Catching a killer: Mechanisms of programmed cell death and immune activation in Amyotrophic Lateral Sclerosis

Abstract

In the central nervous system (CNS), execution of programmed cell death (PCD) is crucial for proper neurodevelopment. However, aberrant activation of these pathways in adult CNS leads to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). How a cell dies is critical, as it can drive local immune activation and tissue damage. Classical apoptosis engages several mechanisms to evoke "immunologically silent" responses, whereas other forms of programmed death such as pyroptosis, necroptosis, and ferroptosis release molecules that can potentiate immune responses and inflammation. In ALS, a fatal neuromuscular disorder marked by progressive death of lower and upper motor neurons, several cell types in the CNS express machinery for multiple PCD pathways. The specific cell types engaging PCD, and ultimate mechanisms by which neuronal death occurs in ALS are not well defined. Here, we provide an overview of different PCD pathways implicated in ALS. We also examine immune activation in ALS and differentiate apoptosis from necrotic mechanisms based on downstream immunological consequences. Lastly, we highlight therapeutic strategies that target cell death pathways in the treatment of neurodegeneration and inflammation in ALS.

Keywords: cell death; innate immunology; neurodegeneration.

Figure 1:. Immunological outcomes of apoptosis versus programmed necrotic mechanisms.

(A) Apoptosis induced either by the intrinsic pathway or by extrinsic death receptor signaling (e.g. TNFR1 and CD95) leads to characteristic morphological changes: nuclear condensation, cell shrinkage, budding of apoptotic bodies. Caspase-mediated inhibition of the ATP11C flippase and activation of Xkr8 scramblase exposes phosphatidyl-serine residues (PS) on the outside of the plasma membrane. Macrophages use several receptors (e.g. TIM4 and MERTK) to bind exposed PS residues (”eat me” signal) and phagocytose debris. (B) Programmed necrotic mechanisms display morphologic features of cellular swelling and loss of plasma membrane integrity. Necrosis culminates in the release of cytokines, chemokines, DAMPs and other intracellular molecules. These cellular contents are sensed by immune cells, which engage innate immune pathways (eg. TLRs, RAGE1, RIGI/MAVS) to promote activation and boost local inflammation.

Figure 2:. Amyotrophic lateral sclerosis (ALS) symptoms and pathogenesis.

Patients presenting with ALS classically exhibit both (A) upper motor neuron (UMN) and (B) lower motor neuron (LMN) symptoms. These include spasticity (UMN) and muscle weakness and fasciculations (LMN). Thirty percent of patients display “bulbar” onset symptoms such as slurred speech, difficulty swallowing and facial weakness. A subset of patients also display concomitant symptoms of frontotemporal dementia (FTD), presenting with cognitive and behavior changes due to cortical degeneration. Ultimately, ALS patients succumb to respiratory insufficiency. (C) In ALS both upper and lower motor neurons display pathology. There are many proposed mechanisms, but aggregation of ALS-associated proteins, such as TDP-43, is considered central to disease onset. Studies employing SOD1 G93A and other mouse models have shown that expression of protein aggregates in neurons is key to disease onset, but that subsequent neuroinflammation drives disease progression. Microglia, astrocytes and oligodendrocytes can sense and respond to protein aggregates by initiating programmed cell death (PCD) pathways. Activation of these cell signaling programs leads to secretion of neurotoxic factors (e.g. IL-1 family cytokines), gliosis and recruitment of peripheral immune cells. Glia can also mediate neuroprotective functions in the context of neurodegeneration. For example, microglia can bind TDP-43 using TREM2 to promote aggregate clearance and neuroprotection. Experimental depletion of microglia (or Trem2 −/−) worsen disease progression in TDP-43 murine models of ALS.

Figure 3:. Evidence for activation of apoptosis in ALS.

