Molecular mechanisms underlying spinal motor neuron diversification and differential vulnerability in vivo and in vitro

Movements involve the tightly regulated interplay of the components of the neuromuscular system. Spinal motor neurons are the elements of the neuromuscular system that regulate muscle fibre contractions. Spinal motor neurons are classified into functional motor neuron types and subtypes based on the muscle fibre type they innervate and they possess distinct morphological as well as electrophysiological properties. Also, the different motor neuron subtypes express specific molecular markers and show differential susceptibility towards neurodegenerative diseases. In this work, two important aspects of the motor neuron diversification in vivo and in vitro were addressed: characterisation of the tissue inhibitor of matrix metalloproteinases 3 (TIMP3) expression as a potential marker of spinal motor neuron subtypes and evaluation of the diversity of mouse embryonic stem cell (ESC)-derived motor neurons on molecular and electrophysiological levels.The first project successfully generated a Timp3 reporter mouse line using state-of-the-art CRISPR/Cas9 technology in combination with homology-directed repair. The analysis of the Timp3 reporter mouse line showed that in adult mice TIMP3 is expressed in gamma motor neurons but not in alpha motor neurons. At early postnatal stages, however, TIMP3 is present in gamma motor neurons and putative alpha S motor neurons. TIMP3 loss-of-function experiments were additionally performed to investigate its function in motor neurons. Preliminary results showed that the absence of TIMP3 does not affect the overall cell soma size distribution of motor neurons and does not lead to the changes in the number of motor neurons expressing matrix metalloproteinase 9 (MMP9). The second project investigated the diversity of ESC-derived motor neurons. It was shown that the established in vitro differentiation protocol results in a highly pure motor neuron cell culture consisting of 90% ChAT-positive cells. ESC-derived motor neurons express postmitotic motor neuron markers, including Chat, Mnx1, Neun, and Isl1, form synaptic connections with C2C12-derived myotube cells of cholinergic nature, and exhibit motor neuron-like behaviour by showing repetitive firing. Moreover, it was proposed that ESC-derived motor neurons possess some degree of diversity in vitro as both, alpha and gamma motor neuron-like phenotypes, are present in cell culture