Molecular mechanisms underlying diversification of spinal motor neurons and neuromuscular synapses

Bian, Yehan; Marquardt, Till (Thesis advisor); Spehr, Marc (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

Motor behaviors rely on the diversity of motor neurons, muscle fibers, and neuromuscular junctions (NMJs). Motor neurons can be classified as "functional" motor neuron subtypes, each of which forms distinctive synapses with the muscle fibers it innervates. However, how motor neurons and NMJs become functionally diversified remains largely unknown. This thesis focused on three transcriptome analysis-derived candidate genes that may be involved in motor unit diversification: protein kinase C delta type (PKCδ) encoding gene Prkcd, and Wnt agonist R-spondin (RSPO) encoding genes Rspo2 and Rspo3. PKCδ was characterized as a molecular marker for α slow motor neurons and a subtype of γ motor neurons based on its expression in cell bodies and motor nerve terminals of NMJs. Interestingly, in mice lacking Err2 and Err3 in motor neurons, in which γ motor neurons fail to gain normal function, PKCδ expression was lost in γ motor neurons but remained preserved in α slow motor neurons. Thus, ERR2/3 could directly or indirectly regulate PKCδ expression in γ motor neurons. Furthermore, in an amyotrophic lateral sclerosis (ALS) mouse model, PKCδ expression persisted in degeneration-resistant α slow motor neurons and γ subtype motor neurons till the late phase of the disease, implying a possible neuroprotective role of PKCδ in ALS disease. Additionally, this thesis investigated the potential contribution of RSPO to the diversification of NMJs. Rspo2 and its paralog Rspo3 were abundant in α fast motor neurons. Since RSPO2 plays a role in the NMJ formation, this thesis hypothesized that RSPO2/3 could contribute to the NMJ specialization in α fast motor units. A floxed Rspo2 mouse line was generated using the CRISPR/Cas9 system and crossed with the Chatcre+; Rosa26fxTomato mouse line where Cre recombinase expression is driven by Chat promoter to inactivate RSPO2 in cholinergic neurons. Preliminarily, heterozygous Chatcre+;Rspo2fll+ mice and Chatcre+;Rspo3fll+ mice showed no obvious phenotype in NMJ formation. It is essential to examine NMJ formation ultimately in homozygous mice lacking RSPO2, RSPO3, and RSPO2/3 in motor neurons. Altogether, these findings assist in the classification of functional motor neuron subtypes and provide insights into some of the molecular mechanisms underlying motor neuron and NMJ diversification in health and disease.

Institutions

• Department of Biology [160000]
• Chair of Neurobiology [164310]