- Email: [email protected]
A novel Alzheimer's disease model based on 3D-cultured human neural progenitor cells
P710
Wednesday, July 17, 2013: Poster Presentations: P4
weeks and then subcutaneously injected with vehicle, Allo (10mg/kg), or 17b-estradiol (E2, 60mg/kg). 24 hours after treatment, mice were sacrificed and brain mitochondria were isolated for mitochondrial respiration and bioenergetic enzyme measurements. Results: Adult NSCs from both nonTg and 3xTgAD mice exhibited a significant age-dependent decline in mitochondrial bioenergetic capacity. Further, aNSC derived from 3xTgAD mice at 6 and 18 months exhibited elevated proton leak. In vitro Allo treatment potentiated mitochondrial respiration in both NSCs, neurons and mixed glia. Further, in vivo Allo treatment restored the OVX-induced decrease in mitochondrial respiration in both nonTg and 3xTgAD mice. Allo treatment increased activity of bioenergetic enzymes such as PDH and aKGDH, and suppressed the OVX-induced increase in lipid peroxidation. Conclusions: There was an age- and Alzheimer’s associated decline in mitochondrial function in aNSCs. Allo treatment potentiated mitochondrial function both in vitro and in vivo. Collectively, these data provided mechanistic basis for developing Allo as a multi-tropic AD therapeutics to sustain and enhance mitochondrial function and to promote neurogenic capacity. P4-014
ALLOPREGNANOLONE AS A REGENERATIVE THERAPEUTIC FOR ALZHEIMER’S DISEASE, 4: IMPACT ON NEURONAL VERSUS GLIOSIS PHENOTYPIC DIFFERENTIATION 1
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Shuhua Chen , Jia Yao , Karren Wong , Roberta Brinton , University of Southern California, Los Angeles, California, United States; 2University of Southern California, Los Angeles, California, United States. Contact e-mail: [email protected] Background: Allopregnanolone (Allo), an endogenous neurogenic steroid, promotes proliferation of human and rodent neural progenitor cells in vitro and in vivo of triple transgenic mouse model of Alzheimer’s disease (3xTgAD). In this study, we investigated the impact of Allo on neural differentiation in vitro and in vivo of 3xTgAD. Methods: Neural stem cells (NSCs) from embryonic E18rats and adult nonTg and 3xTgAD mice were harvested and cultured before the in vitro analyses. E18 rat NSCs were incubated in differentiation medium containing AP a or vehicle for 6-days, fixed and then stained for neuronal, glial and oligodendrocytic markers. To investigate the changes in proliferation and differentiation capacity of adult NSC burdened by age and Alzheimer’s, adult NSCs from 3-, 6- and 18-month 3xTgAD and nonTg were harvested and cultured. NSC proliferation was measured using a neurosphere-forming assay and NSC differentiation capacity was assessed using cell-type specific immunolabeling after 7 days of differentiation. To investigate the impact of Allo on neurogenesis and differentiation in vivo, 3xTgAD mice were treated with Allo once per week for a month and 5 doses of BrdU were injected during the first 5 days. Brain sections were collected and labeled with BrdU, MAP2 and GFAP. Results: Both embryonic E18 rat NSC and adult mouse NSC can differentiate into multiple neural cell types: neurons, astrocytes and oligodendrocytes. However, the proliferation capacity of adult NSCs decreases with age and Alzheimer’s. Furthermore, the majority of NSCs derived from aged brain differentiated into glial cells as they aged, which was magnified or greater in the NSCs from 3xTgAD brain.Allo treatment promoted greater