Trancriptional profile of iGNs and iMNs after TDP-43 knockdown
Description
Research Hypothesis The study hypothesized that while TDP-43 loss-of-function is a near-universal hallmark of ALS, its molecular consequences—specifically regarding gene expression and cryptic splicing—are cell-type specific (glutamatergic induced neurons - iGNs versus cholinergic induced neurons - iMNs) . We further hypothesized that the presence of astrocytes (iAs) would modulate the detectable splicing repertoire of human induced neurons (iNs), providing a more clinically relevant representation of the disease environment. What the Data Shows Subtype-Specific Signatures: The data reveals distinct transcriptomic profiles for iGNs and iMNs. While TDP-43 depletion affected 92 genes in iMNs and 52 in iGNs, only 33 genes overlapped between the two subtypes. Conserved Pathological Core: Despite low overall overlap, a conserved set of ALS-relevant targets—including STMN2, UNC13A, CAMK2B, and ATG4B—were consistently downregulated and mis-spliced across both populations. Glial Influence: The inclusion of astrocytes (iNs+iAs) facilitated the detection of additional cryptic events, most notably in HDGFL2, which matched patient-derived cerebrospinal fluid data . Data organization: Gene counts - "Counts_processed_pure_iGNs_iMNs.csv" (pure iNs) and "Counts_processed_iGNs_iAs.csv" (co-culture iNs+iAs) Splicing variants frequency - "Splicing_iMN_iGN_sgvc_combined.rds" (pure iNs) and "Splicing_ioGN_ioAs_sgvc_combined.rds" (co-culture iNs+iAs)
Files
Steps to reproduce
1. Cell Culture and Maintenance Human induced pluripotent stem cells (iPSC)-derived ioGlutamatergic Neurons and ioMotor Neurons were obtained from Bitbio (Cambridge, UK) and cultured according to the manufacturer's instructions at 37°C in a humidified incubator with 5% CO2. For experiments involving co-culture, ioGlutamatergic and ioMotor Neurons were plated together with iCell Astrocytes (Fuji Film Cellular Dynamics, Inc.) at a ratio of 2:1. Cultures were maintained for a total of three to five weeks and media was exchanged every 2-3 days. For experiments involving microelectrode array (MEA) recordings, cultures were maintained for extended periods up to eight weeks, with regular media changes. 2. TDP43 Knockdown To achieve knockdown of TDP43 cells were treated with 5 µM of a TDP43-targeting antisense oligonucleotide (ASO) on day 4 post-plating, added directly to the culture medium. Control conditions included treatment with a non-targeting ASO at the same concentration and a vehicle control (PBS). Following ASO administration, cultures were maintained under standard conditions for the remainder of the experiment. 3. RNA Extraction and Bulk RNA Sequencing Total RNA was extracted from frozen cell pellets of the iGN and iMN pure cultures or iGN+iAs co-cultures at day 35. Total RNA was quantified with Qubit RNA HS Assay Kit (Thermo Fisher Scientific). RNA integrity was assessed using a Fragment Analyzer. Library preparation was performed using polyA selection for mRNA species to remove ribosomal RNA, generating Illumina-compatible libraries. The libraries were sequenced on an Illumina NovaSeq 6000 system (v2.0.0) to generate 2x150 bp paired-end reads, with an average of 50 million reads per sample. 4. Pre-processing of RNA Sequencing Data Analysis was performed according to methods previously described. Briefly, reads were aligned to the human reference genome GRCh38 using STAR aligner (v2.7.1) with default parameters. Gene counts were calculated from the sorted and indexed BAM files using featureCounts (v2.0.6).
Institutions
- F Hoffmann-La Roche AG Research and Development DivisionBasel-Stadt, Basel
- Universidade Federal do Rio Grande do Norte Instituto do CerebroRio Grande do Norte, Natal