Identification of affected cellular targets, mechanisms and signalling pathways in mouse and cell models for spinal muscular atrophy with respiratory distress type 1 (SMARD1).

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Muscle atrophy, and diaphragmatic palsy are the clinical characteristics of spinal muscular atrophy with respiratory distress type 1 (SMARD1), and are well represented in the neuromuscular degeneration (Nmd2J) mouse, modeling the juvenile form of SMARD1. Both in humans and mice mutations in the IGHMBP2 gene lead to motoneuron degeneration. We could previously demonstrate that treatment with a polyethylene glycol-coupled variant of IGF1 (PEG-IGF1) improves motor functions accompanied by reduced fiber degeneration in the gastrocnemius muscle and the diaphragm of the Nmd2J mouse, but has no beneficial effect on motoneuron survival. IGHMBP2/Ighmbp2 is a ribosome associated ATPase/helicase supposed to be involved in ribosomal events/orchestration and/or translational events. These data raised the question which affected cell autonomous disease mechanisms and signalling pathways contribute to dysfunction and loss of Ighmbp2 deficient motoneurons and muscle fibers. An analysis of primary Ighmbp2 deficient motoneurons and spinal cord tissue from Nmd2J mice exhibited differentiation deficits with excitability failures corresponding to reduced N-type specific voltage-gated calcium channel (Cav2.2) accumulation and a dysregulation in the expression of transient receptor potential channels (TRPCs). In order to discover affected cellular mechanisms and signalling pathways, we will start a detailed morphological and functional analysis of primary Ighmbp2 deficient motoneurons. Primary Ighmbp2 deficient mouse motoneurons under specific culture conditions will be subjected to a Fluorescence-Recovery after Photobleaching (FRAP) and a Calcium Imaging study. We finally want to find out which specific signalling pathways are affected in Ighmbp2 deficient motoneurons leading to functional failures. Simultaneously to the in vitro study, we will analysed in detail Nmd2J mice on a TRPC5 and Nav1.9 knock-out background to discover whether these ion channels are modifiers of the Nmd2J phenotype. This indirect identification of a cellular target will be completed by microarray studies in order to identify other affected cellular targets on RNA and protein levels. Furthermore, a re-differentiation approach with skin fibroblast from SMARD1 patients is planned. The re-differentiated human motoneurons will be functional and morphological compared with primary mouse motoneurons to verify their potency as a reliable tool for the further analysis of functional deficits in Ighmbp2 mutated motoneurons. In a last workpage we want to analyse in detail the cell autonomous mechanisms which lead to myopathic changes of diaphragm and gastrocnemius muscle in the SMARD1 mouse. By means of our project we would like to understand affected cellular mechanisms in Ighmbp2 deficient motoneurons and muscles in order to identify signalling pathways which could be stimulated and thus bypass the affected cellular mechanisms.