Summary Transected axons fail to regrow in the mature central nervous system (CNS). Astrocyte scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or deleting chronic astrocyte scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. In striking contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocyte scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth supporting molecules. Our findings show that contrary to prevailing dogma, astrocyte scar formation aids rather than prevents CNS axon regeneration.

Astroglial scars surround damaged tissue after trauma, stroke, infection, or autoimmune inflammation in the CNS. They are essential for wound repair, but also interfere with axonal regrowth. A better understanding of the cellular mechanisms, regulation, and functions of astroglial scar formation is fundamental to developing safe interventions for many CNS disorders. We used wild-type and transgenic mice to quantify and dissect these parameters. Adjacent to crush spinal cord injury (SCI), reactive astrocytes exhibited heterogeneous phenotypes as regards proliferation, morphology, and chemistry, which all varied with distance from lesions. Mature scar borders at 14 d after SCI consisted primarily of newly proliferated astroglia with elongated cell processes that surrounded large and small clusters of inflammatory, fibrotic, and other cells. During scar formation from 5 to 14 d after SCI, cell processes deriving from different astroglia associated into overlapping bundles that quantifiably reoriented and organized into dense mesh-like arrangements. Selective deletion of STAT3 from astroglia quantifiably disrupted the organization of elongated astroglia into scar borders, and caused a failure of astroglia to surround inflammatory cells, resulting in increased spread of these cells and neuronal loss. In cocultures, wild-type astroglia spontaneously corralled inflammatory or fibromeningeal cells into segregated clusters, whereas STAT3-deficient astroglia failed to do so. These findings demonstrate heterogeneity of reactive astroglia and show that scar borders are formed by newly proliferated, elongated astroglia, which organize via STAT3-dependent mechanisms to corral inflammatory and fibrotic cells into discrete areas separated from adjacent tissue that contains viable neurons.

Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, which display mutant huntingtin in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models is not associated with classical astrogliosis, but is associated with decreased Kir4.1 K+ channel functional expression, leading to elevated in vivo levels of striatal extracellular K+, which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K+, recovered aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte–mediated K+ homeostasis, revealing astrocytes and Kir4.1 channels as novel therapeutic targets.

Differential expression analysis in single-cell transcriptomics enables the dissection of cell-type-specific responses to perturbations such as disease, trauma, or experimental manipulations. While many statistical methods are available to identify differentially expressed genes, the principles that distinguish these methods and their performance remain unclear. Here, we show that the relative performance of these methods is contingent on their ability to account for variation between biological replicates. Methods that ignore this inevitable variation are biased and prone to false discoveries. Indeed, the most widely used methods can discover hundreds of differentially expressed genes in the absence of biological differences. To exemplify these principles, we exposed true and false discoveries of differentially expressed genes in the injured mouse spinal cord.