NLRP3 inflammasome in neurodegenerative disease

Abstract

Neurodegenerative diseases are characterized by a dysregulated neuro-glial microenvironment, culminating in functional deficits resulting from neuronal cell death. Inflammation is a hallmark of the neurodegenerative microenvironment and despite a critical role in tissue homeostasis, increasing evidence suggests that chronic inflammatory insult can contribute to progressive neuronal loss. Inflammation has been studied in the context of neurodegenerative disorders for decades but few anti-inflammatory treatments have advanced to clinical use. This is likely due to the related challenges of predicting and mitigating off-target effects impacting the normal immune response while detecting inflammatory signatures that are specific to the progression of neurological disorders. Inflammasomes are pro-inflammatory cytosolic pattern recognition receptors functioning in the innate immune system. Compelling pre-clinical data has prompted an intense interest in the role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in neurodegenerative disease. NLRP3 is typically inactive but can respond to sterile triggers commonly associated with neurodegenerative disorders including protein misfolding and aggregation, mitochondrial and oxidative stress, and exposure to disease-associated environmental toxicants. Clear evidence of enhanced NLRP3 inflammasome activity in common neurodegenerative diseases has coincided with rapid advancement of novel small molecule therapeutics making the NLRP3 inflammasome an attractive target for near-term interventional studies. In this review, we highlight evidence from model systems and patients indicating inflammasome activity in neurodegenerative disease associated with the NLRP3 inflammasome’s ability to recognize pathologic forms of amyloidβ, tau, and α-synuclein. We discuss inflammasome-driven pyroptotic processes highlighting the potential utility of evaluating extracellular inflammasome-related proteins in the context of biomarker discovery. We complete the report by pointing out gaps in our understanding of intracellular modifiers of inflammasome activity and mechanisms regulating the resolution of inflammasome activation. The literature review and perspectives provide a conceptual platform for continued analysis of inflammation in neurodegenerative diseases through the study of inflammasomes and pyroptosis, mechanisms of inflammation and cell death now recognized to function in multiple highly prevalent neurological disorders.

Background and Canonical Mechanisms

NLR Family Pyrin Domain Containing 3 (NLRP3) was first identified as a 514 base-pair human gene similar to the rat angiotensin/vasopressin receptor in CD34⁺ hematopoietic stem-like cells.¹ Subsequently, genetic studies identified a shared linkage at 1q44 in 2 independently hereditable autoinflammatory disorders, Muckle-Wells Syndrome,² and familial cold autoinflammatory syndrome.³ Based on these linkage studies, Hoffman and others used positional cloning to identify mutations in NLRP3 (originally referred to as CIAS1).⁴ These seminal discoveries expanded our understanding of the Cryopyrin-associated periodic syndromes (CAPS) and provided the initial characterization of NLRP3 as a mediator of inflammation and cell death. Over 2 decades, thousands of reports have detailed mechanisms of NLRP3 inflammasome signaling in health and disease. Among them are many outstanding reviews.^5–10 Here we will provide a brief overview of canonical inflammasome signaling to contextualize mechanisms of translational interest in neurodegenerative disease which we discuss in more detail throughout the review.

The NLRP3 inflammasome exists among multiple families of inflammasomes that include the nucleotide-binding domain-like receptors (NLRs), absent in melanoma 2-like receptors (ALRs), and the pyrin family, each with distinct properties and abilities to respond to unique molecular patterns to initiate formation of a multi-protein inflammasome complex and a pro-inflammatory phenotype. The NLRP3 inflammasome is capable of responding to pathogen-associated molecular patterns (PAMPs). Microbial triggers range from bacterial pathogens¹¹ to the hyperinflammatory SARS-CoV-2 N protein.¹² Interest in the NLRP3 inflammasome in the context of neurodegenerative disorders however likely stems from its ability to respond to endogenous damage-associated molecular patterns (DAMPs). As discussed throughout this review, significant evidence has been generated that the NLRP3 inflammasome is activated by multiple danger signals associated with neurodegeneration. These include normal aging, debris from dying cells, protein aggregates, lysosomal dysfunction, and metabolic stress.

Similar to many inflammasomes, the NLRP3 inflammasome is activated by a “two-hit” mechanism involving priming and stimulus.¹³ Canonical priming is thought to occur after an initial exposure to certain exogenous stimuli, for example lipopolysaccharide (LPS) or other bacterial components, resulting in toll-like receptor (TLR) ligand-receptor interaction. This triggers NF-KB-mediated transcriptional upregulation of inflammasome components and targets, including NLRP3 itself, and the downstream target IL1B.¹⁴ Subsequent exposure to an inflammatory stimulus activates the NLRP3 inflammasome and the formation of the multi-protein inflammasome complex. The NLRP3 inflammasome complex includes the sensor protein NLRP3 , the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), the inactive zymogen pro-caspase-1, and often the NIMA-related kinase NEK7.¹⁵ Formation of the NLRP3 complex drives the auto-catalysis of pro-caspase 1 generating the components of mature caspase-1. Active caspase-1 then processes targets including the pro-inflammatory cytokines IL-1B and IL-18 (Fig 1). Active caspase-1 can also cleave the protein Gasdermin D (GSDMD), the effector of pyroptotic cell death. Once cleaved, the N-terminal fragments of GSDMD are inserted into the plasma membrane whereby oligomeric pores are formed. These pores are important for the secretion of mature IL-1B and IL-18, ionic/osmotic flux, and cellular swelling.^16,17 Pyroptosis is also characterized by vesicular shedding, hypothesized as a means of inter-cellular communication and as a mechanism to remove the harmful pores from the plasma membrane. However, if unresolved, the cell will undergo a lytic, pro-inflammatory demise, releasing all intracellular components into the extracellular environment (Fig 1). The potential to use proteins released during the pyroptotic process as biomarkers of disease is discussed is greater detail later in this review.

Fig 1.

