TNF-α-dependent neuronal necroptosis regulated in Alzheimer's disease by coordination of RIPK1-p62 complex with autophagic UVRAG

doi:10.21203/rs.3.rs-425226/v1

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Research article

TNF-α-dependent neuronal necroptosis regulated in Alzheimer's disease by coordination of RIPK1-p62 complex with autophagic UVRAG

https://doi.org/10.21203/rs.3.rs-425226/v1

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posted 27 Apr, 2021

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Background Neuronal death is a major hallmark of Alzheimer’s disease (AD). Necroptosis, as a programmed necrotic process, is activated in AD. However, what signals and factors initiate necroptosis in AD is basically unknown.

Methods We examined the expression levels of critical molecules in necroptotic signaling pathway by immunohistochemistry (IHC) staining and immunoblotting using brain tissues from AD patients and AD mouse models of APP/PS1 and 5×FAD. We performed brain stereotaxic injection with recombinant TNF-α, anti-TNFR1 neutralizing antibody or AAV-mediated gene expression and knockdown in APP/PS1 brains. For in vitro studies, we used TNF-α combined with zVAD-fmk and Smac mimetic to establish neuronal necroptosis models and utilized pharmacological or molecular biological approaches to study the signaling crosstalk.

Results We find that activated neuronal necroptosis is dependent on upstream TNF-α/TNFR1 signaling in both neuronal cell cultures and AD mouse models. Upon TNF-α stimulation, accumulated p62 recruits RIPK1 and induces its self-oligomerization, and activates downstream RIPK1/RIPK3/MLKL cascade, leading to neuronal necroptosis. We further reveal that ectopic accumulation of p62 is caused by impaired autophagy flux, which is mediated by downregulation of UVRAG during the TNF-α-promoted necroptosis. Notably, UVRAG overexpression can inhibit neuronal necroptosis in cultures and in brains of AD mice.

Conclusions We identify a finely controlled regulation of neuronal necroptosis in AD by coordinated TNF-α signaling, RIPK1/3 activity and autophagy machinery.

Cognitive Neuroscience

Neurobiology of Disease

Alzheimer’s disease

neuronal necroptosis

TNF-α/TNFR1 signaling

RIPK1/RIPK3/MLKL cascade

autophagic flux

p62

UVRAG

Alzheimer’ disease (AD) is a progressive neurodegenerative disorder characterized by clinical symptoms of memory loss, personality changes, and general cognitive decline [1, 2]. The neuropathological alterations of AD brain include Amyloid-β (Aβ) deposit, aggregates of hyperphosphorylated/misfolded tau, neuroinflammation, and neuronal loss [3, 4]. Despite continuing debates about hypotheses for the cause(s) of AD, Aβ-induced neuronal death and brain dystrophy has emerged as the most extensively accepted theory [5]. In the past decades, evidence has shown that extracellular Aβ plaques are directly toxic to adjacent neurons [6], and soluble or aggregated Aβ can also induce inflammatory processes and secondary cell death [7]. Among the various forms of cell death, neuronal apoptosis and necrosis are believed to be the two major death pathways for neuronal loss in AD, but recent findings have also implicated necroptosis, a programmed cell necrosis [8-10]. However, the mechanism initiating necroptosis in AD is not known.

Another process that may be involved in AD pathology is autophagy, a main cellular catabolic process for protein aggregates and damaged organelles, has crucial roles in the maintenance of cell homeostasis [11]. Autophagy primarily acts as a pro-survival role, in some cases the damaged autophagy may lead to distinct autophagic cell death, rather than apoptosis or necrosis [12, 13]. Recent studies also suggest its function in modulating the switching between apoptosis and necroptosis [14]. Interestingly, a considerable amount of investigation has found that impaired autophagy contributes to the accumulation of misfolded protein aggregates, which is featured by most neurodegenerative diseases (NDs), including AD [15]. However, very little is currently known about the possible interplay between autophagy pathway and neuronal necroptosis in AD pathogenesis.

We have turned to investigation of possible action of a candidate critical proinflammatory cytokine in these processes: tumor necrosis factor-α (TNF-α). It is already known to be involved in neuroinflammation and to contribute to AD pathology [16, 17]. TNF-α is elevated in the cerebrospinal fluid (CSF) and plasma in aging, mild cognitive impairment (MCI), and AD patients, and MCI patients with a higher level of TNF-α are much more likely to progress to full AD [18]. Data from animal models further reveal that TNF-α-induced inflammation may have a detrimental effect on neuronal death, and chronic release of TNF-α during AD progression is likely through the activation of microglia, astrocytes and neurons stimulated by aggregated Aβ [7, 19]. Currently, strategies targeting TNF-α inhibition have demonstrated some reduction of brain pathology and alleviation of cognitive decline in both rodent models and in AD patients [17, 19].

TNF-α acts through a classical signaling pathway initiated by binding to its receptor TNFR1. TRADD, TRAF2/5, RIPK1, cIAP1/2 and other subunits are then recruited to form complex I, which in turn leads to ubiquitination of RIPK1 and thus to NF-κB activation [20]. When NF-κB activation is inhibited, e.g., by blocking cIAP1/2, caspase-8 can be activated, which induces RIPK1 cleavage and leads to cell apoptosis. Elimination or inhibition of caspase-8 prompts RIPK1 activation through deubiquitination and leads to its interaction with FADD, TRADD, and RIPK3, to form complex II. Complex II can phosphorylate the pseudokinase MLKL and ultimately promotes necrotic cell membrane disruption and cell death, which is also termed necroptosis [21, 22]. However, whether elevated TNF-α in AD can induce neuronal necroptosis has remained conjectural.

