Wirth, Brunhilde | Piano, Valentina - C 18

Motoneurons (MNs) are highly polarized nondividing cells that innervate muscles via the neuromuscular junctions, the largest synapses in our body.  MNs must be maintained throughout life otherwise neuropathies and muscle atrophy will develop. During development, external cues - such as netrins - direct the axons to their ultimate destination by interacting with specific attractive or repulsive neuronal receptors. To accomplish this unique feature, axons and dendrites rely on highly sophisticated cellular processes - such as efficient local protein translation - that enable a prompt response to internal signals and external cues. In MNs, local protein translation requires a proper transport of mRNAs and cargos, a coordinated autophagy and protein degradation system, and functional mitochondria to provide enough energy locally for all these processes.

In this project we will make use of mouse and cellular models of Spinal Muscular Atrophy (SMA) -the most common recessively inherited MN disorder in humans - to understand why reduced levels of the house-keeping SMN protein primarily led to dysfunction of MNs. In addition, we will take advantage of our knowledge about three SMA protective modifiers (PLS3, NCALD and CHP1), which can counteract SMA development in humans and various SMA species by restoring impaired endocytosis (Ref 1-14). Nevertheless, how endocytosis is affected in SMA MNs and how it ultimately affects MN function, particularly axonal local translation, mitochondrial bioenergetics and neurotransmission, is still poorly understood and it will be the main objective of this project.

In particular, we will focus on these 3 specific questions:

• (WP1) how low SMN levels affect and how the protective modifiers can rescue axonal local translation and vesicle trafficking in MNs;
• (WP2) whether previously identified novel interactors of PLS3 influence receptor-ribosome mediated axonal translation and selective MN vulnerability;
• (WP3) which key biochemical processes are dysregulated in specific sub-compartments of MNs by reduced SMN levels, and can be counteracted by the SMA modifiers

Axonal vesicle trafficking and local translation are key regulatory mechanisms affected in a plethora of MN disorders, including SMA. In SMA, these processes are understudied, especially in the axons and pre-synapses. Recently, three FDA- and EMA-approved therapies, aimed at increasing the levels of functional SMN protein, showed impressive improvement in SMA patients. However, the current therapies are not efficient for severely affected SMA patients, who require very high levels of SMN protein postnatal, and for adult SMA patients, who require only low levels of SMN protein. The identification of new molecular mechanisms that can support MN function is needed to develop new therapies to reduce MN alterations and counteract their loss in SMA patients. Here, we aim to investigate these mechanisms and contribute to the development of SMN-independent therapies for SMA and MN disorders in general.

In this project we will use the following cutting-edge approaches (Figure 1) that will enable us to elucidate outstanding questions in the field of vesicle-trafficking, axonal translation and selective vulnerability in healthy and SMA motor neurons.

• WP1: we will use MNs differentiated from human CRISPR-Cas9 modified iPSCs (Figure 2) and primary murine MNs from SMA and SMA-PLS3 overexpression (OE) embryos as model systems. To study endosomal local translation in proximity to mitochondria and endosomal/lysosomal vesicle distribution/trafficking in health and disease, we will uselive-cell imaging, immunofluorescence staining and high-resolution microscopy.
• WP2: Our PLS3-and CHP1 Co-IP/MS proteome analysis from CRISPR-Cas9-mediated KO of Pls3 and Chp1 versus WT PC12 neuronal-like cells unraveled new possible modifiers. We will assess their role in axonal local translation in SMA, and how their interaction with PLS3 affects local translation, signaling and selective MN vulnerability.
• WP3: To identify impaired components in the relevant sub-compartments of SMA MNs, we will apply a recently developed approach to separate soma from axon in combination with proteome, transcriptome and Ribo-Seq of WT and SMA-or SMA-PLS3 OE human and murine MN.