Abstract

   Parkinson's disease (PD) involves multiple forms of neuronal cell
   death, but the dominant pathway involved in disease progression remains
   unclear. This study employed RNA sequencing (RNA-seq) of brain tissue
   to explore gene expression patterns across different stages of PD.
   Using the Scaden deep learning algorithm, we predicted neurocyte
   subtypes and modelled dynamic interactions for five classic cell death
   pathways to identify the predominant routes of neuronal death during PD
   progression. Our cell type-specific analysis revealed an increasing
   shift towards necroptosis, which was strongly correlated with nitric
   oxide synthase (NOS) expression across most neuronal subtypes. In vitro
   experiments confirmed that nitric oxide (NO) is a key mediator of
   necroptosis, leading to nuclear shrinkage and decreased mitochondrial
   membrane potential via phosphorylation of the PIP1/PIP3/MLKL signalling
   cascade. Importantly, specific necroptosis inhibitors significantly
   mitigated neuronal damage in both in vitro and in vivo PD models.
   Further analysis revealed that NO-mediated necroptosis is prevalent in
   early-onset Alzheimer's disease (AD) and amyotrophic lateral sclerosis
   (ALS) and across multiple brain regions but not in brain tumours. Our
   findings suggest that NO-mediated necroptosis is a critical pathway in
   PD and other neurodegenerative disorders, providing potential targets
   for therapeutic intervention.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s43556-024-00213-y.

   Keywords: Nitric oxide, Necroptosis, Parkinson's disease,
   Neurodegenerative disease

Introduction

   Neurodegenerative diseases are major challenges in medical science and
   are characterized by a gradual decline in nervous system function and
   structure. Common neurodegenerative diseases include Alzheimer's
   disease (AD), Parkinson's disease (PD), and amyotrophic lateral
   sclerosis (ALS) [[40]1–[41]3]. These conditions are characterized by
   various symptoms that progressively impair cognitive and motor
   functions, substantially impacting the life quality of patients [[42]4,
   [43]5]. Current treatment options primarily target symptoms, with
   limited therapies available to halt or slow the underlying pathological
   processes [[44]6–[45]9]. Elucidation of the patterns and underlying
   mechanisms of neuronal cell death has important therapeutic
   implications.

   Recent studies have revealed various mechanisms of cell death involved
   in neurodegeneration, including apoptosis, autophagy, ferroptosis,
   necroptosis, and parthanatos [[46]10–[47]12]. While each pathway can
   independently contribute to neuronal loss, these pathways often
   interact and influence each other. For example, the interplay between
   autophagy and apoptosis can be modulated by the second messenger
   calcium (Ca^2+), which impacts neuronal excitability [[48]13]. Another
   example involves caspase-8 acting as a mediator between the apoptotic
   and necrotic pathways, serving as a pivotal link among diverse cell
   death mechanisms [[49]14]. Our study focuses on these five classic cell
   death pathways because they encompass a spectrum of cell death
   mechanisms, both programmed and unprogrammed. The well-documented
   molecular mechanisms of these pathways provide a robust foundation for
   research and potential therapeutic targets. Additionally, these
   pathways are integral to the pathology of neurodegenerative diseases,
   with apoptosis and autophagy involved in clearing damaged components
   and necroptosis and ferroptosis linked to inflammation and membrane
   damage. Previous research and experiments with inhibitors of these
   pathways have further highlighted their therapeutic potential.
   Investigating the transition and interaction between these death modes
   throughout the progression of neurodegenerative diseases can offer
   valuable insights into the development of these conditions. A deeper
   understanding of these interactions may facilitate the identification
   of effective intervention strategies and therapeutic targets for
   neurodegeneration [[50]15, [51]16].

   Nitric oxide (NO) has been implicated in the pathogenesis of
   neurodegenerative disorders, including PD, where it is suggested to
   mediate disease progression [[52]17, [53]18]. Our preliminary
   investigations revealed that the NO donor GSNO can induce
   necroptosis-like damage in neuronal cells in vitro; necroptosis is an
   important type of neuronal cell death in degenerative diseases. Hence,
   NO may mediate neuronal damage through the induction of necroptosis
   [[54]19].

   In this study, we employ a multitiered research approach to delineate
   the primary pathways of neuronal cell death patterns during the
   progression of degenerative disease and investigate the role and
   mechanism of NO in mediating related illnesses, offering a scientific
   foundation and practical insights for the prevention and treatment of
   neurodegenerative conditions.

Results

Expression characteristics of PD patients at different pathological stages

   Neurodegenerative diseases are characterized by the progressive loss of
   neuronal cells through complex mechanisms [[55]20]. First, we aimed to
   delineate the dynamic transcriptomic landscape during the progression
   of neurodegenerative diseases. We analysed brain tissue samples from
   patients with PD stratified according to the BLB stage. Comparative
   analysis (Fig. [56]1a and Fig. S[57]1) revealed significant differences
   in gene expression profiles among BLB0, BLB5 and BLB6 (Fig. [58]1a).
   With increasing disease severity, the number of differentially
   expressed genes (DEGs) increased from 2,611 to 2,691. Notably, the
   upregulated genes exhibited the most pronounced changes, with 1,327 and
   1,243 genes upregulated in BLB6 and BLB5, respectively (Fig. S[59]1a).
   As the disease progressed, we observed an increase in the Euclidean
   distance of the DEGs of the patients, as shown by principal coordinate
   analysis (PCoA) in Fig. [60]1b. Specifically, patients at BLB5
   presented intermediate distances between stages 0 and 6, indicating
   dynamic and disease severity-dependent progression in PD.

Fig. 1.

   [61]Fig. 1
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   Genomic expression profile of PD at different pathological stages. a
   DEGs at different pathological stages. (Blue dots represent significant
   downregulation, red dots represent significant upregulation,
   |log2FC|≥ 1, P ≤ 0.05). b PCoA at different pathological stages (there
   was a statistically significant difference in the gene expression
   profiles of brain tissues at BLB6 and BLB0, P value < 0.05). c Pathway
   enrichment of DEGs between BLB6 and BLB0; the DEGs are enriched
   predominantly in various pathways associated with the onset of
   neurodegenerative diseases (indicated by the red arrow). d Pathway
   interaction network between BLB6 and BLB0. Pathways of
   neurodegeneration are at the core of the interactive network, indicated
   by the red arrow, each term is represented by a circle node, where its
   size is proportional to the number of input genes fall under that term,
   and its color represent its cluster identity (i.e., nodes of the same
   color belong to the same cluster). Terms with a similarity score > 0.3
   are linked by an edge (the thickness of the edge represents the
   similarity score). The network is visualized with Cytoscape with
   “force-directed” layout and with edge bundled for clarity

