Neuronal ceroid lipofuscinosis (NCL; also known as Batten disease) encompasses a group of autosomal recessive lysosomal storage disorders characterized by the abnormal accumulation of proteins and lipopigments in all cell types (Palmer et al. 1993). Juvenile neuronal ceroid lipofuscinosis (JNCL), the most common inherited neurodegenerative disease in childhood, is caused by a mutation in the CLN3 gene (International Batten Disease Consortium 1995). Approximately, 80–85% of affected children harbor a 1.02 kb deletion on chromosome 16p12, although other point mutations have also been described (International Batten Disease Consortium 1995; Munroe et al. 1997). Despite identifying the causative gene, the normal function of CLN3 remains elusive, which has been complicated by low protein abundance and lack of reliable antibodies (Getty and Pearce 2011). JNCL typically presents in children between the ages of 5–10 years, initiating as blindness and progressing to seizures, advancing cognitive and motor decline, and dementia, with a life expectancy into the late teens to early twenties (Rakheja et al. 2007). Currently, there is no treatment for JNCL, which highlights the need to understand pathologic mechanisms contributing to disease progression for the development of novel therapeutics to extend the survival and quality-of-life of afflicted children.
Microglia are the resident immune cells within the central nervous system (CNS) parenchyma and exert multiple functions, including phagocytosis of cellular debris and pro-inflammatory mediator release (Kettenmann et al. 2011). Abnormal neuron-microglia interactions have been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer's and Parkinson's disease (Huh et al. 2011; Byun et al. 2012). Activated microglia are also observed in the brains of JNCL patients, as well as associated mouse models, and predict areas that will undergo neurodegeneration (Pontikis et al. 2004, 2005). However, the secretory phenotype of activated microglia in the context of CLN3 mutation remains to be defined. This is a critical issue, since activated microglia may either positively or negatively influence neuronal survival via the production of growth factors or pro-inflammatory mediators, respectively (Arai et al. 2004).
Previous work from our laboratory and others has defined the inflammasome as a key molecular pathway responsible for processing the pro-inflammatory cytokine interleukin-1 beta (IL-1β) into its active form in microglia (Halle et al. 2008; Hanamsagar et al. 2011). IL-1β expression is also elevated in the brains of JNCL patients (Dr. Jonathan Cooper, personal communication), which suggests that microglial activation may contribute to the development and progression of neurodegeneration in JNCL. Depending on the initiating stimulus, activated microglia also produce reactive oxygen species (ROS) (Bal-Price and Brown 2001; Block et al. 2007), which have recently been shown to trigger inflammasome activation (Zhou et al. 2011), linking the two processes. Prior studies have revealed oxidative imbalance in the brains of CLN3 knockout (KO) mice (Benedict et al. 2007) and increased sensitivity of CLN3 mutant Drosophila to oxidative stress (Tuxworth et al. 2011), and IL-1β has long been recognized for its neurotoxic properties (Arai et al. 2004; Thornton et al. 2006). Collectively, these observations strongly suggest that these pathways may intersect, which could represent a pathological target in the context of CLN3 mutation.
In addition to pro-inflammatory cytokines, brains of JNCL patients and CLN3 KO mice are characterized by increased levels of ceramide, an important lipid metabolism intermediate (Puranam et al. 1997; Mencarelli and Martinez-Martinez 2013). Elevated ceramide has been shown to induce neuronal apoptosis, neuroinflammation, and demyelination (Cutler et al. 2004; Kim et al. 2012; Wang et al. 2012) and dysregulated ceramide biosynthesis has been implicated in microglial activation in mouse models of neuropathic pain and Alzheimer's disease (Filippov et al. 2012; Kobayashi et al. 2012). Here, we report that treatment of primary CLN3Δex7/8 microglia with C6 ceramide and neuronal lysates induced inflammasome/caspase-1 activation and IL-1β release, whereas wild-type (WT) microglia were largely non-responsive. Low levels of constitutive caspase-1 activity were detected in CLN3Δex7/8 microglia, which when inhibited resulted in significant glutamate release, presumably via hemichannel opening, which was also elevated in CLN3Δex7/8 microglia. CLN3Δex7/8 microglia also displayed increased expression of other pro-inflammatory cytokines and chemokines in response to C6 ceramide and neuronal lysates, indicating a broader effect extending beyond IL-1β release. Similar findings were observed when CLN3Δex7/8 microglia were stimulated with either TNF-α or IL-1β, demonstrating the existence of a conserved core pathway for eliciting heightened microglial activation in the context of CLN3 mutation. Finally, CLN3Δex7/8 neurons were more sensitive to microglial-induced cytotoxicity compared with WT cells. Collectively, these results suggest that CLN3Δex7/8 microglia exist in a primed state to produce inflammatory mediators with known neurotoxic effects, which over time, may contribute to JNCL progression.
