Initiator caspases, like caspase-8 can be activated by dimerization following induced proximity (Shi, 2004), so NCAM clustering may bring caspase-8 molecules together, causing dimerization and activation. Once active, caspase-8 can in turn activate caspase-3. It is proposed that caspase-3 then cleaves spectrin, altering the cytoskeleton and allowing neurite outgrowth. Increased caspase-3 is also seen in retinal neurons responding to the chemotrophic signals lysophosphatidic acid or netrin. Caspase inhibitors prevent this chemotrophic response (Campbell and Holt, 2003).

Caspase-3 and -9 activation has also been detected at the axonal branch points of retinal ganglion cells (Campbell and Okamoto, 2013). The role of caspase-9 in activating caspase-3 in the mitochondrial pathway in other contexts suggests that the mitochondrial pathway is important in this instance too. In support of this idea, axons are misrouted and synapse formation by sensory neurons is impaired in Apaf-1 null mice and caspase-9 null mice. The cleavage by caspase-9 of semaphorin 7A, a protein necessary for proper axonal growth appears to explain these deficits (Ohsawa et al., 2009, 2010). This is particularly interesting as there are relatively few caspase-9 substrates known compared to effector caspases like -3 and -7, and during apoptosis the role of caspase-9 appears to be primarily the activation of these effectors. Cleavage of semaphorin is a rare example of a non-lethal caspase substrate for caspase-9 whose cleavage is functionally important. Interestingly, NCAM and neuron-glia cell adhesion molecule (NgCAM), another protein prevalent on axons and a promoter of neurite growth and axonal fasciculation, are also caspase-3 substrates (Westphal et al., 2010; Weghorst et al., 2020), although the functional significance of this cleavage is not known.

The cleavage by caspase-3 of cytoskeletal growth cone proteins (Campbell and Holt, 2003) as well as proteins like Gap43 that regulate growth cones (Denny, 2006) has also been demonstrated, leading to the suggestion that caspase-mediated remodelling of the cytoskeleton is central to axonal growth and guidance (reviewed by (Kellermeyer et al., 2018)). During apoptosis, caspase-3 cleaves actin, producing a 15 kDa fragment and causing condensation and fragmentation of the actin network (Mashima et al., 1997). Actin remodelling is also seen in the growth cones in the termini of developing neurons (Pacheco and Gallo, 2016). Interestingly, the 15 kDa fragment is detected in non-apoptotic neurons from the aged and from Alzheimer’s disease patients and co-localizes with an active form of caspase-3 (Rossiter et al., 2000), although the significance of this non-apoptotic caspase activation for the disease’s pathology remains uncertain. More recently, proteomic studies of proteins cleaved in the chick auditory brainstem have uncovered a non-apoptotic role for caspase-3 in cell-cell communication (Weghorst et al., 2020). The authors reported that many of the substrates cleaved were associated with non-apoptotic functions of caspases. Analysis of the substrates also revealed a disproportionately high level of proteins found in extracellular vesicles (EVs), a finding corroborated by proteomic analysis of EVs from the auditory brainstem. This analysis also identified NCAM and NgCAM in the EVs. These data suggest that caspase-3 activity may affect development of the auditory brainstem by modifying the cargo of EVs. Thus, despite many uncertainties, it appears that cleavage of proteins by caspase-3, -8 and -9 can regulate different cellular behaviors that are key for axon growth and guidance.

Axon growth and routing also occurs during the regeneration of broken axons. In mammals, caspase-3 inhibitors, but not a caspase-9 inhibitor block axon regeneration in dorsal root sensory neurons by preventing the formation of the growth cone (Verma et al., 2005). Inhibitors of caspase-3 or of calpain (a calcium-activated protease) also block axon regeneration in dorsal root ganglion cells (Öztürk et al., 2013). This process also involves local increases in protein translation at the site of injury (Verma et al., 2005), raising the possibility that a local increase of caspase activators is a key event. However, there are alternative explanations: after axotomy the local concentration of calcium rises into millimolar range (Ziv and Spira, 1995) before falling back to normal levels. These calcium fluxes are likely to activate the calcium sensitive protease, calpain. In apoptotic contexts calpain inhibitors or siRNA against µ-calpain block caspase-3 activation (Varghese et al., 2001), so perhaps calpain may play a role in growth cone formation and regeneration by activating caspase-3.

