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. Author manuscript; available in PMC: 2022 Jun 22.
Published in final edited form as: Neuroscientist. 2012 May 29;19(2):129–136. doi: 10.1177/1073858412447875

Caspase Inhibitors: Prospective Therapies for Stroke

Nsikan Akpan 1, Carol M Troy 1,2
PMCID: PMC9214550  NIHMSID: NIHMS1816428  PMID: 22645109

Abstract

In ischemic stroke, apoptosis persists for days to weeks after the onset of an ischemic event. Cysteine-ASPartic proteASEs (caspases) are key mediators of apoptosis and neurodegeneration in stroke.The impact of caspase activity is not restricted to neuronal death, as caspases can exacerbate inflammation and alter glial function.Thus, caspases are logical therapeutic targets for this disease, but they have never been clinically evaluated due to a paucity of ideal drug candidates. Recent developments in caspase inhibition and drug delivery offer novel neuroprotective strategies for stroke, which are deliberated in this review.

Keywords: apoptosis, caspase, cerebral ischemia, edema, neurodegeneration, stroke

Stroke Neurodegeneration

Trends in mortality and morbidity reveal that modern medicine has failed in the arena of stroke. Although 30-day and long-term survival statistics have moderately improved since the 1960s (Boysen and others 2009; Carandang and others 2006), these gains are largely because of innovations in emergency care. There is still no solution for the progressive neurological destruction that occurs shortly after a stroke. Consequently, stroke remains the 3rd and 10th leading cause of death and chronic disability in the United States, respectively (Centers for Disease Control and Prevention 2001; Centers for Disease Control and Prevention 2009; Dhamoon and others 2009).

Ischemic stroke, which accounts for 85% of all cases, is caused by the occlusion of a cerebral artery by either thrombosis or an embolism. Tissue plasminogen activator (tPA), which degrades blood clots, is the only US Food and Drug Administration–approved neuroprotective drug for ischemic stroke. Unfortunately, tPA use is restricted to less than 3% of the patient pool (American College of Emergency Physicians 2002) because of a short treatment window (<3 h) and medical contraindications, such as hypertension.

Following occlusion, several mechanisms contribute to the development of neuronal injury, including oxidative damage, ionic (Na2+, K+, Ca2+) homeostasis, and inflammation. This extensive list of molecular instigators has been thoroughly reviewed elsewhere (Lipton 1999; Niizuma and others 2010; Ribe and others 2008; Yuan 2009), but we provide a relevant overview for our discussion.

Stroke injury is morphologically divided into two territories (the core and penumbra), which exhibit two prominent modalities of cell death (necrosis and apoptosis). Damage in the core is essentially irreversible. In the core, blood perfusion is lowered below the threshold for viability, and neurons become electrically silent because of complete energy depletion. Necrosis is the prominent mode of cell death in this region, although there is some evidence for apoptosis occurring in the core (Akpan and others 2011; Yuan 2009).

The penumbra circumscribes the core and continues to receive blood from collateral arteries after an ischemic event, albeit at lower than physiological levels. Consequently, neurodegeneration in the penumbra is more gradual and dominated by apoptosis, which is an ATP-dependent process. Markers of apoptosis are found in the penumbra for days to weeks after stroke onset. Thus, a lengthy window of opportunity is available for targeting apoptotic cascades as a means to treat stroke.

Vascular damage accompanies cell death in the stroke infarct (del Zoppo 2006). Disruption of the neurovascular unit (i.e., the dynamic interactions between microvessels, neurons, astrocytes, and microglia) permits the infiltration of inflammatory cells and cytokines that are normally excluded from the central nervous system (CNS) by the blood-brain barrier. Additionally, stroke induces microvascular permeabilization, which causes the extracellular accumulation of fluid, known as tissue edema. Edema is the major cause of mortality within the first 24 h after stroke, as swelling of the brain leads to herniation and compression of neural centers in the brain stem that govern respiration (Strbian and others 2012).

