DAMPs and neurodegeneration.

G Model ARR-547; No. of Pages 12

ARTICLE IN PRESS Ageing Research Reviews xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Ageing Research Reviews journal homepage: www.elsevier.com/locate/arr

Review

DAMPs and neurodegeneration John Thundyil a,∗ , Kah-Leong Lim a,b,c,∗ a

Neurodegeneration Research Laboratory, National Neuroscience Institute, Singapore, Singapore Duke-NUS Graduate Medical School, Singapore, Singapore c Department of Physiology, National University of Singapore, Singapore, Singapore b

a r t i c l e

i n f o

Article history: Received 1 August 2014 Received in revised form 6 November 2014 Accepted 16 November 2014 Available online xxx Keywords: DAMPs Neurodegeneration Neuroinflammation PRRs Inflammasomes

a b s t r a c t The concept of neuroinflammation has come a full circle; from being initially regarded as a controversial viewpoint to its present day acceptance as an integral component of neurodegenerative processes. A closer look at the etiopathogenesis of many neurodegenerative conditions will reveal a patho-symbiotic relationship between neuroinflammation and neurodegeneration, where the two liaise with each other to form a self-sustaining vicious cycle that facilitates neuronal demise. Here, we focus on damage associated molecular patterns or DAMPs as a potentially important nexus in the context of this lethal neuroinflammation-neurodegeneration alliance. Since their nomenclature as “DAMPs” about a decade ago, these endogenous moieties have consistently been reported as novel players in sterile (non-infective) inflammation. However, their roles in inflammatory responses in the central nervous system (CNS), especially during chronic neurodegenerative disorders are still being actively researched. The aim of this review is to first provide a general overview of the neuroimmune response in the CNS within the purview of DAMPs, its receptors and downstream signaling. This is then followed by discussions on some of the DAMP-mediated neuroinflammatory responses involved in chronic neurodegenerative diseases. Along the way, we also highlighted some important gaps in our existing knowledge regarding the role of DAMPs in neurodegeneration, the clarification of which we believe would aid in the prospects of developing treatment or screening strategies directed at these molecules. © 2014 Published by Elsevier B.V.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DAMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Danger hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Defining DAMPs and types of DAMPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. DAMP receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Toll-like receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Receptor for advanced glycation end product (RAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. The nucleotide-binding oligomerization domain receptors or NOD-like receptors (NLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. DAMP-mediated Inflammasome signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. NLRP1 inflammasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. NLRP3 inflammasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DAMPs in chronic neurodegenerative pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. HMGB1 in chronic neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. HMGB1 in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. HMGB1 in PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. HMGB1 in Huntington’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding authors at: Neurodegeneration Research Laboratory, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore. Tel.: +65 63577520. E-mail addresses: John LJ [email protected] (J. Thundyil), Kah Leong [email protected], [email protected] (K.-L. Lim). http://dx.doi.org/10.1016/j.arr.2014.11.003 1568-1637/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Thundyil, J., Lim, K.-L., DAMPs and neurodegeneration. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.11.003


ARTICLE IN PRESS J. Thundyil, K.-L. Lim / Ageing Research Reviews xxx (2014) xxx–xxx

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3.2.

4. 5.

S100 proteins in chronic neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. S100B in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. S100B in PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Circulating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Cell free DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Uric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Adenosine triphosphate (ATP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Closing thoughts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The dramatic increase in average life expectancy is undoubtedly one of the greatest wonders of the last century. With it however, has emerged the problem of age-related pathologies; many of these affecting the central nervous system (CNS) (Cai et al., 2012; Forman et al., 2004). In acute or chronic age-related CNS disorders, the most common denominator is the presence of a neurodegenerative process (Amor et al., 2010). Although the etiology of the majority of these diseases remains unclear, it is generally accepted that the degenerative process involves a myriad of shared pathological events including oxidative stress, mitochondrial dysfunction, excitatory toxicity, and protein aggregation (Campbell, 2004). Another important neurodegenerative mechanism that has gained a lot of scientific traction over recent years is neuroinflammation (Czirr and Wyss-Coray, 2012). In essence, neuroinflammation comprises of a dazzling tapestry of molecular events synchronized within the CNS that are activated in response to noxious stimuli to help the system cope with the deleterious insults (Minghetti, 2005). These events include the activation of microglia and astrocytes, and the release of cytokines. However, during the process of dealing with the harmful stimuli, neuroinflammation may cause collateral damage and result in neurodegeneration (Hohlfeld et al., 2007). Notably, proteinopathies associated with neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) are capable of activating several pro-inflammatory factors within the CNS (Cahill et al., 2009). It is thus reasonable to surmise that the activation of such inflammatory factors could contribute to the progression of the disease by inciting an immune response (Amor et al., 2010; Benarroch, 2013). The fact that activation of the innate immune systems seems to be a common denominator among pathophysiologically divergent diseases like AD, PD and multiple sclerosis (MS), also ratifies this proposition (Wilms et al., 2007; Zipp and Aktas, 2006). Further supporting this, studies conducted in animal models of neurodegeneration and post-mortem brain samples from patients suffering from neurodegenerative disorders often revealed the presence of activated microglia and the accumulation of inflammatory mediators at the lesion sites, which suggests a continuous crosstalk between the brain immune system and the injured neurons during neurodegeneration (Griffin, 2006; Heneka and O’Banion, 2007). Although microglial activation typically occurs as a response to contain the initial triggering insult, their continued presence in large numbers around the lesion areas may actually do more harm than good to neuronal survival (Liu and Hong, 2003). Indeed, the removal of these chronically activated microglia was shown to prevent neurons from progressive degeneration (Liu and Hong, 2003). Further mechanistic studies revealed that the mere sustenance of microglia in their activated states is sufficient for the progression of neuronal death despite the absence of the initial triggering insult (Gao et al., 2011b). Corroborating with this, the degeneration of nigral

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dopaminergic neurons (i.e. neuronal population that are selectively lost in PD) in experimental mouse models of PD ensues long after the clearance of the initial triggering insults [e.g. 1-methyl4-phenyl 1,2,3,6 tetrahydropyridine (MPTP)] amidst the persistent presence of activated microglia (Gao et al., 2011a, 2011b). All these observations implicate microglial activation as a potential contributor of progressive neurodegeneration (Czirr and Wyss-Coray, 2012). However, the pertinent question to ask is what regulates the sustained microglial activation in the absence of the initial triggering insult? A reasonable conjecture is that certain inflammatory factors being released by the dying neurons and/or actively secreted from the activated microglia aid in maintaining the vicious cycle between activated microglia and the damaged neurons (Gao and Hong, 2008; Gorman, 2008). In this regard, damage associated molecular patterns or DAMPs fit well into fulfilling such a role and may represent an important cog in the wheel of chronic progressive neurodegeneration. 2. DAMPs 2.1. Danger hypothesis Although Matzinger coined the term “DAMP” in 2004, its concept was presaged about a decade back in the “danger hypothesis” (Matzinger, 1994, 1998, 2002). The hypothesis basically suggests that the body is capable of launching an immune reaction in response to both an infective stimuli as well as a sterile tissue injury (Land and Messmer, 2012). During a sterile (non-infective) tissue injury, the damaged or dying cells release endogenous molecules that are regarded as “danger signals” by the host’s immune mechanisms. These signals in turn activate the immune system in a fashion analogous to the microbe-associated molecular patterns (MAMPs), also known as PAMPs (pathogen-associated molecular patterns), that are released by pathogenic bacteria or viruses (Bianchi, 2007; Harris and Raucci, 2006). Thus, regardless of the source of tissue injury, the immune system always stages a physiological inflammatory response once it is engaged. This concept formed the basis of future developments in the field of DAMP-mediated neuroinflammatory mechanisms. 2.2. Defining DAMPs and types of DAMPs DAMPs are classically defined as nuclear or cytosolic molecules released extracellularly in response to a non-infectious stimulus (sterile inflammation) that are then capable of triggering and perpetuating an immune response in the body (Tang et al., 2012). However, DAMPs can also act intracellularly. There is a considerable amount of heterogeneity between different DAMP moieties, based on their cellular origin of release (epithelial or mesenchymal) and/or injured tissue. Protein molecules that




