Name: Gait Analysis in an Angelman Syndrome Mouse Model
Uploaded: 2023-10-20T12:10:00
Description: Watch this Scientific Journal Video about Gait Analysis in an Angelman Syndrome Mouse Model at JoVE.com

gait analysis in an angelman syndrome mouse model

行動試験を用いたアンジェルマン症候群のマウスモデルの検証

Angelman syndrome (AS) is a rare neurodevelopmental disease. The most common genetic origin of AS is a large deletion of the 15q11-q13 region of the maternally-derived chromosome, which is found in nearly 74% of patients1. Deletion of this region causes the loss of UBE3A, the main causative gene of AS that encodes an E3 ubiquitin ligase. The paternal allele of the UBE3A gene in neurons is silenced in a process known as imprinting. As a consequence, paternal imprinting of the gene allows only maternal expression in the central nervous system (CNS)2. Therefore, UBE3A gene deletion from the maternally-derived chromosome leads to the development of AS symptoms. In humans, AS manifests at around 6 months of age, with developmental retardation that persists throughout all developmental stages and results in severe debilitating symptoms in affected individuals3,4. The core symptoms of the disorder include the deficit of fine and gross motor skills, including jerky ataxic gait, serious speech impairment, and intellectual disability. Approximately 80% of AS patients also suffer from sleep disturbances and epilepsy. To date, the only available treatment are symptomatic drugs, which reduce epileptic seizures and improve sleep quality1. Therefore, the development of robust animal models with reproducible behavioral phenotypes alongside refined phenotyping analysis will be essential to elucidate the pathophysiological mechanisms of the disorder and discover effective medications and treatments.
The complexity of the human disorder affecting the CNS demands model organisms to possess a comparable genome, physiology, and behavior. Mice are popular as a model organism due to their short reproductive cycle, small size, and relative ease of DNA modification. In 1984, Paul Willner proposed three basic disease model validation criteria: the construct, face, and predictive validity, which are used to determine the model&#39;s value5. Simply, construct validity reflects the biological mechanisms responsible for the disorder development, face validity recapitulates its symptoms, and predictive validity describes the model response to therapeutic drugs.
To adhere to the above principles, we have chosen the most common genetic etiology, a large deletion of the maternal 15q11.2-13q locus including the UBE3A gene, to create AS model mice. We used the CRISPR/Cas9 technique to delete a 76,225 bp long region spanning the entire UBE3A gene, encompassing both the coding and non-coding elements of the gene, in mice from a C57BL/6N background6. We then bred the animals to obtain UBE3A+/&#8722; heterozygous mice. For face validation of the model, we used animals from crosses of UBE3A+/&#8722; females and wild-type males to gain UBE3A+/- progeny (strain named C57BL/6NCrl-UBE3A&#60;em1(IMPC)Ccpcz&#62;/Ph and later assigned as UBE3AmGenedel/+) and control littermates. We tested their fine and gross motor skills, emotionality, and affect to recapitulate core AS symptoms. In a previous article, we also evaluated the animals&#39; cognitive functions, as AS patients also suffer from intellectual disability6. However, we found no cognitive impairments in UBE3AmGenedel/+ mice, perhaps due to the young age of the animals at the time of testing7. Later examination of the older animals, around 18 weeks old, revealed a deficit in behavioral flexibility during reversal learning in the place preference paradigm. However, the complexity of the employed equipment for this analysis requires a separate methodological module and it is not included here.
The behavioral tests presented here belong to the common phenotyping tools in genetic research, thanks to their high predictive value and sufficient construct validity8,9,10. We used these tests to validate a mouse model of AS by recapitulating core symptoms of the human disease in a reproducible, age-independent manner. The emotionality of the animal was evaluated in the elevated plus maze and open field tests. Both of these tests are based on the approach-avoidance conflict, where animals explore a new environment in search of food, shelter, or mating opportunities while simultaneously avoiding anxiogenic compartments11. Additionally, the open field test is used to test a mouse&#39;s locomotor activity8. The tail suspension test is widely used in depression research to screen for new antidepressant drugs or depressive-like phenotypes in mouse knockout models12. This test evaluates the despair that animals develop over time in an inescapable situation. Motor learning and detailed gait characteristics were determined on the rotarod and in DigiGait, respectively. Animal endurance on the accelerating rod characterizes its balance and movement coordination skills, while detailed analysis of a mouse&#39;s step patterns is a sensitive evaluation of neuromuscular impairments connected to many neurogenerative movement disorders13,14,15. The nestlet shredding test is part of the standard methodology for detecting impulsive behavior in rodents, and since it utilizes natural rodent building behavior, it indicates the animal&#39;s well-being16,17.
The size of the experimental groups was a result of a compromise to meet the 3R rule demands and efficient usage of colony breeding performance. However, to obtain statistical power, the groups had no fewer than 10 individuals, due to the establishment of a sufficient amount of breeding pairs. Unfortunately, breeding performance did not always result in a sufficient number of animals.