Mechanism of inflammasome activation and pyroptosis. The NLRP3 inflammasome is activated in a 2-step fashion. First, priming occurs at surface membrane receptors. This can include recognition of pathogen-associated molecular patterns (PAMPS) (eg, viruses and bacteria) at Toll-like receptors (TLRs) and/or danger-associated molecular patterns (DAMPs) (eg, cytokines) at the IL-1 receptor (IL-1R) or TNF-a receptor (TNF-R) for example. Following receptor activation, the transcription factor NF-KB translocates to the nucleus where it causes upregulation of inflammasome components, such as NLRP3 and pro-IL-1B. A secondary stimulus is necessary to cause inflammasome complex formation and activation. This second trigger can be either intra- and extra-cellular. Some common stimuli include both intra- and extra-cellular ATP and misfolded proteins, extracellular particulate matter (eg, silica, uric acid crystals) and components of cellular demise, and intracellular mitochondrial reactive oxygen species (ROS) and lysosomal dysfunction. After NLRP3 inflammasome complex formation, the pro-caspase-1 enzyme is cleaved into its active, mature form and can cleave both pro-inflammatory cytokines, IL-1B and IL-18, and the effector protein of pyroptosis, Gasdermin-D (GSDMD). Once cleaved, the N-terminal fragments of GSDMD form pores in the plasma membrane allowing for the extracellular release of the mature cytokines IL-1B and IL-18. Further, these pores promote osmotic and ionic flux, resulting in cellular swelling and the efflux of potassium (K⁺) and the influx of calcium (Ca²⁺). In a feed-forward mechanism, K⁺ efflux causes further inflammasome activation, while Ca²⁺ influx promotes the release of extracellular vesicles (EVs). Pyroptotic vesicular shedding results in the release of membrane-bound inflammatory proteins, including cytokines, inflammasome components, and even fully formed inflammasome complexes. It is thought that the release of these EVs is a means to resolve the GSDMD pore formation through their budding off the membrane. If unresolved, the osmotic flux and cellular swelling can cause the cell to burst, resulting in the pro-inflammatory release of the cell’s intracellular contents into the extracellular environment and cellular death. Created with BioRender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Mechanistic questions remain related to the adaptability of the priming and stimulus model to explain NLRP3 inflammasome activation in non-microbial neurodegenerative disorders. A significant body of evidence delineates non-microbial or “sterile” induction of NLRP3 inflammasome activity, demonstrating that non-pathogenic danger signals are sufficient to prime the inflammasome.¹⁸ Reactive oxygen species (ROS) are by-products of mitochondrial oxidative metabolism, which in excess, can lead to oxidative stress and damage to structural molecules of the cell.¹⁹ Although classically thought of as an activator of the inflammasome,²⁰ it has been shown that ROS may play a role in its priming as well. NLRP3 expression was decreased in mouse macrophages treated with a ROS scavenger, diphenyliodonium (DPI), and downstream targets, IL-1B and IL-18 were decreased only when macrophages were treated with DPI during the priming step, but not the activation step.²¹ Other sterile inflammatory triggers, including hypoxia,^22,23 tricarboxylic acid (TCA) cycle metabolite itaconate,²⁴ regulators of fatty acid synthesis²⁵ cholesterol metabolism,²⁶ and components of the complement system,²⁷ have been shown to prime the NLRP3 inflammasome for activation. These data have major implications for neurodegenerative diseases where NLRP3 activation and inflammation occur seemingly without a pathogenic trigger. Interestingly, even man-made toxicants have been shown sufficient to drive NLRP3 priming, with rotenone, a mitochondrial respiratory chain inhibitor, exposure eliciting NLRP3 priming in bone-marrow derived macrophages (BMDMs).²⁸ The NLRP3 inflammasome may provide an interesting mechanism by which environmental toxicant exposure may elicit neuroinflammation in neurodegenerative diseases.²⁹ In addition, NLRP3 enters a hypersensitive state reminiscent of priming as a result of normal aging. This hypersensitive state has implicated NLRP3 as a key mediator of “inflammaging” (for review^30,31). Once primed, the NLRP3 inflammasome requires a “second-hit” to be activated and the protein complex formed. Many triggers commonly seen in neurodegenerative-diseased brain, including misfolded proteins,^32,33 extracellular ATP typically caused by nearby cell death,^34,35 and lysosomal dysfunction³⁶ are sufficient to activate the NLRP3 inflammasome. Although most work surrounding ATP and NLRP3 are in the context of extracellular ATP activating the inflammasome via the P2×7R, intracellular ATP has also been shown to mediate inflammasome activation.^37,38 NLRP3 activity is now recognized in a host of sterile inflammatory disorders, including metabolic disorders, monogenic autoinflammatory disorders, and neuroinflammatory diseases that identify non-pathogenic mechanisms sufficient to drive NLRP3 inflammasome activation.¹⁸ Here, we highlight mechanisms of NLRP3 inflammasome activation identified in neurodegenerative disease.

Evidence of Inflammasome Activity in Neurodegenerative Disease

NLRP3 in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, affecting 5.8 million Americans 65 years or older today and is ranked the 6th leading cause of death in the US.³⁹ Although age is the number 1 risk factor for the disease, other possibly modifiable lifestyle factors have been linked to the risk of developing the disorder including general vascular health, educational attainment, mental and physical activity, and depression.⁴⁰ Symptomatically, AD is characterized by memory loss, executive dysfunction, and depression, with more advanced disease presenting with impaired communication, confusion, behavior changes, and difficulty performing everyday tasks.³⁹ Loss of neurons within the hippocampus and neocortical areas resulting in brain atrophy are believed to underlie the clinical manifestations of AD.⁴¹ AD is characterized post-mortem by 2 histopathologic hallmarks: 1) extracellular amyloid beta (Aβ) plaques and 2) intracellular neurofibrillary tau tangles containing hyperphosphorylated tau.⁴² Questions related to toxicity, causality, and therapeutic relevance of the abnormal accumulations of protein observed in AD continue to fuel research efforts. Identification of mutations in amyloid precursor protein (APP), the gene encoding for Aβ itself, and in presenilin 1 (PSEN1/PS1) and 2 (PSEN2/PS2), 2 genes that encode proteins involved in the processing of Aβ, are the only genetic mutations that lead to the autosomal dominant form of AD, called familial AD (fAD). This genetic linkage has been used as evidence that aberrant protein aggregation is indeed a key driver of AD incidence and progression.^43–45

The notion that NLRP3 was related to neurodegenerative disease was accelerated by Halle et al. who identified NLRP3 as a sensor of the fibrillar form of Aβ in microglia.³⁶ Aβ oligomers and protofibrils were later shown to activate the inflammasome suggesting that the NLRP3 inflammasome may be a driver of early-stage disease progression.³² Heneka et al. reported caspase 1 activity in post-mortem brain tissue obtained from patients with mild cognitive impairment (MCI) and AD highlighting the importance of inflammasome signaling in both the early and late stages of AD pathology.⁴⁶ The same group demonstrated that ipsilateral injection of brain homogenates from transgenic mice expressing chimeric mouse/human APP and mutant human PS1 (APP/PS1) increased the number of Aβ immunoreactive deposits compared to the contralateral injection of homogenates derived from wild-type mice in an Nlrp3-dependent manner.⁴⁷ ASC specks were also found to bind Aβ and enhance Aβ aggregation⁴⁸ and a synergistic effect of Aβ and ASC fibril formation exacerbated NLRP3 inflammasome activation and pyroptotic cell death in primary microglia.⁴⁹ ASC cross seeding of amyloids such as Aβ, tau, alpha synuclein, may be a common mechanism by which NLRP3 activation may accelerate disease progression and neurotoxicity and presents a candidate common target for therapeutic intervention possibly via targeting of aggregated ASC with antibody-based therapy. The NLRP3 inflammasome has also been implicated in tau pathology both in AD and primary tauopathies where tau aggregation, but not Aβ aggregation, is the primary pathological hallmark.⁵⁰ Injection of APP/PS1 brain homogenate induced tau hyperphosphorylation in the Tau22 mouse model, but not in Tau22/Asc^−/− or Tau22/Nlrp3^−/− mice indicating a requirement for NLRP3 activity in the Aβ-Tau cascade.⁴⁷ K18 tau fibrils, a fragment containing the central, amyloidogenic, region of tau activates the inflammasome and inhibition of NLRP3 is protective in an in vivo tau seeding model utilizing these aggregated tau fragments.⁵¹ Hyperphosphorylated tau derived from neuronal cultures and mouse or human brains can prime and activate the inflammasome in an ASC-dependent manner and the suppression of ASC in microglia improves cognition in the hTau mouse model of primary tauopathy.⁵² The exact mechanism by which Aβ activates the inflammasome is unclear and there are likely several potential pathways. TLR4 ligation by Aβ has been shown to be a mechanism by which the inflammasome is activated, although this study pretreated cells with LPS which also binds to TL4 as its primary receptor making precise conclusions difficult.⁵³ Conflicting reports suggest P2×7R receptor either mediates⁵⁴ or has no effect⁵⁵ on Aβ mediated activation of the NLRP3 inflammasome. Tau most likely primes the inflammasome through TLR interaction based on the key role of MyD88, a TLR adaptor protein, in mediating NF-κβ induced expression of inflammasome related genes,⁵² although the exact TLR has not been elucidated. The production of ROS and lysosomal rupture induced by aggregates have also been proposed as mechanisms of inflammasome activation in response to tau and Aβ.^56–58

