PD is a neurodegenerative disorder characterized by an insidious onset and gradual progression. The early diagnosis of PD, particularly during its latent period, poses significant challenges but is crucial for effective therapeutic interventions. Traditional diagnostic methods rely heavily on clinical motor symptoms and physical examinations, yet these motor manifestations often lag behind the molecular and pathological changes of PD [16]. It is challenging and critical to realize an early diagnosis of PD before clinical symptoms, given the substantial impact of early intervention on slowing PD progression and improving patient management. Accumulated evidence has revealed great progress in the molecular diagnosis of PD. A series of biomarkers for PD have been proposed in recent years, including alpha-synuclein (aS), amyloid-beta (Aβ), neurofilament light chain (NfL), lysosomal biomarkers, and metabolomics [17–19]. Despite these developments, a universally recognized, efficient, and accurate diagnostic model for PD is still under exploration.
Dopaminergic neuron death is recognized as the most prominent hallmark for PD progression.
Pyroptosis, a form of programmed cell death associated with inflammatory responses, has been implicated in the death of dopaminergic neurons in PD. Pharmacological inhibition of pyroptosis-associated molecules such as NLRP3, caspase-1, and IL-18 alleviated symptoms of PD mice [20, 21]. More specifically, pyroptosis is associated with inflammatory factor release and glial cell activation in PD [22], which contributes to the inflammatory death of dopaminergic neurons. Numerous pyroptosis-related inflammatory factors, such as TNF-α, IL-10, IL-1β, IL-6, IL-2, and NLRP3, have been identified as potential biomarkers for PD, suggesting their potential as diagnostic hallmarks [23, 24]. However, diagnoses based on these factors lack accuracy due to deficiencies in sensitivity, specificity, and variability. Thus, the identification of pyroptosis-related genes suitable for PD diagnosis remains an open question.
Cells are the basic units of life activities. Cell heterogeneity, even among cells of the same genotype or clone, plays a pivotal role in both physiological and pathological processes [25–27]. A large number of studies have revealed that cell heterogeneity plays a pivotal role in PD microenvironment [28–30]. Single-cell sequencing technology makes it possible to reveal gene expression at the level of cell populations. In the previous researches, single-cell RNA sequencing (scRNA seq) was utilized to depict cell population composition and intercellular communication in PD microenvironment [31]. There are multiple cell types identified in midbrain specimens from PD patients, including astrocytes, dopaminergic neurons, endothelial cells, excitatory cells, inhibitory cells, microglial cells, oligodendrocyte precursor cells, oligodendrocytes, and pericytes [32]. Notably, PD patients exhibit distinct cell population composition compared to healthy individuals. For example, idiopathic PD patients have been found to possess an increased number of microglia and astrocytes but fewer oligodendrocytes in the midbrain [14]. In this study, 11 cell types were identified including oligodendrocytes, microglia, astrocytes, metencephalic like cells, oligodendrocyte precursor cells, inhibitory neurons, excitatory neurons, endocelial, pericytes, MHB like cells and ependymal. Among these subpopulations, the metencephalic like cells, inhibitory neurons, excitatory neurons, and MHB like cells were substantially reduced in PD group, as compared with the healthy group. These findings align with the pathological characteristics of PD, namely the loss of dopaminergic neurons in substantia nigra pars compacta. Additionally, the numbers of astrocytes and microglia were almost comparable between the PD and healthy groups, but their proportions were significantly improved in PD than in healthy individuals. It suggested astrocytes and microglia probably play a key role in the pathogenesis of PD. Furtherly, scRNA seq data derived from PD patients and healthy individuals was applied for diagnostic gene identification and immune correlation analysis. POLR2K and TIMM8K were screened and identified as the pyroptosis-associated diagnostic genes for PD. The diagnostic model based on POLR2K and TIMM8K genes showed superior diagnostic performance, although their diagnostic sensitivity and specificity require clinical validation in the future. On the other hand, there were significantly increased immunoinfiltration levels and inflammatory pathway number in PD patients than in healthy individuals. It conformed to the knowledge that inflammation plays an important role in the pathogenesis of neurodegeneration in PD. Alleviating neuroinflammation can reduce symptoms of early PD [33]. The inflammatory neurodegeneration in PD involves activation of microglia, upregulation of pro-inflammatory factors, and gut microbiota, etc. It accorded with the results that the proportions of microglia and astrocytes were significantly increased and inflammatory pathways including GRN, GAS, GALECTIN, WNT, and IL-16 were upregulated in PD group than in healthy group. Additionally, the diagnostic genes POLR2K and TIMM8K were found related to inflammatory signaling pathways including cytokine-receptor interaction, toll-like-receptor, and JAK-STAT pathway, etc. Although immunotherapies have been developed for PD treatment in recent years, few immune-related targets are verified to be clinically beneficial. In this study, the diagnostic genes POLR2K and TIMM8K corralated with many types of infiltrating immune cells, and immune-related signaling pathways. However, whether these two diagnostic genes can be considered as immune-related targets in PD needs further exploration.
This study cleverly established a pyroptosis-related diagnostic model for PD through the analyses of scRNA-seq data combined with transcriptome data, showing innovativeness and clinical translational value. There were several limitations of our study. First, the scRNA-seq data downloaded from the database, instead of experimental data, was applied to identify diagnostic genes, and analyze immunoinfiltration and intercellular communication. Therefore, further experiments will be needed to verify the results in the future. Second, in this study, the molecular phenotypes of activated microglia were not further analyzed. In the following studies, we will identify the gene expression related to microglia activation, so as to reveal molecules or pathways related to PD pathogenesis in the activation process of microglia.
In summary, based on integrated analysis of scRNA-seq and transcriptome data, this study demonstrated the differences in cell cluster composition and gene expression between PD and healthy group. The proportion of microglia and astrocytes, immunoinfiltration, inflammatory signaling pathways and intercellular interaction in PD patients was significantly increased than that in healthy individuals. The pyroptosis-related differentially expressed genes (PDEGs) were screened to determine hub genes using scRNA-seq and WGCNA analyses, and the diagnostic model and nomogram was constructed based on the genes POLR2K and TIMM8B. This diagnostic model showed promising diagnostic performance in verification. We constructed a novel pyroptosis-linked diagnostic model for PD, which improved the understanding of the role of PDEGs in PD and provided new insights into the diagnostic strategies for PD.