To Survive or to Die: How Neurons Deal with it
Unlike the majority of cells in the organism, neurons have only two options during their entire existence, to survive or to die. As a result, they have evolved elaborate mechanisms to determine which path they will follow in response to a multitude of internal and external signals, and to the wear-and-tear associated with the aging process. Until recently, activation of the calcium-dependent protease, calpain, had been traditionally associated with neurodegeneration. This chapter will review recent findings that indicate that two of the major calpain isoforms present in the brain, calpain-1 and calpain-2, play opposite functions in neuronal survival/death. Thus, calpain-1 activation, downstream of synaptic NMDA receptors, is part of a neuronal survival pathway through the truncation of PHLPP1 and the stimulation of the Akt pathway. In contrast, calpain-2 activation is downstream of extrasynaptic NMDA receptors and is neurodegenerative through the truncation of the phosphatase, STEP, and the activation of the p38 protein kinase. These findings have major significance for our understanding of neurological conditions associated with neurodegeneration and for the development of new therapeutic approaches to prevent neuronal death in these disorders.
KeywordsCalpain-1 Calpain-2 Neuronal death Neuronal survival NMDA receptors Akt STEP
Neurons have to perform several basic functions, including growing (from the time of differentiation), migrating, responding and adapting to external and internal stimuli, and surviving or dying, as a result of continuous challenges and the deleterious effects of the aging process. Numerous reviews have discussed the role of calpain in neurodegeneration in general (Vosler et al. 2008; Yildiz-Unal et al. 2015), and in stroke (Anagli et al. 2009; Koumura et al. 2008) and in traumatic brain injury (TBI) (Kobeissy et al. 2015; Liu et al. 2014). Likewise, numerous studies have attempted to use calpain inhibitors to reduce neurodegeneration in both stroke and TBI (Anagli et al. 2009; Bartus et al. 1994a, b; Cagmat et al. 2015; Hong et al. 1994; Li et al. 1998; Markgraf et al. 1998; Siklos et al. 2015; Tsubokawa et al. 2006). While some studies have reported some positive effects of calpain inhibitors in TBI (Thompson et al. 2010), other studies have not confirmed these results. In particular, overexpression of the endogenous calpain inhibitor, calpastatin, was reported to reduce the formation of the Spectrin Breakdown Product (SBDP), resulting from calpain-mediated truncation of spectrin, a widely used biomarker of calpain activation and potentially neurodegeneration (Yan and Jeromin 2012), but had no effect on neurodegeneration (Schoch et al. 2012). Another recent study concluded that even a blood-brain barrier- and cell-permeable calpain inhibitor, SNJ-1945, did not have a sufficient efficacy and a practical therapeutic window in a model of controlled cortical impact (Bains et al. 2013).
Several reasons could account for the failure to develop clinical applications of such inhibitors, including their lack of specificity/potency/selectivity (Donkor 2011), and the incomplete knowledge regarding the functions of the major calpain isoforms in the brain, calpain-1 and calpain-2 (aka μ- and m-calpain). Work from our laboratory over the last 5 years has revealed new features of these two enzymes, which significantly changed our understanding of their functions in the brain. Specifically, we found that calpain-1 and calpain-2 play opposite functions in both synaptic plasticity and neuroprotection/neurodegeneration (Baudry and Bi 2016). Thus, calpain-1 activation is required for theta burst stimulation-induced long-term potentiation (LTP) and for certain types of learning and memory, and is neuroprotective (Wang et al. 2013, 2014). Calpain-1 is neuroprotective due to the degradation of the PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1β) and the resulting activation of the Akt survival pathway. On the other hand, calpain-2 activation limits the magnitude of LTP and restricts learning, and is neurodegenerative due to the cleavage of STEP and the stimulation of death pathways (Wang et al. 2013, 2014). In addition, we found that ischemia-induced damage to retinal ganglion cells was exacerbated in calpain-1 knock-out mice, indicating that calpain-1 inhibition is likely to counteract the potential beneficial effects of calpain-2 inhibition if non-selective calpain inhibitors are used (Wang et al. 2016b). These findings could account for the failure of the previous studies to convincingly demonstrate the role of calpain in neurodegeneration, and for the lack of clear efficacy of the previously tested calpain inhibitors, which did not discriminate between calpain-1 and calpain-2. It is also important to stress that calpain activation has also been implicated in diffuse axonal injury (Wang et al. 2012a), which has been proposed to represent an important component of the pathophysiology of TBI (Xiong et al. 2013), although at this point, there is no information regarding which calpain isoform is involved.
