- Email: [email protected]
COORDINATED ROLES OF COFILIN, SSH1, RANBP9 AND BETA1-INTEGRIN IN ABETA OLIGOMER-INDUCED SYNAPTIC DEFICITS AND APP PROCESSING
Oral Sessions: O3-05: Molecular and Cell Biology: Synaptic Dysfunction
TUESDAY, JULY 15, 2014 ORAL SESSIONS O3-05 MOLECULAR AND CELL BIOLOGY: SYNAPTIC DYSFUNCTION O3-05-01
AN INFLAMMATORY PATHWAY TOWARD SYNAPSE AND COGNITIVE IMPAIRMENT TRIGGERED BY AMYLOID-BETA OLIGOMERS: NOVEL MOLECULAR LINKS BETWEEN ALZHEIMER’S DISEASE AND DIABETES
Mychael V. Lourenco1, Julia Clarke1, Rudimar Luiz Frozza1, Leticia Forny-Germano2, William Klein3, Christian Holscher4, Douglas Munoz5, Sergio Teixeira Ferreira1, Fernanda De Felice1, 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 3Northwestern University, Evanston, Illinois, United States; 4University of Ulster, Coleraine, United Kingdom; 5 Queen’s University, Kingston, Ontario, Canada. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) and diabetes are thought to share similar pathological mechanisms. For instance, brain insulin signaling is disrupted in AD patients and anti-diabetic strategies appear to rescue ADlike pathology in animal models. Double-stranded RNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in a mechanism driven by pro-inflammatory cytokines, such as tumor necrosis factor a (TNF- a). PKR phosphorylates eukaryotic translation initiation factor 2 a (eIF2 a -P) in response to cellular stress, and AD brains exhibit elevated phospho-PKR and eIF2 a -P levels. This mechanism attenuates global protein synthesis in neurons and might impact normal synapse function in Alzheimer’s disease. Methods: Our in vitro model consisted of cultured hippocampal neurons exposed to amyloid-b (A b) oligomers, recognized as central AD toxins, and signaling pathways were manipulated by pharmacological treatments. Morphological and biochemical analyses were then performed. For in vivo studies, adult mice intracerebroventricularly (i.c.v.) injected with A b oligomer preparations and APPSwe/PS1 D E9 transgenic mice were subjected to behavioral assays and their hippocampi were analyzed by immunoblotting. Knockout mice for TNF- a receptor 1 (TNFR1) and PKR were also employed. Finally, we established a non-human primate AD model by i.c.v. injecting cynomolgus monkeys with A b oligomers. Primate hippocampi were assessed for PKR and eIF2 a phosphorylation through immunostaining techniques. Results: We found that PKR responds to A b oligomers and causes eIF2 a -P in the hippocampus in a TNFa -dependent process. Brain PKR and eIF2 a phosphorylation are elevated in AD animal models, including APPSwe/PS1 D E9 transgenic mice and oligomer-injected cynomolgus monkeys. Further, genetic suppression of either TNFR1 or PKR abrogated A b oligomer-dependent brain eIF2a-P and cognitive impairment in mice. Importantly, we found that TNFa/PKR signaling inhibition prevented synapse loss caused by A b oligomers. Conclusions: Our results demonstrate that A b oligomers trigger a pro-inflammatory signaling orchestrated by TNF- a, PKR and eIF2 a -P, ultimately leading to synapse loss and cognitive impairment. These observations reveal a critical pathogenic mechanism connecting Alzheimer’s disease to diabetes, and, in harmony with other recent reports, our findings provide exciting grounds for novel drug targets in AD. O3-05-02
COORDINATED ROLES OF COFILIN, SSH1, RANBP9 AND BETA1-INTEGRIN IN ABETA OLIGOMER-INDUCED SYNAPTIC DEFICITS AND APP PROCESSING
David E. Kang1, Jung A. Woo2, Hirah Khan2, Courtney Uhlar2, Taylor Boggess2, Xinming Wang2, Aurelie Joly-Amado2, George Cappos2, Xingyu Zhao2, Edwin Weeber2, David Morgan3, 1University of South Florida / Byrd Alzheimer’s Institute, Tampa, Florida, United States; 2USF Health Byrd Alzheimer’s Institute, Tampa, Florida, United States; 3 University of South Florida, Tampa, Florida, United States. Contact e-mail: [email protected]
P217
