- Email: [email protected]
Human neural progenitor cells as an in vitro model system to study mitochondrial dysfunction in Alzheimer's disease
Poster Presentations P1 capacity was evaluated in an object recognition test. Here, we used a new method to assess the rat’s preference for the novel object, i.e. recognition memory performance in the Y-maze. A new parameter is used, i.e. time spent in half-arms where the objects are placed, and data are recorded via a video tracking system. Results: First, our results show that the rats’ memory performance measured in the object recognition test performed in the Y-maze is altered when measured 2h after A-beta injection. Second, this acute effect can be reversed by the co-injection of compound A, an A-beta aggregation inhibitor. Conclusions: Altogether, these results indicate that the Y-maze version of the object recognition test is enough sensitive to assess memory capacity in a rodent model of Alzheimer’s disease, and to evaluate efficacy of an inhibitory substance. Therefore, this new method to assess object recognition is as sensitive as the classical object recognition tests, but provides as well advantages when compared with these more classical approaches, e.g. memory assessment is more specific, the parameter used is easier to define, and using a video tracking system makes the task faster to conduct as well as experimenter independent. P1-105
HUMAN NEURAL PROGENITOR CELLS AS AN IN VITRO MODEL SYSTEM TO STUDY MITOCHONDRIAL DYSFUNCTION IN ALZHEIMER’S DISEASE
Angela M. Cacace, Paul Wes, Marianne Flynn, Cathy Kieras, Sethu Sankaranaryanan, Jeremy Toyn, Patricia Poundstone, Lin Chen, Myles Fennell, F. Charles, Albright, Bristol-Myers Squibb, Wallingford, CT, USA. Contact e-mail: [email protected] Background: In Alzheimer’s disease (AD) and Parkinson’s disease (PD), there is mounting evidence for the involvement of mitochondrial dysfunction. In PD, several of the genes mutated in familial PD have been implicated in mitochondrial function and can cause abnormal mitochondrial morphology and function in several systems. In AD, mitochondrial dysfunction similar to PD, including decreased function of the electron transport chain (ETC), has been observed. Decreased ETC can then cause decreased ATP production, generation and accumulation of damaging free-radicals, and impaired calcium homeostasis. Further mitochondrial damage can then occur, including opening of the mitochondrial transition pore and cell death. This continuous cycle of increasing oxidative damage can eventually lead to neuronal cell death, despite the cells ability to induce protective antioxidant enzymes and pathways to protect from these pathological insults. Methods: Here, we report the use of human neural progenitor cells as a model system to study mitochondrial dysfunction as it relates to disrupted calcium homeostasis and accumulation of reactive oxygen species. Results: We describe the pharmacology of compounds that have been reported to reduce reactive oxygen species or protect from calcium mediated insults in functional imaging assays for the mitochondrial transition pore and mitochondrial membrane potential. Conclusions: These results argue against the use of neuroblastoma or cancer derived cell lines for the study of mitochondrial mediated neurodegeneration where key metabolic, protective and anti-apoptotic pathways have been altered. P1-106
DYNAMIC MODELING OF AUTOPHAGY DEFECTS AND THEIR PATHOLOGIC CONSEQUENCES IN ALZHEIMER’S DISEASE AND RELATED DISEASES: CONTRIBUTION OF PROTEOLYSIS IMPAIRMENT AND AXONAL TRANSPORT
Sooyeon Lee1, Ralph A. Nixon2,3, 1New York University Department of Physiology & Neuroscience, New York, NY, USA; 2Nathan Kline Institute, Center for Dementia Research, Orangeburg, NY, USA; 3New York University, Departments of Psychiatry & Cell Biology, New York, NY, USA. Contact e-mail: [email protected] Background: Autophagy, a major pathway for protein and organelle turnover, is markedly impaired in AD as evidenced by the massive and relatively selective accumulation of autophagic vesicles (AVs) in degenerative ‘‘dystrophic’’ neurites especially prominent in senile plaques. We have recently shown that structures resembling AVs in AD accumulate in cultured neurons when lysosomal degradation is pharmacologically slowed but not when
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autophagy is over-stimulated (Boland B, Kumar A, Lee S et al., j. Neurosci. 2008). Methods: To understand how impaired autophagy or autolysosomal/ lysosomal proteolysis seen in AD may lead to AD-related dystrophic neurite pathology, we determined how the normal trafficking dynamics of autophagic and endo-lysosomal compartments are altered when autolysosomal proteolysis during autophagy is impaired. Using fluorescent-tagged-LC3 transfection in combination with markers for endosomes, lysosomes, and mitochondria, we characterized the dynamic itinerary of axonal AVs by liveimaging of primary cortical neurons in culture. Results: Autophagosomes were frequently detected in axons and moved predominantly in the retrograde direction. Lysosomal cysteine protease inhibition by leupeptin treatment, but not rapamycin-induced activation, reduced the mobility of LC3positive compartments and late endosomal/lysosomal compartments (Rab7, LAMP and Lysotracker) leading to the formation of dystrophic swellings containing selective endo-lysosome/AV cargoes similar to those previously described in AD brain. By contrast, mitochondria movement was not affected, implying that general mechanisms of axonal transport remain intact. Conclusions: These findings support the hypothesis that lysosomal system dysfunction, as suspected to be marked in AD, leads to a selective defect in the retrograde transport of specific vesicular cargoes, and provides a possible basis for the selective accumulation of these same cargoes in dystrophic neurites in AD brain. P1-107
BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) REDUCES AMYLOIDOGENIC PROCESSING THROUGH CONTROL OF SORLA GENE EXPRESSION
Michael Rohe1, Michael Synowitz2, Anders Nykjaer3, Thomas E. Willnow1, 1Max-Delbru¨ck-Centrum Berlin, Berlin, Germany; 2 Department of Neurosurgery, Charite´ - Universita¨tsmedizin, Berlin, Berlin, Germany; 3The Lundbeck Foundation Research Center MIND, Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark. Contact e-mail: [email protected] Background: Sortilin-related receptor with A-type repeats (SORLA, also known as LR11) is a genetic risk factor in Alzheimer’s disease (AD), known to be down-regulated in patients with late-onset AD. SORLA acts as an intracellular trafficking receptor for APP regulating its processing into Abeta. The receptor shuttles between Golgi, plasma membrane and endosomes, and thereby affects the residence time of APP in the corresponding intracellular compartments. Overexpression of SORLA in neurons reduces while inactivation of gene (as in knockout mouse models) accelerates amyloidogenic processing and senile plaque formation. Methods: Aim of the current study was the identification of the molecular pathways that control SORLA expression in vivo and that may play a pivotal role in processes leading to low levels of receptor expression in AD. Here, we used primary cortical neurons treated with various growth factors and neurotrophins to identify those factors that induce SORLA expression. Results: In our screen, we identified brain-derived neurotrophic factor (BDNF) as a major inducer of the SORLA gene expression. The induction of Sorla observed in BDNF treated neurons was abolished by inhibiting the extracellular regulated kinase (ERK) pathway, demonstrating that receptor gene induction by BDNF was mediated via this signaling cascade. The role of BDNF as physiological regulator of Sorla was further substantiated in mouse models with genetic (Bdnf -/-) or diseaserelated loss of BDNF activity in the brain (Huntington’s disease). Intriguingly, in both models SORLA expression was significantly reduced. Finally, we demonstrated that exogenous application of BDNF in primary neurons or in the brain of mice in vivo resulted in robust reduction of Abeta production when the wildtype SORLA gene was present. No effect of BDNF on Abeta levels was seen in mice genetically deificient for SORLA, or in primary neurons thereof. Conclusions: Our data document a physiological role for BDNF in regulation of SORLA gene expression. Furthermore they demonstrate that beneficial effects ascribed to BDNF in APP metabolism act through induction of SORLA, a negative regulator of neuronal APP processing. Moreover, these findings implicate altered levels of SORLA gene expression as possible disease mechanisms in conditions of low levels of BDNF activity as in Huntington’s disease.
HUMAN NEURAL PROGENITOR CELLS AS AN IN VITRO MODEL SYSTEM TO STUDY MITOCHONDRIAL DYSFUNCTION IN ALZHEIMER’S DISEASE
Angela M. Cacace, Paul Wes, Marianne Flynn, Cathy Kieras, Sethu Sankaranaryanan, Jeremy Toyn, Patricia Poundstone, Lin Chen, Myles Fennell, F. Charles, Albright, Bristol-Myers Squibb, Wallingford, CT, USA. Contact e-mail: [email protected] Background: In Alzheimer’s disease (AD) and Parkinson’s disease (PD), there is mounting evidence for the involvement of mitochondrial dysfunction. In PD, several of the genes mutated in familial PD have been implicated in mitochondrial function and can cause abnormal mitochondrial morphology and function in several systems. In AD, mitochondrial dysfunction similar to PD, including decreased function of the electron transport chain (ETC), has been observed. Decreased ETC can then cause decreased ATP production, generation and accumulation of damaging free-radicals, and impaired calcium homeostasis. Further mitochondrial damage can then occur, including opening of the mitochondrial transition pore and cell death. This continuous cycle of increasing oxidative damage can eventually lead to neuronal cell death, despite the cells ability to induce protective antioxidant enzymes and pathways to protect from these pathological insults. Methods: Here, we report the use of human neural progenitor cells as a model system to study mitochondrial dysfunction as it relates to disrupted calcium homeostasis and accumulation of reactive oxygen species. Results: We describe the pharmacology of compounds that have been reported to reduce reactive oxygen species or protect from calcium mediated insults in functional imaging assays for the mitochondrial transition pore and mitochondrial membrane potential. Conclusions: These results argue against the use of neuroblastoma or cancer derived cell lines for the study of mitochondrial mediated neurodegeneration where key metabolic, protective and anti-apoptotic pathways have been altered. P1-106