Studies using in vitro and in vivo approaches have found that ALS-associated proteins (e.g. TDP-43, mSOD1, toxic C9ORF72 dipeptide repeat proteins) and mutations in fused-in-sarcoma (FUS) can cause activation of (A) intrinsic apoptosis in neurons. Intrinsic apoptosis begins with cellular stress that activates pro-apoptotic BH3-only proteins. BH3 only proteins such as BIM and BID, sequester anti-apoptotic BCL-2 proteins and promote oligomerization of pro-apoptotic family members (e.g. BAX) in mitochondrial membrane. Following mitochondrial outer membrane permeabilization (MOMP) mediated by BAX and BAK, cytochrome-c (cyt-c) is released from the intermembrane space into the cytosol. There cyt-c binds and activates Apaf-1, allowing for oligomerization and recruitment of pro-caspase-9 via homotypic CARD-CARD interactions. The apoptosome (cyt-c, Apaf-1, active caspase-9) cleaves executioner caspases to mediate apoptosis. (B) Infiltration of CD8+ T cells into the spinal cord, or mutations in TBK1 can sensitize neurons to extrinsic apoptosis. This mode of cell death is triggered by ligation of a death receptor (TNFR1, Fas, or TRAIL-R1/2), and can lead to apoptosis, necroptosis, or cell survival. Following CD95 ligation (by FasL), CD95 binds FADD using death domains (DD). FADD then recurits caspase-8 via death effector domain (DED) interactions, and assembles a caspase-activation platform called the DISC. This platform leads to caspase-8 activation and thus engages the extrinsic apoptotic pathway. Caspase-8 can also process Bid into t-Bid, which then engages the mitochondrial (intrinsic) pathway of apoptosis. Upon ligation to TNF, TNFR1 recruits the adaptor protein TRADD and RIPK1, which in turn mobilizes additional partners, such as the ubiquitin ligases TRAF2 and cIAP1/2 to form complex I. When RIPK1 is deubiquitinated it allows for release of complex 1, which can then bind FADD and caspase-8 (complex II). This complex can induce apoptosis, or necroptosis in situations where FADD/caspase-8 are inhibited. TBK1 restrains TNFR signaling and downstream RIPK1-dependent apoptosis (RDA). Mutations in TBK1 can lead to loss of this inhibition and enhanced caspase-8 activation.

Figure 4:. Signaling mechanisms and outcomes of pyroptosis in the ALS.

(A) Triggers of pyroptosis in the context of neurodegeneration include aggregated proteins such as TDP-43 and mutant SOD1 (ALS), as well as amyloid beta oligomers (AD) and fibrillar alpha-synuclein (PD). There is some evidence to suggest (B) neurons, astrocytes and skeletal muscles can undergo pyroptosis in the context of ALS. Most of the available evidence points to (C) microglia as the chief cells responding to aggregated protein species by engaging pyroptosis. Neurodegeneration associated molecules are detected by sensor molecules (e.g. NLRP3), which recruit and oligomerize adaptor proteins (e.g. ASC). Adaptors such as ASC and pyrin contain CARD domains that can recruit caspase-1 as part of the inflammasome complex. Active caspase-1 can process IL-1 family cytokines and cleave the linker region of full-length GSDMD (Asp 275). (D) When released, N-terminal GSDMD undergoes a conformational change enabling binding to acidic lipid residues and pore formation in the cellular membranes. (i) If these pores form in the plasma membrane of a living cell they act as conduits for IL-1 release and mediate a “hyperactivated” phenotype. (ii) If enough GSDMD pores accumulate in the plasma membrane, cellular swelling and overt pyroptosis can occur. iii) This leads to release of intracellular contents and serves to (iv) recruit peripheral immune cells and activate microglia and astrocytes. High levels of IL-1 release can contribute to neurotoxicity and may exacerbate neuronal injury.

Figure 5:. Necroptotic signaling mechanisms and outcomes in ALS.

(A) Triggers of necroptosis in the context of ALS include aggregated proteins such as TDP-43 and mutant SOD1 (ALS), as well as mutations in negative repressors of the RIPK1 pathway such as optineurin (OPTN) and TBK1. There is evidence to suggest (B) neurons, astrocytes and microglia undergo necroptosis in the context of ALS. However, the strongest evidence points to (C) oligodendrocytes as the chief cells executing necroptosis in ALS. Stimulation of TNFR1 by TNF promotes the formation of an intracellular signaling complex where the death domains (DD) of trimerized TNFR1 binds the DD-containing proteins: TRADD and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). TRADD helps to recruit a number of RIPK1 regulators, including OPTN and TBK1. In situations of caspase-8 down regulation or inhibition, RIPK1 is auto phosphorylated and dimerizes via its C-terminal death domain. This dimerization promotes RIPK3 activation and the subsequent formation of the necrosome (complex IIb) comprised of RIPK1, FADD, Casp8, RIPK3 and mixed-lineage kinase domain-like pseudokinase (MLKL). (D) This complex enables (i) p-MLKL to insert and oligomerize in plasma membrane and iii) execute overt necroptotic cell death. As in other forms of programmed necrosis, release of DAMPs and intracellular contents serves to recruit and activate peripheral immune cells and promote gliosis. In the case of necroptosis in oligodendrocytes, this leads to demyelination and perhaps contributing to the progressive axon loss observed in ALS.