neuroproliferation of NSCs and induced a greater ratio of neuron to glia cells after NSC differentiation in vitro. I n vivo differentiation analyses are ongoing. Conclusions: 1. Allopregnanolone increased NSC proliferation and promoted NSC differentiation into neurons.2. Proliferative and differentiative capacities exhibited age- and disease-dependent deficits in nonTg and 3xTgAD mice.3. Our data suggest that allopregnanolone augmentation of neuronal phenotypic differentiation supports potential therapeutic efficacy for clinical development. P4-015
A NOVEL ALZHEIMER’S DISEASE MODEL BASED ON 3D-CULTURED HUMAN NEURAL PROGENITOR CELLS
Young Hye Kim1, Se Hoon Choi1, Steven Wagner2, Rudolph Tanzi1, Doo Yeon Kim3, 1Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States; 2UCSD School of Medicine, La Jolla, California, United States; 3Massachusetts General
Figure 1. 3D-cultured ReNcell VM (APPSwedish/London, PS1deltaE9) developed extracellular Ab deposits after 40 days of differentiation. The 3D-cultured cells were stained with HRP-conjugated Ab40 antibody (BA-27, DAB staining, brown color) and then counterstained with hematoxylin (blue color). A DAPT treatment dramatically decreased the extracellular Ab deposits. Hospital/Harvard Medical School, Charlestown, Massachusetts, United States. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) research has been hindered by a lack of cellular models that mimic the human nervous system. Advances in stem cell technology make it possible to generate human neurons harboring familial AD (FAD) mutations. However, it is still challenging to fully reproduce neurodegenerative conditions in vitro. Here, we developed a novel cellular AD model based on immortalized human neural progenitor cells (hNPCs) and 3D culture technology. Methods: To accelerate the onset of pathological AD phenotypes, we virally introduced genes harboring multiple familial AD mutations into an immortalized human neural progenitor cell line derived from the fetal brain.Lentiviral IRES constructs were generated to coexpress AD genes including amyloid b precursor protein (APP) with K670N/M671L and V717I FAD mutations and presenilin 1 (PS1) with deltaE9 FAD mutation. The virally transfected cells were FACS sorted, plated in 3D Matrigel, and then differentiated into neuronal and glial cells under growth factor free conditions for about 40 days. AD pathological markers including soluble/aggregated amyloid b peptides (Ab) and phospho tau, were analyzed by ELISA, Western blot and immunohistochemistry. Results: We found that human neural progenitor stem cells (hNPCs) can be genetically modified to harbor multiple FAD mutations and differentiated into neuronal/glial cells. We also found that the overexpression of FAD genes with multiple mutations dramatically elevated soluble Ab40 and 42 levels, which were decreased by either g-secretase inhibitor/modulator or BACE1 inhibitor treatments.Interestingly, by combining with 3D culture techniques, these cells developed strong extracellular Ab aggregate as well as the increased phospho tau pathology. In addition, an experimental g-secretase modulator dramatically decreased TBS-insoluble Ab in 3D-cultured hNPCs expressing FAD genes. Conclusions: Our results strongly indicate that the genetically modified hNPCs, combined with 3D-culture technique, can serve as unique and valid AD models for basic research and drug screening. The strategies we used to accelerate Ab pathology in vitro can also be applicable for developing other neurodegenerative disease models.
