF can modify synaptic plasticity (Synapse 2000; 35:151-9. Eur J Neurosci 2005; 22:2827-32), we performed experiments aimed at understanding if and how TNF affects glutamate receptor-mediated plasticity at hippocampal CA1 synapses. Methods: Whole-cell patch clamp recordings of EPSCs from CA1 pyramidal cells were performed by visualization of the cells with a 40X water immersion lens. The membrane potential was held at either -70 mV for recording AMPAR component of EPSCs or a voltage steps from -70 to ⫹40 mV for recording NMDAR component of EPSCs, with or without CNQX (10 M) or DL-AP5 (100 M). Patch-clamp electrodes were filled with a solution containing (mM): potassium gluconate, 130; KCl, 10; EGTA, 10; CaCl2, 1; MgCl2, 3; Hepes, 20; Mg-ATP, 5; Na-GTP, 0.5; QX 314, 10; pH 7.2 (the osmolality was adjusted to 280 mmol/kg). Data were collected and analyzed using an Axopatch 200B and pCLAMP 8 software (Axon Instruments). All signals were recorded and filtered at 2 kHz and digitized at 10 kHz. Results: Both the NMDA and AMPA components of EPSCs were transiently altered by TNF. A 2 min perfusion with TNF significantly enhanced the NMDAevoked current at ⫹ 40mV and inhibited the AMPA-evoked current at -70mV. A 1 h pre-incubation with GW4869, an inhibitor of nSmase, blocked the ability of TNF to enhance NMDA currents and inhibit AMPA currents. ISP-1, a potent inhibitor of sphingolipid biosynthesis, did not prevent TNF-induced enhancement of NMDA currents or depression of AMPA currents. Conclusions: Our findings reveal rapid, nSmase-mediated, effects of TNF on the function of two types of glutamate receptors that regulate hippocampal synaptic plasticity, and provide a potential explanation for the recently reported rapid improvement in cognitive function of AD patients treated with a TNF inhibitor (Tobnick and Gross, J Neuroinflammation 2008 Jan 9;5:2). P4-267
PROTEOMIC ANALYSIS OF SYNAPSE REVEALS THE PATHOGENIC PROTEIN INTERACTIONS IN ALZHEIMER’S DISEASE
Yuesong Gong, Andrea L. Rosso, Carol F. Lippa, Drexel University College of Medicine, Philadelphia, PA, USA. Contact e-mail: [email protected] Background: Synaptic dysfunction underlies the symptoms of memory loss in AD. The molecular characterization of this synaptic dysfunction is unclear; however, dysfunction of the postsynaptic NMDA receptor has been demonstrated on surviving synapses in AD. The NMDA receptor is a critical component in the protein network at excitatory synapses. Multiple synaptic proteins may be involved in the development of AD beside NMDA receptor. Methods: We used iTRAQ-quantitative proteomics to screen for pathological expression of proteins at the synapse. Synaptic proteins were isolated from human AD and normal control frontal cortexes and were digested by trypsin. The peptides derived from AD and control samples were labeled by iTRAQ114 and 115, and by iTRAQ116 and 117, respectively. iTRAQ labeled peptides were then concentrated, mixed, acidified, and separated by cation exchange chromatography into 30 fractions for LC-MS/MS analysis. Results: One hundred seventy-four of 835 synaptic proteins were abnormally altered in the AD brain. These proteins included receptors (-aminobutyric acid receptor), scaffold proteins (Disks large-associated protein 1), adhesion molecules (immunoglobulin super-
T749
P4-266
MODIFICATION OF SYNAPTIC PLASTICITY BY TNF AND SPHINGOMYELINASE: IMPLICATIONS FOR COGNITIVE IMPAIRMENT IN ALZHEIMER’S DISEASE
