- Email: [email protected]
RIBOSYLATION INDUCES ABETA DEPOSITION, TAU HYPERPHOSPHORYLATION, GLIA ACTIVATION AND COGNITIVE IMPAIRMENT
Poster Presentations: Monday, July 17, 2017 P2-184
14-3-3z: RECRUITING CKIIa AGGRAVATES SET SER-9 PHOSPHORYLATION
Xiaochuan Wang, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: Our previous studies have found that overexpression of
14-3-3z leads to SET retention in the cytoplasm. However, up-regulation of CKII increases SET Ser-9 phosphorylation and enhance SET retention in the cytoplasm. The relationship among 14-3-3z, SET and CKII remains unclear. Methods: AAV 14-3-3z was used to infect C57 mice. LSM and Co-immunoprecipitation was used to investigate the subcellular localization and interaction among SET, 14-3-3z, CKIIain brain slices. Contextual fear condition, Morris water maze test were used to detect the changes in behavior. LSM was used to verify SET subcellular localization in different area of brain slices. 64 array system was used to detect the LTP changes in living brain slices of hippocampal from DG to CA1 region. Primary culture was employed to find the changes in neuronal dendrites and spines. Results: We found that 14-3-3zoverexpression induced SET localization in the cytoplasm while SET, 14-3-3z, CKIIa were interacted with each other. We observed that inhibition of CKII activity by TBB decreased phosphorylation level of SET at Ser-9 in 14-33ztransfectedHEK293/tau cell. si14-3-3zreduced SET pSer-9 in CKII transfectedHEK293/tau cell. Behavioral tests showed that si14-3-3z restored CKII-induced learning and memory impairments. CKIIa led to SET retention in the cytoplasm, while si14-3-3z promoted the SET nuclear importin. si14-3-3z treatment enhanced the reduction of LTP caused by CKIIa. Overexpression of CKIIa reduced neuronal dendrites length, dendritic branches and spine density, while si14-3-3z recovered these changes. We found that CKIIa phosphorylated SET and decreased the levels of synaptotagmin, synaptophysin, synapsin 1, p-synapsin 1, while si14-3-3zrestored these alterations. Conclusions: 14-3-3z interacts with both CKIIa and SET simultaneously, promotes CKII-catalyzed SET phosphorylation, induces neuronal dendritic impairment, LTP and learning and memory damage. si14-3-3z reduces CKIIa phosphorylating SET. Taken together, 14-3-3z recruiting CKIIa aggravates SET Ser-9 phosphorylation and induces downstream events of AD. P2-185
THE STUDY OF SUPPRESSION OF HYPOXIA-INDUCED INFLAMMATION BY TIBETAN MEDICINE RATANASAMPIL IN MICROGLIA CELLS
Xin Zhong1, Junjun Ni2, Jie Meng3, Hiroshi Nakanishi2, Zhou Wu2, Aiqin Zhu1, 1Institute of Geriatric, Qinghai Provincial Hospital, Xining, China; 2Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka, Japan; 3Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. Contact e-mail: [email protected] Background: Ratanasampil (RNSP), a traditional Tibetan medicine,
improves the cognitive function of mild-to-moderate AD patients living at high altitude, as well as learning and memory in an AD mouse model (Tg2576); however, mechanism underlying the effects of RNSP is unknown. Most studies suggested hypoxia facilitates the pathogenesis of AD through accelerating the b-Amyloid (Ab) accumulation. Little is known about the hypoxia induced inflammation in AD pathogenesis. Hypoxia induced microglia activation and animal studies have been shown correlated with neuronal and cognitive decline. To explore the potential protective effect and molecular mechanism of RNSP on hypoxia and reoxygenation induced inflammation using MG6 microglia (MG6 cells).
