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Spinal muscular atrophy (SMA) is a genetic pediatric condition that affects lower motoneurons leading to their degeneration and muscle weakness. It is caused by homozygous loss or mutations in the Survival Motor Neuron 1 (SMN1) gene; however, the pathomechanism leading to motoneuron degeneration is not fully resolved. Cultured embryonic SMA motoneurons display axon elongation and differentiation defects accompanied by collapsed growth cones with a disturbed actin cytoskeleton. Intriguingly, motoneurons cultured from mice deficient for the Tropomyosin-kinase receptor B (TrkB), exhibit similar pathological features. Thus, the question arises whether SMA motoneurons suffer from defective Brain-derived neurotrophic factor (BDNF)/TrkB signaling and whether there is a link to the disturbed actin cytoskeleton. In the recent years, modifier genes such as Plastin 3 (PLS3) were shown to beneficially interfere with SMA pathology. Nevertheless, the mechanism of how the actin-bundler PLS3 counteracts SMN deficiency is not well understood. In this study, we investigated TrkB localization and its activation in cultured SMA motoneurons and neuromuscular junctions (NMJs). While TrkB levels are only mildly affected locally in axon terminals, BDNF-mediated TrkB phosphorylation was massively disturbed. The activity-dependent TrkB translocation to the cell surface and its activation via BDNF were shown to be Pls3-dependent processes, that can be abolished by knockdown of Pls3. In contrast, PLS3 overexpression in SMA motoneurons rescued the defects on morphological and functional level. In particular, the relocation of TrkB to the cell surface after BDNF-induced internalization is disturbed in SMA, which is based on an actin-dependent TrkB translocation defect from intracellular stores. Lastly, AAV9-mediated PLS3 overexpression in vivo in neonatal SMA mice provided further evidence for the capacity of PLS3 to modulate actin dynamics necessary for accurate BDNF/TrkB signaling. In conclusion, we provide a novel role for PLS3 in mediating proper alignment of transmembrane proteins as prerequisite for their appropriate functioning. Hence, PLS3 is required for a key process indispensable for the development and function of motoneurons even beyond the context of SMA.
In highly polarized neurons, endoplasmic reticulum (ER) forms a dynamic and continuous network in axons that plays important roles in lipid synthesis, Ca2+ homeostasis and the maintenance of synapses. However, the mechanisms underlying the regulation of axonal ER dynamics and its function in regulation of local translation still remain elusive. In the course of my thesis, I investigated the fast dynamic movements of ER and ribosomes in the growth cone of wildtype motoneurons as well as motoneurons from a mouse model of Spinal Muscular Atrophy (SMA), in response to Brain-derived neurotrophic factor (BDNF) stimulation. Live cell imaging data show that ER extends into axonal growth cone filopodia along actin filaments and disruption of actin cytoskeleton by cytochalasin D treatment impairs the dynamic movement of ER in the axonal filopodia. In contrast to filopodia, ER movements in the growth cone core seem to depend on coordinated actions of the actin and microtubule cytoskeleton. Myosin VI is especially required for ER movements into filopodia and drebrin A mediates actin/microtubule coordinated ER dynamics. Furthermore, we found that BDNF/TrkB signaling induces assembly of 80S ribosomes in growth cones on a time scale of seconds. Activated ribosomes relocate to the presynaptic ER and undergo local translation. These findings describe the dynamic interaction between ER and ribosomes during local translation and identify a novel potential function for the presynaptic ER in intra-axonal synthesis of transmembrane proteins such as the α-1β subunit of N-type Ca2+ channels in motoneurons. In addition, we demonstrate that in Smn-deficient motoneurons, ER dynamic movements are impaired in axonal growth cones that seems to be due to impaired actin cytoskeleton. Interestingly, ribosomes fail to undergo rapid structural changes in Smn-deficient growth cones and do not associate to ER in response to BDNF. Thus, aberrant ER dynamics and ribosome response to extracellular stimuli could affect axonal growth and presynaptic function and maintenance, thereby contributing to the pathology of SMA.