Parkinson’s disease (PD), the second most prevalent neurodegenerative disorder after Alzheimer’s disease (AD), affects approximately 7 million individuals worldwide (Chi et al., 2019). It is characterized by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra, leading to clinical manifestations such as bradykinesia, myotonia, resting tremor, and postural instability (Samii et al., 2004; Schneider et al., 2017). The exact etiology of PD remains unclear, likely resulting from a complex interplay of genetic and environmental factors (Zhang et al., 2021). Due to its intricate pathogenesis, treatment primarily focuses on symptomatic management, such as dopamine replacement therapy (Antony et al., 2013). However, disabling axial symptoms, particularly postural instability and freezing of gait (FOG), often remain resistant to both medication and surgical interventions (Boonstra et al., 2008; Nieuwboer and Giladi, 2013). Recently, molecular biomarkers have emerged as promising tools for PD diagnosis (Chen-Plotkin et al., 2018). Consequently, there is a pressing need to explore novel methods for early detection and more precise treatments, especially for patients with refractory FOG.

Lysosomes, traditionally known as organelles responsible for cellular digestion, degradation, and recycling of metabolic waste, have recently been shown to play critical roles in cellular metabolism, proliferation, differentiation, apoptosis, immunity, nutrient sensing, protein regulation, and metabolic signaling (Trivedi et al., 2020). The involvement of lysosomal function in PD has been confirmed through both functional and genetic studies (Senkevich and Gan-Or, 2020). PD is characterized by the degeneration of DA neurons and the accumulation of Lewy bodies, primarily composed of misfolded and aggregated α-synuclein proteins (Fares et al., 2021). Lysosomes are the primary site for the degradation of aggregated α-synuclein (Lee et al., 2004; Vogiatzi et al., 2008; Mak et al., 2010). Mutations in lysosomal genes contribute to elevated levels of α-synuclein or its increased tendency to aggregate, thereby enhancing the genetic risk of PD (Ibáñez et al., 2004; Pihlstrøm et al., 2018). Cathepsin B (catB), a proteolytic enzyme of the cysteine cathepsin family with both endopeptidase and exopeptidase activities, is typically localized in the lysosomal cavity (Stoka et al., 2016). Knockdown of the PD risk gene TMEM175 disrupts lysosomal pH and impairs catB activity (Jinn et al., 2017; Hu et al., 2022). Additionally, mutations in LRRK2, a major cause of familial PD, have been shown to inhibit catB expression or activity within lysosomes (Henry et al., 2015; Yadavalli and Ferguson, 2023). Lysosomal dysfunction caused by mutations in these genes can exacerbate the accumulation of α-synuclein in the brain, potentially accelerating the onset of PD (Blumenreich et al., 2020). Although several studies have linked PD onset and progression to lysosome-related genes (LRGs), the precise genetic mechanisms remain poorly understood. Investigating LRGs holds promise for identifying PD biomarkers, aiding in the development of preventive strategies, early diagnosis, and more effective management, while also enhancing the understanding of underlying mechanisms and reducing risks.

In recent years, high-throughput technologies have made significant advances in PD biomarker research, yet critical limitations remain. Current studies primarily follow three directions: First, numerous blood or cerebrospinal fluid transcriptomic studies have screened candidate gene profiles through differential expression analysis. However, over 70% of identified markers lack independent validation (Chen-Plotkin et al., 2018), limiting their clinical translational value. Second, explorations of biomarkers related to specific pathways such as oxidative stress (Zhu et al., 2025) and copper metabolism (Lin et al., 2024) have revealed some pathological mechanisms but have failed to systematically integrate interactions between multiple pathways. Third, while immune-related gene signatures can distinguish PD from healthy controls, they often lack specificity for early diagnosis and disease subtypes (Baird et al., 2019). More notably, although genome-wide association studies have consistently confirmed significant associations between lysosome-related genes (LRGs, such as TMEM175, LRRK2, and GBA) and PD risk (Nalls et al., 2019), functional studies targeting the complete LRG set remain scarce, and their predictive value for PD diagnosis and their links to core motor symptoms have yet to be clarified. Addressing this critical gap, this study, for the first time, systematically identifies lysosome-related biomarkers highly associated with PD phenotypes by integrating weighted gene co-expression network analysis (WGCNA), machine learning algorithms, dual-cohort cross-validation, and qPCR confirmation. It further constructs miRNA-mRNA regulatory networks and performs multidimensional correlation analyses linking the identified biomarkers with FOG-related genes and immune cell infiltration. This work establishes an integrated analytical framework of “lysosomal dysfunction–immune dysregulation–motor symptoms,” offering a novel strategy for early PD diagnosis and precision treatment that combines mechanistic depth with clinical applicability.

