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

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons (MNs) in the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually to death within 3 to 5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the ER-resident protein cerebral dopamine neurotrophic factor (CDNF) in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and etiology: (i) the conditional choline acetyltransferase (ChAT)-tTA/TRE-hTDP43-M337V rat model previously described, (ii) the widely used SOD1-G93A mouse model, and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the ER stress response in MNs, we used three main methods: (i) primary culture of MNs derived from E13 days embryos, (ii) immunohistochemical analyses of spinal cord sections with ChAT as spinal MNs marker, and (iii) qPCR analyses of lumbar MNs isolated via laser microdissection. We show that intracerebroventricular administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. CDNF rescues motor neurons in vitro and in vivo from ER stress-associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis