- Email: [email protected]
Mechanistic studies of antibody mediated clearance of tau aggregates using an ex vivo brain slice model
S276
Poster Presentations P1
neurodegeneration. This is an important area of investigation, which may lead to more timely and effective interventions. Methods: We investigated synaptic plasticity and the dendritic localization of human tau (htau) in the rTg4510 mouse model of tauopathy at an age (4.5M) when the mice are cognitively impaired but exhibit no overt synapse or neuron loss. Subsequently, we examined the dendritic localization of htau and synaptic function in transfected, dissociated rat hippocampal neurons. Finally, we studied surface AMPA receptors in neurons cultured from mice expressing htau. Results: We found higher concentrations of htau in the postsynaptic densities of impaired rTg4510 mice expressing htauP301L than intact rTg21221 mice expressing wild-type htauWT. This mislocalization of tau to dendritic spines also occurs to a greater extent in rat hippocampal neurons transfected with htauP301L than htauWT. Inhibiting proline-directed phosphorylation by substituting alanine at 14 serine and threonine residues blocked the mislocalization of htauP301L to dendritic spines. Conversely, pseudo-hyperphosphorylation of these 14 residues by substituting glutamate mimics htauP301L mislocalization. Spines containing htau showed decreased frequencies and amplitudes of miniature excitatory postsynaptic currents, and reduced levels of surface AMPA receptors. Conclusions: The earliest tau-related neurological deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic dysfunction arising from the mislocalization of tau to dendritic spines, which is accompanied by a reduction in surface AMPA receptors. P1-355
MECHANISTIC STUDIES OF ANTIBODY MEDIATED CLEARANCE OF TAU AGGREGATES USING AN EX VIVO BRAIN SLICE MODEL
Pavan K. Krishnamurthy, Yan Deng, Paul M. Mathews, Einar M. Sigurdsson, New York University School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Our group has shown in two models with tangle pathology that immunization with an AD specific phospho-tau epitope slows the progression of tangle-associated phenotype, reduces tau aggregates and prevents cognitive impairments (Asuni A. et al. J. Neurosci., 2007, Sigurdsson E.M. et al., ICAD Chicago, 2008). Additionally, we demonstrated that immunization against a phospho-tau epitope located within the microtubule binding region could also clear tau aggregates and reduce functional deficits in a mouse model of tauopathy (Krishnamurthy P.K. et al., ICAD Vienna, 2009). A natural follow up of these studies is to understand the cellular mechanisms involved in this phenomenon. Therefore, the uptake and localization of FITC labeled anti-tau antibodies was studied in an organotypic brain slice model derived from adult JNPL3 (P301L) mice with established tau pathology. Methods: Brain slices were prepared, allowed to stabilize and incubated with FITC-IgG for two hours at 35 C with intermittent oxygenation. The IgG was purified from an immunized P301L mouse with a high antibody titre against our phos-tau immunogen (Tau 379-408[pSer396,404]). Slices (400 mm) were fixed in PLP and 40 mm sections from their center were stained with the tau antibodies CP13 (pSer202 epitope) and MC1 (conformational epitope) and a Texas Red conjugated secondary. Sections were also stained with antibodies against LAMP2 and rab5, markers of lysosomes and early endosomes respectively. All sections were counterstained with the nuclear marker Hoescht 33342 and visualized by confocal microscopy. Results: Extensive but partial co-localization was observed between FITC-IgG and the tau antibodies CP13 and MC1. Furthermore, we determined that the FITC-IgG was completely associated with cellular markers of lysosomes, LAMP2, and early endosomes, rab5, with perinuclear vesicles as the main areas of co-staining. In wild-type mice, limited non-specific FITC-IgG binding was observed. Biochemical studies are underway to examine antibody compartmentalization. We are assessing as well the ubiquitin-proteosome system that is probably not involved as it is likely saturated under these conditions. Conclusions: These findings indicate that the endosomal-lysosomal pathway is involved in antibody-mediated clearance of tau aggregates. Disclosure: Patent pending on tau immunotherapy.
