Cutting Both Ways—Besides Death, Caspases Mediate Neurons' LTD

Better known as executioners, caspases have life-giving functions, too. A paper published in the May 28 Cell describes a first-ever role for these proteases in synaptic physiology. Initiated by senior investigator Morgan Sheng and co-lead author Zheng Li while both were at MIT’s Picower Institute for Learning and Memory, the study suggests that hippocampal neurons require caspase-3 activity for long-term depression and AMPA receptor internalization. Though their relevance to neurodegenerative disease is unclear, the data raise the possibility that activation of caspases could upset the normal physiology of neurons in subtler ways before spelling their demise.

When she began this work five to six years ago as a postdoc in Sheng’s lab at MIT, there was no solid evidence for a link between caspases and synaptic plasticity, said Li, who has moved to the National Institute of Mental Health in Bethesda, Maryland. And while caspases had long been implicated in Alzheimer disease and other neurodegenerative disorders, their main role, presumably, was mediating cell death. Yet researchers had detected caspase activation at early stages of AD (Gamblin et al., 2003; Rissman et al., 2004), even at synapses (Yang et al., 1998; Mattson et al., 1998; Mattson et al., 1998), in the absence of apoptosis. These observations led Sheng, who is now at Genentech, South San Francisco, and colleagues to wonder whether caspases may in fact “have nothing to do with cell death in neurons,” Li told ARF. “That’s where I started.”

Several research groups have since published non-apoptotic roles for caspases in the nervous system—in dendrite pruning of Drosophila sensory neurons (Kuo et al., 2006; Williams et al., 2006) and in guidance of growth cones (Campbell and Holt, 2003). Now, Sheng, Li, co-first author Jihoon Jo, University of Bristol, UK, and colleagues provide evidence that caspases are critical for regulating synaptic strength during long-term depression (LTD), a temporary decline in a neuron’s responsiveness to neurotransmitter signal, which is important for development and learning.

The researchers did a series of electrophysiology experiments to examine how caspase activation affects neuronal function. In one analysis, they prepared hippocampal slices from three- to four-week-old rats, treated them with various caspase inhibitors, and measured excitatory post-synaptic potentials in the CA1 region. These studies showed that two inhibitors—DEVD-FMK (selective against caspases 3, 7, and 8) and LEHD-FMK (caspase-9-prefering with weaker activity against caspases 6, 8, and 2)—blocked LTD induced by low-frequency stimulation, but had no effect on long-term potentiation (LTP) or basal synaptic strength. Inhibitors selective for caspases 1 and 8 did not block LTD, suggesting that caspases 3/7 and 9 were likely responsible for the synaptic plasticity effects.

The findings with peptide inhibitors found support in a second set of experiments using molecular approaches to block caspases with greater specificity. Here, the researchers overexpressed several caspase-inhibiting and anti-apoptotic proteins in CA1 neurons of hippocampal slice cultures made from six- to eight-day-old rats. In whole-cell patch-clamp recordings, they found decreased LTD in cells transfected with proteins that block caspase-3/7 (XIAP-Bir3) or caspase-3 and -9 (Bcl-xL). These proteins had no effect on LTP, though, indicating their overexpression did not harm synaptic function and plasticity in a non-specific manner.

The researchers confirmed the pharmacological and overexpression findings by measuring LTD in CA1 neurons of hippocampal slices from two- to four-week-old caspase-3 knockout mice, which reach adulthood with minimal brain pathology (Leonard et al., 2002). Low-frequency stimulation triggered LTD in wild-type but not caspase-3-deficient cells. Both had normal LTP induction and basal synaptic transmission, again suggesting the caspase-mediated effect on neuronal physiology is quite specific.

In line with its impact on LTD, blockage of caspase activity prevented AMPA receptor endocytosis, the molecular phenomenon that makes neurons less activatable. Sheng and colleagues demonstrated this in immunohistochemical experiments on hippocampal neurons either treated with caspase inhibitor peptides or transfected with caspase-inhibiting factors, and in caspase-3-deficient cells. Furthermore, the researchers found that the same NMDA treatment that induces LTD or AMPA receptor internalization in the neurons could activate caspase-3 and caspase-9, its upstream activator in the mitochondrial apoptotic pathway. The activation was modest, transient, not associated with eventual cell death, and able to be blocked with an NMDA receptor antagonist or calcium chelator. Moreover, the scientists showed that the pro-survival kinase Akt3, a substrate of caspase-3 during apoptosis, is also key for caspase-3-mediated LTD. All told, the data suggest that “the upstream signals activating the mitochondrial caspases in cell death and in LTD are very similar. They depend on calcium and on protein phosphatases,” Li said. “Yet the cellular consequences of activating caspases in these contexts are very different. We are curious as to why you have such different outcomes from activation of the same molecule.”

