MLKL inhibition reduces cell death induced by PD stressors in vitro

To investigate the potential involvement of MLKL in Parkinson’s disease (PD) stress conditions, we performed experiments to evaluate the effect of MLKL inhibition on the cytotoxicity of 6-hydroxydopamine (6-OHDA), a neurotoxin commonly used to model PD, in human neuroblastoma cell line SH-SY5Y, and primary mouse embryonic fibroblasts (MEFs) (Fig. 1a and b and Fig. S1a-1d). SH-SY5Y cell line is frequently chosen in current PD research, and primary MEFs are sensitive to phospho-MLKL-triggered necroptotic cell death [24, 25]. Also, an MLKL inhibitor, necrosulfonamide (NSA), was employed with 6-OHDA plus TNF-α-induced cell death. We observed that TNF-α enhanced the sensitivity of both cell types to necrotic cell death in the presence of 6-OHDA (Fig. S1a-S1b). However, the addition of NSA significantly impaired this effect in a dose-dependent manner (Fig. 1a and b). Moreover, the phosphorylated MLKL (p-MLKL) and inducible nitric oxide synthase (iNOS), which are key markers of cell necroptosis, were highly expressed in 6-OHDA/TNF-α-treated cells; however, their expression was significantly reduced when treated with NSA (Fig. 1e and S1c-S1d).

Fig. 1

MLKL inhibition or deficiency decreased cell death in response to 6-OHDA plus TNF-α, or toxic α-Syn PFFs treatments. a-b. SH-SY5Y cells (a) or primary MEF cells (b) were treated with or without 6-OHDA plus TNF-α, followed by adding different concentrations of MLKL inhibitor NSA for 24 h. The cell viability was measured by the CCK8 assay and normalized to untreated cells. c. Primary MEF cells were transfected with GFP-tagged A53T synuclein (A53T) or empty vector (Ev). After 24 h, the transfected cells were treated with or without 6-OHDA, TNF-α, and NSA for another 24 h. d. Mlkl+/+ and Mlkl−/− MEF cells were transfected with GFP-tagged A53T synuclein. After 24 h, the transfected cells were treated with or without 6-OHDA plus TNF-α for another 24 h. e-f. Cells were treated as described in a and c, respectively. Then western blot analysis was performed for inducible nitric oxide synthase (iNOS) and phosphorylated MLKL (p-MLKL). The quantification results were shown in the lower panels. g-h. Mlkl+/+ (WT) and Mlkl−/− MEF cells were treated with GFP-tagged A53T synuclein (A53T) or empty vector (Ev). After 24 h, the transfected cells were treated with or without 6-OHDA plus TNF-α for another 24 h, followed by western blot analysis (g) and cytokine secretion measurement (h). i-k. Assessment of cell viability and protein expression in primary neuronal cells. Primary neuronal cells were treated with or without α-Syn preformed fibrils (PFFs) and NSA for 14 days followed by CCK8 assay and western blotting (i). Mlkl+/+ (WT) and Mlkl−/− primary neuronal cells were treated with or without PFFs over a period of 14 days followed by the CCK8 assay and western blotting (k). Quantitative results from the experiments are presented in two panels. The upper panel displays the quantified data from the treatments in i, and the lower panel presents the quantification corresponding to the treatments in k. l-n. Human induced pluripotent stem cell (iPSC)-derived midbrain organoids (hMOs) were subjected to treatments with PFF, both with and without concurrent AAV9-shMLKL (to facilitate MLKL knockdown). Western blot analysis (l) and immunofluorescence staining (m) were employed to assess the treatments. The quantified expression levels of p-α-Syn are presented in the upper section of l, while the quantifications of p-MLKL intensity are illustrated in n. Scale bars, 10 μm. All data are representative of three independent experiments. The error bars represented the standard deviations (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

