Autolysosomal acidification impairment as a mediator for TNFR1 induced neuronal necroptosis in Alzheimer’s disease

Evridiki Asimakidou, Richard Reynolds, Anna M.Barron, Chih Hung Lo

Neuronal necroptosis–an emerging form of regulated cell death associated with neuroinflammatory signaling: Alzheimer’s disease (AD) is characterized by the presence of extracellular amyloid-β (Aβ) plaques and intracellular tau neurofibrillary tangles as well as progressive neuronal loss.Recent evidence has suggested that prolonged neuroinflammation with increased levels of cytokines, arising from neuronal injury, innate immune responses from glial cells, and peripheral inflammation, leads to neuronal death and AD progression.Neuronal necroptosis is an emerging form of regulated cell death associated with neuroinflammatory signaling.Necroptosis typically occurs in response to sustained inflammation while apoptosis facilitates normal turnover of cellular contents important for growth and development.By combining features of apoptosis and necrosis,necroptosis has been proposed to constitute a more comprehensive mechanistic explanation for neurodegeneration, which is less likely to occur by the immunologically silent apoptotic cell death or by the acute occurrence of necrosis after cellular stress (Jayaraman and Reynolds, 2022).

The tumor necrosis factor (TNF) receptor 1(TNFR1) signaling pathway has been implicated as a central mediator of inflammation and cell death.The binding of TNF cytokine to TNFR1 induces a conformational change in the receptorligand network that enables the recruitment of receptor-interacting protein kinase 1 (RIPK1)and TNFR1-associated death domain (TRADD),and the formation of downstream complexes I and II.While complex I activates the nuclear factor-κB pathway responsible for stimulating cytokine and anti-apoptotic gene expression,complex II mediates cell death signaling including apoptosis and necroptosis.Caspase-8 activity,which is dependent on its expression level and phosphorylation state as well as its interaction with the ubiquitin-proteosome system or the autolysosomal network, determines whether cells undergo apoptosis or necroptosis.Inactivation or absence of caspase-8 results in RIPK1 not being cleaved, leading to subsequent phosphorylation of RIPK1, RIPK3, and pseudokinase mixed lineage kinase domain-like (MLKL) and the initiation of necroptosis.Translocation of phosphorylated MLKL(pMLKL) oligomers and their insertion into the cellular membrane followed by pore formation and the subsequent release of intracellular molecules are key hallmarks of necroptotic cell death.It is worth noting that TNF-independent RIPK3-mediated necroptosis based on the activation of the pattern recognition receptors such as Toll-like receptors 3 and 4 as well as zDNA-binding protein 1 has also been described (Tummers and Green,2022).

In AD brain tissues, TNFR1 expression and signaling responsible for neuronal death is increased, while the expression level of TNFR2 responsible for immune response and neuronal homeostasis is decreased, resulting in increased glial activation and neurodegeneration (Figure 1A).Recent studies have demonstrated that necroptosis or inflammatory cell death plays a major role in AD(Caccamo et al., 2017; Jayaraman et al., 2021;Jayaraman and Reynolds, 2022).In post-mortem AD brain tissues, the expression of RIPK1, RIPK3,MLKL, and their active phosphorylated derivatives was shown to be specific to degenerating neurons(Caccamo et al., 2017).Importantly, we illustrated in our recent study in AD brain tissues that pMLKL-positive hippocampal pyramidal neurons co-expressed phosphorylated tau and were found to be adjacent to Aβ plaques, activated microglia, and cytotoxic T-cells, supporting that a neuroinflammatory micromilieu renders neuronal cells vulnerable to necroptotic death (Jayaraman et al., 2021).In transgenic mouse models of AD,increased levels of proteins that make up the necrosome, including RIPK1, RIPK3, and MLKL,were detected (Caccamo et al., 2017; Xu et al.,2021).Lowering of TNFR1 levels has also been shown to reduce the amount of pMLKL (Xu et al., 2021) and administration of a RIPK1 inhibitor decreased the ratio of pMLKL to MLKL and rescued neuronal death (Caccamo et al., 2017), supporting the mechanistic role of necroptosis in familial AD pathogenesis.