   To elucidate the molecular mechanisms underlying disease progression,
   we subsequently performed pathway annotation (Fig. [63]1 c, Fig.
   S[64]1b) and interaction network analysis (Fig. [65]1d, Fig. S[66]1c).
   The DEGs were enriched predominantly in pathways such as 'Pathways of
   Neurodegeneration—Multiple Diseases', 'Generation of Precursor
   Metabolites and Energy' and 'Membrane Trafficking' (Fig. [67]1c). The
   enrichment of DEGs within the 'Pathways of Neurodegeneration—Multiple
   Diseases' pathway indicates a potential role for these genes as key
   drivers in the aetiology and progression of various neurodegenerative
   conditions. Intriguingly, 'Generation of Precursor Metabolites and
   Energy' and 'Membrane Trafficking' were found at the core of the gene
   regulatory network (Fig. [68]1d, Table S[69]1), which indicates that
   abnormalities in energy metabolism may play critical roles in neuronal
   cell death during PD progression. To test this hypothesis, we examined
   creatine kinase family genes, which are core genes related to cell
   energy storage and buffering [[70]21, [71]22]. We observed significant
   downregulation of CKMT1A and CKMT1B in brain tissues at BLB6 compared
   with BLB5, as shown in Fig. S[72]1D. Given the pivotal roles of these
   molecules in energy metabolism, these results suggest that deficits in
   energy storage may strongly contribute to neuronal cell death and the
   progression of PD.

Necroptosis is the predominant death route in PD progression

   Our objective was to identify the predominant cell death mechanisms
   associated with the progression of PD. By employing deep learning
   algorithms, we classified neural cell types within brain tissues and
   developed dynamic interaction models for five classic modes of cell
   death: apoptosis, autophagy, ferroptosis, necroptosis and parthanatos.
   While we observed no significant disparities in neural cell subtypes
   between the healthy controls and the PD patients (Fig. [73]2a), two
   notable trends emerged. First, with advancing PD, diverse modes of cell
   death were observed in brain tissues (Fig. [74]2b, c). Second, an
   increase in disease severity coincided with a pronounced shift towards
   necroptosis, marked by an increased prevalence of this pathway and a
   concurrent decline in autophagy and ferroptosis (Fig. [75]2b, d). These
   observations indicate that necroptosis may be a predominant regulatory
   mechanism in PD pathogenesis. Furthermore, genes pivotal to
   necroptosis, including FAS, TLR2, and TLR5, were upregulated in
   advanced BLB6 relative to BLB5 (Fig. [76]2e). Conversely, genes linked
   to apoptosis and autophagy were downregulated (Fig. S[77]2), confirming
   that the transition towards necroptosis is a critical pathway in the
   progression of PD.

Fig. 2.

   [78]Fig. 2
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   Dynamic model of the neurocyte death route during the progression of
   PD. a Neurocyte subtype prediction via deconvolution algorithms (the
   heatmap shows the proportion of neural cells; purple indicates no
   significant difference, P > 0.05). b Dynamic interaction model between
   neurocytes and five classic death patterns. (The left side represents
   the mode of death, the right side represents the type of neurocyte, and
   the lines represent interaction effects. The colour of the line
   represents significance, where red represents P < 0.05, and green
   represents P > 0.05. The thickness of the line represents the strength
   of the interaction. The correlation heatmap on the right represents the
   interaction between the death pathway and neurocytes. The colour
   represents the Spearman correlation coefficient.). c Statistics on
   significant interaction numbers during the progression of PD. (As the
   disease progresses, the total number of interactions increases,
   indicated by the red arrow). d Statistics on significant changes in
   interaction numbers during the progression of PD. (As the disease
   progresses, necroptosis increases while ferroptosis decreases, blue
   dots represent decreases, while red dots represent increases, indicated
   by the red arrow). e Expression of marker genes for necroptosis during
   the progression of PD (the x-axis represents different periods, the
   y-axis represents lgTPM, and P < 0.05 indicates a statistically
   significant difference). f Analysis of necroptosis correlation strength
   across different neural cell types at varying BLB grading levels. The
   results revealed a notable increase in necroptosis correlation strength
   as BLB grading progressed across nearly all neural cell types,
   including neurons, astrocytes, VLMCs, tanycytes, OPCs,
   oligodendrocytes, ependymal cells, endothelial cells, microglia, and
   NFOs. Specifically, in astrocytes and endothelial cells, the
   correlation strength decreases at BLB5 but increases again at BLB6.
   Conversely, oligodendrocytes show the highest correlation strength at
   BLB5, which decreases at BLB6 but remains higher than at BLB0. g
   Analysis of ferroptosis correlation strength across the same neural
   cell types during the transition from BLB5 to BLB6. A decrease in
   ferroptosis correlation strength is observed across all cell types,
   supporting the hypothesis that as neurodegenerative diseases progress,
   there is a shift towards necroptosis with increasing disease severity.
   The pattern of cell death mechanism diversity is evident across the
   neural cell types analysed

   Furthermore, we aimed to determine the cell type-specific
   characteristics of necroptosis. Overall, our observations indicate that
   with the progression of BLB grading, there is a notable increase in the
   correlation strength of necroptosis across nearly all neural cell
   types, including neurons, astrocytes, vascular leptomeningeal cells
   (VLMCs), tanycytes, oligodendrocyte progenitor cells (OPCs),
   oligodendrocytes, ependymal cells, endothelial cells, microglia, and
   newly formed oligodendrocytes (NFOs). Specifically, variations among
   cell types revealed that, in astrocytes and endothelial cells, the
   correlation strength decreases at BLB5 but subsequently increases at
   BLB6. Conversely, oligodendrocytes exhibited the highest correlation
   strength at BLB5, which then diminished at BLB6, although it remained
   elevated compared with that at BLB0 (Fig. [80]2f). Furthermore, our
   findings revealed a decrease in the degree of ferroptosis correlation
   across all cell types during the transition from BLB5 to BLB6
   (Fig. [81]2g). These results support our hypothesis that as
   neurodegenerative diseases progress, the diversity of cell death
   mechanisms broadens, and with increased disease severity, there is a
   preferential shift towards necroptosis. This pattern was evident across
   almost all analysed neural cell types.

