Direct neuronal reprogramming is an innovative new technology that involves the conversion of somatic cells to induced neurons (iNs) without passing through a pluripotent state. The capacity to make new neurons in the brain, which previously was not achievable, has created great excitement in the field as it has opened the door for the potential treatment of incurable neurodegenerative diseases and brain injuries such as stroke. These neurological disorders are associated with frank neuronal loss, and as new neurons are not made in most of the adult brain, treatment options are limited. Developmental biologists have paved the way for the field of direct neuronal reprogramming by identifying both intrinsic cues, primarily transcription factors (TFs) and miRNAs, and extrinsic cues, including growth factors and other signaling molecules, that induce neurogenesis and specify neuronal subtype identities in the embryonic brain. The striking observation that postmitotic, terminally differentiated somatic cells can be converted to iNs by mis-expression of TFs or miRNAs involved in neural lineage development, and/or by exposure to growth factors or small molecule cocktails that recapitulate the signaling environment of the developing brain, has opened the door to the rapid expansion of new neuronal reprogramming methodologies. Furthermore, the more recent applications of neuronal lineage conversion strategies that target resident glial cells in situ has expanded the clinical potential of direct neuronal reprogramming techniques. Herein, we present an overview of the history, accomplishments, and therapeutic potential of direct neuronal reprogramming as revealed over the last two decades.

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Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1SA6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1SA6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1SA6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1SA6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1SA6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1SA6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1SA6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1SA6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1SA6 vs. Ascl1, and position Ascl1SA6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.

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Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.