Recent studies have further demonstrated the role of autophagic and lysosomal dysfunctions in mediating TNFR1-activated necroptosis in AD.Besides TNF-induced lysosomal acidification defect and membrane permeabilization (Wang et al., 2015; Wu et al., 2021), there are interactions between the molecules in the TNFR1 signaling axis and the autolysosomal pathway (Liu et al.,2018; Xu et al., 2021).This suggests the existence of crosstalk between these two distinct pathways that can implicate the overall organelle and cellular functions.Importantly, the contribution of lysosomal proteins to cellular signaling has led to lysosomes emerging as signaling hubs that regulate diverse cellular processes, including cell death and survival (Figure 1B), and opening new avenues for therapeutic intervention.Here,we provide new perspectives on the pathogenic mechanisms involved in this new molecular axis and recommend novel therapeutic strategies to target AD.

The pathogenic triad–autophagic-lysosomalmitochondrial dysfunction: Autophagic,lysosomal, and mitochondrial functions form a broad metabolic network in cells, including neurons.Autophagy is an essential process to maintain cellular homeostasis, which executes a pro-survival function under pathological stress.Autophagy is highly dependent on intact lysosomal function, including sufficient luminal acidification,to promote the fusion of autophagosome and lysosome to form autolysosome and complete the degradation of toxic protein aggregates,damaged organelles, and cellular debris.While mitochondria play a key role in supplying adenosine triphosphate, which is required for the maintenance of lysosomal acidification, proper turnover of mitochondria by the autolysosomal system aids in maintaining the bioenergetic equilibrium.Below we will discuss the association of lysosomal acidification defect, autophagy impairment, and mitochondrial dysfunction with TNFR1-mediated neuronal necroptosis in AD.

Figure 1 ｜TNFR1-mediated neuronal necroptosis is associated with the neuroinflammatory microenvironment in Alzheimer’s disease (AD).(A) Intracellular tau tangles, extracellular amyloid plaques, increased levels of cytokines including TNF, as well as glial activation, T-cell activation, and neuronal death are neuropathological hallmarks of AD.Cellular phenotypes with high TNFR1 and low TNFR2 are associated with neurodegenerative pathology and those with low TNFR1 and high TNFR2 may be neuroprotective.(B) Interaction between TNFR1 signaling axis and autolysosomal pathway under neuroinflammatory condition.Pathogenic triad of lysosomal acidification defect, autophagic impairment, and mitochondrial dysfunction contributes to TNFR1-induced neuronal necroptosis.Modulation of the autolysosomal network, including alteration of lysosomal acidification, determines the cell fate to adopt the necroptosis or survival pathway.Created with BioRender.com.FADD: Fas-associated death domain; MLKL: mixed lineage kinase domain-like protein; P: phosphorylated form of proteins; RIPK1/3: receptor-interacting protein kinase 1/3; ROS: reactive oxygen species; TNF: tumor necrosis factor;TNFR1/2: tumor necrosis factor receptor 1/2; TRADD: TNFR1-associated death domain.

It has been shown that TNF stimulation impaired autolysosomal function and reduced autophagic flux in SH-SY5Y neuroblastoma and primary neurons (Xu et al., 2021).This leads to the accumulation of autophagy receptor p62, which is mediated by the downregulation of the UV radiation resistance-associated gene (UVRAG)that is involved in autophagosome formation and maturation.The accumulated p62 interacted and induced RIPK1 oligomerization, activating the downstream RIPK1/RIPK3/MLKL signaling cascade,which could be reversed with overexpression of UVRAG (Xu et al., 2021).Importantly, UVRAG overexpression reduced the accumulation of pMLKL and p62 and mitigated learning and memory deficits in APP/PS1 mice (Xu et al.,2021).It is also worth noting that this study was conducted under the conditions that caspases and inhibitors of apoptosis proteins are inactivated to specifically direct the cellular response towards induction of necroptosis.