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activation by DAMPs engagement (Fig. 1A and B). Although the activation of some of these PRRs is known to play protective roles in host defense against danger, their aberrant activation can contribute in accentuating inflammation. Toll-like receptors (TLRs), Interleukin-1 receptor (IL-1R) and receptor for advanced glycation end product (RAGE), the nucleotide-binding oligomerization domain receptors or NOD-like receptors (NLRs) are some of the more classical DAMP receptors (Burns et al., 2003; Okun et al., 2009; Ramasamy et al., 2009) (Fig. 1B). The TLRs and RAGE are membranebound surface receptors that sense extracellular DAMP moeities, whereas the NLRs are located intracellularly that sense intracellular DAMP signals. Some other examples of intracellular PRRs include the RNA-sensing, RIG-I (retinoic acid-inducible gene I)-like receptors (RLRs; RLHs) or DNA-sensing, AIM2 (absent in melanoma 2) receptors.We discuss briefly a few of these PRRs below. 2.3.1. Toll-like receptors (TLRs) TLRs have been shown to induce and amplify the inflammatory reaction in response to infective pathogens and endogenous molecules alike. TLR2 and TLR4 signaling are known to mediate Nuclear Factor Kappa B (NF-␬B) activation initiated by HMGB1 and serum amyloid A (SAA). The different TLR signaling pathways involved may cross talk at several levels, but all culminate in the activation of NF-␬B (Arroyo et al., 2011; Arumugam et al., 2009). 2.3.2. Receptor for advanced glycation end product (RAGE) RAGE is a multi-ligand receptor, belonging to the immunoglobulin superfamily and is expressed on macrophages, neurons, endothelial cells and a variety of tumor cells. It interacts with a variety of DAMPs, including AGE (advanced glycation end products), HMGB1, S100 proteins and ␤-amyloid (extracellular protein aggregate associated with AD). Stimulation of RAGE induces the activation of NF-␬B and the mitogen-activated protein kinases (MAPKs) like extracellular signal-regulated kinases 1/2 (Erk1/2) and p38 MAPK. RAGE receptors have the unique ability to both initiate and perpetuate the inflammatory immune responses (Chavakis et al., 2003).

Fig. 1. DAMP-mediated signaling. (A) Various DAMPs are released from different sub-cellular components of the neuron following injury to the neurons, which further activate their respective PRRs leading to downstream activation of pro-inflammatory cascades and augmenting neuroinflammation during chronic neurodegeneration. (B) Activation of various PRRs by DAMPs lead to downstream activation of inflammatory mediators; either via the MAP-kinase, NF␬B or inflammasome pathways, which promote cell death and contribute to neurodegenerative mechanisms. (C) Following a cue from DAMPs and consequent priming step of TLR activation the various molecular components (NLRs, Caspase-1, ASC) combine together to form the multiprotein inflammasome complex.

serve as DAMPs include the intracellular proteins like heat-shock proteins, HMGB1 (high-mobility group box 1) and hyaluron fragments (generated from the extracellular matrix following tissue injury). Non-protein DAMPs include Adenosine Triphosphate (ATP), uric acid, heparin sulfate and even DNA materials (Bianchi, 2007; Tang et al., 2012) (Fig. 1A). 2.3. DAMP receptors Typically, the receptors engaged by DAMPs include a group of cellular receptors referred to as pattern recognition receptors (PRRs). PRRs serve as key molecular links between tissue injury and inflammation, by mediating downstream actions following their

2.3.3. The nucleotide-binding oligomerization domain receptors or NOD-like receptors (NLRs) These are intracellular sensors of MAMPs and DAMPs that mediate innate immune inflammatory responses associated with cell stress. They are expressed by several immune and non-immune cells and are subdivided into different classes based on their structures and phylogenetic relationships. The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing receptors (NLRPs) belong to this family of receptor proteins (Lamkanfi and Dixit, 2009). NLR activation promotes the further downstream activation of NF-␬␤ or MAPK signaling pathways consequently leading to the production of cytokines and chemokines. In addition, they play a vital role in the formation of several inflammasome complexes. 2.4. DAMP-mediated Inflammasome signaling DAMP-mediated inflammasome signaling involves the downstream activation of a group of multimeric protein complexes known as inflammasome, which triggers the activation and cleavage of the pro-inflammatory caspase-1, and consequent release of pro-inflammatory cytokines like Interleukin-1␤ (IL-1␤) and Interleukin-18 (IL-18) . Structurally, the inflammasome comprises of an inflammasome sensor moiety, an adaptor protein—apoptosisassociated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 (de Rivero Vaccari et al., 2014) (Fig. 1C). A vast majority of inflammasome sensors, which are




