MicroRNA-375 Is Induced during Astrocyte-to-Neuron Reprogramming and Promotes Survival of Reprogrammed Neurons when Overexpressed.

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. To understand the function of miRNAs during the AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte (HA) cultures upon NeuroD1 overexpression. Bioinformatics analyses showed that NeuroD1 not only activated essential neuronal genes to initiate the reprogramming process but also induced miRNA changes in HA. Among the upregulated miRNAs, we identified miR-375 and its targets, neuronal ELAVL genes (nELAVLs), which encode a family of RNA-binding proteins and were also upregulated by NeuroD1. We further showed that manipulating the miR-375 level regulated nELAVLs' expression during NeuroD1-mediated reprogramming. Interestingly, miR-375/nELAVLs were also induced by the reprogramming factors Neurog2 and ASCL1 in HA, suggesting a conserved function to neuronal reprogramming, and by NeuroD1 in the mouse astrocyte culture and spinal cord. Functionally, we showed that miR-375 overexpression improved NeuroD1-mediated reprogramming efficiency by promoting cell survival at early stages in HA and did not appear to compromise the maturation of the reprogrammed neurons. Lastly, overexpression of miR-375-refractory ELAVL4 induced apoptosis and reversed the cell survival-promoting effect of miR-375 during AtN reprogramming. Together, we demonstrated a neuroprotective role of miR-375 during NeuroD1-mediated AtN reprogramming.

MicroRNA-375 is induced during astrocyte-to-neuron reprogramming and promotes survival of reprogrammed neurons when overexpressed.

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. MicroRNAs (miRNAs), as post-transcriptional regulators of gene expression, play crucial roles during development and under various pathological conditions. To understand the function of miRNAs during AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte (HA) cultures upon NeuroD1 overexpression. Bioinformatics analyses showed that NeuroD1 not only activates essential neuronal genes to initiate reprogramming process but also induces miRNA changes in HA. Among the upregulated miRNAs, we identified miR-375 and its targets, neuronal ELAVL genes ( nELAVLs ), which encode a family of RNA-binding proteins and are also upregulated by NeuroD1. We further showed that manipulating miR-375 level regulates nELAVLs expression during NeuroD1-mediated reprogramming. Interestingly, miR-375/ nELAVLs are also induced by reprogramming factors Neurog2 and ASCL1 in HA suggesting a conserved function to neuronal reprogramming, and by NeuroD1 in the mouse astrocyte culture and spinal cord. Functionally, we showed that miR-375 overexpression improves NeuroD1-mediated reprogramming efficiency by promoting cell survival at early stages in HA even in cultures treated with the chemotherapy drug Cisplatin. Moreover, miR-375 overexpression doesn't appear to compromise maturation of the reprogrammed neurons in long term HA cultures. Lastly, overexpression of miR-375-refractory ELAVL4 induces apoptosis and reverses the cell survival-promoting effect of miR-375 during AtN reprogramming. Together, we demonstrate a neuro-protective role of miR-375 during NeuroD1-mediated AtN reprogramming and suggest a strategy of combinatory overexpression of NeuroD1 and miR-375 for improving neuronal reprogramming efficiency.

Transcriptomic analyses of NeuroD1-mediated astrocyte-to-neuron conversion.

Ectopic expression of a single neural transcription factor NeuroD1 can reprogram reactive glial cells into functional neurons both in vitro and in vivo, but the underlying mechanisms are not well understood yet. Here, we used RNA-sequencing technology to capture the transcriptomic changes at different time points during the reprogramming process. We found that following NeuroD1 overexpression, astroglial genes (ACTG1, ALDH1A3, EMP1, CLDN6, SOX21) were significantly downregulated, whereas neuronal genes (DCX, RBFOX3/NeuN, CUX2, RELN, SNAP25) were significantly upregulated. NeuroD family members (NeuroD1/2/6) and signaling pathways (Wnt, MAPK, cAMP) as well as neurotransmitter receptors (acetylcholine, somatostatin, dopamine) were also significantly upregulated. Gene co-expression analysis identified many central genes among the NeuroD1-interacting network, including CABP7, KIAA1456, SSTR2, GADD45G, LRRTM2, and INSM1. Compared to chemical conversion, we found that NeuroD1 acted as a strong driving force and triggered fast transcriptomic changes during astrocyte-to-neuron conversion process. Together, this study reveals many important downstream targets of NeuroD1 such as HES6, BHLHE22, INSM1, CHRNA1/3, CABP7, and SSTR2, which may play critical roles during the transcriptomic landscape shift from a glial profile to a neuronal profile.

Regeneration of Functional Neurons After Spinal Cord Injury via in situ NeuroD1-Mediated Astrocyte-to-Neuron Conversion.

Spinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimer's disease by overexpressing a single neural transcription factor NeuroD1. Here we demonstrate regeneration of spinal cord neurons from reactive astrocytes after SCI through AAV NeuroD1-based gene therapy. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (~95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate after conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model with a long delay after injury, allowing future studies to further evaluate this in vivo reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift from classical axonal regeneration to neuronal regeneration for spinal cord repair, using in vivo astrocyte-to-neuron conversion technology to regenerate functional new neurons in the gray matter.

In situ structural characterization of ferric iron dimers in aqueous solutions: identification of µ-oxo species.

The structure of ferric iron (Fe(3+)) dimers in aqueous solutions has long been debated. In this work, we have determined the dimer structure in situ in aqueous solutions using extended X-ray absorption fine structure (EXAFS) spectroscopy. An Fe K-edge EXAFS analysis of 0.2 M ferric nitrate solutions at pH 1.28-1.81 identified a Fe-Fe distance at ∼3.6 Å, strongly indicating that the dimers take the µ-oxo form. The EXAFS analysis also indicates two short Fe-O bonds at ∼1.80 Å and ten long Fe-O bonds at ∼2.08 Å, consistent with the µ-oxo dimer structure. The scattering from the Fe-Fe paths interferes destructively with that from paths belonging to Fe(OH2)6(3+) monomers that coexist with the dimers, leading to a less apparent Fe shell in the EXAFS Fourier transform. This might be a reason why the characteristic Fe-Fe distance was not detected in previous EXAFS studies. The existence of µ-oxo dimers is further confirmed by Mössbauer analyses of analogous quick frozen solutions. This work also explores the electronic structure and the relative stability of the µ-oxo dimer in a comparison to the dihydroxo dimer using density function theory (DFT) calculations. The identification of such dimers in aqueous solutions has important implications for iron (bio)inorganic chemistry and geochemistry, such as understanding the formation mechanisms of Fe oxyhydroxides at molecular scale.