Caspases: Focused Overview

Emblematic of their importance in stroke, Cysteine-ASPartic proteASEs (caspases, “casp”) contribute to the inflammatory, apoptotic, and vascular processes that dominate stroke neurodegeneration (Table 1).

Table 1.

Caspases Implicated in Stroke Pathogenesis

Caspase Paradigm (Species) Measure Reference
1 tMCAo (mouse) Genetic Knockout (infarct size, cerebral blood flow, brain edema) Schielke and others (1998)
1 pMCAo (mouse) Ac-YVAD-AFC (catalytic activity) Benchoua and others (2001)
1 pMCAo (rat) mRNA levels Harrison and others (2001)
2 Global ischemia (rat) Caspase cleavage (WB, IHC) Niizuma and others (2008)
3 tMCAo (mouse) Genetic Knockout (infarct volume, neuron density, TUNEL) Le and others (2002)
3 pMCAo (rat) Caspase cleavage (WB) Velier and others (1999)
3 pMCAo (mouse) Ac-DEVD-AFC (catalytic activity), caspase cleavage (WB, IHC) Benchoua and others (2001)
3 pMCAo (rat) mRNA levels Harrison and others (2001)
6 pMCAo (rat) mRNA levels Harrison and others (2001)
6 tMCAo (mouse, rat) Caspase cleavage (IHC), knockout mice (infarct volume, motor behavior) Akpan and others (2011)
7 pMCAo (rat) mRNA levels Harrison and others (2001)
8 pMCAo (rat) mRNA levels Harrison and others (2001)
pMCAo (mouse) Ac-IETD-AFC (catalytic activity), caspase cleavage (WB, IHC) Benchoua and others (2001)
pMCAo (rat) Caspase cleavage (WB) Velier and others (1999)
9 tMCAo (rat, mouse) Caspase cleavage (IHC), biochemical isolation of active caspase-9 Ferrer and Planas (2003), Akpan and others (2011)
tMCAo (canine) Release of caspase 9 from mitochondria Krajewski and others (1999)
Human stroke tissue IHC Duan and others (2010)
11 pMCAo (rat) mRNA levels Harrison and others (2001)
pMCAo (mouse) Genetic knockout (TUNEL-positive cells) Kang and others (2000)
Translational intervention Caspase Paradigm Inhibitor Delivery method Reference
Catalytic Pan-caspase tMCAo (mouse) Z-VAD-fmk Intracerebro-ventricular Endres and others (1998)
Expression 1 tMCAo (mouse) Dominant-negative caspase 1 Transgenic expression Friedlander and others (1997), Hara and others (1997)
Expression 1 tMCAo (rat)/global (gerbil) Minocycline Intraperitoneal Yrjanheikki and others (1999)
Catalytic 3 tMCAo (mouse) Z-DEVD-fmk Intracerebro-ventricular Endres and others (1998)
Expression 3 Endothelin-1 (mouse, rat) Carbon nanotubes w/caspase-3 RNAi Intracerebral, stereotaxis Al-Jamal and others (2011)
Catalytic 9 tMCAo (mouse, rat) Penetratin1-BIR3 Intranasal Akpan and others (2011)
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Short peptide substrates: Ac-YVAD-AFC, Ac-DEVD-AFC, Ac-IETD-AFC. Short peptide enzymatic inhibitors: Z-VAD-fmk, Z-DEVD-fmk.

Although there are 13 mammalian caspase family members, this update will focus on caspases that have been implicated in ischemic stroke (Fig. 1; casp-1, casp-2, casp-3, casp-6, casp-7, casp-8, casp-9, and casp-11). For a comprehensive review of caspases and neurodegeneration, consult Ribe and others (2008).

Figure 1.

Figure 1.

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Mammalian caspases implicated in stroke. Caspases are sorted by their roles in inflammation and apoptosis. Structural details are stressed.Yellow lines indicate cleavage sites

Caspases are classified on the basis of

  1. Structure

  2. Mechanism of activation

  3. Cellular function

  4. Apoptotic activation pathways

Structure

Caspases possess a prodomain (Fig. 1) that is either short (casp-3, casp-6, and casp-7) or long (casp-1, casp-2, casp-8, casp-9, and casp-11). The size and composition of the prodomain determines whether a caspase requires cleavage for activation (see “Mechanism of Activation”).