We now report that elevated TNF-α signaling can indeed induce the activation of neuronal necroptosis in AD. TNF-α-triggered neuronal necroptosis is further modulated by impaired autophagic flux, in which a key regulator UVRAG tightly regulates signaling transduction. These findings thus implicate crosstalk between neuronal necroptosis and autophagy machinery in AD pathology, and may exemplify a more general paradigm for cell death regulation in other NDs.

Human brain tissues

Paraffin sections of the human cerebral cortex were kindly provided by NIH NeuroBioBank. These materials include samples from 8 AD patients (Braak VI) and 7 age-matched healthy elderly.

Mice

APP/PS1 (JAX, Stock # 034848) and 5×FAD (JAX, Stock # 034848) transgenic mice were purchased from The Jackson Laboratory and generously provided by Dr. Guobing Chen (Jinan University, China). In this study, we crossed the APP/PS1 or 5×FAD with C57BL/6 mice (GemPharmatech, China) to obtain daughter mice for further study. All mice were maintained under standard laboratory conditions with free access to food and water unless otherwise indicated. All experiments were conducted in accordance with the regulations for the Administration of Affairs Concerning Experimental Animals (China) and approved by the China Pharmaceutical University Animal Ethics Committee. Mice were genotyped by One Step Mouse Genotyping Kit (Vazyme, Cat# PD104-01), according to manufacturer's instructions.

Immunohistochemistry (IHC), immunofluorescence (IF) and autophagic flux analysis

For DAB staining of paraffin sections, 5 µm slices were fixed in 10% formaldehyde followed a protocol as described [23]. For Immunofluorescence staining of cryostat sections, 30 µm frozen slices were sectioned by a Leica CM1950 microtome and followed a protocol as previously described [23]. All antibodies used in this study were listed (Table S1). Images were analyzed by Olympus IX73 microscopy.

Images of cellular autophagic flux analysis were captured by the Olympus FV1000 laser scanning confocal microscope. For quantitative analysis of the fluorescence intensity, the integral optical density was measured by using Image-Pro Plus 6.0 software.

Brain stereotaxic injection

Brain stereotaxic injection was performed as described [24, 25] with modification. In brief, for lateral ventricle injection of TNF-α, 2 μL recombinant murine TNF-α (5 μg in 2 μl of PBS) or 2 μL PBS (Ctrl) was injected into the lateral ventricle at -0.4 mm from bregma, mediolateral 1.0 mm, depth 2.5 mm of mouse brains using a digital pump (ZS Dichuang Company, China) at a speed of 0.25 μl/min with a microinjection syringe. After injection, the syringe was remained in the injection point for additional 5 min before it was slowly removed. For hippocampal CA1 injection, same instruments and procedure applied, except 4 μL TNFR1 antibody (Thermo Fisher Scientific, Cat# 16-1202-85) or 2 μL viral particles (10¹²~10¹³ v.g./ml) was injected into the hippocampal CA1 region at -2.2 mm from bregma, mediolateral 1.7 mm, depth 2.4 mm of mouse brains. All mice were placed on a heating pad until they recovered from the surgery. One week after injection of TNF-α (or) TNFR1 antibody, while three weeks after virus injection, mice were sacrificed for further experiments.

Cell culture

Human SH-SY5Y cells were cultured in DMEM with 10% FBS. Rat PC12 cells were cultured in DMEM with 5% FBS (Gibco) plus 10% horse serum (Hyclone). The primary mouse cortical neuron culture was performed as described [26], In brief, cortical neurons were isolated from E16-E18 mouse brains and cultured in Neurobasal (Gibco, Ca# 21103049) medium supplemented with B-27 (Gibco, Ca# 17504044) on Poly-D-Lysine (Sigma Aldrich, Ca# P7405) coated 6-well or 96-well plates. Cells were maintained at 37 °C in an incubator containing 5% CO₂. The cells were pretreated with or without zVAD-fmk (MCE, Ca# HY-16658B), Necrostatin-1 (MCE, Ca# HY-15760) or Chloroquine (MCE, Ca# HY-17589) for 1 h followed by adding recombinant human or murine TNF-α (or PBS) (PeproTech, Cat# 300-01A or 315-01A) in combination with (or without) SMAC mimetic (MCE, Ca# HY-12600) for 8 h for protein extraction, RNA/DNA extraction, IHC and cellular autophagic flux analysis. In some cases, cells were cultured for 24 h for cell viability analysis, Propidium Iodide (PI) staining, and Acridine Orange (AO) staining.

Preparation of adeno-associated virus (AAV), lentivirus and adenovirus

The AAV vector expressing two shRNA sequences and a separated RFP (pAV-2in1shRNA-CMV-RFP) was obtained from Vigene Biosciences, Inc. The structure of AAV vector can be found in Supplementary information (Fig S2). The below is shRNA sequences. TNFR1 shRNA-1: 5’-CCTCGTGCTTTCCAAGATGA-3’; TNFR1 shRNA-2: 5’-CCCGAAGTCTACTCCATCAT-3’; p62 shRNA-1: 5’-TAGTACAACTGCTAGTTATT-3’, p62 shRNA-2: 5’-GAGGTTGACATTGATGTGGA-3’. The AAV vector expressing GFP (pAV-CAG-P2A-GFP) was obtained from Vigene Biosciences, Inc. HA-tag fused UVRAG was cloned into an AAV vector named pAV-CAG-P2A-GFP (Vigene Biosciences) (Fig. S2). The viral particles (10¹²~10¹³ v.g./ml) were packaged and purified by Vigene Biosciences for further use.

