TRANsCre-DIONE transdifferentiates scar-forming reactive astrocytes into functional motor neurons

Abstract

SUMMARYIn spinal cord injury (SCI), the scar-forming reactive astrocytes with upregulated GFAP proliferate aberrantly near the injury site, allowing themselves as a prime target for transdifferentiation into neurons to replenish dead neurons. However, the conventional use of GFAP promoter to target reactive astrocytes has two inherent problems: inadvertent conversion of normal astrocytes and low efficiency due to progressive weakening of promoter activity during transdifferentiation. Here, we report that the scar-forming reactive astrocytes are selectively transdifferentiated into neurons with 87% efficiency and 96% specificity via TRANsCre-DIONE, a combination of the split-Cre system under two different promoters of GFAP and Lcn2 and a Cre-loxP-dependent inversion and expression of Neurog2 under the strong EF1α promoter. After SCI, TRANsCre-DIONE caused transdifferentiation into Isl1-positive motor neurons, reduced astrogliosis, enhanced regeneration in surrounding cells, and a significant motor recovery. Our study proposes TRANsCre-DIONE as the next-generation therapeutic approach for patients suffering from SCI.HighlightsTRANsCre-DIONE converts reactive astrocyte into neuron by over-expression of Neurog2 Reactive astrocytes are targeted using split-Cre under two promoters, GFAP and Lcn2 TRANsCre-DIONE reduces reactivity, replaces dead neurons and alleviates symptom of SCI Transdifferentiated-neurons are GABA+ in the striatum and Isl1+ in the spinal cord

Longitudinal intravital imaging of cerebral microinfarction reveals a dynamic astrocyte reaction leading to glial scar formation

“…LCN2 has been reported as an autocrine factor for reactive astrocytosis (Lee et al, 2009), has a neurotoxicity, and can be secreted from GFAP + reactive astrocytes in neurodegenerative disease animal model (Bi et al, 2013). Additionally, severe reactive astrocytes recognized by co-expression of LCN2 and GFAP were characterized as scar-forming reactive astrocytes in spinal cord injury animal model (An et al, 2020).…”

Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar

“…Custom-built video-rate laser-scanning confocal and two-photon microscope system previously implemented (Ahn, Kong et al, 2019, Ahn, Choe et al, 2017, Choe, Hwang et al, 2013, Lee, Kong et al, 2020, Park, Choe et al, 2018, Park, Kim et al, 2019, Seo, Hwang et al, 2015) was used. Four continuous-wave laser modules with output wavelengths at 405nm (OBIS 405, Coherent), 488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt) and 640 nm (MLD640, Cobolt) were used as an excitation sources for confocal microscope.…”

Challenges and Opportunities of Targeting Astrocytes to Halt Neurodegenerative Disorders

“…A complementary intriguing option that is recently coming to the spotlight is to restore lost functions by exploiting the potential of the astrocytes to trans-differentiate into neurons. In particular, it has been shown that forced expression of selected transcription factors can transform astrocytes into glutamatergic [240][241][242][243][244], GABAergic [243,245,246], dopaminergic [247], retinal [248,249], and motor neurons [250] in mice. Intriguingly, astrocyte trans-differentiation into neurons can be achieved also by downregulating the expression of the polypyrimidine tract binding protein 1 gene (Ptbp1) by either a short hairpin RNA [251] or by expression of an ortholog of CRISPR-Cas13d (CasRx) and two suitable guide RNAs [252].…”