PD is a complex neurodegenerative disorder in which the pathogenesis involves an interplay between genetic and environmental factors, leading to dysregulation of essential biological processes such as lysosomal dysfunction. This dysfunction impairs α-synuclein degradation and accelerates DA neuron death (Kalia and Lang, 2015; Van Veen et al., 2020). Research has revealed significant overlap between LRGs and PD pathogenic genes, highlighting the urgent need for population-based LRG screening and novel PD biomarkers (Robak et al., 2017). This study integrates bioinformatics and experimental validation to identify two lysosomal function-related biomarkers in PD and elucidates their clinical significance and regulatory networks. Specifically, FGD4 and MAN2B1 consistently demonstrated robust diagnostic performance across dual cohorts (AUC >0.8), with their elevated expression in peripheral blood further confirmed by qPCR. More importantly, these two biomarkers are associated with FOG, a core motor symptom of PD. FGD4 is co-localized with LRRK2 on chromosome 12 and shows a significant positive correlation, while MAN2B1 cooperatively regulates the dopamine metabolism pathway with COMT. Concurrently, immune infiltration analysis revealed their differential regulatory roles in the immune microenvironment: MAN2B1 is strongly associated with monocytes, whereas FGD4 shows a strong correlation with activated memory effector CD4⁺ T cells, thereby establishing a “lysosome-immune” interaction axis. Furthermore, this study predicted compounds such as Tetrachlorodibenzodioxin that can target these two biomarkers. Collectively, these multidimensional findings support lysosomal dysfunction as a novel target for early diagnosis and progression prediction in PD.

Focusing on the close relationship between lysosomal function and PD, this study highlights two critical biomarkers: FGD4 and MAN2B1. FGD4 (FYVE, RhoGEF, and PH domain-containing 4) is a protein that regulates the cytoskeleton and cell shape (Obaishi et al., 1998; Delague et al., 2007). Aberrant expression of FGD4 disrupts the polarity, proliferation, and morphology of myelin sheaths, impairing nerve conduction (Obaishi et al., 1998; Ono et al., 2000). Moreover, FGD4 is implicated in lysosomal encapsulation, endocytosis, and degradation processes (Delague et al., 2007). MAN2B1, another key biomarker, is closely linked to lysosomal accumulation responses (Wood et al., 2013), with proteomic analysis of cerebrospinal fluid identifying it as a potential PD biomarker (Karayel et al., 2022). Both FGD4 and MAN2B1 contribute to lysosomal membrane stability, endocytosis, and degradation. Overexpression of these genes may result in excessive protease release, autophagy system instability, or inflammatory response activation (Taylor et al., 2018). Further drug prediction analysis in this study indicates that Tetrachlorodibenzodioxin and Bisphenol A could intervene with FGD4 and MAN2B1, providing novel therapeutic references for PD treatment. Bisphenol A has been shown to regulate autophagy through pathways such as AKT-mTOR (Zhang et al., 2023), while Tetrachlorodibenzodioxin, an immunosuppressive compound (Dooley and Holsapple, 1988), can reduce inflammatory cytokines like IFN-γ (Ciftci and Ozdemir, 2011). Tetrachlorodibenzodioxin may also inhibit the autophagy system’s phagocytosis-degradation function (Podechard et al., 2009). Therefore, these two predicted drugs are essential for targeting the immune-autophagy pathways associated with FGD4 and MAN2B1 in patients with PD. In conclusion, this study identified two LRGs with differential expression in patients with PD, predicting alterations in various lysosomal autophagy functions. These findings provide new insights into the pathogenic mechanisms related to lysosomal damage in PD, opening avenues for further exploration.