P1-356
EFFECT OF CHOLESTEROL IN CSF ON TAU PHOSPHORYLATION
Yi Li, Mental Health Center, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China. Contact e-mail: psyyee@gmail. com Background: In Alzheimer’s disease brain, imbalance of cholesterol usually attends by neurofibrillary tangles (NFTs), which mainly consist of hyperphosphorylated tau. Some researchers reported that cholesterol overload colocalized with NFTs. Others alleged that the reduction of cholesterol caused tau hyperphosphorylation. It shows the relationship between cholesterol and tau phosphorylation is still not clear. The present study investigated different level of cholesterol in cerebrospinal fluid (CSF) on tau phosphorylation. Methods: We carried out Morris water maze to test the rats’ memory deficit 24 h after 2, 8, 20 mg/ ml of cholesterol was delivered into lateral ventricles via stereotactic brain injection respectively. Tau hyperphosphorylation and distribution were assayed by immunohistochemistry using sites specific antibodies (pS 396, Tau-1 and total tau antibody R134d). Results: 24 hours post-injection, AD-like spatial memory retention disturbance was observed in 2, 20 mg/ ml of cholesterol rats, but not in 8 mg/ ml of cholesterol rats. Meanwhile, immunohistochemistry staining showed that the immunoreaction of tau at pS 396 epitope was enhanced, and the reaction at Tau-1 epitope was weakened significantly at 24 h after injection of 2, 20 mg/ ml of cholesterol, suggesting hyperphosphorylation of tau at Ser396 (pS 396) and Ser199/Ser202 (Tau-1) sites, while 8mg/ ml of cholesterol didn’t alter tau phosphorylation. Conclusions: Either high or low level of cholesterol all can induce tau hyperphosphorylation, thus result in the memory deficits. P1-357
TAU GENE POLYMORPHISM INFLUENCES RISK OF SPORADIC TAUOPATHY BY ALLELE-SPECIFIC CHANGES IN TRANSCRIPTION AND ALTERNATIVE SPLICING
Victoria Kay, Jana Vandrovcova, Alan Pittman, Andrew Lees, Rohan de Silva, Institute of Neurology, London, United Kingdom. Contact e-mail: [email protected] Background: Tauopathies, including Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP), are a group of neurodegenerative diseases characterised neuropathologically by brain lesions comprising aggregates of the microtubule associated protein, tau. Common variation in the gene encoding tau (MAPT) affects the production of tau protein and its six isoforms and has been shown to modulate disease risk. Objective: To study the activity of three common MAPT promoter variants (H1b, H1c and H2) in vitro in order to ascertain whether the genetic differences in disease risk are conferred by differences in MAPT expression and alternative splicing. Methods: Combinations of the allelic variants of three regions of the MAPT promoter - the minimal core promoter and an upstream and a downstream putative enhancer domain - were cloned into luciferase reporter plasmids. Reporter gene expression was analysed in vitro in SH-SY5Y and BE(2)-M17 neuroblastoma cell cultures. We have now extended this to studies of co-transcriptional regulation of splicing using MAPT minigenes. Results: The H1c promoter variant had a 4.2-fold increase in transcriptional activity over the H2 variant (p < 0.0001). When only the core promoter and the downstream putative enhancer domain were included, the H1c variant had 2.7-fold greater transcriptional activity than the H1b variant (p < 0.01) and 4.2-fold greater than the H2 variant (p < 0.01). We also observed coincident H1c allele-specific changes in alternative splicing of MAPT exon 10 in vivo with increased 4R-tau transcripts. Conclusions: The H1c MAPT variant, previously associated with increased risk of neurodegenerative disease, has higher transcriptional activity in vitro than the neutral (H1b) and protective (H2) variants and this appears to be driven by the putative downstream enhancer. This is coupled with H1cspecific elevation of mRNAs coding for the more fibrillogenic 4R-tau variants. We are currently conducting an independent replication of this study, and also an investigation of other conserved putative regulatory promoter regions in order to obtain a better picture of the allele-specific differences in MAPT expression and the effect of promoter strength on alternative splicing. The results of this further work will also be presented.
