Abstract
The master neuronal transcription factor NeuroD1 can directly reprogram astrocytes into induced neurons (iNeurons) after stroke. Using viral vectors to drive ectopic ND1 expression in gliotic astrocytes after brain injury presents an autologous form of cell therapy for neurodegenerative disease. Cultured astrocytes transfected with ND1 exhibited reduced proliferation and adopted neuronal morphology within 2–3 weeks later, expressed neuronal/synaptic markers, and extended processes. Whole-cell recordings detected the firing of evoked action potentials in converted iNeurons. Focal ischemic stroke was induced in adult GFAP-Cre-Rosa-YFP mice that then received ND1 lentivirus injections into the peri-infarct region 7 days after stroke. Reprogrammed cells did not express stemness genes, while 2–6 weeks later converted cells were co-labeled with YFP (constitutively activated in astrocytes), mCherry (ND1 infection marker), and NeuN (mature neuronal marker). Approximately 66% of infected cells became NeuN-positive neurons. The majority (~80%) of converted cells expressed the vascular glutamate transporter (vGLUT) of glutamatergic neurons. ND1 treatment reduced astrogliosis, and some iNeurons located/survived inside of the savaged ischemic core. Western blotting detected higher levels of BDNF, FGF, and PSD-95 in ND1-treated mice. MultiElectrode Array (MEA) recordings in brain slices revealed that the ND1-induced reprogramming restored interrupted cortical circuits and synaptic plasticity. Furthermore, ND1 treatment significantly improved locomotor, sensorimotor, and psychological functions. Thus, conversion of endogenous astrocytes to neurons represents a plausible, on-site regenerative therapy for stroke.
Highlights
Ischemic stroke is a devastating disease with limited therapies available (Liu et al, 2014; Ginsberg, 2016; Catanese et al, 2017)
Among single transcription factor-mediated direct reprogramming candidate genes, both ND1 and Ngn2 demonstrated an ability to reprogram reactive astrocytes but ND1 was selected based on both efficiency and efficacy (Supplementary Figure 1)
By 4 weeks, 58.6% of astrocytes were positive for the immature neuronal marker Tuj1 (Figure 1F)
Summary
Ischemic stroke is a devastating disease with limited therapies available (Liu et al, 2014; Ginsberg, 2016; Catanese et al, 2017). Stem cell transplantation has emerged as a promising regenerative therapy for stroke due to its potential for repairing damaged brain structures and improving functional recovery (Liu et al, 2014; Wei et al, 2017). Resident astrocytes in the brain remain mitotic throughout the lifespan and undergo rapid gliosis in response to injury. This characteristic response provides a rich source of cells adjacent to the site of injury (Liu and Chopp, 2016). The accumulation of reactive astrocytes and astrogliosis during later phases after stroke is viewed as a major obstacle for regenerative therapy involving either endogenous or transplanted cells
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