DYNAMIC MODELING OF AUTOPHAGY DEFECTS AND THEIR PATHOLOGIC CONSEQUENCES IN ALZHEIMER’S DISEASE AND RELATED DISEASES: CONTRIBUTION OF PROTEOLYSIS IMPAIRMENT AND AXONAL TRANSPORT
Sooyeon Lee1, Ralph A. Nixon2,3, 1New York University Department of Physiology & Neuroscience, New York, NY, USA; 2Nathan Kline Institute, Center for Dementia Research, Orangeburg, NY, USA; 3New York University, Departments of Psychiatry & Cell Biology, New York, NY, USA. Contact e-mail: [email protected] Background: Autophagy, a major pathway for protein and organelle turnover, is markedly impaired in AD as evidenced by the massive and relatively selective accumulation of autophagic vesicles (AVs) in degenerative ‘‘dystrophic’’ neurites especially prominent in senile plaques. We have recently shown that structures resembling AVs in AD accumulate in cultured neurons when lysosomal degradation is pharmacologically slowed but not when
S205
autophagy is over-stimulated (Boland B, Kumar A, Lee S et al., j. Neurosci. 2008). Methods: To understand how impaired autophagy or autolysosomal/ lysosomal proteolysis seen in AD may lead to AD-related dystrophic neurite pathology, we determined how the normal trafficking dynamics of autophagic and endo-lysosomal compartments are altered when autolysosomal proteolysis during autophagy is impaired. Using fluorescent-tagged-LC3 transfection in combination with markers for endosomes, lysosomes, and mitochondria, we characterized the dynamic itinerary of axonal AVs by liveimaging of primary cortical neurons in culture. Results: Autophagosomes were frequently detected in axons and moved predominantly in the retrograde direction. Lysosomal cysteine protease inhibition by leupeptin treatment, but not rapamycin-induced activation, reduced the mobility of LC3positive compartments and late endosomal/lysosomal compartments (Rab7, LAMP and Lysotracker) leading to the formation of dystrophic swellings containing selective endo-lysosome/AV cargoes similar to those previously described in AD brain. By contrast, mitochondria movement was not affected, implying that general mechanisms of axonal transport remain intact. Conclusions: These findings support the hypothesis that lysosomal system dysfunction, as suspected to be marked in AD, leads to a selective defect in the retrograde transport of specific vesicular cargoes, and provides a possible basis for the selective accumulation of these same cargoes in dystrophic neurites in AD brain. P1-107
BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) REDUCES AMYLOIDOGENIC PROCESSING THROUGH CONTROL OF SORLA GENE EXPRESSION
Michael Rohe1, Michael Synowitz2, Anders Nykjaer3, Thomas E. Willnow1, 1Max-Delbru¨ck-Centrum Berlin, Berlin, Germany; 2 Department of Neurosurgery, Charite´ - Universita¨tsmedizin, Berlin, Berlin, Germany; 3The Lundbeck Foundation Research Center MIND, Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark. Contact e-mail: [email protected] Background: Sortilin-related receptor with A-type repeats (SORLA, also known as LR11) is a genetic risk factor in Alzheimer’s disease (AD), known to be down-regulated in patients with late-onset AD. SORLA acts as an intracellular trafficking receptor for APP regulating its processing into Abeta. The receptor shuttles between Golgi, plasma membrane and endosomes, and thereby affects the residence time of APP in the corresponding intracellular compartments. Overexpression of SORLA in neurons reduces while inactivation of gene (as in knockout mouse models) accelerates amyloidogenic processing and senile plaque formation. Methods: Aim of the current study was the identification of the molecular pathways that control SORLA expression in vivo and that may play a pivotal role in processes leading to low levels of receptor expression in AD. Here, we used primary cortical neurons treated with various growth factors and neurotrophins to identify those factors that induce SORLA expression. Results: In our screen, we identified brain-derived neurotrophic factor (BDNF) as a major inducer of the SORLA gene expression. The induction of Sorla observed in BDNF treated neurons was abolished by inhibiting the extracellular regulated kinase (ERK) pathway, demonstrating that receptor gene induction by BDNF was mediated via this signaling cascade. The role of BDNF as physiological regulator of Sorla was further substantiated in mouse models with genetic (Bdnf -/-) or diseaserelated loss of BDNF activity in the brain (Huntington’s disease). Intriguingly, in both models SORLA expression was significantly reduced. Finally, we demonstrated that exogenous application of BDNF in primary neurons or in the brain of mice in vivo resulted in robust reduction of Abeta production when the wildtype SORLA gene was present. No effect of BDNF on Abeta levels was seen in mice genetically deificient for SORLA, or in primary neurons thereof. Conclusions: Our data document a physiological role for BDNF in regulation of SORLA gene expression. Furthermore they demonstrate that beneficial effects ascribed to BDNF in APP metabolism act through induction of SORLA, a negative regulator of neuronal APP processing. Moreover, these findings implicate altered levels of SORLA gene expression as possible disease mechanisms in conditions of low levels of BDNF activity as in Huntington’s disease.