For lentivirus production, HEK293T cells were transfected with lentiviral vectors and packing plasmids using a Lentiviral Packaging Kit (Yeasen, Ca# 41102ES20). The structure of lentiviral vectors (Yeasen) used were listed in Supplementary information (Fig. S2). The shRNA sequences were listed as below. RIPK1 shRNA: 5’-CGGAACAGATTCTGGTGTCT-3’; p62 shRNA: 5’-CCTCTGGGCATTGAAGTTGA-3’. shRNA-resistant wild type p62 (p62-wt), zz domain deleted p62 (p62-zz△), and uvrag genes were cloned into indicated vectors, respectively (Fig. S2). SH-SY5Y cells were transduced with above lentiviral particle-containing supernatant in the presence of 8 mg/ml polybrene for 24 h, and then exposed to 2 mg/ml puromycin. The lentivirus encoding eGFP was used as a control.

The adenoviral particles expressing mCherry-GFP-LC3B protein (Ad-mCherry-GFP-LC3) and GFP-p62 fusion protein (Ad-GFP-p62) were purchased from Beyotime (Ca# C3011 and C3015). The control adenoviral particles expressing β-glactosidase (Ad-LacZ) were purchased from Sciben Biotech (Ca# AD1002). SH-SY5Y cells or primary neurons were transduced with the individual adenoviral particles for 24 h at 5 of multiplicity of infection (MOI=5). Ad-LacZ was used as a control.

Cell viability analysis

Cells were seeded in 96-well plates before indicated treatment. The cells were then loaded with Enhanced Cell Counting Kit-8 (CCK-8) (Beyotime, Ca# C0046). Subsequently, the cell viability was determined by measuring the absorbance at 460nm.

Propidium iodide (PI) staining and Acridine orange (AO) staining

Cells were seeded in 6-well plates before indicated treatment. Then cells were incubated with 2 μg/ml propidium iodide (PI) (Sigma Aldrich, Ca# P4170) for 10 min or 200 μM Acridine orange (AO) (Sigma Aldrich, Ca# A6014) for 15 min as described [27]. All stained wells were rinsed three times with PBS, followed by direct image capture using Olympus IX73 microscope.

Immunoblotting, immunoprecipitation and insoluble protein extraction

Immunoblotting and immunoprecipitation were performed as described [28]. For soluble and insoluble protein extraction, cells were lysed in cold PBS/1% Triton X-100 buffer (PBS containing 1% Triton X-100 and cocktail protease inhibitor) for 30 min on ice. After centrifugation at 15,000×g for 30 min at 4 °C, the supernatants were collected as soluble protein fractions. Pellets were washed with cold PBS/1% Triton X-100 buffer, and then re-suspended in 8 M urea containing 1% SDS, 1% Triton X-100 and cocktail protease inhibitor. After sonicated, the samples were centrifuged at 15,000 × g for 15 min at 4 °C and the supernatants were collected as insoluble fractions. All the antibodies used were listed (Table S1).

Quantitative real-time PCR (qPCR)

Cells were homogenized in Trizol reagent (Vazyme, Ca# R701-01) for extraction of total RNA based on the manufacturer’s protocol. Purified RNA was reverse transcribed to cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Ca# R312-01). mRNA levels were determined by qPCR using AceQ qPCR SYBR Green Master Mix kit (Vazyme, Ca# Q131-02). All primers used are listed in Table 2.

Core promoter analysis and chromatin immunoprecipitation

Core promoter analysis of Uvrag gene was performed using a computational program ConTra v2 (http://bioit.dmbr.ugent.be/contrav2/index.php). Chromatin was extracted and immunoprecipitated using a Chromatin-IP (ChIP) kit (CST, #9004) and an anti-RelA antibody (CST, #8242) according to the manufacturer’s protocol. Then separated and purified DNA was amplified by qPCR. All primers used are listed in Table 2.

Data and statistical analyses

All data were expressed as mean ± SEM. Student's t-test for nonpaired replicates was used to identify statistically significant differences between treatment means. Group variability and interaction were compared using either one-way or two-way ANOVA followed by Bonferroni's post-tests to compare replicate means. Significance was accepted at p < 0.05.

TNF-α signaling promotes neuronal necroptosis in AD mice

We hypothesized that elevated TNF-α signaling in AD brain may lead to neuronal necroptosis. To test this hypothesis, we first examined the level of MLKL, a marker of cell necroptosis, in the brains of AD patients. Immunohistochemical (IHC) staining demonstrated MLKL upregulation in samples of cerebral cortex from AD patients at Braak VI stage compared to age-matched healthy controls (Ctrl) (Fig. 1a). We then checked the levels of total MLKL and phosphorylated-MLKL (p-MLKL) in 10-month old APP/PS1 and 5×FAD transgenic AD mouse models. IHC images of hippocampal CA1 regions showed that compared to wild type (WT) littermate controls, p-MLKL levels were greatly increased in either APP/PS1 or 5×FAD mice, although total MLKL levels were only mildly augmented (Fig. 1b, c). We noted that 5×FAD showed stronger activation of MLKL compared to APP/PS1 mice, possibly due to their more severe AD pathology [29]. In addition to the IHC data, immunoblotting of whole brain lysate also showed a higher p-MLKL level in 10-month old APP/PS1 mice compared to WT littermates (Fig. 1d). These data indicate that in line with previous findings [8], necroptosis is indeed activated in AD.

