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A rapid gene delivery-based mouse model for early-stage Alzheimer disease-type tauopathy.

Siman R, Lin YG, Malthankar-Phatak G, Dong Y - J. Neuropathol. Exp. Neurol. (2013)

Bottom Line: However, perforant pathway function has not been assessed in experimental models of AD, and a therapeutic agent that protects its structure and function has not yet been identified.Expression of pathologic human tau but not enhanced green fluorescent protein led to specific dose-dependent apoptotic death of perforant pathway neurons and loss of synapses in as little as 2 weeks.This novel adeno-associated virus-based method elicits rapid tauopathy and tau-mediated neurodegeneration localized to the mouse perforant pathway and represents a new experimental approach for studying tau-driven pathogenic processes and tau-based treatment strategies in a highly vulnerable neural circuit.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Neurosurgery and Center for Brain Injury and Repair, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

ABSTRACT
The perforant pathway projection from the entorhinal cortex (EC) to the hippocampal dentate gyrus is critically important for long-term memory and develops tau and amyloid pathologies and progressive degeneration starting in the early stages of Alzheimer disease (AD). However, perforant pathway function has not been assessed in experimental models of AD, and a therapeutic agent that protects its structure and function has not yet been identified. Therefore, we developed a new adeno-associated virus-based mouse model for perforant pathway tauopathy. Microinjection into the lateral EC of vectors designed to express either human tau bearing a pathogenic P301L mutation or enhanced green fluorescent protein as a control selectively drove transgene expression in lateral EC layer II perikarya and along the entire rostrocaudal extent of the lateral perforant pathway afferents and dentate terminal field. After human tau expression, hyperphosphorylated tau accumulated only within EC layer II perikarya, thereby modeling Braak stage I of transentorhinal AD tauopathy. Expression of pathologic human tau but not enhanced green fluorescent protein led to specific dose-dependent apoptotic death of perforant pathway neurons and loss of synapses in as little as 2 weeks. This novel adeno-associated virus-based method elicits rapid tauopathy and tau-mediated neurodegeneration localized to the mouse perforant pathway and represents a new experimental approach for studying tau-driven pathogenic processes and tau-based treatment strategies in a highly vulnerable neural circuit.

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Identification of an AAV vector that drives gene expression focally throughout the mouse perforant pathway. At 3 weeks after intraentorhinal microinjection of 1 × 109 particles of AAV2/9–synapsin I promoter–eGFP–WPRE, enhanced green fluorescent protein (eGFP) is expressed predominantly in entorhinal cortex (EC) layer II neurons and along the entire rostrocaudal extent of the lateral perforant pathway projection. Low-power (A) and high-power (B) photomicrographs taken immediately rostral to the injection site in the lateral entorhinal area (LEA) visualizing eGFP in green and counterstained nuclei in blue. Enhanced GFP expression is confined to layer II of the LEA and the dendritic processes of these neurons but is absent from the medial entorhinal area (MEA) and cells in other layers of entorhinal cortex. (C) In the hippocampus, 3 weeks after intraentorhinal vector delivery, eGFP expression is present in the perforant pathway afferents traversing the hippocampal stratum lacunosum-moleculare (SLM, inset) and strong in the terminal field for the lateral perforant pathway in the dentate gyrus outer molecular layer (OML) but is absent from the middle and inner molecular layers (MML and IML), respectively, the granule cell layer (GCL), and the adjacent regions of the hippocampus. Enhanced GFP expression in the lateral perforant pathway terminal field is strong at caudal (D), intermediate (E), and rostral (F) levels of the hippocampal formation. Scale bars = (A) 400 μm; (B, C) 150 μm; (D–F) 250 μm.
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Figure 2: Identification of an AAV vector that drives gene expression focally throughout the mouse perforant pathway. At 3 weeks after intraentorhinal microinjection of 1 × 109 particles of AAV2/9–synapsin I promoter–eGFP–WPRE, enhanced green fluorescent protein (eGFP) is expressed predominantly in entorhinal cortex (EC) layer II neurons and along the entire rostrocaudal extent of the lateral perforant pathway projection. Low-power (A) and high-power (B) photomicrographs taken immediately rostral to the injection site in the lateral entorhinal area (LEA) visualizing eGFP in green and counterstained nuclei in blue. Enhanced GFP expression is confined to layer II of the LEA and the dendritic processes of these neurons but is absent from the medial entorhinal area (MEA) and cells in other layers of entorhinal cortex. (C) In the hippocampus, 3 weeks after intraentorhinal vector delivery, eGFP expression is present in the perforant pathway afferents traversing the hippocampal stratum lacunosum-moleculare (SLM, inset) and strong in the terminal field for the lateral perforant pathway in the dentate gyrus outer molecular layer (OML) but is absent from the middle and inner molecular layers (MML and IML), respectively, the granule cell layer (GCL), and the adjacent regions of the hippocampus. Enhanced GFP expression in the lateral perforant pathway terminal field is strong at caudal (D), intermediate (E), and rostral (F) levels of the hippocampal formation. Scale bars = (A) 400 μm; (B, C) 150 μm; (D–F) 250 μm.