Steve Barger of the University of Arkansas for Medical Sciences, Little Rock, was similarly intrigued. “Here’s something that seems, on the face of it, to be degradative and harmful, and yet it’s an important component of physiology,” he said of the newly proposed function of caspases in LTD. One could imagine caspases playing a role in removing spines or whole branches of dendrites, which may be important for certain kinds of learning and memory but “wouldn’t lead to death of the entire cell,” Barger said.

Before this presumed killing of spines, it is conceivable that activated caspases have subtler physiological effects that might eventually contribute to neurodegenerative disease. Though the current study does not directly address this, Li raised the possibility that in AD, caspases may be active in early, pre-symptomatic stages, but “instead of leading to cell death, they may be involved in decreasing synaptic transmission.” If so, early intervention with caspase inhibitors “may help slow the deterioration of neuronal function,” she suggested. (Rohn, 2010 describes potential AD therapeutic approaches targeting caspases.)

A recent report suggests that soluble Aβ facilitates LTD (Li et al., 2009 and ARF related news story), consistent with the authors’ speculation that caspase-3-mediated LTD may contribute to synapse loss in AD. And in multiphoton imaging studies, caspase activity was dissociated from cell death and showed up prior to tangle formation in the brains of pathogenic tau-overexpressing transgenic mice, supporting the idea of caspase activation as an early, non-apoptotic event in AD (de Calignon et al., 2010 and ARF related news story).

Elliott Mufson, Rush University Medical Center, Chicago, speculated that, given all the molecular dysfunction in AD (e.g., hyperphosphorylation of tau, overexpression of neurotrophins), there could be a “shift in the equilibrium of how caspases are held in check as you get older, that might even get more out of whack when you develop AD.”

Andrea LeBlanc thinks the real test for whether caspase-3 is indispensable for LTD would be to check behavior and cognition in caspase-3 knockout mice. However, these animals have vision and hearing problems (Takahashi et al., 2001) that prevent their use in behavioral studies, Li said.—Esther Landhuis

Comments

  1. The recent study by Li et al. is an intriguing one that provides a mechanism for long-term depression (LTD) in neurons. LTD is a phenomenon by which neurons lose AMPA receptors from the post-synaptic membrane, leading to a diminished ability to activate this excitatory pathway. Thus, LTD is one important mechanism underlying plasticity, a vital process for learning and development. Although LTD has been well characterized, the exact mechanism of this process has remained elusive. In this study, the authors use both pharmacological and molecular approaches to indicate the activation of caspases (namely, caspase-3 and -9) is critical for the internalization of AMPA receptors and, therefore, the process of LTD. Moreover, the authors demonstrate that Akt1 is a key player in this process. Akt1 has a role in preventing LTD by inhibiting the activity of GSK3β, whose function is critical for LTD. Li et al. now show that caspase activation leads to Akt1 cleavage and inactivation, thus providing a mechanism by which caspase-3 initiates LTD.

    What makes this study even more provocative is that activation of caspases in this manner mediates a physiological function, and does not lead to overt cell death by apoptosis. In the developing brain, anywhere from 20-80 percent of neurons die through apoptosis as the CNS is sculpted to its final appearance and function (Oppenheim, 1991). For years it was believed that in the adult brain, this pathway remains inactive (for good reason) except during disease states whereby activation of caspases could contribute to neurodegeneration (i.e., Alzheimer disease; see Rohn, 2010). However, Li et al. now provide strong experimental evidence that caspases are playing a non-apoptotic role in neurons and in fact are critical for synaptic plasticity. Previous studies have suggested additional roles in neurons beyond executing apoptosis. For example, caspase activation may be required for neuronal differentiation, dispersal, and correct morphology (Rohn et al., 2004; Yan et al., 2001). Although caspases are indispensable for the proper execution of apoptosis, the study by Li et al. clearly shows that the actions of caspases can “cut” both ways—either physiologically in carrying out LTD, or pathologically when activated during certain neurodegenerative diseases.

    References:

    Oppenheim RW. Cell death during development of the nervous system. Annu Rev Neurosci. 1991;14:453-501. PubMed.

    Rohn TT. The role of caspases in Alzheimer's disease; potential novel therapeutic opportunities. Apoptosis. 2010 Nov;15(11):1403-9. PubMed.

    Rohn TT, Cusack SM, Kessinger SR, Oxford JT. Caspase activation independent of cell death is required for proper cell dispersal and correct morphology in PC12 cells. Exp Cell Res. 2004 Apr 15;295(1):215-25. PubMed.

    Yan XX, Najbauer J, Woo CC, Dashtipour K, Ribak CE, Leon M. Expression of active caspase-3 in mitotic and postmitotic cells of the rat forebrain. J Comp Neurol. 2001 Apr 23;433(1):4-22. PubMed.

    View all comments by Troy Rohn

  2. The neurotrophin receptor p75 has been reported to facilitate NMDAR-dependent long-term depression of Schaeffer collateral-CA1 synapses (Woo et al., 2005) and (in p75-mediated apoptosis of various cell types including neurons) to induce activation of caspase-3. The lack of LTD of Schaeffer collateral-CA1 synapses in caspase-3 knockout mice observed by Li et al. should correlate with the lack of LTD in p75 knockout mice described by Woo et al., indicating that caspase-3 is essential for p75-induced LTD and that LTD of Schaeffer collateral-CA1 synapses is under the control of p75.

