- Email: [email protected]
REGULATION OF APP SPLICING BY DYRK1A IN CELLS AND MICE BRAINS
P472
Poster Presentations: P2
kit (Pierce) as per manufacturer’s instructions. The serum samples and the samples from different brain regions including olfactory lobes (OL), cortex (CTX), hippocampus (HP), and cerebellum (CBM) were collected at different time-points (30 min, 2h, 8h, 12h, 24h) following a single bolus IN and IPadministration. The brain regions were homogenized in sterile saline (mg brain tissue/ml sterile saline) and 100ml of homogenate equating 100mg of brain tissue and 100ml of serum samples was read in the gamma counter (RRC Common Resource-UIC). The data were expressed as cpm/100mg. Results: Results show that the brain uptake of IN delivered therapeutics was w4-5 times greater than that after IP delivery. Although the entry of test therapeutics in different brain regions was observed to be spaced out according to the distance of brain region from the site of administration, the peak resident time of all test therapeutics for all brain regions was retained between 4-8h both after IN administration. Currently observed fading of radioactive counts beyond 12h post IN administration and further decrease incounts to the base levels by 24h post IN administration, was consistent with the known mouse brain ventricular bulk-flow of 24h. Approximately, 1/5 th of the total IN dose was found to enter the blood. IP delivered test therapeutics exhibited much higher serum values than the brain uptake values at all experimental time points. Conclusions: Intranasal administration is an effective means of delivering therapeutics to the brain. P2-004
Ab-INDUCED CALCIUM FLUX IS DEPENDENT ON GLUTAMATE AND COPPER
Timothy Johanssen1, Paul S. Donnelly2, Stewart Nuttall3, Andrew F. Hill4, Kevin Jeffery Barnham5, 1The University of Melbourne, Melbourne, Australia; 2Department of Chemistry, Parkville, Australia; 3CSIRO Molecular and Health Technologies, Parkville, Australia; 4University of Melbourne, Parkville, Australia; 5Mental Health Research Institute, Parkville, Australia. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterised by progressive cognitive impairment resulting from synaptic degeneration and neuronal loss. Oligomeric forms of the beta amyloid (Ab) peptide have been implicated in the aetiology of AD by disrupting calcium homeostasis rendering human neurons vulnerable to calcium flux and excitotoxicity mediated through the NMDA receptor (NMDAr). Recent work has demonstrated Ab induces excitotoxicity. We sought to determinewhethercopper was involved in enhanced glutamate toxicity caused by Ab that has previously been reported. Methods: Mouse cortical neurons were cultured for 9 days in vitro prior to performing calcium flux and toxicity assays. In order to observe Ab42-induced calcium flux cortical neurons were loaded with the Flou4 dye (Invitrogen) for 1 hr prior to addition of Ab42, glutamate or copper or a combination of these components. Neurons were treated for 48 hours with a combination of Ab42, glutamate and copper prior to measuring cell viability by CCK8 assay based on reduction of the tetrazolium salt, WST-8 (Dojindo). Results: Treatments of neurons with various Ab42 preparations alone were not sufficient to induce a calcium flux above background. However, co-treatment with glutamate and copper significantly increased calcium flux and this increase was dose-dependent with regard to Ab42. Cell viability experiments demonstrated that the resulting toxicity from Ab42, glutamate and copper treatment was mediated via the NMDA receptor. Finally the observed increase in calcium flux was shown to be dependent on metals following treatment with chelators. Conclusions: Here we have demonstrated that Ab-induced NMDAr-mediated toxicity is irrespective of aggregation and dependent on Ab42 interacting with glutamate through a metal dependent mechanism. These results support the use of metal complexing agents as potential therapies for AD. P2-005
REGULATION OF APP SPLICING BY DYRK1A IN CELLS AND MICE BRAINS
Xiaomin Yin1, Wei Qian2, Nana Jin3, Fei Liu4, 1Capital Medical University, Beijing, China; 2Nantong University, Nantong, China; 3Key Laboratory of Neuroregeneration, Nantong University, Nantong, China; 4Nantong University, Nantong, China. Contact e-mail: [email protected]
