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Optic neuritis (ON), which is an acute inflammatory autoimmune demyelinating disease of the central nervous system (CNS), often occurs in multiple sclerosis (MS). ON is an early diagnostic sign in most MS patients caused by damage to the optic nerve leading to visual dysfunction. Various features of both MS and ON can be studied following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in Lewis rats. Inflammation and cell death in the optic nerve, with subsequent damage to the retinal ganglion cells in the retina, are thought to correlate with visual dysfunction. Thus, characterizing the pathophysiological changes that lead to visual dysfunction in EAE animals may help develop novel targets for therapeutic intervention. We treated EAE animals with and without the calpain inhibitor calpeptin (CP). Our studies demonstrated that the Ca2+-activated neutral protease calpain was upregulated in the optic nerve following induction of EAE at the onset of clinical signs (OCS) of the disease, and these changes were attenuated following treatment with CP. These reductions correlated with decreases in inflammation (cytokines, iNOS, COX-2, and NF-κB), and microgliosis (i.e. activated microglia). We observed that calpain inhibition reduced astrogliosis (reactive astroglia) and expression of aquaporin 4 (AQP4). The balance of Th1/Th2 cytokine production and also expression of the Th1-related CCR5 and CXCR3 chemokine receptors influence many pathological processes and play both causative and protective roles in neuron damage. Our data indicated that CP suppressed cytokine imbalances. Also, Bax:Bcl-2 ratio, production of tBid, PARP-1, expression and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated after treatment with CP. Our results demonstrated that CP decreased demyelination [loss of myelin basic protein (MBP)] and axonal damage [increase in dephosphorylated neurofilament protein (de-NFP)], and also promoted intracellular neuroprotective pathways in optic nerve in EAE rats. Thus, these data suggest that calpain is involved in inflammatory as well as in neurodegenerative aspects of the disease and may be a promising target for treating ON in EAE and MS.
Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are neurodegenerative diseases with characteristic inflammation and demyelination in the central nervous system (CNS) areas, including the optic nerve (Potter and Bigazzi 1992; Guyton et al. 2005a). Neuronal and axonal damage is considered the main cause of long-term disability in patients with MS. Optic neuritis (ON), inflammation of the optic nerve, is the first diagnosed sign in approximately 20% of patients with MS and as many as 50% of patients with ON eventually develop the disease. Electroretinogram (ERG) studies, which assess retinal function and visual-evoked potential (VEP) data to determine optic nerve abnormalities, have demonstrated that visual function is impaired in MS patients and in animals with EAE (Kornek et al. 2000; Meyer et al. 2001; Fisher et al. 2006). MS and EAE are caused by an immune attack on the CNS by auto-reactive T cells and activated macrophages. As a result, there is a significant increase in proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), as well as other mediators (Williams et al. 1994). However, current research suggests that MS and EAE are also neurodegenerative diseases, leading to axonal degeneration and neuronal death (Trapp et al. 1999; Guyton et al. 2009, 2010). As pathophysiological changes that occur in EAE spinal cord also occur in EAE optic nerve, the EAE animal model is ideal for studying ON (Davie et al. 1995; Shields and Banik 1998; Shields et al. 1998). In the CNS, aquaporin 4 (AQP4) expression promotes inflammation and causes demyelinating lesions. This idea is a subject of intense speculation. Recently, increased AQP4 expression was found in brain and spinal cord in EAE, providing further support for the possible involvement of AQP4 in CNS inflammation (Misu et al. 2007; Miyamoto et al. 2009). No data were available about modifications in the expression of both AQP4 and glial fibrillary acidic protein (GFAP) in the reported EAE models in the optic nerve. A recent study in a myelin oligodendrocyte glycoprotein (MOG)-induced EAE model showed the attenuation of disease progression in AQP4 knockout mice (Misu et al. 2007; Li et al. 2009). Other experimental data were obtained from the MOG-induced EAE models in non-obese diabetic (NOD)/Lt and C57BL/6 mice, known to develop a diffuse inflammatory process not limited to the opticospinal location. Furthermore, complete Freund's adjuvant (CFA) and Pertussis toxin, which are known to alter the blood–brain–barrier permeability, are largely used (Namer et al. 1994; Lu et al. 2008).