In C. elegans, CED-4 (which is the nematode homolog of Apaf-1) and CED-3, (the homolog of caspase-9) are key proteins in axonal regeneration after axotomy of ALM mechano-sensory neurons (Pinan-Lucarre et al., 2012). The axonal regeneration is not dependent on EGL-1, the upstream regulator that controls CED-4 during apoptosis (Figure 3). Instead, CED-4 appears to be locally activated by signals arising from axonal damage and involves increased levels of intracellular calcium. These calcium fluxes activate the conserved Dual Leucine Zipper Kinase-1 (DLK-1) regeneration pathway to induce axonal regeneration (Hammarlund et al., 2009; Yan et al., 2009; Shin et al., 2012) upstream of CED-4/CED-3. As DLK-1 can induce local protein translation, it is possible that this is important for CED-3 activation. While DLK-1 is part of a conserved regeneration pathway in mammals, millimolar levels of calcium are known to bind and inhibit Apaf-1 (Bao et al., 2007) and Apaf-1 proteolysis is triggered by increases in calcium (Reimertz et al., 2001) so the role of Apaf-1 in mammalian axonal regeneration is not clear. Which proteins are cleaved by CED-3 during non-canonical processes is poorly understood, but cleavage of LIN-14, LIN-28 and DISL-2, which are key regulators of development involved in microRNA processing, is seen in non-apoptotic context (Weaver et al., 2014).

During apoptosis in Drosophila DRONC is activated by the Apaf-1 homolog, DARK. Kang et al. (2017) reported DRONC-dependent axon pruning of ddaC neurons and that this occurred in DARK null flies, suggesting a DARK-independent activation mechanism. The basal level of DRONC activity is held in check by Drosophila homolog of X-linked inhibitor of apoptosis protein (XIAP), DIAP-1. DIAP-1 itself is antagonized by the pro-apoptotic proteins Grim, Reaper, Hid and Scythe (Silke and Meier, 2013). Basal levels of DRONC activation are known to play roles in Drosophila development being freed of DIAP-1 inhibition (Shinoda et al., 2019) and pruning also relies on the basal levels of activated DRONC. Consistent with this idea, the non-apoptotic remodelling of sensory neurons is dependent on the DIAP-1 inhibitors, Reaper, Hid and Grim (Mukherjee et al., 2021), and the tight control of Hid levels is vital for the process (Bhogal et al., 2016). The DARK-independent mechanism for sub-lethal activation of DRONC during pruning involves Tango7 (Kang et al., 2017), a protein that has also been linked to apoptosome regulation during spermatogenesis in Drosophila (D’Brot et al., 2013). Thus the DRONC activation occurs by distinct mechanisms depending on whether an apoptotic or non-apoptotic process is underway, the first involving an Apaf-1 homolog and the second critically dependent on inhibitor of apoptosis proteins.

Apaf-1-independent but caspase-dependent pruning also occurs in mammalian cells (Cusack et al., 2013), but how the caspases are activated is less well understood. In mammals, caspase-9 can be activated independently of Apaf-1 in a pathway triggered by the Patched receptor (Fombonne et al., 2012) or by the Deleted in Colorectal Cancer (DCC) dependence receptor (Forcet et al., 2001). There is also evidence that basal levels of active caspase-3 held in check by XIAP are freed to drive axon degeneration by XIAP degradation (Schoenmann et al., 2010; Ertürk et al., 2014). This is supported by several lines of evidence: XIAP regulates axonal degeneration (Unsain et al., 2013), and XIAP null mice have fewer synapses and learning and memory deficits (Gibon et al., 2016). These findings may be relevant to neurodevelopmental abnormalities: overexpression of E6AP is linked to autism and leads to significant loss of dendritic arborization. E6AP ubiquitinylates XIAP, increasing caspase activity and causing local degeneration and retraction at the tips of dendritic branches (Khatri et al., 2018). XIAP is also regulated by Smac/Diablo and Omi/Htr2A, two proteins that are released from mitochondria during apoptosis and that bind XIAP, relieving caspase inhibition (Ekert and Vaux, 2005). Although loss of Omi/Htr2A is neuroprotective (Martins et al., 2002), and Omi/Htr2A has been linked to Parkinson’s (Strauss et al., 2005) suggesting important roles in controlling neuron apoptosis, it is not clear whether non-apoptotic caspase activity involves the release of Omi/Htr2A (or Smac/Diablo). However, Omi/Hrt2A does play a non-apoptotic role as a serine protease in neurons, controlling the distribution of mitochondria along axons by cleaving vimentin (Lucotte et al., 2015). Caspase dysregulation has been implicated in Down Syndrome (DS), but has largely been linked to cell death through traditional apoptotic signaling. However, there are changes in the dendritic arbor of hippocampal pyramidal cells in the Ts65Dn mouse model of DS (Uguagliati et al., 2021) that suggest a non-apoptotic mechanism. The protein DYRK1A regulates caspase-9 in the developing retina (Laguna et al., 2008) and the gene is amplified in DS (Laguna et al., 2013). This constellation of observations suggests a caspase-mediated pathway may be central to abnormal connectivity seen in DS neurodevelopment.