To assess whether TNF-α signaling is involved in neuronal necroptosis in the hippocampus, we carried out lateral ventricle injection of murine TNF-α as described [30] (Fig. 1e left). Notably, 5 μg TNF-α compared to PBS control injection induced p-MLKL levels ~3 fold in the cells of CA1 pyramidal layers (Fig. 1f, g), suggesting that a high level of TNF-α is sufficient to activate necroptosis in mouse hippocampus. In addition, TNF-α injection into APP/PS1 hippocampus only slightly increased the levels of p-MLKL (~1.47 fold), perhaps because the level of p-MLKL activated in APP/PS1 mice was already so high (Fig 1f, g).

To further study the function of TNF-α signaling in necroptosis, we used adeno-associated virus (AAV) vector-mediated small hairpin RNA (shRNA) to knockdown (KD) TNFR1 (Fig. S2a), a TNF-α receptor mediating cell death signaling. Assessed by IHC and immunoblotting at least 70% reduction of TNFR1 efficiency were seen (Fig. S1a, c). Using intrahippocampal injection (Fig. 1e middle and right), we transduced CA1 cells with AAV particles expressing a TNFR1 shRNA or a scrambled control (sc) shRNA in 10-month old APP/PS1 mice. After 3 weeks, p-MLKL levels were then examined by IHC. Data showed that TNFR1 shRNA reduced p-MLKL levels by 55.5% compared to sc shRNA (Fig. 1h, J left). By injection of an anti-TNFR1 neutralizing antibody that can block TNF-α signaling [31], we also observed an average 45.5% reduction of p-MLKL levels in CA1 neurons (Fig. 1i, j right).

In sum, we infer TNF-α/TNFR1 signaling is indispensable for the neuronal necroptosis activated in AD patients and AD mouse models.

TNF-α can trigger neuronal necroptosis in cell culture

To explore the molecular mechanism of TNF-α-mediated neuronal necroptosis, we performed cell culture experiments using various neuronal cell types including SH-SY5Y, PC-12 and primary cortical neurons. We examined the effects of TNF-α on cell viability in all above cell types. The pan-caspase inhibitor zVAD-fmk (zVAD, Z) and cIAPs inhibitor Smac mimetic (S), two inducers of cell necroptosis [32], were also utilized. We found that 40 ng/ml TNF-α alone (T) induced a loss of cell viability in all three cell types, with a percentage of 16.4% (SH-SY5Y), 21.5% (PC12), and 22.4% (primary neurons), but zVAD did not fully block cell death (Fig. 2a and Fig. S3a). We further observed that Smac mimetic remarkably increased TNF-α-induced cell death, which could only be partially inhibited by zVAD, with a percentage of 5.5% in SH-SY5Y or 27% in PC12. Moreover, zVAD even provoked cell death in primary neurons with a percentage of 4.9% (Fig. 2a). These data imply that TNF-α or TSZ treatment can induce type(s) of cell death besides common apoptosis.

Propidium iodide (PI) staining and cell morphology images showed that TNF-α, especially combined with Smac mimetic and zVAD (TSZ) treatment, indeed triggered the characteristic features of necrosis/necroptosis [27], including PI-positive labeling and cell swelling (Fig. 2b). Quantitation indicated that TSZ increased cell necrosis with a percentage of 51% in SH-SY5Y, 53.2% in PC12 or 54% in primary neurons; TNF-α treatment also slightly increased necrosis with a percentage of 11.7% in PC12, 14.7% in SH-SY5Y, and 14% in primary neurons (Fig. 2c).

To confirm the involvement of necroptosis in TNF-α-induced cell death, we used Acridine Orange (AO) staining, a unique technique used to distinguish necroptosis from apoptosis [27]. After staining, cultured SH-SY5Y cells as well as primary neurons showed flattened morphology with intact cytoplasm (red fluorescence) and nuclei (green fluorescence) (Fig. 2d). TNF-α treatment induced cell contraction and nuclear condensation, while TS triggered features of apoptotic cells, with nuclear fragmentation. Particularly, zVAD yielded necrotic cell morphology in TS treated cells with PI-positive nuclei, loss of plasma membrane integrity, and translucent cytosol. zVAD also slightly induced cell necrosis in TNF-α treated cells (Fig. 2b-d). To verify that TNF-α-induced cell necrosis is indeed necroptosis, we examined the levels of the specific marker p-MLKL with different combination treatment of T/S/Z. We found that TNF-α or Smac mimetic alone both mildly activated caspase-8 and caspase-3 in SH-SY5Y cells but not in primary neurons, and the activation was inhibited by zVAD (Fig. 2e). Interestingly, TS powerfully activated caspase-8 and caspase-3, an action entirely blocked by zVAD (Fig. 2e). In addition to the apoptotic caspase activation, TNF-α or TZ treatment indeed upregulated p-MLKL, while TSZ treatment had a sharp effect on p-MLKL (Fig. 2e and Fig. S3b).

