Projection neurons in the anterolateral part of entorhinal cortex layer II (alEC LII) are the predominant cortical site for hyperphosphorylation of tau (p-tau) and formation of neurofibrillary tangles (NFTs) in brains of subjects with early-stage Alzheimer Disease (AD). A majority of alEC LII-neurons are unique among cortical excitatory neurons by expressing the protein reelin (Re+). In AD patients, and a rat model for AD overexpression mutated human APP, these Re+ excitatory projection-neurons are prone to accumulate intracellular amyloid-β (iAβ). Biochemical pathways that involve reelin-signaling regulate levels of p-tau, and iAβ has been shown to impair such reelin-signaling. We therefore used the rat model and set out to assess whether accumulation of iAβ in Re+ alEC LII projection neurons relates to the fact that these neurons express reelin. Here we show that in Re+ alEC LII-neurons, reelin and iAβ42 engage in a direct protein-protein interaction, and that microRNA-mediated lowering of reelin-levels in these neurons leads to a concomitant reduction of non-fibrillar iAβ ranging across three levels of aggregation. Our experiments are carried out several months before plaque pathology emerges in the rat model, and the reduction of iAβ occurs without any substantial associated changes in human APP-levels. We propose a model positioning reelin in a sequence of changes in functional pathways in Re+ alEC LII-neurons, explaining the region and neuron-specific initiation of AD pathology.
Sweden Graphical abstractFunctional neuromuscular junction in a microfluidic chip (a) Overview of microfluidic chip. Human iPS cell-derived motor neuron aggregates (spheroids indicated by black arrows) are seeded in the three lateral compartments of the chip, while human myotubes (white arrows) are seeded in the middle compartment. (b) Directed connectivity and retrograde virus tracing.Outgrowing axons (yellow arrow) from the motor neuron aggregate enter the directional axon tunnels (grey rectangles) and form connections with the myotubes (white arrow) within the opposite compartment. Addition of a designer monosynaptic pseudotyped ΔG-rabies virus to the myotube compartment, infects the myotubes (green) expressing an exogenous receptor (TVA) and rabies glycoprotein (G), subsequently making infectious viruses that are retrogradely transported through the motor neuron axons (green arrow) back to the neuronal cell bodies within the aggregate, validating neuromuscular junction functionality. AbstractCompartmentalized microfluidic culture systems provide new perspectives in in vitro disease modelling as they enable co-culture of different relevant cell types in interconnected but fluidically isolated microenvironments. Such systems are thus particularly interesting in the context of in vitro modelling of mechanistic aspects of neurodegenerative diseases such as amyotrophic lateral sclerosis, which progressively affect the function of neuromuscular junctions, as they enable the co-culture of motor neurons and muscle cells in separate, but interconnected compartments. In combination with cell reprogramming technologies for the generation of human (including patient-specific) motor neurons, microfluidic platforms can thus become important research tools in preclinical studies. In this study, we present the application of a microfluidic chip with a differentially-perturbable microenvironment as a platform for establishing functional neuromuscular junctions using human induced pluripotent stem cell derived motor neurons and human myotubes. As a novel approach, we demonstrate the functionality of the platform using a designer pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing.
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