As a high expression level of α-synuclein (α-Syn) has been reported to produce PD-like cellular and axonal pathologies in the nigrostriatal region [26, 27], we also investigated the effect of NSA on cell death induced by α-Syn aggregates under PD stress conditions. We transfected primary MEF cells with human A53T α-Syn-GFP and observed that NSA exhibited neuroprotective effects against 6-OHDA plus TNF-α, accompanied by downregulated expressions of p-MLKL and iNOS (Fig. 1c and f). Additionally, in A53T α-Syn-GFP-transfected MEF cells, knocking out Mlkl caused a significant decrease in 6-OHDA plus TNF-α-triggered cell death (Fig. 1d). Consistently, the expression of p-MLKL and iNOS were detectable in wild-type (WT) MEF cells, but not in Mlkl−/− cells, even with 6-OHDA/TNF-α treatment (Fig. 1g). These results suggest that MLKL-mediated necroptosis may be positively correlated with oxidative stress responses.

Further enzyme-linked immunosorbent assay (ELISA) analyses revealed that 6-OHDA and TNF-α co-stimulation triggered the robust secretion of proinflammatory cytokines IL-6, IP-10, MCP-1, CCL3, CCL5, CXCL1, CXCL2, and CXCL5 (Fig. 1h). In line with these observations, the expression levels of chemokines, particularly IL-6 and MCP-1, were much higher in Mlkl+/+ MEF cells after 6-OHDA/TNF-α treatment but were lower in 6-OHDA/TNF-α-treated Mlkl−/− MEF cells (Fig. 1h). These findings suggest that MLKL-mediated inflammatory signaling is highly associated with the 6-OHDA or α-Syn-induced PD model.

Further investigations into MLKL’s role in PD were conducted using primary neuronal cultures derived from the embryonic mesencephalon, a region that develops into the substantia nigra pars compacta (SNpc) during brain maturation. We utilized α-Syn preformed fibrils (PFFs) as a relevant neurotoxic stimulus for PD. Our findings revealed significant neuroprotective effects of NSA against PFFs, marked by decreased p-α-Syn and increased TH expression (Fig. 1i and j). Additionally, MLKL knockout markedly reduced PFFs-induced neuronal cell death (Fig. 1j and k). Correspondingly, p-α-Syn expression was significantly elevated in Mlkl+/+ primary neuronal cells upon α-Syn PFFs treatment, whereas reduced in PFFs-treated Mlkl−/− primary neuronal cells (Fig. 1j and k).

To further elucidate the role of MLKL in PD pathology, we extended our investigations to human induced pluripotent stem cell (iPSC)-derived midbrain organoids (hMOs) subjected to the PFF-toxicity assays. The data revealed a pronounced upregulation of p-α-Syn expression in hMOs post-PFF treatments, which occurred concurrently with a reduction in MLKL expression and increased activation of p-MLKL within neuronal cells (Fig. 1l and n and S1e-S1f). Intriguingly, the targeted knockdown of MLKL significantly bolstered the resilience of hMOs to PFF-induced toxicity (Fig. 1l and n). Comparative analysis showed that hMOs treated with PFFs in conjunction with AAV9-shMLKL displayed a substantial decline in p-α-Syn levels and neuronal p-MLKL expression (Fig. 1l and n). These findings underscore that the lack or suppression of MLKL expression imparts a protective effect, mitigating the cellular impacts of diverse PD-related stressors in murine and human models.

MLKL deficiency ameliorates motor symptoms in the A53T transgenic mice

The inhibition or deficiency of MLKL was observed to reduce cell death and proinflammatory cytokine secretion in the presence of 6-OHDA-induced PD traits, as depicted in Fig. 1. Next, we investigated the role of MLKL in motor capability and anxiety-like behaviors in progressive PD using Tg-Mlkl−/− mice generated by crossbreeding Mlkl−/− mice with human A53T α-Syn transgenic (denoted hereafter as Tg) mice (Fig. 2a) [10, 28]. Western blot analysis of different tissues of the Tg-Mlkl−/− mice revealed the absence of MLKL and overexpression of human A53T α-Syn protein (Fig. 2b and c). As the Tg-Mlkl+/+ mice exhibit typical PD characteristics, including abnormal motor activities at around ten months old [10], we subjected the Tg-Mlkl−/− mice and control groups (WT and Tg-Mlkl+/+ mice) to perform behavioral tests (Fig. 2d-m).

