Reprogramming Glial Cells into Functional Neurons for Neuro-regeneration: Challenges and Promise.

The capacity for neurogenesis in the adult mammalian brain is extremely limited and highly restricted to a few regions, which greatly hampers neuronal regeneration and functional restoration after neuronal loss caused by injury or disease. Meanwhile, transplantation of exogenous neuronal stem cells into the brain encounters several serious issues including immune rejection and the risk of tumorigenesis. Recent discoveries of direct reprogramming of endogenous glial cells into functional neurons have provided new opportunities for adult neuro-regeneration. Here, we extensively review the experimental findings of the direct conversion of glial cells to neurons in vitro and in vivo and discuss the remaining issues and challenges related to the glial subtypes and the specificity and efficiency of direct cell-reprograming, as well as the influence of the microenvironment. Although in situ glial cell reprogramming offers great potential for neuronal repair in the injured or diseased brain, it still needs a large amount of research to pave the way to therapeutic application.

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo.

In vivo induction of non-neuronal cells into neurons by transcription factors offers potential therapeutic approaches for neural regeneration. Although generation of induced neuronal (iN) cells in vitro and in vivo has been reported, whether iN cells can be fully integrated into existing circuits remains unclear. Here we show that expression of achaete-scute complex homolog-like 1 (Ascl1) alone is sufficient to convert dorsal midbrain astrocytes of mice into functional iN cells in vitro and in vivo. Specific expression of Ascl1 in astrocytes by infection with GFAP-adeno-associated virus (AAV) vector converts astrocytes in dorsal midbrain, striatum, and somatosensory cortex of postnatal and adult mice into functional neurons in vivo. These iN cells mature progressively, exhibiting neuronal morphology and markers, action potentials, and synaptic inputs from and output to existing neurons. Thus, a single transcription factor, Ascl1, is sufficient to convert brain astrocytes into functional neurons, and GFAP-AAV is an efficient vector for generating iN cells from astrocytes in vivo.

Tlx3 controls cholinergic transmitter and Peptide phenotypes in a subset of prenatal sympathetic neurons.

The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.

Tlx1/3 and Ptf1a control the expression of distinct sets of transmitter and peptide receptor genes in the developing dorsal spinal cord.

Establishing the pattern of expression of transmitters and peptides as well as their receptors in different neuronal types is crucial for understanding the circuitry in various regions of the brain. Previous studies have demonstrated that the transmitter and peptide phenotypes in mouse dorsal spinal cord neurons are determined by the transcription factors Tlx1/3 and Ptf1a. Here we show that these transcription factors also determine the expression of two distinct sets of transmitter and peptide receptor genes in this region. We have screened the expression of 78 receptor genes in the spinal dorsal horn by in situ hybridization. We found that receptor genes Gabra1, Gabra5, Gabrb2, Gria3, Grin3a, Grin3b, Galr1, and Npy1r were preferentially expressed in Tlx3-expressing glutamatergic neurons and their derivatives, and deletion of Tlx1 and Tlx3 resulted in the loss of expression of these receptor genes. Furthermore, we obtained genetic evidence that Tlx3 uses distinct pathways to control the expression of receptor genes. We also found that receptor genes Grm3, Grm4, Grm5, Grik1, Grik2, Grik3, and Sstr2 were mainly expressed in Pax2-expressing GABAergic neurons in the spinal dorsal horn, and their expression in this region was abolished or markedly reduced in Ptf1a and Pax2 deletion mutant mice. Together, our studies indicate that Tlx1/3 and Ptf1a, the key transcription factors for fate determination of glutamatergic and GABAergic neurons in the dorsal spinal cord, are also responsible for controlling the expression of two distinct sets of transmitter and peptide receptor genes.

c-Maf is required for the development of dorsal horn laminae III/IV neurons and mechanoreceptive DRG axon projections.

Establishment of proper connectivity between peripheral sensory neurons and their central targets is required for an animal to sense and respond to various external stimuli. Dorsal root ganglion (DRG) neurons convey sensory signals of different modalities via their axon projections to distinct laminae in the dorsal horn of the spinal cord. In this study, we found that c-Maf was expressed predominantly in the interneurons of laminae III/IV, which primarily receive inputs from mechanoreceptive DRG neurons. In the DRG, c-Maf⁺ neurons also coexpressed neurofilament-200, a marker for the medium- and large-diameter myelinated afferents that transmit non-noxious information. Furthermore, mouse embryos deficient in c-Maf displayed abnormal development of dorsal horn laminae III/IV neurons, as revealed by the marked reduction in the expression of several marker genes for these neurons, including those for transcription factors MafA and Rora, GABA(A) receptor subunit α5, and neuropeptide cholecystokinin. In addition, among the four major subpopulations of DRG neurons marked by expression of TrkA, TrkB, TrkC, and MafA/GFRα2/Ret, c-Maf was required selectively for the proper differentiation of MafA⁺/Ret⁺/GFRα2⁺ low-threshold mechanoreceptors (LTMs). Last, we found that the central and peripheral projections of mechanoreceptive DRG neurons were compromised in c-Maf deletion mice. Together, our results indicate that c-Maf is required for the proper development of MafA⁺/Ret⁺/GFRα2⁺ LTMs in the DRG, their afferent projections in the dorsal horn and Pacinian corpuscles, as well as neurons in laminae III/IV of the spinal cord.

