Engineering neurogenesis: Direct conversion of resident oligodendrocyte progenitor cells to neurons in the adult mammalian cortex

Neurons fail to regenerate in the adult cerebral cortex following their loss to neurological injury or disease. This may be due to either the inhibition of cortical neurogenesis or the absence of a progenitor cell population with the capacity to adopt a neuronal fate. The goal of this thesis was to elucidate neurogenic cells in the cortex under normal conditions and in response to injury and to specify a neuronal fate for cortical oligodendrocyte progenitor cells using developmental factors that are pro-neuronal. Outcomes from a number of studies suggest that brain injury may initiate signals supporting neurogenesis. Nevertheless, new neurons are not generated as a result of injury. The neurogenic capacity of the entorhinal cortex was examined under normal conditions and following experimental lesion of the perforant pathway. Adult-derived hippocampal progenitor cells failed to differentiate following engraftment to normal or injured entorhinal cortex suggesting the lack of neurogenic signals. The expression profile of the injured entorhinal cortex was examined and revealed that factors promoting glial fate or maintaining cell phenotype were elevated.;The capacity to reprogram cell lineage using developmental transcription factors has recently emerged as a potential therapeutic strategy. Re-specifying lineage of resident cells to a new fate in the context of their normal tissue environment may confer advantages for complete phenotypic maturation and function. Previous work reported limited success with phenotypic maturation of converted non-neuronal cells, suggesting a failure to initiate the full neuronal program. The limitation to full maturation may be due to the type of non-neuronal cell being targeted for conversion and the appropriateness of the factors used to drive conversion. Oligodendrocyte Progenitor Cells (OPCs) are the most abundant proliferating resident neural cell population in the adult CNS. Furthermore, there is evidence that OPCs maintain population homeostasis, suggesting the functional population of resident OPCs would not be depleted following lineage respecification to neurons. Adult rat cortical OPCs were isolated using magnetic activated cell sorting for O4 antigen selection and maintained as a primary culture for screening combinations of putative neurogenic transcription factors including neurogenin2 (ngn2), achaete-scute complex homolog 1 (ascl1), distal-less homeobox 2 (dlx2), SRY (sex determining region Y)-box 2 (sox2), neurogenic differentiation 1 (neuroD1), paired box 6 (pax6), and a dominant negative form (see Table 3.1 caption) of oligodendrocyte transcription factor 2 (Olig2). Beta-III-tubulin-expressing cells were observed by 10 days post transduction (dpt) of OPCs with retroviral supernatants of ngn2, ascl1, ascl1/dlx2, or neuroD1, with ngn2 and neuroD1 exhibiting the most robust response. Ngn2-transduced cells co-cultured with postnatal primary neurons expressed synaptophysin and displayed neuron specific functions including sodium currents, repetitive action potentials and spontaneous synaptic activity.;As ngn2 also drove very high levels of neuroD1 expression, we delivered ngn2 to adult rat motor cortex and entorhinal cortex in the absence of experimental lesion. Delivery of vectors for GFP alone and an unsuccessful factor, pax6, validated that only proliferating OPCs had been transduced. By one week, cells transduced with ngn2 expressed doublecortin (DCX), weak NeuN, and displayed an elaborate, but still immature neuronal morphology. By three weeks, ngn2-converted cells displayed robust NeuN expression and subtype specification to glutamatergic neurons indicated by Tbr1 and CamKII? staining. These newly-generated neurons displayed elaborate cytoarchitecture appropriate for their cortical location with abundant, but immature, dendritic spines with frequent synaptophysin-positive contacts.;Forced expression of the single factor, ngn2, is sufficient to autonomously overcome the non-neurogenic barriers of the cerebral cortex through the direct conversion of OPCs to mature cortical neurons with subtype specification and synaptic contacts in two, phylogenetically distinct cortical regions. The direct conversion of resident glia may provide an autologous alternative to cell transplantation, foregoing the time, labor, and expense of ex vivo cell preparation with the potential for minimal invasive delivery.