Transcriptome analysis by RNA sequencing of primary mouse sensory neurons of the dorsal root ganglia (DRGs) treated with glucosylceramide (GlcCer 24:1) versus vehicle
Glucosylceramides (GlcCer) are endogenous lipids which are generated in the ER from ceramides via the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) and are required for the  generation of gangliosides. They are metabolized and degraded in the lysosome via the lysosomal enzyme glucocerebrosidase (GBA1). Biallelic mutations of GBA1 lead to Gaucher disease, which is a the lysosomal storage disease. Monoallelic mutations of GBA1 are associated with a high risk of sporadic Parkinson's Disease (PD). GBA1 dysfunctions increase the accumulation and aggregation of alpha synuclein. We have previously shown that accumulation of GlcCer is associated with sensory neuropathy and pain in PD (Mov Disord. 2020 Oct;35(10):1822-1833. doi: 10.1002/mds.28186) and in the DRGs and sciatic nerve of Pink1-/-SNCA A53T double mutatnt mice (Neuropathol Appl Neurobiol. 2021 Dec;47(7):1060-1079. doi: 10.1111/nan.12734). Here, we assessed the effects of GlcCer 24:1 (the most abundant GlcCer) on primary sensory neurons of the dorsal woot ganglia of C57BL6 mice via RNA-sequencing in four biological replicates per group. DRGs were were rapidly excised and collected in HBSS. Primary neurons were prepared via tissue digestion in collagenase/dispase and DNAse and collection via passaging through a BSA filter. They were seeded on Poly-L-Lysine and laminin coated cover slips in serum-free Neurobasal medium containing 1x B27 supplement , 1x Pen/Strep, 200 ng/ml nerve growth factor nd 2 mM L-glutamine at 37 °C and 5% CO2 and 95% humidity. The day after cell seeding, GlcCer 24:1 was added at a final concentration of 1 µM. Control cells received the vehicle treatment which was 1:10000 dilution of a 2:1 mixture of chloroform and methanol. After two days of culture cells were collected and total RNA was isolated using a single cell RNA isolation kit (picoPure Thermo Scientific). Quantity of total RNA was assessed with a Qubit 2.0 fluorometer and quality was checked using Agilent's bioanalyzer. Indexed sequencing libraries were prepared with TAKARA SMARTer Ultra Low RNA Kit for sequencing compatible with Illumina NGS. The alignment was performed with SeqMan NGen 17 (Lasergene) and with CLC genomic workbench (Qiagen) using the reference genome mm10 provided from UCSC (GRCm38) as template, a minimum read length of 35 bp and automatic adapter trimming. Sequence reads were normalized as TMM (Trimmed Mean of the M-values) using the EdgeR algorithm. Differential gene expression was analyzed with CLC genomic workbench and with ArrayStar 17 (Lasergene) using general linear models and adjustment of P-values according to Benjamini Hochberg. Overall design: Total RNA was prepared from primary neurons of the dorsal root ganglia of 4 mice per group. DRG cultures were treated with GlcCer 24:1 (1 µM) or vehicle (1:10000 of 2:1 mixture of chloroform and methanol). RNA sequencing was  performed of 4 biological replicates per group using an Illumina NextGen 2000 system.