NLRP3 activation has also been shown in neurons within the context of AD. Aβ_1-42 has been found to induce NLRP3-caspase-1 dependent pyroptosis in primary cortical neurons.⁵⁹ Further, injection of Aβ_1-42 was found to induce pyroptosis in the hippocampus of a rat model of AD that was ameliorated with administration of the biflavonoid antioxidant amentoflavone.⁶⁰ There are conflicting reports as to whether astrocytes express functional NLRP3. Using primary murine cell cultures, Gustin et al. (2015) showed Aβ peptides activated the NLRP3 inflammasome in microglia but not astrocytes.⁶¹ Later work by Couturier et al. (2016) found that astrocyte cultures treated with Aβ showed IL-1B production in an ASC-dependent manner.⁶² Discrepancies in these two reports could stem from the treatment paradigms and Aβ peptide species used. Gustin et al. (2015) stimulated cells for 6 hours with 10 ng/mL LPS followed by 20 uM Aβ_25-35 peptide exposure for 5 hours, while Couturier et al. (2016) treated cells for 3 hours with 1 ug/mL LPS prior to 10 uM Aβ_1-42 peptide exposure for 3 hours. Supportive of astrocytic expression of NLRP3, Freeman et al. (2017) showed NLRP3 activation in both microglia and astrocytes following the sterile trigger lysophosphatidylcholine⁶³ and Ebrahimi et al. 2018⁶⁴ found Aβ_1-42 to upregulate NLRP3 mRNA/protein and caspase-1 activity in primary murine cortical astrocytes. Infiltrative immune cells provide an important source of inflammation in the diseased brain.⁶⁵ Not only do these cells respond to immune changes within the periphery, but they traverse into the CNS, bringing with them vital information to influence the inflammatory microenvironment within this highly regulated system.⁶⁶ Saresella et al. (2016) investigated NLRP3 pathway activation in peripheral monocytes derived from healthy control, MCI, mild AD, and severe AD patients. Following LPS-priming and stimulation with AB_1-42, they saw increased inflammasome transcript and protein expression in mild and severe AD monocytes, suggesting global changes in NLRP3 activation in AD.⁶⁷

NLRP3 in Parkinson’s disease

Parkinson’s disease (PD) is the most common neurodegenerative motor disorder, with an estimated 1.2 million Americans living with the disease by 2030.⁶⁸ Pathologically, PD is characterized by the loss of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpC). These DA neurons project to the striatum, releasing DA from their terminals which allows for proper motor function. When these neurons are lost, less DA is produced, resulting in severe motor impairment.⁶⁹ PD is defined clinically by 4 cardinal motor symptoms: bradykinesia, rest tremor, rigidity, and postural instability.⁷⁰ However, non-motor symptoms including olfactory dysfunction, constipation, sleep disturbances, and depression are common in PD and can manifest decades before the onset of motor symptoms.⁷¹ Definitive diagnosis of PD can be made post-mortem and is largely based on the observation of dopaminergic neuronal cell loss in the substantia nigra and the presence of intracellular Lewy bodies and Lewy neurites.⁷² The observation of early-stage alterations and the estimation that 30%–70% of DA neurons are already lost when a patient clinically presents with the disease underly considerations of a “prodromal” period of PD during which neuroprotective measures may be able to modify the disease course.^73,74 Lewy histopathology is highly immunoreactive for misfolded and post-translationally modified alpha-synuclein (α-syn), encoded by SNCA. SNCA mutations,⁷⁵ duplications,⁷⁶ and triplications⁷⁷ observed in familial PD suggest a causal role for SNCA in PD etiology. In animal models, pre-formed exogenous α-syn fibrils can seed endogenous α-syn, promoting widespread deposition of α-syn aggregates in mice, associated with the development of PD-like pathologies.⁷⁸ Neuroinflammation co-occurs with proteinopathy in PD.⁷⁹ Reactive microglia were first described within the SNpC of PD patients post-mortem by McGeer et al. in 1988.⁸⁰ The use of certain non-steroidal anti-inflammatory drugs has been linked to a decreased risk of PD,^81,82 and genetic mutations in various immune genes, including in the human leukocyte antigen (HLA) family^83–85 have been shown to alter PD risk. The presence of cell death, self-proteins that may take on pathologic conformations, normal aging, and the observation of inflammasome activity under similar condition in AD has prompted the study of the NLRP3 inflammasome in PD.

Evidence of inflammasome signaling in PD patient tissue was first described by Wang et al. who reported co-localization of caspase-1 and α-syn in Lewy bodies derived from PD tissues.⁸⁶ These findings were supported by our laboratory and others demonstrating enhanced NLRP3 immunoreactivity in mesencephalic tissues obtained from PD patients,⁸⁷ an increased abundance of the activated caspase-1 p20 fragment and ASC adapter within the PD SNpC,⁸⁸ and evidence of enhanced NLRP3 expression in dopaminergic neurons from PD patients compared to healthy controls.^89,90 The designer drug MPTP caused dopaminergic neuron death and PD symptomatology in humans and animal models.^91,92 In the MPTP-based mouse model of PD, loss of Nlrp3 results in decreased microglial activation, sparing of DA neurons, and rescue of motor deficits.⁹³ Loss of caspase-1 also has been shown to prevent DA loss and motor dysfunction in the MPTP-model of PD.⁹⁴ Attenuation of hepatic NLRP3 alone can be sufficient to reduce MPTP-induced microglial activation and DA neuron loss in vivo.⁹⁵ Further, a recent study shows that MPTP causes DA cell loss through pyroptotic mechanisms, and inhibition of this pathway ameliorates MPTP-induced DA death.⁹⁶ Conversely, DA-neuron specific expression of a hyperactivating NLRP3-allele results in neuroinflammation and motor deficits in aged mice.⁹⁷ Although controversial, some have reported astrocytic NLRP3 activation (see above), with 1 study finding the dopamine D2 receptor (Drd2) inhibited NLRP3 activation in cultured astrocytes treated with LPS and ATP. In the same study, they showed a Drd2 agonist inhibited inflammasome activation in the midbrain of mice treated with MPTP.⁹⁸ Further work is needed to delineate the role of neurons and astrocytes in NLRP3-mediated pathogenesis in PD.