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essentially cytosolic PRRs, belong to the members of the NLRP family (Vajjhala et al., 2012). These include the NLRP1, NLRP3, NLRP6, NLRP7, NLRP12 or NLRC4 (Ting et al., 2008). Other inflammasome sensors molecules include pyrin and HIN domain-containing protein (PYHIN) family members, AIM2 and IFN␥-inducible protein 16 (IFI16) and RIG-I (Proell et al., 2013). The adaptor protein ASC consists of two death-fold domains; the pyrin and the CARD (caspase activation and recruitment domain) domains (Fernandes-Alnemri et al., 2007). The pyrin domain facilitates interaction with the upstream inflammasome sensor molecules. This interaction facilitates the assembly of ASC dimers into a large multimeric protein (Fernandes-Alnemri and Alnemri, 2008). The CARD domain on the other hand, promotes the close positioning of ASC with monomers of pro-caspase-1 (the precursor molecule of active caspase-1), thereby initiating caspase-1 self-cleavage and the formation of the active caspase-1 (Proell et al., 2013). Active caspase-1 plays a vital role in the initiation of pyroptosis, which is a rapid form of cell death following caspase-1 activation. Morphologically, the cellular attributes of pyroptosis bear semblance to those of apoptosis (such as DNA fragmentation) and necrosis (such as cell swelling and rupture). A range of substances that emerge during infections, tissue damage or metabolic imbalances can trigger the formation of inflammasome complexes (Martinon et al., 2009). Once activated, the inflammasome sensor molecules binds to pro-caspase-1, either homotypically via its own CARD domain or via the CARD of the adaptor protein ASC (Chakraborty et al., 2010). Caspase-1 then assembles into its active form consisting of two heterodimers with a p20 and p10 subunit each. Once activated, caspase-1 proteolytically activates a number of pro-inflammatory cytokines like pro-IL-1␤ and pro-IL-18 (Freche et al., 2007). These potent pro-inflammatory cytokines are controlled by two important checkpoints: the transcription stage and its maturation/release stage (Dinarello, 2009). While the transcription of pro-IL-1␤ is mediated via NF-␬B induction, pro-IL-18 is constitutively expressed and demonstrates increased activity following cellular activation (de Rivero Vaccari et al., 2014) (Fig. 1B). Following their activation, the members of the IL-1␤ cytokine family promotes the recruitment and the activation of other immune cells, such as neutrophils, at the site of infection and/or tissue damage (Lamkanfi et al., 2011). Thus, analogous to the apoptosome that activates apoptotic cascades, the inflammasome activates an inflammatory cascade. The exact composition of an inflammasome and its downstream activation is dependent on the activator that initiates inflammasome assembly (Becker and O’Neill, 2007). A descriptive mention of each of the inflammasome cascade is beyond the scope of this review. However, a brief mention about activation of NLRP1 and NLRP3 inflammasomes is relevant in the context of chronic neurodegenerative pathologies. 2.5. NLRP1 inflammasome The neuronal NLRP1 inflammasome comprises of caspases1 and -11, NLRP1, the adaptor protein ASC and the X-linked inhibitor of apoptosis protein (XIAP). Assembly and activation of the NLRP1 inflammasome eventually leads to the maturation and secretion of IL-1␤ and IL-18 (de Rivero Vaccari et al., 2008, 2009). Once secreted, these cytokines initiate inflammatory processes throughout the CNS. These cytokines have been reported to contribute to the pathology of different neurodegenerative diseases such as AD and PD (Chiarini et al., 2006; Di Bona et al., 2008; Koprich et al., 2008). However, the involvement of NLRP1 activation in these pathologies still remains unclear. Interestingly, Mawhinney et al. showed that aging induced increased expression and altered cellular distribution of critical components of the NLRP1 inflammasome in hippocampal neurons. Increased levels of inflammatory cytokines (IL-1␤ and IL-18), along with elevated

levels of different components of NLRP1 inflammasome including caspase-1, caspase-11 and XIAP, were found in the hippocampal protein lysates of aged animals as compared to the younger ones. Furthermore, these changes also corresponded to age-related cognitive deficits in spatial learning in the aged animals (Mawhinney et al., 2011). These findings suggest that aging-related activation of the NLRP1 inflammasome and its resultant inflammation may contribute to age-related cognitive decline in the growing elderly population. Conceivably, the inhibition of the heightened NLRP1 inflammasome activity induced by the natural aging process may be beneficial in impeding memory impairment. Overall, the findings by Mawhinney and colleagues provided a good roadmap for further investigations using age-related disease models. 2.6. NLRP3 inflammasome The best characterized neuronal inflammasome is the NLRP3 (also known as NALP3 and cryopyrin). NLRP3 oligomerization is activated by a large number of stimuli. In addition, its activity has been shown to be induced and/or increased by low intracellular potassium (K+ ) concentrations. Pore-forming toxins and ATP-activated pannexin-1 may trigger K+ efflux and result in a reduction of its intracellular level and/or grant access of toxins into the cell to directly activate NLRP3. In addition, MAMPs like viruses, bacterial toxins, and most notably molecules associated with stress or danger, including crystalline and particulate substances such as monosodium urate (MSU), alum, silica and asbestos can also activate NLRP3 (Menu and Vince, 2011). NLRP3 activation is unique among the NLRs in that its basal expression is not sufficient for inflammasome activation in resting cells. Its activation involves two distinct signals: The first signal includes the cellular priming and upregulation of NLRP3 and pro-IL-1␤ expression via Toll-like receptor activation. The second ‘activation checkpoint’ results in the assembly of the NLRP3 inflammasome, caspase-1 activation and IL-1␤ secretion (de Rivero Vaccari et al., 2014; Martinon et al., 2006; Pelegrin and Surprenant, 2007). Several mechanisms seem to play a role in the assembly of the NLRP3 inflammasome. These include membrane damage leading to release of DAMPs, the efflux of intracellular potassium and the generation of reactive oxygen species (ROS) (Zheng et al., 2014). Notably, several groups have reported higher expression of IL1␤ in microglia surrounding ␤-amyloid plaques associated with AD (Apelt and Schliebs, 2001; Lue et al., 2001), suggesting the possible involvement of NLRP3 in AD pathogenesis. The participation of NLRP3 inflammasome in these AD-related microglia was later demonstrated Halle et al. who also showed that NLRP3 activation leads to the production and secretion of IL-1␤ that in turn triggers further microgliosis resulting in an apparent vicious inflammatory cycle (Halle et al., 2008). An elegant study reported recently by Heneka et al. provided to date the clearest support for the role of NLRP3 inflammasome in AD (Heneka et al., 2013). In this study, the authors found that APP/PS1 mice (a model for AD), when crossed with NLRP3-deficient (as well as capase-1 null) mice, are largely protected from the loss of spatial memory and other cognitive deficiencies that are otherwise exhibited by APP/PS1 mice alone. Correlating with this improved phenotype, they also found that NLRP3 deficiency reduces the ␤-amyloid load and at the same time increases the phagocytosis of ␤-amyloid by microglia. These findings suggest that blocking NLRP3 inflammasome activity may be a rational therapeutic approach for AD. 3. DAMPs in chronic neurodegenerative pathologies Several endogenous molecules serve as DAMPs in the CNS and mediate innate immune responses by engaging PRRs on local




