NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes

Abstract

SummaryCurrently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier without injury, to deliver NEUROD1 to astrocytes through an intravascular route. Interestingly, we found that a small, but significant number of non-reactive astrocytes converted to neurons in the striatum, but not the cortex. Moreover, astrocytes cultured to minimize their proliferative potential also exhibited limited neuronal transdifferentiation with NEUROD1 expression. Our results show that a single transcription factor can induce astrocyte-to-neuron conversion under physiological conditions, potentially facilitating future clinical approaches long after the acute injury phase.

Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion

“…We first established mouse astrocytes in vitro and cultured them without growth factors ( Figure S5A), a condition under which they display properties closely resembling those of non-reactive astrocytes in the physiological state (White et al, 2011). In accordance with our earlier findings (Brulet et al, 2017), we observed few Map2ab-positive cells in ND1-transduced non-reactive astrocytes at 7 dpt ( Figures S5B and S5C). This result is in marked contrast to a previous study in which astrocytes were grown in the presence of epidermal growth factor (EGF) and fibroblast Figure 3.…”

“…These findings suggest that ND1 instructs reprogramming of the epigenetic landscape to silence microglial cell identity. Because ND1 does not always effectively induce neuronal reprogramming in cells such as non-reactive astrocytes (Brulet et al, 2017), we believe that future studies (e.g., stepwise analysis of the epigenetic profile and chromatin modifier dynamics during neuronal reprogramming from different types of somatic cells) will yield insights into how and when ND1 achieves the rewriting of the epigenetic landscape.…”

“…To assess whether microglia can be converted into neurons, we first prepared and cultured CD11b-, CD68-, and Iba1-positive microglia in vitro and confirmed that they did not contain bIIItubulin-positive neurons, Nestin-positive neural stem and precursor cells (NS/PCs), glial gibrillary acidic protein (GFAP)-positive astrocytes, or Olig2-positive oligodendrocyte precursor cells, indicating that they were highly enriched microglia (Figures S1A and S1B). We selected eight transcription factors (Ascl1, Brn2, Myt1l, Olig2, Zic1, Sox2, Neurog2, and ND1), one microRNA (miR124), and one epigenetic factor (MBD3) as candi-dates based on their known roles in reprogramming (Ambasudhan et al, 2011;Brulet et al, 2017;Heinrich et al, 2010;Niu et al, 2013;Pang et al, 2011;Rais et al, 2013;Vierbuchen et al, 2010). We used lentiviruses encoding each of these factors or short hairpin RNA (shRNA) for MBD3 and expressed them under the control of the doxycycline (Dox)-inducible tetracycline response element (TRE) promoter ( Figures 1A and S1C).…”

“…Cultured mouse astrocytes have also been converted to neurons with a single transcription factor, such as ND1 or Neurog2 (Berninger et al, 2007;Guo et al, 2014;Heinrich et al, 2010). More recent studies indicate that exogenous Sox2 or ND1 expression converts endogenous astrocytes in the mouse brain and spinal cord to neurons (Brulet et al, 2017;Guo et al, 2014;Niu et al, 2013Niu et al, , 2015Su et al, 2014;Wang et al, 2016). Because glial cells, including astrocytes, proliferate in response to injury in the CNS and eventually form a glial scar (Sofroniew, 2009), in vivo neuronal conversion from astrocytes raises the possibility of modifying gliotic tissues to provide a cellular source for replenishing impaired neuronal circuits.…”

Regeneration of dorsal spinal cord neurons after injury viain situNeuroD1-mediated astrocyte-to-neuron conversion

“…Since certain serotypes of AAV can cross the blood-brain-barrier (BBB), intravenous delivery of conversion factors has make it possible to reach a broader area of the CNS (Chan et al, 2017;Foust et al, 2009). Following our earlier report of efficient conversion of reactive astrocytes into functional neurons by NeuroD1 through retroviral infection (Guo et al, 2014), intravenous injection of AAV9 expressing NeuroD1 has been reported to infect a small but significant number of resting astrocytes in the striatum and convert them into neurons (Brulet et al, 2017). On the other hand, our intracranial injection of AAV9 expressing NeuroD1 in stab injury model (Zhang et al, 2018) and ischemic injury model (Chen et al, 2018) have both resulted in high conversion efficiency, suggesting that reactive astrocytes are more likely converted into neurons than the resting astrocytes.…”