D EVELOPMENT OF THE N EUROMUSCULAR SYSTEM

As briefly mentioned in section 1.1.5.2, the neuromuscular junction (NMJ) is the connection between axon and muscle. The communication between nerve and muscle is ensured through neurotransmitter release from the presynaptic nerve terminal and receptor mediated signal transduction at the postsynapse. The most striking disease features in SMA mice are malformation and dysfunction of the NMJ. These defects are then followed by MN loss in the ventral horns of the spinal cord (Kariya et al. 2008; Kong et al. 2009). It has been reported that severely affected Taiwanese mice displayed no MN loss before P8 (Fayzullina

and Martin 2016). Nonetheless, as there is also evidence of MN loss starting at P4 in very severely affected Δ7 SMA mice (Mentis et al. 2011; Le et al. 2005), it still remains unclear whether this ‘dying-back mechanism’ should be considered as the only mechanism in SMA pathology or whether MN loss can proceed or occur in parallel to NMJ pathology. In SMA pathology, NMJ abnormalities include both pre- and postsynaptic alterations. Among these are abnormal neurofilament (NF) accumulation in the presynaptic nerve terminals, pronounced axon routing and arborization deficits (Rajendra et al. 2007), decreased amount of neurotransmitter released per action potential (quantum content, QC), reduced postsynaptic size with immature plaque-like AChRs (Kariya et al. 2008) and reduced post-synaptic currents (Torres-Benito et al. 2012). Therefore, an overview about fundamental processes driving axon growth and specification (axonal pruning) as well as NMJ development and refinement will be given below.

1.2.1 Axonal pruning and NMJ refinement

A delay of axon withdrawal from the NMJ during the process of axonal pruning in a PLS3 overexpressing SMA mouse has been described by (Ackermann et al. 2013). The underlying mechanism of this developmental process is briefly summarized below. Axonal pruning is a mechanism to selectively remove exuberant neuronal branches and connections during developmental stages of the nervous system to ensure structural of functional properties of the neuronal circuitry (Low and Cheng 2006). During NMJ development, numerous nerve terminals innervate individual AChR clusters. For proper axonal pruning, all but one presynaptic connection are removed, until every endplate gets innervated by only one single motor nerve ending (Lichtman and Colman 2000). There are two possible mechanisms, which have been described previously, to explain the process of axonal pruning. One of them is the so-called axon retraction, in which axonal contents are believed to be shuttled to other axon branches but so far, it has never been reported to occur at the neuromuscular junction (Bishop et al. 2004). Interestingly, NMJs in neonatal mice showing signs of abnormal ultrastructure were observed during the period of synapse elimination (Rosenthal and Taraskevich 1977). The second possible process is the classical Wallerian-type degeneration. During this kind of mechanism the entire axon arbor is removed rather than a subset of axons, which also has not been observed at the NMJ. Another concept of this process is termed axosome shedding (Bishop et al. 2004), in which axons are retreated and leave behind dismantled bulbs at their tips, which get removed as they get subdivided into

smaller remnants, termed axosomes. Previous findings of Schwann cells, which incorporated axosome material, support the idea of axosome shedding as they actively drive axonal recycling (Bishop et al. 2004). Nonetheless, on molecular level the process of axonal pruning is still poorly understood and further investigations are inevitable (Vanderhaeghen and Cheng 2010).

1.2.2 Influence of Actin on the Motor Neuron Integrity

Neurotransmitter release at the presynaptic terminals is a very complex process controlled by cytoskeletal dynamics. The actin cytoskeleton is important for cell shape, movement, signalling and transport of molecules into the cell (Blanchoin et al. 2014). Within the neuronal circuitry actin is involved in transport of synaptic vesicles (SVs) towards the active zone, as well as endocytic and exocytic pathways within the synapse. Overexpression of PLS3 – an actin-binding and -bundling protein – results in increased presynaptic nerve arborization and AChR clustering at the NMJ. Furthermore, PLS3 delays axonal pruning, indicating that PLS3 positively influences the axonal integrity (Ackermann et al. 2013). As PLS3 has an actin-binding and -bundling function, it is highly interesting to understand the connection between actin and the MN integrity, which in turn also highlights some of the physiological improvements of our mouse model (5.3 and 5.4).

Growth cones are the starting points of axonal outgrows. Actin polymerization is the main trigger for axonal outgrowth at the growth cones. Cofilin, which is also present at the growth cones has an actin destabilizing effect, as it localizes to the pointed (-) end of actin filaments, thereby depolymerizing actin (Pak et al. 2008). Previous studies have demonstrated, that axonal outgrowth is strongly dependent on the condensation state of the actin cytoskeleton at the growth cones. Indeed, less condensed actin supports axonal outgrowth (Bradke and Dotti 1999; Kunda et al. 2001). As already mentioned above (1.1.5.2), β-actin mRNA has been shown to be decreased in the growth cones of MNs derived from SMA mice and SMN-depleted PC12 cells (Rossoll et al. 2003). This indicates that the amount of actin molecules is significantly decreased in SMA growth cones, which are moreover significantly smaller compared to controls. Additionally, the connection between actin filaments, which can be mediated by additional proteins through actin-binding and -bundling abilities, can be important for cytoskeleton dynamics and thus for axonal growth. Besides the actin- bundling protein PLS3, there are several additional bundling proteins that are located in the growth cones including α-actinin, fascin, CamKII, Myosin, AbLIM, Filamin (Oprea et al. 2008; Dent

and Gertler 2003; Letourneau and Shattuck 1989; Okamoto et al. 2007). Therefore, the actin cytoskeleton and its network are fundamental for axonal outgrowth and maintenance.