Amyotrophic lateral sclerosis (ALS) represents the major adult-onset neuron disease leading to motor neuron degeneration, alteration in the neuromuscular junction (NMJ), muscle atrophy, paralysis, and death. In the 20% of cases that show familial inheritance, ALS is linked to mutations in the gene encoding the superoxide dismutase 1 (SOD1). Recent studies on SOD1 G93A mice showed that the communication between nerve and muscle is highly impaired, indicating that Neuromuscular Junction (NMJ) dysfunction precedes the clinical phase of the disease. However, mechanistic insight into how NMJ dysfunction relates to muscle-nerve alteration is incomplete. This is, in part, caused by a lack of robust in vitro models. The main goal of this research is to use a 3D NMJ system to study the physiopathological interplay between nerve and muscle occurring during ALS disease. We propose to define the potential cellular and molecular players involved in the functional short circuit that contributes to the so-called dying-back process. We will define: i) whether altered skeletal muscle impinges motor neuron homeostasis or ii) whether the maintenance of healthy muscle counteracts motor neuron decline. To this purpose, we will use a custom build microfluidic device, containing the main elements forming the NMJ architecture, namely the ex vivo muscle engineered tissue (X-MET), already developed in our laboratory, coupled with motor neuron cell cultures and the terminal Schwann cells (TSC). Based on the comparison between muscle contractile response to direct membrane stimulation and muscle response to indirect nerve stimulation, we will be able to evaluate NMJ functional properties, allowing a deeper analysis of synaptic transmission in the 3D ALS model. This study will provide new insights into the mechanisms that trigger functional denervation and offer the possibility of developing the specific intervention to attenuate NMJ loss and nerve-muscle dysfunction in ALS.
This research project aims at using an innovative three-dimensional (3D) neuromuscular junction model to clarify the physio-pathological interplay between muscle and nerve occurring during Amyotrophic Lateral Sclerosis (ALS). Tissue engineering is an emerging field of research focused on the development of bioartificial substitutes for organs and tissues, which can, in turn, be applied in regenerative medicine, pharmaceutical, diagnostic, and basic research. Despite the generation of different 3D muscle models, to date, no effective model is integrating engineered skeletal muscle with motor neurons to reproduce in vitro the neuromuscular junction (NMJ). X-MET is a 3D skeletal muscle engineered tissue obtained through an in vitro culture of heterogeneous populations of cells without the use of any scaffold that successfully demonstrated robust contracting properties over time[1]. Interestingly, scaffoldless constructs allow for accurate measurement of their contractile force and power [1],[2] and better mimic in vivo conditions of skeletal muscle. The innovation of this research project can be explained in different points. 1) The development of 3D skeletal muscle tissue from SOD1G93A transgenic mice, constituted by a heterogeneous population of cells and obtained without a scaffold, will offer the advantage of studying the pathological and physiological conditions affecting the striated muscle during ASL pathology, from both a functional [3] and molecular [4] point of view. 2) The use of a microfluidic device that allows for the coupling of motor neurons with skeletal muscle construct, will yield to the formation of the entire NMJ architecture (the presynaptic motor nerve terminal, the intrasynaptic basal lamina, and the post-synaptic muscle membrane) permitting a better investigation of the NMJ dysfunctionality. 3) Through the use of this device, we will be able to better clarify the short circuit that contributes to the dying-back process and test the saving back hypothesis in ALS progression analyzing i) the effects of SOD1G93A-derived X-MET on wild type-derived motor neurons and ii) the effects of wild type-derived X-MET on SOD1G93A-derived motor neurons. Within this context, it is expected that the proposed study will have a direct impact on the field of neuromuscular junction functionality and will contribute to highlighting fundamental issues related to the coupling alterations between muscle and nerve in neuromuscular diseases.
[1] S. Carosio et al. Sci. Rep., 2013.
[2] S. Pisu et al., IEEE Trans. Instrum. Meas., 2019.
[3] E. Rizzuto et al., Ann. Biomed. Eng., 2014.
[4] A. Natarajan et al., ACS Omega, 2019.