Background: The defining pathological hallmark of Alzheimer’s disease (AD) is the accumulation of A b in brain associated with tau pathology, synapse loss, cytoskeletal aberrations, mitochondrial dysfunction, and cognitive decline. However, it is not clear how the A b oligomer neurotoxic signals are transduced intracellularly to induce synaptic impairment and eventual neurodegeneration. An early and consistent impairment secondary to A b oligomer exposure in primary neurons is the shrinkage of dendritic spines involving the rearrangement of F-actin cytoskeleton in spines and loss of spine-associated proteins such as PSD95 and Drebrin. The F-Actin-severing protein Cofilin has been implicated in A b -induced loss of postsynaptic proteins, although the mechanistic basis of such phenotype is unknown. Methods: In this study, we utilized HT22 neuroblastoma cells, primary neurons, acute hippocampal slices, and genetically modified mice to examine the coordinated roles of Beta1-Integrin, RanBP9, Slingshot homolog 1 (SSH1), and Cofilin in Abeta oligomer-induced synaptic deficits (synaptic proteins & synaptic plasticity), Integrin-dependent focal adhesion complex disruption, APP processing, Abeta pathology, and contextual learning/memory. Results: We demonstrate that A b oligomers rapidly induce the loss of cell surface b 1-Integrin, reorganization of Integrin-associated focal complexes (Talin/Vinculin), depletion of postsynaptic proteins (PSD95, Drebrin, & F-Actin), and activation of Cofilin in vitro and in vivo. Such phenotypes require the engagement of A b oligomers with b 1-integrin complexes, while the scaffolding protein RanBP9 promotes Cofilin activation via SSH1 regulation. Further, Cofilin and its activator SSH1 are essential for A b oligomer-induced disruption of Talin/Vinculin focal complexes, loss of synaptic proteins, and apoptosis. Finally, endogenous Cofilin and SSH1 are also indispensible for A b generation as well as mediating the deficits in long-term potentiation and contextual memory associated with the APP/PS1 mouse model. Conclusions: Taken together, these results demonstrate the essential involvement of the b 1-Integrin-RanBP9-SSH1-Cofilin molecular pathway in both A b -induced neurotoxicity and amyloidogenic processing of APP.
O3-05-03
SYNAPTIC ALTERATIONS IN APP KNOCKOUT NEURONS
Gunnar K. Gouras1, Mathilde Faideau1, Estibaliz Capetillo-Zarate2, Katarina Willen1, Susanne Frykman3, Lars Tjernberg4, 1Lund University, Lund, Sweden; 2Weill Cornell Medical College, New York, New York, United States; 3Karolinska Institute, Stockholm, Sweden; 4Karolinska Institute, Huddinge, Sweden. Contact e-mail: [email protected] Background: Mutations in the amyloid precursor protein (APP) and its metabolite Ab are linked to familial forms of Alzheimer’s disease (AD). However, the normal function of APP, in particular in brain, remains unclear, although it was reported to be involved in synaptic pruning during development. We previously reported differential reductions in selective synaptic proteins and receptors in AD transgenic compared to wild-type neurons (Almeida CG et al., 2005) and now used similar methods to look for synaptic changes in APP knockout versus wild-type neurons. We aim to examine knockout as well as later reduction (RNA interference) of APP on neurite morphology and to characterize alterations in synaptic composition when APP is absent before compared to after the formation of mature synapses. Methods: Selective pre- and post-synaptic proteins and receptors (synaptophysin, synapsin I, GluA1, PSD-95, drebrin) are analysed by Western blot (WB) or immunofluorescence (IF) microscopy in APP KO compared to wild-type primary neurons or brain. Dendritic arborisation and number of synapses are compared in the primary neurons. APP interference is used to compare the effect of APP knockdown before and after synapse maturation. WB, RT-PCR and IF are used to evaluate for APP knock-down. Results: APP KO neurons show selective alterations in synaptic proteins and receptors important in synaptic plasticity that seem to be almost opposite to changes we previously reported in APP AD mutant neurons. Interestingly, we see an up-regulation in selective synaptic proteins despite reduced dendritic arborisation with APP KO. Conclusions: APP plays a role in the synaptic composition of neurons that appears to be almost opposite to that of mutant APP and aberrant Ab accumulation in AD.
