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THE ALZHEIMER’S DISEASE SUSCEPTIBILITY GENE CD2AP REGULATES PRESYNAPTIC FUNCTION
Podium Presentations: Monday, July 17, 2017
A442A disrupts a splicing enhancer binding site and reduces PLD3 splicing in human brains. Differentiation of PLD3 A442A and isogenic control iPSCs into cortical neurons produced cells that were morphologically similar. At the molecular level, PLD3 A442A neurons displayed a similar defect in PLD3 splicing as was observed in human brains and a significant increase in Ab42/ Ab40 compared with isogenic control lines. Thus, PLD3 A442A is sufficient to alter PLD3 splicing and Ab metabolism. PLD3 expression was significantly lower in AD brains compared with controls, and PLD3 expression was highly correlated with expression of lysosomal genes. Thus, we sought to determine whether PLD3 contributes to Ab accumulation in AD via disrupted Ab metabolism. We found that overexpression of PLD3 in immortalized cells decreased Ab levels while shRNA silencing of PLD3 increased Ab levels. In an AD mouse model, overexpression of PLD3 in hippocampal neurons produced decreased interstitial fluid (ISF) Ab levels and accelerated Ab turnover. Conversely, PLD3 silencing increased ISF Ab and reduced Ab turnover. Thus, reduced turnover of ISF Ab may lead to Ab accumulation. To begin to determine whether PLD3 influences Ab turnover via the lysosome, we isolated lysosomal fractions from human AD and control brains. PLD3 was enriched in lysosomal subfractions and PLD3 distribution in these subfractions was altered in AD. Furthermore, PLD3 stability in the lysosomal fractions was disrupted in AD brains. Conclusions: These findings demonstrate that PLD3 may contribute to AD risk via altered Ab clearance through the lysosome.
O2-18-03
TAU OLIGOMERS MEDIATE RIBOSOMAL DYSFUNCTION AT THE SYNAPSE
Sarah N. Fontaine, Grant Nation, Shelby E. Meier, Joe F. Abisambra, University of Kentucky, Lexington, KY, USA. Contact e-mail: [email protected] Background: Tau is known to interact with ribosomes both under
normal and Alzheimer’s disease conditions. We have shown that oligomeric tau, a conformer associated with tau toxicity, impairs ribosomal activity and attenuates nascent protein translation. We hypothesize the tau mediated impairment of ribosome function is a key process in the pathogenesis of Alzheimer’s disease and other tauopathies. Methods: The impact of tau-mediated impairment of ribosomal function on synaptic proteins was assessed by treating primary neurons with tau oligomers and collecting synaptic fractions. These fractions were analyzed with surface sensing of translation (SUnSET) to determine ribosomal function at the synapse. Changes in the levels and types of mRNA and protein were also determined. The in vitro data was expanded by examining protein translation in rTg4510 tau transgenic mice using an innovative approach of adapting SUnSET in vivo. Protein synthesis as measured by immunohistochemistry and biochemical analysis of synaptosome fractions. Broad neuronal function measures were performed using manganese-enhanced MRI with R1 mapping, a novel MRI approach developed in our laboratory. Cognitive status was evaluated using a battery of behavioral tests. Results: Tau oligomers impaired translation of key synaptic proteins both in primary neurons and rTg4510 mice. Further, tau-mediated impaired protein
P603
synthesis in rTg4510 mice was readily detectable by SUnSET. Finally, we identified rescue of broad neuronal function and cognition in tau transgenic when tau transgenic mice were treated with compounds that uncouple the tau-ribosome association. Conclusions: These data suggest that a key pathway contributing to cognitive deficits characteristic of tauopathies such as AD may be taumediated impairment of synaptic protein synthesis. Future efforts will focus on identifying and validating therapeutic strategies to rescue dysfunctional synaptic protein synthesis.