CNS cells. These include high mobility HMGB1, HSPs, uric acid, chromatin, adenosine and ATP, galectins , thioredoxin, surfactant proteins A and D, hyaluronan, fibrinogen and aggregated, modified or misfolded proteins such as ␤-amyloid, alpha synuclein (␣-synuclein) and microtubule associated protein-tau, mutant huntigtin and mutant superoxide dismutase (SOD1). However, only some of them have been reported in the pathology of chronic neurodegenerative conditions. We discuss below some of these molecules that are pertinent to CNS pathologies. 3.1. HMGB1 in chronic neurodegeneration The high-mobility group box (HMGB) proteins are non-histone nuclear proteins that act as DNA chaperones to regulate various chromatin processes such as DNA transcription and replication. They are so named so because of their high mobility in electrophoretic polyacrylamide gels. The HMGB family consists of three members – HMGB1, HMGB2 and HMGB3 – that share about 80% amino acid homology with each other. Amongst the three members, HMGB1 is the most highly conserved through evolution. The HMGB1 protein sequence is 99% identical among mammalian species with only two amino acid residues (out of 215) being substituted between its rodent and human versions. Structurally, HMGB1 has two DNA-binding domains—the A-box (a.a. 1–79) and the Bbox (a.a. 89–163) and a highly acidic, repetitive C-terminal tail (a.a. 186–215). Although HMGB1 is a nuclear protein, it can be secreted into the extracellular milieu as a signaling molecule when cells are under stress as a cytokine mediator of inflammation. The cytokine-inducing part of the HMGB1 molecule comprises of the first 20 amino acids of the B-box domain (Ulloa and Messmer, 2006). HMGB1 can also be found in the cytosol. It may be fair to consider HMGB1 as the prototypical DAMP within the CNS, as it is perhaps the most extensively studied DAMP within the context of CNS pathologies. HMGB1 is ubiquitously expressed in neurons and glial cells (Fang et al., 2012). Functionally, neural HMGB1 either serves as a nuclear factor important for the regulation of DNA architecture or when secreted, as an inflammatory factor or DAMP. The architectural roles of HMGB1 are assumed early on during development, where its complex temporal and spatial distribution pattern within the CNS facilitates neurite outgrowth and cell migration. During adulthood however, HMGB1 acts as a danger signal and promotes neuroinflammation following injury in the spinal cord or the brain (Fossati and Chiarugi, 2007). To signal as an inflammatory mediator, HMGB1 must be released extracellularly. This extracellular release is primarily carried out by two principally different mechanisms: the first being an active secretion mode from living inflammatory cells or secondly being passively released from necrotic cells. The passive release of HMGB1 acts as an early initiator of inflammation whereas its active secretion via living cells like macrophages helps promote the inflammation. Hence, HMGB1 bears a unique ability to both initiate neuroinflammation and also perpetuate its progress. Within the context of chronic neurodegenerative disorders, the roles of HMGB1 are still being explored continually (Degryse and de Virgilio, 2003; Erlandsson Harris and Andersson, 2004; Gardella et al., 2002; Lotze and Tracey, 2005; Sun and Chao, 2005). We discuss the role of HMGB1 in a few of these diseases below. 3.1.1. HMGB1 in AD Consistent with the pathogenic role of HMGB1 in AD, the level of the protein is significantly increased in both the cytosolic and particulate fractions of AD brains (Takata et al., 2004). Further, HMGB1 tends to co-localize with amyloid beta (A␤) in senile

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plaques associated with activated microglia. In rat hippocampi injected with kainic acid and A␤42 (i.e. the toxic form of A␤), HMGB1 immunoreactivity is detected in senile plaques formed by A␤42 and around dying neurons along with activated microglia. When these rats were co-injected with HMGB1 and A␤42, HMGB1 not only mediated the oligomerization of A␤42, but also delayed its clearance. This delay in clearance was the result of HMGB1 induced inhibition of microglial A␤42 phagocytosis. These results suggest that HMGB1 promotes A␤ toxicity by causing a dysfunction in microglial phagocytosis and thereby, accelerating neurodegeneration in these rats (Takata et al., 2004). Not surprisingly, mice injected with HMGB1 intracerebroventricularly display impairments in encoding for long term memory (Mazarati et al., 2011). Interestingly, this phenotype may be rescued by the introduction of a TLR-4 antagonist in RAGE-deficient mice, suggesting that TLR-4 and RAGE are responsible for HMGB1-mediated memory impairments. In another rodent model of AD (Tg2576 mice), Jang et al. (2013) showed that deletion of the p35 gene, which codes for an activator of CDK5 (cyclin-dependent kinase 5), leads to higher mortality rates and impaired spatial learning and memory at 6 months of age. Immunohistochemical and biochemical analyses showed a dramatic increase in the number of microglial cells in the region of the hippocampus, which resulted in an elevated secretion of the soluble HMGB1 in response to A␤. HMGB1 was found to accelerate ER-mediated stress and consequent cell death in the p53 KO/Tg2576 double mutant mice. Taken together, these findings suggest that secretion of HMGB1 by activated microglia in response to A␤ promotes neuronal death, synaptic destruction in the hippocampus, thereby leading to pronounced behavioral deficits.

3.1.2. HMGB1 in PD Abnormal accumulation of ␣-synuclein (SNCA) filaments in Lewy bodies is a neuropathological hallmark of PD and its sequestration into these protein aggregates has been shown to contribute to the degenerative process. HMGB1 preferentially binds to aggregated ␣-synuclein and shows positive co-localization with ␣-synuclein in Lewy bodies from post-mortem PD brain samples (Erlandsson Harris and Andersson, 2004; Fang et al., 2012; Gao et al., 2011b). Because of its known function in the nucleus, the sequestration of HMGB1 within ␣-synuclein aggregates suggests that ␣-synuclein could potentially disturb cellular gene expression. As mentioned earlier, besides its nuclear functions, HMGB1 also has roles to play in the cytosol and one of these is being a regulator of autophagy. Cytosolic HMG-B1 binds to the autophagy protein Beclin1 and displaces Bcl-2 (whose interaction with Beclin serves to inhibit autophagy) (Tang et al., 2010). Interestingly, SNCA and its rare mutations have also been implicated as culprit proteins in autophagy dysregulation in PD. In a cultured cell model, Song et al., demonstrated that overexpression of both wild-type (WT) and mutated SNCA (A53T) inhibits autophagy in a time-dependent manner (Song et al., 2014). Investigations into the underlying mechanisms showed that SNCA binds to cytosolic and nuclear HMGB1. This binding impaired the cytosolic translocation of HMGB1, consequently blocking HMGB1-Beclin1 binding and strengthening Beclin1–Bcl2 association, which leads to autophagy inhibition. While siRNA knockdown of HMGB1 in these cells inhibits basal autophagy, its overexpression restores autophagy, thus suggesting that SNCA-induced impairment of autophagy in PD may partly be dependent on HMGB1. On the other hand, HMGB1 obviously could promote neuroinflammation in the PD brain. Attempts to examine the inflammatory mechanisms incited by HMGB1 in animal models of PD revealed an interaction between HMGB1 and Mac1, a microglial PRR. Furthermore, HMGB1–Mac1–NADPH oxidase signaling axis assisted




