6.4 Effects of PLS3V5 overexpression at NMJ level
6.4.3 Innervation defects in Hung SMA mice and hyperinnervation of endplates in
PLS3V5 expressing animals
Besides delayed axonal pruning, hyperinnervated endplates were found at P4 in SMA +
PLS3V5 and HET + PLS3V5 mice compared to SMA and HET mice (Figure 40, A).
Furthermore, SMA + PLS3V5 animals displayed highly arborized nerve terminals at the same time point, while nerve terminals of age matched SMA littermates exhibited significantly reduced terminal sprouting when compared to HET controls (Figure 40, B). Most strikingly, the observed effects of PLS3V5 overexpression at NMJ level seem to be functional, since mice specifically overexpressing PLS3V5 in the motor neurons showed a highly significant increase in muscle fiber size.
The finding of reduced endplate occupancy in Hung SMA mice is consistent with previous findings from other mouse models (Murray et al., 2008, Kong et al., 2009). In 2008, Kariya et al. have been the first to extensively investigate NMJ pathology in the SMNΔ7 SMA mouse model (Kariya et al., 2008). In their studies, they have been able to show that SMA mice display severe structural abnormalities at the NMJ level in Gastrocnemius muscle, including massive neurofilament accumulations and a failure of axons to form fine terminal arbors. However, Kariya et al. were unable to observe complete endplate denervation in the distal
Gastrocnemius muscle of postnatal SMNΔ7 SMA mice. In humans, SMA usually affects the
proximal muscles first. In line with this, others have detected the presence of 51 % unoccupied AChR clusters already at E18.5 and 49 % at P2 in the proximal intercostal muscles of the SMNΔ7 mouse (McGovern et al., 2008). Similarly, in late symptomatic SMA mice of the very severe Monani model (Smn-/-;SMN2(89Ahmb)tg/tg, survival ~ 5 d, (Monani et al., 2000)), ~15 % of endplates have been shown to be completely denervated in proximal TVA muscle (Murray et al., 2008). Interestingly, the authors have observed denervated endplates also in distal muscles of the lumbricals in Monani SMA animals, however, at significantly lower percentage. Therefore, as above findings suggest, NMJs display highly variable phenotypic severity among diverse muscles in one and the same SMA model, but also between different SMA mouse models. Despite a reduction in presynaptic coverage of
endplates, completely denervated endplates were more or less absent in proximal TVA muscle of Hung SMA mice in the present study (Figure 40). Surprisingly, however, it has been published before that in the distal Gastrocnemius muscle of Hung SMA mice around 9 % of endplates showed a denervation phenotype (Riessland et al., 2010). Together, these observations are not in line with the findings from human, where proximal rather than distal muscles are severely affected by SMA. One possible explanation for these findings is that the Hung SMA mice analyzed here were on pure C57BL/6N background, while Riessland et al. have used Hung SMA mice on pure FVB background in their work. As was found in the present study, Hung SMA mice on C57BL/6N background show a five days prolonged mean life span (15.5 d) compared to Hung SMA mice on pure FVB background (9.9 d). As it was discussed before (chapter 6.2), the C57BL/6N background obviously exerts an ameliorative effect on disease severity, which might explain the observation of better nerve connectivity in proximal TVA (C57BL/6N) compared to distal Gastrocnemius muscle (FVB) in Hung SMA mice.
As mentioned, P4 SMA and HET animals expressing PLS3V5 displayed increased endplate occupancy and extensive sprouting of nerve terminals compared to SMA and HET littermates, respectively (Figure 40). What implication do these observations have with regards to NMJ maturation and better neurotransmission? Since reduced nerve occupancy is a hallmark of SMA, stronger nerve coverage of endplates has frequently been taken as positive criteria to verify beneficial effects of various drugs, e.g. in studies investigating the therapeutic potential of the HDACi VPA, SAHA or TSA (Narver et al., 2008, Tsai et al., 2008, Riessland et al., 2010). In the present work, an increase in endplate occupancy and nerve terminal arborization was observed at P4, however, PLS3V5 expressing mice at earlier (P1) or later (P8, P11) time points showed similar effects at the NMJ. Therefore, observed effects at the NMJ might impact on both endplate maturation and signal transduction of established NMJs. It is well known that synaptogenic factors, such as Agrin and ACh, direct endplate maturation by refining areas of AChR clustering (Wu et al., 2010). While Agrin positively triggers the recruitment, assembly and maintenance of AChR at sites of innervation, ACh negatively impacts on AChR clustering, suppresses AChR expression and destabilizes AChR clusters globally in entire muscle fibers. During endplate maturation, the patch-like structure of AChR clusters gets perforated and clusters only remain at sites of presynaptic innervation. In the present work, motor neuron specific PLS3V5 expressing animals showed an increase in endplate size (Figure 45). Therefore, one possible explanation for the bigger endplates could be that the increased nerve occupancy and terminal arborization leads to a higher release of pro-AChR-clustering factors such as Agrin.
In line with this idea, PLS3V5 overexpression and concomitant cytoskeletal rearrangements might have an influence on the location and the release of synaptic vesicles
and thus on neurotransmission. In two studies using the SMNΔ7 mouse model, an abnormal synaptic vesicle number and location was accompanied with defects in neurotransmission as assessed by detailed electrophysiological analyzes (Kariya et al., 2008, Kong et al., 2009). Two pools of vesicles exist in motoric presynapses: Readily releasable vesicles (RRVs), which are in proximity to or already docked with the presynaptic membrane and vesicles of the reserve pool (RPV), which are located farther from the membrane (Dillon and Goda, 2005). Kong et al. have observed a 36 % reduction of RRVs in TVA muscle of the SMNΔ7 mouse (Kong et al., 2009). Moreover, the transmitter per single vesicle (quantal content, QC) QA) was unchanged between SMA and control mice, while a significantly reduced number of synaptic vesicles was released after nerve stimulation in SMA mice (quantal amplitude, QA). Therefore, the authors assumed that reduced density of synaptic vesicles may contribute to declined QC at SMA NMJs. As mentioned, not only the number of vesicles was found to be reduced, but also overall location of vesicles in the presynapse was disturbed. Therefore, the authors further speculated whether the commonly observed neurofilament (NF) accumulations are indicative for a more generally disturbed neuronal cytoskeleton. A disruption of cytoskeletal structures, in turn, might then result in displacement of vesicles, and consequentially in impaired neurotransmission. Interestingly, the idea of cytoskeletal disorganization as a pathological feature of SMA also matches the observation of significantly impaired β-actin mRNA transport into growth cones of SMA motor neurons (Rossoll et al., 2002). Additionally, also many other findings highlight the importance of a functional actin cytoskeleton for presynaptic function: E.g., it is well accepted that F-actin plays important roles in both recycling and transport of synaptic vesicles (Shupliakov et al., 2002, Bloom et al., 2003). Furthermore, after a current model synaptic vesicles are tethered near the membrane by F-actin via phosphorylation-dependent interaction with the vesicular molecule synapsin (Dillon and Goda, 2005). Upon depolymerization, Synapsin undergoes a conformational change, thereby freeing synaptic vesicles for release (Li et al., 2010). Last not least, F-actin regulates the availability of RPV by surrounding the vesicle cluster, thus providing a corralling function by forming a physical barrier to impede vesicle dispersion (Dillon and Goda, 2005). Taken together, these observations underline the importance of F- actin in synaptic vesicle organization and release. Therefore, by influencing F-actin dynamics PLS3V5 might act as a regulator of synaptic release, compensating for presynaptic dysfunction in SMA nerve terminals.