Parkinson's disease (PD) is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra and the accumulation of α-synuclein (α-syn) into Lewy bodies. Patients with PD exhibit motor and nonmotor symptoms (NMSs), including bradykinesia, resting tremor, rigidity, postural instability, hyposmia, sleep disorders, depression, anxiety and constipation (Warnecke et al., 2023).

The pathogenesis of PD involves a cascade of interconnected molecular pathways. Neuroinflammation serves as a pivotal driver, subsequently triggering nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. This activation leads to mature IL-1β secretion, amplification of inflammatory responses, and accelerated α-syn aggregation-collectively contributing to DA neuronal injury (Shah et al., 2024; Singh and Khatri, 2024). Concurrently,  autophagy dysfunction leads to the accumulation of autophagolysosomes and triggers apoptosis of DA neurons (Khot et al., 2023). Furthermore, BNIP3-PINK1-Parkin-mediated mitophagy dysfunction prevents the clearance of damaged mitochondria, enhancing the accumulation of dysfunctional mitochondria and thereby exacerbating neurodegeneration (Khot et al., 2023; Pinjala et al., 2024; Rajan et al., 2024). Damaged mitochondria activate caspase-1 via inflammatory signaling, releasing IL-1β and IL-18. These interconnected pathways ultimately drive neuroinflammation and PD pathogenesis (Ebadpour et al., 2024).

Disability in the late stages of PD is mostly related to NMSs and motor complications, such as dementia, psychosis, dysautonomia, and levodopa-induced dyskinesia, particularly chorea, dystonia, ballism, myoclonus, and akathisia (Kwon et al., 2022). As for levodopa-induced dyskinesia, less common than peak dose dyskinesia was diphasic dyskinesia, which presented as chorea and dystonia in the legs at both the beginning and end of the dosing period. Dystonia may be caused by dysfunction in an integrated circuit that involves the cortex, basal ganglia, thalamus, and cerebellum. Dystonia is also caused by dysregulation of striatal cholinergic signaling and abnormalities in striatal synaptic plasticity (Loens et al., 2021). Few effective PD animal models that simulate dystonia have been reported.

Cognitive impairment has a significant impact on the quality of life of PD patients. Approximately 20 % of people with PD have mild cognitive impairment (MCI) at the time of diagnosis, and PD patients have an increased risk of developing MCI and dementia as the disease progresses (Gavelin et al., 2022). In late-stage PD models, observing alterations in cognitive impairment might be helpful for determining the mechanism underlying the development of dementia.

The action of gut microbiota has been implicated in modulating neuroinflammation, which is associated with the onset and progression of PD. A previous study indicated that oral administration of rotenone (ROT) induced dysbiosis of the gut microbiota (Yan et al., 2022). Moreover, ROT increased the abundance of Verrucomicrobia and decreased the abundance of Bacteroidetes, which is consistent with the alterations observed in fecal samples from patients with PD (Zhao et al., 2021). The composition of the gut microbiome in late-stage PD models remains to be further studied.

The NLRP3 inflammasome was reported to trigger neurotoxic astrocytes in a mouse model of depression (Li et al., 2022). In rats with ROT-induced PD, activation of the caspase-1 pathway in astrocytes is involved in the regulation of α-syn-specific T cells (Wang et al., 2024). The immune response of α-syn-specific T-cells contributes to neuroinflammation in patients with PD. Some studies have indicated that α-syn activates T-cells through antigen presentation via major histocompatibility complex II (MHC-II) (Dressman and Elyaman, 2022). In PD mice, α-syn antigen presentation by astrocytes has not been reported frequently.

Brain parenchymal CD4+T cells may play a critical role in dopaminergic neurodegeneration. CD4+T cells are activated through the interaction of the T-cell receptor (TCR) with peptide complexes, consisting of MHC and antigen peptides. MHC-II-mediated antigen presentation of the α-syn peptide to CD4+T cells by microglia and astrocytes induces an immune response in PD pathology (Wang et al., 2024). Previous studies have indicated that caspase-1 cleaves full-length α-syn (α-syn FL) to generate a C-terminal 19-residue truncated α-syn (α-syn121). α-Syn121 forms amorphous aggregates that activate caspase-1, leading to the cleavage of α-syn FL and the generation of additional α-syn121. This process could promote the development of PD (Ma et al., 2018). In this study, the activation of α-syn-specific T-cells, which are associated with antigen presentation by astrocytes, was investigated in an ROT-based progressive PD model.

Dopamine analogs such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), and ROT are the most widely used compounds for establishing PD models. The MPTP model struggles to generate α-syn aggregation and exhibits significant interspecies variability in responses. The 6-OHDA model requires intracerebral injection to selectively damage the nigrostriatal pathway and induce acute motor deficits but cannot cross the blood-brain barrier and fails to form Lewy bodies. ROT is versatile across experimental cells and organisms, potently inducing α-syn aggregation. ROT models recapitulate non-motor PD features, such as dopaminergic neurodegeneration-associated intestinal wall abnormalities, and better mimic PD's progressive pathological trajectory (Ibarra-Gutiérrez et al., 2023).

Because of peripheral toxicity caused by systemic administration of ROT (Innos and Hickey, 2021), stereotactic injection of ROT into the right ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) replicate the behavioral changes and neuropathological features of hemiparkinsonism in rats (Chuproski et al., 2023). However, non-motor symptoms and dystonia have rarely been investigated in late-stage PD models. In the current investigation, longitudinal assessment of motor dysfunction and non-motor comorbidities was conducted at 4- and 36-week post-intervention intervals following stereotaxic ROT microinjection, utilizing a low-dose regimen with abbreviated exposure period to minimize systemic toxicity.