著者スポットライト:アンジェルマン症候群マウスモデルを検証するための再現性の高い一連の行動試験 

アンジェルマン症候群モデルマウスの行動特性

Our goal is to elucidate the physiological roles of genes using mice as an experimental model. We also focus on developing mouse models of specific diseases that scientists can use to investigate disease causation and develop effective treatments. An example of such activity is the mouse model of Angelman syndrome.Arguably, the most promising new technology with potential is integrating artificial intelligence based on deep learning techniques in animal research. This approach can be used to monitor animal behavior in the home cage directly without need for the experimenter to disturb the animals. The biggest challenge in behavioral studies currently is reproducibility.Therefore, we introduce a battery of tests that validate the Angelman syndrome of model in a way that does not depend on the model's age or species. We use established behavioral tests to validate our AS model. However, with the novel approach for the model development closely resembling the human condition with a large deletion.Using CRISPR technology, we deleted the entire UBE3A gene from the mouse genome. In contrast to other models where the deletion is limited to 3KB. We're working on developing a genetic therapy that could restore a symptoms in a mouse model.

Watch this Scientific Journal Video about Gait Analysis in an Angelman Syndrome Mouse Model at JoVE.com

Gait Analysis in an Angelman Syndrome Mouse Model  

アンジェルマン症候群マウスモデルにおける歩行解析

トレッドミルの電源を入れ、機器パネルでベルト速度を手動で毎秒20センチメートルに設定してから、機器のライトをオンにします。DigiGait Imagerソフトウェアを起動し、シャッタースピードをアルビノマウスの場合は100、フィールド内の黒または暗いマウスの場合はシャッタースピードを130に設定します。マウスをトレッドミルベルトに置き、約3秒間ゆっくりとした歩行速度に設定し、マウスを観察し続けながら停止することで、マウスがテストを実行できることを確認します。スタートボタンを押してベルトを始動し、10秒間録音します。少なくとも10〜15ステップの明確で流暢な移動が観察可能であることを確認してください。停止ボタンを押してベルトを停止し、マウスを一時保持ケージに戻します。流暢な手順で一連の画像の記録をスクリーニングするには、[再生] をクリックし、編集モードのビジュアル コントロールで記録を確認します。10〜15の流暢な動きを選択するには、開始フレーム番号と終了フレーム番号を関連するフィールドに手動で書き込みます。動物ID、生年月日、性別、体重、ベルト速度、ベルト角度などの動物情報を入力します。[保存] をクリックしてファイルを保存します。次に、DigiGait解析ソフトウェアを開き、動物の足跡のビデオ録画の完全自動分析に基づいて歩行分析を実行します。UBE3A変異動物の歩行指数を改変した。UBE3A変異動物は、スイングとスタンスが長く、ストライドの持続時間と長さが長くなりました。マウスの後肢、推進力、持続時間、および減速も増加した。これらの発見はまた、ピークスタンスでより大きな細孔面積を明らかにしました。

Research

JoVE Journal

Behavior

Gait Analysis in an Angelman Syndrome Mouse Model

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

行動学

Elevated Plus Maze Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおける上昇プラス迷路テスト

Open Field Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおけるオープンフィールド試験

Tail Suspension Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおける尾懸垂試験

Rotarod Test in an Angelman Syndrome Mouse Model

アンジェルマン症候群マウスモデルにおけるロータロッド試験

Nestlet Shredding: Nest Building Test in an Angelman Syndrome Mouse Model

ネストレットシュレッダー:アンジェルマン症候群マウスモデルにおける巣作り試験