Neuropathological studies of GFAP, a specific marker of reactive astrocytes, and AQP4, particularly abundant in the optic nerves, spinal cord, and periependymal regions (Nagelhus et al. 1998; Wujek et al. 2002; Trip et al. 2005; Vitellaro-Zuccarello et al. 2005; Pittock et al. 2006; Tsoi et al. 2006; Takano et al. 2008), have demonstrated decreases in expression of GFAP and AQP4 in the optic nerve and spinal cord of neuromyelitis optica (NMO) patients prior to demyelination (Misu et al. 2007; Roemer et al. 2007). This pattern of reactivity seems different from that encountered in MS where loss of AQP4 has been found mainly in strongly demyelinated regions in intensive and acute MS lesions, whereas overexpression of AQP4 has mostly been observed in other chronic demyelinated tissue due to astrogliosis. The inflammatory nature of ON implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell-associated IFN-γ, the Th2 cell-related interleukin-4 (IL-4), and the immune response-down-regulating cytokine transforming growth factor-beta (TGF-β), but proof for their involvement in ON has been lacking (Guan et al. 2006). Moreover, patients with ON had elevated percentages of CXCR3 and CCR5 expressing T cells compared with patients with other non-inflammatory neurological diseases (OND). Greater chemokine receptor expression may be one pre-requisite for Th1 cells to migrate to the CNS.
Studies in our laboratory have demonstrated that calpain expression is upregulated in splenic cells isolated from EAE animals before onset of clinical signs of disease (Schaecher et al. 2002; Guyton et al. 2005b; Das et al. 2008a). Calpain degrades myelin proteins into immunogenic fragments that can potentially activate myelin-specific autoreactive T cells. Calpain also degrades many cytoskeletal substrates, including myelin basic protein (MBP), neurofilament protein (NFP), and spectrin, which can lead to cellular damage. In addition, calpain cleaves many signaling proteins that are involved in the regulation of apoptosis (Ray and Banik 2003). Also, studies in our laboratory have demonstrated increased calpain activity in immune cells and astrocytes in the optic nerves of animals with EAE (Shields et al. 1998; Guyton et al. 2005b; Shindler et al. 2006); however, we have not previously examined if calpain is involved in cell death in EAE optic nerve before the onset of clinical signs. As calpain is involved in myelin degradation in MS and as optic nerve damage arises before appearance of EAE signs in animal models of EAE, we have hypothesized that increase in calpain activity, as a result of increased Ca2+ influx, may correlate with cell death in EAE optic nerve, thus resulting in impaired visual function. Our studies demonstrated that calpain expression and cell death were increased in the optic nerve at the onset of clinical symptoms of disease and persisted during the acute EAE attack. These findings suggest that early treatment with calpain inhibitors may be of therapeutic value in preserving retinal function in EAE. ON manifests as an acute, self-limited episode of optic nerve inflammation with decrease in vision that recovers over several weeks in the majority of patients (Beck et al. 1992; Berkelaar et al. 1994; Bettelli et al. 2003; Shao et al. 2004; Arnold 2005). However, some level of permanent vision loss occurred in approximately 40% of patients in the ON Treatment Trial (Beck et al. 1992), and subsequent studies have shown that retinal nerve fiber layer thinning, which is used as a surrogate marker for retinal ganglion cell (RGC) axonal loss, correlates with vision loss after an episode of ON. Inhibition of chemokine receptor expression may constitute a potentially important therapeutic effect.
In the current study, we examined the timing and extent of several parameters (e.g. cell death, inflammation, anti-apoptotic factors, cytokines, chemokines) in optic nerve damage during acute ON in EAE rats, and we also examined whether cell death occurred by an apoptotic mechanism. Our current results suggest that inflammatory cells infiltrating into the optic nerve mediate the mechanisms of apoptotic cell death and CP attenuates these deleterious effects.
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The most important findings from our current investigation indicated that calpain played an undeniable role in the pathogenesis of ON in EAE rats and CP treatment significantly attenuated both inflammatory factors and molecular alterations for prevention of apoptosis in optic nerve in EAE animals (Fig. 8). ON is one of the first presenting clinical signs in many MS patients. While not all ON patients develop MS, at least 50% of MS patients will experience visual dysfunction. Therefore, understanding the timing and mechanisms of cell death associated with the visual problem is an utmost important factor for developing potential therapies to prevent permanent vision loss from ON. Reactive oxygen species-mediated cell damage has been demonstrated in EAE optic nerves as early as 3–6 days after immunization (Qi et al. 2007), suggesting that the onset of ON may vary. Previous studies have demonstrated that assaults on the optic nerve occur before the appearance of clinical signs of disease in rodents with EAE (Shields and Banik 1998; Guyton et al. 2005a). The purpose of this study was to determine if up-regulation of calpain activity was a prime pathophysiological event in ON. Our study has established that calpain activity is upregulated in optic nerve during the development of acute EAE with increased axonal damage and increased infiltration of T cells and macrophages. These findings are in agreement with our previous investigations carried out in acute EAE spinal cord and retina (Schaecher et al. 2002; Guyton et al. 2005b; Smith et al. 2011). Our previous studies prompted us to investigate now the efficacy of calpain inhibition using CP for attenuation of optic nerve damage before or after onset of clinical signs. Administration (i.p.) of CP (50–250 μg/kg) twice daily in EAE Lewis rats significantly attenuated cell death, calpain and caspase activities, inflammation, apoptotic factors, cytokines, and chemokines when compared with untreated EAE animals. Also, increases in MBP degradation and axonal damage were significantly decreased in EAE animals after CP treatment.