O2-18-04
THE ALZHEIMER’S DISEASE SUSCEPTIBILITY GENE CD2AP REGULATES PRESYNAPTIC FUNCTION
Shamsideen A. Ojelade1,2, Tom V. Lee1,2, Nikolaos Giagtzoglou2, Joshua M. Shulman1,2, 1Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA; 2Baylor College of Medicine, Houston, TX, USA. Contact e-mail: [email protected] Background: Genome-wide association studies have identified
numerous loci associated with Alzheimer’s disease (AD) risk, but the mechanisms remain largely unknown. We previously found that cindr, a conserved, Drosophila homolog of the AD susceptibility gene, CD2AP, modulates Tau-mediated neurodegeneration. In order to further understand the potential contribution of CD2AP in AD pathogenesis, we are investigating the function of cindr in the Drosophila nervous system. Methods: We have generated cindr null mutant files, and examined for evidence of neuronal dysfunction and/or degeneration. We have also characterized Cindr protein expression and potential neuronal binding partners. Neurophysiologic studies at the larval neuromuscular junction (NMJ) were performed to address requirements for cindr in synaptic plasticity. Results: In Drosophila, Cindr is expressed robustly in the nervous system and enriched at presynaptic boutons. Consistent with our prior studies using RNAi-knockdown, cindr loss of function (cindr-/-) enhances Tau-induced progressive neuronal loss in the adult fly brain. In the absence of Tau, cindr-/- flies have markedly reduced survival, but development and maintenance of the adult brain appear normal. Consistent with a role in synapse maturation, cindr loss-of-function leads to an increased number of undifferentiated presynaptic boutons (ghost boutons) at the larval NMJ. Furthermore, NMJ electrophysiology reveals reduced synaptic strength and altered short-term plasticity in cindr-/- animals, consistent with requirement for both synaptic structure and function. Cindr co-immunoprecipitates with synaptic vesicle recycling factors, such as synapsin and synaptotagmin, as well as proteins important for synaptic proteostasis, including Leonardo (leo), the fly ortholog of 14-3-3z. Interestingly, in the absence of cindr, leo protein levels are reduced and Synapsin reciprocally increases. Additional genetic experiments suggest that cindr and leomay interact to regulate Synapsin turnover by the proteasome, resulting in altered synaptic plasticity. In preliminary studies, CD2AP is robustly expressed in the mouse brain and loss of CD2AP similarly impacts Synapsin protein levels. Conclusions: We postulate that CD2AP/cindr dysfunction affects synaptic efficacy, and leads to enhanced vulnerability to Tau-mediated toxicity in AD.
A442A disrupts a splicing enhancer binding site and reduces PLD3 splicing in human brains. Differentiation of PLD3 A442A and isogenic control iPSCs into cortical neurons produced cells that were morphologically similar. At the molecular level, PLD3 A442A neurons displayed a similar defect in PLD3 splicing as was observed in human brains and a significant increase in Ab42/ Ab40 compared with isogenic control lines. Thus, PLD3 A442A is sufficient to alter PLD3 splicing and Ab metabolism. PLD3 expression was significantly lower in AD brains compared with controls, and PLD3 expression was highly correlated with expression of lysosomal genes. Thus, we sought to determine whether PLD3 contributes to Ab accumulation in AD via disrupted Ab metabolism. We found that overexpression of PLD3 in immortalized cells decreased Ab levels while shRNA silencing of PLD3 increased Ab levels. In an AD mouse model, overexpression of PLD3 in hippocampal neurons produced decreased interstitial fluid (ISF) Ab levels and accelerated Ab turnover. Conversely, PLD3 silencing increased ISF Ab and reduced Ab turnover. Thus, reduced turnover of ISF Ab may lead to Ab accumulation. To begin to determine whether PLD3 influences Ab turnover via the lysosome, we isolated lysosomal fractions from human AD and control brains. PLD3 was enriched in lysosomal subfractions and PLD3 distribution in these subfractions was altered in AD. Furthermore, PLD3 stability in the lysosomal fractions was disrupted in AD brains. Conclusions: These findings demonstrate that PLD3 may contribute to AD risk via altered Ab clearance through the lysosome.
