In vivo analysis of gene expression changes in mouse astrocytes after traumatic spinal cord injury

Central nervous system (CNS) lesions become surrounded by neuroprotective borders of newly proliferated reactive astrocytes. Fundamental features of these cells are poorly understood. Here, using temporal transcriptome analysis of Aldh1l1-expressing local astrocytes we showed that after CNS injury, local mature astrocytes dedifferentiated, proliferated, and become transcriptionally reprogrammed to permanently altered new functional states, with persisting downregulation of molecules associated with astrocyte-neuron interactions, and upregulation of molecules associated with wound healing, microbial defence, and interactions with stromal and immune cells. Our findings show that at CNS injury sites, local mature astrocytes proliferate and adopt canonical features of essential wound repair cells that persist in adaptive states and are the predominant source of neuroprotective borders that re-establish CNS integrity by separating neural parenchyma from stromal and immune cells as occurs throughout the healthy CNS. Overall design: We conducted genome-wide RNA sequencing of spinal cord astrocyte specific ribosome-associated mRNA using RiboTag methods. To generate the mice for this study, mice expressing RiboTag (JAX: 029977) were crossed either with Aldh1l1-CreERT2 (JAX: 031008) or with mGfap-Cre-73.12 (JAX: 012886). Astrocyte specific mRNA as well as non-enriched flow through samples were collected and analyzed from Aldh1l1 CreERT2-RiboTag mice at 2, 5, 14, 28, 42, and 70 days after crush spinal cord injury. Astrocyte specific mRNA as well as non-enriched flow through samples were collected and analyzed from mGfap-Cre-73.12 RiboTag mice at 14 days after crush spinal cord injury. Healthy uninjured astrocyte specific mRNA as well as non-enriched flow through samples were collected and analyzed from Aldh1l1 CreERT2-RiboTag and mGfap-Cre-73.12 RiboTag mice. Astrocyte specific mRNA was also collected and analyzed from healthy uninjured mGfap-RiboTag mice at postnatal days P0, P3, P7, P14, P21, P35, P63. RiboTag transgene expression for each sample was confirmed by genotyping of collected tail samples prior to processing for astrocyte specific RNA. For SCI surgery, a laminectomy of a single vertebra was performed at spinal cord level T10. A timed (5 second) lateral compression, complete crush SCI was made using No. 5 Dumont forceps with a tip width of 0.5 mm. Uninjured mice, or mice at various time points after complete crush SCI, were perfused with ice cold heparinized saline that was prepared using RNAse- and DNAse- free water and 10X PBS for 2 minutes at 7ml/min for blood clearance. Spinal cords were rapidly dissected on ice chilled blocks. For SCI samples, the lesion core center was identified and a tissue block of 1 mm rostral and caudal was then rapidly removed. Anatomically equivalent regions of spinal cord were taken from uninjured mice including from postnatal samples. Tissue samples were rapidly snap frozen in microcentrifuge tubes maintained in a dry ice bath and stored at -80°C until further processing. Frozen spinal cord tissue was processed by RiboTag Immunoprecipitation (IP). For specific animals, two RNA samples were generated: (i) Sample A (RiboTag) = RiboTag immunoprecipitation (IP) using Anti-HA.11 Epitope Tag Antibody to enrich for border astrocyte Ribosomes with mRNA extracted from those ribosomes; (ii) Sample B (Flow through samples) = mRNA extracted from the supernatant solution remaining after RiboTag IP to provide a characterization of the other cells in the tissue and enable astrocyte gene enrichment analysis. For the RiboTag procedure, dounce homogenization was used to generate tissue lysates from frozen spinal cord segments and centrifuged to remove tissue debris. IP of HA-positive ribosomes was performed by incubating with Anti-HA.11 Epitope Tag Antibody (Biolegend, Cat# 901515) for 4 hours in a microcentrifuge tube on a microtube rotator kept at 4°C. IP solutions were combined with Pierce A/G Magnetic Beads (Thermofisher, #PI88803) and incubated overnight on a microtube rotator at 4°C. On the second day, the solution was separated from the magnetic beads and processed as the “flow through” sample representing mRNA of other cells not from RiboTag-positive cells. Magnetic beads were washed three times with high salt solution (50 mM Tris pH 7.4, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM Dithiothreitol (DTT), 100 mg/ml Cyclohexamide). Unpurified RNA was collected from the magnetic beads by addition of RLT Plus buffer with BME and vigorous vortexing. RNA was then purified using RNeasy Plus Mini (for in vitro cell pellets) or Micro Kits (for spinal cord tissue) (QIAGEN Cat# 74134 and 74034). Total mRNA derived from the RiboTag IP was quantified using a 2100 Bioanalyzer (Agilent) with RNA samples having an RNA integrity numbers (RIN) greater than seven being processed for RNA-Sequencing. Sequencing was performed on poly-A selected libraries using Illumina NovaSeq S2 (UCLA Technology Center for Genomics & Bioinformatics) using pair end reads (2x50 – 50bp length) with an average of 50-100M reads per sample split over two lanes of the S2 flow cell.