Figure 8. A schematic presentation of changes in factors pathways due to calpain up-regulation and calpeptin (CP) treatment in experimental autoimmune encephalomyelitis (EAE)-induced optic neuritis (ON). EAE results in increased Ca2+ in the optic nerve, which in turn triggers activation of calpain. Increased calpain is associated with elevation of cell death markers, inflammatory mediators, and Th1 cytokine and chemokine elevations, which are in turn implicated in activated microglia, reactive astroglia, axonal damage, and ultimately cell death. CP therapy attenuates these changes and results in converse increases in Th2 cytokines, anti-inflammatory mediators, and prosurvival markers that prevent cell death.
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Increases in calpain expression and activity were demonstrated in splenic cells before the onset of the disease (Shields and Banik 1998; Shields et al. 1998), and similar increases were also evident in infiltrating immune cells and glial cells of the spinal cord and optic nerve (Shields and Banik 1998a) even after the appearance of clinical signs. The current study confirmed our earlier findings (Schaecher et al. 2002; Guyton et al. 2005b) of increased calpain expression and activity in EAE optic nerve in a time-dependent manner. Interestingly, these increases are seen in the optic nerve of animals before they are seen in spinal cord and before appearance of clinical signs. The optic nerve degeneration and visual dysfunction occurring in EAE prior to disease onset are important determinants to MS, where the optic nerve is affected first. The increased calpain expression and activity correlated well with a decrease in expression of calpastatin, the endogenous inhibitor of calpain. This finding also suggests that calpain is involved in the neurodegenerative process in EAE and perhaps in MS.
Astrogliosis observed in this study was demonstrated by increased expression of GFAP. Also, astrocytes selectively express AQP4, a channel important for transport of both water and solutes across the plasma membrane (Das et al. 2012). Normal AQP4 activity is critical for maintenance of both cell volume and ionic gradients in the CNS. The astrocytic water channel AQP4 plays a major role in the physiopathology of ON. While animal models of EAE showing optic nerve demyelination have been developed, the involvement of astrocytes and AQP4 in these models is poorly documented. This is one of the important aspects of our current investigation in ON in EAE rats. During the disease stage, we observed that levels of expression of both GFAP and AQP4 were significantly increased in the demyelinated optic nerves. The enhanced expression of AQP4 in optic nerve astrocytes following elevation of inflammation may explain the astrocytic hypertrophy that is normally seen in MS patients. CP therapy is capable of attenuating the expression of both GFAP and AQP4 in optic nerve in EAE rats.
NF-κB is an important transcription factor that regulates both innate and adaptive immune responses. Deregulated activation of NF-κB or its regulatory kinases is associated with chronic inflammation, autoimmunity, and cancers (Bernes and Karin 1997). Our long-range goals of current investigation were to dissect the signaling pathway mediating NF-κB activation, elucidate the role of NF-κB in regulating inflammatory response, and examine expression of COX-2, iNOS, and anti-apoptotic proteins in EAE rats. Improper up-regulation of COX-2 and/or iNOS has been associated with pathophysiology of inflammatory disorders (Stolina et al. 2000). Our results demonstrated a possible mechanistic target of calpain inhibition for down regulating the inflammatory responses for prevention of apoptosis in optic nerve in EAE animals. Calpain inhibition by CP is associated with the inhibition of synthesis and release of proinflammatory mediators and inhibition of activated immune cells and COX-2, which are responsible for the synthesis of proinflammatory mediators. The varying and multiple protective characteristics of CP may clinically also hold a respectable position for it as a better alternative than other anti-inflammatory drugs. These data provide evidence that CP exhibits potent anti-inflammatory activity and explain the underlying mechanism of protection of optic nerve in ON. Recent studies have indicated that inflammation plays important roles in neurodegenerative diseases (Das et al. 2008b). In EAE, factors released from damaged neurons induce reactive gliosis, especially microgliosis, which is the hallmark of inflammation (Ponomarev et al. 2007). Reactive microgliosis may enhance the neurotoxicity by producing excessive proinflammatory factors, including cytotoxic superoxide, NO, and proinflammatory cytokines (Das et al. 2008b). Our current data demonstrated that calpain inhibition attenuated microgliosis and microglia-dependent inflammatory process that caused the progressive and self-propelling nature of EAE.