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- Materials and methods
- Supporting Information
Activated microglia are observed in the brains of CLN3 mutant mouse models (Pontikis et al. 2004, 2005); however, relatively little information is known about the biological consequences of activated microglia in the context of disease progression. Here, we report that CLN3Δex7/8 microglia exist in a primed state, where exposure to stimuli encountered in the JNCL brain elicit exaggerated cytokine and chemokine production, inflammasome/caspase-1 activation, and hemichannel activity, leading to glutamate release. In contrast, WT microglia were largely non-responsive, emphasizing the importance of mutant CLN3 in controlling the microglial pro-inflammatory state. The precise signaling pathways and molecular mechanisms responsible for these exaggerated responses remain to be completely defined; however, our studies have established a central role for inflammasome/caspase-1 activation (Fig. 7). In particular, CLN3Δex7/8 microglia displayed enhanced caspase-1 activity, which translated into elevated IL-1β secretion, a cytokine that requires proteolytic processing by caspase-1 for its release (Franchi et al. 2009). In addition, caspase-1 inhibition significantly reduced the expression of additional inflammatory mediators that do not rely on enzymatic processing, suggesting an important role for autocrine/paracrine actions of IL-1β in promoting the pro-inflammatory phenotype of CLN3Δex7/8 microglia. Surprisingly, treatment of CLN3Δex7/8 microglia with a caspase-1 inhibitor alone led to heightened glutamate release, implying that constitutive caspase-1 activity is critical for maintaining glutamate homeostasis. This possibility was strengthened by the finding that inhibition of constitutive caspase-1 activity led to increased hemichannel opening, which is a known mechanism for glutamate release (Bennett et al. 2012; Mika and Prochnow 2012). The molecular identity of the hemichannel that is affected in CLN3Δex7/8 microglia remains to be determined, but could involve traditional proteins (i.e. Cx43 or pannexin 1) or other channels that are capable of transporting small molecular weight compounds (i.e. P2X7R)(Mika and Prochnow 2012). Interestingly, CLN3Δex7/8 microglia displayed increased caspase-1 activation without any perturbation, which supports an adaptive role for caspase-1 activity in the context of CLN3 mutation. The degree of caspase-1 activation becomes more evident when CLN3Δex7/8 microglia are challenged with inflammatory stimuli, where the enzyme contributes to exaggerated cytokine release. Interestingly, caspase-1 activation was also observed in WT microglia following treatment with C6 ceramide and neuronal lysate, yet no IL-1β release could be detected. This finding suggests that the threshold to trigger cytokine release was not achieved or IL-1β levels were below the limit of detection, whereas this was clearly not the case for CLN3Δex7/8 microglia that produced high levels of IL-1β and other pro-inflammatory mediators. We predicted that lysates prepared from primary CLN3Δex7/8 neurons would elicit more inflammation in CLN3Δex7/8 microglia compared to WT lysates. Instead, we found that lysates from both CLN3Δex7/8 and WT neurons were equally capable of triggering similar inflammatory responses in CLN3Δex7/8 microglia (data not shown). However, it is important to note that we utilized embryonic neurons that harbor few lysosomal inclusions compared to adult neurons in vivo; therefore, this may be one reason why we did not observe a striking difference between the neuronal lysate preparations. It is technically challenging to isolate neurons from adult mice; therefore, we are not able to address this issue in greater detail.