NOS is involved in the regulation of necroptosis in PD, but it does not have
a role in brain tumours

   Our research revealed that GSNO, a donor of NO, induced a form of
   neural cell death resembling necroptosis, marked by disrupted cell
   membranes observable 5–6 h post-treatment rather than immediately after
   the stimulus (Fig. S[82]3). These findings indicated that NO may
   mediate neuronal necroptosis in the pathogenesis of PD. To validate
   this finding, we initially assessed the relationship between NOS family
   genes and those pivotal in necroptosis. We observed that with PD
   progression, the correlation between NOS genes and necroptotic genes,
   particularly NOS3, significantly increased. Moreover, the correlation
   between NOS1 and necroptotic genes generally increased, although the
   difference did not reach statistical significance (Fig. [83]3a).
   Furthermore, statistical analysis of the interactions between the NOS
   family and necroptotic genes revealed a significant increase in
   positive interactions and a corresponding decrease in negative
   interactions with PD progression (Fig. [84]3b). These results suggest
   that NO-mediated necroptotic cell death could be a central mechanism in
   the progression of PD.

Fig. 3.

   [85]Fig. 3
   [86]Open in a new tab

   Association analysis of NOS with key genes involved in necroptosis. a
   Correlation heatmap showing the associations between NOS and key genes
   involved in necroptosis. Orange represents a positive correlation, and
   blue represents a negative correlation. (*, P < 0.05; **, P < 0.01;
   ***, P < 0.001). b Statistics of significantly positively and
   negatively correlated numbers. (As the disease progresses, the positive
   association between NO and necroptosis increases, while the negative
   association decreases, indicated by the red arrow). c In glioblastoma,
   the correlation between NO-mediated necroptosis and tumour progression
   significantly decreases as the tumour develops. d In pituitary
   adenomas, the correlation between NO-mediated necroptosis is more
   common in invasive tumours than in non-invasive tumours. e In
   meningiomas, the correlation strength of NO-mediated necroptosis
   gradually decreases with increasing WHO grade, indicating a reduced
   association in more aggressive forms of the tumour

   Moreover, we conducted a comparative analysis using human brain
   tumours, as these diseases also exhibit chronic progression and are
   related to the nervous system. We found that NO-mediated necroptosis is
   not conserved in brain tumours and that most of these processes are not
   significant. In glioblastoma, the correlation between NO-mediated
   necroptosis significantly decreased following tumour development
   (Fig. [87]3c). In pituitary adenomas, the correlation was greater in
   invasive tumours than in noninvasive tumours (Fig. [88]3d). In
   meningiomas, this correlation gradually diminished with increasing WHO
   grade (Fig. [89]3e). These findings provide counterevidence that
   NO-mediated necroptosis is universally prevalent in classic
   neurodegenerative diseases but may not be conserved in other brain
   diseases.

NO-mediated necroptosis is the central mechanism of neural cell death

   We identified NO-mediated necroptosis as the principal mechanism
   underlying neural cell death in PD. To substantiate our findings
   through computational analysis, we developed an in vitro PD model in
   which the neural cell line SH-SY5Y was exposed to rotenone to establish
   that NO-induced necroptosis is a central pathway for cell death.

   Initially, we utilized various inhibitors—Z-VAD for apoptosis, 3-MA for
   autophagy, Fer-1 for ferroptosis, Ola for parthanatos, and Nec-1s for
   necroptosis—to delineate the predominant cell death pathway.
   Additionally, we introduced GSNO, a NO donor, to validate the role of
   NO in cellular injury (Fig. [90]4a). Our observations revealed a marked
   decrease in cell viability in the rotenone-induced PD model
   (Fig. [91]4). Notably, the application of Nec-1s, 3-MA, and Fer-1, but
   not Ola, significantly restored cell viability, suggesting that the
   cell death mechanisms involved predominantly autophagy, ferroptosis,
   and necroptosis rather than parthanatos. The apoptotic inhibitor Z-VAD
   did not affect cell viability, and the ineffective cleavage of caspase
   3 (Fig. [92]4b), an apoptotic marker, indicated that apoptosis is
   likely not the primary route of cell death in PD pathogenesis.

Fig. 4.

   [93]Fig. 4
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   NO-mediated necroptosis in a PD cell model. a Experimental workflow:
   The neural cell line SH-SY5Y was treated with rotenone, with or without
   the NO donor GSNO, to establish a cellular model for PD. Specific
   inhibitors targeting various cell death pathways were utilized to
   identify the pathways involved in the death of the cell line. b Western
   blot analysis of the cleavage of the apoptotic marker protein caspase3.
   c NO-induced cell death pathways were distinguished via the use of
   specific inhibitors targeting different cell death pathways, and the
   inhibitor of necroptosis has the strongest effect, indicated by the red
   arrow. (*, P < 0.05; **, P < 0.01; ***, P < 0.001)

   These findings are congruent with our bioinformatics-derived cell death
   models, which revealed increased interactions between neurocytes and
   pathways of autophagy, ferroptosis, and necroptosis during the BLB0 to
   BLB5 and BLB6 progression stages, with no interaction observed for
   apoptosis and parthanatos (Fig. [95]2b). Furthermore, we discovered
   that NO not only directly induces neurocyte damage but also strongly
   amplifies the neurotoxic effects of rotenone, indicating a role for NO
   in mediating neural cell death in PD. Among the inhibitors tested,
   Nec-1s demonstrated the most efficacious rescue of cell viability
   (Fig. [96]4c), suggesting that NO-driven necroptosis is the predominant
   cell death mechanism in the progression of PD models. Collectively, our
   in vitro experiments corroborate the bioinformatics model, reinforcing
   the notion that NO-mediated necroptosis is a central mechanism in
   neural cell death associated with PD.

Abnormal chromatin condensation and MMP collapse in neural cells triggered by
NO-mediated necroptosis

   Specifically, a reduction in the MMP and the condensation of nuclear
   DNA are characteristic indicators of neuronal degeneration in PD,
   whereas increased cell membrane permeability is a defining feature of
   necroptosis. Our subsequent investigations focused on these features of
   cellular damage within the context of NO-induced necroptotic pathways.

   We observed notable aberrations in the chromatin structure of neural
   cells in a rotenone-induced PD model characterized by pronounced DNA
   condensation and nuclear shrinkage. The introduction of NO exacerbated
   these chromatin structural anomalies, which were accompanied by
   cellular oedema and elevated staining with Trypan blue (Fig. [97]5a, b,
   Fig. S[98]3). Utilization of Nec-1s, an inhibitor of necroptosis,
   significantly reduced the proportion of neural cells exhibiting nuclear
   damage and compromised cell membrane integrity (Fig. [99]5b, Fig.
   S[100]3). Concurrently, we noted substantial alterations in the MMP of
   the affected neural cells. While the MMP was initially reduced in the
   rotenone-induced PD model, NO resulted in a more pronounced reduction
   in the MMP. Importantly, Nec-1s significantly attenuated this decrease
   in the MMP (Fig. [101]5c).