In this chapter, we will first discuss how calpain-1 and calpain-2 activation appear to be closely related to the stimulation of synaptic and extra-synaptic NMDA receptors, respectively. We will then review the mechanisms underlying the neuroprotective effects of calpain-1 activation, which will be followed by a discussion of the mechanisms involved in calpain-2-mediated neurodegeneration. These two aspects will be illustrated by studies using intra-ocular NMDA injection to produce acute neurodegeneration of retinal ganglion cells. Finally, we will discuss the potential clinical implications of these findings and our current efforts to develop selective calpain-2 inhibitors as a new approach for neuroprotection in conditions associated with acute neurodegeneration.
2.2 Calpains and NMDA Receptors
NMDARs play critical roles in both physiological and pathological conditions, and several studies have shown that NMDA receptor localization is responsible for opposite consequences of NMDA receptor stimulation for neuronal survival or death; thus, synaptic NMDAR activation provides neuroprotection, while extrasynaptic NMDARs are linked to pro-death pathways (Hardingham and Bading 2010). The Akt and MAP kinase/extracellular signal-regulated kinase (ERK1/2) pathways are two key pro-survival pathways downstream of synaptic NMDARs (Hardingham et al. 2001; Papadia et al. 2005; Wang et al. 2012b). Akt phosphorylates and inhibits various pro-apoptotic substrates, such as glycogen synthase kinase-3 (GSK3), forkhead box O (FOXO) (Soriano et al. 2006), apoptosis signal-regulating kinase 1 (ASK1) (Kim et al. 2001), p53 (Yamaguchi et al. 2001), and Bcl2-associated death promoter (BAD) (Downward 1999). On the other hand, ERK1/2 activates the survival nuclear transcription factor, cyclic-AMP response element binding protein (CREB) (Hardingham et al. 2001). Although some protein kinases linking NMDARs to Akt and ERK have been found (Krapivinsky et al. 2003; Perkinton et al. 2002), how Akt and ERK1/2 were activated by synaptic but not extrasynaptic NMDARs was not clearly understood until recently.
PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1) exhibits two splice variants, PHLPP1α and PHLPP1β, which share amino acid sequence similarity but have different sizes (140 kDa and 190 kDa, respectively). PHLPP1α dephosphorylates Akt at Ser473 in cancer cells (Gao et al. 2005) and neurons (Jackson et al. 2010), and its down-regulation is related to cell survival in CNS (Chen et al. 2013; Liu et al. 2009; Saavedra et al. 2010). PHLPP1β inhibits ERK1/2 by binding and trapping its activator Ras in the inactive form (Shimizu et al. 2003). PHLPP1β was previously shown to be degraded by calpain in hippocampus, and its degradation contributes to novel object recognition memory (Shimizu et al. 2007). Thus, PHLPP1 was a good candidate to link NMDA receptor stimulation to Akt and ERK regulation.
As previously reported (Xu et al. 2009), we found that calpain activated by extrasynaptic NMDAR stimulation cleaved STEP and caused neuronal death (Wang et al. 2013). It had previously been proposed that prolonged or excessive activation of calpain was responsible for calpain-mediated neurotoxicity, whereas brief and limited calpain activation could be involved in the regulation of synaptic plasticity. However, prolonged activation of synaptic NMDARs (by Bic and 4-AP treatment) for as long as 3 days did not result in STEP cleavage, nor in neuronal damage, but produced neuroprotection against starvation and oxidative stress. On the other hand, activation of extrasynaptic NMDARs did not affect PHLPP1 or its downstream pathways, strongly suggesting that there are two separate pools of calpain downstream of synaptic and extrasynaptic NMDARs, which regulate different substrates and therefore exert separate functions.