Mentions: Several AAV vectors of varying serotypes, gene promoters, and regulatory elements were evaluated for transduction of the mouse perforant pathway initially using an eGFP reporter transgene. Based on preliminary findings, we focused on AAV2/1 and 2/9 serotypes and the synapsin I gene promoter. All 3 of the eGFP vectors shown schematically in Figure 1 generated reporter expression exclusively in neurons and to varying magnitudes after stereotaxic convection-enhanced delivery into the lateral entorhinal area (27). Based on reports that the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) can markedly stabilize viral vector–encoded mRNA (36) and enhance transgene expression in a neuronal cell type–dependent manner (37, 38), we examined its effect on entorhinal transgene expression in the context of the AAV9 serotype and synapsin I promoter. The WPRE dramatically increased eGFP expression confined to the layer II neurons of origin for the mouse perforant pathway. By 3 weeks pi of 3 × 109 or fewer virus particles, eGFP expression was restricted to a band of lateral entorhinal neuronal perikarya in layer II ventral to the rhinal fissure (Fig. 2A, B), to the dendrites of these neurons, to perforant pathway afferents (39) in the hippocampal stratum lacunosum-moleculare (Fig. 2C inset), and to the lateral perforant pathway terminal field in the dentate gyrus outer molecular layer (OML) (Fig. 2C). Transgene expression was strong along the entire rostrocaudal extent of the synaptic field of the lateral perforant pathway (Fig. 2D–F).


A rapid gene delivery-based mouse model for early-stage Alzheimer disease-type tauopathy.

Siman R, Lin YG, Malthankar-Phatak G, Dong Y - J. Neuropathol. Exp. Neurol. (2013)

