- Email: [email protected]
AN HERBAL EXTRACT (HE238) SUPPRESSES AMYLOID-β42-ELICITED NEUROTOXICITY IN MICE THROUGH ACTIVATION OF AUTOPHAGY
P658 P2-135
Poster Presentations: Monday, July 17, 2017 AN HERBAL EXTRACT (HE238) SUPPRESSES AMYLOID-b42-ELICITED NEUROTOXICITY IN MICE THROUGH ACTIVATION OF AUTOPHAGY
Yung-Feng Liao, Rita Chen, Chang-Jen Huang, Academia Sinica, Taipei, Taiwan. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly in the world. Compelling evidence has shown that dysfunction in autophagic clearance of amyloid-b (Ab) and Tau could significantly contribute to the manifestation of the two most distinctive neuropathological hallmarks, the extracellular deposition of amyloid plaques and the intracellular aggregation of fibrillary tangles, in AD brain. We thus seek to identify novel autophagy-enhancing agents from herbal extracts that can reduce Ab-elicited neurotoxicity and cognitive impairment through promoting the autophagic clearance of Ab. Methods: We established a cell-based Renilla luciferase reporter assay for p62-dependent selective autophagy, which was employed to screen a collection of 650 distinct herbal extracts. Effective herbal extracts that exhibit autophagy-inducing activity in lowering the p62-dependent Renilla luciferase signal, indicative of autophagy activation, were further pursued. The autophagy-inducing potency was validated in the secondary confirmation assay, in which the conversion of LC3-I to LC3-II and the level of p62 were determined. An intracerebral Ab42-injection mouse model of AD was employed to assess the biological efficacy of effective herbal extracts on cognitive improvement. Results: Our data demonstrated that an herbal extract (HE238) can effectively induce autophagy by promoting the LC3-I to LC3-II conversion and the clearance of p62 in cellular models. Oral administration of HE238 to an Ab42-injection mouse model of AD for 2 months can significantly improves the cognitive function of the AD mice by Morris water maze test. Biochemical analysis of brain homogenates derived from HE238-treated animals revealed a significant increase in autophagic activity and neuronal viability. Conclusions: The present findings suggest that the active ingredients of HE238 could contain an enormous resource for AD-alleviating agents.
P2-136
KINESIN-1, AMYLOID PRECURSOR PROTEIN (APP)-VESICLE TRANSPORT MOTOR INTERACTS WITH FUN14 DOMAIN CONTAINING 1 (FUNDC1) VIA THE TETRATRICOPEPTIDE REPEAT DOMAIN OF KLC1
Dae-Hyun Seog, Sang-Jin Kim, Inje University College of Medicine, Busan, Republic of South Korea. Contact e-mail: [email protected] Background: Mutations of the amyloid precursor protein (APP) cause for the formation of amyloid-b peptides. These peptides play a key role in Alzheimer’s diseases. The tetratricopeptide repeat (TPR) domain of kinesin light chain 1 (KLC1) may be responsible for binding APP either directly or via interaction with C-jun N-terminal kinase-interacting protein 1 (JIP1). However, the binding partners of the TPR domain of KLCs have not yet been fully identified. Methods: We were used the yeast two-hybrid system to identify the binding proteins that interact with the TPR domain of KLC1. The binding affinity was quantified by measuring b-galactosidase activity in liquid cultures of yeast transformed cells. Direct
interaction between binding proteins and KLC1 in mammalian cells as well as in vitro was assayed using the co-immunoprecipitation with the antibodies. The cellular co-localization in cells was used the immunocytochemistry. Results: We revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin-1) and KIF3A (a motor subunit of kinesin-2). The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Conclusions: These results suggest that KLC1 may modulate mitophagy through the interaction with FUNDC1.
P2-137
PROTEASOME ACTIVITY INVOLVED IN THE MODIFICATIONS OF APP METABOLISM AND THE INDUCTION OF ALZHEIMER PATHOLOGY
Matias Alloatti1, Ivan Fernandez Bessone1, Alan Hallberg1, Maria Gabriela Otero1, Trinidad Saez1,2, Stevens Rehen3, Tomas Falzone1,2, 1IBCN (UBA-CONICET), Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina; 2IBYME (CONICET), Buenos Aires, Argentina; 3D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil. Contact e-mail: tfalzone@fmed. uba.ar Background: Long lasting cells such as neurons depend on the cor-
rect protein homeostasis achieved by protein synthesis and degradation. Distant local defects in protein accumulation lead to synaptic decline and neurodegeneration. The ubiquitin–proteasome pathway is a master regulator of protein degradation within neurons. Interestingly proteasome inhibition has been associated with abnormal protein accumulation and aggregation in Alzheimer Disease. Therefore, it is important to understand the role of the proteasome in APP metabolism and the pathogenic mechanisms induced by its defects. Methods: The effect of proteasome inhibition on APP were studied in primary mouse cultures related with its axonal transport, processing and local accumulation within neurons. rtPCR were performed to evaluate the levels of APP mRNA associated with proteasome inhibition. Cerebral organoids generated from human derived neurons were generated and characterized to be used as a model for understanding disease and pathology. 40 days-old cerebral organoids were evaluated for APP metabolism and tau behavior in control condition and after proteasome is inhibited. Results: Proteasome inhibition induces a marked and selective decrease in full length APP levels. These changes in APP levels are correlated with significant defects in APP axonal transport under proteasome inhibition. mRNA analysis do not revealed significant changes in APP expression under this treatment. Human cerebral organoids can be used as system to test for drug treatment to understand disease pathology or pathogenic mechanisms associated with changes in APP and tau protein levels or modifications. Conclusions: Our experiments revealed that proteasome impairments can induce similar impairments both at the level of APP metabolism or by tau changes that are associated with the exacerbation of pathology in many neurodegenerative diseases such as AD.