Background: The deposition of amyloid b(A b) in brains is a common feature in Alzheimer’s disease (AD). A b peptide is a cleaved product of amyloid precursor protein (APP).In adult human brain, APP gene undergoes an alternative splicing regulation, which leads to the generation of six isoforms. Among all these isoforms, APP-KPI and APP695 are reported to be correlated tightly to the A b production. Dyrk1A is a potential SR protein kinase and was shown to play a role in regulating the biogenesis of the splicing speckle compartment of several genes. Here, we investigated the role of Dyrk1A in APP alternative splicing. Methods: By PCR amplification, we generated an APP mini-gene comprising exon 6 to exon 9 and their flanking introns to mimic APP exon 7 and 8 splicing in vivo. GST-Pull down and Co-immunoprecipitation assays were conducted to determine the interaction between Dyrk1A and SR proteins. Ts65Dn transgenic mice were genotyped and the APP splicing pattern were examined by RT-PCR during early development. Results: We transfected APP mini-gene and Dyrk1A plasmid together into HEK293FT cells and detectedthat Dyrk1A increased APP exon 7 and 8inclusion. In addition, we compared the ratio of APP-KPI/APP695 during early development between Ts65Dn and its euploid control mice. We found the ratio was increased in Ts65Dn mice brains. Next we detected that Dyrk1A was pull downed by ASF, SC35, 9G8 and SRp55 in vitro and could be immunoprecipitated by these SR proteins in situ. Further phosphsorylation assay indicated that Dyrk1A phosphorylated SR proteins in a dose-dependent manner in vitro. Dyrk1A specific inhibitors Harmine and Tg003 suppressed 32 Pincorporation onto SR proteins in HEK293FT cells, indicating Dyrk1A phosphorylated SR proteins in situ. Conclusions: Dyrk1A is a SR protein kinase and regulates APP exon 7 and 8 alternative splicing. Dyrk1A modulates APP splicing process by phosphorylating SR proteins. Overexpression of Dyrk1A may disrupt APP exon 7 and 8 exclusion/inclusion switching during early mice brain development. P2-006
BIOCHEMICAL ANALYSIS OF ABETA TOXIC CONFORMER IN HUMAN AUTOPSIED BRAINS AND CEREBROSPINAL FLUID
Yasushi Tomidokoro1, Kazuhiro Ishii2, Akira Tamaoka3, 1Univ. of Tsukuba, Faculty of Medicine, Tsukuba, Japan; 2University of Tsukuba, Faculty of Medicine, Tsukuba, Japan; 3Tsukuba University, Tsukuba, Japan. Contact e-mail: [email protected] Background: It remains a big challenge to develop a therapy targeting Abto modify the process of Alzheimer disease (AD), which is closely associated with the development of AD pathology. A b molecules contain turn structure at Gly25-Ser26 or Glu22-Asp23; A b with turn structure at Glu22-Asp23 is reported to be more toxic and has a higher potential to induce oxidative stress [Murakami K, et al., ACS Chem Neurosci. 2010]. Here we tried to identify toxic A b conformer in human brains and cerebrospinal fluid (CSF). Methods: Eight cases of AD (63-87 years old) and 2 cases of non-AD (ALS-D, 59-66 years old) autopsied brains were used to analyze Ab in human brains. CSF obtained from 3 cases of communicating hydrocephalus, 2 cases of AD and 2 cases of control subjects were also used. Diagnosis of AD and control subjects was confirmed by the amounts of A b 42 in CSF. To identify toxic A b conformer in human brains, molecules were sequentially extracted from frozen tissues into PBS, SDS and then formic acid. Toxic A b conformer was identified by Western blot or immunoprecipitation using mouse monoclonal 11A1 which specifically recognizes turn structure in toxic conformer of A b. Monoclonal 4G8, whose epitope is from Val18 to Glu22 residues in A b peptide, was used as a control. Synthetic peptides corresponding to A b were used to find out the effect of SDS to maintain toxic conformation. Results: The structure of toxic Abconformer was shown to be sensitive against SDS. In PBS extracts obtained from autopsied AD brains, toxic conformer isolated by 11A1 was successfully visualized as monomers, dimmers, trimers and high molecular weight smears, identical pattern to blots ofAbmolecules isolated by 4G8. The amounts ofAbisolated by 11A1 were smaller than, but comparable toAbisolated by 4G8. NoAbwas detected in non-AD brain extracts. In CSF (<10mL) obtained from 3 communicating hydrocephalus, mainly monomers were identified. Blots using antibodies against N- or
