Validation of Functional Connectivity of Engineered Neuromuscular Junction With Recombinant Monosynaptic Pseudotyped ΔG-Rabies Virus Tracing

Abstract

Current preclinical models of neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS), can significantly benefit from in vitro neuroengineering approaches that enable the selective study and manipulation of neurons, networks, and functional units of interest. Custom-designed compartmentalized microfluidic culture systems enable the co-culture of different relevant cell types in interconnected but fluidically isolated microenvironments. Such systems can thus be applied for ALS disease modeling, as they enable the recapitulation and study of neuromuscular junctions (NMJ) through co-culturing of motor neurons and muscle cells in separate, but interconnected compartments. These in vitro systems are particularly relevant for investigations of mechanistic aspects of the ALS pathological cascade in engineered NMJ, as progressive loss of NMJ functionality may constitute one of the hallmarks of disease related pathology at early onset, in line with the dying back hypothesis. In such models, ability to test whether motor neuron degeneration in ALS starts at the nerve terminal or at the NMJ and retrogradely progresses to the motor neuron cell body largely relies on robust methods for verification of engineered NMJ functionality. In this study, we demonstrate the functionality of engineered NMJs within a microfluidic chip with a differentially perturbable microenvironment using a designer pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing.

Microdissection and culturing of adult lateral entorhinal cortex layer II neurons from APP/PS1 Alzheimer model mice

“…Aside from the relevance of Re+ lLEC-LII neurons in the onset of AD, it is likely that culturing of adult neurons offers a more relevant model for the disease, because it removes potential confounds owing to developmental factors operating in embryonic or early postnatal neurons. The ability to extract and move Re+ lLEC-LII neurons into an in vitro setting furthermore enables direct monitoring of their network properties and offers ease of access with respect to chemogenetic (Bauer et al, 2022; Westhaus et al, 2020), molecular-, (Valderhaug et al, 2021) and pharmacological tools (Han et al, 2017). Our approach may thus represent a valuable tool for studying very early proteinopathy and offers neuron- and network specific modelling of initiating AD pathology in vitro .…”

Human fetal membrane-mesenchymal stromal cells generate functional spinal motor neurons in vitro

“…To confirm that the contractile activity observed was effectively because of the presence of a functional NMJ, we treated the co-culture with the α-BTX, which binds AChR with high affinity blocking their post-synaptic activity causing muscle paralysis ( Bauer et al., 2022 ). Data showed that the contractions of myotubes co-cultured with hFM-MSC-derived MNs were completely abolished after the incubation with α-BTX ( Video S3 ).…”

“…The α-BTX assay was performed as previously reported ( Saini et al., 2021 ; Bauer et al., 2022 ). Briefly, a video of contraction activity was recorded for 18s, then it was stopped and α-BTX 1:300 (Thermo Fisher Scientific) was added to the cell culture and incubated for 10 min at 37°C.…”

Evolving alterations of structural organization and functional connectivity in feedforward neural networks after induced P301L tau mutation

“…Such networks develop with progressively increasing structural and functional complexity over time, recapitulating fundamental aspects of neural network behavior as seen in the brain (Collingridge et al, 2010;Valderhaug et al, 2021;van de Wijdeven et al, 2019;Winter-Hjelm N, 2023). Engineered in vitro models thus enable longitudinal studies of dynamic network behavior and allow for selective perturbation and monitoring of network responses at the micro-and mesoscale level (Bauer et al, 2022;Bruno et al, 2020;Fiskum et al, 2021;Gribaudo et al, 2019;Nonaka et al, 2011;Valderhaug et al, 2021;Valderhaug et al, 2024;Weir et al, 2023).…”