TUESDAY, JULY 15, 2014 ORAL SESSIONS O3-05 MOLECULAR AND CELL BIOLOGY: SYNAPTIC DYSFUNCTION O3-05-01
AN INFLAMMATORY PATHWAY TOWARD SYNAPSE AND COGNITIVE IMPAIRMENT TRIGGERED BY AMYLOID-BETA OLIGOMERS: NOVEL MOLECULAR LINKS BETWEEN ALZHEIMER’S DISEASE AND DIABETES
Mychael V. Lourenco1, Julia Clarke1, Rudimar Luiz Frozza1, Leticia Forny-Germano2, William Klein3, Christian Holscher4, Douglas Munoz5, Sergio Teixeira Ferreira1, Fernanda De Felice1, 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 3Northwestern University, Evanston, Illinois, United States; 4University of Ulster, Coleraine, United Kingdom; 5 Queen’s University, Kingston, Ontario, Canada. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) and diabetes are thought to share similar pathological mechanisms. For instance, brain insulin signaling is disrupted in AD patients and anti-diabetic strategies appear to rescue ADlike pathology in animal models. Double-stranded RNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in a mechanism driven by pro-inflammatory cytokines, such as tumor necrosis factor a (TNF- a). PKR phosphorylates eukaryotic translation initiation factor 2 a (eIF2 a -P) in response to cellular stress, and AD brains exhibit elevated phospho-PKR and eIF2 a -P levels. This mechanism attenuates global protein synthesis in neurons and might impact normal synapse function in Alzheimer’s disease. Methods: Our in vitro model consisted of cultured hippocampal neurons exposed to amyloid-b (A b) oligomers, recognized as central AD toxins, and signaling pathways were manipulated by pharmacological treatments. Morphological and biochemical analyses were then performed. For in vivo studies, adult mice intracerebroventricularly (i.c.v.) injected with A b oligomer preparations and APPSwe/PS1 D E9 transgenic mice were subjected to behavioral assays and their hippocampi were analyzed by immunoblotting. Knockout mice for TNF- a receptor 1 (TNFR1) and PKR were also employed. Finally, we established a non-human primate AD model by i.c.v. injecting cynomolgus monkeys with A b oligomers. Primate hippocampi were assessed for PKR and eIF2 a phosphorylation through immunostaining techniques. Results: We found that PKR responds to A b oligomers and causes eIF2 a -P in the hippocampus in a TNFa -dependent process. Brain PKR and eIF2 a phosphorylation are elevated in AD animal models, including APPSwe/PS1 D E9 transgenic mice and oligomer-injected cynomolgus monkeys. Further, genetic suppression of either TNFR1 or PKR abrogated A b oligomer-dependent brain eIF2a-P and cognitive impairment in mice. Importantly, we found that TNFa/PKR signaling inhibition prevented synapse loss caused by A b oligomers. Conclusions: Our results demonstrate that A b oligomers trigger a pro-inflammatory signaling orchestrated by TNF- a, PKR and eIF2 a -P, ultimately leading to synapse loss and cognitive impairment. These observations reveal a critical pathogenic mechanism connecting Alzheimer’s disease to diabetes, and, in harmony with other recent reports, our findings provide exciting grounds for novel drug targets in AD. O3-05-02
COORDINATED ROLES OF COFILIN, SSH1, RANBP9 AND BETA1-INTEGRIN IN ABETA OLIGOMER-INDUCED SYNAPTIC DEFICITS AND APP PROCESSING
David E. Kang1, Jung A. Woo2, Hirah Khan2, Courtney Uhlar2, Taylor Boggess2, Xinming Wang2, Aurelie Joly-Amado2, George Cappos2, Xingyu Zhao2, Edwin Weeber2, David Morgan3, 1University of South Florida / Byrd Alzheimer’s Institute, Tampa, Florida, United States; 2USF Health Byrd Alzheimer’s Institute, Tampa, Florida, United States; 3 University of South Florida, Tampa, Florida, United States. Contact e-mail: [email protected]
P217