Poster Presentations: P2
kit (Pierce) as per manufacturer’s instructions. The serum samples and the samples from different brain regions including olfactory lobes (OL), cortex (CTX), hippocampus (HP), and cerebellum (CBM) were collected at different time-points (30 min, 2h, 8h, 12h, 24h) following a single bolus IN and IPadministration. The brain regions were homogenized in sterile saline (mg brain tissue/ml sterile saline) and 100ml of homogenate equating 100mg of brain tissue and 100ml of serum samples was read in the gamma counter (RRC Common Resource-UIC). The data were expressed as cpm/100mg. Results: Results show that the brain uptake of IN delivered therapeutics was w4-5 times greater than that after IP delivery. Although the entry of test therapeutics in different brain regions was observed to be spaced out according to the distance of brain region from the site of administration, the peak resident time of all test therapeutics for all brain regions was retained between 4-8h both after IN administration. Currently observed fading of radioactive counts beyond 12h post IN administration and further decrease incounts to the base levels by 24h post IN administration, was consistent with the known mouse brain ventricular bulk-flow of 24h. Approximately, 1/5 th of the total IN dose was found to enter the blood. IP delivered test therapeutics exhibited much higher serum values than the brain uptake values at all experimental time points. Conclusions: Intranasal administration is an effective means of delivering therapeutics to the brain. P2-004
Ab-INDUCED CALCIUM FLUX IS DEPENDENT ON GLUTAMATE AND COPPER
Timothy Johanssen1, Paul S. Donnelly2, Stewart Nuttall3, Andrew F. Hill4, Kevin Jeffery Barnham5, 1The University of Melbourne, Melbourne, Australia; 2Department of Chemistry, Parkville, Australia; 3CSIRO Molecular and Health Technologies, Parkville, Australia; 4University of Melbourne, Parkville, Australia; 5Mental Health Research Institute, Parkville, Australia. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterised by progressive cognitive impairment resulting from synaptic degeneration and neuronal loss. Oligomeric forms of the beta amyloid (Ab) peptide have been implicated in the aetiology of AD by disrupting calcium homeostasis rendering human neurons vulnerable to calcium flux and excitotoxicity mediated through the NMDA receptor (NMDAr). Recent work has demonstrated Ab induces excitotoxicity. We sought to determinewhethercopper was involved in enhanced glutamate toxicity caused by Ab that has previously been reported. Methods: Mouse cortical neurons were cultured for 9 days in vitro prior to performing calcium flux and toxicity assays. In order to observe Ab42-induced calcium flux cortical neurons were loaded with the Flou4 dye (Invitrogen) for 1 hr prior to addition of Ab42, glutamate or copper or a combination of these components. Neurons were treated for 48 hours with a combination of Ab42, glutamate and copper prior to measuring cell viability by CCK8 assay based on reduction of the tetrazolium salt, WST-8 (Dojindo). Results: Treatments of neurons with various Ab42 preparations alone were not sufficient to induce a calcium flux above background. However, co-treatment with glutamate and copper significantly increased calcium flux and this increase was dose-dependent with regard to Ab42. Cell viability experiments demonstrated that the resulting toxicity from Ab42, glutamate and copper treatment was mediated via the NMDA receptor. Finally the observed increase in calcium flux was shown to be dependent on metals following treatment with chelators. Conclusions: Here we have demonstrated that Ab-induced NMDAr-mediated toxicity is irrespective of aggregation and dependent on Ab42 interacting with glutamate through a metal dependent mechanism. These results support the use of metal complexing agents as potential therapies for AD. P2-005
REGULATION OF APP SPLICING BY DYRK1A IN CELLS AND MICE BRAINS
Xiaomin Yin1, Wei Qian2, Nana Jin3, Fei Liu4, 1Capital Medical University, Beijing, China; 2Nantong University, Nantong, China; 3Key Laboratory of Neuroregeneration, Nantong University, Nantong, China; 4Nantong University, Nantong, China. Contact e-mail: [email protected]