Significant attention has been devoted to the determination of whether pathologic α-syn is a direct inflammasome activator. α-syn fibrils or monomers can prime and activate the inflammasome in human monocytes via interactions with TLR2 in association with α-syn fibril phagocytosis, ROS production, and cathepsin B release from the lysosome into the cytosol.⁹⁹ The differential ability of various forms of α-syn to prime and activate the NLRP3 inflammasome was further elucidated in a recent paper from Scheiblich et al. where they showed that α-syn monomer and non-fibrillar oligomers are able to the prime and activate the inflammasome in a 1-step process in primary mouse microglia without the addition of the microbial priming agent LPS whereas sonicated α-syn fibrils require priming using LPS. Findings indicted that activation was primarily mediated by TLR2, and to a lesser extent TLR5 interactions. In this model, inhibition of the inflammasome increased the degradation rate of α-syn oligomers whereas no change in degradation efficiency is seen for monomers or sonicated fibrils.³³ A second study provides evidence that sonicated α-syn fibrils benefit from an LPS priming step to achieve robust NLRP3 activation, but the authors found, in contradiction to the previous study, that sonicated α-syn alone could induce NLRP3 activation to a lesser extent than if a priming step was used.⁸⁸ Finally, another study showed that α-syn fibrils can prime and activate the inflammasome in a 1 step process,¹⁰⁰ although these fibrils were not sonicated to fragment them into smaller fibrils as compared to the study by Scheiblich et al.³³ and Gordon et al.⁸⁸. α-syn induction of NLRP3 activation was also shown in primary human microglia derived from post-mortem brains where fibrils, but not monomers, induced inflammasome priming and activation in a 1-step process.¹⁰¹ Similar findings were reported in microglia derived from human induced pluripotent stem cells (hIPSCs) where activation was mediated by TLR2. The study demonstrated that oligomers contaminated with proto-fibrils prime and activate the inflammasome via TLR2 ligation and mitochondrial ROS production whereas monomers have no effect. The authors then depleted α-syn in a hiPSC-derived microglia/hiPSC-derived dopaminergic neuron co-culture system in a paradigm that models anti-α-syn antibody-based therapies for PD currently in clinical trials. Unexpectedly, the authors found that the α-syn antibody complex exacerbated inflammasome activation instead of attenuating it which has significant implications in the context of α-syn antibodies being developed for clinical use.¹⁰²

The heterogeneity observed across these studies regarding the specific proteoform of α-syn used to prime and activate the inflammasome highlights a distinct difficulty across studies addressing α-syn, Aβ, and tau induced NLRP3 priming and activation. That is the fact that none of these studies are directly comparable due to differences in oligomer and fibril preparation, specifically relating to if the α-syn fibril is or is not sonicated, how the oligomeric species are prepared, and the specific assembly conditions used to aggregate protein into oligomers or fibrils. It is difficult to define the oligomeric state of amyloidogenic proteins, including α-syn and Aβ, in general due to their disordered nature and tendency to sample thousands of conformational states along a continuum of aggregation from monomer to mature fibril^103–105 thus making both the identification of a distinct neurotoxic oligomeric species and the reliable preparation of similar oligomers virtually impossible. Whether the mature fibrils are sonicated or not is also suggested to play a key role in NLRP3 activation dynamics where sonicated fibrils require priming the microglia with LPS³³ and unsonicated fibrils appear to not require priming with LPS.¹⁰⁰ This distinction in sonication state is important because one of the primary in vivo mouse models of PD and synucleinopathies is the injection of WT mice with sonicated, fibrillar α-syn fibrils which directly templates the aggregation of endogenous mouse α-syn, neuroinflammation, and neuronal loss.^78,106,107 Thus, it seems that in vitro models of microglia reactivity to aberrant α-syn species and accompanying NLRP3 activation should follow the widely used in vivo model even if that requires priming with LPS, which is not required in the in vivo model but appears to be required in vitro. Finally, the conditions in which protein is aggregated to form oligomers or fibrils also plays a large role in the final ultrastructure of the aggregates and the level of toxicity that these aggregates have. A recent report used 2 preparations of α-syn fibrils using different buffers to create distinct fibrillar polymorphs that had differential abilities to activate the inflammasome in primary microglia³³ highlighting the strain specific differences between different protein assemblies. Other parameters of aggregation assembly including ionic composition and strength, pH, protein concentration, agitation, and temperature also play a role in determining the ultimate fibril structure and resultant toxicity.^108–110 Taken together, these 3 qualities of describing the specific protein species used to activate the inflammasome are important to keep in mind when performing in vitro studies and parity in the preparations used across the field will increase the reproducibility and rigor when probing NLRP3 dynamics in response to amyloids.

It should also be noted that inflammasome activation itself may directly modulate α-syn pathology. Caspase-1 can directly cleave α-syn producing a species more prone to aggregation.⁸⁶ Gordon et al., also showed the converse is true, demonstrating that blocking NLRP3 inflammasome activation with the small molecular inhibitor MCC950 reduces α-syn aggregation and motor deficits in several in vivo mouse models including the α-syn PFF model.^78,88 The mice used in this study were treated with the NLRP3 inhibitor MCC950 for the entire duration of their exposure to α-syn fibrils and were sacrificed at a single time point at 8- months post injection of α-syn. This time point is somewhat late in the progression of the model and the report does not determine whether changes are observable at earlier time points after α-syn administration.¹¹¹ It would be of interest to measure NLRP3 associated changes at different stages of disease progression in the model both with and without treatment with MCC950 as distinct neuroimmune progression has been observed in this model over time.¹⁰⁷ It is possible that early NLRP3 activation may be protective and as the burden of aberrant α-syn grows, the NLRP3 inflammasome becomes over-activated and eventually neurotoxic. This highlights the importance of temporal profiling of NLRP3 dynamics in this model and others. Although pathogenic, viral infections have been suggested as triggers of neurodegenerative disease.¹¹² A recent report found that the SARS-CoV-2 spike glycoprotein can act as both a priming and an activation signal of the NLRP3 inflammasome in microglia and that the presence of α-syn amplified its activation.¹¹³ This data is intriguing in the context of case reports describing individuals who suddenly developed PD following infection with SARS-CoV-2.^114–116

Peripheral immune involvement in PD and other neurodegenerative disorders is a topic of increasing interest. Notably, NLRP3 activation is reported in peripheral blood mononuclear cells (PBMCs) of PD patients. Fan et al. (2020) collected PBMCs from PD patients and healthy controls for NLPR3 pathway transcript and protein expression profiling. They discovered elevated levels of NLRP3 pathway components at both the RNA and protein level in in PD PBMCS. Further, they found elevated IL-18 in the plasma of PD patients which correlated to cognitive and motor clinical scores. Circulating plasma-borne α-syn also correlated to plasma IL-1B levels, suggestive of a link between PD pathology and inflammasome activation.¹¹⁷ Data from our lab found elevated levels of circulating plasma-borne NLPR3 in PD patients compared to healthy controls⁸⁷ prompting our discussion of pyroptosis as a platform for biomarker discovery in later sections.

NLRP3 in amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder that primarily affects upper- and lower-motor neurons and is the third most prevalent progressive neurodegenerative disease after AD and PD.¹¹⁸ Like other neurodegenerative diseases, there is considerable genetic heterogeneity, with the most common genetic causes of ALS attributable to mutations in the proteins: chromosome 9 open reading frame 72 (C9orf72), superoxide dismutase 1 (SOD1), TAR DNA binding protein 43 (TDP-43), fused in sarcoma (FUS), and TANK-binding kinase 1 (TBK1).¹¹⁹ Together, mutations to these 5 proteins account for 15% of all ALS cases with the majority of ALS patients displaying no clear familial link to disease.¹¹⁹ The primary pathological feature of ALS is the presence TDP-43 positive inclusions bodies in the brain and spinal cord.¹²⁰ Neuroinflammation is readily observed in ALS patients. Using a positron emission tomography (PET) tracer specific for reactive microglia, Turner et al. showed widespread microglia activation in human ALS patients where the magnitude of microglial activation correlates with the severity of motor neuron degeneration.¹²¹ A recent study shed light on the role of the NLRP3 inflammasome specially in ALS. Van Schoor et al. showed that expression of NLRP3 was increased in microglia located in the white matter of the motor cortex of ALS patients and this was accompanied by GSDMD antibody reactivity, indicating pyroptotic NLRP3 activation. Interestingly, this was isolated to the cortex and was not observed in microglia located in the spinal cord of ALS patients.¹²² Another recent study suggests that dysregulation of NLRP3 assembly, causing an increased propensity to assemble, results in reduced cognitive resiliency to non-motor symptoms in ALS patients.¹²³