Background: The defining pathological hallmark of Alzheimer’s disease (AD) is the accumulation of A b in brain associated with tau pathology, synapse loss, cytoskeletal aberrations, mitochondrial dysfunction, and cognitive decline. However, it is not clear how the A b oligomer neurotoxic signals are transduced intracellularly to induce synaptic impairment and eventual neurodegeneration. An early and consistent impairment secondary to A b oligomer exposure in primary neurons is the shrinkage of dendritic spines involving the rearrangement of F-actin cytoskeleton in spines and loss of spine-associated proteins such as PSD95 and Drebrin. The F-Actin-severing protein Cofilin has been implicated in A b -induced loss of postsynaptic proteins, although the mechanistic basis of such phenotype is unknown. Methods: In this study, we utilized HT22 neuroblastoma cells, primary neurons, acute hippocampal slices, and genetically modified mice to examine the coordinated roles of Beta1-Integrin, RanBP9, Slingshot homolog 1 (SSH1), and Cofilin in Abeta oligomer-induced synaptic deficits (synaptic proteins & synaptic plasticity), Integrin-dependent focal adhesion complex disruption, APP processing, Abeta pathology, and contextual learning/memory. Results: We demonstrate that A b oligomers rapidly induce the loss of cell surface b 1-Integrin, reorganization of Integrin-associated focal complexes (Talin/Vinculin), depletion of postsynaptic proteins (PSD95, Drebrin, & F-Actin), and activation of Cofilin in vitro and in vivo. Such phenotypes require the engagement of A b oligomers with b 1-integrin complexes, while the scaffolding protein RanBP9 promotes Cofilin activation via SSH1 regulation. Further, Cofilin and its activator SSH1 are essential for A b oligomer-induced disruption of Talin/Vinculin focal complexes, loss of synaptic proteins, and apoptosis. Finally, endogenous Cofilin and SSH1 are also indispensible for A b generation as well as mediating the deficits in long-term potentiation and contextual memory associated with the APP/PS1 mouse model. Conclusions: Taken together, these results demonstrate the essential involvement of the b 1-Integrin-RanBP9-SSH1-Cofilin molecular pathway in both A b -induced neurotoxicity and amyloidogenic processing of APP.
O3-05-03
SYNAPTIC ALTERATIONS IN APP KNOCKOUT NEURONS
Gunnar K. Gouras1, Mathilde Faideau1, Estibaliz Capetillo-Zarate2, Katarina Willen1, Susanne Frykman3, Lars Tjernberg4, 1Lund University, Lund, Sweden; 2Weill Cornell Medical College, New York, New York, United States; 3Karolinska Institute, Stockholm, Sweden; 4Karolinska Institute, Huddinge, Sweden. Contact e-mail: [email protected] Background: Mutations in the amyloid precursor protein (APP) and its metabolite Ab are linked to familial forms of Alzheimer’s disease (AD). However, the normal function of APP, in particular in brain, remains unclear, although it was reported to be involved in synaptic pruning during development. We previously reported differential reductions in selective synaptic proteins and receptors in AD transgenic compared to wild-type neurons (Almeida CG et al., 2005) and now used similar methods to look for synaptic changes in APP knockout versus wild-type neurons. We aim to examine knockout as well as later reduction (RNA interference) of APP on neurite morphology and to characterize alterations in synaptic composition when APP is absent before compared to after the formation of mature synapses. Methods: Selective pre- and post-synaptic proteins and receptors (synaptophysin, synapsin I, GluA1, PSD-95, drebrin) are analysed by Western blot (WB) or immunofluorescence (IF) microscopy in APP KO compared to wild-type primary neurons or brain. Dendritic arborisation and number of synapses are compared in the primary neurons. APP interference is used to compare the effect of APP knockdown before and after synapse maturation. WB, RT-PCR and IF are used to evaluate for APP knock-down. Results: APP KO neurons show selective alterations in synaptic proteins and receptors important in synaptic plasticity that seem to be almost opposite to changes we previously reported in APP AD mutant neurons. Interestingly, we see an up-regulation in selective synaptic proteins despite reduced dendritic arborisation with APP KO. Conclusions: APP plays a role in the synaptic composition of neurons that appears to be almost opposite to that of mutant APP and aberrant Ab accumulation in AD.