Poster Presentations P1
neurodegeneration. This is an important area of investigation, which may lead to more timely and effective interventions. Methods: We investigated synaptic plasticity and the dendritic localization of human tau (htau) in the rTg4510 mouse model of tauopathy at an age (4.5M) when the mice are cognitively impaired but exhibit no overt synapse or neuron loss. Subsequently, we examined the dendritic localization of htau and synaptic function in transfected, dissociated rat hippocampal neurons. Finally, we studied surface AMPA receptors in neurons cultured from mice expressing htau. Results: We found higher concentrations of htau in the postsynaptic densities of impaired rTg4510 mice expressing htauP301L than intact rTg21221 mice expressing wild-type htauWT. This mislocalization of tau to dendritic spines also occurs to a greater extent in rat hippocampal neurons transfected with htauP301L than htauWT. Inhibiting proline-directed phosphorylation by substituting alanine at 14 serine and threonine residues blocked the mislocalization of htauP301L to dendritic spines. Conversely, pseudo-hyperphosphorylation of these 14 residues by substituting glutamate mimics htauP301L mislocalization. Spines containing htau showed decreased frequencies and amplitudes of miniature excitatory postsynaptic currents, and reduced levels of surface AMPA receptors. Conclusions: The earliest tau-related neurological deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic dysfunction arising from the mislocalization of tau to dendritic spines, which is accompanied by a reduction in surface AMPA receptors. P1-355
MECHANISTIC STUDIES OF ANTIBODY MEDIATED CLEARANCE OF TAU AGGREGATES USING AN EX VIVO BRAIN SLICE MODEL
Pavan K. Krishnamurthy, Yan Deng, Paul M. Mathews, Einar M. Sigurdsson, New York University School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Our group has shown in two models with tangle pathology that immunization with an AD specific phospho-tau epitope slows the progression of tangle-associated phenotype, reduces tau aggregates and prevents cognitive impairments (Asuni A. et al. J. Neurosci., 2007, Sigurdsson E.M. et al., ICAD Chicago, 2008). Additionally, we demonstrated that immunization against a phospho-tau epitope located within the microtubule binding region could also clear tau aggregates and reduce functional deficits in a mouse model of tauopathy (Krishnamurthy P.K. et al., ICAD Vienna, 2009). A natural follow up of these studies is to understand the cellular mechanisms involved in this phenomenon. Therefore, the uptake and localization of FITC labeled anti-tau antibodies was studied in an organotypic brain slice model derived from adult JNPL3 (P301L) mice with established tau pathology. Methods: Brain slices were prepared, allowed to stabilize and incubated with FITC-IgG for two hours at 35 C with intermittent oxygenation. The IgG was purified from an immunized P301L mouse with a high antibody titre against our phos-tau immunogen (Tau 379-408[pSer396,404]). Slices (400 mm) were fixed in PLP and 40 mm sections from their center were stained with the tau antibodies CP13 (pSer202 epitope) and MC1 (conformational epitope) and a Texas Red conjugated secondary. Sections were also stained with antibodies against LAMP2 and rab5, markers of lysosomes and early endosomes respectively. All sections were counterstained with the nuclear marker Hoescht 33342 and visualized by confocal microscopy. Results: Extensive but partial co-localization was observed between FITC-IgG and the tau antibodies CP13 and MC1. Furthermore, we determined that the FITC-IgG was completely associated with cellular markers of lysosomes, LAMP2, and early endosomes, rab5, with perinuclear vesicles as the main areas of co-staining. In wild-type mice, limited non-specific FITC-IgG binding was observed. Biochemical studies are underway to examine antibody compartmentalization. We are assessing as well the ubiquitin-proteosome system that is probably not involved as it is likely saturated under these conditions. Conclusions: These findings indicate that the endosomal-lysosomal pathway is involved in antibody-mediated clearance of tau aggregates. Disclosure: Patent pending on tau immunotherapy.
