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Activation of microglia and inflammation-mediated neurotoxicity are suggested to play a decisive role in the pathogenesis of several neurodegenerative disorders. Activated microglia release pro-inflammatory factors that may be neurotoxic. Here we show that the orderly activation of caspase-8 and caspase-3/7, known executioners of apoptotic cell death, regulate microglia activation through a protein kinase C (PKC)-δ-dependent pathway. We find that stimulation of microglia with various inflammogens activates caspase-8 and caspase-3/7 in microglia without triggering cell death in vitro and in vivo . Knockdown or chemical inhibition of each of these caspases hindered microglia activation and consequently reduced neurotoxicity. We observe that these caspases are activated in microglia in the ventral mesencephalon of Parkinson’s disease (PD) and the frontal cortex of individuals with Alzheimer’s disease (AD). Taken together, we show that caspase-8 and caspase-3/7 are involved in regulating microglia activation. We conclude that inhibition of these caspases could be neuroprotective by targeting the microglia rather than the neurons themselves. Caspases and neurotoxicity Brain inflammation is a typical feature of neurodegenerative diseases and acute forms of brain injury. Microglia are thought to play a part in the pathogenesis of such disorders by secreting neurotoxic cytokines. Experiments in cell and animal models of inflammation show that microglia activation requires the orderly activation of caspase-8 and caspase-3/7 — well known as agents of cell death. Inhibition of the caspase cascade prevents activation of microglia and protects against neurotoxicity. Caspase activation also occurs in microglia in the brains of patients with Parkinson's disease and Alzheimer's disease, raising the prospect that caspase inhibitors may have therapeutic potential.

Members of the caspase family of cysteine proteases play central roles in coordinating the stereotypical events that occur during apoptosis. Because the major executioner caspases, caspase-3 and caspase-7, exhibit almost indistinguishable activity toward certain synthetic peptide substrates, this has led to the widespread view that these proteases occupy functionally redundant roles within the cell death machinery. However, the distinct phenotypes of mice deficient in either of these caspases, as well as mice deficient in both, is at odds with this view. These distinct phenotypes could be related to differences in the relative expression levels of caspase-3 and caspase-7 in vivo, or due to more fundamental differences between these proteases in terms of their ability to cleave natural substrates. Here we show that caspase-3 and caspase-7 exhibit differential activity toward multiple substrate proteins, including Bid, XIAP, gelsolin, caspase-6, and cochaperone p23. Caspase-3 was found to be generally more promiscuous than caspase-7 and appears to be the major executioner caspase during the demolition phase of apoptosis. Our observations provide a molecular basis for the different phenotypes seen in mice lacking either caspase and indicate that these proteases occupy nonredundant roles within the cell death machinery.

Crohn’s disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn’s disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296–299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3 , or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn’s disease. The Crohn’s disease risk-conferring T300A variant in the autophagy protein ATG16L1 increases its sensitivity to caspase-3-mediated cleavage; this decreases the induction of autophagy in response to metabolic stress or pathogen infection, leading to increased secretion of inflammatory cytokines. The genetics of Crohn's disease The Thr 300-to-Ala (T300A) polymorphism in the autophagy gene ATG16L1 has been recognized as a significant susceptibility factor for Crohn's disease, a chronic inflammatory bowel disease that is emerging as a significant health problem in industrialized countries. This study demonstrates that Thr 300 resides at the P1′ position of a caspase-cleavage site in human ATG16L, where it increases ATG16L1 sensitivity to caspase-3-mediated cleavage. This decreases the induction of autophagy in response to metabolic stress or death receptor stimulation leading to increased secretion of inflammatory cytokines. These observations raise the possibility that therapeutic inhibition of caspase 3 activation pathways may restore autophagy and gut homeostasis in part by stabilizing ATG16L1.

During brain development, synapses are initially formed in excess and are later eliminated in an activity-dependent manner. Weak synapses are preferentially removed, but the mechanism linking neuronal activity to synapse removal is unclear. Here, we show that, in the developing mouse visual pathway, inhibiting synaptic transmission induces postsynaptic activation of caspase-3. Caspase-3 deficiency results in defects in synapse elimination driven by both spontaneous and experience-dependent neural activity. Notably, caspase-3 deficiency blocks activity-dependent synapse elimination, as evidenced by reduced engulfment of inactive synapses by microglia. Furthermore, in a mouse model of Alzheimer’s disease, caspase-3 deficiency protects against synapse loss induced by amyloid-β deposition. Our results reveal caspase-3 activation as a key step in activity-dependent synapse elimination during development and synapse loss in neurodegeneration.

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.

Approximately 40% of people under 30 and over 90% of people 55 or older suffer from moderate-to-severe levels of degenerative intervertebral disc (IVD) disease in their lumbar spines. Surgical treatments are sometimes effective; however, the treatment of back pain related to IVD degeneration is still a challenge; therefore, new treatments are necessary. Apoptosis may be important in IVD degeneration because suppressing cell apoptosis inside the IVD inhibits degeneration. Caspase-3, the primary effector of apoptosis, may be a key treatment target. We analyzed caspase-3’s role in two different types of IVD degeneration using caspase-3 knockout (Casp-3 KO) mice. Casp-3 KO delayed IVD degeneration in the injury-induced model but accelerated it in the age-induced model. Our results suggest that this is due to different pathological mechanisms of these two types of IVD degeneration. Apoptosis was suppressed in the IVD cells of Casp-3 KO mice, but cellular senescence was enhanced. This would explain why the Casp-3 KO was effective against injury-induced, but not age-related, IVD degeneration. Our results suggest that short-term caspase-3 inhibition could be used to treat injury-induced IVD degeneration.

Synthetic biological circuits that can generate outputs with distinct expression dynamics are useful for a variety of biomedical and industrial applications. We present a method to control output dynamics by altering output mRNA decay rates. Using oscillatory expression of the transcription factor p53 as the circuit regulator, we use two approaches for controlling target gene transcript degradation rates based on the output gene’s 3′-untranslated region (3′-UTR): introduction of copies of destabilizing AU-rich elements into the 3′-UTR or swapping in naturally occurring 3′-UTRs conferring different transcript stabilities. As a proof of principle, we apply both methods to control the expression dynamics of a fluorescent protein and visualize the circuit output dynamics in single living cells. We then use the naturally occurring 3′-UTR approach to restore apoptosis in a tunable manner in a cancer cell line deficient for caspase-3 expression. Our method can be readily adapted to regulate multiple outputs each with different expression dynamics under the control of a single naturally occurring or synthetically constructed biological oscillator.

Caspase-3 is the best known executioner caspase in apoptosis. We generated caspase-3 knockout (C3KO) and knockdown human colorectal cancer cells, and found that they are unexpectedly sensitized to DNA-damaging agents including 5-fluorouracil (5-FU), etoposide, and camptothecin. C3KO xenograft tumors also displayed enhanced therapeutic response and cell death to 5-FU. C3KO cells showed intact apoptosis and activation of caspase-7 and -9, impaired processing of caspase-8, and induction of necrosis in response to DNA-damaging agents. This form of necrosis is associated with HMGB1 release and ROS production, and suppressed by genetic or pharmacological inhibition of RIP1, MLKL1, or caspase-8, but not inhibitors of pan-caspases or RIP3. 5-FU treatment led to the formation of a z-VAD-resistant pro-caspase-8/RIP1/FADD complex, which was strongly stabilized by caspase-3 KO. These data demonstrate a key role of caspase-3 in caspase-8 processing and suppression of DNA damage-induced necrosis, and provide a potentially novel way to chemosensitize cancer cells.

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster . However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo . Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3 , but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.