Engineered neurogenesis in naïve adult rat cortex by Ngn2-mediated neuronal reprogramming of resident oligodendrocyte progenitor cells

Abstract

Adult tissue stem cells contribute to tissue homeostasis and repair but the long-lived neurons in the human adult cerebral cortex are not replaced, despite evidence for a limited regenerative response. However, the adult cortex contains a population of proliferating oligodendrocyte progenitor cells (OPCs). We examined the capacity of rat cortical OPCs to be re-specified to a neuronal lineage both in vitro and in vivo. Expressing the developmental transcription factor Neurogenin2 (Ngn2) in OPCs isolated from adult rat cortex resulted in their expression of early neuronal lineage markers and genes while downregulating expression of OPC markers and genes. Ngn2 induced progression through a neuronal lineage to express mature neuronal markers and functional activity as glutamatergic neurons. In vivo retroviral gene delivery of Ngn2 to naive adult rat cortex ensured restricted targeting to proliferating OPCs. Ngn2 expression in OPCs resulted in their lineage re-specification and transition through an immature neuronal morphology into mature pyramidal cortical neurons with spiny dendrites, axons, synaptic contacts, and subtype specification matching local cytoarchitecture. Lineage re-specification of rat cortical OPCs occurred without prior injury, demonstrating these glial progenitor cells need not be put into a reactive state to achieve lineage reprogramming. These results show it may be feasible to precisely engineer additional neurons directly in adult cerebral cortex for experimental study or potentially for therapeutic use to modify dysfunctional or damaged circuitry.

Keywords: NG2 cell; NeuroD1 transcription factor; Neurogenin 2; neural stem/progenitor cells; neuronal replacement; oligodendrocyte precursor cell; reprogramming and differentiation.

Figure 1

Derivation of adult rat cortical oligodendrocyte progenitor cells (OPCs). Experimental Design: (A) Genes encoding relevant developmental transcription factors were cloned into a retroviral vector with a fluorescent reporter gene. (B) Adult rat cortical gray matter was dissociated and O4-positive oligodendrocyte progenitor cells (OPCs) were selected, cultured, and transduced by retroviral delivery. β-III-tubulin expression of reporter-labeled cells served as a readout of neuronal induction. (C) Retrovirus containing successful constructs is then delivered to the cortex of a naïve animal to infect resident proliferating OPCs and induce neuronal reprogramming. Characterization of Adult Cortical OPCs: (D) Cultured O4-selected phase-bright multipolar cells with morphology consistent with an OPC identity. (E) Cells stain positive for pan-oligodendroglial lineage markers Olig2 and Sox10. (F) Isolated OPCs are NG2-positive and demonstrate a concomitant loss of nestin and increased O4 expression as cells progressed from early bipolar to later multipolar morphologies. (G) Under growth conditions, only the most elaborate O4-positive cells contained detectable staining for the premyelinating oligodendrocyte marker RIP. However, under oligodendrocyte differentiation media, (H) nearly all OPCs matured into RIP-positive oligodendrocytes, while maintaining expression of Olig2.

Figure 2

In vitro screen for reprogramming to phenotypic neurons. Screening Assay: OPC cultures received retroviral vectors for neurogenic transcription factors and were evaluated for neuronal induction based upon expression of the early neuronal marker β-III-tubulin. (A) GFP alone or (B) Pax6-GFP transduced OPCs but did not induce β-III-tubulin. Co-delivery of (C) Ascl1-dsRed and Dlx2-dsRed or (D) NeuroD1-GFP did induce β-III-tubulin. (E) Ngn2-GFP strongly induced β-III-tubulin, and induced neurons also expressed Map2. (F) Only non-transduced OPCs (non-GFP cells) continue to express Olig2 or O4 (arrowheads), indicating successful lineage respecification by Ngn2. Efficiency of Ngn2-induction: (G) The total number of cells in culture (DAPI) was reduced by about 20% over the course of the screening assay (14 days). Not all cells were transduced and the number of GFP-positive cells declined by about 90% over the course of the assay. Ngn2 successfully induced neurons, but the number of β-III-tubulin cells decreased by 80% by day 14. At all times, nearly 100% of β-III-tubulin cells were GFP-positive. Thus, loss of GFP positive cells occurred mainly in the portion that did not successfully convert into neurons. Values are mean ± standard deviation. (H) When stated in terms of efficiency, retroviral-Ngn2 transduced 60% of the cells and induced β-III-tubulin in 25% of those cells (i.e., 15% of total cells). Thus, over time, as the number of GFP cells declined, the apparent conversion efficiency increased.