Creutzfeldt-Jakob’s disease. Recent evidence suggests that rather than amyloid plaques, pre-fibrillar small oligomeric amyloid intermediates may be the most neurotoxic species. Notably oligomers from diverse diseaserelated amyloids share a remarkably similar quaternary structure and are equally cytotoxic, suggesting common mechanisms of action. With this in mind, we investigated the role of calcineurin (CaN), a protein phosphatase abundant in neurons and involved in synaptic plasticity and neuronal death, in mediating the cytotoxic and cognitive effects of various amyloid oligomers. Methods: The effect of monomeric, oligomeric and fibrillar A, ␣-synuclein, prion, Huntington’s polyQ and amylin (IAPP) were assessed on 1) CaN activity, CREB-mediated transcription and CaN-dependent cell death in human SY5Y neuroblastoma cells; 2) CaN-dependent long term potentiation (LTP) in hippocampal slices and 3) CaN-dependent cognitive function after icv injection in mice. Results: We found that all oligomeric amyloids tested induced CaN and evoked CaN-dependent cell death, whereas their monomeric and fibrillar counterparts were ineffective. We also found that oligomeric A, ␣-synuclein and prion decreased CREBdriven transcription, an event important for LTP and negatively regulated by CaN. Indeed, we found CaN activity to be increased and LTP to be depressed by oligomeric A and ␣-synuclein and this effect was prevented by inhibition of CaN. Lastly, oligomeric A and ␣-synuclein similarly induced CaN and CaN-dependent memory deficits when acutely injected icv in mice. Conclusions: Collectively, these results suggest that there may be a dysfunctional signaling pathway commonly elicited by oligomeric amyloid proteins that involves CaN hyper-activity and produces decreased LTP, memory impairments and eventually cell death, all events playing a central role in such amyloid diseases as AD, PD and Creutzfeldt-Jacob disease. These results further suggest that inhibition of CNS CaN shall be considered as a possible pharmacological target for the treatment of cognitive deficits and neurodegeneration in amyloid diseases. P4-265
CHANGES OF SYNAPTIC ULTRASTRUCTURE, SYNAPTOPHYSIN AND CHOLINE ACETYLTRANSFERASE IN HIPPOCAMPUS OF AGE-INCREASING RATS
Rong Wang, Lin Li, Yu Tang, Lan Zhang, Beijing Xuan-Wu Hospital, Capital Medical University, Beijing, China. Contact e-mail: [email protected] Background: The aim of the present study is to investigate the changes in hippocampal synapses and their relation with learning-memory abilities at different ages, and evaluate the effects of 2,3,5,4’-tetrahydroxystilbene-2O--D-glucoside (TSG), which is one of the major components of a traditional Chinese herb Polygonum multiflorum, on brain aging. Methods: SD rats at the age of 1, 3, 6, 18 and 24 months were used. TSG at doses of 30 and 60mg/kg/day was intragastrically administered to 21-month-old rats for 3 months, respectively. Learning-memory abilities were determined by Morris water maze (MWM) and passageway water maze test. The ultra-structure of synapses in hippocampal CA1 region was observed by electronic microscopy. The expression of synaptophysin (SYP) in whole hippocampus was measured by using immunohistochemistry. Results: Compared with rats at 6 months of age, both the 1-month-old rats and 24-month-old rats showed longer escape latency and swimming distance in MWM test, while more errors are detected in the passageway water maze test, with smaller number of synapses and synaptic vesicles and less expression of SYP in the hippocampus. Treatment with high-dose TSG in rats at 24 months of age had significant improvement in the learningmemory abilities in the water maze tests associated with an increase in the number of synapses and synaptic vesicles, and an elevation of expression of SYP in the hippocampus. Conclusions: In conclusion, hippocampal synapses count and SYP expression decreased in aged rats, which may be one of the mechanisms involved in learning-memory deficit. TSG reversed the above changes in aged rats, suggesting that TSG may be beneficial for the treatment of Alzheimer disease or cognitive impairment in old people.