The primary rodent model of ALS expresses a familial mutation in SOD1 where glycine is changed to alanine at position 93 (SOD1-G93A) which results in decreased stability of SOD1 and an increased propensity to aggregate.¹²⁴ Inflammasome components including IL-1β, NLRP3, ASC, and Caspase-1 have all been shown to be upregulated in the SOD1-G93A mouse model,¹²⁵ in addition to TLR4 and NF-κβ.¹²⁶ Blockade of IL-1β signaling in SOD1-G93A mice using an IL-1 receptor antagonist prolonged the lifespan of the mice and reduced inflammation,¹²⁷ suggesting that IL-1β is the primary driver of neuroinflammation in ALS. ALS-associated proteins including aggregated TDP-43 and SOD1 readily activate the NLRP3 inflammasome in primary microglia.^125,128 SOD1-G93A may also cause increased oxidative stress through increased peroxynitrite formation that directly leads to increased inflammasome activation.¹²⁹ Astrocytic NLRP3 activation has also been suggested as a driver of neuroinflammation in ALS mice,¹³⁰ although these results have since been called into question.¹²⁵

NLRP3 in multiple sclerosis

Multiple sclerosis (MS) is the most common progressive neurodegenerative disorder affecting younger adults.¹³¹ A recent study estimated approximately 1 million Americans were living with the disorder in 2017.¹³² This disease disproportionally affects women, whose risk of developing the disorder is increased 3-fold compared with men.¹³² Genetic mutations in HLA genes account for approximately 20-30% of all MS genetic susceptibly.¹³³ Environmental exposures and lifestyle factors influence disease development, with vitamin D deficiency,¹³⁴ cigarette smoking,¹³⁵ obesity,¹³⁶ and exposure to the Epstein-Barr virus^137,138 identified as risk modifiers. MS is an auto-inflammatory disorder, driven by an immune response targeting myelin sheaths in the central nervous system (CNS), resulting in axonal demyelination and eventual neuronal cell death. This neuronal loss results in slowly progressive brain atrophy.¹³⁹ The adaptive and innate arms of the immune system play a role in MS pathology (for review¹⁴⁰). Pathologic demyelination manifests as periods of acute neurologic deficit, with the symptomology dependent upon the location of the inflammatory lesion.¹⁴¹ Microglial activation and subsequent release of pro-inflammatory cytokines is believed to play a role in lesion formation.¹⁴² The clear association between MS and inflammation alongside the production of sterile triggers resulting from demyelination and cell death provides a platform for evaluation of DAMP sensitive pattern recognition receptors such as the NLRP3 inflammasome.

Multiple lines of evidence support a role for the NLRP3 inflammasome in MS. Polymorphisms in NLRP3,^143,144 IL1B,^145,146 and IL18¹⁴⁷ genes confer an increased risk of MS. Increased levels of IL-1B have been detected in the blood^148,149 and cerebrospinal fluid (CSF)^149,150 of MS patients. CSF IL-1B levels correlate with the severity of demyelination within the brain¹⁵¹ and disease progression.¹⁵² NLRP3,¹⁵³ caspase-1,¹⁵⁴ IL-1B,^153,155 have all been detected in MS lesions, while elevated mRNA levels of caspase-1 and IL-18 were seen in peripheral blood mononuclear cells (PBMCs) isolated from MS patients.¹⁵⁶ A 2020 study suggests an inflammasome signature may be present in MS patients’ PBMCs that differs from healthy controls. Immunophenotyping and RNASeq showed an IL-1B signature in monocytes of patients with primary progressive MS. Monocytes from these individuals had higher baseline levels of ASC specks and upon LPS and ATP treatment had increased IL-1B production. Further, individuals with high IL-1B transcript levels had faster disease progression.¹⁵³

Pronounced NLRP3 inflammasome activity is observed in the autoimmune encephalomyelitis (EAE) model of MS.¹⁵⁷ The immune response in the EAE system is dependent on ASC,¹⁵⁸ caspase-1,^158,159 NLRP3,^159,160 IL-18,^159,160 and Gasdermin D (GSDMD), a key effector of pyroptosis.¹⁶¹ Enhancing the anti-inflammatory cytokine interferon-β (IFN-β) has been suggested as a treatment for MS. IFN-β inhibits NLRP3 inflammasome function¹⁶² and reduces IL-1B production¹⁶³ in the EAE model while in variations of the model that negate the reliance on the NLRP3 inflammasome, there is no response to IFN-β.¹⁶⁴ Direct inhibition of the NLRP3 inflammasome with small molecule MCC950 decreases IL-1B production, disease severity,¹⁶⁵ relapse,¹⁶⁶ astrocyte activation, and cognitive deficits¹⁶⁷ in the EAE model. MCC950 in combination with rapamycin, an autophagy inducer, reduced the clinical symptomology and cytokine burden in this model as well.¹⁶⁸ Other newly developed drugs JC-171¹⁶⁹ and OLT1177 (Dapansutrile)¹⁷⁰ which directly target the NLRP3 inflammasome, delayed the progression, reduced the severity, and ameliorated clinical symptomology of disease in the EAE model.

Oligodendrocytes (OLs) are the major cell type at risk in MS. There is some evidence to suggest that OLs express inflammasome components.^171,172 In the context of AD, OLs from AD patients and the 5xFAD mouse model of AD showed immunoreactivity for GSDMD, the pyroptotic effector protein, alongside axonal damage and demyelination. In vitro, the treatment of OLs with AB_1-42 caused an increase in NLRP3, cleaved GSDMD, and IL-1B expression, suggestive of inflammasome activation.¹⁷² Studies exploring NLRP3 activation in OLs in the context of MS are lacking, but we can infer from these data that OLs may be capable of mounting their own NLRP3-based response.

The influence of peripheral immune involvement is well-described in MS.¹⁷³ Inflammasomes have been well-characterized within circulating cells of the peripheral immune system and they are most likely contributing to the inflammatory signature within not only the periphery, but also within the CNS. Interestingly, pyroptotic signaling within the periphery has proven vital for neuroinflammation within the EAE model of MS. Loss of GSDMD within peripheral myeloid cells in the EAE mouse model led to reduced inflammation and demyelination in the CNS. Further, it impaired the infiltration of peripheral T cells into the CNS.¹⁶¹ In a similar study, Inoue et al. (2012) found that in the EAE model NLRP3 played a role in T-cell migration to the CNS whereby inflammasome-activated cells primed CD4+ T-cells to upregulate chemotaxis-related proteins.¹⁷⁴

