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AAV-Tau(AAV-Tau蛋白病)模型概述

对于这种进行性核上性麻痹和皮质基底变性模型,我们会在约2-3个月大的C57BL/6小鼠的黑质中单侧立体定向接种过表达野生型人类tau蛋白(MAPT)的AAV。这种小鼠模型可复制人类tau蛋白病的一些关键特征,包括

  • 黑质致密部多巴胺能神经元减少
  • 同侧纹状体多巴胺能神经元脱神经
  • 细胞体和神经突起的磷酸化tau聚集
  • 活化的微胶质细胞
  • 反应性星形胶质细胞
  • 运动功能障碍
  • 通过活体核磁共振扫描测量的大脑萎缩(黑质、中脑、尾状核-壳核)

AAV-Tau模型生成

模型生成的一般方案是:

带有啮齿动物的立体定向手术装置

对于这种特定模型,我们使用8-12周龄的C57BL/6小鼠。然后,我们将AAV载体立体定向注射到黑质附近。我们使用带有自动微量注射器的数字立体定向设备,以实现高精度和精确度。

使用该模型的研究可以迅速启动。研究的体内阶段通常持续约6周。因此,可以在相对较短的时间内提供读数,尤其是与阿尔茨海默病和tau蛋白病变的传统tau转基因模型相比。

我们的验证措施

  • 后肢夹紧试验
  • 尾悬挂摆动测试
  • 圆筒测试
  • 旋转木马测试
  • 核磁共振成像脑萎缩
  • IHC和多重免疫荧光

 

注射 SNc 的 AT8 免疫荧光染色
注射鼻窦中的 Iba-1 和 GFAP 双重免疫荧光染色
同侧大脑半球的酪氨酸羟化酶染色显著减少
多巴胺能神经元在注射的窦房结中严重缺失
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注射 SNc 的 AT8 免疫荧光染色 

注射鼻窦中的 Iba-1 和 GFAP 双重免疫荧光染色

同侧大脑半球的酪氨酸羟化酶染色显著减少

多巴胺能神经元在注射的窦房结中严重缺失

模型特征

下面的交互式演示可让您了解我们对AAV-Tau小鼠模型的表征,包括体内数据和整个多重免疫荧光组织切片的高分辨率图像。

您只需使用左侧面板浏览图像故事”即可。

您可以使用鼠标左键在高清显微镜图像中平移您可以使用 鼠标/触控板(上/下)或左上角的+和-按钮放大和缩小 。您可以在右上角的控制面板中 切换(开/关)、更改颜色以及调整通道和分割的图像设置。

们建议使用 全屏模式,以获得 最佳交互体

Characterization of a Novel AAV-hTau Mouse Model of Tauopathies with Parkinsonian Features

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Biospective Preclinical Logo

Tauopathies, such as Progressive Supranuclear Palsy and Corticobasal Degeneration, are rare diseases with prominent Parkinsonian features, including motor symptoms such as postural instability, vertical gaze palsy, rigidity, slowed movement (bradykinesia), muscle contractions (dystonia), and sudden jerks (myoclonus). Furthermore, individuals may face difficulties with speech and swallowing, cognitive decline, and loss of sensory perception at the cortical level. These neurodegenerative diseases are often rapidly progressing and pathologically characterized by phosphorylated tau inclusions in neurons and glia.

A significant problem for the development of disease-modifying therapeutics for tauopathies is the lack of animal models that recapitulate the human disease. To address this issue, Biospective has developed and characterized an adeno-associated virus (AAV) vector-induced mouse model that is well-suited for preclinical therapeutic efficacy studies for Progressive Supranuclear Palsy and Corticobasal Degeneration.

This Interactive Presentation illustrates some of the key motor function, neuroimaging, and pathologic features of Biospective's AAV human tau model of tauopathies with Parkinsonian features.

This model was generated by injecting 2 month-old C57BL/6 mice with AAV-hTau (wild-type 2N4R human tau) or AAV-null (control) vectors unilaterally into the left substantia nigra pars compacta (SNc) using a digital stereotaxic device with an automated microinjector.