Additionally, the catalytic site of each caspase has an optimal substrate motif (5 amino acid; P4-P3-P2-P1-P1′) for cleavage specificity (O’Brien 2007; Pop and Salvesen 2009). P1-P1′ is the scissile bond target of catalytic Cys, where P1 is Asp and P1′ is a small, uncharged residue (Gly, Ser, Ala). P4-P3-P2 residues interact with the corresponding amino acids within the catalytic groove. Additionally, tertiary structure (i.e., loops or folds) influences the proteolysis of natural substrates.

Mechanism of Activation

Caspases with long prodomains are present in the cell as zymogen monomers (Fig. 2). Activation occurs on dimerization because structural changes in the longer intersubunit linker permit the exposure of the catalytic active site. Long prodomains also contain CARD or DED sequences (Fig. 1). These subdomains interact with complementary regions within an adaptor platform molecule (discussed below), which mobilizes caspase dimerization/activation. Following proximity-induced dimerization, initiator caspases may autocleave, but cleavage alone does not result in activation (Pop and Salvesen 2009).

Figure 2.

Figure 2.

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Mechanisms of caspase activation. Activation models for initiator and effector caspases are shown. Blue indicates inactive caspase, whereas orange denotes activated caspase. Some of the activated caspases (i.e., casp-3, casp-7, casp-9) are subject to regulation by inhibitor of apoptosis proteins (IAPs)

In contrast, short prodomain caspases exist as inactive zymogen dimers (Fig. 2). Proteolytic cleavage within an intersubunit linker is an absolute requirement for activation of this group.

Cellular Function

Caspases are further generalized into three functional groups: inflammatory, apoptotic initiator, or apoptotic effector.

Caspase-1 contributes to inflammation in stroke by processing the proinflammatory cytokine interleukin-1β (IL-1β) into its active form (Friedlander 2000). During ischemic stroke in rodents, IL-1β levels rapidly increase in the CNS. Transgenic expression of a caspase-1 dominant negative mutant prevents IL-1β maturation and reduces ischemic brain injury (Friedlander and others 1997).

Recently, genetic knockout of murine caspase-11 was shown to be protective against lipopolysaccharide exposure (Green 2011), which suggests a role in the innate immune response. In a separate study, caspase-11 knockout mice were protected from stroke (Kang and others 2000). Caspase-11 is the probable ortholog of human caspase-4 and caspase-5 (59% and 54% homology, respectively); however, the latter enzymes have not been directly studied in stroke pathogenesis.

Coined “Death by a Thousand Cuts” (Stroh and Schulze-Osthoff 1998), caspase-mediated apoptosis involves the selective cleavage of a tremendous volume of substrates to promote cellular demolition (Luthi and Martin 2007). Apoptotic caspases are classified as either “initiator” or “effector,” which is based on their hierarchal nature of activation. Initiators cleave a limited a number of substrates, including effector caspases. In turn, effector caspases are charged with propagating cell death by targeting a wider variety of protein substrates (e.g., cytoskeleton, kinases, organelles, etc.).

Notice that caspase structure (short vs. long prodomain) dictates apoptotic function (effector vs. initiator). Caspase-2 is the one exception. It possesses a long prodomain but can act as an initiator or effector, depending on the death signal. To add to its uniqueness, caspase-2 is also a zymogen dimer (Schweizer and others 2003).

Apoptotic Activation Pathways

Apoptotic caspases are organized into two canonical signaling cascades. The “extrinsic” pathway is dependent on the formation of the death-inducing signaling complex (DISC). On ligand binding, a death receptor recruits an adaptor protein, such as the Fas-Associated protein with Death Domain, to the cell membrane. This adaptor protein sequesters two caspase-8 monomers into close proximity to induce structural changes that free the catalytic site. This trimeric complex (death receptor, adaptor protein, and caspase-8) defines the DISC and proceeds to cleave effector caspases. Autocleavage of caspase-8 occurs, which enhances its activity.

