Synthetic development is a nascent field of research that uses the tools of synthetic biology to design genetic programs directing cellular patterning and morphogenesis in higher eukaryotic cells, such as mammalian cells. One specific example of such synthetic genetic programs was based on cell–cell contact-dependent signaling using synthetic Notch pathways and was shown to drive the formation of multilayered spheroids by modulating cell–cell adhesion via differential expression of cadherin family proteins in a mouse fibroblast cell line (L929). The design method for these genetic programs relied on trial and error, which limited the number of possible circuits and parameter ranges that could be explored. Here, we build a parameterized computational framework that, given a cell–cell communication network driving changes in cell adhesion and initial conditions as inputs, predicts developmental trajectories. We first built a general computational framework where contact-dependent cell–cell signaling networks and changes in cell–cell adhesion could be designed in a modular fashion. We then used a set of available in vitro results (that we call the “training set” in analogy to similar pipelines in the machine learning field) to parameterize the computational model with values for adhesion and signaling. We then show that this parameterized model can qualitatively predict experimental results from a “testing set” of available in vitro data that varied the genetic network in terms of adhesion combinations, initial number of cells, and even changes to the network architecture. Finally, this parameterized model is used to recommend novel network implementation for the formation of a four-layered structure that has not been reported previously. The framework that we develop here could function as a testing ground to identify the reachable space of morphologies that can be obtained by controlling contact-dependent cell–cell communications and adhesion with these molecular tools and in this cellular system. Additionally, we discuss how the model could be expanded to include other forms of communication or effectors for the computational design of the next generation of synthetic developmental trajectories.

Alpha-synuclein (aSyn)-rich aggregates propagate in neuronal networks and compromise cellular homeostasis leading to synucleinopathies such as Parkinson's disease. Aggregated aSyn spread follows a conserved spatio-temporal pattern that is not solely dependent on connectivity. Hence, the differential tropism of aSyn-rich aggregates to distinct brain regions, or their ability to amplify within those regions, must contribute to this process. To better understand what underlies aSyn-rich aggregates distribution within the brain, we generated primary neuronal cultures from various brain regions of wild-type mice and mice expressing a reduced level of aSyn, and exposed them to fibrillar aSyn. We then assessed exogenous fibrillar aSyn uptake, endogenous aSyn seeding, and endogenous aSyn physiological expression levels. Despite a similar uptake of exogenous fibrils by neuronal cells from distinct brain regions, the seeded aggregation of endogenous aSyn differed greatly from one neuronal population to another. The different susceptibility of neuronal populations was linked to their aSyn expression level. Our data establish that endogenous aSyn expression level plays a key role in fibrillar aSyn prion-like seeding, supporting that endogenous aSyn expression level participates in selective regional brain vulnerability.

Microfluidic devices for controlling neuronal connectivity in vitro are extremely useful tools for deciphering pathological and physiological processes occurring in neuronal networks. These devices allow the connection between different neuronal populations located into Methods in Cell Biology,

Prions are infectious agents that cause fatal neurodegenerative diseases. Current evidence indicates that they are essentially composed of an abnormally folded protein (PrP). These abnormal aggregated PrP species multiply in infected cells by recruiting and converting the host PrP protein into new PrP. How prions move from cell to cell and progressively spread across the infected tissue is of crucial importance and may provide experimental opportunity to delay the progression of the disease. In infected cells, different mechanisms have been identified, including release of infectious extracellular vesicles and intercellular transfer of PrP-containing organelles through tunneling nanotubes. These findings should allow manipulation of the intracellular trafficking events targeting PrP in these particular subcellular compartments to experimentally address the relative contribution of these mechanisms to in vivo prion pathogenesis. In addition, such information may prompt further experimental strategies to decipher the causal roles of protein misfolding and aggregation in other human neurodegenerative diseases.

β-Methylamino-L-alanine (BMAA) is implicated in neurodegeneration and neurotoxicity, particularly in ALS-Parkinson Dementia Complex. Neurotoxic properties of BMAA have been partly elucidated, while its transcellular spreading capacity has not been examined. Using reconstructed neuronal networks in microfluidic chips, separating neuronal cells into two subcompartments-(1) the proximal, containing first-order neuronal soma and dendrites, and (2) a distal compartment, containing either only axons originating from first-order neurons or second-order striatal neurons-creates a cortico-striatal network. Using this system, we investigated the toxicity and spreading of BMAA in murine primary neurons. We used a newly developed antibody to detect BMAA in cells. After treatment with 10 μM BMAA, the cyanotoxin was incorporated in first-degree neurons. We also observed a rapid trans-neuronal spread of BMAA to unexposed second-degree neurons in 48 h, followed by axonal degeneration, with limited somatic death. This in vitro study demonstrates BMAA axonal toxicity at sublethal concentrations and, for the first time, the transcellular spreading abilities of BMAA. This neuronal dying forward spread that could possibly be associated with progression of some neurodegenerative diseases especially amyotrophic lateral sclerosis.

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The trans-neuronal spread of protein aggregates in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. This hinders the development of therapeutic approaches aiming at preventing the spatial progression of intracellular inclusions in neural networks. To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic culture system which allows for the first time to reconstruct in vitro fully oriented and synaptically connected neural networks. This is achieved by filtering axonal growth with unidirectional "axon valves" microchannels. We exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size: Fibrils and Oligomers. Both aggregates were transferred to postsynaptic neurons through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates. By reconstructing chimeric networks, we found that neuron characteristics, such as the brain region from which they originate or aSyn expression levels, did not significantly impact aggregates transfer, and observed no trans-neuronal seeding where the presynaptic population did not express aSyn. Overall, we demonstrate that this novel platform uniquely allows the quantitative interrogation of original aspects of the trans-neuronal propagation of seeding pathogenic entities.

la SLA (ARSLA), the NeuroDis Fundation and the AFM. LC received for his PhD an ARC2 "Qualité de vie et Viellissement" Grant from the région Auvergne Rhône-Alpes.