miRNAs have become a central focus in biological research, with several miRNAs linked to DEGs in various diseases. This study identified MIR342, MIR339, and MIR933 for the first time as miRNAs significantly associated with key biomarkers of PD-LRGs. MIR342 is known to be involved in telomerase activity and has a strong association with neurodegenerative diseases (Likonen et al., 2022). MIR339 has been recognized as a biomarker in atypical PD syndrome (Bougea, 2022), while MIR933 has been implicated in AD, where it interferes with nerve growth factor translation, leading to neuroinflammation (Dias et al., 2018). Notably, MIR342-3p promotes autophagy by inhibiting MAP1LC3B (Zhang et al., 2020), MIR339-5p significantly affects the phagocytic and degradative functions of immune cells (Hakimzadeh et al., 2017), and the upregulation of MIR933 has been shown to induce autophagy dysregulation (Mohammadi et al., 2018). In summary, this is the first study to identify the critical roles of multiple miRNAs in PD, providing new avenues for exploring the genetic pathogenic factors of the disease.

Immune cell infiltration analysis, combined with the overlay analysis of DE-IRGs for each biomarker, revealed that MAN2B1 was most strongly associated with monocyte infiltration. A strong positive correlation was found between MAN2B1 and RHOG. Upregulation of MAN2B1 causes functional divergence in lysosomal α-mannosidase, affecting glycoconjugate modifications, which may facilitate monocyte infiltration by binding to the cell membrane (Urbanelli et al., 2011). RHOG, in turn, integrates multiple receptor signals during the phagocytic process of monocytes/macrophages (Tzircotis et al., 2011). Another analysis revealed that memory effector CD4 T cell infiltration was most strongly associated with FGD4, showing a strong positive correlation between FGD4 and TIMP1. It is well established that α-synuclein accumulation in patients with PD acts as a primary antigen for memory effector CD4 T cells (Christiansen et al., 2016), which are linked to TIMP1 expression (Ding et al., 2022). The dopamine receptors on these cells are closely associated with FOG (Kustrimovic et al., 2016; Diener et al., 2023). Based on these findings, the activation of memory effector CD4 T cells in PD may lead to the upregulation of FGD4, thereby enhancing macropinocytosis in lysosomal encapsulation, endocytosis, and degradation (Charpentier et al., 2020). This hypothesis establishes a link between the adaptive immune response and the regulation of lysosomal function in PD pathogenesis. However, the causal relationships and underlying molecular mechanisms need further validation through functional experiments.