The possibility that calpain-1 and calpain-2 could exert different roles in CNS had not been extensively discussed. However, the discovery that calpain-2 could be activated by phosphorylation (Zadran et al. 2010), coupled with the identification of PTEN as a specific calpain-2 substrate (Briz et al. 2013), raised the possibility that calpain-1 and calpain-2 could play distinct functions. Interestingly, synaptic NMDAR activation did not result in the degradation of PTEN, a specific calpain-2 substrate, further supporting the idea that synaptic NMDAR activation does not activate calpain-2. The use of calpain-1 and calpain-2 specific inhibitors also confirmed this idea, as a calpain-2 specific inhibitor did not affect synaptic NMDAR-dependent PHLPP1 cleavage and neuroprotection but blocked extrasynaptic NMDAR-dependent STEP cleavage and neurotoxicity. In contrast, a calpain-1 specific inhibitor blocked synaptic NMDAR-mediated effects but not extrasynaptic NMDAR-mediated neurotoxicity. Down-regulation of calpain-1 and calpain-2 by specific siRNAs in cultured neurons also indicated that only calpain-1 knockdown blocked synaptic NMDAR-mediated neuroprotective pathways. In addition, knockdown of calpain-2 but not calpain-1, by AAV-shRNA transfection increased survival of primary hippocampal neurons following NMDA treatment (Bevers et al. 2009). Results obtained in cultured neurons were further confirmed using a model of NMDA-induced neurotoxicity in acute hippocampal slices from young mice, which had previously indicated that NMDA treatment of acute hippocampal slices caused neurotoxicity in young but not adult rats (Zhou and Baudry 2006), probably because young rats have more NR2B-containing NMDARs, which are preferentially localized extrasynaptically (Tovar and Westbrook 1999). In hippocampal slices prepared from young calpain-1 knock-out mice, NMDA induced the degradation of STEP but not PHLPP1, and exacerbated neurotoxicity, as compared to slices prepared from wild-type mice. On the other hand, calpain-2 specific inhibition by applying either a selective calpain-2 inhibitor in slices from wild-type mice or a non-selective calpain inhibitor in slices from calpain-1 knock-out mice blocked NMDA-induced degradation of STEP and suppressed neurotoxicity (Wang et al. 2013).
Together, these results demonstrate that calpain-1 is preferentially activated by synaptic NMDAR stimulation, whereas calpain-2 is preferentially activated by extrasynaptic NMDAR stimulation. Calpain-1 was shown to be localized in synaptic compartments (Perlmutter et al. 1988), where it could regulate synaptic function through its action on synaptic elements such as cytoskeletal and scaffolding proteins, as well as glutamate receptors (Liu et al. 2008). Little is known regarding the ultrastructural localization of calpain-2 in neurons. One of the newly discovered physiological roles of calpain-2 is to regulate activity-dependent local protein synthesis (Briz et al. 2013; Wang and Huang 2012), which takes place not in synapses but in nearby extrasynaptic areas (Frey and Morris 1998; Steward and Wallace 1995). In addition, calpain-2 has been reported to control synaptogenesis in dendritic shafts through constitutive proteolysis of the cytoskeletal protein, cortactin (Mingorance-Le Meur and O’Connor 2009). These findings would suggest that calpain-2 is localized, at least in part, in extrasynaptic domains (Fig. 2.1).
The existence of separate signaling pathways for calpain-1 and calpain-2 suggested that these two calpain isoforms belong to different protein scaffolds, which could segregate them in different neuronal compartments. PHLPP1 could be cleaved by both purified calpain-1 and calpain-2 in membrane fractions, yet it was cleaved only by calpain-1 following synaptic NMDAR activation in hippocampal slices, suggesting that substrate specificity for calpains depends not only on amino acid sequences within substrates, but also on localization and scaffolding of both substrates and calpains in neurons. Co-immunoprecipitation experiments confirmed that NR2A-containing NMDARs, PSD95, calpain-1 and PHLPP1, form a complex in neurons. Furthermore, synaptic NMDAR activity recruited calpain-1 to this NMDAR multi-protein complex; such recruitment could facilitate the proteolysis of PHLPP1 and possibly other calpain-1 substrates in the complex. In contrast, calpain-2 was not present in this complex under basal conditions nor was it recruited by activity, consistent with the absence of calpain-2 activation following synaptic NMDAR activation. It is likely that a calpain-2-containing multi-protein complex is associated with extrasynaptic NMDARs. How could activation of extrasynaptic NMDARs results in calpain-2 activation? It has been repeatedly shown that NR2B subunits are enriched in extrasynaptic NMDARs (Papouin and Oliet 2014), and that their activation is critical for excitotoxicity (Chazot 2004). Interestingly, NR2B directly binds RasGRF1, which provides a link between NMDAR activation and ERK activation (Krapivinsky et al. 2003). As we have shown that ERK activation directly phosphorylates and activates calpain-2 (Zadran et al. 2010), this pathway is likely responsible for the prolonged activation of calpain-2 following stimulation of extrasynaptic NMDA receptors (Fig. 2.1). In addition, we discussed elsewhere the existence of different PDZ binding domains in the C-terminal of calpain-1 and calpain-2, which could account for their differential subcellular distribution (Baudry and Bi 2016).
