Cellular and Transcriptomic Analyses Identify the Source and Lineage of Regenerated Neurons in the Adult Zebrafish Telencephalon following Blunt-Force Traumatic Brain Injury

Traumatic brain injuries (TBIs) represent a large global disease burden and can result in several short- and long-term deficits. Treatment for TBIs are limited, however stem cell therapies involving either transplantation of exogenous stem cells or stimulation of endogenous stem cells have been proposed. These approaches possess issues with survival and differentiation of stem/progenitor cells, which highlights the need to elucidate mechanisms necessary for complete regeneration. Zebrafish exhibit a robust neuronal regenerative capacity following damage in various parts of the central nervous system. In this study, we use a modified blunt-force TBI (bfTBI) model, the most common type of TBI seen in the human population, to damage the adult zebrafish telencephalon and examine cellular and molecular consequences. This model induces a variety of TBI-related phenotypes across different injury severities, which mimic the pathologies observed following bfTBI in humans. Additionally, we show that after bfTBI, cells in the zebrafish telencephalon, including radial glia (RGC), non-glial neural progenitors (NPs), and neuroblasts, proliferate and differentiate into various neural cell types. We performed a snRNA-Seq analysis of the damaged and regenerating telencephalon, which revealed heterogeneity among the progenitor cells. Further, the snRNA-Seq demonstrated that quiescent radial glia became activated upon injury and yielded neural progenitor cells. To functionally assess how the regenerative process is regulated, we knocked down the expression of PdgfrB and demonstrated that it played a role in maintaining RGC quiescence. Collectively, this study provides a foundation for future studies of neuronal regeneration in the adult zebrafish telencephalon following a bfTBI. Telencephalons of undamaged and severe bfTBI damaged fish at 5, 12, 18, 24, 48, 72, 96, 120, 144, and 168 hpi were dissected as described above. Six telencephalons per group were placed in a Low-Bind Tube (Eppendorf) and snap frozen using liquid nitrogen. Nuclei were extracted using the Frankenstein Protocol as previously described (Martelotto et al. 2020) with some modification. Briefly 500 µL of Lysis buffer (1% Tris-HCl/0.2% NaCl/0.3%MgCl/0.1% NP40 in nuclease-free water) was added to brains. Brains were homogenized using a dounce homogenizer (10-20 times). Homogenate was passed through a 70 µm mesh strainer (Miltenyi Biotech, Auburn, CA, USA) and spun down at 500 xg at 4˚C for 5 minutes. Supernatant was discarded and the nuclear pellet was resuspended in Nuclei Wash Buffer (1% BSA in PBS with 0.2U/µL RNase inhibitors (Roche)). Nuclei were washed and resuspended 2 additional times as described above, and cells were resuspended to a concentration of ~1,000-1,500 nuclei/µL. Nuclei samples were passed through a 35 µm mesh strainer (Miltenyi Biotech). Cell suspension concentration and percent viability were determined by staining with Trypan Blue Stain (0.4%) (Thermo-Fisher Scientific) and Countess II Automated Cell Countess. Nuclei were loaded into Chromium Next GEM Chip G (10X Genomics, Pleasanton, CA, USA) with a targeted recovery of 10,000 nuclei per well and processed with Chromium Controller or Chromium iX (10X Genomics). Single-nuclei RNA-Seq libraries were prepared with Chromium Single Cell 3' Reagent Kit (v3.1 - Dual Index; 10X Genomics PN-1000269 | PN-1000215) at the Genomics and Bioinformatics Core Facility at the University of Notre Dame, following the standard throughput manual workflow and pooled at equal molar ratios. Libraries were sequenced on NovaSeq 6000 System at Center for Medical Genomics at Indiana University.