Assistant professor Binghamton University, SUNY Binghamton, New York, United States
Introduction: : Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by prion-like α-synuclein aggregation. As the disease progresses, these aggregates lead to neuronal dysfunction and loss, spreading across brain regions and ultimately causing both motor and non-motor symptoms including tremors, bradykinesia, rigidity and cognitive decline. Given that these symptoms are closely tied to neurodegeneration, most PD research has focused on delineating the neuronal basis of the disease. However, there is growing recognition of vascular abnormalities and their potential role in disease progression.
The cerebral vasculature is essential for maintaining neural function by delivering nutrients and oxygen, removing metabolic waste, regulating inflammation, and protecting the brain from harmful substances. In PD, vascular pathologies such as blood-brain barrier leakage, reduced capillary density, and vascular degeneration may disrupt these functions, exacerbating neuronal dysfunction as the disease progresses. Despite their pathological significance, the causes and progression of vascular abnormalities in PD remain poorly understood, largely due to the lack of disease models that can precisely capture the diverse vascular pathologies characteristic of PD alongside dopaminergic neurodegeneration.
To address this technical challenge, we proposed a vascularized midbrain-on-a-chip model that can accurately emulate the physiological functions and anatomical structure of the 3D vascular network interfaced with dopaminergic neurons of the human midbrain (Figs 1a and b). Using this model, we investigated how α-synuclein fibrils affect the temporal dynamics of vascular pathology and dopaminergic neuronal dysfunction in PD. This study provides new insights into the interplay between α-synuclein-rich inclusions and the diverse neuronal and vascular pathologies in PD.
Materials and
Methods: : Our mid-brain tissue culture platform was soft-lithographically fabricated using polydimethylsiloxane (PDMS) and 3D-printed master molds. The microfabricated device includes a central neuron-chamber, surrounding vascular-channel and two side channels positioned laterally to the vascular-channel (Fig 1b). These compartments are partially separated by a capillary pinning design, which leverages surface tension to guide cellular flows, thereby establishing spatially distinct tissue components.
The cell culture process involved human neuroblastoma cells (SH-SY5Ys) with a vasculogenic co-culture of primary human umbilical vein endothelial cells (HUVECs) and normal human lung fibroblasts (NHLFs) to create the vascular network. For vasculogenesis, a fibrin gel solution containing HUVECs and NHLFs was first introduced into the vascular-channel, followed by gelation process and maintained with EGM-2 (Endothelial Growth-2, Lonza) media. After one day, SH-SY5Ys were introduced into the neuron-chamber with their growth media to support initial adhesion and survival. On Day 2, the growth media was replaced with stage 1 differentiation media (DMEM supplemented with hiFBS (2.5%), glutamax-I (1%), penicillin/streptomycin (1%) and retinoic acid (10 μM)) to initiate neuronal differentiation. After five days, the media was changed to stage 2 differentiation media (Neurobasal-A media supplemented with brain-derived neurotrophic factor, potassium chloride (20 mM), B27 (1%), glutamax-I (1%) and penicillin/streptomycin (1%)) to further promote neurite process and was maintained for five days more.
On Day 12 post-seeding, α-synuclein fibril seeds were introduced into the neuron-chamber and the side channels (Fig 2b). The device was then incubated with the fibril seeds for 48 hours before further analysis.
Results, Conclusions, and Discussions:: The human midbrain features dopaminergic neurons integrated with a vasculature, which are essential for motor-related brain functions (Fig 1a). To recapitulate neurovascular environment, we developed a microengineered 3D mid-brain model (Fig 1b) by coculturing neuronal cells with a surrounding vascular network established through vasculogenesis (Fig 1c). Immunofluorescence staining for CD31 confirmed the self-organization of the vasculature (Fig 1c). MAP2 and GAP43 staining indicated the presence of functional dendrites and axons in our neuronal cells, respectively. Furthermore, tyrosine hydroxylase (TH) staining validated the dopaminergic identity of the neuronal cells (Fig 1d).
To model PD, our mid-brain device was treated with pathological α-synuclein preformed fibrils (α-syn PFFs), specifically generated using disease-associated cellular membrane components (Figs 2a and b). We first demonstrated the neuronal dysfunction in PD model, by increased levels of Ser129-phosphorylated α-synuclein and decreased expression of TH following treatment with PD-associated α-syn PFFs (Fig 2c). Moreover, PFF-treated neuronal cells exhibited elevated levels of reactive oxygen species (ROS) compared to controls, indicating higher oxidative stress (Fig 2d), a key driver of neuroinflammation which was further supported by nuclear translocation of NF-κB, observed in treated devices (Fig 2e).
Vascular integrity was progressively disrupted upon α-syn PFF-treatment (Fig 2b). VE-cadherin, showed reduced expression beginning at 24 hours and was substantially decreased by 72 hours, indicating endothelial barrier impairment (Fig 3a). Structural analysis revealed mild vessel thinning initially, followed by pronounced vascular disintegration and reduced vessel density at 72 hours (Fig. 3b). These observations were further supported by our quantification (Fig. 3c). Notably, we observed an increased formation of acellular, string-like vessels after 48 hours, resembling the “string vessels” reported in PD patient brain tissues (Fig 3b). CD31 staining also indicated significant vascular regression due to α-syn PFF treatment (Data not shown).
Our PD model successfully recapitulates key pathological features, particularly dopaminergic neuronal dysfunction. A notable outcome of this study is the model’s enhanced ability to reproduce progressive vascular disruption and string vessel formation in vitro. The string vessels indicate the structural collapse of the vasculature and thus compromised vascular function, suggesting that α-synuclein pathology can directly impair vascular integrity and potentially exacerbate neurodegeneration.
Acknowledgements and/or References (Optional):: This work was supported by the National Institutes of Health (NIH) (grant no. 1R21NS139178-01); Binghamton University (grant nos. TAE 1182867, ADLG258).
