Caspases—Guilty of Murder in First, and Now Second Degrees

Murderers often incite others to do the same. Molecular killers may be no exception. Executioner caspases don’t just kill cells directly, according to a study posted online March 9 in Nature. These proteases incite microglia to attack other cells, including nearby neurons. Furthermore, activated apoptotic caspases are found in microglia of Alzheimer’s and Parkinson’s disease patients, leading the authors to suggest the data “should revitalize interest in caspase inhibitors as potential therapeutic agents in disorders of the central nervous system.” The research was led by Bertrand Joseph of Karolinska Institute in Stockholm, Sweden, and Jose Venero of the Universidad of Sevilla, Spain.

By and large, activation of caspases—which triggers apoptosis, or programmed cell death—is considered a cellular death sentence. That’s why the scientists were shocked to find that stimulating BV2 microglial cells with lipopolysaccharide (LPS) induced caspase-3 cleavage without the expected cellular demise. “We saw activated caspases, but the microglia were not dying,” Joseph told ARF. “If [the caspases] are not killing the cells, what are they doing?”

To find out, first author Miguel Burguillos and colleagues transfected BV2 microglia cells with small interfering RNAs to knock down endogenous apoptotic caspase-3 or -7. The scientists then discovered that LPS treatment was not as effective at spurring the phagocytes’ usual barrage of pro-inflammatory molecules. Compared to BV2 cells treated with control siRNAs, microglia with caspase-3 or -7 knockdown produced just a trickle of the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and made less inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κ B kinase β subunit (IKK-β), and reactive oxygen species. Another key readout of LPS-induced microglial activation is nuclear translocation of nuclear factor κB (NF-κB), and the researchers detected less of this in microglia with caspase knockdown.

Fleshing out the molecular players a bit further, the team found that the LPS-induced increase in caspase-3 activity acts through protein kinase C-Δ and relies on caspase-8 (but not on caspase-1), and that Toll-like receptor signaling is needed to activate caspase-8. Caspase-8 is thought to active caspase-3/7 as an upstream regulator of the apoptotic pathway mediated by Toll-like receptors. Caspase-1 also plays a key role in LPS-mediated inflammation.

Moving toward in vivo settings, the scientists injected LPS into rat substantia nigra and found that it caused microglia to not only release the usual torrent of cytokines and pro-inflammatory molecules, but also induce caspase-8, leading to a ramp-up of caspase-3 activity. When the researchers used pharmacological agents to block caspase-3/7 activity in the microglia, the LPS effects were much weaker. Similarly, in a PD model where injection of the chemical MPTP activates microglia and kills nearby dopamine neurons, the scientists were able to alleviate MPTP-mediated induction of reactive microglia and neurotoxicity using caspase-8 inhibitors. And, in immunohistochemical analyses of postmortem human brain, strong staining of cleaved (active) forms of caspase-3 and -8 appeared in microglia of PD ventral mesencephalon and AD frontal cortex, but not in age-matched control brains.

The study has “two major novelties,” said Robert Friedlander, who recently moved from Brigham and Women’s Hospital, Boston, to the University of Pittsburgh, Pennsylvania. “First, it demonstrates that executioner caspases have a non-cell death function in microglia. Second, it provides further support that caspase inhibitors would be a fruitful avenue for treatment of neurodegenerative disease.” Though human studies of caspase blockade have been limited, Friedlander and others have shown that caspase inhibitors promote survival and delay disease progression in mouse models of spinal cord injury (Li et al., 2000), brain trauma (Fink et al., 1999), and stroke (Hara et al., 1997).

In AD, caspase inhibitors have not yet been tried in patients, but curbing inflammation using non-steroidal anti-inflammatory drugs (NSAIDs) did not appear beneficial in a randomized controlled trial (ADAPT), noted Terrence Town, Cedars-Sinai Medical Center, Los Angeles, in an e-mail to ARF (see ARF related news story). In fact, a recent study (Sonnen, et al., 2010) “showed greater neuritic plaque load in brains of NSAID users that converted to AD,” Town wrote (see full comment below). “It is therefore unclear that the principle of inhibiting inflammation to provide AD prophylaxis is a valid one.” Ben Barres of Stanford University, Palo Alto, California, agreed, commenting that the current study does not address several key issues—namely, the purpose of microglial activation, and whether it is good or bad, for neurodegenerative disease processes. Indeed, recent studies have described various forms of microglial activation, some of which are deleterious (see Fan et al., 2007), and others, beneficial (see El Khoury et al., 2007). “So the question becomes, Which form does caspase-3/7/8 endorse?” said Town.—Esther Landhuis

