**Background:** Parkinson's disease (PD) is characterized by dopaminergic neuron loss and α-synuclein (α-syn) aggregation. Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder with elevated cholestanol, often presents with parkinsonism. The study investigates whether cholestanol contributes to sporadic PD pathogenesis by activating asparagine endopeptidase (AEP), which cleaves α-syn at N103, promoting aggregation.
**Methods:** Serum cholestanol was measured by LC-MS in 20 sporadic PD patients (Hoehn & Yahr I-II) and 19 healthy controls. In vitro, SH-SY5Y cells, α-syn-HEK293 cells, and primary cortical neurons were treated with cholestanol (10 μM) ± α-syn preformed fibrils (PFFs) ± AEP inhibitor CP11 (2 μM) or shRNA knockdown of LGMN (AEP). Cell viability (CCK8), ROS (flow cytometry), mitochondrial complex activities (kits), AEP activity (fluorogenic substrate), and protein levels (Western blot) were assessed. In vivo, 3-month-old WT and AEP knockout (Lgmn-/-) mice were fed cholestanol (2/1000 wt/wt) or chow for 1 month, then intrastriatally injected with α-syn PFFs (10 μg) or PBS. Six months post-injection, behavioral tests (rotarod, pole, beam-walking) were performed. Brains were analyzed by immunohistochemistry (pS129, TH), Western blot, and Nissl staining. A separate WT group received CP11 (10 mg/kg/d i.p.) or vehicle starting 1 month before PFF injection.
**Key Results:** Serum cholestanol was significantly higher in PD patients vs controls (exact values not clearly reported in abstract; Supplemental Figure 2G). In vitro, cholestanol (10 μM) decreased SH-SY5Y cell viability in a concentration-dependent manner (Figure 1A), increased ROS (Figure 1B), and reduced mitochondrial complex II, III, IV, and V activities (Supplemental Figure 3). Cholestanol upregulated C/EBPβ, p-C/EBPβ, AEP, α-syn N103 fragment, and p-α-syn (Figure 1E). shRNA knockdown of LGMN or CP11 (2 μM) attenuated these effects (Figure 1F,G). In α-syn-HEK293 cells, cholestanol increased pS129-positive aggregates (Figure 2A,B) and insoluble p-α-syn (Figure 2C). Primary neurons showed increased pS129 and AEP activity (Figure 2D,E). AEP KO (Lgmn-/-) or N103A mutation blocked cholestanol-induced aggregation (Figure 2F-H; Supplemental Figure 5). In vivo, cholestanol-fed WT mice injected with α-syn PFFs showed worse motor performance (rotarod: ~50% shorter latency; pole test: ~2-fold longer time; beam-walking: ~2-fold longer time; Figure 4A-C) and more severe p-α-syn spreading in cortex, substantia nigra, and hippocampus (Figure 4D,E). Cholestanol reduced TH+ neurons in SNpc by ~40% and TH+ fibers in striatum by ~50% (Figure 4G-J). AEP KO abolished these effects (Figure 4A-J). CP11 (10 mg/kg/d) similarly attenuated motor deficits, p-α-syn pathology, and TH loss (Figure 5A-J).
**Clinical Implications:** Elevated cholestanol in sporadic PD patients suggests a novel risk factor. Cholestanol activates the C/EBPβ/AEP pathway, driving α-syn aggregation and spreading. AEP inhibition (genetic or pharmacological) blocks these effects, positioning AEP as a therapeutic target. CP11, a selective AEP inhibitor, shows promise for PD, especially in patients with high cholestanol. Limitations include small human sample size (n=20 PD, n=19 controls) and need for validation in larger cohorts. Future studies should explore AEP activity as a biomarker and test CP11 in clinical trials.