Fig. 2

MLKL deficiency improves motor capability in homozygous A53T synuclein transgenic (Tg) mice. Wild-type (WT, n = 10), Tg-Mlkl+/+ (n = 7), and Tg-Mlkl−/− mice (n = 13) around 10–12 months old were examined for different motor activities as described in the Methods section. a. Schematic of the crossbreeding between A53T transgenic mice (expressing mutant human α-synuclein) and MLKL KO mice. b-c. Immunoblotting analysis of the protein expression levels of α-synuclein (α-Syn) and MLKL in multiple tissues from the Tg-Mlkl+/+ and Tg-Mlkl−/− mice. d-f. The autonomous trajectory map of WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice in the open field test. Tg-Mlkl−/− mice exhibited more entries (e) and times (f) in the center region than Tg-Mlkl−/− mice. g. The average time required for the WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice to descend the pole. h. WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice were scored for latency to fall on the accelerating rotarod. i-l. Performance of mice in the elevated plus-maze test (EPMT). i. The autonomous trajectory maps of WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice in the EPMT were recorded. j. The total distance traveled in 5 min was shown. The Tg-Mlkl−/− mice had more entries into the open arms (k) and spent more time in the anxiety-provoking open arms (l) than the Tg-Mlkl−/−mice. m. Detection of depression with the tail suspension test. The Tg-Mlkl−/− mice showed a significant reduction in the duration of immobility compared with the control mice. n. Long-term pole test results from 2–18 months, comparing WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice. o. Survival curves for Tg-Mlkl+/+ (n = 19) and Tg-Mlkl−/− (n = 37) mice. All data are representative of three independent experiments. The error bars represented the standard deviations (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance

The Tg-Mlkl−/− mice showed similar locomotor activities in the open field with the WT mice, while more active than the Tg-Mlkl+/+ mice (Fig. 2d). Moreover, the Tg-Mlkl−/− mice exhibited reduced anxiety-like behavior compared to the Tg-Mlkl+/+ mice, as evidenced by their increased entries and longer time spent in the center zone, indicating a decrease in avoidance of the center (Fig. 2d and f). Additionally, the pole test revealed that the Tg-Mlkl−/− mice exhibited a significant decrease in the time taken to reach the pole base compared to the control Tg-Mlkl+/+ group (Fig. 2g), demonstrating an improvement in movement disorder caused by striatal dopamine depletion.

Furthermore, the Tg-Mlkl−/− mice exhibited significantly reduced behavioral deficits observed in the Tg-Mlkl+/+ mice as assessed by the accelerating rotarod test (Fig. 2h). The elevated plus-maze test (EPMT) showed that the Tg-Mlkl−/− mice spent a higher percentage of arm entries and time in the open arm compared to the Tg-Mlkl+/+ mice, as depicted in Fig. 2i L; however, there was no significant difference observed in the total distance traveled between the two groups (Fig. 2i and j). Finally, the tail suspension test was utilized to evaluate the depressive-like symptoms of mice, where the Tg-Mlkl−/− mice exhibited decreased immobility times when compared to the Tg-Mlkl+/+ mice (Fig. 2m). Long-term motor function assessment using the pole test highlighted that Tg-Mlkl−/− mice, especially at 18 months, took significantly less time to reach the pole base compared to Tg-Mlkl+/+ mice (Fig. 2n). Survival analysis revealed a prolonged lifespan in Tg-Mlkl−/− mice compared to Tg-Mlkl+/+ mice, with a median survival time of 19 months (Fig. 2o). In conclusion, our results suggest that Mlkl knockout can effectively improve motor capabilities and alleviate depressive symptoms in Tg-Mlkl+/+ mice exhibiting PD.

