After a brain lesion, highly specialized cortical astrocytes react, supporting the closure or replacement of the damaged tissue, but fail to regulate neural plasticity. Growing evidence indicates that repair response leads astrocytes to reprogram, acquiring a partially restricted regenerative phenotype in vivo and neural stem cells (NSC) hallmarks in vitro. However, the molecular factors involved in astrocyte reactivity, the reparative response, and their relation to adult neurogenesis are poorly understood and remain an area of intense investigation in regenerative medicine. In this context, we addressed the role of Notch1 signaling and the effect of Galectin-3 (Gal3) as underlying molecular candidates involved in cortical astrocyte response to injury. Notch signaling is part of a specific neurogenic microenvironment that maintains NSC and neural progenitors, and Gal3 has a preferential spatial distribution across the cortex and has a central role in the proliferative capacity of reactive astrocytes. We report that in vitro scratch-reactivated cortical astrocytes from C57Bl/6J neonatal mice present nuclear Notch1 intracellular domain (NICD1), indicating Notch1 activation. Colocalization analysis revealed a subpopulation of reactive astrocytes at the lesion border with colocalized NICD1/Jagged1 complexes compared with astrocytes located far from the border. Moreover, we found that Gal3 increased intracellularly, in contrast to its extracellular localization in non-reactive astrocytes, and NICD1/Gal3 pattern distribution shifted from diffuse to vesicular upon astrocyte reactivation. In vitro, Gal3–/– reactive astrocytes showed abolished Notch1 signaling at the lesion core. Notch1 receptor, its ligands (Jagged1 and Delta-like1), and Hes5 target gene were upregulated in C57Bl/6J reactive astrocytes, but not in Gal3–/– reactive astrocytes. Finally, we report that Gal3–/– mice submitted to a traumatic brain injury model in the somatosensory cortex presented a disrupted response characterized by the reduced number of GFAP reactive astrocytes, with smaller cell body perimeter and decreased NICD1 presence at the lesion core. These results suggest that Gal3 might be essential to the proper activation of Notch signaling, facilitating the cleavage of Notch1 and nuclear translocation of NICD1 into the nucleus of reactive cortical astrocytes. Additionally, we hypothesize that reactive astrocyte response could be dependent on Notch1/Jagged1-Hes5 signaling activation following brain injury.

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The effects of neuroinvasion by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) become clinically relevant due to the numerous neurological symptoms observed in Corona Virus Disease 2019 (COVID‐19) patients during infection and post‐COVID syndrome or long COVID. This study reports the biofabrication of a 3D bioprinted neural‐like tissue as a proof‐of‐concept platform for a more representative study of SARS‐CoV‐2 brain infection. Bioink is optimized regarding its biophysical properties and is mixed with murine neural cells to construct a 3D model of COVID‐19 infection. Aiming to increase the specificity to murine cells, SARS‐CoV‐2 is mouse‐adapted (MA‐SARS‐CoV‐2) in vitro, in a protocol first reported here. MA‐SARS‐CoV‐2 reveals mutations located at the Orf1a and Orf3a domains and is evolutionarily closer to the original Wuhan SARS‐CoV‐2 strain than SARS‐CoV‐2 used for adaptation. Remarkably, MA‐SARS‐CoV‐2 shows high specificity to murine cells, which present distinct responses when cultured in 2D and 3D systems, regarding cell morphology, neuroinflammation, and virus titration. MA‐SARS‐CoV‐2 represents a valuable tool in studies using animal models, and the 3D neural‐like tissue serves as a powerful in vitro platform for modeling brain infection, contributing to the development of antivirals and new treatments for COVID‐19.

After a Traumatic Brain Injury (TBI), the neural network activates a reparative response seeking to restore homeostasis. Astrocyte reactivation is an essential component of this response. The injury creates a temporal microenvironment where neurogenic signaling molecules regulate cell fate decisions of neocortical neural progenitors. Likewise, astrocyte reactivation triggers a transcriptional-proliferative program where neurogenic signaling molecules play crucial roles. However, precise molecular mechanisms are context-specific and are not fully understood. Here we studied cellular and molecular aspects of reactive astrocytes response after Notch-Wnt neurogenic signaling modulation. Our results provide new evidence of cortical Notch-Wnt signaling activation after TBI. Reactive astrocytes in the core of Notch signaling showed a differential aggregated distribution. In vitro, Notch inhibition promoted a neural precursor profile and might increase the number of cells committed in a proliferative response. Finally, we found an indirect co-regulation of Wnt-Shh signaling in BHLH-Notch target genes and a Notch-supportive effect in Wnt-Shh signaling activation.