Background: The deposition of amyloid b(A b) in brains is a common feature in Alzheimer’s disease (AD). A b peptide is a cleaved product of amyloid precursor protein (APP).In adult human brain, APP gene undergoes an alternative splicing regulation, which leads to the generation of six isoforms. Among all these isoforms, APP-KPI and APP695 are reported to be correlated tightly to the A b production. Dyrk1A is a potential SR protein kinase and was shown to play a role in regulating the biogenesis of the splicing speckle compartment of several genes. Here, we investigated the role of Dyrk1A in APP alternative splicing. Methods: By PCR amplification, we generated an APP mini-gene comprising exon 6 to exon 9 and their flanking introns to mimic APP exon 7 and 8 splicing in vivo. GST-Pull down and Co-immunoprecipitation assays were conducted to determine the interaction between Dyrk1A and SR proteins. Ts65Dn transgenic mice were genotyped and the APP splicing pattern were examined by RT-PCR during early development. Results: We transfected APP mini-gene and Dyrk1A plasmid together into HEK293FT cells and detectedthat Dyrk1A increased APP exon 7 and 8inclusion. In addition, we compared the ratio of APP-KPI/APP695 during early development between Ts65Dn and its euploid control mice. We found the ratio was increased in Ts65Dn mice brains. Next we detected that Dyrk1A was pull downed by ASF, SC35, 9G8 and SRp55 in vitro and could be immunoprecipitated by these SR proteins in situ. Further phosphsorylation assay indicated that Dyrk1A phosphorylated SR proteins in a dose-dependent manner in vitro. Dyrk1A specific inhibitors Harmine and Tg003 suppressed 32 Pincorporation onto SR proteins in HEK293FT cells, indicating Dyrk1A phosphorylated SR proteins in situ. Conclusions: Dyrk1A is a SR protein kinase and regulates APP exon 7 and 8 alternative splicing. Dyrk1A modulates APP splicing process by phosphorylating SR proteins. Overexpression of Dyrk1A may disrupt APP exon 7 and 8 exclusion/inclusion switching during early mice brain development. P2-006
BIOCHEMICAL ANALYSIS OF ABETA TOXIC CONFORMER IN HUMAN AUTOPSIED BRAINS AND CEREBROSPINAL FLUID
Yasushi Tomidokoro1, Kazuhiro Ishii2, Akira Tamaoka3, 1Univ. of Tsukuba, Faculty of Medicine, Tsukuba, Japan; 2University of Tsukuba, Faculty of Medicine, Tsukuba, Japan; 3Tsukuba University, Tsukuba, Japan. Contact e-mail: [email protected] Background: It remains a big challenge to develop a therapy targeting Abto modify the process of Alzheimer disease (AD), which is closely associated with the development of AD pathology. A b molecules contain turn structure at Gly25-Ser26 or Glu22-Asp23; A b with turn structure at Glu22-Asp23 is reported to be more toxic and has a higher potential to induce oxidative stress [Murakami K, et al., ACS Chem Neurosci. 2010]. Here we tried to identify toxic A b conformer in human brains and cerebrospinal fluid (CSF). Methods: Eight cases of AD (63-87 years old) and 2 cases of non-AD (ALS-D, 59-66 years old) autopsied brains were used to analyze Ab in human brains. CSF obtained from 3 cases of communicating hydrocephalus, 2 cases of AD and 2 cases of control subjects were also used. Diagnosis of AD and control subjects was confirmed by the amounts of A b 42 in CSF. To identify toxic A b conformer in human brains, molecules were sequentially extracted from frozen tissues into PBS, SDS and then formic acid. Toxic A b conformer was identified by Western blot or immunoprecipitation using mouse monoclonal 11A1 which specifically recognizes turn structure in toxic conformer of A b. Monoclonal 4G8, whose epitope is from Val18 to Glu22 residues in A b peptide, was used as a control. Synthetic peptides corresponding to A b were used to find out the effect of SDS to maintain toxic conformation. Results: The structure of toxic Abconformer was shown to be sensitive against SDS. In PBS extracts obtained from autopsied AD brains, toxic conformer isolated by 11A1 was successfully visualized as monomers, dimmers, trimers and high molecular weight smears, identical pattern to blots ofAbmolecules isolated by 4G8. The amounts ofAbisolated by 11A1 were smaller than, but comparable toAbisolated by 4G8. NoAbwas detected in non-AD brain extracts. In CSF (<10mL) obtained from 3 communicating hydrocephalus, mainly monomers were identified. Blots using antibodies against N- or