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AAVタウオパチー(AAV-Tau)モデルの概要

進行性核上性麻痺および大脳基底核変性症のこのモデルでは、生後2~3ヶ月のC57BL/6マウスの黒質に、野生型ヒトタウ(MAPT)を過剰発現するAAVを片側から定位注入します。このマウスモデルは、ヒトのタウオパチーのいくつかの重要な特徴を再現しており、以下が含まれます。

  • 黒質緻密部のドーパミン作動性神経細胞の減少
  • 同側の線条体のドーパミン作動性神経の脱神経
  • 細胞体および神経突起におけるリン酸化タウの凝集体
  • 活性化されたミクログリア
  • 反応性アストロサイト
  • 運動機能障害
  • 生体MRIスキャンで測定した脳萎縮(黒質、中脳、尾状核)

AAV-Tauモデルの生成

モデル生成の一般的なスキームは次のとおりです。

齧歯類用の定位固定装置

この特定のモデルでは、生後8~12週のC57BL/6マウスを使用します。次に、AAVベクターを黒質近辺に定位注入します。 高い精度と正確性を実現するために、自動マイクロインジェクター付きのデジタル定位装置を使用します。

このモデルを用いた研究は迅速に開始することができます。生体内試験は通常、約6週間続きます。そのため、特にアルツハイマー病やタウオパチーの従来のタウ遺伝子導入モデルと比較すると、比較的短期間で結果を得ることができます。

検証済みの対策

  • 後肢把持テスト
  • テールサスペンションのスイングテスト
  • シリンダー試験
  • ロータロッド試験
  • MRI脳萎縮
  • IHCおよび多重免疫蛍光法

 

注射したSNcにおけるAT8免疫蛍光染色。
注射したSNcにおけるIba-1とGFAPの二重免疫蛍光染色
同側半球におけるチロシン水酸化酵素染色の劇的な減少
注射されたSNcにおけるドーパミン作動性ニューロンの重度の消失
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注射したSNcにおけるAT8免疫蛍光染色。

注射したSNcにおけるIba-1とGFAPの二重免疫蛍光染色

同側半球におけるチロシン水酸化酵素染色の劇的な減少

注射されたSNcにおけるドーパミン作動性ニューロンの重度の消失

モデルの特性評価

以下のインタラクティブなプレゼンテーションでは、生体内データや多重免疫蛍光組織切片の全体像の高解像度画像など、当社のAAV-Tauマウスモデルの特性評価についてご覧いただけます。

左側のパネルを使って、この「イメージストーリー」を簡単にナビゲートすることができます。

高解像度の顕微鏡画像は、マウスの左ボタンで移動できます。 マウスまたはトラックパッド(上/下)または左上隅の + および - ボタンを使用して、拡大/縮小が可能です 右上隅のコントロールパネル では、チャンネルとセグメンテーションの切り替え(オン/オフ)、色の変更、画像設定の調整が可能です。

最高のインタラクティブ体験 をお楽しみいただくには、フルスクリーンモード のご利用をお勧めします。

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 and (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.

Table of Contents
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Section: SNc Section 1
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当社のAAV-Tauマウスモデルの特性評価、検証済みの測定方法、および前臨床神経科学CROサービスについて、さらに詳しく知ることができます。

アルツハイマー病とタウオパチーのモデルをもっと見る

よくある質問

MRIによる脳萎縮は、翻訳バイオマーカーとして使用できるでしょうか?


脳の萎縮は主にタウタンパク質またはアミロイドβによって引き起こされるのでしょうか?


このタウオパチーモデルにおける尾状核・被殻のドーパミン作動性神経の変性を定量的に評価することはできますか?


「テールサスペンションスイングテスト」とは何ですか?


AAV-Tauモデルにおける「反応性」アストロサイトの定量化はどのように行いますか?


「アデノ随伴ウイルス(AAV)」ベクターとは何ですか?


関連コンテンツ

アルツハイマー病およびタウオパチーに関する最新情報と、動物モデルにおける治療薬の評価のための翻訳バイオマーカーの使用に関するベストプラクティス。

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