Exploring caspase functions in mouse models

Abstract

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

Keywords: Animal model; Apoptotic; Caspases; Deficiency; Mouse; Non-apoptoic.

Fig. 1

Conserved caspase signalling cascades in eukaryotic organisms. In C. elegans, the antagonist EGL-1 inhibits CED-9, leading to the release of CED-4 from the CED-9–CED-4 complex. This liberation promotes the activation of CED-3. In Drosophila, the inhibitors of apoptosis (IAPs) Reaper, Hid, and Grim facilitate the degradation of DIAP1, thereby freeing Drice and Dcp-1. This process also involves the interaction of Dronc with Ark and the formation of the apoptosome, which activates executioner caspases. The activation of the apoptosome might be regulated by proteins such as Buffy. In mammals, Bcl-2 and BH3-only proteins regulate BAX- and BAK-dependent release of cytochrome c from the mitochondria. Cytochrome-c then binds to APAF1 to form the apoptosome. In parallel, IAP antagonists, including DIABLO, HTRA2 and ARTS, translocate from the mitochondria and release caspases from their negative regulation by IAPs. Caspase-9 is subsequently liberated from XIAP and activated by the apoptosome, triggering executioner caspases-3 and − 7. Green: caspase-9 like, yellow: Apaf-1 like, blue: executor caspases, dark grey: Bcl-2 like, light grey: apoptotic inhibitors, pink: BH3-only like, purple: IAP binding. Figure based on Bell and Megeney [284], Fuchs and Steller [285]

Fig. 2

Overview of caspase modulation, analysis, and functions. Downstream caspase pathways may be studied with the help of the caspase downregulation at several levels, including inhibition of caspase gene expression by siRNA and inhibition of caspase activity by inhibitors in vitro. Alternatively, recombinant caspases may be used for specification of caspase functions. In vivo investigation relies on deficient mice with null or targeted caspase deletion. Analysis of caspases includes quantification of caspase expression by PCR-based techniques and activity assessment (e.g., western blot, bioluminescence, bioimaging) applied in vitro and in vivo. Detection of caspases by specific antibodies in situ provides information about caspase importance in individual cell types. With the help of these approaches, caspases have been associated with multiple functions such as programmed apoptotic cell death [286], programmed non-apoptotic cell death [287], inflammation and immune system [288], differentiation [16], proliferation [289], regulation of stem cells maintenance [43], non-apoptotic regulation of malignancies [290], modification of ECM [181], migration [28]

Fig. 3

Classification of caspases. Blue/Grey: long pro-domain containing CARD/DED, pink: long domain L, green: short domain S. The asterisk is used to highlight human/mouse caspase orthologue caspase-4, caspase-5/caspase-11. C12L and C12S stands for full-length and truncated versions, respectively

Fig. 4

Apoptotic and non-apoptotic caspase signalisation. The extrinsic pathway is regulated by death receptors, leading to the activation of caspase-8 and -10. The intrinsic pathway is usually initiated in a cell-autonomous manner, resulting in expression of BH3-only proteins that inhibit anti-apoptotic proteins such as Bcl-2, permeabilization of the mitochondrial outer membrane, formation of apoptosome, and activation of caspase-9. Both pathways aim to activate caspase-3 (or other executors: caspase-6, -7). The extrinsic and intrinsic pathways are often interconnected (e.g. caspase − 8 (and also − 2) cleaves Bid into tBid, which impacts mitochondria). Caspase-12 contributes to Ca²⁺-dependent apoptosis. Caspase-2 activation occurs in response of both intrinsic and extrinsic stimuli