Axonal transcriptome reveals upregulation of PLK1 as a protective mechanism in response to increased DNA damage in FUSP525L spinal motor neurons

Mutations in the gene fused-in-sarcoma (FUS) have been implicated in the motor neuron disease Amyotrophic lateral sclerosis (ALS). However, it is poorly understood how these gene mutations lead to selective motor neuron (MN) degeneration and if there are common pathomechanisms across disease etiology. To address the biological impact of the FUS-P525L mutation on the transcriptome of neurons, we applied RNA-sequencing (RNA-seq) method, using microfluidic chambers, to generate axonal as well as soma compartment specific profiles from isogenic induced pluripotent stem cells (iPSCs)-derived motor neurons. We demonstrate high purity of axonal and soma fractions, and further show that the axonal transcriptome is very specific from somas with fewer number of transcripts. Notably, functional enrichment analysis revealed a unique and distinct transcriptional landscape in both axonal and soma compartments including specific transcript types, most of which were required for RNA metabolism and DNA damage/cell cycle related processes, that were likely altered by FUS mutation. Among altered transcripts, we identified robust upregulation of polo-like kinase 1 (PLK1), a master regulator of cell cycle-related events, in FUS-P525L mutant MNs and confirmed that inhibition of PLK1 increases late apoptotic or necrosis-induced neuronal cell death in mutant neurons compared to healthy controls. Taken together, our findings provide insights into compartment-specific transcriptomics in FUS-ALS neuronal model and we propose that PLK1 activation is a consequence of MN-specific upregulation, possibly contributing to the DNA-damage response and other associated pathways including cell cycle and cytoskeleton machinery. Patient-derived isogenic spinal motoneurons (MNs) expressing either normal (FUS WT) or mutant P525L FUS-eGFP (FUS P525L) were matured for 14 DIV using microfluidic chambers, where somas (n=6) and axons (n=6) were harvested and total RNA was isolated for RNA-sequencing. The main cell model used in this study were neurons derived from FUS-WT-EGFP and FUS-P525L-EGFP that were generated as a part of previous study (Naumann, Pal et al. 2018). Briefly, fibroblasts carrying R521C FUS mutation obtained from a 58-year-old female were reprogrammed into iPSC using cDNA of OCT4, SOX2, KLF4 and cMYC delivered via retroviral vectors. Next, CRISPR/Cas9 genome editing was used to i) correct the R521C mutation into WT FUS and to tag it with EGFP to generate the FUS WT EGFP line or ii) replace the R521C mutation into P525L FUS mutation and to tag it with EGFP to generate FUS P525L EGFP line. Differentiation of hiPSC into spinal motor neurons (MNs) was carried out based on the protocol from Reinhardt et al. 2013. RNA-sequencing was performed on 2 cell lines (isogenic control and FUSP525L) resulting in the following 4 groups:control somas (n=3), control axons (n=3), FUSP525L somas (n=3) and FUSP525L axons (n=3). A total of 12 samples were analyzed here.