Comments

  1. On the basic biology front, Burguillos et al. present a thought-provoking idea that the caspase-3/7/8 pathway regulates innate immune activation in microglia. Until now, the dogma has been that caspases are critical cogs in the apoptosis wheel, where they have been thought to function almost exclusively. In the immunology world, our thinking began to shift with the discovery of the inflammasome, an intracellular pathogen recognition system that requires caspase-1 activation to cleave pro-interleukin 1β to the active cytokine. This paper shows that caspases 3, 7, and 8 are enablers of Toll-like receptor activation in microglia (and likely other innate immune cells, e.g., macrophages and dendritic cells). This is certainly interesting and will no doubt lead to a flurry of activity in search of the molecular mechanism for these findings.

    The authors speculate that the caspase-3/7/8 pathway may represent an attractive candidate for therapeutic intervention in Alzheimer’s and Parkinson’s diseases, given that this pathway is upregulated in patients’ brains. Their idea is to block this cascade and thereby shut down neuroinflammation as a treatment for these diseases. In the context of AD, though, it deserves mentioning that generally inhibiting inflammation via NSAIDs did not produce a positive signal for primary prevention in a randomized controlled trial (ADAPT). In fact, recent evidence from Tom Montine and John Breitner actually showed greater neuritic plaque load in brains of NSAID users that converted to AD (Sonnen et al., 2010). It is therefore unclear that the principle of inhibiting inflammation to provide AD prophylaxis is a valid one. We, and others, have revised our thinking to a more contemporary view: There are multiple forms of microglial “activation,” some of which are deleterious, and others, beneficial. So the question becomes, Which form does caspase-3/7/8 endorse?

    References:

    Sonnen JA, Larson EB, Walker RL, Haneuse S, Crane PK, Gray SL, Breitner JC, Montine TJ. Nonsteroidal anti-inflammatory drugs are associated with increased neuritic plaques. Neurology. 2010 Sep 28;75(13):1203-10. PubMed.

    View all comments by Terrence Town

  2. The experiments in the paper by Burguillos et al. focus mainly on in vitro work with BV-2 cells, myc-raf immortalized mouse microglia. Stimulating these cells with LPS, the authors showed that caspases, enzymes involved in apoptosis and inflammation, signal via PKC-Δ to mediate standard inflammatory endpoints such as NFκB activation. This finding is novel. PD or AD relevance would need to come from clinical trials targeting one of these enzymes. The blocking of apoptosis-mediating enzymes, however, would be fraught with concerns about induction of tumors or autoimmunity. The in vivo studies here involved intrastriatal injection of LPS to confirm that in vitro biochemistry could be recapitulated in vivo.

    View all comments by Richard Ransohoff

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References

News Citations

  1. NSAIDs in AD: Epi and Trial Data at Odds—Again

Paper Citations

  1. Li M, Ona VO, Chen M, Kaul M, Tenneti L, Zhang X, Stieg PE, Lipton SA, Friedlander RM. Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury. Neuroscience. 2000;99(2):333-42. PubMed.
  2. Fink KB, Andrews LJ, Butler WE, Ona VO, Li M, Bogdanov M, Endres M, Khan SQ, Namura S, Stieg PE, Beal MF, Moskowitz MA, Yuan J, Friedlander RM. Reduction of post-traumatic brain injury and free radical production by inhibition of the caspase-1 cascade. Neuroscience. 1999;94(4):1213-8. PubMed.
  3. Hara H, Friedlander RM, Gagliardini V, Ayata C, Fink K, Huang Z, Shimizu-Sasamata M, Yuan J, Moskowitz MA. Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):2007-12. PubMed.
  4. Sonnen JA, Larson EB, Walker RL, Haneuse S, Crane PK, Gray SL, Breitner JC, Montine TJ. Nonsteroidal anti-inflammatory drugs are associated with increased neuritic plaques. Neurology. 2010 Sep 28;75(13):1203-10. PubMed.
  5. Fan R, Xu F, Previti ML, Davis J, Grande AM, Robinson JK, Van Nostrand WE. Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid. J Neurosci. 2007 Mar 21;27(12):3057-63. PubMed.
  6. El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med. 2007 Apr;13(4):432-8. PubMed.

Further Reading

Primary Papers

  1. Burguillos MA, Deierborg T, Kavanagh E, Persson A, Hajji N, Garcia-Quintanilla A, Cano J, Brundin P, Englund E, Venero JL, Joseph B. Caspase signalling controls microglia activation and neurotoxicity. Nature. 2011 Apr 21;472(7343):319-24. PubMed.

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