Wnt1-cre-mediated conditional loss of Dicer results in malformation of the midbrain and cerebellum and failure of neural crest and dopaminergic differentiation in mice.

The involvement of microRNAs (miRNAs) in the development of the neural crest (NC) cells and other neuronal differentiation is still poorly understood. Here, we investigated the global function of miRNAs in embryonic development by examining the Wnt1-cre-mediated Dicer knockout mice. Dicer ablation resulted in malformation of the midbrain and cerebellum and failure of NC and dopaminergic differentiation. First, the Dicer mutant fetuses exhibited dramatic malformation of the tectum and cerebellum and the eyelids were open. Second, the skeletal structures that are derived from the cranial NC were lost or mostly ablated in Dicer mutant mice. Third, deletion of Dicer in the NC cells resulted in the malformation of the dorsal root ganglia, enteric nervous system and sympathetic ganglia. Interestingly, the expression of neuropeptide Y and its potential regulators TrkA, AP-2alpha and AP-2beta was largely abolished in sympathetic neurons of Dicer mutant mice. Fourth, in situ hybridization data revealed that the expression of miR-9, miR-124 and miR-218 in the midbrain and rostral hindbrain area was mostly eliminated in the Dicer mutant mice. We then demonstrated that the development of dopaminergic neurons was impaired in Dicer-deleted mice. Our studies therefore suggest that miRNAs contribute to the embryonic development in multiple locations.

Different transcription factors regulate nestin gene expression during P19 cell neural differentiation and central nervous system development.

Nestin is a molecular marker for neural progenitor cells. Rat and human nestin genes possess a central nervous system-specific enhancer within their second introns. However, the transcription factors that bind to the nestin enhancer have not been fully elucidated. Here, we show that the second intron of the mouse nestin gene is sufficient to drive reporter gene expression in the developing nervous system. The core sequence of this central nervous system-specific enhancer localizes to the 3' 320-bp region. The cis-elements for Sox and POU family transcription factors and the hormone-responsive element are essential for nestin expression during embryonic carcinoma P19 cell neural differentiation and in the developing chick neural tube. Interestingly, different transcription factors bind to the nestin enhancer at different stages of P19 cell neural differentiation and central nervous system development. Sox2 and SF1 may mediate basal nestin expression in undifferentiated P19EC cells, whereas Sox2, Brn1, and Brn2 bind to the enhancer in P19 neural progenitor cells. Similarly, in vivo, Oct1 binds to the nestin enhancer in embryonic day 8.5 (E8.5) mouse embryos, and Oct1, Brn1, and Brn2 bind to this enhancer in E10.5 and E12.5 mouse embryos. Our studies therefore suggest a temporal coordination of transcription factors in determining nestin gene expression.

Regulation of apoptosis of rbf mutant cells during Drosophila development.

Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.

Second intron of mouse nestin gene directs its expression in pluripotent embryonic carcinoma cells through POU factor binding site.

Nestin, an intermediate filament protein, is expressed in the neural stem cells of the developing central nervous system. This tissue-specific expression is driven by the neural stem cell-specific enhancer in the second intron of the nestin gene. In this study, we showed that the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19 and F9 cells, not in the differentiated cell types. This cell type-specific expression was conferred by the enhancer in the second intron. Mutation of the conserved POU factor-binding site in the enhancer abolished the reporter gene expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed with nestin in EC cells. Electrophoretic mobility-shift assays and supershift assays showed that a unique protein-DNA complex was formed specifically with nuclear extracts of EC cells, and Oct4 protein was included. Together, these results suggest the functional relevance between the conserved POU factor-binding site and the expression of the nestin gene in pluripotent EC cells.

Characterization and promoter analysis of the mouse nestin gene.

The intermediate filament protein nestin is expressed in the neural stem cells of the developing central nervous system (CNS). Promoter analysis revealed that the minimal promoter of the mouse nestin gene resides in the region -11 to +183 of the 5'-non-coding and upstream flanking region, and that two adjacent Sp1-binding sites are necessary for promoter activity. Electrophoretic mobility-shift assays (EMSA) and supershift assays showed that Sp1 and Sp3 proteins selectively bind to the upstream Sp1 site. These results demonstrate an important functionality of Sp1 and Sp3 in regulating the expression of the mouse nestin gene.

Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates.

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

Proper development of relay somatic sensory neurons and D2/D4 interneurons requires homeobox genes Rnx/Tlx-3 and Tlx-1.

Trigeminal nuclei and the dorsal spinal cord are first-order relay stations for processing somatic sensory information such as touch, pain, and temperature. The origins and development of these neurons are poorly understood. Here we show that relay somatic sensory neurons and D2/D4 dorsal interneurons likely derive from Mash1-positive neural precursors, and depend on two related homeobox genes, Rnx and Tlx-1, for proper formation. Rnx and Tlx-1 maintain expression of Drg11, a homeobox gene critical for the development of pain circuitry, and are essential for the ingrowth of trkA+ nociceptive/thermoceptive sensory afferents to their central targets. We showed previously that Rnx is necessary for proper formation of the nucleus of solitary tract, the target for visceral sensory afferents. Together, our studies demonstrate a central role for Rnx and Tlx-1 in the development of two major classes of relay sensory neurons, somatic and visceral.