The intrinsic pathway is triggered by a variety of cellular stressors (e.g., genotoxicity, reactive oxygen species, endoplasmic reticulum stress). The first critical event is the permeabilization of the outer mitochondrial membrane (OMM) (Westphal and others 2011), which is triggered by the Bcl-2 family members Bax and Bak. In healthy cells, Bak resides in the OMM, while Bax is cytosolic. Death signals cause Bax to translocate to the OMM and induce conformational changes in Bax and Bak that lead to their homo-oligomerization. Although the precise crystal structure of these oligomers is unknown, it is hypothesized that Bax and Bak form pores in the OMM that release cytochrome c, the principal killing factor in apoptosis, into the cytosol. In an ATP-dependent fashion, cytochrome c forms an activation platform with APAF-1, which recruits caspase-9 monomers, which subsequently dimerize and activate. This caspase 9–based complex, termed the apoptosome, cleaves and activates effector caspases. Although caspase-9 also undergoes autocleavage, this action does not appreciably increase caspase-9 activity.

Endogenous inhibitors can also modulate the intrinsic mitochondrial pathway. Inhibitor of Apoptosis Proteins (IAPs), particularly XIAP, inhibits active casp-3, casp-7, and casp-9 (discussed below). In turn, Diablo/SMAC or HtrA2/Omi, which are released from permeabilized mitochondria, are IAP antagonists that block the IAP inhibition of caspases. Thus, there are multiple levels of natural regulation for the intrinsic pathway.

Translational Strategies for Caspase Inhibition

There are three major strategies for inhibiting caspases in stroke.

  1. Death receptor blockade

  2. Genetic manipulation

  3. Catalytic inhibitors

Death Receptor Blockade

Inhibiting death receptors can disrupt the execution of the extrinsic pathway. When administered 30 min after stroke, antibodies against tumor necrosis factor–receptor-1 (TNF-R1) and CD95, members of the TNF receptor superfamily, can reduce neural injury (Martin-Villalba and others 2001).

Genetic Manipulation

Ischemic stroke induces caspase expression (mRNA and/or protein) within the first 24 h for all the caspases mentioned above (Ribe and others 2008) (Table 1). Furthermore, genetic knockout of casp-1, casp-3, casp-6, or casp-11 is neuroprotective against stroke (Akpan and others 2011; Ribe and others 2008).

Obstacles for an RNAi-based gene therapy for caspases include sufficient distribution of the therapeutic siRNA/miRNA to susceptible CNS regions and efficient delivery into neuronal cells. A proof-of-concept study recently overcame these issues with a nonviral-based RNAi system in a rodent ischemia model (Al-Jamal and others 2011). Immediately before the induction of stroke, carbon nanotubes loaded with caspase-3 siRNA were stereotactically delivered into motor cortices. Neurons in the treated regions were protected against stroke, and neurological function was preserved.

Certain drugs can also mollify caspase expression. Minocycline inhibits casp-1 expression during stroke (Yrjanheikki and others 1999) and has the additional benefit of inducing the expression of anti-apoptotic Bcl-2 (Wang and others 2004). However, drug-based knockdown often lacks complete specificity, with compounds such as minocycline modifying a variety of gene targets.

Gene therapy is an effective prophylactic strategy, but the true litmus test for any stroke treatment is potency after ischemia has begun. In this regard, gene therapy might have limited utility even in the best scenarios. Caspases are expressed at modest levels in the CNS, which means there is an available pool of death molecules at stroke onset. Therefore, caspase activation could be abundant before a gene therapy could significantly curtail expression.

For further review on the potential of gene therapies for stroke, consult Chu and others (2007).