Taken together, our data show that TNF-α alone can mildly activate neuronal necroptosis; but combined with Smac mimetic and zVAD (TSZ), it triggers strong activation of neuronal necroptosis.

TNF-α-induced neuronal necroptosis is dependent on RIPK1 action

We aimed to clarify whether TNF-α-induced neuronal necroptosis is dependent on RIPK1, a known crucial control center in regulating TNF-α-induced NF-κB signaling pathway as well as apoptosis and necroptosis [21]. In cultured SH-SY5Y and primary neurons, similar to MLKL activation (Fig. 2e), TNF-α alone lightly induced the phosphorylation of RIPK1 on Ser166 -used as a marker of RIPK1 activation (Fig. 3a). TNF-α also increased the phosphorylation of RIPK3, a RIPK1 binding partner, on its Ser227 (human) or Thr231/Ser232 (mouse) site. Smac mimetic, however, markedly repressed RIPK1/3 phosphorylation, while TSZ strongly activated RIPK1 and RIPK3 (Fig. 3a). Of note, we observed that zVAD effectively prevented TS induced RIPK1 cleavage (Fig. 3a), suggesting a possible promotion of RIPK1 oligomerization and aggregation, additional features of RIPK1-dependent necroptosis [8, 33]. As shown in Fig. 3b, TNF-α alone or TSZ did not change the RIPK1 levels in the soluble Triton X-100 fractions, but strongly elevated RIPK1 levels in the insoluble urea fractions, indicating an increased RIPK1 aggregation by TSZ treatment. Using an RIPK1 inhibitor Necrostain-1 (Nec-1), we found that Nec-1 effectively suppressed the activation of RIPK3 and MLKL, as well as subsequent cell necroptosis induced by TNF-α or TSZ (Fig. 3c-e). Furthermore, RIPK1 reduction by lentiviral vector mediated shRNA distinctly blocked phosphorylation of RIPK1/3 and MLKL, and thereby also blocked cell necroptosis (Fig. 3f, g and Fig. S2b). Thus, our data suggest that TNF-α-induced neuronal necroptosis is dependent on the activation of an RIPK1/RIPK3/MLKL cascade.

p62 mediates TNF-α-induced necroptosis via its interaction with RIPK1

Previous studies have found that RIPK1 interacts directly with p62/SQSTM1, a molecule responsible for the clearance of aggregated proteins [34]. We inferred that p62 might participate in RIPK1-dependent neuronal necroptosis in AD. To test this notion, we first examined the protein complex formation of RIPK1, RIPK3 and p62 in neuronal necroptotic cells. Immunoprecipitation data showed that p62 indeed complexed with RIPK1, RIPK3 and MLKL when cells were treated with TSZ (Fig. 4a). Endogenous p62 was also upregulated in both SH-SY5Y cells and primary neurons with TNF-α or TSZ treatment (Fig. 4b, c).

To evaluate the function of p62 in necroptosis, we transduced SH-SY5Y cells with recombinant adenoviral particles encoding GFP-tagged p62 (Ad-p62-GFP) or encoding LacZ as a control (Ad-LacZ) (Fig. S2c). Interestingly, overexpression of p62-GFP evidently upregulated the total protein levels of RIPK1, increased the phosphorylation levels of RIPK1 as well as MLKL, and augmented the percentage of neuronal necroptosis (Fig. S4a, b). Consistently, p62 shRNA transduced into cells with lentiviral particles reversed the effect of p62 overexpression (Fig. S2b, S4c, d). Furthermore, necroptosis was restored by expression of p62-GFP (shRNA-resistant) in p62 shRNA transfected cells (Fig. 4d, e), implying a critical role of p62 in TNF-α-induced neuronal necroptosis.

The ZZ domain (amino acid 122-167) of p62 is the binding domain with RIPK1 [35]. Here, a ZZ domain deleted p62 (p62-ZZΔ, 3×Flag-tagged) expressing plasmid (Fig. S2b) was used to determine whether necroptosis elevation by p62 expression is due to its interaction with RIPK1. Data showed that the expression of p62-ZZΔ in p62 KD cells failed to recover the activation of RIPK1/RIPK3/MLKL pathway and subsequent necroptosis (Fig. 4d, e). We conclude that p62 promotes neuronal necroptosis by complexing with RIPK1 and activating MLKL.

To test the possible promotion role of p62 for neuronal necroptosis in AD, we then assessed p62 levels in the brains of AD patients. IHC staining showed higher p62 levels in samples of cerebral cortex from AD patients compared to healthy controls (Fig. 4f). Similarly, IHC images of hippocampal CA1 regions also showed remarkable upregulation of p62 in APP/PS1 mice compared to WT littermates (Fig. 4f, g). Next, we used AAV vector-mediated shRNA to knock down p62 (Fig. S1b, d, S2a) and transduced cells in CA1 regions with AAV particles in APP/PS1 mice by intrahippocampal injection. After three weeks, IHC of p62 and p-MLKL was performed. Our data indicated that p62 shRNA diminished p-MLKL levels by 47% compared to sc shRNA (Fig. 4h, i).