Coronal Atlas with AAV Injection Site

Coronal Atlas View of SNc Injection Site

Multiplex immunofluorescence (mIF) images were generated by immunostaining for phosphorylated Tau (AT8), GFAP, Iba-1, Tyrosine Hydroxylase, Dopaminergic Nuclei, and counterstained with the DAPI nuclear stain. Tissue sections were digitized using a high-throughput slide scanner and were processed using Biospective's PERMITSTM software platform.

To navigate though this Image Story, you can use the arrows and/or the Table of Contents icon in the upper right corner of this panel.

Navigation Panel with Tooltips

You can also interact with the microscopy image in the viewer on the right at any time to further explore this high-resolution data.

Phosphorylated Human Tau Pathology

This microscopy image shows AT8 immunostaining for pTau. The ipsilateral (left hemisphere) midbrain shows extensive staining in the vicinity of the SNc and slightly beyond. For anatomical reference, an illustration with atlas labels for this brain level is provided below.

Coronal Brain Atlas at the Level of the Substantia Nigra

Coronal Mouse Brain Section (Bregma -3.2) with Neuroanatomy Labels

Tau Pathology in Neuronal Cell Bodies & Processes

This high magnification image shows extensive pTau staining in both the soma and processes of neurons in the SNc.

Neurodegeneration in the Substantia Nigra

As can be seen in this microscopy image, there is substantial loss of TH-positive dopaminergic neurons in the ipsilateral SNc compared to the contralateral hemisphere. For reference, an illustration with atlas labels for this brain level is provided below.

Coronal Mouse Brain Section (Bregma -3.2) with Neuroanatomy Labels

Using our PERMITSTM quantitative analysis software, we have quantified the TH staining in the SNc. The plot below shows a highly significant reduction in the ipsilateral hemisphere of the AAV-Tau compared to the AAV-null (control) mice.

Tyrosine Hydroxylase Staining in the Substantia Nigra

TH stain density for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

Brain Atrophy in the SNc and Midbrain

Regional brain atrophy is a key feature of tauopathies. Magnetic Resonance Imaging (MRI) is clinically used for non-invasive neuroimaging of Progressive Supranuclear Palsy (see our Resource) and Corticobasal Degeneration (see our Resource). Our team at Biospective has investigated the spatiotemporal pattern of brain atrophy in tauopathies (see MRI Measures of Disease Progression for Progressive Supranuclear Palsy Clinical Trials and MRI & Corticobasal Degeneration). We have found significant atrophy in multiple brain areas, including the midbrain and striatum in both diseases.

Given that MRI is a “translational biomarker”, we have acquired high-resolution in vivo anatomical 3D MR images from the AAV-hTau and AAV-null (control) mice using a 7T preclinical MRI scanner. We performed fully-automated image processing using our proprietary NIGHTWINGTM software and found highly significant brain atrophy in the SNc and midbrain. This data corresponds nicely to the loss of TH-positive neurons seen in the microscopy image.

MRI Brain Atlas and Volume Data for the SNc Level

Anatomical MRI with segmented SNc and midbrain, as well as plots of relative difference between ipsilateral and contralateral hemispheres for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

Dopaminergic Neurons in the Contralateral SNc

This microscopy image shows the contralateral (right hemisphere) SNc which demonstrates TH-positive cell bodies and processes in red. The nuclei of the dopaminergic neurons are shown in blue.

Loss of Dopaminergic Neurons in the Ipsilateral SNc

This microscopy image shows the ipsilateral (left hemisphere) SNc which demonstrates a profound reduction of TH-positive cell bodies and processes (in red) compared to the contralateral hemisphere. The dopaminergic neuron nuclei are shown in blue.

Neurodegeneration in the Caudate-Putamen & Dopaminergic Motor Deficits

This microscopy image shows severe dopaminergic denervation of the ipsilateral (left hemisphere) caudate-putamen (loss of TH-positive terminals). For reference, an illustration with atlas labels for this approximate brain level is provided below.

Atlas Section at the Striatum Level

Coronal Mouse Brain Section (Bregma +0.86) with Neuroanatomy Labels

Using our PERMITSTM quantitative analysis software, we have quantified the TH staining in the Caudate-Putamen. The plot below shows a highly significant reduction in the ipsilateral hemisphere.