Fig. 5.

   [102]Fig. 5
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   Modulation of nuclear morphology and the MMP by NO-mediated
   necroptosis. a NO-mediated necroptosis triggered nuclear condensation
   and a decrease in the MMP (blue represents nuclear staining, while red
   represents the MMP, bar = 50μm). b Statistics of nuclear condensation
   in the neural cells. Nuclei were stained with Hoechst 33342, and the
   morphology of the nuclei was statistically analysed by randomly
   selecting ten microscopic fields of view (*, compared with the control
   group; **, P < 0.01; ***, P < 0.001. #, compared with the
   rotenone + GSNO- group; ##, P < 0.01). c Statistics of the MMP of
   neural cells. The MMP was characterized by staining with MitoTracker™
   Red CMXRos dye, and the relative fluorescence intensity indicated the
   strength of the MMP. d Phosphorylation detection of the necroptotic
   marker RIP1 via western blotting

   Moreover, we observed a significant increase in the phosphorylation of
   receptor-interacting protein kinase 1 (RIP1), a recognized marker of
   necroptotic cell death. Notably, a marked increase in phosphorylation
   was observed exclusively following supplementation with NO, and this
   increase was significantly abrogated in the presence of the necroptosis
   inhibitor Nec-1s. These findings indicate that NO plays a mediating
   role in the induction of necroptosis (Fig. [104]5d).

Validation of NO-mediated necroptosis in primary neurons

   Primary neurons are considered more representative of the physiological
   state of nerve cells, as they retain a greater extent of their original
   biological properties [[105]23, [106]24]. Therefore, we aimed to
   further validate the occurrence of NO-induced necroptosis in these
   cells. Initially, we isolated foetal mouse midbrain neurons
   (Fig. [107]6a) and subjected them to NO stimulation and inhibitor
   treatment. Our findings confirmed that NO-mediated necroptosis is
   replicable in primary neuronal cultures. Upon NO stimulation, there was
   a significant increase in the incidence of nuclear shrinkage and a
   marked increase in cell death, and importantly, the necroptosis
   inhibitor Nec-1s effectively reversed this deleterious process
   (Fig. [108]6b, c, d).

Fig. 6.

   [109]Fig. 6
   [110]Open in a new tab

   Validation of NO-mediated necroptosis in mouse primary neurons. a
   Experimental workflow: Primary mouse midbrain neurons were cultured and
   utilized for experimentation, in which the cells were treated with
   GSNO, either in the presence or absence of the necroptosis-specific
   inhibitor Nec-1s. The role of necroptosis in primary neurons was
   confirmed through methodologies including cellular morphological
   assessment and western blot analysis. b Necroptosis in GSNO-treated
   neurons was detected. Microscopic images of cells under visible light
   and the distribution of DNA, as revealed by Hoechst 33342 staining,
   revealed morphological changes in the cell body and nucleus following
   GSNO treatment. The cells stained with Trypan blue exhibited
   compromised plasma membrane integrity, bar = 20μm. c Statistics of the
   nuclear condensation rate. Following a 12-h exposure to GSNO, the cells
   were stained with Hoechst 33342. A total of ten random fields of view
   were assessed for quantification, and the proportion of condensed
   nuclei was determined relative to the total cell count within each
   field. (*, compared with the GSNO group; **, P < 0.01). d Statistics of
   the necroptosis rate. One hour following stimulation with GSNO, the
   cells were rapidly stained with Trypan blue and photographed under a
   microscope. The proportion of stained cells, indicative of necroptotic
   injury, was compared with that of the entire group of cells (*,
   compared with the GSNO group; **, P < 0.01). e Detection of the
   phosphorylation cascade of the PIP1/PIP3/MLKL signalling pathway

   We subsequently aimed to elucidate the molecular mechanisms of
   NO-mediated necroptosis, with a particular focus on phosphorylation
   events within the RIP1/RIP3/MLKL cascade, which is known to be a
   critical juncture in the execution of necroptotic cell death [[111]25,
   [112]26]. Our results from primary neuron cultures demonstrated that
   NO-induced necroptosis is facilitated through the activation of the
   RIP1/RIP3/MLKL cascade. The initiation of necroptosis is NO dependent,
   as evidenced by the fact that the phosphorylation cascade occurs only
   after the addition of the NO donor GSNO (Fig. [113]6e). On the basis of
   these findings, we propose that NO-mediated necroptosis is executed via
   activation of the RIP1/RIP3/MLKL phosphorylation cascade.

Nec-1s administration mitigates MPTP-induced motor dysfunction and
neurodegeneration

   To investigate the neuroprotective effects of Nec-1s against
   MPTP-induced parkinsonism, we conducted behavioural assessments and
   analysed neurodegeneration markers in the substantia nigra. In the
   rotarod test (Fig. [114]7a, b), the MPTP-treated mice presented a
   significant reduction in retention time compared with the
   saline-treated mice, indicating motor dysfunction. However, Nec-1s
   treatment significantly improved retention times in the MPTP-treated
   mice, suggesting partial recovery of motor function. Similarly, in the
   pole test (Fig. [115]7a, c), the MPTP-treated mice took significantly
   longer to descend than the control mice did. Nec-1s administration
   significantly reduced the descent time in the MPTP-treated mice,
   further supporting the protective effect of Nec-1s on motor
   coordination. Histological analysis (Fig. [116]7d, e) revealed that
   MPTP treatment resulted in a marked decrease in TH-positive neurons in
   the substantia nigra, indicative of dopaminergic neuron loss. Nec-1s
   treatment significantly preserved the number of TH-positive cells in
   the MPTP-treated mice, demonstrating its neuroprotective effect.
   Western blot analysis (Fig. [117]7g, h) of necroptosis markers revealed
   that MPTP treatment increased the phosphorylation levels of RIP1, RIP3,
   and MLKL, key proteins involved in the necroptosis pathway. Treatment
   with Nec-1s significantly reduced the phosphorylation of these
   proteins, indicating that Nec-1s inhibits MPTP-induced necroptosis.

Fig. 7.