O2-18-03
TAU OLIGOMERS MEDIATE RIBOSOMAL DYSFUNCTION AT THE SYNAPSE
Sarah N. Fontaine, Grant Nation, Shelby E. Meier, Joe F. Abisambra, University of Kentucky, Lexington, KY, USA. Contact e-mail: [email protected] Background: Tau is known to interact with ribosomes both under
normal and Alzheimer’s disease conditions. We have shown that oligomeric tau, a conformer associated with tau toxicity, impairs ribosomal activity and attenuates nascent protein translation. We hypothesize the tau mediated impairment of ribosome function is a key process in the pathogenesis of Alzheimer’s disease and other tauopathies. Methods: The impact of tau-mediated impairment of ribosomal function on synaptic proteins was assessed by treating primary neurons with tau oligomers and collecting synaptic fractions. These fractions were analyzed with surface sensing of translation (SUnSET) to determine ribosomal function at the synapse. Changes in the levels and types of mRNA and protein were also determined. The in vitro data was expanded by examining protein translation in rTg4510 tau transgenic mice using an innovative approach of adapting SUnSET in vivo. Protein synthesis as measured by immunohistochemistry and biochemical analysis of synaptosome fractions. Broad neuronal function measures were performed using manganese-enhanced MRI with R1 mapping, a novel MRI approach developed in our laboratory. Cognitive status was evaluated using a battery of behavioral tests. Results: Tau oligomers impaired translation of key synaptic proteins both in primary neurons and rTg4510 mice. Further, tau-mediated impaired protein
P603
synthesis in rTg4510 mice was readily detectable by SUnSET. Finally, we identified rescue of broad neuronal function and cognition in tau transgenic when tau transgenic mice were treated with compounds that uncouple the tau-ribosome association. Conclusions: These data suggest that a key pathway contributing to cognitive deficits characteristic of tauopathies such as AD may be taumediated impairment of synaptic protein synthesis. Future efforts will focus on identifying and validating therapeutic strategies to rescue dysfunctional synaptic protein synthesis.
O2-18-04
THE ALZHEIMER’S DISEASE SUSCEPTIBILITY GENE CD2AP REGULATES PRESYNAPTIC FUNCTION
Shamsideen A. Ojelade1,2, Tom V. Lee1,2, Nikolaos Giagtzoglou2, Joshua M. Shulman1,2, 1Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA; 2Baylor College of Medicine, Houston, TX, USA. Contact e-mail: [email protected] Background: Genome-wide association studies have identified
numerous loci associated with Alzheimer’s disease (AD) risk, but the mechanisms remain largely unknown. We previously found that cindr, a conserved, Drosophila homolog of the AD susceptibility gene, CD2AP, modulates Tau-mediated neurodegeneration. In order to further understand the potential contribution of CD2AP in AD pathogenesis, we are investigating the function of cindr in the Drosophila nervous system. Methods: We have generated cindr null mutant files, and examined for evidence of neuronal dysfunction and/or degeneration. We have also characterized Cindr protein expression and potential neuronal binding partners. Neurophysiologic studies at the larval neuromuscular junction (NMJ) were performed to address requirements for cindr in synaptic plasticity. Results: In Drosophila, Cindr is expressed robustly in the nervous system and enriched at presynaptic boutons. Consistent with our prior studies using RNAi-knockdown, cindr loss of function (cindr-/-) enhances Tau-induced progressive neuronal loss in the adult fly brain. In the absence of Tau, cindr-/- flies have markedly reduced survival, but development and maintenance of the adult brain appear normal. Consistent with a role in synapse maturation, cindr loss-of-function leads to an increased number of undifferentiated presynaptic boutons (ghost boutons) at the larval NMJ. Furthermore, NMJ electrophysiology reveals reduced synaptic strength and altered short-term plasticity in cindr-/- animals, consistent with requirement for both synaptic structure and function. Cindr co-immunoprecipitates with synaptic vesicle recycling factors, such as synapsin and synaptotagmin, as well as proteins important for synaptic proteostasis, including Leonardo (leo), the fly ortholog of 14-3-3z. Interestingly, in the absence of cindr, leo protein levels are reduced and Synapsin reciprocally increases. Additional genetic experiments suggest that cindr and leomay interact to regulate Synapsin turnover by the proteasome, resulting in altered synaptic plasticity. In preliminary studies, CD2AP is robustly expressed in the mouse brain and loss of CD2AP similarly impacts Synapsin protein levels. Conclusions: We postulate that CD2AP/cindr dysfunction affects synaptic efficacy, and leads to enhanced vulnerability to Tau-mediated toxicity in AD.