Figure 7. CLN3 mutation leads to aberrant inflammasome activation. CLN3Δex7/8 microglia exhibit exaggerated caspase-1 activation and IL-1β secretion in response to stimuli that are elevated in the juvenile neuronal ceroid lipofuscinosis (JNCL) brain, representing ‘signal 1’ (neuron lysates, presumably via Toll-like receptor (TLR) signaling) and ‘signal 2’ (ceramide) for inflammasome/caspase-1 activation. In addition, CLN3Δex7/8 microglia display constitutive caspase-1 activity, which when inhibited leads to exaggerated glutamate release and hemichannel opening. This suggests an altered baseline status of CLN3Δex7/8 microglia in the context of CLN3 mutation. A role for reactive oxygen species (ROS) and cathepsin B in regulating inflammasome activation in CLN3Δex7/8 microglia was not apparent based on data where each was pharmacologically inhibited. Neurons are sensitive to CLN3Δex7/8 microglia-induced cytotoxicity, with potential cytotoxic candidates including IL-1β, TNF-α, and glutamate based on the results presented in the current report, the JNCL literature, and well-characterized modes of microglial-mediated neuronal toxicity.
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Currently, the factor(s) responsible for triggering inflammasome activation in CLN3Δex7/8 microglia remain unknown. We treated CLN3Δex7/8 microglia with inhibitors for both ROS and cathepsin B, but neither had an effect on IL-1β release (data not shown), suggesting that inflammasome/caspase-1 activation proceeds independently of both pathways that have been reported to elicit caspase-1 activation in macrophages (Lamkanfi 2011). Alternative possibilities include cytoplasmic mitochondrial DNA release or cellular osmotic disturbances (Petrilli et al. 2007; Shimada et al. 2012), which remain to be investigated. Recently, Ca2+ and cAMP have been shown to control inflammasome activation in macrophages (Lee et al. 2012; Murakami et al. 2012; Rossol et al. 2012) and we are currently evaluating these signaling intermediates as potential molecular triggers for inflammasome activity in CLN3Δex7/8 microglia. A prevailing concept in the field is that inflammasome activation results from a generalized ‘stress signal’ elicited following lysosomal membrane damage (Hornung and Latz 2010). The connection with lysosome dysfunction in JNCL is intriguing and may explain why inflammasome activation is heightened in CLN3Δex7/8 microglia and why it occurs in response to diverse stimuli. Another outstanding question is what type of inflammasome is triggered in CLN3Δex7/8 microglia, since we only evaluated the last step in the pathway (i.e. caspase-1). Based on its widespread involvement in eliciting caspase-1 activation in numerous cell types, the NLRP3 inflammasome is a likely candidate (Franchi et al. 2009), although other inflammasomes (i.e. NLRP1) cannot be disregarded at the present time.
Our studies revealed a broader involvement of caspase-1 activation in CLN3Δex7/8 microglia. Specifically, not only was IL-1β processing affected, but other pro-inflammatory cytokines and glutamate release were modulated. It is important to note that caspase-1 is capable of cleaving a wide range of substrates besides IL-1β, including the vATPase that acidifies lysosomes (Dix et al. 2008). It is intriguing to speculate that caspase-1-mediated inactivation of the vATPase may be responsible, in part, for the lysosome acidification defect in JNCL, although this remains to be determined.
Several studies have demonstrated immune system involvement during the course of JNCL. For example, autoantibodies to brain and retinal proteins are present in JNCL patients and CLN3 KO mice, and attenuation of autoantibody production leads to improvements in motor function (Pearce et al. 2004; Seehafer et al. 2011). Currently, the mechanism(s) responsible for triggering autoantibody formation are unknown; however, it was interesting to note that IL-15 production was dramatically elevated in CLN3Δex7/8 microglia, whereas little cytokine was released from WT cells. IL-15 is a key B cell cytokine that facilitates plasma cell differentiation and antibody secretion (Fehniger and Caligiuri 2001; Steel et al., 2012). Therefore, it is possible that activated CLN3Δex7/8 microglia may contribute to autoantibody formation during JNCL via IL-15 release, although this remains speculative.