Overall, our results demonstrate that p62 elevates neuronal necroptosis by interacting with RIPK1 and promoting phosphorylation of RIPK1/RIPK3/MLKL cascades, both in TNF-α-treated neuronal cultures and in AD mouse brain.

Impaired autophagic flux regulates neuronal necroptosis

p62 is a critical molecule participating in several processes of autophagic flux including the initiation/assembly of autophagosome, docking/fusion of autophagosomes with lysosomes, and cargo degradation [36]. We asked whether p62 upregulation during neuronal necroptosis is caused by impaired autophagic flux. We checked the state of autophagic flux by monitoring the protein levels and cellular localization of LC3, a key marker of autophagy. In all three cell culture models mentioned above, TNF-α treatment upregulated LC3-II, a standard indicator for autophagosomes, in a dose-dependent manner (Fig. S5a). Strikingly, the levels of LC3-II and p62 were much higher with TSZ compared to TNF-α treatment (Fig. 4b, 5a). Moreover, p62 levels in the insoluble fraction was sharply increased in TSZ treated cells (Fig. 5b), suggesting that the formation of p62 aggregates is correlated with the inhibition of autophagy-mediated protein degradation.

To confirm this notion, we used recombinant adenoviral particles encoding GFP and mCherry tagged LC3 (Ad-GFP&mCherry-LC3) to evaluate autophagic flux [36]. As shown in Fig. S5b, c, TNF-α gradually reduced the number of red dots (mCherry without GFP fluorescence puncta, representing autolysosomes) and increased the number of yellow dots (co-localization of mCherry with GFP fluorescence puncta, representing autophagosomes). This change was notably further amplified by treatment with zVAD and Smac mimetic (Fig. 5c, d), suggesting that TNF-α can impair neuronal autophagic flux. To clarify whether the impaired autophagic flux contributes to necroptosis, we used an autophagic flux inhibitor chloroquine (CQ) that can effectively raise the lysosomal pH and inhibit proteases [36]. Of note, CQ sharply increased the upregulation of p62 and LC3-II triggered by TNF-α or TSZ treatment and enhanced the activation of RIPK1 and MLKL, as well as the consequent neuronal necrosis (Fig. 5e, f).

Our data thus clearly show that impaired neuronal autophagic flux contributes to TNF-α-induced neuronal necroptosis.

UVRAG down-regulation by TSZ leads to neuronal necroptosis

We extended our study to the molecular mechanism of autophagy in TNF-α-mediated neuronal necroptosis. We examined mRNA expression levels of a series of genes responsible for the recognition and fusion of autophagosome and lysosome [37]. Real-time quantitative PCR (RT-qPCR) data showed that among the tested genes, UVRAG mRNA, was the most significantly downregulated by TSZ treatment (Fig. 6a and Fig. S5d), suggesting that TSZ may disrupt neuronal autophagic flux by down-regulating UVRAG. To test this possibility, we constructed a SH-SY5Y cell line stably expressing UVRAG. Data showed that UVRAG overexpression reversed TSZ-induced autophagic flux impairment and necroptosis activation by preventing LC3-II and p62 upregulation as well as RIPK1/MLKL phosphorylation (Fig. 6b-e). Hence, our data infer that down-regulation of UVRAG transcription by TSZ impairs neuronal autophagic flux, leading to p62 accumulation and the onset of necroptosis.

UVRAG transcription is reduced through inactivation of NF-κB

cIAP1/2 facilitates TNF-α-induced transcription factor NF-κB activation [38]. Given that Smac mimetic(S), as an inhibitor of cIAP1/2, can induce strong neuronal necroptosis (Fig. 2), we speculated that TSZ-induced UVRAG downregulation resulted from NF-κB inhibition. Therefore, we checked the level of RelA (p65), a key subunit of NF-κBs, before and after TSZ treatment. Compared to TNF-α alone, TSZ did not alter the levels of RelA in cytoplasm, but reduced the levels in nuclei, indicative of an inhibition of NF-κB activation (Fig. 6f).

Next we analyzed the core promoter regions of Uvrag gene across species using ConTra v2 software and found a high-scoring NF-κB binding site (Fig. 6g, h). Chromatin-IP (ChIP) qPCR showed that NF-κB indeed bound to the indicated region of the Uvrag promoter (Fig. 6i). Importantly, TSZ significantly reduced the binding of RelA to the Uvrag promoter (Fig. 6i). We thus conclude that Uvrag is a direct target of TNF-α/TNFR1/NF-κB signaling and TSZ reduces UVRAG transcription by inhibiting NF-κB action.

Upregulation of UVRAG reduced neuronal necroptosis in AD

Based on our findings, we considered that UVRAG upregulation may inhibit neuronal necroptosis in AD mice. To test this hypothesis, we first utilized AAV-mediated overexpression of UVRAG (Fig. S2a) in primary neuronal cultures. UVRAG overexpression did markedly suppress TNF-α-induced MLKL phosphorylation (Fig. 7a, b). We next transduced cells in hippocampal CA1 regions with AAV expressing UVRAG or control GFP in APP/PS1 mice at 10-month age by intrahippocampal injection. After 3 weeks, we sacrificed the APP/PS1 mice and measured p62 and p-MLKL levels in transfected neurons. IHC images showed the average level of p62 in UVRAG-overexpressing neurons was 34.5% lower than that in control GFP expressing neurons (Fig. 7c, e). Notably, UVRAG overexpression also lowered MLKL phosphorylation 36.5% (Fig. 7d, f). These results support the notion that upregulation of UVRAG inhibits neuronal necroptosis in AD mice, and that UVRAG may thus be a potential target for AD intervention.