This study found that FGD4 expression is significantly positively correlated with LRRK2. LRRK2 is a key gene regulating lysosomal phagocytosis and degradation, and its mutations can inhibit cathepsin activity (Yadavalli and Ferguson, 2023). We speculate that FGD4, as a cytoskeletal regulator, may functionally synergize with LRRK2 in the lysosomal protein clearance pathway or be co-transcriptionally regulated by influencing lysosomal membrane stability or endocytic efficiency (Abe et al., 2024; Hughes et al., 2025). Additionally, both FGD4 and MAN2B1 in this study showed positive correlations with COMT, which is involved in dopamine metabolism and whose genotype is associated with the severity of motor symptoms in PD (Williams-Gray et al., 2009). This correlation suggests that lysosomal dysfunction may exacerbate metabolic stress within dopaminergic neurons. We hypothesize that abnormal release of lysosomal proteases (such as cathepsin B) may affect the activity of dopamine-metabolizing enzymes, thereby synergizing with COMT to aggravate motor symptoms (Williams-Gray et al., 2009). Interestingly, FGD4 was negatively correlated with CYP2R1, a gene involved in vitamin D metabolism, providing a potential molecular link to the clinical observation of vitamin D deficiency and increased fall risk in PD patients—namely, that lysosomal stress may interfere with the expression of genes related to vitamin D metabolism (Wang et al., 2010; Ding et al., 2013; Mahanty et al., 2019). In summary, this study reveals associations between FGD4/MAN2B1 and FOG-related genes, suggesting that lysosomal dysfunction may engage in molecular crosstalk with PD motor symptoms, particularly FOG. These associations offer new clues for understanding the lysosome-neuroinflammation-motor regulation network in PD. It is important to note that these findings are derived from bioinformatics correlation analyses and have not yet been functionally validated for causality. Therefore, future studies should employ functional experiments—such as knocking down or overexpressing FGD4/MAN2B1 in cellular or animal models and assessing their effects on the expression of LRRK2 and COMT, as well as on lysosomal function and motor behavior—to verify whether these correlations possess a causal and mechanistic basis. In summary, this study identified FGD4 and MAN2B1 as biomarkers related to lysosomal dysfunction in PD, revealing their roles in disrupting transcriptional networks and contributing to the progression of FOG through neuroinflammatory cascades. These findings offer novel insights into the lysosomal-autophagy-immune axis in PD. However, several limitations must be acknowledged: First, due to sample size constraints, the gene modules initially identified through WGCNA may lack stability. Additionally, conducting WGCNA, differential expression analysis, and subsequent internal validation within the same discovery cohort raises the risk of circular analysis. Similarly, the relatively small sample size used for qPCR validation may limit the statistical validity and generalizability of our findings. So the results should be interpreted with caution. Future studies will include larger independent cohorts to validate these modules’ reliability. This study used blood samples for their accessibility, facilitating clinical translation. However, gene expression changes in blood may reflect systemic alterations rather than the specific intracranial pathological processes of PD. Caution is needed when directly linking these findings to central lysosomal dysfunction in PD. That said, considering the peripheral-central interplay characteristic of PD pathology—such as peripheral immune abnormalities, like increased monocyte infiltration, potentially crossing the blood-brain barrier and exacerbating central microglial activation and neuroinflammation, and gut inflammation transmitting peripheral signals to the central nervous system through the gut-brain axis, affecting lysosomal-autophagy function (Morris et al., 2024)—the observed overexpression of FGD4 and MAN2B1 in blood may indirectly contribute to central inflammatory pathology in PD by modulating peripheral monocyte infiltration. Alternatively, they may serve as surrogate markers reflecting the systemic pathological state of PD. To further clarify this, future studies will involve correlating FGD4 and MAN2B1 expression in both brain tissue and peripheral blood using PD animal models to determine how well peripheral biomarkers reflect central pathology. While this study has computationally identified and experimentally validated FGD4 and MAN2B1 as lysosome-related biomarkers for PD, and preliminary PCR experiments have investigated their expression trends in clinical samples, the functional mechanisms remain to be explored. Further cellular functional assays, such as gene overexpression/knockdown using techniques like siRNA or CRISPR, will be conducted to assess their specific roles in PD pathogenesis, including their impact on core PD pathological markers such as lysosomal pH. Additionally, co-immunoprecipitation (Co-IP) will be performed to determine whether direct PPIs exist between FGD4, LRRK2, and COMT. PD animal models will also be developed to evaluate FGD4 and MAN2B1 expression, with an emphasis on FOG-like behaviors, to assess whether FGD4 and MAN2B1 influence PD progression and FOG phenotypes through regulation of lysosomal function or FOG-related genes like LRRK2 and COMT. Moreover, leveraging the validated gene associations, the findings of this study may have potential clinical applications in diagnostic panels. Large-scale prospective cohort studies are needed to validate these diagnostic models, which could incorporate multi-gene blood test panels featuring FGD4, MAN2B1, and other novel markers. Depending on clinical scenarios, methods like qPCR or digital PCR may be applied for non-invasive early screening.

In conclusion, FGD4 and MAN2B1 function as lysosomal biomarkers associated with PD and show significant correlations with genes linked to PD-related freezing of gait. This study offers new perspectives for the diagnosis and understanding of PD.