Numerous studies have established that pro-inflammatory mediators, such as TNF-α and IL-1β, can induce glutamate release and neuronal death (Stoll et al. 2000; Rothwell 2003; Allan et al. 2005; McCoy and Tansey 2008). In addition, TNF-α regulates synaptic strength by modulating AMPA receptors and excessive TNF-α release contributes to hippocampal seizure activity (Beattie et al. 2002; Stellwagen and Malenka 2006; Huie et al. 2012; Santello et al. 2012). Importantly, defects in AMPA receptor activity have been implicated in early JNCL (Kovacs and Pearce 2008; Kovacs et al. 2011). Our results have demonstrated exaggerated TNF-α release in addition to other pro-inflammatory cytokines known to impact neuronal homoeostasis, including IL-1β and IL-6 in CLN3Δex7/8 microglia. We propose that CLN3 mutation leads to a progressive pro-inflammatory state within the JNCL brain, which over time contributes to heightened glutamate levels and neuronal death. Accordingly, we found that conditioned medium collected from either activated CLN3Δex7/8 or WT microglia induced cell death in primary CLN3Δex7/8 neurons, whereas WT neurons were resistant to cytotoxicity. These findings demonstrate that intrinsically diseased CLN3Δex7/8 neurons are less equipped to withstand inflammatory insults generated by activated microglia. In addition, elevated glutamate has been demonstrated by MR spectroscopy in the brains of JNCL patients as well as CLN3 KO mice (Salek et al. 2011) and neuronal loss during the disease has been attributed, in part, to glutamate-induced excitotoxicity (Kovacs et al. 2006, 2011; Finn et al. 2011). Therefore, a better understanding of the inflammatory feedback loop and interactions between these factors may reveal novel therapeutic mechanisms to delay JNCL disease progression.
Interestingly, unlike the heightened pro-inflammatory responses observed in CLN3Δex7/8 microglia following treatment with C6 ceramide and neuronal lysates, TNF-α, or IL-1β, this was not the case when CLN3Δex7/8 microglia were exposed to the gram-negative cell wall component LPS, where cells were significantly less responsive compared to WT microglia (Fig. 1 and data not shown). Microglia were treated with a combination of LPS and ATP to provide the requisite ‘signal 1 and signal 2’, respectively, for inflammasome activation (Ferrari et al. 1997). It is currently not clear why LPS and ATP induced opposite results compared to the other stimuli tested here; however, it is important to note that LPS does not represent a stimulus that microglia would encounter during the course of JNCL. We are planning to exploit these differences in future studies to identify disparities in signal transduction pathways that may shed new light on novel disease mechanisms. Interestingly, there is correlative evidence to suggest that JNCL patients are more susceptible to infections (Castaneda et al. 2008); however, no studies have been performed in CLN3 mouse models to determine whether peripheral inflammation leads to perturbations within the CNS during disease.
Based on the currently available data in mouse models of JNCL, it is envisioned that microglial activation precedes neuronal deficits because activated microglia are observed as early as post-natal day 7 in CLN3 mouse models, yet neuronal death is not evident until 5–7 months of age (Pontikis et al. 2004, 2005). Therefore, it is likely that this early microglial response affects the CNS homeostatic milieu and, by extension, neuronal viability. The precise mechanism whereby CLN3Δex7/8 microglia affect neuronal integrity/function remains unknown; however, the primed pro-inflammatory state of CLN3Δex7/8 microglia identified in this study, along with the proposed opening of microglial hemichannels by cytokines, such as TNF-α, may disturb neurons via excessive glutamate release. Indeed, we show here that constitutive caspase-1 activity in CLN3Δex7/8 microglia is involved in preventing glutamate release, and glutamate excitotoxicity is a leading mechanism to account for neuronal loss in JNCL (Kovacs and Pearce 2008; Finn et al. 2011; Kovacs et al. 2011). Therefore, although we cannot conclude at the present time that the primed inflammatory state of CLN3Δex7/8 microglia described in this report is a causal factor leading to neuronal demise in vivo, this possibility is supported by our in vitro studies where conditioned medium from CLN3Δex7/8 microglia induced the death of CLN3Δex7/8 neurons.
Collectively, these studies suggest that CLN3Δex7/8 microglia exist in a primed state as evident by constitutive caspase-1 activation and exaggerated pro-inflammatory responses to stimuli that microglia encounter in the JNCL brain. Caspase-1 appears to play a central role in these responses, as inhibiting the enzyme has broad-reaching implications on cytokine/chemokine production, glutamate release, and hemichannel activity. Therefore, it is intriguing to consider the potential of targeting caspase-1 activity as a potential novel JNCL therapeutic; however, the complex nature of this disease will likely require intervention with compounds that can simultaneously affect multiple aberrant pathways to achieve maximal improvements in survival and quality-of-life for children suffering from this fatal neurodegenerative disease.