Restricciones a la Libertad de Expresión

The motor neurons are widely accepted as the primary cell type affected by SMN deficiency, although the reason of their susceptibility to low SMN levels is not fully clear and remains the subject of many studies in the SMA research (Saal et al. 2014, Fallini et al. 2016). One possible explanation could be that motor neurons express lower SMN from SMN2 that other cell populations in the spinal cord due to a particularly inefficient splicing of exon 7 (Ruggiu et al. 2012). Other hypotheses view the high energy demand of neurons as critical for their vulnerability, especially as mitochondrial dysfunction has been reported in SMA (Malkki 2016), as well as the polarity of neurons which is coupled with their high demand of local translation (Liu-Yesucevitz et al. 2011, Fallini et al. 2016).

NCALD was previously identified as localizing to axons of sensory and hippocampal neurons (Yamatani et al. 2010); additionally, our group could pinpoint the localization of NCALD to the soma and growth cones of motor neurons (Svenja Schneider, unpublished data). With this important indication that NCALD may play a role in the tissue of interest, we studied two characteristics of motor neurons that are impaired under low SMN levels:

axonal length and number of glutamatergic inputs on the motor neuron soma as a component of spinal motoneuronal circuitry (Rossoll et al. 2003, Mentis et al. 2011). Yamanati and colleagues identified NCALD and another protein of the NCS family – VILIP1 – in a proteomic screen as highly upregulated during axon development, which suggested a prominent function of neuronal calcium sensors in the growing axon. Furthermore, both NCALD and VILIP1 localized to the growth cone which is a dynamic structure orchestrating the axonal growth (Tamariz and Varela-Echavarria 2015). Overexpression studies showed detrimental effect of excessive NCALD on neurite outgrowth – thus highlighting that an alteration of NCALD levels is able to affect the homeostasis of axon development (Yamatani et al. 2010). We showed that NCALD suppression counteracts the axonal outgrowth impairment in SMA, strengthening the evidence of NCALD as an important regulator of axonal growth (5.2.5). The mechanism how this happens remains elusive; it is feasible, however, that the reported interaction of NCALD with tubulin and actin, two key components of the cytoskeleton, plays a role as the growth cone and axonal growth are directly dependent on the dynamics of the cytoskeleton (Gordon-Weeks and Fournier 2014). Another hypothesis would involve a possible restoration of Ca2+_{homeostasis, which is known to be altered in SMA, by altering the Ca}2+ sensing (Ruiz et al. 2010). The precise nature of Ca2+ _{disturbance is SMA is still under} discussion, as human and mouse studies yield incongruous results (McGivern et al. 2013), therefore intensive research would be required to verify this hypothesis.

Recently, disturbed neuronal circuits are increasingly recognized as contributing to and potentially even initiating various neurodegenerative disorders, e.g. Parkinson’s and Huntington’s disease (Palop et al. 2006). Importantly, as also other cell types directly interacting with motor neurons, such as interneurons and astrocytes, were reported to show alterations when SMN is depleted, not only cell-autonomous effects, but the entire neuronal network needs to be taken into consideration in SMA research (Zhou et al. 2016). This is particularly relevant for motor neurons, as their function is not only dependent on intrinsic qualities, but also reflects the excitatory and inhibitory inputs that the motor neurons receive: when the overall numbers or firing frequency of these inputs or the ratio of excitatory to inhibitory ones are changed, this directly affects the activity of the target neuron and ultimately its output – in this case the activation of skeletal muscle (Kitzmann 2010). In SMA, it has been shown that specifically the excitatory, but not the inhibitory inputs are reduced (Simon 2016). The excitatory inputs are prevalently of glutamatergic nature and provide the motor neuron with sensory information with respect to touch, proprioception, mechanoreception and nociception as well as integrate information from different levels of CNS (cortex, brain stem and spinal cord). We analyzed the glutamatergic

inputs on spinal motor neurons by immunohistochemical staining of spinal cord sections and in both SMA models under study: the severe and the intermediate one, we could observe that reducing NCALD lead to the increase of the number of glutamatergic inputs on motor neuron soma (see 5.2.7 and 5.3.5), implicating that the positive effect of lower NCALD levels is not limited to motor neurons but is capable of modifying the spinal circuitry. Other studies found that the loss of excitatory inputs accompanied by a block in their synaptic transmission leads to hyperexcitability of motor neurons (Simon et al. 2016). This manifested in an alteration of two intrinsic membrane properties: input resistance and time constant, that were reported to be increased in SMA motor neurons. As other neuronal sensor proteins have been shown to play a role in membrane trafficking and calcium channel regulation (Burgoyne and Haynes 2010, Weiss et al. 2010), and defective clustering of calcium channels has been reported to disturb the excitability of SMA motor neurons (Jablonka et al. 2007), one could envisage that reducing NCALD possibly interferes with these SMA defects and restores some provisory balance at the membrane. The limitation of the anatomical studies is that when an increase in excitatory inputs to motor neurons is detected by immunolabeling, this does not necessarily implicate an actual increase in glutamate release. Therefore, electrophysiological analysis of the properties of SMA motor neurons along the methodology presented in (Mentis et al. 2011, Gogliotti et al. 2012) would be technically challenging, yet highly interesting in order to make a conclusive statement about the functional impact of NCALD reduction.