Pyroptosis as a platform for biomarker discovery

Inflammasome activation drives pyroptosis, an inflammatory form of programmed cell death characterized by chromatin condensation, osmotic flux/cellular swelling, vesicular shedding, and potentially membrane rupture.¹⁷⁵ Pyroptosis shares similarities and distinctions with apoptosis and necrosis, including the release of pro-inflammatory cytosolic proteins (Fig 2).¹⁶ This is of interest because it broadens the pool of potential extracellular biological indicators of inflammasome activation to include virtually the entire intracellular proteome. Pyroptosis is mediated by a cascade of molecular events which ultimately result in the cleavage of the protein effector of pyroptosis, GSDMD. For this reason, pyroptosis has been referred to as “GSDMD-mediated programmed necrosis.”¹⁷⁶ GSDMD is a member of a family of related proteins that include GSDMA, GSDMB, GSDMC, GSDMD, GSDME (aka DFNA5) and PJVK (aka DFNB59), all of which play a role in inflammation and cell death.¹⁷ Three labs simultaneously discovered GSDMD as the pyroptosis effector protein.^177–179 In canonical inflammasome signaling (Fig 1), DAMPs or PAMPs drive inflammasome complex formation leading to caspase 1 activation and resultant GSDMD cleavage and activation.¹⁸⁰ Non-canonical activation of caspase 4/5 (caspase-11 in mice) through pathogenic stimuli (e.g., gram-negative bacteria) can also cause cleavage of GSDMD and pore formation.^181,182 Regardless of its initial upstream modulator, GSDMD is cleaved within its central linker domain to produce a 31-kDa N-terminal fragment and a 22- kDa C-terminal fragment.^177–179 The function of the 22 kDa fragment is to halt cell death, as it acts in an autoinhibitory fashion binding to the N-terminal fragment to prevent insertion into the plasma membrane. Once uncoupled from its inhibitory partner, 31-kDa N-terminal fragments of GSDMD associate with the inner leaflet of the plasma membrane, where they assemble into oligomeric pores.^183–186 The N-terminal of GSDMD can directly interact with the plasma membrane, as it has a high affinity for acidic phospholipids, such as phosphoinositides,¹⁸⁴ which are only found in the cytoplasmic leaflet of the plasma membrane. GSDMD pores range in size from 10 to 20 nm,^187,188 however these pores are large enough for mature IL-1B (~5 nm in diameter) to be released upon canonical NLRP3 inflammasome activation.^189,190 Although the entire mechanism of GSDMD membrane pore formation has not been fully delineated, recent studies suggests that monomeric N-terminal GSDMD fragments insert into the plasma membrane where they begin to form intermediate structures before eventually forming ring-shaped membrane-spanning pores.¹⁸⁷ A recent study sought to determine the timeline of events which ultimately lead to pyroptosis. They report that GSDMD-mediated ion channels are the first to open, with a much smaller diameter than previously reported for GSDMD-pores. These channels allow the flux of ions, with Ca²⁺ influx being cited as occurring before complete membrane permeabilization. Osmotic swelling, mitochondrial depolarization, and lysosome leakage occur before the plasma membrane integrity of the cells is lost, which coincides late-stage nuclear condensation and release of intracellular components.¹⁹¹ Morphologically, end-stage pyroptosis is characterized by membrane blebbing, loss of plasma membrane integrity, and release of cytosolic material (Fig 1).

Fig 2.

Comparison of necrosis, apoptosis, and pyroptosis. Apoptosis and pyroptosis, 2 forms of programmed cell death, share many commonalties with each other and with an accidental form of cell death, necrosis. These are not meant as absolutes, as there are many examples where these statements might not necessarily hold true, dependent on cell type, deadly trigger, and molecular platforms available. However, this framework is helpful to break these forms of cell death into distinct categories with general characteristics. Pyroptosis is typically inflammasome- and gasdermin-mediated, resulting in the release of inflammatory cytokines. Similar to apoptosis, it is highly regulated by caspase enzymes and can be triggered by both endogenous and exogenous triggers. However, apoptosis is immunologically silent resulting in apoptotic body formation, unlike the pro-inflammatory pyroptotic phenotype. Further, apoptosis can occur as a consequence of normal physiology, such as during development and normal cellular turnover, or in response to a harmful trigger or pathologic insult. Necrosis shares its greatest commonalties to pyroptosis as it is lytic, resulting in membrane rupture, and results in the pro-inflammatory release of intracellular contents into the extracellular environment. However, unlike either pyroptosis or apoptosis, it is accidental, not resulting from mechanistic regulation. Necrosis is exogenously induced from physical, chemical, or mechanical stimuli causing immediate cellular demise from pathologic injury. Interestingly, a process known as secondary necrosis can follow apoptosis if the apoptotic bodies cannot be phagocytosed, which is characterized by lytic release of intracellular material. Created with BioRender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The formation of GSDMD pores, although classically linked to membrane rupture and cellular lysis, is part of a protracted process that does not always translate into cellular demise. Lysis-independent GSDMD pore formation has been found to occur following inflammasome activation and is critical for the release of mature pro-inflammatory cytokines without causing cell death.^{189,190,192,193} During the sublytic phase of pyroptosis, GSDMD pore formation results in the secretion of pro-inflammatory cytokines, IL-1B and IL-18. There is some evidence to suggest that GSDMD pore formation, during non-canonical pyroptosis, can cause K⁺ efflux, resulting in NLRP3 inflammasome formation as well.^178,179 Further, there has been some work to show that damage to the plasma membrane can be repaired and therefore is not an inherently fatal event.¹⁹⁴ One of the major pyroptotic events – vesicular shedding – has in fact been shown to repair GSDMD-pore perforation of the plasma membrane. In a recent publication, endosomal sorting complexes required for transport (ESCRT) machinery were found to transport to the plasma membrane in response to GSDMD-pore induced Ca²⁺ influx and cause vesicular shedding of the perforated membrane.¹⁹⁵ More recently, the release of such inflammasome-related vesicles was discovered to be dependent on the activation of GSDMD.¹⁹⁶ Vesicular release from inflammasome-activated and pyroptotic cells provides an interesting avenue for potential biomarker investigation. There have been several studies showing release of IL-1B-containing vesicles following NLRP3 inflammasome activation from monocytes.^197–199 Secretion of inflammasome-component containing extracellular vesicles (EVs) following NLRP3 inflammasome activation has been shown in a variety of cell types including macrophages^200–202 and microglial cells.²⁰³ Although pyroptosis can be resolved, in most cases, the lytic phase eventually occurs resulting in plasma membrane rupture and release of cytosolic material, which includes the individual components of the inflammasome as well as some evidence to suggest, fully formed inflammasome complexes. Overall, pyroptosis provides 3 potential avenues by which intracellular proteins can be released into the extracellular environment: (1) through GSDMD pores (eg, IL-1B, IL-18), (2) EV shedding, and (3) bursting of the cell (Fig 1).

The conceptual platform of pyroptosis has fueled a significant effort to identify evidence of plasma-borne inflammasome indicators. Elevated expression of inflammasome-related transcripts in PBMCs and whole blood has been seen in multiple diseases including AD,²⁰⁴ PD,¹¹⁷ and ALS.²⁰⁵ Elevated levels of circulating IL-1B protein have been identified in a host of inflammatory disorders²⁰⁶ including PD.^117,207,208 More recently, typically cytosolic inflammasome- or pyroptosis-related proteins and structures have been observed in the biofluids of patients in multiple disorders. Baroja-Mazo et al. identified oligomeric ASC particles in the serum of patients with active CAPS.²⁰⁹ Elevated circulating inflammasome proteins have been seen in the biofluids of patients with diabetes,²¹⁰ human immunodeficiency virus (HIV-1),²¹¹ chronic obstructive pulmonary disease (COPD) and pneumonia,²¹² myelodysplastic syndromes,²¹³ psoriasis/ psoriatic arthritis,²¹⁴ and macular degeneration.²¹⁵ Many disorders of the CNS also exhibit elevations in these circulating pyroptotic proteins. These include traumatic brain injury (TBI),²¹⁶ stroke,²¹⁷ MS,²¹⁸ AD,²¹⁹ and PD.^87,117 Specifically, higher levels of ASC were found in the serum of MCI and AD patients compared to healthy controls, suggesting ASC as a novel biomarker of dementia.²¹⁹ Elevated caspase-1 and NLRP3 have been reported in PD plasma.^87,88 It is noteworthy that EVs are avidly released following inflammasome activation. Refinement of sampling to include isolation of the EV fraction may improve the precision of bioassays for inflammasome-related proteins. Elevated EV-bound ASC was observed in individuals who had suffered a stroke.²¹⁷ Our laboratory reported higher levels of plasma-borne EV-contained NLRP3 in PD patients compared to healthy controls. Deeper characterization determined that ASC, the cleaved forms of CASP1 and GSDMD, and pro-IL-1B were also enriched in the EV fraction.⁸⁷ These data suggest that inflammasome activation and pyroptosis result in the release of a subset of typically cytosolic proteins into the extracellular environment, that may be contained within EVs. Such evidence suggests that inflammatory, pathologic, neurodegenerative disease processes could be monitored through evaluation of pyroptotic EVs in peripheral biofluids.