P677
Methods: MG6 cells were seeded in 24-well plated for 24h before experiment. The control or 1h pre-treatment of RNSP in MG6 were maintained in hypoxia chamber (1% O2, 5%CO2, 95%N2) for 6h. After 6h hypoxia, MG6 were cultured in normal condition for reoxygenation for 12h(H6/R12). The cell viability assay was conducted by MTT assay; The mRNA level of inflammatory cytokines was measured by real-time PCR. HIF-1a protein expression level was measured by western blotting. Results: The hypoxia significantly reduced the cell viability of MG6 cells, and RNSP significantly inhibited the hypoxia (12h)-reduced the cell viability of MG6 cells, respectively, which can be significantly protected by pre-treatment of RNSP. RNSP significantly inhibited the hypoxia reduced the cell viability of MG6 cells after 6h of hypoxia followed by 12h reoxygenation (H6/R12), respectively. The mRNA level of Interleukin6 (IL-6), Interleukin-1b (IL-1b) and Inducible Nitric Oxide Synthase (iNOS) were significantly up-regulated by MG6 cells during hypoxia-reoxygenation(H6/R12). The up-regulation of inflammatory cytokines were suppressed by pretreatment of RNSP. Furthermore, RNSP was found to significantly reduce the hypoxia-upregulated expression of HIF-1a in MG6 cells. Conclusions: Our data suggest that RNSP may inhibit the microglia-mediated neuroinflammation, thus provide a potential mechanism of the RNSP in improving the cognitive function in AD during hypoxia and H/R. The suppression of inflammation by RNSP may provide therapeutic strategy for AD treatment. The on-going work of HIF-1a expression assay may also represent the potential therapeutic target of RNSP on the inflammation induced by hypoxia.
P2-186
RIBOSYLATION INDUCES ABETA DEPOSITION, TAU HYPERPHOSPHORYLATION, GLIA ACTIVATION AND COGNITIVE IMPAIRMENT
Yan Wei1, Rongqiao He2, 1Institue of Biophysics, Chinese Academy of Sciences, Beijing, China; 2Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. Contact e-mail: [email protected] Background: D-ribose in cells and human serum participates in the
glycation of proteins resulting in advanced glycation end products (AGEs) that affect cell metabolism and induce cell death. AGEs have been shown to be implicated with diabetes mellitus and also found in the senile plaques and neurofibrillary tangles in the brain of Alzheimer’s patients. Abnormally high levels of ribose, along with glucose were found in the urine of patients with type 2 diabetic mellitus. The role of D-ribose in neurodegenerative diseases needs to be clarified. Methods: The effects of D-ribose on protein, cell and animal were investigated by biochemical, cellular and animal experiments. Results: Glycation with D-ribose induces protein to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. Intraperitoneal injection, oral administration and tail intravenous injection of D-ribose caused impairment of spatial learning and memory ability in animals, along with Abeta; deposition, tau hyperphosphorylation and glia activation, one of the key pathological features. The involved mechanisms were also studied. Conclusions: These data demonstrate that there may be some mechanistic link between ribosylation and tau hyperphosphorylation related cognitive impairment. Targeting ribosylation could be a promising therapeutic strategy to prevent Alzheimer’s disease and diabetic encephalopathies.
14-3-3z: RECRUITING CKIIa AGGRAVATES SET SER-9 PHOSPHORYLATION
Xiaochuan Wang, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: Our previous studies have found that overexpression of
14-3-3z leads to SET retention in the cytoplasm. However, up-regulation of CKII increases SET Ser-9 phosphorylation and enhance SET retention in the cytoplasm. The relationship among 14-3-3z, SET and CKII remains unclear. Methods: AAV 14-3-3z was used to infect C57 mice. LSM and Co-immunoprecipitation was used to investigate the subcellular localization and interaction among SET, 14-3-3z, CKIIain brain slices. Contextual fear condition, Morris water maze test were used to detect the changes in behavior. LSM was used to verify SET subcellular localization in different area of brain slices. 64 array system was used to detect the LTP changes in living brain slices of hippocampal from DG to CA1 region. Primary culture was employed to find the changes in neuronal dendrites and spines. Results: We found that 14-3-3zoverexpression induced SET localization in the cytoplasm while SET, 14-3-3z, CKIIa were interacted with each other. We observed that inhibition of CKII activity by TBB decreased phosphorylation level of SET at Ser-9 in 14-33ztransfectedHEK293/tau cell. si14-3-3zreduced SET pSer-9 in CKII transfectedHEK293/tau cell. Behavioral tests showed that si14-3-3z restored CKII-induced learning and memory impairments. CKIIa led to SET retention in the cytoplasm, while si14-3-3z promoted the SET nuclear importin. si14-3-3z treatment enhanced the reduction of LTP caused by CKIIa. Overexpression of CKIIa reduced neuronal dendrites length, dendritic branches and spine density, while si14-3-3z recovered these changes. We found that CKIIa phosphorylated SET and decreased the levels of synaptotagmin, synaptophysin, synapsin 1, p-synapsin 1, while si14-3-3zrestored these alterations. Conclusions: 14-3-3z interacts with both CKIIa and SET simultaneously, promotes CKII-catalyzed SET phosphorylation, induces neuronal dendritic impairment, LTP and learning and memory damage. si14-3-3z reduces CKIIa phosphorylating SET. Taken together, 14-3-3z recruiting CKIIa aggravates SET Ser-9 phosphorylation and induces downstream events of AD. P2-185
THE STUDY OF SUPPRESSION OF HYPOXIA-INDUCED INFLAMMATION BY TIBETAN MEDICINE RATANASAMPIL IN MICROGLIA CELLS
Xin Zhong1, Junjun Ni2, Jie Meng3, Hiroshi Nakanishi2, Zhou Wu2, Aiqin Zhu1, 1Institute of Geriatric, Qinghai Provincial Hospital, Xining, China; 2Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka, Japan; 3Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka, Japan. Contact e-mail: [email protected] Background: Ratanasampil (RNSP), a traditional Tibetan medicine,
improves the cognitive function of mild-to-moderate AD patients living at high altitude, as well as learning and memory in an AD mouse model (Tg2576); however, mechanism underlying the effects of RNSP is unknown. Most studies suggested hypoxia facilitates the pathogenesis of AD through accelerating the b-Amyloid (Ab) accumulation. Little is known about the hypoxia induced inflammation in AD pathogenesis. Hypoxia induced microglia activation and animal studies have been shown correlated with neuronal and cognitive decline. To explore the potential protective effect and molecular mechanism of RNSP on hypoxia and reoxygenation induced inflammation using MG6 microglia (MG6 cells).