   [118]Fig. 7
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   Nec-1s alleviates MPTP-induced motor dysfunction and neurodegeneration
   in a mouse model of PD. a Schematic diagram showing the experimental
   design. The mice were treated with MPTP (20 mg/kg, i.p.) or saline,
   followed by Nec-1s (10 mg/kg, i.p.) administration. Behavioural tests
   (rotarod and pole tests) were conducted to assess motor function. b
   Rotarod test results indicated that MPTP significantly reduced
   retention time compared with the saline control, whereas Nec-1s
   treatment partially restored motor function. c Pole test results
   showing that MPTP treatment increased the time taken to descend, which
   was reduced by Nec-1s administration. d Schematic diagram of the
   experimental design for histological analysis. The mice were treated
   with MPTP (20 mg/kg, i.p.) or saline, followed by Nec-1s (10 mg/kg,
   i.p.) administration, followed by immunohistochemical staining and
   detection of necroptosis markers. e Representative images of TH
   staining in the substantia nigra showing significant loss of
   TH-positive neurons in the MPTP-treated mice, which was alleviated by
   Nec-1s treatment (20x). f Quantification of TH-positive cells revealed
   that Nec-1s significantly preserved dopaminergic neurons compared with
   MPTP treatment alone. g Western blot analysis of necroptosis markers,
   including p-RIP1, p-RIP3, and p-MLKL. MPTP treatment increased the
   phosphorylation levels of these markers, indicating activation of the
   necroptosis pathway, which was mitigated by Nec-1s. h Densitometric
   analysis of western blots showing the relative levels of p-RIP1/t-RIP1,
   p-RIP3/t-RIP3, and p-MLKL/t-MLKL. Nec-1s significantly reduced the
   phosphorylation of these necroptosis markers in MPTP-treated mice. The
   data are presented as the means ± SEMs. Statistical significance:
   *P < 0.05, **P < 0.01, ***P < 0.001

The universality of NO-mediated necroptosis in AD, PD, and ALS patients and
in multiple brain regions

   Next, we sought to ascertain the universality of NO-mediated
   necroptosis across various neurodegenerative conditions, with the goal
   of informing clinical applications. We conducted a comprehensive
   analysis of patient-derived samples stratified by disease onset and
   type, encompassing early-onset and late-onset sporadic AD, as well as
   ALS, and corresponding control samples from a diverse array of brain
   regions. We also included a new set of PD brain tissue validation
   samples to verify the reliability of the results. We investigated the
   correlation between the NOS gene family and pivotal genes implicated in
   necroptosis, assessing the uniformity of this association across a
   spectrum of brain tissues. Our findings revealed the universal nature
   of NO-mediated necroptosis. In the context of AD, patients with
   early-onset sporadic AD presented a greater positive correlation
   between NOS expression and markers of necroptosis. In contrast,
   patients with late-onset sporadic AD presented a diminished
   correlation, suggesting a potential link between NO-mediated
   necroptosis and disease progression. (Fig. [120]8a). In the PD
   validation samples, we likewise observed an increase in association as
   the disease progressed (Fig. [121]8b). In the individuals with ALS, the
   correlation between NO-induced necroptosis and disease onset was
   significantly greater than that in the healthy controls. Additionally,
   we conducted an in-depth analysis to determine the relationship between
   necroptosis and various cell types within distinct brain regions,
   including the frontal cortex and motor cortex. This evaluation included
   neurons, oligodendrocyte lineage cells, and other glial cells. Our
   findings revealed a pronounced correlation, suggesting a potential role
   of NO-mediated necroptosis in the cellular pathology of ALS. Although
   some of the associations were not statistically significant, we still
   found trends indicative of a correlation, such as an increase in the
   number of correlations and a rise in the magnitude of the correlation
   coefficients (Figs. [122]8c, S4).

Fig. 8.

   [123]Fig. 8
   [124]Open in a new tab

   Universality of NO-mediated necroptosis in AD, PD and ALS. a
   Association analysis of NOS with key necroptotic genes in both
   early-onset and late-onset sporadic AD. b Association analysis of NOS
   with key necroptotic genes in the PD verification cohort. c Association
   analysis of NOS with key necroptotic genes in ALS. Orange represents a
   positive correlation, and blue represents a negative correlation. *,
   P < 0.05; **, P < 0.01; ***, P < 0.001; × represents not statistically
   significant. d ROC curve analysis of necroptosis markers as potential
   biomarkers for disease stratification in PD, AD, and ALS patients. e
   NO-mediated necroptosis is the primary death route of neurocytes. Our
   research indicates that as neurodegenerative diseases progress, the
   diversity of cell death patterns initially increases. In the final
   stages, necroptosis becomes the predominant pathway, a process that is
   accompanied by the phosphorylation of the PIP1/PIP3/MLKL cascade
   signalling pathway

   Collectively, these observations indicate that NO-mediated necroptosis
   is a fundamental mechanism underlying the aetiology and progression of
   classic neurodegenerative diseases, including AD, PD, and ALS.

   Finally, we conducted multifactorial logistic regression analyses to
   assess the efficacy of necroptosis markers as potential biomarkers for
   disease stratification in PD, AD, and ALS patients via ROC curves
   (Fig. [125]8d). In the context of PD, the combined application of
   necroptosis markers indicates a moderate classification capacity and
   presents a low sensitivity and high specificity, suggesting that this
   method is highly effective at identifying negative samples but less
   adept at detecting positive samples. These results indicate a need for
   further optimization to increase the sensitivity and overall
   classification capability of this method. In contrast, for AD,
   necroptosis markers demonstrate robust classification performance,
   positioning them as excellent biomarkers for disease stratification
   with high sensitivity and specificity, reflecting their strong ability
   to distinguish various disease states. Conversely, in ALS, although the
   classification capacity of these markers is detectable, it is
   comparatively less pronounced than their utility in AD and PD.

Discussion

   Our study revealed three important findings: 1) We revealed the
   diversity of neuronal cell death mechanisms, particularly highlighting
   necroptosis as the primary pathway involved in the progression of
   neurodegenerative diseases. 2) We validated the critical role of NO in
   mediating necrotic apoptosis. This mechanism involves the
   PIP1/PIP3/MKLK phosphorylation cascade (Fig. [126]8e). 3) We confirmed
   the universality of NO-mediated necroptosis across various types of
   classic neurodegenerative diseases and multiple brain regions.

   Currently, research on the mechanisms of neuronal cell death
   encompasses multiple aspects, such as apoptosis, autophagy,
   necroptosis, and ferroptosis, which are crucial for the development of
   neurodegenerative diseases [[127]10, [128]20, [129]27, [130]28].
   However, only by clearly delineating the macroscopic spectrum of
   neuronal cell death patterns can we comprehensively understand the
   temporal and spatial characteristics of disease progression [[131]29].

   Our findings indicate that during the progression of neurodegenerative
   diseases, neural cells exhibit a convergent death
   pattern—necroptosis—regulated by NO. As BLB grades increase,
   necroptosis becomes more pronounced across all neural cell types.
   Astrocytes and endothelial cells initially show a temporary reduction
   in necroptosis at BLB5 but increase at BLB6. Oligodendrocytes peak in
   necroptosis at BLB5, with a decline at BLB6 still above baseline. In
   contrast, ferroptosis decreases across all cell types from BLB5 to
   BLB6.

   Based on these findings, we speculate in the early stages, cells may
   primarily clear damaged components through cell death mechanisms, such
   as apoptosis and autophagy, which help maintain neuronal function.
   However, as the disease progresses and the pathological environment
   deteriorates, cell death pathways such as necroptosis and ferroptosis
   gradually become dominant. This shift may be closely related to factors
   such as neuroinflammation and oxidative/nitrosative stress, with a
   significant increase likely due to their association with inflammatory
   responses and cellular membrane damage. Although our experiments cannot
   explain why cells utilize this strategy, it may represent a
   characteristic feature of exacerbated neurodegenerative diseases.
   Alternatively, this convergent death pattern may serve as a
   compensatory self-rescue process for the organism, as studies suggest
   that glial cells are sensitive to iron death, that reversing iron death
   can reduce glial cell death [[132]30], and that microglial ferroptosis
   is regulated by SEC24B and contributes to neurodegeneration [[133]31].
   Therefore, cells may actively select death modes to avoid further
   exacerbation of self-injury [[134]32–[135]34]. However, constrained by
   changes in the brain microenvironment and organismal ageing, this
   compensatory death mode adjustment may not reverse the progression of
   the disease [[136]35–[137]37].

   Precise modulation of NO can ameliorate oxidative stress status and
   signal transduction in neural cells, thereby alleviating neuronal
   degeneration and synaptic loss during the progression of relative
   diseases [[138]38, [139]39], and have good value in the formulation of
   clinical treatment strategies [[140]40]. We have performed both
   cellular and mouse experiments and confirmed that NO is an important
   executor of necroptosis. Based on our results, this process is
   initiated by the phosphorylation of the PIP1/PIP3/MLKL signalling
   cascade, which underscores the intricate molecular mechanisms of
   NO-mediated necroptosis.

   Neuroinflammation is a key feature of PD, where inflammatory cytokines
   like TNF-α and IL-1β activate the RIPK1/RIPK3 complex, enhancing the
   phosphorylation of the PIP1/PIP3/MLKL cascade. This activation triggers
   MLKL, leading to plasma membrane destruction and further inflammation,
   creating a cycle of damage. Mitochondrial dysfunction and oxidative
   stress, common in PD, may also affect PIP1/PIP3 through pathways like
   AMPK and mTOR, driving necroptotic neuronal death and worsening
   neuroinflammation as the disease progresses [[141]41–[142]43]. The
   development of specific inhibitors targeting the RIP1/RIP3/MLKL pathway
   holds promise for delaying the process of necroptosis and associated
   diseases. Current necroptosis inhibitor development primarily focuses
   on RIP1, with some targeting RIP3 or MLKL [[143]43]. Several
   necroptosis inhibitors, including GSK2982772 and GSK3145095, have
   entered clinical trials but face issues like off-target effects,
   metabolic instability, and limited efficacy, with GSK2982772 failed in
   trials and caused severe rheumatoid arthritis [[144]43]. Future
   development should focus on enhancing efficacy while minimizing side
   effects and improving drug stability and administration.

   Lastly, our study demonstrates the widespread involvement of
   NO-mediated necroptosis in the pathogenesis of PD, AD, and ALS, with a
   consistent trend observed across various brain regions. ROC analysis
   further confirmed that necroptosis-related molecules could serve as
   potential biomarkers for predicting disease progression, offering new
   avenues for early diagnosis and personalized treatment. The development
   of blood-based biomarkers to reflect tissue conditions may represent a
   promising direction for early diagnosis in the future.

   Our study has limitations, including insufficient samples from BLB1 to
   BLB4 levels to fully capture early neurodegenerative patterns in PD.
   Additionally, while we propose and validate the role of NO-mediated
   necroptosis, further investigation into its detailed molecular and
   cellular mechanisms is needed for a more comprehensive understanding of
   neuronal cell death regulation.

   In summary, our study reveals the universality and mechanism of
   NO-mediated necroptosis in neurodegenerative diseases. Targeting the
   NO-mediated necroptosis pathway may offer promising avenues for the
   development of novel treatments to combat neurodegenerative diseases.

Methods and materials

Data source and preprocessing

   The present study was based on public data from the Gene Expression
   Omnibus (GEO) database [[145]44, [146]45]. We conducted a keyword
   search using AD, PD, and ALS, ultimately identifying three viable brain
   tissue cohorts for our study. These datasets were [147]GSE216281,
   featuring PD samples with Braak Lewy body (BLB) stages of 0–6 (n = 84);
   [148]GSE203206, comprising early-onset sporadic AD, late-onset sporadic
   AD, and control samples (n = 47); and [149]GSE219278, encompassing ALS
   and control samples from multiple brain regions (n = 84). We also used
   [150]GSE168496 as a validation set for PD (n = 16), the glioblastoma
   multiforme dataset [151]GSE263588 (n = 49), the meningioma dataset
   [152]GSE252291 (n = 279), and the pituitary adenoma dataset
   [153]GSE260487 (n = 32) as reverse validation sets. Upon integrating
   the data, we noted that the [154]GSE216281 cohort had a limited number
   of samples in BLB stages 1–4, with the lowest representation being only
   two sample. This lack of samples posed a potential risk of reduced
   statistical power; therefore, we removed these data while retaining all
   the samples from the other datasets.

Differential expression gene analysis and pathway annotation

   We removed low-quality counts and lncRNAs, and the counts were then
   converted to transcripts per million (TPM) values for subsequent
   analyses. Differential gene expression analysis was conducted via the
   EdgeR package in R [[155]46], with the criteria for significance set at
   |log2FoldChange (FC)|≥ 1 and P ≤ 0.05. Volcano plots were generated to
   visualize these differences. The significant genes were subsequently
   subjected to pathway enrichment analysis and gene interaction network
   construction via Metascape [[156]47], providing comprehensive insight
   into the molecular mechanisms underlying the observed gene expression
   changes.