Brain deterioration caused by neuronal loss is the most consequential feature of AD pathogenesis [39]. The mechanistic study of neuronal loss is an object of even more intense study because neuronal loss starts at the early stage of AD, when Aβ plaques and neurofibrillary tangles have not yet appeared [40, 41]. Therefore, targeting neuronal loss might slow or even prevent the progression of AD. According to the amyloid cascade hypothesis, Aβ accumulation serves as an initial signal in AD, which leads to neurofibrillary tangles and facilitates neuronal death and cognitive defects [42]. A huge body of evidence shows that different forms of Aβ, either insoluble aggregates or soluble oligomers, can cause neuronal death by a rapid toxic effect on neurons [6] or by triggering chronic neuroinflammation and toxic cytokine release [7].

As for the mechanism of neuronal cell death, distinct processes including apoptosis, necrosis, autophagy, and parthanatos have been described in AD as well as other NDs [43]. Necroptosis, a programmed cell necrosis, is a more recently discovered pathway to cell death detected in several NDs [44]. In AD, neuronal necroptosis activation has been observed in the brains of AD patients and rodent AD models [8, 45]. In line with these findings, our data also find upregulated protein levels of MLKL or p-MLKL, a necroptotic marker, in the brains of AD patients, APP/PS1 and 5xFAD mice (Fig. 1a-d).

Various extracellular stimuli can activate necroptosis, including TNF, interferon, and Toll-like receptor (TLR) signaling as well as viral infection [46]. But the question has then been raised, how is neuronal necroptosis in AD initiated? Given the accumulated evidence of elevated TNF-α in the CSF and brains of AD patients [18, 47], we hypothesized that upregulated TNF-α in AD brain might lead to the activation of neuronal necroptosis. Using injection of recombinant TNF-α, an anti-TNFR1 neutralizing antibody, and AAV particles expressing TNFR1 shRNA, we provide evidence that activated neuronal necroptosis in APP/PS1 hippocampus is dependent on TNF-α/TNFR1 signaling (Fig. 1f-j). In addition, using three different types of neuronal cell culture models, we also successfully observed that TNF-α treatment could induce cell necroptosis in vitro (Fig. 2). Thus, TNF-α, as a critical proinflammatory cytokine, also activates neuronal necroptosis in AD. Our data also support the notion that neuronal necroptosis is likely a secondary source of cell death induced by Aβ-mediated neuroinflammation at a later stage in AD, consistent with previous findings that necroptosis inhibitor Nec-1 can block Aβ-induced cell death in APP/PS1 mice, but with limited efficacy at early stages [48].

Turning to details of the inferred pathway, Fig. 7g outlines the cascade of interacting molecules and processes involved in the initiation and level of necroptosis. In the overall fate of neurons, RIPK1 is a key regulator of survival signaling, inflammation response, and pro-death stimuli in many human diseases such as autoimmune and NDs [49]. TNF-α/TNFR1 mediated RIPK1 activation is the most comprehensively studied signaling pathway; it decides whether cells live or die by tightly regulating pro-survival NF-κB action, pro-apoptotic caspase activity, and necroptotic MLKL activation [50]. We used our neuronal culture models and confirmed that RIPK1/RIPK3/MLKL cascade was activated upon TNF-α or TZS stimulation (Fig. 3a). Consistently, RIPK1 inhibitor Nec-1 and RIPK1 KD both prevented phosphorylation of RIPK1/RIPK3/MLKL cascade (Fig. 3c-g). We also saw the increased oligomerization of RIPK1 in TNF-α-induced necroptosis (Fig. 3b), possibly due to the inhibition of cIAP1/2 by Smac mimetic and blocking of ubiquitylation at the K377 site targeting RIPK1 degradation [21, 38].

To further explore the mechanism of RIPK1-dependent neuronal necroptosis in AD, we focused on p62/SQSTM1, a RIPK1 binding partner that participates in both the ubiquitin-proteasome system (UPS) and autophagic lysosomal degradation process in many diseases [51]. In our cell culture models, we verified the complex formation of p62, RIPK1, RIPK3 and MLKL (Fig. 4a). Some literature has reported lower p62 expression in the brains of AD patients and rodent models of AD [52, 53]; but one group reported increased p62 levels in the hippocampus of AD patients [54]. This discrepancy may result from biological differences among patients or in different animal models. In our own study, data consistently show higher levels of p62 in TNF-α-treated cells in cultures, hippocampal CA1 cells in APP/PS1 mice, and neuronal cells in the cortexes of AD patients (Fig. 4b, c, f, g). We further find that p62 KD suppresses the activation of neuronal necroptosis in both cell cultures and in APP/PS1 mice (Fig, 4d, e, h, i). Our results thus imply that p62 inhibition, a therapeutic modality in prostate cancer [55], could be a possible candidate for AD intervention.