During axonal degeneration of mouse TRKA + sensory neurons a neuronal protein called RUFY3 is cleaved by caspase-3. Deletion of RUFY3 protects the axon, even when caspase-3 is activated, suggesting that RUFY3 is the key substrate in caspase-3-mediated axonal degeneration. Phosphorylation of RUFY3 at Ser34 prevents axonal degeneration, so local dephosphorylation of RUFY3 may provide a mechanism to spatially limit axon degeneration (Hertz et al., 2019). RUFY3 is also known to limit axonal growth, and loss of RUFY3 results in multiple axons that are shorter (Mori et al., 2007; Wei et al., 2014). This may be explained mechanistically by RUFY3 forming complexes with Fascin, an actin-bundling protein, and Drebrin 1 (DBN1) (Wei et al., 2014). DBN1 can regulate actin too (G. Wang et al., 2015), but it also binds CXCR4, a chemokine that regulates neuronal migration (Shan et al., 2021). These data hint at multiple roles for RUFY3 in both axonal growth and axonal degeneration although caspase-3 cleavage of RUFY3 during axonal growth has not been reported.

The primacy of caspase-3 in axonal degeneration is not clear: Nikolaev et al. (2009) showed that caspase-6 was present and activated in dorsal root ganglion axons after withdrawal of NGF in a Bax-dependent manner, and that this activation was required for axonal degeneration. The dependence on Bax suggests that caspase-6 was activated by the mitochondrial pathway (see Figure 1). Sympathetic neurons in caspase-6 null mice also show less axonal degeneration (Uribe et al., 2012). This role of caspase-6 may have disease relevance as caspase-6 has been linked to Alzheimer’s (Gervais et al., 1999; Guo et al., 2004; Albrecht et al., 2007) and to Huntington’s disease (Graham et al., 2006; Graham et al., 2010). Caspase-6 inhibitors reverse the cognitive impairment caused by caspase-6 overexpression and loss of caspase-6 reduces other disease aspects in a mouse model of Huntington’s (Wong et al., 2015; Pakavathkumar et al., 2017). Bax-dependent caspase-6 activity during axonal degeneration was also implicated in another study (Schoenmann et al., 2010), but a more complex picture was presented. On NGF withdrawal both caspase-3 and caspase-6 activity increased, but blocking caspase activity alone was insufficient to protect the axons. However combining caspase inhibition with NAD⁺ did protect the axons, suggesting a caspase-dependent pathway working in parallel with an NAD⁺ sensitive pathway. Because loss of Bax provided a more profound protection, it appears that it lies upstream of both the caspase-dependent and the NAD⁺ sensitive pathway.

The Bax-dependence of caspase-6 activity during axonal degeneration suggests that Apaf-1, caspase-9 and caspase-3 lie upstream of caspase-6. However, caspase-6 can also be activated by other pathways. For example, axonal degeneration in primary cultures of human neurons involves activation of neuronal NLRP1, which then forms an inflammasome to activate caspase-1, and caspase-1 cleaves and activates caspase-6 (Kaushal et al., 2015). However the crystal structure of caspase-6 suggests that unlike other effector caspases (-3 and -7), caspase-6 could also be activated by self-cleavage (Wang et al., 2010). This autocatalytic activation is controlled by caspase-6 phosphorylation, which locks caspase-6 in an inactive state (Cao et al., 2012).