Tyrosine Hydroxylase Staining in the Caudate-Putamen

TH stain density for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

This loss of dopaminergic innervation corresponds well with unilateral motor deficits in these mice, including a highly significant increase in use of the ipsilateral paw during the Cylinder Test, decreased latency to fall in the Rotarod Test, increased swings to the contralateral side in the Tail Suspension Swing Test (TSST), and increased Hindlimb Clasping.

Illustration of Motor Tests and Plots of AAV-Tau vs. AAV-null

Cylinder Test, Rotarod Test, Tail Swing Suspension Test (TSST), and Hindlimb Clasping data for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

Loss of Dopaminergic Terminals in the Ipsilateral Caudate-Putamen

This high magnification view shows the severe extent of loss of dopaminergic (TH-positive) terminals in the ipsilateral striatum. There are some remaining (albeit dystrophic) axons present.

We have also identified significant brain atrophy in the caudate-putamen on MRI scans, which aligns well with our analysis of human MRI data from Progressive Supranuclear Palsy and Corticobasal Degeneration populations. This data supports the “translatability” of this tauopathy model.

MRI Atlas and Volume Data at Striatum Level

Anatomical MRI with segmented striatum, as well as plot of relative difference between ipsilateral and contralateral striatum. ****p<0.0001.

Microgliosis in Response to Human 2N4R Tau Expression

In this low magnification image, one can readily appreciate the higher density of Iba-1-positive microglia in the ipsilateral (left) hemisphere (indicated by the box) relative to the contralateral hemisphere.

The plot below shows the Iba-1 stain density in the SNc.

Plots of Iba-1 staining for AAV-Null and AAV-Tau Injected Mice

Iba-1 stain density for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, *** p<0.001.

We have performed a morphological analysis of microglia using a novel computer vision & machine learning approach developed by our team. This fully-automated algorithm classifies non-activated (ramified) and activated (non-ramified) microglia.

Image of non-activated and activated microglia

The plot below shows the microglial activation in the SNc, with highly significant increased microglial activation in the AAV-Tau mice.

Plot of PERMITS Data Showing Activated Microglia in SNc

Microglial activation for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, *** p<0.0001.

Iba-1 Staining in Proximity to Phosphorylated Tau

This high magnification view shows the increased density of Iba-1-stained microglia in areas with phosphorylated tau aggregates.

Astrogliosis & Human Tau Pathology

This low magnification microscopy image show a higher density of GFAP-positive astrocytes in the ipsilateral hemisphere (indicated by the box). The plot below shows the GFAP stain density in the SNc.

Plot of GFAP Staining Density in SNc

GFAP stain density for AAV-Tau compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

GFAP Staining in Proximity to p-Tau

This high magnification view shows the increased density of GFAP-stained astrocytes in areas with phosphorylated Tau aggregates.

Summary

This novel mouse model of tauopathies with Parkinsonian features recapitulates many of the hallmark features of Progressive Supranuclear Palsy and Corticobasal Degeneration, including the development of asymmetric motor dysfunction (due to unilateral injection), and associated loss of TH+ SNc neurons and striatal TH expression.

AAV-hTau regionally results in highly significant brain atrophy, elevated microglial density and activation levels, and increased astrocyte density and hypertrophy. Further studies are planned to continue to investigate the pathologic changes in this model.

This inducible and rapidly progressing mouse model is well-suited for drug discovery with quantitative in vivo and ex vivo readouts, and possesses distinct advantages over existing transgenic models as a screening method for novel treatment options targeting tau-related pathology.

Please feel free to further explore the microscopy image in the viewer.

We would be happy to discuss this model and our characterization if you would like to Contact Us.

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了解更多关于我们AAV-Tau小鼠模型的表征、我们经过验证的措施以及我们的临床前神经科学合同研究组织服务。

了解更多阿尔茨海默病和牛磺酸病模型

常见问题解答

核磁共振成像(MRI)脑萎缩是否可以作为转化生物标志物?


脑萎缩主要是由tau蛋白还是淀粉样蛋白和β蛋白引起的?


您能否定量评估这种tau蛋白病模型中尾状核-壳核的多巴胺能神经支配?


什么是“尾部悬挂摆动测试”?


如何量化AAV-Tau模型中的反应性星形胶质细胞?


什么是“腺相关病毒”(AAV)载体?


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