p62 is also implicated in the interacting pathway of autophagy. Impaired autophagic flux has been found to be able to induce cell death by disabling degradation of protein aggregates in NDs [15, 56]. As a critical regulator in autophagic flux as well, p62 was accumulated in both soluble form and in insoluble aggregates after TNF-α treatment (Fig. 5b). Notably, autophagic flux was seen to be inhibited, also mediating RIPK1-dependent necroptosis (Fig. 5 and Fig. S5a-c). TNF-α can damage autophagic flux by disrupting lysosome acidification and blocking the degradative function of autolysosomes [57]. These hints led us to search for possible molecules involved in regulating autophagic flux. We examined the mRNA levels of major genes participating in autophagosome recognition and autolysosome fusion. Among the tested targets, UVRAG mRNA was the most one strikingly reduced in level by TSZ treatment (Fig. 6a and Fig. S5d). UVRAG is in fact a critical modulator of several steps in autophagic flux, including autophagosome-lysosome fusion [37, 58]. In agreement with these findings, our data clearly show that UVRAG overexpression can alleviate the impairment of autophagic flux and reduce RIPK1-dependent necroptosis (Fig. 6b-e). UVRAG downregulation can likely disrupt autophagosome-lysosome fusion, resulting in p62 accumulation, which then further recruits RIPK1 to autophagosome and facilitates its oligomerization and auto-phosphorylation (Fig. 7g).

To understand how UVRAG expression was down-regulated during TSZ-induced cell necroptosis, we investigated the status of NF-κB, a key transcriptional factor acting downstream of TNF-α. Because NF-κB transcriptional activity can be blocked by Smac mimetic-mediated inhibition of cIAP1/2, we predicted that UVRAG is a transcriptional target of NF-κB. Indeed, we found a putative NF-κB binding site within the core promoter region of Uvrag gene, and ChIP-qPCR confirmed UVRAG as a direct transcriptional target of NF-κB (Fig. 6g-i). Strikingly, AAV-mediated UVRAG overexpression reduced TNF-α-induced p-MLKL upregulation in both neuronal cultures and in APP/PS1 animal mice (Fig. 7a-f). These findings thus provide evidence that upregulation of UVRAG may be another route to suppress neuronal necroptosis in AD.

Overall, in the picture of TNF-α/TNFR1-dependent neuronal necroptosis in AD, there appear to be several key successive events, including UVRAG downregulation and autophagic flux impairment and p62 accumulation, which further leads to RIPK1 activation and subsequent neuronal necroptosis (Fig. 7g). It remains unknown how other molecules and signaling pathways are modulated to precisely set levels of cell necroptosis in AD. However, in a broader view, our results have demonstrated that inflammatory TNF-α signaling, pro-survival NF-κB action, autophagic machinery, and RIPK1 activity are all tightly controlled and unbalanced during neuronal necroptosis in AD. This may provide a general outline of the mechanism of neuronal necroptosis in other NDs as well.

AAV: Adeno-associated viral vectors; Aβ: Amyloid beta; AD: Alzheimer’s disease; Ad: adenoviral; AO: Acridine Orange; Ctrl: Control; CQ: chloroquine; CSF: cerebrospinal fluid; IF: immunofluorescence; IHC: Immunohistochemistry; KD: knockdown; MCI: mild cognitive impairment; MLKL: Mixed Lineage Kinase Domain Like Pseudokinase; Nec-1: Necrostain-1; NDs: neurodegenerative diseases; PI: Propidium iodide; p62/SQSTM1: Sequestosome 1; RIPK1: Receptor Interacting Serine/Threonine Kinase 1; RIPK3: Receptor Interacting Serine/Threonine Kinase 3; S: Smac mimetic; sc: scrambled control; shRNA: small hairpin RNA; T: TNF-α; TNF-α: Tumor necrosis factor alpha; TNFR1: Tumor necrosis factor receptor 1; UPS: ubiquitin-proteasome system; UVRAG: UV Radiation Resistance Associated; zVAD/z: zVAD-fmk.

Acknowledgements

The authors thank the Harvard Brain Tissue Resource Center, funded through the NIH NeuroBioBank, and brain donors and their families for tissue samples used in this study; Dr. David Schlessinger for support on human sample application and preliminary study; Dr. David Schlessinger for critical reading and comments on the manuscript.

Author contributions

J.S. designed and supervised the project; C.X., J.W., Y.W., Z.R. and Y.Y. performed the experiments; G.C. provided transgenic mice; L.W. and X.C. provided resource and technical support; C.X. and J.S. analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the High-Level Talents Start-up Funding of China Pharmaceutical University (No.3150120042); the National Natural Science Foundation of China (No.82001144; No.82003375); the Natural Science Foundation of Jiangsu Province (No.BK20180562); and the Fundamental Research Funds for the Central Universities (No.2632018PY02).

Availability of data and materials

All data used and analyzed for the current study are available from the corresponding author on reasonable request.

Ethical Approval and Consent to participate

All experiments were conducted in accordance with the regulations for the Administration of Affairs Concerning Experimental Animals (China) and approved by the China Pharmaceutical University Animal Ethics Committee.

Consent for publication

All authors have given their consent for publication.

Competing interests

The authors declare no competing interests.

Author details

¹Laboratory of Aging Neuroscience and Neuropharmacology, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, China. ²Institute of Geriatric Immunology, School of Medicine, Jinan University, Guangzhou, 510632, China. ³Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing, 210009, China. ⁴Clinical Pharmacokinetics Laboratory, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 211198, China.

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TNF-α-dependent neuronal necroptosis regulated in Alzheimer's disease by coordination of RIPK1-p62 complex with autophagic UVRAG

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