    References:

    Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B. Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nat Neurosci. 2005 Aug;8(8):1069-77. PubMed.

    View all comments by Rudolf Bloechl

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References

News Citations

  1. Neuronal Glutamate Fuels Aβ-induced LTD
  2. Who’s on First? Multiphoton Imaging Suggests Caspases, Not Tangles

Paper Citations

  1. Gamblin TC, Chen F, Zambrano A, Abraha A, Lagalwar S, Guillozet AL, Lu M, Fu Y, Garcia-Sierra F, Lapointe N, Miller R, Berry RW, Binder LI, Cryns VL. Caspase cleavage of tau: linking amyloid and neurofibrillary tangles in Alzheimer's disease. Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):10032-7. PubMed.
  2. Rissman RA, Poon WW, Blurton-Jones M, Oddo S, Torp R, Vitek MP, Laferla FM, Rohn TT, Cotman CW. Caspase-cleavage of tau is an early event in Alzheimer disease tangle pathology. J Clin Invest. 2004 Jul;114(1):121-30. PubMed.
  3. Yang F, Sun X, Beech W, Teter B, Wu S, Sigel J, Vinters HV, Frautschy SA, Cole GM. Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease. Am J Pathol. 1998 Feb;152(2):379-89. PubMed.
  4. Mattson MP, Partin J, Begley JG. Amyloid beta-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 1998 Oct 5;807(1-2):167-76. PubMed.
  5. Mattson MP, Keller JN, Begley JG. Evidence for synaptic apoptosis. Exp Neurol. 1998 Sep;153(1):35-48. PubMed.
  6. Kuo CT, Zhu S, Younger S, Jan LY, Jan YN. Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning. Neuron. 2006 Aug 3;51(3):283-90. PubMed.
  7. Williams DW, Kondo S, Krzyzanowska A, Hiromi Y, Truman JW. Local caspase activity directs engulfment of dendrites during pruning. Nat Neurosci. 2006 Oct;9(10):1234-6. PubMed.
  8. Campbell DS, Holt CE. Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones. Neuron. 2003 Mar 27;37(6):939-52. PubMed.
  9. Leonard JR, Klocke BJ, D'Sa C, Flavell RA, Roth KA. Strain-dependent neurodevelopmental abnormalities in caspase-3-deficient mice. J Neuropathol Exp Neurol. 2002 Aug;61(8):673-7. PubMed.
  10. Rohn TT. The role of caspases in Alzheimer's disease; potential novel therapeutic opportunities. Apoptosis. 2010 Nov;15(11):1403-9. PubMed.
  11. Li S, Hong S, Shepardson NE, Walsh DM, Shankar GM, Selkoe D. Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake. Neuron. 2009 Jun 25;62(6):788-801. PubMed.
  12. de Calignon A, Fox LM, Pitstick R, Carlson GA, Bacskai BJ, Spires-Jones TL, Hyman BT. Caspase activation precedes and leads to tangles. Nature. 2010 Apr 22;464(7292):1201-4. PubMed.
  13. Takahashi K, Kamiya K, Urase K, Suga M, Takizawa T, Mori H, Yoshikawa Y, Ichimura K, Kuida K, Momoi T. Caspase-3-deficiency induces hyperplasia of supporting cells and degeneration of sensory cells resulting in the hearing loss. Brain Res. 2001 Mar 16;894(2):359-67. PubMed.

Further Reading

Papers

  1. Rohn TT. The role of caspases in Alzheimer's disease; potential novel therapeutic opportunities. Apoptosis. 2010 Nov;15(11):1403-9. PubMed.
  2. de Calignon A, Fox LM, Pitstick R, Carlson GA, Bacskai BJ, Spires-Jones TL, Hyman BT. Caspase activation precedes and leads to tangles. Nature. 2010 Apr 22;464(7292):1201-4. PubMed.
  3. Spires-Jones TL, de Calignon A, Matsui T, Zehr C, Pitstick R, Wu HY, Osetek JD, Jones PB, Bacskai BJ, Feany MB, Carlson GA, Ashe KH, Lewis J, Hyman BT. In vivo imaging reveals dissociation between caspase activation and acute neuronal death in tangle-bearing neurons. J Neurosci. 2008 Jan 23;28(4):862-7. PubMed.
  4. Li S, Hong S, Shepardson NE, Walsh DM, Shankar GM, Selkoe D. Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake. Neuron. 2009 Jun 25;62(6):788-801. PubMed.

Primary Papers

  1. Li Z, Jo J, Jia JM, Lo SC, Whitcomb DJ, Jiao S, Cho K, Sheng M. Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization. Cell. 2010 May 28;141(5):859-71. PubMed.

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