Yue Wang, NIA/NIH, Baltimore, MD, USA. Contact e-mail: [email protected] Background: Levels of the pro-inflammatory cytokine tumor necrosis factor-␣ (TNF), and the sphingomyelin metabolite ceramide, are increased in association with the A and neurofibrillary pathology of AD (Exp Neurol 1999;160:289-99. PNAS 2004; 101:2070-5). In response to TNF and oxidative stress, sphingomyelin can be rapidly cleaved by neutral sphingomyelinase (nSmase) to produce ceramide. Because recent findings suggest that TNF can modify synaptic plasticity (Synapse 2000; 35:151-9. Eur J Neurosci 2005; 22:2827-32), we performed experiments aimed at understanding if and how TNF affects glutamate receptor-mediated plasticity at hippocampal CA1 synapses. Methods: Whole-cell patch clamp recordings of EPSCs from CA1 pyramidal cells were performed by visualization of the cells with a 40X water immersion lens. The membrane potential was held at either -70 mV for recording AMPAR component of EPSCs or a voltage steps from -70 to ⫹40 mV for recording NMDAR component of EPSCs, with or without CNQX (10 M) or DL-AP5 (100 M). Patch-clamp electrodes were filled with a solution containing (mM): potassium gluconate, 130; KCl, 10; EGTA, 10; CaCl2, 1; MgCl2, 3; Hepes, 20; Mg-ATP, 5; Na-GTP, 0.5; QX 314, 10; pH 7.2 (the osmolality was adjusted to 280 mmol/kg). Data were collected and analyzed using an Axopatch 200B and pCLAMP 8 software (Axon Instruments). All signals were recorded and filtered at 2 kHz and digitized at 10 kHz. Results: Both the NMDA and AMPA components of EPSCs were transiently altered by TNF. A 2 min perfusion with TNF significantly enhanced the NMDAevoked current at ⫹ 40mV and inhibited the AMPA-evoked current at -70mV. A 1 h pre-incubation with GW4869, an inhibitor of nSmase, blocked the ability of TNF to enhance NMDA currents and inhibit AMPA currents. ISP-1, a potent inhibitor of sphingolipid biosynthesis, did not prevent TNF-induced enhancement of NMDA currents or depression of AMPA currents. Conclusions: Our findings reveal rapid, nSmase-mediated, effects of TNF on the function of two types of glutamate receptors that regulate hippocampal synaptic plasticity, and provide a potential explanation for the recently reported rapid improvement in cognitive function of AD patients treated with a TNF inhibitor (Tobnick and Gross, J Neuroinflammation 2008 Jan 9;5:2). P4-267
PROTEOMIC ANALYSIS OF SYNAPSE REVEALS THE PATHOGENIC PROTEIN INTERACTIONS IN ALZHEIMER’S DISEASE
Yuesong Gong, Andrea L. Rosso, Carol F. Lippa, Drexel University College of Medicine, Philadelphia, PA, USA. Contact e-mail: [email protected] Background: Synaptic dysfunction underlies the symptoms of memory loss in AD. The molecular characterization of this synaptic dysfunction is unclear; however, dysfunction of the postsynaptic NMDA receptor has been demonstrated on surviving synapses in AD. The NMDA receptor is a critical component in the protein network at excitatory synapses. Multiple synaptic proteins may be involved in the development of AD beside NMDA receptor. Methods: We used iTRAQ-quantitative proteomics to screen for pathological expression of proteins at the synapse. Synaptic proteins were isolated from human AD and normal control frontal cortexes and were digested by trypsin. The peptides derived from AD and control samples were labeled by iTRAQ114 and 115, and by iTRAQ116 and 117, respectively. iTRAQ labeled peptides were then concentrated, mixed, acidified, and separated by cation exchange chromatography into 30 fractions for LC-MS/MS analysis. Results: One hundred seventy-four of 835 synaptic proteins were abnormally altered in the AD brain. These proteins included receptors (-aminobutyric acid receptor), scaffold proteins (Disks large-associated protein 1), adhesion molecules (immunoglobulin super-