Microengineering of human vascularized brain tissue for modeling Lewy pathology in Parkinson's Disease and related Synucleinopathies

Introduction: α-synuclein is a major component of intraneuronal inclusions known as Lewy bodies, which are a hallmark of synucleinopathies [1,2] including Parkinson’s disease (PD). As the disease progresses, the monomeric protein undergoes pathological misfolding, aggregation, and propagation across susceptible brain regions, causing selective neurodegeneration that characterizes clinically distinct synucleinopathies [3,4]. While the prion-like behavior of α-synuclein fibrils provides insights into the spread of Lewy pathology, the disease etiology and fundamental mechanisms regulating its propagation still remain elusive [5]. However, research efforts to bridge this knowledge gap have been largely hampered by technical challenges in creating human brain models capable of accurately replicating potential pathophysiological factors, including cellular diversity and the complex anatomical features of the human brain (Fig. 1a).

Motivated by this, here we introduce a novel bioengineering strategy to produce a human vascularized brain-on-a-chip through the co-culture of neuronal cells with vasculature within a microengineered tissue culture platform. By incorporating midbrain neurons into a self-organized vascular bed, we developed a 3D multicellular mid-brain model that can accurately recapitulate the specialized functions and dynamic structures of the human brain, including the blood-brain-barrier and neuronal networks (Fig. 1b). Following the creation of this model, we applied PD-associated α-synuclein fibril seeds to establish an in vitro PD model capable of faithfully reproducing key pathological features such as the intraneuronal formation of α-synuclein-rich inclusions and neuroinflammation. This work represents a significant advance in our ability to model Lewy pathology of synucleinopathies and has the potential for broad impact in neurodegenerative disease research.

Materials and

Methods: Our mid-brain model is constructed in an elastomeric cell culture device made out of poly(dimethylsiloxane) (PDMS) that features a micro-chamber and multiple microchannels necessary for in vitro culture of neuronal cells and 3D microvascular networks. This microengineered device consists of a neuron culture micro-chamber which is open to an upper medium reservoir and flanked by microchannel bifurcations (Fig. 2a). Due to its open-top design, the hollow micro-chamber permits direct feeding of embedded neuronal cells (SH-SY5Y, ATCC) for their growth and differentiation achieved by modifying the protocol reported in a previous study [6] (Fig. 2b). Surrounding microchannels contain a vascularized hydrogel scaffold, which is maintained by vasculogenic culture media supplied through dedicated outermost side channels (Fig. 2c). Briefly, a fibrin gel precursor solution mixed with a suspension of primary human umbilical vascular endothelial cells (HUVECs, Lonza) and lung fibroblasts (NHLFs, Lonza) is injected into the microchannel bifurcations and enzymatically solidified to form a cell-laden fibrin gel scaffold. After gelation, endothelial cell growth media (EGM-2, Lonza) is introduced into two side channels to induce the vasculogenic formation of microvessels.

After establishing vascularized brain tissue in vitro with differentiated neuronal cells, our model was exposed to PD-associated α-synuclein fibril seeds to simulate PD conditions. On day 10 of cell culture within our model, α-synuclein fibrils at a concentration of 0.5 μM were introduced into both the neuron culture media and the vascular growth media. This mixture was then applied to the culture within our model and maintained for 48 hours before analysis.

Results, Conclusions, and Discussions: Building upon the creation of our brain tissue model, we investigated whether this model could be utilized to create a specialized in vitro brain disease model capable of recapitulating intraneuronal α-synuclein-rich inclusions, a key pathological feature of PD. To this end, we first exposed our model to pathological α-synuclein fibril seeds generated in the presence of cellular membrane components associated with PD (Fig. 3a). After a 48-hour treatment, we assessed the neuronal internalization of these PD-associated fibrils using Thioflavin T (ThT) fluorescence [7]. The resulting fluorescent images clearly demonstrated the presence of intraneuronal PD-associated fibrils compared to the control group, confirming our model’s ability to emulate the initial processes of Lewy pathology in PD (Fig. 3b).

We then examined the impact of internalized PD-associated α-synuclein fibrils on disease progression, focusing specifically on neuroinflammation which is a critical pathological feature of synucleinopathic brain tissue [8]. Given the established role of reactive oxygen species (ROS) as key mediators of neuroinflammation [9], we analyzed their production levels within our PD model. Consistent with previous studies, the treatment and subsequent internalization of PD-associated α-synuclein fibrils in our model led to a robust increase in ROS production compared with the control group (Fig. 3c,e). The toxicity of PD-associated α-synuclein fibrils was further evidenced by the noticeable activation of caspase-dependent apoptosis within our PD model, indicating the implication of fibrillar α-synuclein in the neuronal loss observed in PD patients (Fig. 3d,e). This observation aligns well with previous findings that link α-synuclein fibrilization to neuron death [10].

While this study demonstrates the feasibility and potential of our approach, it has yet to fully capture the organ-specific cellular heterogeneity of the cerebro-vasculature and its perivascular environment. Thus, further research efforts are necessary to engineer the vasculogenic co-culturing process involving brain endothelial cells, pericytes, and astrocytes, aiming to more accurately replicate the brain-specific blood vessel network and its perivascular region [11]. Despite this challenge, we believe that our work embodies significant conceptual and technical innovations as crucial initial steps towards our ultimate goal of effectively modeling complex physiological and pathological systems for biomedical and pharmaceutical applications in neurodegenerative diseases.

Acknowledgements (Optional): This work was supported by the Binghamton University Transdisciplinary Area of Excellence (TAE) Seed Grant award.