The mechanistic study of ON pathogenesis is currently seeking to understand the effects of immune responses on cell damage and protection. Cytokines are the factors that mediate most of the biological effects in both the immune and non-immune systems (Kennedy and Karpus 1999). CD4-expressing T-helper (Th) cells are a major source of cytokine production and regulation. Th type 1 (Th1, inflammatory effect) cells are characterized by the production of proinflammatory cytokines such as IL-1α/β/ra, IL-2, IL-3, IFN-γ, and TNF-α while Th type 2 (Th2, anti-inflammatory effect) cells are characterized by the production of IL-4, IL-6, and IL-10. The balance of Th1/Th2 cytokine production influences many pathological processes and plays both destructive and protective roles in a disease process. Growing evidence indicates that imbalances of Th1/Th2 cytokine production are involved in neural damage or protection in many neurological diseases. In the current investigation, we analyzed the possible roles of Th1/Th2 cytokine production and imbalance of Th1/Th2 cytokines in optic nerve in EAE rats. Our results demonstrated a significant increase in Th1 cytokines and decrease in Th2 cytokines in acute EAE. In contrast, treatment with the calpain inhibitor CP reversed this change so that Th1-associated cytokines were decreased, while Th2-associated cytokines were increased. These results suggest that calpain is involved in Th1/Th2 deregulation in EAE and CP has potential as a therapeutic in ameliorating this deregulation. In addition, it is possible that CP therapy reduces peripheral inflammatory activation via systemic immunomodulatory effects and thus attenuation or prevents the localized inflammatory cascades-mediated degenerative parameters.
A role for chemokines as mediators of Th1 cell recruitment to the CNS in MS has previously been suggested by several groups (Samson et al. 1996; Qin et al. 1998; Quinones et al. 2008). Therefore, we studied expression of Th1-related CCR5 and CXCR3 chemokine receptors in EAE animals. The chemokine receptor CXCR3 is a Gαi protein-coupled receptor in the CXC chemokines receptor family (Clark-Lewis et al. 2003). CXCR3 binds to the CXC chemokines CXCL9 (MIG), and CXCL-10 (IP-10) and particularly the CXCR3/CXCL10 system has been implicated in inflammatory process in the CNS. This receptor is expressed on astrocytes throughout human brain and has been speculated to be up-regulated in MS lesions. The CCR5 chemokine ligands that bind to this receptor are RANTES (a chemotactic cytokine protein also known as CCL5) and macrophage inflammatory protein (MIP) 1α and 1β (also known as CCL3 and CCL4). CCR5 is predominantly expressed on T cells, macrophages, dendritic cells, and microglia. It is likely that CCR5 plays an essential role in inflammatory responses, although its exact role in normal immune function remains unclear. We observed that optic nerve of EAE-ON rats had elevated levels of CCR5 and CXCR3 expressing cells. Such elevations disappeared in EAE rats due to the treatment with the calpain inhibitor.