MLKL deficiency protects dopaminergic neurons in A53T transgenic mice

To investigate whether MLKL deficiency improves motor capability by regulating α-Syn function, we conducted immunoblotting, immunofluorescence, and immunohistochemical staining for phosphorylated α-Syn at serine 129 (p-α-Syn129S), a specific pathological form associated with α-Syn aggregation in PD [29]. Our results demonstrated that p-α-Syn129S was present in high abundance in the cortex, striatum, and substantia nigra regions of Tg-Mlkl+/+ mice; however, they markedly decreased in the corresponding regions of Tg-Mlkl−/− mice (Fig. 3a and b). Additionally, immunofluorescence staining confirmed that MLKL deficiency significantly reduced phosphorylated α-Syn inclusions in the striatum region of the A53T transgenic mice (Fig. 3c). In humans with Parkinson’s disease, the substantial loss of dopaminergic (DA) neurons in the SN is closely related to motor dysfunction [30]. Although there was no significant difference in dopaminergic neurodegeneration in the striatum among the three mouse groups (Fig. 3d and e), we observed a higher level of TH-positive neurons in the SN of Tg-Mlkl−/− mice and WT mice, but not in Tg-Mlkl+/+ mice (Fig. 3d and e). Furthermore, the DA neurons exhibited different morphologies in the SN regions of Tg-Mlkl+/+ and Tg-Mlkl−/− mice. DA neurons in the pars compacta of the SN in Tg-Mlkl−/− mice exhibited a high density of TH-positive fibers and contained a denser cell mass than those in Tg-Mlkl+/+ mice (Fig. 3d). Immunoblot analysis also demonstrated that TH accumulation was significantly elevated, accompanied by a remarkable reduction of p-α-Syn and iNOS in the cortex, striatum, and/or SN of Tg-Mlkl−/− mice (Fig. 3f g). Furthermore, we segregated the whole brain tissues of the three mouse cohorts into soluble and insoluble components for Immunoblot analysis. This revealed that the whole brain tissues of Tg-Mlkl+/+ mice exhibited significantly higher levels of α-Syn and p-α-Syn compared to WT mice (Fig. 3h and i). Notably, the deletion of the Mlkl gene substantially diminished the p-α-Syn expression in Tg-Mlkl+/+ mouse brains (Fig. 3h and i). Additionally, in Tg-Mlkl−/− mice, the levels of p-α-Syn were consistently lower in soluble and insoluble fractions than in Tg-Mlkl+/+ mice (Fig. 3h and i).

Fig. 3

MLKL deficiency protects dopaminergic neuron loss and decreases hyperphosphorylated α-synuclein in the A53T transgenic mice. a. Representative immunostaining for phosphorylated α-Syn (p-α-Syn129S) in the cortex, striatum, and substantia nigra (SN) regions of WT, Tg-Mlkl+/+ and Tg-Mlkl−/− mice. Scale bars, 100 μm. The whole-brain sections were shown in the top right corner. The solid rectangles were zoomed from the selected dashed rectangles in the cortex, striatum, and SN regions, respectively. The quantification results of p-α-Syn129S are shown in b. c. Representative immunofluorescence image of the striatum region in the frozen brain sections of WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice staining with DAPI (blue, denoting the nuclear signal) and anti-p-α-Syn antibody (green). Scale bars, 50 μm. d. Dopaminergic neurons were determined by tyrosine hydroxylase (TH) staining. Representative TH immunostaining images of the cortex and SN sections from WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice were shown in d. The whole-brain sections were shown in the bottom left corner. The solid rectangles were zoomed from the selected dashed rectangles in the striatum and SN regions. Scale bars, 100 μm. e. Quantification of the total number of TH-positive cells in the entire striatum and SN regions, corresponding with d. f-g. Representative Western blot results of the expression levels of TH, α-Syn, p-α-Syn, and iNOS in the cortex, striatum, and SN sections of the WT, Tg-Mlkl+/+ and Tg-Mlkl−/− mice (f). Quantifications of the expressions for TH, α-Syn, p-α-Syn, and iNOS were shown in g. h-i, Western blot results displaying α-Syn and p-α-Syn levels in whole brain tissue (Whole), soluble fractions, and insoluble fractions from WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice. The soluble fraction contains cytoplasmic proteins, while the insoluble fraction includes membrane-bound proteins, organelle-associated proteins, and nuclear proteins. Quantifications of the results for α-Syn and p-α-Syn were shown in i. All data are representative of three independent experiments. The error bars represented the standard deviations (SD). ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance

Given that the homozygous line M83+/+ A53T mice naturally manifest a pronounced motor phenotype between 8 and 16 months linked with the accumulation of α-Syn inclusions in areas such as the spinal cord, brain stem, thalamus, periaqueductal gray, mesencephalon (surrounding the substantia nigra), and dorsal cochlear nucleus (DCN) [10], we further investigated the expression of p-α-Syn129S and NeuN (a neuron-specific nuclear protein) in the spinal cord and DCN of Tg-Mlkl+/+ and Tg-Mlkl−/− mice. Our findings illustrate that Mlkl gene knockout significantly reduces the expression of p-α-Syn129S in the spinal cord, mesencephalon, and DCN brain regions of Tg mice, while notably augmenting NeuN expression in the spinal cord (Fig. S2a-2b), consistent with above results (Fig. 3a and e). Additionally, double immunofluorescence staining of p-α-Syn129S and NeuN in the cortical area of both mouse groups indicated that Mlkl gene knockout genuinely diminishes the accumulation of p-α-Syn129S in neuronal cells (Fig. S2c). Consequently, these findings suggest that MLKL-mediated signaling is intricately associated with dopaminergic neurodegeneration and α-Syn aggregation in mice.

MLKL deficiency attenuates neuroinflammation in A53T transgenic mice

Microglia activation, as evidenced by increased expression of Iba-1, can indirectly indicate a neuronal abnormality in A53T transgenic (Tg-Mlkl+/+) mice. To investigate this, we assessed the expression of Iba1 and found that Mlkl knockout significantly reduced Iba1 immunoreactivity in the cortex and substantia nigra (SN) of Tg-Mlkl+/+ mice (Fig. 4a and b). Additionally, MLKL deficiency resulted in a significant decrease in the expression of CD11b, a surface receptor that is upregulated on activated microglia and is involved in the neuroinflammatory response in the brain [31], in the cortex and ventricle regions of Tg-Mlkl+/+ mice (Fig. 4e and f).

Fig. 4

MLKL deficiency attenuates neuroinflammation in the A53T transgenic mice. a-b. Representative immunostaining for the microglial marker Iba1 in the cortex, striatum, and SN regions of WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice (a). The quantification results of Iba1 intensity and relative soma perimeter are shown in b. The whole-brain sections were shown in solid rectangles. The dashed rectangle regions were zoomed in and shown in the middle. Scale bars, 200 μm. c-d. Immunohistochemistry staining and quantification of GFAP expression. Representative images of GFAP immunoreactivity from WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice were shown in c, and the according quantification results were shown in d. e-f. Representative immunostaining for CD11b in the cortex and ventricle of WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice (e). The quantification results of CD11b are shown in f. Scale bars, 50 μm. The whole-brain sections were shown in solid rectangles. The dashed rectangle regions were zoomed in and shown in the middle. Scale bars, 200 μm. g. The heat map depicted the average baseline serum cytokine concentration for the Tg-Mlkl+/+ (n = 3) and Tg-Mlkl−/− mice (n = 3). All data are representative of three independent experiments. The error bars represented the standard deviations (SD). ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance

The pathological marker, glial fibrillary acidic protein (GFAP), was prominently accumulated in the cortex and SN regions of Tg-Mlkl+/+ mice. In contrast, it was significantly decreased in the cortex and SN regions of Tg-Mlkl−/− mice (Fig. 4c and d). Notably, microglia and astrocytes showed enlarged somas in Tg-Mlkl+/+ mice, indicating morphological activation; however, microglia and astrocytes shrank when Mlkl was knocked out (Fig. 4b and d). These findings indicate that MLKL deficiency significantly attenuated microglia and astrocyte activation, ameliorating Parkinson’s symptoms in A53T transgenic (Tg-Mlkl+/+) mice.