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chronic inflammation and progressive dopaminergic neurodegeneration, hinting at the possibility that HMGB1 perhaps bridges the gap between facilitating persistent neuroinflammation and mediating chronic neurodegeneration (Lindersson et al., 2004; Song et al., 2014; Zhang et al., 2013a, 2013b). 3.1.3. HMGB1 in Huntington’s disease In Huntington’s disease (HD) models, HMGB1 seems to protect neurons against the toxicity of polyglutamine (polyQ) repeats. Apparently, HMGB1 also exhibits chaperone-like activity. It directly interacts with polyQ repeats and reduces their propensity to aggregate (Min et al., 2013). Interestingly, proteome analysis of soluble nuclear proteins prepared from neurons expressing mutant huntingtin or ataxin-1 (another polyQ containing protein) revealed a significant reduction in the level of HMGB1 (and HMGB2). Further analysis by immunohistochemistry demonstrated that the reduction of HMGB proteins occur in the nuclear region outside of inclusion bodies in affected neurons. Importantly, ectopic expression of HMGBs mitigated the pathology associated with polyQ in both primary neurons and Drosophila polyQ models (Qi et al., 2007). Taken together, it appears that the role of HMGB1 in neurodegenerative diseases less than straightforward. In some instances, it could serve as a risk factor for the disease while in other instances it has protective functions. However, in all of these diseases, HMGB1 has the capacity to assume a pro-inflammatory role. Understanding the function of HMGB1 in different contexts would thus be important in positioning it as a potential therapeutic target for neurodegenerative diseases. 3.2. S100 proteins in chronic neurodegeneration S100 proteins, also known as calgranulins are small (10–12 kDa), calcium-binding proteins that are expressed exclusively in vertebrates. First identified by B.W. Moore in 1951, the S100 family currently consists of 24 members that are characterized into three subgroups, i.e. those that only exert intracellular regulatory effects, those that mainly exert extracellular regulatory effects and the third that have both intracellular and extracellular functions. Hence, these proteins are not just restricted to the intracellular milieu; rather they are also secreted and/or released extracellularly in a paracrine or autocrine manner where they act to regulate target cell functions. As alluded to earlier, these proteins are calcium sensors that modulate biological activity via calcium binding. They are expressed in a diverse spectrum of tissues where they regulate several physiological cellular functions such as cell growth and differentiation, structural organization of membranes, dynamics of cytoskeleton constituents, and protection from oxidative cell damage, protein phosphorylation and secretion (Deloulme et al., 2002; Donato et al., 2013; Heizmann et al., 2002; Schafer and Heizmann, 1996). During pathological states, some of the S100 family members are secreted extracellularly where they function as alarmins or DAMP factors that principally mediate functions of the innate immune systems. Examples include the S100A8, S100A9, and S100A12, S100B that are secreted at sites of inflammation (Donato et al., 2009). With regards to pathological conditions in the CNS, information about the regulation of expression of most S100 proteins is fragmentary. The S100B is one of the S100 family members that are documented to exert its actions in the CNS. It is secreted or released principally from astrocytes (Van Eldik and Wainwright, 2003). Depending on its concentration, it either has trophic or toxic effects on neurons, astrocytes and microglia. In the nanomolar range, S100B mediates neurotrophic actions protecting neuronal

cells against toxic stimuli by stimulating the p42/44 MAP kinase and/or NF-␬B-mediated upregulation of the anti-apoptotic Bcl-2 gene (Sorci et al., 2010). On the other hand, micromolar doses lead to lethal effects on neuronal integrity via excessive ERK1/2 stimulation and ROS production and/or potentiation of neurotoxic effects of ␤-amyloid, mediated by RAGE receptor activation. S100B also promotes inflammatory activities in astrocytes and microglia at high-doses in a RAGE-dependent manner. 3.2.1. S100B in AD Alteration in the level of S100B is associated with AD. In postmortem AD brains, the expression of S100B correlates with the sites of lesion, being highest in the most severely affected regions (Van Eldik and Griffin, 1994). Consistent with this, S100B has been found to associate with plaques. The level of S100B in serum or CSF is also an indirect indicator of the cognitive status of AD individuals. In this case, the relationship is inversely proportional, i.e. patients with higher S100B levels exhibit lower cognitive scores (Chaves et al., 2010). Further supporting a relationship between S100B and AD is the identification of a single nucleotide polymorphism in S100B gene (rs2300403) that is linked to impaired cognitive function and AD (Lambert et al., 2007). Thus, S100B appears to drive AD pathology. Supporting this, S100B null mice were demonstrated to exhibit enhanced spatial and fear memories as well as enhanced longterm potentiation (LTP) in the hippocampal CA1 region, whereas perfusion of hippocampal slices prepared from these mice with S100B reverses the levels of LTP (Kleindienst et al., 2005; Mori et al., 2010). Further, when the S100B gene is ablated in a mouse model of AD, the double mutant mice exhibit regionally selective reductions in plaque deposition (Roltsch et al., 2010). Although the mechanism by which S100B contributes to AD pathogenesis is not well understood, there seems to exist a reciprocal relationship between them whereby S100B expression promotes A␤ biogenesis and tau hyperphosphorylation and its expression is in turn enhanced by A␤ (Esposito et al., 2008). Notwithstanding this, it is likely that the progression of AD is also accelerated by S100B-mediated neuroinflammation. Notably, overexpression of S100B has been shown to accelerate AD-like pathology with enhanced astrogliosis and microgliosis (Businaro et al., 2006). Collectively, these studies suggest that S100B may be a relevant therapeutic target for AD. Indeed, pharmacological inhibition of S100B expression by arundic acid reduces plaque load and gliosis in the hippocampus and cortex of Tg2576 AD mice (Mori et al., 2006). 3.2.2. S100B in PD As with the case in AD, increased S100B protein level is also detected in the brains of PD patients, especially in the degenerating substantia nigra region, and also in the CSF (Sathe et al., 2012). MPTP, which causes neurological and pathological changes comparable to those observed in PD, increases S100B expression in mice treated with the toxin. Conversely, mice ablated of S100B gene exhibit protection against MPTP-induced neurotoxicity that is accompanied by a reduction in microgliosis as well as reduced expression of RAGE and TNF␣ receptor (Sathe et al., 2012). Consistent with this, the culture medium of MPTP-treated astrocytes reduces PC12 neuronal cell viability, an effect counteracted by an S100B neutralizing antibody. Although the above studies suggest that S100B is playing a detrimental role in PD pathogenesis, some studies have also shown that elevated S100B levels can exert neuroprotective effects (Sorci et al., 2010). For example, MPTP-treated mice injected with the antiepileptic drug, zonisamide (which improves PD symptoms) show enhanced S100B expression in astrocytes (Choudhury et al., 2011). This amelioration of clinical signs of the disease was speculated to be due to an




augmented secretion of the neurotrophic S100B. Thus, the verdict regarding the role of S100B in PD remains open. The heterogeneity (i.e. detrimental vs. beneficial) of S100B-mediated effects appears to be dependent on an S100-RAGE activity gradient, which is logical given that S100B effects are dependent on RAGE. So, any change in the activation intensities of RAGE, duration of RAGE stimulation and/or different extents of S100B-induced upregulation of RAGE expression could influence the outcome of S100B-mediated effects (Donato et al., 2013; Sorci et al., 2011). 3.3. Heat shock proteins Heat shock proteins (HSPs) are molecular chaperones that facilitate the proper folding and assembly of nascent polypeptides and mediate the re-folding and stabilization of damaged polypeptides. The HSP family of proteins is classified into six major families: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small HSPs (sHSPs, e.g., ␣B crystallin and Hsp27). The HSPs are constitutively and inducibly expressed in the nervous system and have been shown to function intracellularly (Stetler et al., 2010). The role of HSPs as DAMPs during neurodegeneration has not yet been fully elucidated. Nonetheless, there have been reports implicating them as DAMPs in several tissue injury models (Broere et al., 2011; Ganter et al., 2006; LoCicero et al., 1999). In this regard, the roles of extracellular HSPs, including Hsp72 (Gelain et al., 2011), Hsp27 (HSPB1), Hsp90 (HSPC), Hsp60 (HSPD) and Chaperonin/Hsp10 (HSPE) have been shown to be particularly relevant in sepsis. These studies demonstrated the extracellular release of HSPs from damaged or stressed cells propagated local “danger signals”, thereby activating stress response programs in surrounding cells (Adachi et al., 2009; Giuliano et al., 2011). However, these reports have been met with a fair share of skepticism by the community; the reason being that HSPs were shown to bind to several MAMPs and enhanced TLR ligand stimulation, making the interpretation of published results inconclusive (Broere et al., 2011; Chen and Nunez, 2010; Habich et al., 2005; Osterloh et al., 2007; Warger et al., 2006). In addition, even the pure forms of HSP preparation showed the presence of microbial products. The depletion of microbial products from HSP preparations reduced or completely abolished HSP-induced inflammatory responses thus weakening the role HSPs acting as DAMPs (van Eden et al., 2012). 3.4. Circulating DNA Several studies have previously demonstrated baseline levels of plasma DNA in normal, healthy populations, albeit at very low levels (Jylhava et al., 2011). The level of circulating cell-free (cfDNA) and mitochondrial DNA is however elevated in the plasma of critically ill patients, including those with sepsis, myocardial infarction, trauma and even chronic conditions like cancer. Increased plasma levels of mitochondrial and cf-DNA seem to be inherently linked with underlying systemic inflammatory conditions, oxidative stress or robust tissue damage (Jylhava et al., 2012; Pinti et al., 2014). Potentially, they might also play a role in aging and neurodegeneration. 3.4.1. Mitochondrial DNA Intra-mitochondrial components, including mitochondrial DNA (mt-DNA), N-formyl peptides, and lipids such as cardiolipin, can be released extracellularly, which then enter the blood flow to act as DAMP agents, triggering the same pathways that respond to MAMPs, thereby causing inflammation (Zhang et al., 2010b). Mt-DNA can bind TLR-9 and activate its downstream pathway. Many studies have previously shown that immune cells, including