2.3 Calpain-1 Activation and Neuroprotection
As discussed above, calpain-1 is downstream of synaptic NMDARs and as such, we postulated that it has a neuroprotective function. This notion was supported by results obtained in cultured neurons, where we demonstrated that calpain-1 activation following stimulation of synaptic NMDARs was neuroprotective against starvation- and oxidative stress-mediated neurotoxicity (Wang et al. 2013). Previous studies have shown that normal stimulation of synaptic NMDA receptors is required to limit the extent of apoptotic neuronal death during the postnatal period, as blockade of these receptors during this period increases the extent of apoptotic neuronal death (Monti and Contestabile 2000). Calpain activity is higher in cerebellum than in cortex or hippocampus across different mammalian species (Baudry et al. 1986). An immunohistochemical study revealed that the major calpain isoform expressed in cerebellar neurons is calpain-1 (Hamakubo et al. 1986). Calpain-1 activity in cerebellum during prenatal and early postnatal period is high, as compared to that in adulthood (Simonson et al. 1985), suggesting a potential role for calpain-1 in cerebellar development. Interestingly, a CAPN1 missense mutation in the Parson Russell Terrier dog breed has been associated with spinocerebellar ataxia (Forman et al. 2013).
Loss of cerebellar granule cells (CGCs) induced by different mechanisms results in ataxia (Hashimoto et al. 1999; Kim et al. 2009; Pennacchio et al. 1998; Shmerling et al. 1998). NMDAR activity is essential for CGC survival during the critical stage of cerebellar development (Monti and Contestabile 2000; Balazs et al. 1988; Monti et al. 2002; Moran and Patel 1989), although the underlying mechanism has remained elusive. NMDAR-induced activation of the nuclear factor CREB is required (Monti et al. 2002), and CREB is a target of the pro-survival kinase Akt (Du and Montminy 1998).
As discussed above, synaptic NMDAR-mediated calpain-1 activation results in the degradation of PHLPP1. PHLPP1 dephosphorylates and inhibits Akt, and is involved in tumorigenesis (Chen et al. 2011), circadian clock (Masubuchi et al. 2010), learning and memory process (Wang et al. 2014; Shimizu et al. 2007), and autophagy (Arias et al. 2015). Calpain-1-mediated degradation of PHLPP1 activates Akt and promotes neuronal survival (Wang et al. 2013), and we postulated that calpain-1 mediated regulation of PHLPP1 and Akt could be involved in NMDAR-dependent CGC survival during postnatal development.
Thus the NMDAR/calpain-1/PHLLP1/Akt pro-survival pathway is active in developing CGCs, where it limits the extent of CGC apoptosis. Increased PHLPP1 and decreased pAkt levels were found in cerebellar homogenates of calpain-1 KO mice, indicating that calpain-1 activity normally reduces PHLPP1 levels and maintains Akt activated during the postnatal period in cerebellum. The density of pAkt-positive puncta was reduced in cerebellar granular layer but not in Purkinje or molecular layer of calpain-1 KO mice, suggesting that calpain-1-dependent regulation of Akt only takes place in CGCs but not in other cerebellar cell types. Down-regulation of PHLPP1 restored normal levels of pAkt in developing cerebellum of calpain-1 KO mice, indicating that PHLPP1 is downstream of calpain-1 and that its level is important for Akt regulation. Finally, reduced Akt activity was associated with enhanced CGC apoptosis in calpain-1 KO mice, while increased Akt activity was associated with reduced CGC apoptosis in bpV-injected WT and in mice lacking both calpain-1 and PHLPP1.
NMDAR- and calpain-1-mediated neuronal survival during brain development was not limited to CGCs, as enhanced apoptosis was present in other brain regions such as cortex, striatum and hippocampus in developing calpain-1 KO mice (Fig. 2.2). Importantly, calpain-1-mediated neuroprotection is also present in human brain, as calpain-1 mutations resulting in lack of function are associated with cerebellar ataxia (Wang et al. 2016a; Gan-Or et al. 2016). Furthermore, the important roles of calpain-1 in hippocampal neuronal survival during development and in synaptic plasticity in the adult (Wang et al. 2014; Zhu et al. 2015) may contribute to the cognitive decline found in ataxia patients with CAPN1 mutations.
