Calnexin controls TrkB cell surface transport and ER-phagy in mouse cerebral cortex development.

Transactivation of Tropomyosin receptor kinase B (TrkB) by EGF leads to cell surface transport of TrkB, promoting its signaling responsiveness to brain-derived neurotrophic factor (BDNF), a critical process for proper cortical plate development. However, the mechanisms that regulate the transport of TrkB to the cell surface are not fully understood. Here, we identified Calnexin as a regulator for targeting TrkB either to the cell surface or toward autophagosomal processing. Calnexin-deficient mouse embryos show impaired cortical plate formation and elevated levels of transactivated TrkB. In Calnexin-depleted mouse neuronal precursor cells, we detected an impaired cell surface transport of TrkB in response to EGF and an impaired delivery to autophagosomes. Mechanistically, we show that Calnexin facilitates the interaction of TrkB with the ER-phagy receptor Fam134b, thereby targeting TrkB to ER-phagy. This mechanism appears as a critical process for fine-tuning the sensitivity of neurons to BDNF.

A 3D cell culture system for bioengineering human neuromuscular junctions to model ALS.

The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.