The substrates that caspase-6 cleaves to cause axonal degeneration are not clear. Proteomic and other approaches have identified substrates cleaved during apoptosis, such as Lamin A (Ruchaud, 2002), or during neuronal degeneration such as CBP/p300 (Rouaux et al., 2003). Others have identified ∼24 substrates in neurons (Klaiman et al., 2008) and ∼47 substrates in other cell types (Cho et al., 2013). The identified substrates have roles in the cytoskeleton, signaling, chaperones, protein synthesis regulation, metabolism, proteolysis and membrane and lipid binding, but none are tied specifically to axonal degeneration.

In mammals at least, Apaf-1 is controlled by cytochrome c release from mitochondria, an event that is regulated by pro- and anti-apoptotic members of the Bcl-2 family. Axons in sensory and sympathetic neurons are protected from pruning in Bax null animals (Nikolaev et al., 2009) or by overexpression of the anti-Bcl-2 family member, Bcl-X_L (Vohra et al., 2010). While permeabilization of the outer mitochondrial membrane does more than simply release cytochrome c, the observations that Bcl-2 family members also play roles in axonal regeneration and guidance is consistent with a role for Apaf-1 and caspase-9. Similarly, axon pruning in mammals requires Bax (Nikolaev et al., 2009; Schoenmann et al., 2010; Simon et al., 2012), caspase-9 (Simon et al., 2012), and caspase-3 (Schoenmann et al., 2010), which suggests apoptosome involvement, even though a role for Apaf-1 was not tested directly in these studies. Axonal degeneration has also been indirectly linked to the mitochondrial pathway (Maor-Nof et al., 2016): the dual specificity phosphatase-16 (DUSP-16) is required for the preservation of sensory axons in vivo, and its loss accelerated axonal degeneration. DUSP-16 negatively regulates the pro-apoptotic BH3-only protein PUMA which causes axonal degeneration (Simon et al., 2016) and is required for the degeneration caused by loss of DUSP-16.

However, the Bcl-2 proteins also have non-apoptotic roles that are separate from their ability to regulate caspases (reviewed in (Glab et al., 2020)), so concluding there is a role for the mitochondrial pathway in a non-apoptotic process simply on the basis it is regulated by Bcl-2 proteins is inappropriate. For example, Bad^−/− mice are resistant to acute seizures induced by either kainic acid or pentylenetetrazole (Giménez-Cassina et al., 2012) and a DGN-specific Bad knockout is sufficient to reduce seizure-like activity. Bad functions to control neuronal excitability by regulating cellular metabolism: Bad increases glucose metabolism and cellular ATP in response to phosphorylation on Ser155 (mouse Bad) or Serine 118 (human Bad). Phosphorylation on Ser155 also blocks Bad’s apoptotic activity (Virdee et al., 2000). In contrast, the pro-apoptotic effect of Bad is increased by phosphorylation on serine 128 (Konishi et al., 2002). The change in intracellular ATP regulates an ATP sensitive potassium channel, thus influencing neuronal excitability (Ramón Martínez-François et al., 2018). The reduced ATP levels affect the likelihood of K_ATP channel opening in neurons, decreasing the excitability of the neurons and reducing the risk of seizures (Giménez-Cassina et al., 2012).

The BH3-only members of the Bcl-2 proteins, most typically described as inducers of apoptosis, can also play non-apoptotic roles in a range of cellular contexts (Esposti et al., 2001; Danial et al., 2003, 2008; Sinha and Levine, 2008; Giménez-Cassina et al., 2012) including in the nervous system. For example, the reactive (and non-apoptotic) astrocytes that contribute to disease pathology in a mouse model of Amyotrophic Lateral Sclerosis (ALS) have increased expression of the BH3-only proteins, Bid, Hrk and Bnip3L. These data suggest that the BH3-only proteins are contributing to the development of ALS (Duval et al., 2018). There are also roles reported in non-mammalian models: in C. elegans EGL-1 expression increases in the URX pair of sensory neurons after changes in oxygen levels without killing the cells (Cohn et al., 2019).

In the fly, the sensory organ precursor cell (SOP) gives rise to mechano-sensory organs in the peripheral nervous system by dividing asymmetrically to make the shaft, socket, and sheath cells, and a neuron that comprise each sensory organ. The formation of SOPs is negatively regulated by DARK-dependent non-apoptotic caspase activation (Kanuka et al., 2005). Mechanistically, DRONC regulates this development by cleaving Shaggy, a negative regulator of Wingless signalling, which is required for SOP formation (Kanuka et al., 2005). DRONC activity is controlled by IKK-related kinase (DmIKKε)-mediated phosphorylation of DIAP-1. DIAP-1 phosphorylation increases DIAP-1 ubiquitinylation, which leads to a decrease in DIAP-1 levels through proteasome-mediated degradation (Kuranaga et al., 2006).