Figure 3

Trajectory analysis of Ngn2-expressing OPCs during the process of neuronal reprogramming. Trajectory analysis: Following retroviral delivery of Ngn2-eGFP (0 days post-infection or DPI), OPC cultures were observed by time-lapse imaging. A video summarizing the changes with neuronal reprogramming is included as Supplementary Material and can also be found at https://doi.org/10.5281/zenodo.8189544. (A–C) Four cells (a–d) are shown at 24 h intervals as they move, change morphology, and remodel their neurite extensions (see video for a dynamic view). Cells a and b exhibit a multipolar morphology at 4 DPI, while cells c and d are already showing more bipolar processes at this time. By 6 DPI, all four cells exhibit rounded cell bodies and distinct neurite processes that connect with neighboring induced neurons. Neuronal phenotype confirmation: (D–F) Cultures were fixed and immunostained at 7 DPI, revealing that nearly all GFP-positive cells expressed the neuronal marker, NeuN. Morphological progression and NeuN-positive cells were not observed in control cultures receiving retroviral eGFP-only delivery.

Figure 4

Ngn2 Expression Reprograms OPCs into Functional Neurons. Neuronal Induction-Gene Expression: (A) Control GFP-only transduced cells exhibited a largely similar gene expression profile to OPCs in a neurogenesis pathway PCR array, while Ngn2-induced neurons demonstrate distinct gene expression changes. (B) There was greater than a 4-fold expression increase in Ngn2-induced neurons for selected genes relevant to neuronal lineage, including NeuroD1 and DCX. Olig2, a pan-oligodendroglial lineage marker was actually reduced by more than 4-fold. Neuronal Induction-Membrane Properties: (C) Ngn2-GFP induced neurons (green) co-cultured with neonatal primary rat cortical neurons elaborate Map2-positive processes (blue). Primary neurons stain only with Map2. (D) There were extensive synaptophysin-positive contacts (red) on Ngn2-GFP dendrites. Inset shows synaptophysin staining alone. (E) Synaptic contacts were also found on dendrites near the soma and frequently on the soma itself. In vitro patch clamp recordings demonstrate (F) strong sodium currents (G) repetitive firing of action potentials upon depolarizing injection, and (H) spontaneous excitatory post-synaptic current activity that is (I) largely blocked by the glutamatergic AMPA receptor antagonist CNQX, indicative of synaptic input. Values are mean ± SEM, F (2, 6) = 23.28, p = 0.0015.

Figure 5

In vivo retroviral delivery of GFP reporter-only control infects proliferating OPCs but does not induce any detectable neuronal phenotype. In Vivo Retroviral Delivery-GFP Reporter Only Control: As retrovirus infects only dividing cells and the vast majority of proliferating cells in the naïve brain are OPCs, it was expected that this population would be targeted by retroviral delivery. By 7 days following in vivo cortical delivery of retroviral-GFP control vector, a distinct population of transduced cells was observed. (A) GFP-positive cells with a complex branching morphology consistent with NG2 Glia were uniformly distributed. A systematic, quantitative examination of coexpression with GFP revealed that 99.7% of GFP-positive cells observed in the control condition were positive for the OPC marker Olig2 (red) or Sox10 (not shown) by 7 DPI. (B) GFP-positive cells were distributed amongst mature neurons (NeuN, blue) and were often closely associated with, but distinct from, neurons. NeuN-positive cells were never observed to coexpress GFP. To verify that no GFP-positive cells were other neural cell types, adjacent sections were stained for (C) Iba1 (red) to detect microglia or (D) S100β (red) to detect astrocytes. In no case were GFP-positive microglia or astrocytes observed, as would be predicted for a naïve brain without previous injury where these cells are not actively proliferating. Staining for the early neuronal marker DCX revealed an absence of DCX expression in GFP-only positive cells (DCX detection in the hippocampal dentate gyrus within the same section was used as a positive control for validation of imaging parameters).

Figure 6

Reprogramming in vivo OPCs by Ngn2 induces neuronal lineage commitment. In Vivo Retroviral Delivery-Ngn2-GFP: Within 7 days, delivery of Ngn2-GFP resulted in a population of cells (four of which are identified by a, b, c, and d) expressing the GFP reporter (A,B). Ngn2-induction also initiated neuronal lineage commitment evidenced by their co-labeling with DCX (D) and in some cells there may be some weak expression of NeuN (C), indicating a further progression toward neuronal maturation. Ngn2-induced Transitional Morphology: (E) Ngn2-GFP-positive cells exhibited elaborate process extension with the adoption of defined cell polarity, similar to the dynamic morphological transition observed in vitro in Figure 4. Cells indicated in the boxed regions are shown at right with their co-expression of DCX. The elaboration of polarized cell morphology, extensive varicose process extension, weak NeuN expression, and discontinuous DCX staining suggests adoption of early neuronal lineage with eventual transition from DCX-positive neuroblasts to NeuN-positive neurons with a mature morphology. While some very weak DCX staining was observed at 7 days following retroviral GFP control delivery, this distinct DCX-positive phenotype and polarized cell morphology was only observed following retroviral Ngn2-delivery.