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monocytes and granulocytes, express TLR-9, and that triggering of TLR-9 in these cells by mt-DNA, induces the release of type I IFNs and TNF-␣, through the activation of IRAK-1, IRAK-2, and IRAK-4 and the phosphorylation of p38 MAP kinase (Hauser et al., 2010; Manfredi and Rovere-Querini, 2010; Zhang et al., 2010a). During aging, the human body is progressively exposed to a variety of antigens. Consequently, the burden to cope up with this life-long antigenic stress accumulates on the body’s defense mechanism—the immune system (Giunta, 2006). The immune system in turn, responds to this aging-related antigenic stress by initiating a chronic low-grade inflammation characterized by mildly elevated plasma levels of pro-inflammatory cytokines. This old-age related inflammatory status is now being recognized as “inflamm-aging” (Zheng, 2014). In order to investigate whether circulating mt-DNA could significantly contribute to the onset of inflamm-aging in human subjects, Pinti et al. evaluated mt-DNA content was evaluated in plasma samples of 800 Caucasian subjects aged between 1 and 104 years (Pinti et al., 2014). They also evaluated a possible genetic correlation to the circulating level of mt-DNA, by comparing their levels in a large cohort of ultra-nonagenarian siblings. Their results showed that mt-DNA plasma levels increased gradually after the fifth decade of life, indicating an age-dependency (Pinti et al., 2014). In the ultranonagenarian cohort, mt-DNA values highly correlated within sibling pairs, highlighting a possible role of genetic background in controlling the levels of circulating mt-DNA. Further evaluation revealed a strong correlation between plasma levels of mt-DNA and pro-inflammatory cytokines in older subjects and 90+ siblings. The subjects with the highest mt-DNA plasma levels had the highest amounts of TNF-␣, IL-6, RANTES, and IL-1R; while the subjects with the lowest mt-DNA levels had the lowest levels of the same cytokines (Pinti et al., 2014). Tests to evaluate the capacity of mt-DNA to stimulate the production of pro-inflammatory cytokines in vitro showed that monocytes stimulated with mt-DNA concentrations similar to the highest levels observed in subjects, secrete increased levels of TNF-␣, thereby suggesting that mtDNA can modulate the production of pro-inflammatory cytokines. Investigations into the suspected PRR mediating these inflammatory signals showed that mt-DNA was able to trigger the pathway downstream of TLR-9 and mediate the release of TNF-␣. Thus, mitochondria not only participate in danger signaling inside the cell, but are also a major source of DAMP molecules able to activate an innate immune response. These findings suggest an age-dependent increase in circulating mt-DNA, which can also be significant contributor toward the low-grade, chronic inflammation observed in elderly people. Moreover targeting TLR-9 receptor signaling and/or interfering with soluble mitochondrial DAMPs could perhaps reduce harmful immune activation during agingrelated pathologies. At the same time, it is also attractive to speculate that mt-DNA-mediated inflammation may trigger neurodegeneration in the brains of susceptible elderly individuals. Supporting this, Mathew et al. recently demonstrated that primary astrocytes with oxidant-initiated degraded mitochondrial polynucleotides, which they termed DeMPs, induce the production of pro-inflammatory cytokines and the activation of the inflammasome. They further showed the presence of DeMPs in human CSF and plasma and that DeMPs are produced in response to oxidative stress (Mathew et al., 2012). 3.4.2. Cell free DNA Circulating cell-free DNA (cf-DNA) is currently thought to arise from apoptotic or necrotic cells and may thus reflect systemic inflammatory conditions or tissue damage. Interestingly, a study by Jylhävä revealed that aging is associated with quantitative and qualitative changes in circulating c-f DNA (Jylhava et al., 2012).




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They observed that the concentration of cf-DNA is significantly higher in aged individuals (>90 years) as compared to younger controls (22–37 years) and that the older group tends to display a fragmented pattern of low molecular weight cf-DNA compared to their younger counterparts, whose cf-DNA was intact and assumed a high molecular weight appearance. More recently, the same group found an association between cf-DNA and inflammation in the nonagenarians but not in the young controls, suggesting that cf-DNA might aggravate immunoinflammatory activity in elderly individuals (Jylhava et al., 2013). Mechanistically, cf-DNA can act as a DAMP by activating TLR and non-TLR receptors. TLR-9 acts as a ubiquitous receptor for endocytosed DNA. Intracellular signal transduction of the DNA-induced TLR9-pathway is mediated through the MyD88 and a series of activation events, resulting in an IFN-regulatory factor 7 (IRF7)-mediated type-I IFN response, or in NF-kB or mitogen-activated protein kinase-dependent cytokine and chemokine production (Barbalat et al., 2011). AIM2 is another PRR capable of recognizing cytoplasmic DNA, independent of TLRsignaling that can lead to caspase-1 mediated pyroptosis. However this list of DNA sensing molecules is still expanding with many uncharacterized DNA sensing PRRs and downstream pathways still waiting to be discovered. Given the presence of a damaged BBB in chronic neurodegenerative conditions, it may be quite possible that the DNA material release from dead neuronal cells may perhaps find its way into systemic circulation and incite an inflammatory reaction by binding and activation of PRRs (McRae and Dahlstrom, 1992; Su and Federoff, 2014). The elevated cf-DNA concentrations in the blood also increase the viscosity of blood and could often stimulate autoimmune response—a phenomenon often encountered in aged individuals (Ellinger et al., 2012; Kohler et al., 2009; Pyle et al., 2010). Future studies should clarify the role of cf-DNA in neurodegeneration.