Catalytic Inhibitors

For nearly 20 years, short peptide inhibitors have been the principal tools for studying and manipulating caspase activity. Based on the optimal substrate motif (P4, P3, P2, P1) of an individual caspase, these synthetic inhibitors can be modified by adding chemical moieties to determine their reversibility and membrane permeability. The pan-caspase inhibitor zVAD (benzyloxycarbonyl-Val-Ala-Asp) and putative caspase-3 inhibitor DEVD confer neuroprotection in rodent models of stroke (Endres and others 1998). Although they have never been tested in clinical trials for stroke, trials are underway for the use of these peptide-based inhibitors for liver injury and psoriasis (O’Brien 2007).

However, multiple studies have demonstrated that short peptide inhibitors are highly promiscuous (Benkova and others 2009; McStay and others 2008). Given that certain caspases have non–death roles in synaptic plasticity and microglial activation (Huesmann and Clayton 2006; Venero and others 2011), this lack of specificity reduces the overall usefulness of short peptide-based inhibitors in stroke.

Natural protein biologics that inhibit caspases offer higher target specificity and an alternative to short peptide inhibitors. Given their larger size, most biologics require assistance to cross the plasma membrane. Trojan peptides (e.g., Tat or Penetratin1) can be used toward this goal.

Recently, we applied this delivery strategy to a biologic derived from a metazoan caspase inhibitor (Akpan and others 2011). X-linked inhibitor of apoptosis protein (XIAP) is composed of three baculoviral IAP repeat (BIR) domains. The third BIR domain from XIAP (XBIR3) is a potent inhibitor of caspase-9, the initiator of the intrinsic apoptotic pathway.

Penetratin1 (Pen1) was used as a Trojan peptide and linked to XBIR3 via a disulfide bond. On cellular entry, this transient linkage is reduced by the cytoplasmic pH. When given intranasally, as a prophylactic or therapeutic, Pen1-XBIR3 prevented activation of the intrinsic pathway (i.e., subsequent caspase-6 activation) and provided substantial neuroprotection against stroke. Additionally, it was revealed that caspase-9 inhibition reduces cerebral edema. As previously stated, edema is a major cause of mortality within the first 24 h after stroke.

Trojan peptides could usher in a new era in resolving caspase mechanisms and treating cell death. For Pen1, the upper limit for the molecular size of cargo is ˜100 kDa. Other natural inhibitors require evaluation with this method. Alternatively, endogenous substrates or catalytic dominant negatives can be attached to Trojan peptides and used as competitive inhibitors.

CNS Delivery Methods

The blood-brain barrier (BBB) evolved to protect the CNS from molecular insults, but it also restricts the passage of many neuroprotective agents administered into the bloodstream.

Routes for bypassing the BBB do exist. Intranasal delivery is proving efficacious for accessing the CNS and treating neurodegenerative disease (Dhuria and others 2010). For example, cognition is significantly improved after intranasal administration of insulin in early clinical trials for Alzheimer’s disease and mild cognitive impairment (Craft and others 2012). This method provides 100-fold higher CNS concentrations relative to intravenous administration (Thorne and others 2004). Alternatively, more invasive methods that use direct injection into the brain (e.g., intracerebral and intraventricular) are clinically viable, although less favorable from a patient’s viewpoint.

Conclusion

The world is careening toward a major crisis, as millions of “baby boomers” reach the age where nearly three-quarters of all strokes occur (>65 years old). Currently, there is no exemplary therapy for this impending public health disaster.

Neuroprotection is one answer for stroke, but this idea has encountered repudiation in recent years. It is frequently quoted that more than 1000 neuroprotective treatments have been tested and failed in clinical trials (Ginsberg 2008). These nihilistic sentiments are unjust, as these setbacks speak to the difficulties in selecting a meaningful drug target for this complex medical disorder.

Given the myriad roles for caspases in stroke neurodegeneration, it is surprising that direct caspase inhibition has never been explored in clinical trials. Expression- or activity-based strategies, which can be used in isolation or combination, have proven effective in model systems and preclinical trials. These innovations in caspase inhibition highlight new potentials for stroke therapy.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This article was funded by NIH NINDS grant NS033689.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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