Figure 7

Morphologically mature neurons induced by in vivo Ngn2-reprogramming. Superficial cortical layers: by 3 weeks, Ngn2-GFP induced neurons showed an advancement in neuronal lineage commitment as evidenced by a more mature and cytoarchitecturally appropriate neuronal morphology. Superficial cortical layers contained induced neurons exhibiting a continuum of morphological maturation, but also showing coexpression of the mature neuronal marker NeuN (blue). (A) Some Ngn2-GFP-positive cells evidenced less maturation by 3 weeks, exhibiting incomplete process polarity, varicosities in processes and a lack of dendritic spines, but still expressing NeuN as evidence of neuronal lineage commitment. However, Ngn2-induced neurons did not co-express Iba1 (red), indicating the absence of non-specific uptake of GFP by microglia. (B) Other Ngn2-induced neurons exhibited a distinctly mature neuronal morphology with dendritic branching and a descending axon that left the plane of section within 50 μm from the soma along with strong NeuN co-expression. (C) Yet other Ngn2-induced neurons exhibited a less complex morphology, but still strongly expresses NeuN. GFP signal outside of the boxed region belongs to other induced neurons and their processes that lay beyond the focal planes included in this image. Images are presented as a maximum projection of a number of focal planes to provide a three-dimensional representation. Deep cortical layers: Deeper cortical layers also contained Ngn2-induced newly-generated neurons with layer-appropriate cytoarchitecture including an Ngn2-induced neuron (D) with an inverted pyramidal morphology only seen in cortical layer V neurons. Orthogonal projections in the XZ and YZ planes at the level indicated by the yellow lines are included for each maximum projection image to validate coexpression of staining in three-dimensions. Confocal image stacks showing three examples of Ngn2-induced neurons (D–F) confirm these cells are NeuN positive (blue); arrowheads indicate removal of GFP-signal. Panels (D,E) also illustrate the absence of GFP coexpression in GFAP-positive astrocytes (red). In fact, no GFAP-positive astrocytes were observed to express GFP. Entorhinal cortex (Allocortex): In addition to the motor cortex, OPCs can be induced by retroviral Ngn2 delivery to convert to a neuronal lineage in other cortical regions. Retroviral delivery of Ngn2-GFP to the entorhinal cortex (allocortex) also induced new neurons by 3 weeks, including this example in entorhinal cortical layer III (G,H). However not all infected cells survived the reprogramming instruction as shown by the dying cell to the left of a cytoarchitecturally appropriate new neuron. The successfully reprogrammed new neuron on the right also expresses NeuN (I) as evidenced by the three-dimensional image shown digitally sectioned at the planes indicated at the blue lines. The boxed region in inset validates the colocalization with NeuN following the removal of the GFP-signal overlay (arrow). Neuronal subtype specification: The subtype specification of GFP-positive Ngn2-induced new neurons (J; arrow) to a glutamatergic phenotype is shown by their co-expression of Tbr1 (blue) and Cam-II-kinase (red). This expression is identical to adjacent pre-existing neurons (asterisk). (K) Inset shows digital resectioning of a confocal image stack at the planes indicated at the blue lines with digital removal of the GFP signal (arrow) leaving the combined Tbr1 (blue) and Cam-II-kinase (red) signal. (L) Digital removal of the Tbr1 signal leaves only the Cam-II-kinase expression, cumulatively demonstrating the coexpression of these glutamatergic phenotype markers in the induced neuron. Dendritic spine and synaptic contact formation: Apical and basal dendrites of all newly-generated neurons contain abundant small spines (M,N) shown in different orientations from a confocal image stack. Three-dimensional rendering of dendrites and spines (O) reveals that both spines (arrowheads) and dendritic shafts (arrows) receive synaptophysin-positive contacts (red) indicating that preexisting axons are making synaptic contact with the newly-generated neurons.

Figure 8

Stereological quantitation of OPC infection by retroviral delivery and subsequent neuronal reprogramming outcomes. (A) The volume of cortex containing GFP-positive cells following retroviral delivery did not differ between the control, GFP-only vector, and the experimental, Ngn2-GFP vector, at any time point. However, the reduction in cell survival over time is reflected in the declining volume occupied by GFP-positive cells over time. (B) The determination of total number of GFP-positive cells likewise showed no difference between GFP-only and Ngn2-GFP infection at any time point, although a decline in total number is evident over time. (C) GFP-positive cells were quantified for coexpression of the early neuronal lineage marker DCX at 7 days or 14 days, or coexpression with the mature neuronal lineage marker NeuN at 21 days. The GFP-only control group contained only few cells expressing some weak DCX labeling at early time points and only a single cell was observed to weakly express NeuN at 21 days. The number of GFP-positive cells expressing neuronal markers in the Ngn2-GFP group is significantly higher at all time points. (D) When expressed as a percentage of GFP-positive cells for each condition at each time point, retroviral delivery of Ngn2 resulted in a high percentage of all remaining GFP-positive cells being induced neurons at all time points.