Additional Topics

Resolution

Inflammation is an inherently beneficial process despite the prevailing focus on pathologic outcomes. Induction of the inflammatory response is critical for elimination of microbial pathogens, restoration of homeostasis in stressed or damaged tissues, and the elaboration of the adaptive response and immune memory. Resolution is defined by a series of cellular and molecular transitions that reduce the initial pro-inflammatory response and promote clearance of debris in damages tissues (for review,^220–222). Mechanisms resolving the NLRP3-driven inflammatory response are poorly characterized, however, several lines of evidence have provided a framework for understanding this process.

The IκB kinase (IKK) complex subunit IKKa is a negative regulator of inflammation that contributes to resolution by inhibiting NF-kB interactions with pro-inflammatory gene promoters.²²³ IKKa also interacts with the inflammasome scaffold ASC, limiting NLRP3 inflammasome activation by sequestering ASC in the nucleus. Loss of IKKα kinase activity in murine bone marrow-derived macrophages (BMDMs) results in inflammasome hyperactivation.²²⁴ Ubiquitination and degradation of NLRP3 is a reproducibly observed mechanism of inflammasome inactivation. Bile acid driven signaling has been implicated in the control of inflammation and reported to inhibit NLRP3 inflammasome activation via TGR5-cAMP-PKA axis driven NLRP3 ubiquitination.²²⁵ Acetate inhibits NLRP3 inflammasome activation in BMDMs by activating soluble adenylyl cyclase, inducing PKA-mediated ubiquitination of NLRP3 and subsequent degradation through autophagic pathways.²²⁶ Resolvins are a well characterized family of pro-resolving lipid mediators with protective functions in neurodegenerative diseases (for review²²⁷). Resolvin D2 (RvD2) promotes the degradation of NLRP3 through autophagic pathways, and the inhibition of autophagy reversed RvD2-mediated suppression of NLRP3 inflammasome in murine peritoneal macrophages.²²⁸ Indirect mechanisms of inflammasome resolution are suggested in studies of downstream inflammasome effectors. RGS10 is a GTPase activating protein best characterized as an inhibitor of platelet activation, providing a “brake” critical for regulation of clotting and hemostasis.²²⁹ Anti-inflammatory and neuroprotective functions for RGS10 have been delineated in myeloid cells and PD models.²³⁰ Lee et al. demonstrated that Rgs10^−/− macrophages produced higher levels of pro-inflammatory cytokines, including IL-1B, in response to inflammatory stimulus suggesting the potential of an antagonistic relationship between RGS10 and NLRP3.²²⁹ Taken together evidence indicates that there are active cellular mechanisms functioning to regulate and resolve the NLRP3 inflammasome-mediated inflammatory response. Further characterization of mechanisms of resolution specific to NLRP3 in the context of neurological disorders and aging has a high likelihood if identifying pathways of interest for novel treatments and preventative strategies.

Drug development

Pre-clinical studies have defined a key role for the NLRP3 inflammasome in neurodegenerative diseases, including PD, AD, and MS. Evidence also suggests that the inflammasome may function during the earliest stages of disease pathogenesis making the NLRP3 inflammasome an attractive target for therapeutic development. A rapid expansion of drug discovery efforts followed reports by Coll and others. These studies reinvigorated a molecule initially described as CRID3, a diarylsulfonylurea-containing compound among the Cytokine Release Inhibitory Drug (CRID) class.²³¹ Collectively, these groundbreaking reports determined that CRID3 (more recently described as MCC950) directly targeted NLRP3 itself, interacting with the Walker B motif within the NLRP3 NACHT domain, blocking ATP hydrolysis, and subsequent NLRP3 activation (^165,232 for additional review²³³).

Preclinical models have advanced the development of known NLRP3 targeting drugs specifically in neurodegenerative disease paradigms. Pharmacological inhibition of NLRP3 with MCC950 ameliorated inflammation, reduced Aß plaque burden, and improved behavioral outcomes in the APP/PS1 mouse model of AD.²³⁴ Dapansutrile (OLT1177) is well-tolerated in humans and effective in the treatment of gout (EU Clinical Trials Register, EudraCT 2016-000943-14).²³⁵ Oral administration of Dapansutrile (OLT1177) was tested in the APP/PS1 model of AD and it was found that disease phenotype was attenuated, although CNS pharmacokinetics (PK) were not performed so it is still unknown how much of the drug enters the CNS.²³⁶ Another group used a small molecule inhibitor, NIC7, which specifically targets the NLRP3 protein. The authors found that NIC7, administered via oral gavage, successfully ameliorated disease phenotype in an AD mouse model.²³⁷ Glibenclamide (glyburide), an ATP-sensitive K⁺ channel inhibitor used clinically to treat type 2 diabetes,²³⁸ has been shown to inhibit NLRP3 in vitro.²³⁹ The drug was used to treat a pesticide-induced PD mouse model by intraperitoneal injection and was shown to attenuate neuronal loss and inhibit the NLPR3 inflammasome and overall microglial reactivity.²⁴⁰ None of these animal studies explicitly tested the PK of their respective drug candidate so it remains unclear whether the compounds were able to enter the CNS.

Clinical studies of novel inflammasome inhibitors have advanced in the context of CAPS family of monogenic inflammatory disorders. Although rare, these disorders are a meaningful indication in early-stage testing of novel NLRP3 inhibitors due to a clear genetic link between CAPS and NLRP3 hyperactivation. Canakinumab, an antibody therapy targeting IL-1β was shown to significantly attenuate CAPS symptoms (NCT01302860)²⁴¹ and there are several other ongoing clinical trials investigating the utility of therapies designed to inhibit ASC oligomerization (ZYIL1, NCT05186051) or target the NLRP3 protein itself (DFV890, NCT04868968; Inzomelid/IZD174, NCT04015076) in CAPS patients. Recent efforts are underway to target NLRP3 components specifically in diseases of the CNS. Treatment of CNS specific diseases is challenging due to lack of CNS bioavailability of many candidate compounds stemming from a failure to cross the blood brain barrier (BBB).²⁴² Progress in overcoming this field-wide hurdle includes Inzomelid/IZD174 (NCT04015076), now known as Emlenoflast, a drug candidate that received promising results in phase I trials but has yet to enter stage II trials.

