The Role And Mechanism Of α-Synuclein Fibril-Mediated Cerebral Microvascular Damage In Parkinson’s Disease-Related Cognitive Impairment

[Background]Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease,and is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and pathological aggregation ofα-synuclein（αSyn）to form Lewy bodies.In addition to motor symptoms such as resting tremor,rigidity,and bradykinesia,cognitive impairment is one of the most prominent non-motor symptoms of Parkinson’s disease,with high morbidity and mortality.In Parkinson’s disease,cognitive impairment presents as a complex pathogenesis,and the clinical diagnosis lags behind,as does treatment.Studies have shown that cognitive decline caused by pathologicalαSyn deposition in cortical regions is closely related to vascular risk factors and vascular degeneration.However,how the brain microvasculature regulatesαSyn pathology and neuronal degeneration is unclear,and the underlying mechanisms are rarely investigated.[Objective]This study aimed to investigate the role of brain capillaries in Parkinson’s disease cognitive impairment and to elucidate the roles and mechanisms of brain microvascular endothelial cells in regulating cognition-relatedαSyn pathology and neuronal degeneration.[Methods]In this study,a Parkinson’s disease model was constructed by injectingαSyn preformed fibrils（αSyn PFF）into the cerebral cortex and striatum.After six months of receivingαSyn PFF injection,mice were tested for motor function using the pole test,wire hang test,and/or rotarod test,and for cognitive function using the Morris water maze and/or Y maze.（1）In terms of pathology,neuronal changes,cerebral microvascular damage and its correlation with the pathological deposition ofαSyn in Parkinson’s disease mice with cognitive impairment were observed using Evans blue perfusion and immunofluorescence staining.（2）The intracellular domain（ICD）of low-density lipoprotein receptor-related protein 1（LRP1）in cortical capillaries and isolated mouse brain microvascular endothelial cells was detected using its C-terminal antibody.The human brain microvascular endothelial cell line HBEC-5i was treated with biotin-conjugatedαSyn PFF(αSyn PFF^GFP)to explore endothelialαSyn fibrillation and accumulation of LRP1-ICD.LRP1-ICD has a binding site for poly（adenosine 5’-diphosphate ribose）polymerase 1（PARP1）,andαSyn PFF-induced PARP1 activation was observed by overexpressing LRP1-ICD in brain microvascular endothelial cells.The PARP1 pathway was inhibited by ABT-888.The effect of LRP1-ICD/PARP1 on the structural and functional changes of brain microvascular endothelial cells was verified by the following experiments:Western blotting to detect endothelial tight junction protein expression,immunocytochemistry to detect endothelial phosphorylatedαSyn deposition,transepithelial electrical resistance and permeability assay to detect endothelial barrier function,angiogenesis assay to detect endothelial tube formation ability,migration assay to detect endothelial migration ability,as well as mitochondrial membrane potential and the ratio of nicotinamide adenine dinucleotide and its reduced coenzyme I to detect endothelial metabolism.To elucidate the effect of endothelial LRP1-ICD/PARP1 on neuronal survival,brain microvascular endothelial cells overexpressing LRP1-ICD were co-cultured with neurons.Levels of tyrosine hydroxylase（TH）and cleaved Caspase3 in neurons were detected using western blotting.Levels of pro-brain-derived neurotrophic factor（pro BDNF）released from endothelial cells was measured using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.（3）Prior toαSyn PFF injection,LRP1-ICD was overexpressed in mouse brain vascular endothelium using adeno-associated virus carrying an endothelial-specific promoter.Mice were continuously and intermittently fed the PARP1inhibitor ABT-888.The effects of vascular endothelial LRP1-ICD/PARP1 onαSyn PFF-induced motor and cognitive impairment as well as vascular damage,αSyn aggregation and propagation,and neuronal death in the cerebral cortex and hippocampus were observed.（4）To elucidate the role of PARP1 inαSyn PFF-induced vascular endothelial injury,PARP1endothelial-specific knockout mice（PARP1 CKO）were constructed.αSyn PFF was injected into wild type（WT）and PARP1 CKO mouse brains.The effects of vascular endothelial knockout of PARP1 onαSyn PFF-induced motor and cognitive decline as well as phosphorylatedαSyn deposition and PAR synthesis were observed using behavioral,pathological,and biochemical experiments.RNA sequencing was used to reveal differentially expressed genes and functional enrichment in brain microvascular endothelial cells isolated from WT and PARP1 CKO mice.[Results]（1）Six months after injection ofαSyn PFF,mice showed significant impairments in motor and spatial learning and memory.αSyn PFF led to the loss of TH+neurons in the substantia nigra pars compacta and microvascular damage to the cerebral cortex and striatum.Damaged brain capillaries exhibited morphological abnormalities,such as shorter lengths and fewer junctions.In mice treated withαSyn PFF,there was a negative correlation between vessel density and phosphorylatedαSyn deposition in the cerebral cortex.（2）αSyn PFF led to high expression of the C-terminal chain of LRP1 in cortical capillaries.In isolated brain microvascular endothelial cells isolated fromαSyn PFF-treated mice,we observed elevated levels of LRP1-ICD.LRP1-ICD aggregated and colocalized with Rab11 in brain microvascular endothelial cells treated withαSyn PFF.LRP1-ICD acceleratedαSyn fibrillation and increased the accumulation of Rab11+recycling vesicles and intracellularαSyn PFF uptake.In the presence ofαSyn PFF,overexpression of LRP1-ICD exacerbated PARP1activation,while the PARP1 inhibitor ABT-888 inhibited PAR production.We validated the effects of LRP1-ICD/PARP1 in vitro on brain microvascular endothelial structure and function.LRP1-ICD exacerbatedαSyn PFF-induced reduction in endothelial tight junction protein expression,and impaired endothelial barrier function,angiogenesis,migratory motility,and energy metabolism.Furthermore,endothelial LRP1-ICD increased the release of pro BDNF and induced early neuronal apoptosis in an endothelial-neuron co-culture system.（3）Overexpression of endothelial LRP1-ICD promoted pathologicalαSyn propagation in cerebral cortex,hippocampus,hypothalamus,substantia nigra and striatum.Endothelial LRP1-ICD exacerbatedαSyn PFF-induced vascular damage,pathologicalαSyn aggregation,and neuronal death in the cerebral cortex and hippocampus,resulting in severe motor and cognitive impairment.However,feeding mice with ABT-888 effectively alleviated vascular-related neurodegeneration by inhibiting PARP1 activation.（4）Compared with WT mice,endothelial-specific knockout of PARP1 partially preventedαSyn PFF-induced motor deficits and long-term memory impairment,and prevented working memory impairment.Pathologically,PARP1depletion significantly alleviated PAR upregulation in brain microvascular endothelial cells and reduced phosphorylatedαSyn deposition.The protective mechanism was further investigated using transcriptome sequencing.PARP1 was found to be involved in endothelial proliferation through FGF10 and inflammation through CSF1R to modulate LRP1-ICD/αSyn PFF-induced endothelial injury.[Conclusion]This study confirmed that LRP1-ICD in brain microvascular endothelial cells promotedαSyn PFF-induced endothelial damage,αSyn pathological aggregation and propagation,and neurodegeneration by activating PARP1,and accelerated Parkinson’s disease progression.This study demonstrates a novel vascular mechanism of Parkinson’s disease-related cognitive impairment,reveals the role of endothelial LRP1-ICD/PARP1 inαSyn pathology and neuronal damage,and provides evidence for vascular protection strategies in Parkinson’s disease treatment.