Neural cell subtype prediction through the scaden algorithm

   The inherent challenge in using bulk-tissue RNA sequencing (RNA-seq) is
   its aggregation of gene expression data from various cell types,
   yielding only a mean expression level. In our research, we aimed to
   accurately estimate cell-type proportions within actual bulk-tissue
   RNA-seq samples and identify death patterns among multiple neural cell
   subtypes in brain tissue, so we employed the deep learning-based Scaden
   algorithm [[157]48]. This algorithm, specifically developed for
   deconvolution, is trained on simulated bulk-tissue RNA-seq data, which
   are derived from tissue-specific single-cell RNA-seq data [[158]49]. We
   utilized brain-specific training data generated by simulating
   artificial bulk RNA-seq samples. Prior to training the Scaden model for
   5000 steps, a measure to prevent overfitting was used, as recommended
   by the creators. We ensured gene and feature scale consistency within
   our datasets. Subsequently, cell-type proportion predictions were
   conducted via Scaden. The data obtained from this process will be
   instrumental in analysing cell types and constructing models to
   understand the patterns of neural cell death.

Construction of a dynamic interactive model between cell death patterns and
cell types

   To construct a dynamic model elucidating the interactions between
   multiple neural cell types and various cell death modalities, we
   established four distinct phases. Initially, we generated five datasets
   encompassing core genes associated with apoptosis, autophagy,
   ferroptosis, necroptosis, and parthanatos. These gene sets were curated
   from Cell Snapshot ([159]https://www.cell.com/snapshots) [[160]50],
   which are considered by the current scientific community to be the most
   important regulatory routes in neurodegenerative diseases,serving as
   the death mode dataset for later analysis. In the second phase, we
   extracted the TPM values of these genes, forming the basis for
   subsequent interaction network construction. This extraction process
   resulted in a comprehensive gene expression matrix. The third phase
   involved integrating the cell death-related gene matrix with the matrix
   of neural cell subtypes. This integration was accomplished through a
   Mantel test [[161]51]; the Mantel test was conducted via 1,000
   permutations to ensure robustness, and the Pearson correlation
   coefficient was used as the distance measure. The resulting interactive
   signature model was further refined by incorporating Pearson
   correlation coefficients, Mantel test correlation values (R), and
   adjusted P values (p.adjust) calculated via the Benjamini‒Hochberg
   procedure to control for multiple comparisons. We defined a P
   value ≤ 0.05 as statistically significant, with a Mantel test R
   value > 0.5 indicative of a strong positive correlation. Finally, we
   quantified the interactions within the network to elucidate the dynamic
   interplay between neural cell types and cell death patterns throughout
   the progression of neurodegenerative diseases. This comprehensive
   approach helped elucidate the intricate relationships governing
   neuronal survival and death under these conditions.

The correlation between nitric oxide synthase (NOS) and necroptosis

   To investigate the correlation between NO and necroptosis, we performed
   correlation analysis of NOS1, NOS2, and NOS3, which are enzymes
   involved in NO production and may be the core genes involved in
   necroptotic cell death. The Pearson correlation coefficient was
   calculated via the ggcor R package.

Mouse PD modelling

   Healthy adult mice with body weights ranging from 20 to 25 g were
   randomly divided into the MPTP group, MPTP + Necrostatin-1s (Nec-1s)
   group, and saline group. All the animals were housed under standard
   conditions, including a constant temperature and humidity environment
   as well as a 12-h light‒dark cycle. The mice in the MPTP group received
   continuous intraperitoneal injections of MPTP
   (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) starting from the first
   day of the experiment, with an injection dose of 20 mg/kg body weight.
   After the initial injection, subsequent injections were administered
   every 2 h for a total of four injections. The mice in the MPTP + Nec-1s
   group received Nec-1s intraperitoneal injections 3 days before MPTP
   administration, with the first dose being 10 mg/kg, followed by daily
   injections of 5 mg/kg until the behavioural experiments. The animals in
   the saline group received an equivalent volume of physiological saline,
   and on the 7th day, the success of the model was determined by tyrosine
   hydroxylase (TH) staining.

Behavioural testing

   On the 7th day after injection, behavioural testing was conducted on
   all surviving animals to assess the impact of MPTP injection on motor
   function. The tests included the rotarod test, which was used to
   evaluate the motor coordination and balance of the animals. The animals
   were placed on a rotating rod, and the time they were able to stay on
   the rod or until they fell off was recorded. The pole test was used to
   assess the movement disorders in mice. The animals were placed at the
   top of a vertical pole, and recorded the time taken for them to descend
   to the ground.

TH staining of the substantia nigra

   After the completion of the behavioural experiments, the animals were
   deeply anaesthetized to ensure painless euthanasia. The whole brain
   tissue was immediately removed and rinsed with physiological saline to
   remove blood and residual tissue. The brain tissue was fixed in a 4%
   paraformaldehyde solution and stored at 4°C for 24 h. After fixation,
   the brain tissue underwent stepwise dehydration, clarification, and
   paraffin embedding, followed by embedding in paraffin. The brain tissue
   was sectioned into 3.5 μm thick continuous slices via a microtome, and
   the slices were mounted on glass slides for immunohistochemical
   staining. After deparaffinization and antigen retrieval, the slices
   were stained with the dopaminergic neuron-specific antibody TH. The
   stained slices were observed under a light microscope, and the number
   of TH-positive cells was recorded and analysed. The potential
   protective effect of Nec-1s on MPTP-induced symptoms was assessed on
   the basis of the intensity of TH staining.

Foetal mouse midbrain tissue isolation and primary neuron culture

   Pregnant C57BL/6 mice at 14 days of gestation were euthanized swiftly
   with CO[2] and placed on a prechilled dissection dish. Following rapid
   disinfection and uterus separation, the uterus was transferred to
   prechilled D-Hank's solution. With a stereomicroscope, foetal mouse
   midbrain tissue was meticulously dissected via microinstruments, and
   the soft meninges and vascular membranes were removed from the brain
   tissue. The dissected tissue was fully minced into a slurry on ice. The
   thoroughly minced midbrain tissue was then thoroughly mixed with 1 ml
   of 0.05% trypsin and incubated in a 37°C CO[2] cell culture incubator
   for 15 min. The digestion process was halted by addition of Dulbecco's
   modified Eagle’s medium (DMEM) containing 10% foetal bovine serum and
   then by low-speed centrifugation (1000 rpm for 5 min), followed by
   washing and settling. The tissue was aspirated with DMEM, transferred
   to an appropriate volume of neurobasal medium supplemented with 2% B27,
   and repeatedly pipetted to generate a single-cell suspension. The final
   density was adjusted to 5 × 10^5 cells/ml on the basis of the cell
   count results, and an appropriate amount of the cell suspension was
   seeded into a 6-well plate (coated with poly-L-lysine) and placed in a
   37°C CO[2] cell culture incubator. After 8 h, the culture medium was
   replaced entirely, and subsequently, every two days, half the
   neurobasal medium volume was replenished to maintain cell culture. The
   cells were ready for experimentation after 8 days [[162]52].