Concluding Remarks

Neurodegenerative disorders such as AD, PD, MS, and ALS represent major public health burdens. Despite years of study, the establishment of clear causes for these disorders remains elusive. In many cases, the initial degenerative processes are selective, however, as diseases progress, neuronal degeneration can become widespread,²⁴³ further complicating the identification of key pathologic mechanisms. Co-occurring pathobiologies offer inroads to understanding the degenerative process that may be leverages to prevent or slow disease progression. Chronic inflammation, both within the CNS²⁴⁴ and the peripheral immune system²⁴⁵ are common to many age-related neurodegenerative disorders. Glial cell activation,²⁴⁶ enhanced pro-inflammatory cytokine profiles,²⁴⁷ and infiltration of peripheral immune cells into the CNS⁶⁶ are now recognized as key pathologies within many neurodegenerative diseases but an incomplete understanding of the molecular mechanisms underlying the inflammatory responses has impeded the development of anti-inflammatory therapies designed to treat neurodegenerative disorders. NLRP3 is clearly active in neurodegeneriative diseases yet the degree to which the inflammatory response causes, contributes to, or is a bystander to neurodegeneration remains unclear. This is especially important in the conundrum of proteinopathy. More work is needed to clarify whether the inflammatory milieu drives protein aggregation, responds to already misfolded proteins as it might to other pathogenic molecular patterns, or both. Understanding these mechanisms will have a transformative impact on our understanding of disease progression and the development of diagnostic and neuroprotective strategies to improve the care of aging and at-risk populations.

The rationale for consideration of pyroptosis as a key mechanism in neurodegeneration emerges from the recent realization that inflammasomes are active in neurodegeneration-associated inflammation. Inflammasome activation has been demonstrated in a variety of neurodegenerative diseases including AD,^46,67 PD,^87–89 and MS,¹⁵⁷ and ALS.^122,123 Inflammasome activity has been detected in multiple cell types in the CNS including microglia,^36,248, neurons,^89,97 and peripheral immune cells known to infiltrate the brain including monocytes.²⁴⁹ These findings are exciting because they provide a platform for exploring the prediction that pyroptosis links cell death and concomitant inflammation observed in neurodegenerative diseases. Studies of pyroptotic processes including the processing and release of vesicles, release of normally intracellular proteins into the extracellular environment, and pro-inflammatory cellular rupture, provides an interesting area of study towards understanding the complex multi-cellular interactions and stress processes observed in during neurodegeneration. In this review, we highlighted evidence of NLRP3 inflammasome activity and pyroptosis in the CNS and neurodegenerative disease, and discussed pyroptotic-extracellular vesicles (EVs) as a tractable means of detecting inflammation in neurological disorders.

NLRP3 has been largely characterized within peripheral immune cells, such as monocytes and macrophages, and resident immune cells of the CNS, namely microglia. Only recently has data emerged to suggest NLRP3 activation within neurons.^87,89,90 Despite the NLRP3 inflammasome being the most widely studied inflammasome, other inflammasome complexes play important roles within the CNS, especially within non-immune cells. In particular, the NLRP1 inflammasome has largely been implicated in neuronal inflammasome activation. For example, the knockdown of NLRP1 in a mouse model of AD resulted in reduced neuronal pyroptosis and reversed cognitive impairments.²⁵⁰ Further, elevated levels of NLRP1 and caspase-1 have been seen in resected hippocampal sections from patients with intractable mesial temporal lobe epilepsy (mTLE) and knock-down of NLRP1 and caspase-1 in a rat model of epilepsy reduced neuronal pyroptosis.²⁵¹ Also, while not explicitly linked to neuronal cell death, following ischemia-like conditions, such as glucose and oxygen-glucose deprivation, NLRP1 and NLRP3 proteins and cleaved caspase-1 levels were increased, and IL-1B and IL-18 precursor proteins matured in cultured cortical neurons, suggesting evidence of pyroptosis.²⁵² The AIM2 inflammasome has also been widely associated with neuronal damage. Embryonic cortical neurons induced with aberrant synthetic DNA were found to undergo pyroptosis after AIM2 inflammasome activation of caspase-1.²⁵³ AIM2 and caspase-1 up-regulation resulting in inflammasome-induced pyroptosis has also been determined as a cell death mechanism in neurons after Enterovirus-A71 infection.²⁵⁴ Knockdown of the AIM2 inflammasome attenuated inflammasome activation and pyroptosis in a mouse model of ischemia-reperfusion injury.²⁵⁵ The AIM2 inflammasome has also been implicated in astrocyte²⁵⁶ and microglial²⁵⁷ activation in the EAE model of MS. For comprehensive reviews on inflammasomes and neurodegenerative disease, please refer to these resources.^258,259

In conclusion, the NLRP3 inflammasome remains a focus of foundational and translational research while pointing towards other inflammasome classes as new frontiers for discovery in the context of neurological disorders. Novel bioassays and drugs provide an opportunity to conduct informed interventional trials aimed at modifying the progression of age-related disorders associated with chronic sterile inflammation. Based on the current knowledgebase, it is feasible that subsets of patients suffering from neurodegenerative disorders with detectably enhanced NLRP3 inflammasome activity could be treated with NLRP3 inhibitors (Fig 3). It stands to reason that these treatments could then be monitored over time to determine whether management of sterile inflammation could modify the disease course in debilitating conditions such as AD, PD, MS, and ALS, among others. More work will need to be done before this aspiration can be safely realized. This includes the optimization of universal biomarker assays that can reliably and efficiently detect inflammasome activation in living patients. Drug development hinges on the understanding of the strengths and weaknesses of peripheral versus central acting agents administered in oftentimes complex chronic disorders impacted by systemic factors such as aging and environmental exposure. Perhaps most importantly, foundational studies aimed at understanding secretory pathways, mechanisms of resolution, and cell and tissue specific mechanisms and modifiers of inflammasome activity merit prioritization to increase the scope of efforts to understand disease progression and target this critical pathway.

Fig 3.

Using pyroptotic proteins as biomarkers for disease. We suggest identification of at-risk individuals and populations based on environmental exposures, familial history, and genetic testing. Collection and screening of patient biofluids for inflammasome-related proteins will allow for the generation of unique pyroptotic signatures. This information can be used by physicians to inform treatment strategies. With the development of new inflammasome pathway modulators, targeted therapies might one day exist for the direct treatment of pyroptosis-related disorders. Created with BioRender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Abbreviations:

ALRs

absent in melanoma 2-like receptors

α-syn

alpha-synuclein

AD

Alzheimer’s disease

Aβ

amyloid beta

APP

amyloid precursor protein

ALS

amyotrophic lateral sclerosis

ASC

apoptosis-associated speck-like protein containing a CARD

BBB

blood brain barrier

BMDMs

bone-marrow derived macrophages

CARD

caspase activation and recruitment domain

CNS

Central nervous system

CSF

cerebrospinal fluid

COPD

chronic obstructive pulmonary disease

CRID

cytokine release inhibitory drug

DAMPs

danger associated molecular patterns

DPI

diphenyliodonium

DA

dopaminergic

Drd2

dopamine D2 receptor

ESCRT

endosomal sorting complexes required for transport

EAE

experimental autoimmune encephalomyelitis

EVs

extracellular vesicles

GSDMD

Gasdermin D

hIPSCs

human induced pluripotent stem cells

HIV-1

human immunodeficiency virus

HLA

human leukocyte antigen

IKK

IκB kinase

IFN-β

interferon-β

LPS

lipopolysaccharide

MCI

mild cognitive impairment

MS

Multiple Sclerosis

MVBs

multivesicular bodies

NLRP3

NLR family pyrin domain containing 3

NLRs

nucleotide-binding domain-like receptors

PD

Parkinson’s disease

PAMPs

pathogen associated molecular patterns

PET

positron emission tomography

PK

pharmacokinetics

PTMs

post-translational modifications

PSEN1/PS1

presenilin 1

PSEN2/PS2

presenilin 2

ROS

Reactive oxygen species

RvD2

Resolvin D2

SOD1

superoxide dismutase 1

SNpC

substantia nigra pars compacta

TDP-43

TAR DNA binding protein 43

TBI

traumatic brain injury

TLR

toll-like receptor

TCA

tricarboxylic acid