3.5. Uric acid Uric acid is the final metabolic product of purine metabolism in humans. Normally, uric acid disposal occurs via the kidneys and is excreted in urine. The lack of uricase enzyme makes it impossible for humans to oxidize uric acid to the more soluble compound allantoin. Thus hyperuricemia, defined as high levels of blood uric acid, has been increasingly linked with pathology of several systemic diseases and can be detrimental to the body (Kutzing and Firestein, 2008). For example, uricase gene deficient mice have a 10 fold increase in the serum uric acid level, and are found to have urate nephropathy with infiltration of plasma cells, lymphocytes, and macrophages (Jin et al., 2012). Additionally, monosodium urate (MSU) crystals formed in the blood, as a result of uric acid levels exceeding 6.8 mg/dL, have been associated with inflammatory pathologies like gout, and several vascular diseases. MSU crystals activate the TLRs, which then activate NLRP3 inflammasome (Jin et al., 2012). MSU has been shown to interact with TLR2 and TLR4. Using antibody-blocking and transfection approaches, MSU was shown to interact with TLR2 on chondrocytes to induce nitric oxide (Liu-Bryan et al., 2005b). MSU mediated TLR2/TLR4 activation also led to IL-1␤ production (Liu-Bryan et al., 2005a). MSU also triggers neutrophil activation and further produces immune mediators, which lead to a pro-inflammatory response. Hence, uric acid crystals can acts as DAMP, mediating an inflammatory response in several cardiac, kidney and joint pathologies (Jounai et al., 2012). However, hyperuricemia also seems to have beneficial effects in the nervous system, as evidenced by favorable outcomes observed in some neurological diseases (Romanos et al., 2007). Reduced levels of serum uric acid have been associated with PD, HD, and MS occurrence. Annanmaki et al. (2007, 2008, 2011) showed that patients with PD have significantly lower plasma uric acid levels

when compared with matched controls. In addition, men showed a significant inverse correlation between uric acid levels and disease duration, with lower levels associated with longer duration of PD (Andreadou et al., 2009). These results suggested that patients with hyperuricemic levels seen in gout may perhaps have a protective effect in PD. In a prospective case-controlled study to determine the association between gout and the risk of developing PD, Alonso et al. (2007), Alonso and Sovell (2010) found that individuals with a history of gout have significantly lower risk of developing PD than those without a history of gout. However, this finding was only significant for men, but not for women. De Vera et al. later published that over an 8-year median follow up, there was a 30% reduction in the risk of developing PD in both male and female patients with a history of gout, independent of age, prior co-morbid conditions, and non-steroidal anti-inflammatory drugs (NSAIDs) or diuretic use (Alonso et al., 2007; Alonso and Sovell, 2010; De Vera et al., 2008). In a 6-OHDA PD model in SH-SY5Y cells, Huang et al. (2012) showed that the toxin induced cell injury is attenuated by uric acid. The underlying mechanisms may involve the up-regulation of Akt and the reduction of GSK-3␤ activity. Similarly, Auinger et al. (2010) also found an association between higher serum uric acid levels and slower HD progression (Auinger et al., 2010). In addition, there was a trend of decreased worsening of motor function with increasing uric acid levels, hinting that uric acid may aid as a therapeutic target for the slowing of the motor component of HD progression. However, cognitive, behavioral, and neuropsychological functions did not correlate to uric acid levels. 3.6. Adenosine triphosphate (ATP) ATP is a purine base that mediates almost all physical responses such as glucose metabolism, muscle contraction, biosynthesis, and molecular transfer within the cell. Despite its roles as an almost indispensible intracellular molecule, ATP released extracellularly from dead or injured cells can trigger the activation of NLRP3 and caspase-1 (Communi et al., 2000). In addition, other ion channel molecules, namely, P2X7 and pannexin-1, can also induce extracellular ATP-mediated caspase-1 activation following IL-1␤ maturation (Ferrari et al., 2006; Kanneganti et al., 2006). Although extracellular ATP has been suggested to act as a DAMP molecule, there is no however correlation between high amounts of extracellular ATP acting as DAMPs under physiological conditions in vivo. Eckle et al. (2007) suggested that most extracellular ATP may undergo hydrolysis by ectonucleotidases. Notwithstanding this, extracellular ATP per se is toxic for primary neuronal as well organotypic CNS cultures. Additionally, P2 receptors can also mediate and aggravate hypoxic signaling in many CNS neurons. Taken together, there is a potential role for extracellular ATP to promote neurodegeneration via its DAMP-like functions although this nonconventional function of ATP needs to be examined in more depth. 4. Discussion Normal aging is known to lead to the development of a chronic low-grade inflammatory state within the body (Noyan-Ashraf et al., 2005). Even the immune privileged brain is not spared of this age-associated inflammatory phenomenon, which is likely to precipitate or augment neurodegeneration in susceptible elderly individuals. Clinical studies in elderly patients suffering from infections revealed an increase in the occurrence of deliriums and development of dementia (Wofford et al., 1996). These results point toward an on-going low level inflammatory response in the aged brain that tends to accentuate following an antigenic stimulus (involving MAMPs in the case of an infective state). The consequent amplified production of inflammatory molecules in the brain




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Fig. 2. DAMPs—A nexus in neuroinflammation–neurodegeneration liason. Following its release from injured neurons, DAMPs activate the PRRs on adjacent neurons and microglia which help in sustaining microglial activation and thereby perpetuating the progress of neuroinflammation-neurodegeneration cycle.

may then result in frank neuronal loss leading to cognitive and perhaps other neurological impairments (Barrientos et al., 2006). An analogy to this inflammatory process could perhaps be applied to delineate the contribution of DAMPs during chronic neurodegenerative states. Whilst the ageing process primes the microglia via a mild but sustained inflammatory stimulus, an exposure to stressors, either in the form of environmental toxins or mutationsinduced protein aggregates could then trigger them to launch a potent inflammatory onslaught that could prove detrimental to neuronal health in the afflicted brain regions (Golde et al., 2013; Pan et al., 2014). The common pathological hallmark of a majority of the neurodegenerative conditions is the abnormal accumulation of proteins, some of which can operate as endogenous danger signals or DAMPs. Additionally, other DAMP moieties like HMGB1, S100B, mt-DNA are also elevated in AD and PD and also MS (Donato et al., 2013; Gao et al., 2011b; Heizmann et al., 2002). The majority of these DAMPs incite the release of pro-inflammatory cytokines like IL-1␤ and IL18, thereby further contributing to the neurodegenerative process. IL-1␤ is synthesized and secreted both, by neuronal and glial cells (Lechan et al., 1990). These cells also express PRRs like RAGE, NODs and NLRPs. In addition, ASC, caspase-1 and caspase-11 are present in astrocytes, oligodendrocytes and microglial cells (Mawhinney et al., 2011). Thus, neurons and glial cells possess all the components that could facilitate their engagement in a positive feedback mechanism to potentiate pro-inflammatory responses during neurodegenerative conditions. This mechanism could be initiated with the release of endogenous proteins and cytokines by dead neurons in the affected regions, which could then engage PRRs on neighbouring neurons and microglial cells to incite activation of inflammasome complexes and cause a further release of IL-1␤ and 1L-18 (Fig. 2). Although a number of reports suggest the involvement of inflammasomes (NLRP3 and NLRP1) in chronic neurodegeneration, several important questions still remain unanswered (Mawhinney et al., 2011; Tan et al., 2013). Firstly, how many inflammasome complexes exist within the CNS and what are their specific roles? Secondly, pertaining to their molecular mechanisms of activation; how is the assembly of different inflammasome complexes triggered during neurodegeneration? Furthermore, is there any