The notion of TNF-induced autolysosomal dysfunction is supported by another study evaluating the neurotoxic effect of triclosan (TCS),an antimicrobial agent used in healthcare products that has been identified to generate neurological conditions.This study found that TCS treatment in Neuro-2a mouse neuroblastoma increases TNF expression and activates TNFR1 signaling,leading to the necroptotic pathway with enhanced phosphorylation of RIPK1, RIPK3, and MLKL (Katiyar et al., 2022).It has been suggested that TCS contributes to neuronal necroptosis by preventing lipidation of autophagosome bilayers, impairing lysosomal acidification, reducing the formation of autolysosomes, and abrogating the autophagic process (Katiyar et al., 2022).This is consistent with another study, which reported that lysosomal degradation is blocked under the condition of TNF-induced necroptosis and established the association between autophagy and necroptosisregulating kinases (Wu et al., 2021).Strikingly, TCS also induces a three-fold increase in tau expression as well as pathogenic phosphorylation, alluding to the presence of crosstalk between pathogenic tau proteins, neuroinflammatory signaling, and necroptosis in AD neurodegeneration.

These observations are complemented by a study in PC12 cells derived from rat pheochromocytoma,where TNF released from activated microglia elevated lysosomal pH and disrupted autophagic flux, causing reduced degradation and increased accumulation of toxic α-synuclein (Wang et al.,2015).Importantly, this study also suggests that TNF-induced autophagic impairment mainly results from the suppression of autophagic flux and lysosome degradation but not from blocking of autophagy initiation and induction.This is further supported by the finding that activation of transcription factor EB and promotion of lysosome biogenesis with mammalian target of rapamycin inhibitor treatment restored toxic protein degradation and clearance from the cells(Wang et al., 2015).Another study supports the idea that RIPK1 and RIPK3 are typically degraded by the lysosomal pathway and lysosomal inhibition sensitizes PC12 cells and rat cortical neurons to TNF-induced necroptosis in the presence of caspase inhibitors (Liu et al., 2018).Under the condition of lysosomal acidification inhibition,it was also observed that caspase inhibition alone, independent of TNF stimulation, was able to induce neuronal necroptosis, suggesting that the accumulation of RIPK1 and RIPK3 in impaired lysosomes may be sufficient for necrosome formation to trigger necroptosis (Liu et al., 2018).Altogether, these studies exemplify the detrimental implication of poorly acidified lysosomes in mediating necroptosis.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for lysosome membrane repair to preserve lysosomal acidification (Radulovic et al., 2018).Recent reports have highlighted that dysfunctional ESCRT-III proteins, in particular charged multivesicular body proteins (CHMPs), lead to inhibition of autophagy and are associated with neurodegeneration (Lee et al., 2007).Specifically,frontotemporal dementia-associated mutant CHMP2B failed to dissociate from ESCRT-III and induced dysfunctional machinery, leading to primary cortical neuronal death (Lee et al., 2007).In AD brain tissues, elevated levels of CHMP2B proteins were expressed in pMLKL-positive AD hippocampal neurons (Jayaraman et al., 2021),supporting the idea that upregulation of ESCRTIII machinery exerts a compensatory role in necroptosis in an effort to decelerate or interrupt necroptotic cell death.Nonetheless, the role of ESCRT proteins as well as changes in their expression levels to impart neurodegenerative or neuroprotective effects remain to be investigated.Disrupted lysosome-mitochondria crosstalk with increased accumulation of damaged mitochondria is detrimental to AD progression.Mitochondrial dysfunction generates elevated levels of reactive oxygen species (ROS), which cannot be counteracted by antioxidant defense and triggers neuroinflammation.In a neuronal model of necroptosis, the formation of the necrosome and the subsequent necroptotic signaling were associated with mitochondrial damage and ROS production (Jayaraman et al., 2021).Defective lysosomal acidification and necroptosis in Neuro-2a cells have also been linked to a decrease in mitochondrial membrane potential and integrity(Katiyar et al., 2022).In another study, Aβ peptide has been shown to elevate TNF levels and induce neuroinflammation through stimulating JNK signaling pathways and mediating necroptosis through RIPK1 signaling.This observation is linked to mitochondrial damage and oxidative stress in both BV2 mouse microglial cells and in the hippocampus of a rat model of AD (Tu et al., 2020).Furthermore, it has been shown that, besides permeabilizing the cell membrane, pMLKL can also translocate to mitochondria, activate pyruvate dehydrogenase, and increase ROS production,forming a positive feedback loop to further promote necrosome formation and necroptosis activation (Yang et al., 2018).These observations are critical as they underline the importance of inter-organelle interaction and functional crosstalk between lysosomes and mitochondria in necroptosis.