Caspase-3 is activated in non-apoptotic cells during mammalian neuronal differentiation in a cultured neurosphere model, and preventing this activation disrupts differentiation (Fernando et al., 2005). There is also evidence for the mitochondrial pathway playing a role during neuronal cell differentiation an siRNA against caspase-9 reduced neuronal differentiation in a cell culture model (Pistritto et al., 2012). Changes in calcium levels during the differentiation of neuronal cells have also been implicated in caspase activation (Rebellato et al., 2019). The mechanism of caspase activation is not clear, but it involves the low-voltage-dependent T-type calcium (Ca²⁺) channel Cav3.2 which mediates increased levels of Ca²⁺ that are required to activate caspase-3-dependent neurogenesis during cortical development (Rebellato et al., 2019).

In Drosophila salivary cells undergoing remodelling there is a 30-fold increase in Reaper expression, compared to a 1000-fold increase in dying cells (Kang et al., 2017). The implication is that there is two orders of magnitude more caspase activity in dying cells, and that the decision between a non-apoptotic fate and an apoptotic fate is made at the level of Reaper expression. Low level caspase activity is involved in the emergence of neural precursor cells in Drosophila (Kanuka et al., 2005; Kuranaga et al., 2006). In mammalian cells control of XIAP and expression of Apaf-1 are reported to be important in determining the choice between death and differentiation (Wright et al., 2004; Potts et al., 2005; Johnson et al., 2007; Yin et al., 2007; Smith et al., 2009). At least one mechanism for controlling XIAP in non-apototic processes has been described (see above, (Moubarak et al., 2013; Coccia et al., 2020)), but in neurons it appears that the level of Apaf-1 limits the caspase activity (Wright et al., 2004).

How levels of Apaf-1 are controlled during differentiation is unresolved. In cancer cells Apaf-1 transcription is controlled by E2F-1 (Furukawa et al., 2002) and by TP53 (Rozenfeld-Granot et al., 2002), so the decrease in E2F-1 that accompanies exit from the cell cycle and that precedes differentiation may provide a straight forward explanation for a decrease in Apaf-1 that limits caspase activation during differentiation. Apaf-1 levels are also decreased in some cancers by microRNAs. Notably one miRNA that targets Apaf-1, miR-221, is increased during neuronal differentiation (Hamada et al., 2012) and during myogenesis (Liu et al., 2018), both systems where a decrease in Apaf-1 level appears to be important step during differentiation (Fernando et al., 2005; Dehkordi et al., 2020). Other microRNAs are reported to regulate Apaf-1 in cancers, but their roles in neurogenesis and myogenesis are not well described.

There is evidence that caspase localization is important in apoptosis. For example, in lung cancer cells the nuclear localization of active caspase-3 is linked to sensitivity to cancer chemotherapy (Joseph et al., 2001). During spermatid individualization caspases are activated by DARK to remodel the spermatid without apoptosis (Arama et al., 2003; Arama et al., 2007; Huh et al., 2004). This activity is localized through the interplay of an E3-ligase inhibitor, Soti and the IAP-like dBruce protein (Kaplan et al., 2010). In a different Drosophila model, Amcheslavsky et al. (2018) used cells lacking Drice (which are unable to undergo apoptosis) to show that Myo1D localizes DRONC to the plasma membrane where DRONC increases expression of NADPH-oxidase Duox, generating eROS that drives apoptosis induced cell proliferation. In DRONC-dependent salivary cell remodeling, Tango7 is required for the cortical localization of active DRONC (Kang et al., 2017). These data are very interesting as they show the activation of caspases in distinct sub-cellular domains being driven by different caspase-activation pathways. In Drosophila neurons caspase activity is restricted to dendrites during pruning, but is not seen in the soma or axon (Kuo et al., 2006; Williams et al., 2006). Nuclear localization of active caspase-3 is seen in non-apoptotic Bergmann Glial cells (Noyan-Ashraf et al., 2005). This activity appears to be important for the differentiation of these cells (Oomman et al., 2006).