P1-356
EFFECT OF CHOLESTEROL IN CSF ON TAU PHOSPHORYLATION
Yi Li, Mental Health Center, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China. Contact e-mail: psyyee@gmail. com Background: In Alzheimer’s disease brain, imbalance of cholesterol usually attends by neurofibrillary tangles (NFTs), which mainly consist of hyperphosphorylated tau. Some researchers reported that cholesterol overload colocalized with NFTs. Others alleged that the reduction of cholesterol caused tau hyperphosphorylation. It shows the relationship between cholesterol and tau phosphorylation is still not clear. The present study investigated different level of cholesterol in cerebrospinal fluid (CSF) on tau phosphorylation. Methods: We carried out Morris water maze to test the rats’ memory deficit 24 h after 2, 8, 20 mg/ ml of cholesterol was delivered into lateral ventricles via stereotactic brain injection respectively. Tau hyperphosphorylation and distribution were assayed by immunohistochemistry using sites specific antibodies (pS 396, Tau-1 and total tau antibody R134d). Results: 24 hours post-injection, AD-like spatial memory retention disturbance was observed in 2, 20 mg/ ml of cholesterol rats, but not in 8 mg/ ml of cholesterol rats. Meanwhile, immunohistochemistry staining showed that the immunoreaction of tau at pS 396 epitope was enhanced, and the reaction at Tau-1 epitope was weakened significantly at 24 h after injection of 2, 20 mg/ ml of cholesterol, suggesting hyperphosphorylation of tau at Ser396 (pS 396) and Ser199/Ser202 (Tau-1) sites, while 8mg/ ml of cholesterol didn’t alter tau phosphorylation. Conclusions: Either high or low level of cholesterol all can induce tau hyperphosphorylation, thus result in the memory deficits. P1-357
TAU GENE POLYMORPHISM INFLUENCES RISK OF SPORADIC TAUOPATHY BY ALLELE-SPECIFIC CHANGES IN TRANSCRIPTION AND ALTERNATIVE SPLICING
Victoria Kay, Jana Vandrovcova, Alan Pittman, Andrew Lees, Rohan de Silva, Institute of Neurology, London, United Kingdom. Contact e-mail: [email protected] Background: Tauopathies, including Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP), are a group of neurodegenerative diseases characterised neuropathologically by brain lesions comprising aggregates of the microtubule associated protein, tau. Common variation in the gene encoding tau (MAPT) affects the production of tau protein and its six isoforms and has been shown to modulate disease risk. Objective: To study the activity of three common MAPT promoter variants (H1b, H1c and H2) in vitro in order to ascertain whether the genetic differences in disease risk are conferred by differences in MAPT expression and alternative splicing. Methods: Combinations of the allelic variants of three regions of the MAPT promoter - the minimal core promoter and an upstream and a downstream putative enhancer domain - were cloned into luciferase reporter plasmids. Reporter gene expression was analysed in vitro in SH-SY5Y and BE(2)-M17 neuroblastoma cell cultures. We have now extended this to studies of co-transcriptional regulation of splicing using MAPT minigenes. Results: The H1c promoter variant had a 4.2-fold increase in transcriptional activity over the H2 variant (p < 0.0001). When only the core promoter and the downstream putative enhancer domain were included, the H1c variant had 2.7-fold greater transcriptional activity than the H1b variant (p < 0.01) and 4.2-fold greater than the H2 variant (p < 0.01). We also observed coincident H1c allele-specific changes in alternative splicing of MAPT exon 10 in vivo with increased 4R-tau transcripts. Conclusions: The H1c MAPT variant, previously associated with increased risk of neurodegenerative disease, has higher transcriptional activity in vitro than the neutral (H1b) and protective (H2) variants and this appears to be driven by the putative downstream enhancer. This is coupled with H1cspecific elevation of mRNAs coding for the more fibrillogenic 4R-tau variants. We are currently conducting an independent replication of this study, and also an investigation of other conserved putative regulatory promoter regions in order to obtain a better picture of the allele-specific differences in MAPT expression and the effect of promoter strength on alternative splicing. The results of this further work will also be presented.