crosstalk between different inflammasome complexes? Another facet of DAMP-mediated neuroinflammation is the concept of pyroptosis, which is a form of cell death that is uniquely dependent on caspase-1. Although reports about neuronal death via pyroptosis in vivo are rare, its occurrence in these degenerative states cannot be ruled out. This is because the activation of caspase-1 has been shown in models of chronic neurodegeneration (Friedlander, 2003). Moreover, pyroptotic cell death leads to osmotic swelling and subsequent rupture of the cytoplasmic membrane that result in the release of cytoplasmic contents (Fernandes-Alnemri et al., 2007). This then could perhaps be one of the mechanisms underlying the release of DAMPs in the brain during chronic neurodegeneration. Thus, it is important to investigate the role of caspase-1 in the context of chronic neurodegeneration. As mice deficient only for caspase-1 are now available, the specific contribution of caspase1 to chronic neuroinflammation can now be addressed to some extent. However, owing to the obvious differences between mice and men, particularly at the immunologic level, knock down studies with human (primary) cells could be more relevant in the analysis of the function of caspase-1 in humans. Finally, it is not clear at present whether all the protein aggregates functions as DAMPs and, if so, do they all behave in similar or distinct fashions? Addressing these gaps in our understanding could perhaps facilitate in identifying receptor targets and/or downstream mechanisms that may be a common feature of the downstream degenerative cascades in multiple CNS proteinopathies, which may in turn lead to the development of common therapeutic targets for multiple CNS disorders. 5. Closing thoughts In recent years, much has been learnt about the roles of DAMPs in mediating immune responses during acute and chronic neurodegenerative pathologies. While the basic understanding of these innate immune activators in neurodegeneration is growing, their clinical implications in the context of human patients are still largely unclear. It is perhaps intuitive to devise ways to integrate this newfound knowledge and understanding regarding DAMPs into clinical applications pertaining to chronic neurodegenerative pathologies. In this regard, understanding the implications of DAMP-mediated neuroinflammation could provide us with


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valuable hints at exploring possible diagnostic and therapeutic approaches aimed at mitigating the progress of neurodegenerative pathologies. The mechanistic requirement for intracellular endogenous moieties to be released extracellularly in many cases in order to function as DAMPs provides important opportunities to probe their potential as biological markers in neurodegenerative pathologies. However, the non-specificity of these moieties with regards to the CNS dampens any prospects of developing these DAMPs as serum bio-markers of chronic neurodegenerative pathologies. For instance, S100B, which is normally found in pM amounts in human serum under normal physiological state, is elevated in AD as well as other non-neurological clinical conditions such as cardiac ischemia and extracerebral infections. A similar problem is also seen with the use of HMGB1, uric acid, cell-free and mitochondrial DNA as biomarkers of neurodegeneration. While their elevated levels are indicative of an inflammatory phenotype, they do not specifically imply a neurodegenerative state (or a specific neurodegenerative disorder). This lack of specificity could make the interpretation of elevated levels of DAMPs rather confounding, especially in patients having multiple systemic pathologies. Perhaps their incorporation with other specific disease markers along with a more robust assessment and large patient cohorts may increase their effective use as a diagnostic marker. Aside from being biomarkers, DAMPs may also be positioned as disease targets. Accumulating evidences show that therapeutic strategies that modulate the expression of DAMPs and its downstream signaling could aid in the treatment of neurodegenerative diseases. Small molecule inhibitors or antibodies against circulating mt-DNA, extracellular DAMPs, or microglial PRRs may prove to be novel strategy in therapy directed toward neurodegenerative diseases. However, the majority of these DAMPs tend to assume the “Jekyll and Hyde” roles within the CNS. Whilst they mediate several physiological cellular functions inside the cell, they assume a cytokine-like character with multiple signaling properties (for e.g. HMGB1, S100B) upon their release into the extracellular milieu. The challenge would be to develop specific and powerful inhibitors with an acceptable degree of selectivity and pharmacokinetic profile so that only the harmful extracellular DAMPs are targeted. Another Achilles’ tendon in deriving effective therapies is the impact of these drugs in modulating the innate immune response. The innate immune systems are primarily activated as a defense mechanism. Too much or too little modulation of these responses could prove to be counterproductive in terms of long-term side effects in patients. Hence, a detailed knowledge of DAMP-mediated signaling mechanisms is required to judge the timing and degree of intervention so that maximum hit rate could be achieved with minimum side effects. Acknowledgements This work was supported by grants from the National Medical Research Council-CBRG (0049-2013) and the Translational Clinical Research Programme in Parkinson’s disease. We thank Ms. Hang Liting for illustrations. References Adachi, H., Katsuno, M., Waza, M., Minamiyama, M., Tanaka, F., Sobue, G., 2009. Heat shock proteins in neurodegenerative diseases: pathogenic roles and therapeutic implications. Int. J. Hyperther 25, 647–654 (the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group). Alonso, A., Rodriguez, L.A., Logroscino, G., Hernan, M.A., 2007. Gout and risk of Parkinson disease: a prospective study. Neurology 69, 1696–1700. Alonso, A., Sovell, K.A., 2010. Gout, hyperuricemia, and Parkinson’s disease: a protective effect? Curr. Rheumatol. Rep. 12, 149–155. Amor, S., Puentes, F., Baker, D., van der Valk, P., 2010. Inflammation in neurodegenerative diseases. Immunology 129, 154–169.

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DAMPs from Cell Death to New Life.

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Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death.

DAMPs activating innate and adaptive immune responses in COPD.

Gut mucosal DAMPs in IBD: from mechanisms to therapeutic implications.

The effectors of innate immunity: DAMPs, DAMEs, or DIMEs?

Mortalin, apoptosis, and neurodegeneration.

Metals and neurodegeneration.

Vitamin E and neurodegeneration.

Tau protein and neurodegeneration.

Commentary: neurodegeneration and sport.

Dopamine Receptors and Neurodegeneration.

Autophagy and neurodegeneration.

Neurodegeneration and regeneration.

The Role of Damage-Associated Molecular Patterns (DAMPs) in Human Diseases: Part II: DAMPs as diagnostics, prognostics and therapeutics in clinical medicine.

The role of danger-associated molecular patterns (DAMPs) in trauma and infections.

Neurodegeneration and Neuroprotection in Glaucoma.

Neurodegeneration: etiologies and new therapies.

Defective pantothenate metabolism and neurodegeneration.

Neurodegeneration and mirror image agnosia.

Neurodegeneration and the Circadian Clock.

S100A8 and S100A9: DAMPs at the crossroads between innate immunity, traditional risk factors, and cardiovascular disease.

Fructans As DAMPs or MAMPs: Evolutionary Prospects, Cross-Tolerance, and Multistress Resistance Potential.