Trypan blue staining

   A 4% stock solution of Trypan blue was prepared with Locke's buffer,
   and the solubilization process was facilitated by vertexing.
   Thereafter, the solution was subjected to filtration and stored at 4°C.
   Prior to staining, the stock solution was diluted with cell culture
   medium to a final concentration of 0.4% to create the working staining
   solution. The cells seeded in a 6-well plate were treated with the
   appropriate agents, after which the spent medium was removed. Then, 1
   ml of 0.4% Trypan blue staining solution was added, and the cells were
   stained for 5 min. The staining outcome was subsequently immediately
   observed under a microscope and photographed.

Neural cell line culture and drug treatment

   The human neuroblastoma cell line SH-SY5Y was purchased from the China
   Typical Culture Collection Center of the School of Life Sciences, Wuhan
   University. The cells were cultured in high-glucose DMEM (12800, Gibco,
   USA) supplemented with 10% foetal bovine serum (A5669701, Gibco, USA),
   100 units/ml penicillin and 100 μg/ml streptomycin (15070063, Gibco,
   USA) at 37°C, 5% CO[2] and 95% humidity. We established a PD cellular
   model by treating SH-SY5Y cells with 100 nmol/l rotenone (R8875;
   Sigma‒Aldrich, USA) for 24 h [[163]53], which has been reported to
   effectively simulate neuronal damage in PD [[164]53–[165]56]. For
   analysis of the role of NO in the mechanism of neurodegeneration, the
   exogenous NO donor GSNO (57564–91-7, Sigma, USA) was applied to SH-SY5Y
   cells at a stimulating concentration of 500 μmol/l, either alone or in
   combination with rotenone, to elicit specific cellular effects. For the
   primary neuron assay, 100 µM GSNO was used to treat midbrain neurons
   derived from mice to elucidate the role of NO-induced necroptosis.

Determination of cell death patterns in PD cellular models

   We treated neural cells with specific pharmacological inhibitors,
   including an apoptosis inhibitor [Z-VAD-FMK (Z-VAD), 219007, Sigma,
   USA] at a stimulating concentration of 2.5 mmol/l; an autophagy
   inhibitor [3-methyladenine (3-MA), SAE0107, Sigma, USA] at a
   stimulating concentration of 5 μmol/l; a ferroptosis inhibitor
   [ferrostatin-1 (Fer-1), 347174–05-4, Sigma, USA] at a stimulating
   concentration of 100 μmol/l; a necroptosis inhibitor (Nec-1s,
   852391–15-2, Sigma, USA) at a stimulating concentration of 50 μmol/l;
   and a parthanatos inhibitor [olaparib (Ola), 763113–22-0, Sigma, USA]
   at a stimulating concentration of 10 μmol/l, to validate the cellular
   damage pathways of apoptosis, autophagy, ferroptosis, necroptosis, and
   parthanatos.

MTT assays to evaluate cell viability

   To determine cell viability, we conducted an MTT (3580GR001, BioFroxx,
   China) assay on each group. The cells were subsequently centrifuged at
   1,000 rpm for 5 min and seeded in a 96-well plate at a density of
   1 × 10^5 cells per well with 200 μl of culture medium. Twenty-four
   hours after drug treatment, 20 μl of MTT solution (0.5 g/l) was added
   to each well, followed by a 3-h incubation. The supernatant was then
   removed, and 150 μl of DMSO was added. The plate was shaken at 130 rpm
   for 5 min. The absorbance was measured at 565 nm.

Mitochondrial membrane potential (MMP) assay and nuclear staining

   To assess the energy charge status of the cell, we conducted MMP
   measurements. The cells in each group were washed twice with Locke's
   buffer after stimulation. Then, 1 ml of MitoTracker™ Red CMXRos dye
   (M7512, Invitrogen, USA) was added to each group, and the cells were
   incubated at 37°C for 30 min. After the dye was removed, the cells were
   stained with Hoechst 33342 (CB2261, Sigma, USA) diluted in liquid
   culture medium, followed by incubation in the dark for 10 min. The
   staining solution was then discarded, and the cells were fixed with
   prechilled (-20°C) methanol for 30 min. After the methanol was
   discarded, each group was washed with 1 ml of Locke's solution. The
   cells were subsequently observed under a fluorescence microscope, and
   images were captured for further analysis.

Western blot analysis

   Protein lysates were prepared by lysing the cells in each well of a
   6-well plate with 200 μl of RIPA buffer. The protein concentration was
   determined via the BCA method. The lysates were subsequently mixed with
   5 × SDS loading buffer and boiled for later use. The samples were then
   loaded into separate lanes for SDS‒PAGE, which was initially run
   through the stacking gel at 80 V and then through the separating gel at
   120 V. After electrophoresis, the proteins were transferred to a PVDF
   membrane, which was then blocked with 5% skim milk. The membranes were
   incubated overnight at 4°C with specific primary antibodies:
   anti-caspase-3 (dilution 1:1000, ab32351, Abcam, UK), anti-cleaved
   caspase-3 (1:1000, ab32351, Abcam, UK), anti-RIP1, anti-phospho-RIPK1,
   anti-RIP3, anti-phospho-RIP3, anti-phospho-MLKL, anti-MLKL, and
   anti-beta-actin antibodies included in the Necroptosis Antibody Sampler
   Kit (#98110, Cell Signaling Technology, USA). Later, the membrane was
   washed three times with TBST and incubated with an HRP-linked secondary
   anti-rabbit antibody (dilution 1:5000, ab288151, Abcam, UK) for 1 h.
   After three further TBST washes, the membrane was developed with an
   enhanced chemiluminescence (ECL) substrate, and the protein bands were
   visualized. Semiquantitative analysis of the bands was performed via
   ImageJ.

Statistical analysis

   All the statistical analyses were performed via R and SPSS 24.0. The
   means ± SDs were used to describe continuous variables that were
   normally distributed. P < 0.05 indicated statistical significance. To
   evaluate the performance of the necroptosis molecular diagnostic test
   and its efficacy for a disease stratification biomarker across PD, AD,
   and ALS, we conducted multifactorial logistic regression analyses. The
   receiver operating characteristic (ROC) curve was subsequently plotted
   to calculate the area under the curve (AUC), sensitivity, and
   specificity.

Supplementary Information

   [166]Supplementary Material 1.^ (4MB, docx)
   [167]Supplementary Material 2.^ (26.3KB, xlsx)
   [168]Supplementary Material 3.^ (3.1MB, pptx)

Acknowledgements