Intrinsic apoptosis is evolutionary divergent among metazoans

Apoptosis is characterised by an analogous set of morphological features1 that depend on a proteolytic multigenic family, the caspases. Each apoptotic signalling pathway involves a specific initiator caspase, upstream of the pathway regulation, which finally converges to common executioner caspases. Intrinsic apoptosis, previously known as the mitochondrial apoptotic pathway, is often considered as ancestral and evolutionary conserved among animals. First identified in the nematode Caenorhabditis elegans, intrinsic apoptosis was next characterised in fruit fly Drosophila melanogaster and mammals. Intrinsic apoptosis depends on the key initiator caspase-9 (named Ced-3 and Dronc in Caenorhabditis and Drosophila, respectively), the activator Apaf-1 and the Bcl-2 multigenic family. Many functional studies have led to a deep characterisation of intrinsic apoptosis based on those classical models. Nevertheless, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspases activation remain highly heterogeneous and hide profound molecular divergences among apoptotic pathways in animals. Independent of functional approaches, the phylogenetic history of the signal transduction actors, mostly the caspase family, is the Rosetta Stone to shed light on intrinsic apoptosis evolution. Here, after exhaustive research on CARD-caspases, we demonstrate by phylogenetic analysis that the caspase-9, the fundamental key of intrinsic apoptosis, is deuterostomes-specific, while it is the caspase-2 which is ancestral and common to bilaterians. Our analysis of Bcl-2 family and Apaf-1 confirm the high heterogeneity in apoptotic pathways elaboration in animals. Taken together, our results support convergent emergence of distinct intrinsic apoptotic pathways during metazoan evolution.

Genuine Selective Caspase-2 Inhibition with new Irreversible Small Peptidomimetics

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics shows that these new compounds, such as LJ2 or its specific isomer LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. According to Casp2 role in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~ 5 500 000 M-1s-1) and the most selective inhibitor, LJ3a, has a near to 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that spatial configuration of C[alpha]; at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with [beta]-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

Effects of intravitreal injection of siRNA against caspase-2 on retinal and optic nerve degeneration in air blast induced ocular trauma

Ocular repeated air blast injuries occur from low overpressure blast wave exposure, which are often repeated and in quick succession. We have shown that caspase-2 caused the death of retinal ganglion cells (RGC) after blunt ocular trauma. Here, we investigated if caspase-2 also mediates RGC apoptosis in a mouse model of air blast induced indirect traumatic optic neuropathy (b-ITON). C57BL/6 mice were exposed to repeated blasts of overpressure air (3 × 2 × 15 psi) and intravitreal injections of siRNA against caspase-2 (siCASP2) or against a control enhanced green fluorescent protein (siEGFP) at either 5 h after the first 2 × 15 psi (“post-blast”) or 48 h before the first blast exposure (“pre-blast”) and repeated every 7 days. RGC counts were unaffected by the b-ITON or intravitreal injections, despite increased degenerating ON axons, even in siCASP2 “post-blast” injection groups. Degenerating ON axons remained at sham levels after b-ITON and intravitreal siCASP2 “pre-blast” injections, but with less degenerating axons in siCASP2 compared to siEGFP-treated eyes. Intravitreal injections “post-blast” caused greater vitreous inflammation, potentiated by siCASP2, with less in “pre-blast” injected eyes, which was abrogated by siCASP2. We conclude that intravitreal injection timing after ocular trauma induced variable retinal and ON pathology, undermining our candidate neuroprotective therapy, siCASP2.