Unveiling synapse pathology in spinal bulbar muscular atrophy by genome-wide transcriptome analysis of purified motor neurons derived from disease specific iPSCs

Abstract

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients' pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9 e438 ::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.

Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS

“…The length of CAG repeats was determined by fragment analysis and Sanger sequencing of the PCR products. 43 NSC34 motor neuron-like cells stably expressing AR24Q or AR97Q were established as previously described. 44 Cells were maintained in DMEM supplemented with 10% FBS.…”

C9ORF72 dipeptide repeat proteins disrupt formation of GEM bodies and induce aberrant accumulation of survival of motor neuron protein

“…The origins of the iPSC clones used in this analysis are described in Table 1. Differentiation of iPSCs into MNs was performed as previously described [33][34][35] (Fig. 4a).…”

“…iPSCs were maintained on mitomycin-C-treated SNL murine fibroblast feeder cells in 0.1% gelatine-coated tissue culture dishes in hESC medium. iPSCs were differentiated into spinal MNs as previously described [33][34][35] . Briefly, iPSC colonies were detached using a dissociation solution (0.25% trypsin, 100 μg/ml collagenase IV (Thermo Fisher Scientific, USA), 1 mM CaCl2, and 20% KnockOut TM Serum Replacement (KSR, Thermo Fisher Scientific, USA)) and cultured in suspension in bacteriological dishes in standard hESC medium.…”

Cell-Clearing Systems Bridging Repeat Expansion Proteotoxicity and Neuromuscular Junction Alterations in ALS and SBMA

“…It is noteworthy that regardless of the causative mechanism, disease symptoms in ALS and SBMA are bound to a failure of neurotransmission which may precede protein aggregation [33][34][35]. In particular, early alterations within the skeletal muscle, NMJ, and sensory/motor axons are emerging as a primum movens in both ALS and SBMA [33,[35][36][37][38]. In line with this, both diseases are being redefined as neuromuscular synaptopathies, remarking the importance of proteostasis within the sensory-motor system as a whole [36].…”