P4-016
ALLOPREGNANOLONE AS A REGENERATIVE THERAPEUTIC FOR ALZHEIMER’S DISEASE, 2: BRIDGING SUBCUTANEOUS TO INTRAVENOUS FORMULATIONS FOR CLINICAL TRIALS
Ronald Irwin1, Shuhua Chen1, Jimmy To1, Muye Zhu2, Jia Yao1, Kathleen Rodgers1, Michael Bolger3, Roberta Brinton1, 1University of Southern California, Los Angeles, California, United States; 2USC, Los Angeles, California, United States; 3Simulations Plus, Inc., Lancaster, California, United States. Contact e-mail: [email protected] Background: Allopregnanolone (Allo) stimulates proliferation of rodent and human neural progenitor cells in vitro and in triple transgenic Alzheimer’s disease (3xTgAD) mice in vivo. Simultaneously, Allo reduces AD pathology and improves cognitive performance in 3xTgAD mice. Methods: Allo was administered to mice subcutaneously (SC; 10 mg/kg) or
Wednesday, July 17, 2013: Poster Presentations: P4
weeks and then subcutaneously injected with vehicle, Allo (10mg/kg), or 17b-estradiol (E2, 60mg/kg). 24 hours after treatment, mice were sacrificed and brain mitochondria were isolated for mitochondrial respiration and bioenergetic enzyme measurements. Results: Adult NSCs from both nonTg and 3xTgAD mice exhibited a significant age-dependent decline in mitochondrial bioenergetic capacity. Further, aNSC derived from 3xTgAD mice at 6 and 18 months exhibited elevated proton leak. In vitro Allo treatment potentiated mitochondrial respiration in both NSCs, neurons and mixed glia. Further, in vivo Allo treatment restored the OVX-induced decrease in mitochondrial respiration in both nonTg and 3xTgAD mice. Allo treatment increased activity of bioenergetic enzymes such as PDH and aKGDH, and suppressed the OVX-induced increase in lipid peroxidation. Conclusions: There was an age- and Alzheimer’s associated decline in mitochondrial function in aNSCs. Allo treatment potentiated mitochondrial function both in vitro and in vivo. Collectively, these data provided mechanistic basis for developing Allo as a multi-tropic AD therapeutics to sustain and enhance mitochondrial function and to promote neurogenic capacity. P4-014
ALLOPREGNANOLONE AS A REGENERATIVE THERAPEUTIC FOR ALZHEIMER’S DISEASE, 4: IMPACT ON NEURONAL VERSUS GLIOSIS PHENOTYPIC DIFFERENTIATION 1
1
2
2 1
Shuhua Chen , Jia Yao , Karren Wong , Roberta Brinton , University of Southern California, Los Angeles, California, United States; 2University of Southern California, Los Angeles, California, United States. Contact e-mail: [email protected] Background: Allopregnanolone (Allo), an endogenous neurogenic steroid, promotes proliferation of human and rodent neural progenitor cells in vitro and in vivo of triple transgenic mouse model of Alzheimer’s disease (3xTgAD). In this study, we investigated the impact of Allo on neural differentiation in vitro and in vivo of 3xTgAD. Methods: Neural stem cells (NSCs) from embryonic E18rats and adult nonTg and 3xTgAD mice were harvested and cultured before the in vitro analyses. E18 rat NSCs were incubated in differentiation medium containing AP a or vehicle for 6-days, fixed and then stained for neuronal, glial and oligodendrocytic markers. To investigate the changes in proliferation and differentiation capacity of adult NSC burdened by age and Alzheimer’s, adult NSCs from 3-, 6- and 18-month 3xTgAD and nonTg were harvested and cultured. NSC proliferation was measured using a neurosphere-forming assay and NSC differentiation capacity was assessed using cell-type specific immunolabeling after 7 days of differentiation. To investigate the impact of Allo on neurogenesis and differentiation in vivo, 3xTgAD mice were treated with Allo once per week for a month and 5 doses of BrdU were injected during the first 5 days. Brain sections were collected and labeled with BrdU, MAP2 and GFAP. Results: Both embryonic E18 rat NSC and adult mouse NSC can differentiate into multiple neural cell types: neurons, astrocytes and oligodendrocytes. However, the proliferation capacity of adult NSCs decreases with age and Alzheimer’s. Furthermore, the majority of NSCs derived from aged brain differentiated into glial cells as they aged, which was magnified or greater in the NSCs from 3xTgAD brain.Allo treatment promoted greater neuroproliferation of NSCs and induced a greater ratio of neuron to glia cells after NSC differentiation in vitro. I n vivo differentiation analyses are ongoing. Conclusions: 1. Allopregnanolone increased NSC proliferation and promoted NSC differentiation into neurons.2. Proliferative and differentiative capacities exhibited age- and disease-dependent deficits in nonTg and 3xTgAD mice.3. Our data suggest that allopregnanolone augmentation of neuronal phenotypic differentiation supports potential therapeutic efficacy for clinical development. P4-015