2.4 Calpain-2 and Neurodegeneration
Six days after intravitreal injection of NMDA or PBS to WT mice, frozen retinal sections were prepared and H&E staining was performed to evaluate cell numbers in the ganglion cell layer (GCL) and the thickness of the Inner Plexiform Layer (IPL), which contains RGC dendrites. NMDA injection (NMDA plus Vehicle) significantly reduced cell numbers in the GCL and IPL thickness, while PBS injection (PBS plus Vehicle) had no effect on these parameters (Fig. 2.3d–f). Systemic injection of C2I 30 min before and 6 h after NMDA injection significantly suppressed the reduction in GCL cell numbers and IPL thickness (Fig. 2.3d–f), suggesting that calpain-2 activation contributes to NMDA-induced cell death in GCL.
In calpain-1 KO mice, GCL cell number and IPL thickness were not affected by vehicle injection. However, the effects of NMDA injection on GCL cell number and IPL thickness were larger than in WT mice (Fig. 2.3g–i). GCL cell death in calpain-1 KO mice after NMDA injection was significantly more severe than that in WT mice (Fig. 2.3j), suggesting that calpain-1 supports cell survival in GCL after NMDA injection. Systemic injection of C2I to calpain-1 KO mice partially but significantly reversed NMDA-induced decrease in GCL cell number and IPL thickness (Fig. 2.3g–i). A very similar pattern of results was obtained in a different model of acute glaucoma, consisting in a brief period of increased intraocular pressure (Wang et al. 2016b). Furthermore, recent studies in a mouse model of TBI also support the notion that calpain-2 activation is prolonged and responsible for neuronal death, while calpain-1 activation is neuroprotective (Wang et al 2017).
2.5 Clinical Implications of Specific Calpain-2 Inhibition and Calpain-1 Activation
Our results clearly demonstrate that calpain-1 and calpain-2 have opposite functions in both synaptic plasticity and neuronal survival/death after acute insults. Thus, calpain-1 activation is required for LTP induction and for hippocampus-dependent learning and is neuroprotective both during the postnatal developmental period and in adulthood following acute insults. On the other hand, calpain-2 activation limits the extent of hippocampus-dependent learning and is neurodegenerative following acute insults, and in particular excitotoxicity. Our results have important implications for the development of new approaches for treating diseases associated with excitotoxicity, such as epilepsy, stroke, Alzheimer’s and Parkinson’s disease, Huntington disease and ischemia. In all these cases, it has been suggested that extrasynaptic NMDAR activation and STEP degradation are involved in neurodegeneration. Our results would, therefore, suggest that specific inhibition of calpain-2 but not calpain-1 would have neuroprotective effects under these conditions. Conversely, overexpression or activation of calpain-1, by cleaving PHLPP1 and stimulating pro-survival cascades, could also have beneficial effects. In addition, calpain-2 activation is involved in regulating the magnitude of long-term potentiation (LTP) in hippocampus, due to the existence of a molecular brake consisting in calpain-2-mediated PTEN degradation and stimulation of m-TOR dependent PHLPP1β synthesis (Wang et al. 2014). We also showed that low doses of a selective calpain-2 inhibitor facilitate learning in normal mice, while higher doses, which inhibit calpain-1, impair learning. Thus, a selective calpain-2 inhibitor could be extremely beneficial for preventing neurodegeneration, while facilitating certain forms of learning and memory. As discussed above, a selective calpain-2 inhibitor prevented death of retinal ganglion cells and maintained vision in a mouse model of acute glaucoma (Wang et al. 2016b). Calpain inhibitors have previously been proposed to represent potential treatments for a variety of eye disorders, including glaucoma and macular degeneration (Azuma and Shearer 2008; Paquet-Durand et al. 2007), and further studies are needed to assess the potential use of selective calpain-2 inhibitors for these disorders. Calpain inhibition has been proposed to represent a therapeutic approach for stroke and TBI, although this notion has not been supported by a variety of experiments. We postulate that the use of selective calpain-2 inhibitors might overcome the problems associated with that of non-selective calpain inhibitors. Our results in a mouse model of TBI supports this notion, as we have found that post-treatment with a selective calpain-2 inhibitor provides a highly significant degree of neuroprotection and facilitates behavioral recovery (Wang et al 2017). The potential use of selective calpain-2 inhibitors for chronic neurodegenerative disorders needs to be further evaluated. It is important to note that calpain has been proposed to participate in neurodegeneration associated with Parkinson’s disease as well as Alzheimer’s disease, and it is tempting to speculate that selective calpain-2 inhibitors might also be beneficial in these disorders.
This work was supported by grant P01NS045260-01 from NINDS (PI: Dr. C.M. Gall), grant R01NS057128 from NINDS to M.B., and grant R15MH101703 from NIMH to X.B. X.B. is also supported by funds from the Daljit and Elaine Sarkaria Chair.
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