Poster Presentations: Monday, July 17, 2017 AN HERBAL EXTRACT (HE238) SUPPRESSES AMYLOID-b42-ELICITED NEUROTOXICITY IN MICE THROUGH ACTIVATION OF AUTOPHAGY
Yung-Feng Liao, Rita Chen, Chang-Jen Huang, Academia Sinica, Taipei, Taiwan. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly in the world. Compelling evidence has shown that dysfunction in autophagic clearance of amyloid-b (Ab) and Tau could significantly contribute to the manifestation of the two most distinctive neuropathological hallmarks, the extracellular deposition of amyloid plaques and the intracellular aggregation of fibrillary tangles, in AD brain. We thus seek to identify novel autophagy-enhancing agents from herbal extracts that can reduce Ab-elicited neurotoxicity and cognitive impairment through promoting the autophagic clearance of Ab. Methods: We established a cell-based Renilla luciferase reporter assay for p62-dependent selective autophagy, which was employed to screen a collection of 650 distinct herbal extracts. Effective herbal extracts that exhibit autophagy-inducing activity in lowering the p62-dependent Renilla luciferase signal, indicative of autophagy activation, were further pursued. The autophagy-inducing potency was validated in the secondary confirmation assay, in which the conversion of LC3-I to LC3-II and the level of p62 were determined. An intracerebral Ab42-injection mouse model of AD was employed to assess the biological efficacy of effective herbal extracts on cognitive improvement. Results: Our data demonstrated that an herbal extract (HE238) can effectively induce autophagy by promoting the LC3-I to LC3-II conversion and the clearance of p62 in cellular models. Oral administration of HE238 to an Ab42-injection mouse model of AD for 2 months can significantly improves the cognitive function of the AD mice by Morris water maze test. Biochemical analysis of brain homogenates derived from HE238-treated animals revealed a significant increase in autophagic activity and neuronal viability. Conclusions: The present findings suggest that the active ingredients of HE238 could contain an enormous resource for AD-alleviating agents.
P2-136
KINESIN-1, AMYLOID PRECURSOR PROTEIN (APP)-VESICLE TRANSPORT MOTOR INTERACTS WITH FUN14 DOMAIN CONTAINING 1 (FUNDC1) VIA THE TETRATRICOPEPTIDE REPEAT DOMAIN OF KLC1
Dae-Hyun Seog, Sang-Jin Kim, Inje University College of Medicine, Busan, Republic of South Korea. Contact e-mail: [email protected] Background: Mutations of the amyloid precursor protein (APP) cause for the formation of amyloid-b peptides. These peptides play a key role in Alzheimer’s diseases. The tetratricopeptide repeat (TPR) domain of kinesin light chain 1 (KLC1) may be responsible for binding APP either directly or via interaction with C-jun N-terminal kinase-interacting protein 1 (JIP1). However, the binding partners of the TPR domain of KLCs have not yet been fully identified. Methods: We were used the yeast two-hybrid system to identify the binding proteins that interact with the TPR domain of KLC1. The binding affinity was quantified by measuring b-galactosidase activity in liquid cultures of yeast transformed cells. Direct
interaction between binding proteins and KLC1 in mammalian cells as well as in vitro was assayed using the co-immunoprecipitation with the antibodies. The cellular co-localization in cells was used the immunocytochemistry. Results: We revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin-1) and KIF3A (a motor subunit of kinesin-2). The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Conclusions: These results suggest that KLC1 may modulate mitophagy through the interaction with FUNDC1.
P2-137
PROTEASOME ACTIVITY INVOLVED IN THE MODIFICATIONS OF APP METABOLISM AND THE INDUCTION OF ALZHEIMER PATHOLOGY
Matias Alloatti1, Ivan Fernandez Bessone1, Alan Hallberg1, Maria Gabriela Otero1, Trinidad Saez1,2, Stevens Rehen3, Tomas Falzone1,2, 1IBCN (UBA-CONICET), Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina; 2IBYME (CONICET), Buenos Aires, Argentina; 3D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil. Contact e-mail: tfalzone@fmed. uba.ar Background: Long lasting cells such as neurons depend on the cor-
rect protein homeostasis achieved by protein synthesis and degradation. Distant local defects in protein accumulation lead to synaptic decline and neurodegeneration. The ubiquitin–proteasome pathway is a master regulator of protein degradation within neurons. Interestingly proteasome inhibition has been associated with abnormal protein accumulation and aggregation in Alzheimer Disease. Therefore, it is important to understand the role of the proteasome in APP metabolism and the pathogenic mechanisms induced by its defects. Methods: The effect of proteasome inhibition on APP were studied in primary mouse cultures related with its axonal transport, processing and local accumulation within neurons. rtPCR were performed to evaluate the levels of APP mRNA associated with proteasome inhibition. Cerebral organoids generated from human derived neurons were generated and characterized to be used as a model for understanding disease and pathology. 40 days-old cerebral organoids were evaluated for APP metabolism and tau behavior in control condition and after proteasome is inhibited. Results: Proteasome inhibition induces a marked and selective decrease in full length APP levels. These changes in APP levels are correlated with significant defects in APP axonal transport under proteasome inhibition. mRNA analysis do not revealed significant changes in APP expression under this treatment. Human cerebral organoids can be used as system to test for drug treatment to understand disease pathology or pathogenic mechanisms associated with changes in APP and tau protein levels or modifications. Conclusions: Our experiments revealed that proteasome impairments can induce similar impairments both at the level of APP metabolism or by tau changes that are associated with the exacerbation of pathology in many neurodegenerative diseases such as AD.