A NOVEL ALZHEIMER’S DISEASE MODEL BASED ON 3D-CULTURED HUMAN NEURAL PROGENITOR CELLS
Young Hye Kim1, Se Hoon Choi1, Steven Wagner2, Rudolph Tanzi1, Doo Yeon Kim3, 1Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts, United States; 2UCSD School of Medicine, La Jolla, California, United States; 3Massachusetts General
Figure 1. 3D-cultured ReNcell VM (APPSwedish/London, PS1deltaE9) developed extracellular Ab deposits after 40 days of differentiation. The 3D-cultured cells were stained with HRP-conjugated Ab40 antibody (BA-27, DAB staining, brown color) and then counterstained with hematoxylin (blue color). A DAPT treatment dramatically decreased the extracellular Ab deposits. Hospital/Harvard Medical School, Charlestown, Massachusetts, United States. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) research has been hindered by a lack of cellular models that mimic the human nervous system. Advances in stem cell technology make it possible to generate human neurons harboring familial AD (FAD) mutations. However, it is still challenging to fully reproduce neurodegenerative conditions in vitro. Here, we developed a novel cellular AD model based on immortalized human neural progenitor cells (hNPCs) and 3D culture technology. Methods: To accelerate the onset of pathological AD phenotypes, we virally introduced genes harboring multiple familial AD mutations into an immortalized human neural progenitor cell line derived from the fetal brain.Lentiviral IRES constructs were generated to coexpress AD genes including amyloid b precursor protein (APP) with K670N/M671L and V717I FAD mutations and presenilin 1 (PS1) with deltaE9 FAD mutation. The virally transfected cells were FACS sorted, plated in 3D Matrigel, and then differentiated into neuronal and glial cells under growth factor free conditions for about 40 days. AD pathological markers including soluble/aggregated amyloid b peptides (Ab) and phospho tau, were analyzed by ELISA, Western blot and immunohistochemistry. Results: We found that human neural progenitor stem cells (hNPCs) can be genetically modified to harbor multiple FAD mutations and differentiated into neuronal/glial cells. We also found that the overexpression of FAD genes with multiple mutations dramatically elevated soluble Ab40 and 42 levels, which were decreased by either g-secretase inhibitor/modulator or BACE1 inhibitor treatments.Interestingly, by combining with 3D culture techniques, these cells developed strong extracellular Ab aggregate as well as the increased phospho tau pathology. In addition, an experimental g-secretase modulator dramatically decreased TBS-insoluble Ab in 3D-cultured hNPCs expressing FAD genes. Conclusions: Our results strongly indicate that the genetically modified hNPCs, combined with 3D-culture technique, can serve as unique and valid AD models for basic research and drug screening. The strategies we used to accelerate Ab pathology in vitro can also be applicable for developing other neurodegenerative disease models.
P4-016
ALLOPREGNANOLONE AS A REGENERATIVE THERAPEUTIC FOR ALZHEIMER’S DISEASE, 2: BRIDGING SUBCUTANEOUS TO INTRAVENOUS FORMULATIONS FOR CLINICAL TRIALS
Ronald Irwin1, Shuhua Chen1, Jimmy To1, Muye Zhu2, Jia Yao1, Kathleen Rodgers1, Michael Bolger3, Roberta Brinton1, 1University of Southern California, Los Angeles, California, United States; 2USC, Los Angeles, California, United States; 3Simulations Plus, Inc., Lancaster, California, United States. Contact e-mail: [email protected] Background: Allopregnanolone (Allo) stimulates proliferation of rodent and human neural progenitor cells in vitro and in triple transgenic Alzheimer’s disease (3xTgAD) mice in vivo. Simultaneously, Allo reduces AD pathology and improves cognitive performance in 3xTgAD mice. Methods: Allo was administered to mice subcutaneously (SC; 10 mg/kg) or