P677
Methods: MG6 cells were seeded in 24-well plated for 24h before experiment. The control or 1h pre-treatment of RNSP in MG6 were maintained in hypoxia chamber (1% O2, 5%CO2, 95%N2) for 6h. After 6h hypoxia, MG6 were cultured in normal condition for reoxygenation for 12h(H6/R12). The cell viability assay was conducted by MTT assay; The mRNA level of inflammatory cytokines was measured by real-time PCR. HIF-1a protein expression level was measured by western blotting. Results: The hypoxia significantly reduced the cell viability of MG6 cells, and RNSP significantly inhibited the hypoxia (12h)-reduced the cell viability of MG6 cells, respectively, which can be significantly protected by pre-treatment of RNSP. RNSP significantly inhibited the hypoxia reduced the cell viability of MG6 cells after 6h of hypoxia followed by 12h reoxygenation (H6/R12), respectively. The mRNA level of Interleukin6 (IL-6), Interleukin-1b (IL-1b) and Inducible Nitric Oxide Synthase (iNOS) were significantly up-regulated by MG6 cells during hypoxia-reoxygenation(H6/R12). The up-regulation of inflammatory cytokines were suppressed by pretreatment of RNSP. Furthermore, RNSP was found to significantly reduce the hypoxia-upregulated expression of HIF-1a in MG6 cells. Conclusions: Our data suggest that RNSP may inhibit the microglia-mediated neuroinflammation, thus provide a potential mechanism of the RNSP in improving the cognitive function in AD during hypoxia and H/R. The suppression of inflammation by RNSP may provide therapeutic strategy for AD treatment. The on-going work of HIF-1a expression assay may also represent the potential therapeutic target of RNSP on the inflammation induced by hypoxia.
P2-186
RIBOSYLATION INDUCES ABETA DEPOSITION, TAU HYPERPHOSPHORYLATION, GLIA ACTIVATION AND COGNITIVE IMPAIRMENT
Yan Wei1, Rongqiao He2, 1Institue of Biophysics, Chinese Academy of Sciences, Beijing, China; 2Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. Contact e-mail: [email protected] Background: D-ribose in cells and human serum participates in the
glycation of proteins resulting in advanced glycation end products (AGEs) that affect cell metabolism and induce cell death. AGEs have been shown to be implicated with diabetes mellitus and also found in the senile plaques and neurofibrillary tangles in the brain of Alzheimer’s patients. Abnormally high levels of ribose, along with glucose were found in the urine of patients with type 2 diabetic mellitus. The role of D-ribose in neurodegenerative diseases needs to be clarified. Methods: The effects of D-ribose on protein, cell and animal were investigated by biochemical, cellular and animal experiments. Results: Glycation with D-ribose induces protein to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. Intraperitoneal injection, oral administration and tail intravenous injection of D-ribose caused impairment of spatial learning and memory ability in animals, along with Abeta; deposition, tau hyperphosphorylation and glia activation, one of the key pathological features. The involved mechanisms were also studied. Conclusions: These data demonstrate that there may be some mechanistic link between ribosylation and tau hyperphosphorylation related cognitive impairment. Targeting ribosylation could be a promising therapeutic strategy to prevent Alzheimer’s disease and diabetic encephalopathies.