Moreover, as MLKL-mediated necroptosis exacerbates multiple neurodegenerative diseases by triggering cell death and neuroinflammation [32], we evaluated the production of multiple serum cytokines using the ELISA method. Our results demonstrated that many proinflammatory cytokines, including IL6 and MCP-1, were significantly reduced in Tg-Mlkl−/− mice compared to Tg-Mlkl+/+ mice (Fig. 4g), which was consistent with the aforementioned results (Fig. 1h). In addition, we performed IHC analyses on six consecutive brain sections from each mouse group. The analyses showed that, in comparison to WT mice, Tg-Mlkl+/+ mice exhibited a notable increase in phosphorylated MLKL (p-MLKL) within the cortex, striatum, and substantia nigra (Fig. 5). This increase coincided with reduced levels of TH and NeuN, and elevated levels of p-α-Syn, Iba1, and GFAP. In contrast, Mlkl knockout in Tg mice significantly lowered p-MLKL expression in these brain regions, accompanied by a reduction in p-α-Syn, Iba1, and GFAP, and an upregulation of TH and NeuN expressions (Fig. 5). These results imply a significant association between MLKL expression in the mouse brain and the extent of neuronal damage and neuroinflammatory activity.

Fig. 5

MLKL deficiency reduces MLKL and p-MLKL levels in A53T transgenic mice. Seven consecutive tissue sections were obtained from the same brain region (cortex, striatum, and SN) of individual WT, Tg-Mlkl+/+, and Tg-Mlkl−/− mice. These sections underwent IHC to determine the expression levels of MLKL, p-MLKL, NeuN, TH, p-α-Syn, Iba1, and GFAP. Scale bars, 25 μm. All data are representative of three independent experiments

Single-cell RNA sequencing (scRNA-seq) analysis reveals the upregulated synaptic-related neurons and downregulated microglia in the SN region of the Tg-Mlkl −/− mice

To investigate the role of MLKL in advanced PD, we conducted scRNA-seq on nuclei isolated from substantia nigra regions of Tg-Mlkl−/− and Tg-Mlkl+/+ mice (Fig. S3). In the SN region of Tg-Mlkl+/+ mice (n = 3), we generated 3,563 single nuclei gene expression profiles, with a median of 405 genes and 100,993 transcripts per nucleus. For the SN region of Tg-Mlkl−/− mice (n = 3), we generated 8,466 single nuclei gene expression profiles, with a median of 497 genes and 43,481 transcripts per nucleus (Fig. S4a-S4b). In addition, we utilized Uniform Manifold Approximation and Projection (UMAP) visualization to separate nuclei into distinct clusters (Fig. 6a). Next, we annotated these clusters using cell-type-specific markers to identify oligodendrocytes (e.g., Ptgds, Gm16233, Anln, Ndrg1, and Gng11), oligodendrocyte precursor cells (e.g., Vcan, Cspg5, Thr, Neu4, and Pdgfra), astrocytes (e.g., Atp1a2, Gm3764, Slc4a4, Slc1a2, and Rorb), neurons (e.g., Meg3, Snhg11, Ahi1, Ube3a, and Syt1), Bergman glial cells (e.g., Atp13a5, Pdgfrb, Kcnj8, Igfbp7, and Vtn), type II spiral ganglion neurons (e.g., H2-D1, H2-K1, Kif2, Cd52, and Ly6c1), and microglia (e.g., C1qb, Arhgap45, C1qc, C1qa, and Ctss) (Fig. 6b).

Fig. 6

Single-cell RNA-seq analysis reveals the upregulated synaptic-related neurons and downregulated microglia in the substantia nigra region of the Tg-Mlkl−/− mice. a. UMAP visualization showing the clustering of single nuclei colored by cell types (upper) or individuals (bottom) in the SN regions of the Tg-Mlkl+/+ and Tg-Mlkl−/− mice. b. Heatmap showing the top 5 markers for each of the 7 clusters. c. The proportions of different types of cells in the Tg-Mlkl+/+ and Tg-Mlkl−/− mice. d. Heatmap depicting the z-scores of the common differential expressed signature genes in neuron, microglia, and astrocyte clusters. Representative genes were highlighted on the right side. The colors represent the cluster’s mean expression (transcripts per million). e. Top biological pathways enriched for DEGs were identified across neuron cells in the SN region. f. Beanplot showed different cytokine genes expression in microglia of the Tg-Mlkl+/+ (WT) and Tg-Mlkl−/− (KO) mice

We observed significant differences in the cluster sizes between the two groups, with a marked increase in the proportions of neurons and astrocytes in the Tg-Mlkl−/− mice, while oligodendrocytes and microglia were more frequent in the Tg-Mlkl+/+ mice (Fig. 6a and c). Additionally, we identified three clusters (neuron, microglia, and astrocyte) that exhibited both cell-type-specific and common gene expression patterns between the Tg-Mlkl−/− and Tg-Mlkl+/+ mice (Fig. 6e).