Concluding remarks and future perspectives:Increasing evidence points to a pivotal role of necroptosis in neurodegeneration particularly in AD, although further studies are required to fully understand the molecular signals that trigger and modulate this process.The TNF-TNFR1 signaling pathway plays a fundamental role and acts as a major mediator of necroptotic neuronal death.Downstream molecules that are crucial in this cascade encompass RIPK1, RIPK3, and MLKL and their phosphorylated forms, with the latter being regarded as a marker for necroptosis.An interplay between autolysosomal dysfunction and TNFR1-activated necroptosis has been established, while novel concepts underlying important roles of ESCRT machinery and mitochondrial function in necroptosis have emerged.Moreover, early detection and targeting of lysosomal acidification dysfunction could have both prognostic and therapeutic values (Lo and Zeng, 2023).Future directions include: (1) investigating the role of autolysosomal dysfunction and associated neuronal death in other neuroinflammatory diseases such as multiple sclerosis; (2) dissecting the role of lysosome-mitochondria crosstalk in driving necroptosis; (3) expanding lysosome reacidifying pharmacological agents in addition to the existing efforts in developing receptorspecific inhibitors of TNFR1 signaling; and (4)understanding the brain-body interaction between peripheral inflammation and neuroinflammation in driving neurodegeneration.Taken together,understanding the pathogenic mechanisms of TNFR1-mediated neuronal necroptosis holds promise for the development of novel therapeutic strategies targeting AD and related dementias.

The authors thank Jialiu Zeng from Nanyang Technological University Singapore for technical discussion and proofreading the manuscript.

This work was supported by a Lee Kong Chian School of Medicine Dean’s Postdoctoral Fellowship(021207-00001) from Nanyang Technological University Singapore and a Mistletoe Research Fellowship (022522-00001) from the Momental Foundation USA (to CHL).

Evridiki Asimakidou, Richard Reynolds,Anna M.Barron, Chih Hung Lo*

Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK (Asimakidou E)Department of Brain Sciences, Faculty of Medicine,Imperial College London, London, UK (Reynolds R)Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore(Barron AM, Lo CH)

*Correspondence to: Chih Hung Lo, PhD,chihhung.lo@ntu.edu.sg.https://orcid.org/0000-0003-2717-4484(Chih Hung Lo)

Date of submission: August 19, 2023

Date of decision: October 28, 2023

Date of acceptance: November 5, 2023

Date of web publication: December 15, 2023

https://doi.org/10.4103/1673-5374.390979 How to cite this article:Asimakidou E, Reynolds R,Barron AM, Lo CH (2024) Autolysosomal acidification impairment as a mediator for TNFR1 induced neuronal necroptosis in Alzheimer’s disease.Neural Regen Res 19(9):1690-1870.

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