As mitochondria play a key role both in maintaining cellular homeostasis and in triggering the activation of cell death pathways in EAE-ON, we evaluated the effect of timing of CP treatment in EAE rats on expression of proapoptotic Bax and anti-apoptotic Bcl-2. Our results showed that CP could protect cells from apoptosis by decreasing the Bax:Bcl-2 ratio. Ischemic and hypoxic injuries to the nervous system have been previously shown to involve the release of cell death-inducing cytokines and the activation of death receptors. It is likely that these events involve caspase 8-mediated cleavage of the BH3-only protein Bid to tBid. This pathway provides evidence that activation of the receptor mediated pathway of apoptosis. Once Bid is cleaved by caspase 8, tBid translocates from the cytosol to the mitochondria to cause mitochondrial damage, release of cytochrome c, increase in caspase activity, nuclear condensation, and apoptotic cell shrinkage (Das et al. 2008a, b; Li et al. 2011). In addition, tBid is able to activate Bax to form Bax multimers in the mitochondria, thereby inducing the release of cytochrome c. In this study, our results showed that caspase 8 activation correlated well with Bid cleavage to tBid in acute EAE, again demonstrating induction of cell death signals early in disease process. Our results also suggested that CP treatments (50–250 μg/kg) attenuated the processing of caspase 8 (degradation of pro-caspase 8 and appearance of active caspase 8) and Bid cleavage to tBid. Similar mechanisms of cell death may also exist in spinal cord and retina in EAE (Smith et al. 2011). Calpain interacts and modifies caspase pathways as well. Specifically, calpain has been shown to activate caspase 3 (Das et al. 2006, 2008b). Caspase also has been shown to cleave calpastatin, indicating that caspase activity may maintain calpain as an active proteolytic enzyme, to promote apoptosis. Activation of caspase 3, the final executioner in apoptosis, which degrades the cellular target protein PARP-1, is elevated early in EAE. These results in particular indicate a pivotal co-operative role of calpain and caspase system, generating crucial cell death signals early in disease development. Calpain inhibition following treatment with CP has been found to inhibit caspase 3 and decrease apoptotic indicators, including proapoptotic proteins and internucleosomal DNA fragmentation. We observed increase in DNA laddering (the hallmark of apoptosis) following induction of EAE (Das et al. 2008a), indicating occurrence of apoptosis before onset (days 6–9) of EAE. This also suggested that the apoptotic process in optic nerve was initiated early in the inflammatory phase in EAE development, even before the appearance of clinical signs. In contrast, treatment of EAE animals with CP at all three doses that we used significantly attenuated DNA fragmentation. This observation is supported by earlier studies demonstrating the role of calpain in other neurodegenerative disorders, including ischemia, spinal cord injury, brain trauma, Alzheimer's disease, Parkinson's disease, and epilepsy (Ray and Banik 2003). Our results also suggest that calpain may be a potential therapeutic target for the amelioration of neurodegenerative process in MS.
Axonal damage is a key determinant in neurological disability in MS patients (Costello et al. 2006). Calpain, which is present in axon, degrades myelin and axonal cytoskeletal proteins including NFP (Li and Banik 1995; Schaecher et al. 2002). The degeneration of axons and damage to cells were attenuated by calpain inhibitor treatment suggesting that preservation of axon–myelin structure and cell could reduce the disease severity. Calpain inhibitor treatment prevented degeneration and protected optic nerve, indicating the importance of calpain inhibition as a promising therapeutic option (Das et al. 2006, 2008b; Guyton et al. 2009, 2010). In the development and progression of diseases, calpain activation can induce apoptotic death through multiple mechanisms (Maier et al. 2004, 2007; Sättler et al. 2004; Das et al. 2006, 2008b; McKernan et al. 2007; Malemud and Miller 2008; Sharma et al. 2011). The finding that redistribution of Bad and suppression of apoptosis by an inhibitory mutant of calcineurin suggests that calcineurin is a significant mediator of apoptotic death signals (Li et al. 2011). Thus, the possibility that Bad activation by calcineurin activity might be involved in cell death in EAE-ON was explored. Our results suggested that calcineurin-induced apoptosis by dephosphorylating Bad in EAE-ON, which was attenuated after CP treatment indicating that CP protected the optic nerve largely by interfering with the participation of calcineurin in the apoptotic process. To determine the intracellular signal transduction cascade, which could be involved in the optic nerve survival promoting effect of CP, we investigated whether CP treatment would promote optic nerve cell survival via intracellular signal transduction cascades. The current study revealed the down regulation of p-Akt, p-Bcl-2, p44/42-MAPK, p-STAT-3, and p-Bad in EAE-ON. We analyzed whether CP could antagonize these proapoptotic mechanisms involved in optic nerve damage. Our results showed significant increases in the protein levels of p-Akt, p-Bcl-2, p44/42-MAPK, p-STAT-3, and p-Bad in EAE-ON due to CP treatment (Fig. 7). The amounts of the inactive, dephosphorylated proteins were comparable in all protein lysates, indicating that not the expression level but the degree of Akt phosphorylation could promote growth factor-mediated cell survival both directly and indirectly (data not shown). Bad is a proapoptotic protein of the Bcl-2 family. Akt can phosphorylate Bad on Ser136, which makes Bad dissociate it from the Bcl-2/Bcl-X complex and lose the proapoptotic function. STAT-3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (Sharma et al. 2011). Our results demonstrated that CP protected optic nerve by potentiating the Akt/Bcl-2/MAPK/STAT-3/Bad signaling pathways (Fig. 7).
In conclusion, our current study directly and significantly demonstrated that calpain inhibition could prevent inflammation, apoptosis, and axonal degeneration in optic nerve in EAE rats (Fig. 8). Our investigation also implies that calpain inhibition may also block auto-reactive T cell-mediated inflammatory and neurodegenerative events in EAE and possibly in MS patients as well.