Differential expression of genes (DEGs) was identified in neuron, microglia, and astrocyte clusters between Tg-Mlkl−/− and Tg-Mlkl+/+ mice, followed by gene ontology (GO) term enrichment analysis of biological processes (Fig. 6d and e and S5a-S5b). Notably, Tg-Mlkl−/− neuronal cells exhibited upregulation of nervous system processes (e.g., Efnb3) and downregulation of nitrogen compound metabolic processes (e.g., Tsix) (Fig. 6d and e). Furthermore, downregulated genes in Tg-Mlkl−/− neuronal cells were enriched in functions related to cytokine production and apoptotic signaling pathways, indicating reduced inflammation and cell death (Fig. 6d and f). Similarly, microglia from Tg-Mlkl−/− mice showed upregulation of DEGs related to neurogenesis and downregulation of DEGs that are positive regulators of inflammation and secretion (Fig. S5a). Additionally, upregulated genes were enriched in neuron projection development and morphogenesis, while DEGs related to innate immune response were downregulated in Tg-Mlkl−/− astrocytes (Fig. S5b).

Furthermore, by employing 15 markers (Fig. S6b), we differentiated the neuron cluster into a dopaminergic neuron cluster, designated as Thpos, and the remaining cells formed the Thneg cluster (Fig. S6a). Notably, in Tg-Mlkl−/− mice, the neuronal cells showed a higher ratio of Thpos compared to Tg-Mlkl+/+ mice (Fig. S6c). Upon conducting GO analysis on these differential genes, it was observed that Thpos cells from Tg-Mlkl−/− mice demonstrated an upregulation in DEGs associated with the regulation of signaling receptor activity and GABAergic synapse (Fig. S6d). Conversely, genes showing a decrease were predominantly involved in pathways related to Parkinson’s disease, oxidative phosphorylation, ribosome, and mitochondrial functions (Fig. S6d).

The differentially expressed genes (DEGs) within the neuron, microglia, and astrocyte clusters were combined to identify shared up-regulated and down-regulated genes. A heatmap displaying the clustering analysis of these 180 common DEGs is presented in Fig. 6e. Among them, the mt-Co1, mt-Co2, mt-Co3, mt-Cytb, mt-Atp6, Ptgds, AC149090.1, Tsix, Gm47283, and Xist genes exhibited the highest expression changes (Fig. 6e). Notably, five out of the ten DEGs (mt-Co1, mt-Co2, mt-Co3, mt-Cytb, and mt-Atp6) are critical components of the mitochondrial electron transport chain (ETC) and mitochondrial respiratory chain [33, 34]. Mitochondrial dysfunction has long been implicated in the pathogenesis of PD [35], and several studies have identified mutations in mt-Co2, mt-Co2, mt-Co3, and mt-Atp6 associated with various clinical brain disorders [36]. Glutathione-independent prostaglandin D synthase (PTGDS), a prostaglandin involved in pain and sleep, was also identified as a unique blood-based signature capable of differentiating between idiopathic PD patients and controls [37]. A recent study demonstrated that PTGDS was upregulated in PD patients and could serve as an optimal biomarker for PD diagnosis [37]. In our case, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted, confirming a 2.8-fold decrease in PTGDS mRNA expression in Tg-Mlkl−/− compared to Tg-Mlkl+/+ mice (Fig. 7a). IHC also confirmed that MLKL deficiency increased PTGDS protein levels in the cortex, striatum, and SN regions of the A53T transgenic (Tg) mice (Fig. 7b and c).

Fig. 7