Toll-like Receptor Signalling in Neurodegenerative Disease

The innate immune response has come under the spotlight in recent years due to its central role in propagating the pathogenesis of several diseases, and specifically in driving neurodegenerative aetiology (1-3). Toll-like receptors (TLRs), the mammalian homologue of the Drosophila melanogaster Toll, are highly conserved innate immune receptors and master regulators of the cellular innate immune response (4-6). Research by several groups in the early 1990s discovered the pivotal role of TLRs in the initiation and propagation of the inflammatory signalling in response to bacterial, viral or microbial nucleic acids, known as Pathogen Associated Molecular Patterns (PAMPs) or Danger Associated Molecular Pattern (DAMPs) (5, 7, 8), primarily focusing on what is now termed TLR4. To date, 13 murine and 11 human TLRs identified (9, 10), with several intracellularly expressed on endosomes (TLR3, TLR7, TLR8 and TLR9), whereas others are characterized as transmembrane receptors (all other TLRs, including TLR2 and TLR4).

It is widely established that reactive gliosis, a term used to describe the activation of glial cells in response to pathogens (11), is concurrent with neuronal death in the Central Nervous System (CNS) (12-14), with microglial cells eliciting the primary response to pathogenic insult (15, 16) and driving chronic neuroinflammation (17-19). Importantly, microglia highly express TLRs (20, 21). TLR signalling, specifically increased levels of TLR2 and TLR4, is reported in multiple neurodegenerative pathologies (22, 23), with mounting evidence for misfolded protein aggregate induced activation of TLR2 and TLR4 in Alzheimer’s disease (24, 25), Amyotrophic Lateral Sclerosis (2), and Prion disease (26). In the CNS, glial cell crosstalk potentiates a TLR pro-inflammatory feedback loop, with evidence for TLR signaling in initiating the adaptive immune response (27). These studies highlight the importance of TLR-induced inflammation in the chronic neuroinflammatory cascade seen in neurodegenerative aetiology.

All TLRs signal as dimers, with stimulation of TLR2 and TLR4 leading to the recruitment of extracellular co-receptors CD14 (28) and MD2 (TLR4 only) (29), preceding homodimerization (TLR4/TLR4), or heterodimerization (TLR1/TLR2) (30) and subsequent assembly of a cytoplasmic Toll/Interleukin-1 Receptor (TIR) adaptor domain (31-33). The TIR-adaptor domain is comprised of MyD88 (Myeloid Differentiation Primary Response Gene 88), Mal (MyD88 adaptor-like), TRIF (TIR-domain-containing adapter-inducing interferon-β) and TRAM (34-36). MyD88 facilitates downstream signalling in all TLRs, with the exception of TLR3 (37). Mal (also known as TIRAP) bridges MyD88 to TLR2 and TLR4 (38), with TRAM adapting signals from TLR4 to TRIF (39).

Activation of both TLR2 and TLR4 leads to MyD88-dependent signal propagation, whereas TLR4 can additionally initiate a MyD88-independent, also termed TRIF-dependent, signalling pathway, ultimately culminating in the activation of transcription factors, such as NF-κB, Mitogen Activating Protein Kinase (MAPK) and Interferon Regulatory Factor 3 (IRF3), promoting inflammatory gene transcription [reviewed in (21)]. TLR signalling is mediated by a multifaceted network of adaptor proteins, kinases and E3 ubiquitin ligases (40). Ubiquitination is a post-translational modification executed by the formation of polyubiquitin chains on target proteins at different lysine residues modifying the function or stability of the target protein (41-44), and is tightly regulated by a number of E3 ubiquitin ligases that facilitate the polyubiquitination of several critical components inducing TLR-induced pro-inflammatory signalling (42, 45, 46).

MyD88-dependent signalling results in the activation of the Interleukin-1 Receptor Associated Kinase (IRAK) complex at the membrane. IRAK1, a member of the multi-domain protein kinase IRAK complex containing IRAK4, is phosphorylated upon activation which facilitates the ubiquitin- and phosphorylation- dependent activation of the IκB (IKK) kinase complex, leading to NF-κB transcription (47). Both the kinase activity and adaptor functions of IRAK1 facilitate IRAK4-MyD88 interactions, and are essential for TLR-mediated NF-κB activation (48). The E3 ubiquitin ligase TRAF6 induces ubiquitination of the IRAK complex activating NF-κB (41, 49). Additionally, TRAF6 can initiate NF-κB activation by facilitating the recruitment of the TAK1 (Transforming growth factor beta-activated kinase 1) – TAB1 (TGF-Beta Activated Kinase 2) – TAB2 (TGF-Beta Activated Kinase 2) complex (42) and can directly form ubiquitin chains with the IKK subunit NEMO (also referred to as IKKγ) (50).

The association of Pellino proteins and IRAK1, specifically Peli1-induced K63-linked polyubiquitination of IRAK1, facilitates IRAK activation and mediates downstream signalling to NF-κB and MAPK (43, 51). TLR4-induced dissociation and cytoplasmic translocation of the TRAF6-Peli1-IRAK complex from the membrane bound MyD88-receptor complex is essential for MAPK activation, and is regulated by TRAF3 degradation (52). Peli1-mediated K63-linked polyubiquitination of Cellular Inhibitor of Apoptosis (cIAP2) induces the degradation of TRAF3 via cIAP2-mediated K48-linked polyubiquitination of TRAF3 (53).

The presence of a MyD88-independent signalling pathway was first reported using MyD88-/- mice (36) stimulated with Lipopolysaccharide (LPS), a well-defined potent agonist of TLR4 (54, 55), and is mediated through engagement of the TIR adaptor domain protein TRIF (31). TRIF interacts with Receptor interacting Protein 1 (RIP1) and Tumour Necrosis Factor α receptor associated factor 6 (TRAF6), to activate NF-ҡB upon and TLR4 stimulation (56, 57). Both TRAF6 and RIP1 facilitate the polyubiquitination of NEMO eliciting downstream phosphorylation and activation of the IKK complex (58).

Additionally, TRIF initiates TRAF3-dependent signalling to TANK-binding kinase 1 (TBK1) and the IKK subunit IKKε, inducing activation of the IRF3 transcription factor (59). Interestingly, TRAF3 has a differential role in the regulation of the MyD88 and TRIF-dependent pathway dictated by the alternative ubiquitination of TRAF3 (52), negatively regulating MyD88 signalling (53), and positively regulating the TRIF-dependent pathway to IRF3 (52). Peli1 has a critical role in the modulation of TRIF-dependent signalling, with Peli1-deficient mice shown to be resistant to TLR4 induced septic shock (60). Furthermore, it was demonstrated that Peli1 interacts with and promotes the phosphorylation of TBK1 resulting in bidirectional communication and enhanced activation of Peli1 (61, 62).

Targeting the immune system by developing therapeutics for the amelioration of neurodegeneration has been a central focus in recent years; however it has proven difficult due to the ablation of the protective effects associated with the pro-inflammatory response. Recent studies have highlighted the therapeutic potential of targeting TLR4, demonstrating reduced neuronal degeneration upon TLR4 attenuation (63), and misfolded protein induced motoneuron death rescued by TLR4 inhibitors in vitro (64). However, the dual roles of the immune response in pathogenic insult and misfolded proteins need to be considered carefully for further design of immunotherapeutics targeting the CNS. Notwithstanding, TLR signalling holds a fascinating promise for therapeutic modulation of chronic pro-inflammatory signalling in disease.

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