Identification of an AAV vector that drives gene expression focally throughout the mouse perforant pathway. At 3 weeks after intraentorhinal microinjection of 1 × 109 particles of AAV2/9–synapsin I promoter–eGFP–WPRE, enhanced green fluorescent protein (eGFP) is expressed predominantly in entorhinal cortex (EC) layer II neurons and along the entire rostrocaudal extent of the lateral perforant pathway projection. Low-power (A) and high-power (B) photomicrographs taken immediately rostral to the injection site in the lateral entorhinal area (LEA) visualizing eGFP in green and counterstained nuclei in blue. Enhanced GFP expression is confined to layer II of the LEA and the dendritic processes of these neurons but is absent from the medial entorhinal area (MEA) and cells in other layers of entorhinal cortex. (C) In the hippocampus, 3 weeks after intraentorhinal vector delivery, eGFP expression is present in the perforant pathway afferents traversing the hippocampal stratum lacunosum-moleculare (SLM, inset) and strong in the terminal field for the lateral perforant pathway in the dentate gyrus outer molecular layer (OML) but is absent from the middle and inner molecular layers (MML and IML), respectively, the granule cell layer (GCL), and the adjacent regions of the hippocampus. Enhanced GFP expression in the lateral perforant pathway terminal field is strong at caudal (D), intermediate (E), and rostral (F) levels of the hippocampal formation. Scale bars = (A) 400 μm; (B, C) 150 μm; (D–F) 250 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Identification of an AAV vector that drives gene expression focally throughout the mouse perforant pathway. At 3 weeks after intraentorhinal microinjection of 1 × 109 particles of AAV2/9–synapsin I promoter–eGFP–WPRE, enhanced green fluorescent protein (eGFP) is expressed predominantly in entorhinal cortex (EC) layer II neurons and along the entire rostrocaudal extent of the lateral perforant pathway projection. Low-power (A) and high-power (B) photomicrographs taken immediately rostral to the injection site in the lateral entorhinal area (LEA) visualizing eGFP in green and counterstained nuclei in blue. Enhanced GFP expression is confined to layer II of the LEA and the dendritic processes of these neurons but is absent from the medial entorhinal area (MEA) and cells in other layers of entorhinal cortex. (C) In the hippocampus, 3 weeks after intraentorhinal vector delivery, eGFP expression is present in the perforant pathway afferents traversing the hippocampal stratum lacunosum-moleculare (SLM, inset) and strong in the terminal field for the lateral perforant pathway in the dentate gyrus outer molecular layer (OML) but is absent from the middle and inner molecular layers (MML and IML), respectively, the granule cell layer (GCL), and the adjacent regions of the hippocampus. Enhanced GFP expression in the lateral perforant pathway terminal field is strong at caudal (D), intermediate (E), and rostral (F) levels of the hippocampal formation. Scale bars = (A) 400 μm; (B, C) 150 μm; (D–F) 250 μm.
Mentions: Several AAV vectors of varying serotypes, gene promoters, and regulatory elements were evaluated for transduction of the mouse perforant pathway initially using an eGFP reporter transgene. Based on preliminary findings, we focused on AAV2/1 and 2/9 serotypes and the synapsin I gene promoter. All 3 of the eGFP vectors shown schematically in Figure 1 generated reporter expression exclusively in neurons and to varying magnitudes after stereotaxic convection-enhanced delivery into the lateral entorhinal area (27). Based on reports that the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) can markedly stabilize viral vector–encoded mRNA (36) and enhance transgene expression in a neuronal cell type–dependent manner (37, 38), we examined its effect on entorhinal transgene expression in the context of the AAV9 serotype and synapsin I promoter. The WPRE dramatically increased eGFP expression confined to the layer II neurons of origin for the mouse perforant pathway. By 3 weeks pi of 3 × 109 or fewer virus particles, eGFP expression was restricted to a band of lateral entorhinal neuronal perikarya in layer II ventral to the rhinal fissure (Fig. 2A, B), to the dendrites of these neurons, to perforant pathway afferents (39) in the hippocampal stratum lacunosum-moleculare (Fig. 2C inset), and to the lateral perforant pathway terminal field in the dentate gyrus outer molecular layer (OML) (Fig. 2C). Transgene expression was strong along the entire rostrocaudal extent of the synaptic field of the lateral perforant pathway (Fig. 2D–F).

Bottom Line: However, perforant pathway function has not been assessed in experimental models of AD, and a therapeutic agent that protects its structure and function has not yet been identified.Expression of pathologic human tau but not enhanced green fluorescent protein led to specific dose-dependent apoptotic death of perforant pathway neurons and loss of synapses in as little as 2 weeks.This novel adeno-associated virus-based method elicits rapid tauopathy and tau-mediated neurodegeneration localized to the mouse perforant pathway and represents a new experimental approach for studying tau-driven pathogenic processes and tau-based treatment strategies in a highly vulnerable neural circuit.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Neurosurgery and Center for Brain Injury and Repair, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

ABSTRACT
The perforant pathway projection from the entorhinal cortex (EC) to the hippocampal dentate gyrus is critically important for long-term memory and develops tau and amyloid pathologies and progressive degeneration starting in the early stages of Alzheimer disease (AD). However, perforant pathway function has not been assessed in experimental models of AD, and a therapeutic agent that protects its structure and function has not yet been identified. Therefore, we developed a new adeno-associated virus-based mouse model for perforant pathway tauopathy. Microinjection into the lateral EC of vectors designed to express either human tau bearing a pathogenic P301L mutation or enhanced green fluorescent protein as a control selectively drove transgene expression in lateral EC layer II perikarya and along the entire rostrocaudal extent of the lateral perforant pathway afferents and dentate terminal field. After human tau expression, hyperphosphorylated tau accumulated only within EC layer II perikarya, thereby modeling Braak stage I of transentorhinal AD tauopathy. Expression of pathologic human tau but not enhanced green fluorescent protein led to specific dose-dependent apoptotic death of perforant pathway neurons and loss of synapses in as little as 2 weeks. This novel adeno-associated virus-based method elicits rapid tauopathy and tau-mediated neurodegeneration localized to the mouse perforant pathway and represents a new experimental approach for studying tau-driven pathogenic processes and tau-based treatment strategies in a highly vulnerable neural circuit.

Show MeSH
Related in: MedlinePlus