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.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Altered expression and function of astroglial gap junction protein Connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). While earlier studies have examined the effect of increased amyloid-β (Aβ) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aβ modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aβ can alter Cx43 gap junctions. We show that Aβ25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aβ25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aβ25-35 exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aβ-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aβ25-35 exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aβ25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aβ can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aβ modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

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.

Schwann cells are the principal glial cells of the peripheral nervous system, and their development into myelinating glia is critically dependent on MEK/ERK signaling. Ets-domain transcription factors (Etv1, Etv4, Etv5) are common downstream effectors of MEK/ERK signalling, but so far, only Etv1 has been ascribed a role in Schwann cell development, and only in non-myelinating cells. Here, we examined the role of Etv5, which is expressed in Schwann cell precursors, including neural crest cells and satellite glia, in Schwann cell lineage development. We analysed Etv5tm1Kmm mutants (designated Etv5−/−) at embryonic days (E) 12.5, E15.5 and E18.5, focusing on dorsal root ganglia. At these embryonic stages, satellite glia (glutamine synthetase) and Schwann cell markers, including transcriptional regulators (Sox10, Sox9, Tfap2a, Pou3f1) and non-transcription factors (Ngfr, BFABP, GFAP), were expressed in the DRG of wild-type and Etv5−/− embryos. Furthermore, by E18.5, quantification of Sox10+ Schwann cells and NeuN+ neurons revealed that these cells were present in normal numbers in the Etv5−/− dorsal root ganglia. We next performed peripheral nerve injuries at postnatal day 21, revealing that Etv5−/− mice had an enhanced injury response, generating more Sox10+ Schwann cells compared to wild-type animals at five days post-injury. Thus, while Etv5 is not required for Schwann cell development, possibly due to genetic redundancy with Etv1 and/or Etv4, Etv5 is an essential negative regulator of the peripheral nerve injury repair response.SIGNIFICANCE STATEMENTOur study sought to determine whether the ets domain transcription factor, Etv5, plays a role in regulating Schwann cell development and nerve repair. By using an embryonically and postnatally viable hypomorphic Etv5 mutant allele, we demonstrated that Etv5 is not required for the development of Schwann cells or other neural crest derivatives in the dorsal root ganglia, including satellite glia and neurons. Surprisingly, loss of Etv5 had a direct impact on the Schwann cell repair response post-injury, resulting in more Schwann cells populating the distal injured nerve site compared to wild-type animals. Thus, this work describes for the first time a role for Etv5 in regulating the Schwann cell repair response after peripheral nerve injury.

Oligodendrogliomas are lower-grade, slow-growing gliomas that are ultimately fatal. Although driver mutations are known, the mechanisms underlying their signature slow growth rates are poorly understood. We found evidence for intra-tumoral interactions between neoplastic and nonneoplastic cells in oligodendroglioma tissues. To further study these cell interactions, we used two patient-derived oligodendroglioma cell lines of lower and higher aggressivity. Both oligodendroglioma cell lines released extracellular vesicles that had cytotoxic effects on nonneoplastic and neoplastic cells, but each had distinct vesicular proteomes. Consistent with extracellular vesicles mediating growth inhibitory effects in oligodendrogliomas, higher expression levels of several extracellular vesicle biogenesis genes (SMPD3,TSG101, STAM1) correlates with longer survival in oligodendroglioma patients. Furthermore, SMPD3 overexpression slows oligodendroglioma cell growth in culture. Conversely, SMPD3 knockdown enhances oligodendroglioma proliferation in vitro, in murine xenografts, and in human cerebral organoid co-cultures. Oligodendroglioma-derived extracellular vesicles thus mediate tumor cell microenvironmental interactions that contribute to low aggressivity.

Oligodendrogliomas are lower-grade, slow-growing gliomas that are ultimately fatal. Although driver mutations are known, the mechanisms underlying their signature slow growth rates are poorly understood. We found evidence for intra-tumoral interactions between neoplastic and non-neoplastic cells in oligodendroglioma tissues. To further study these cell interactions, we used two patient-derived oligodendroglioma cell lines of lower and higher aggressivity. Both oligodendroglioma cell lines released extracellular vesicles that had cytotoxic effects on non-neoplastic and neoplastic cells, but each had distinct vesicular proteomes. Consistent with extracellular vesicles mediating growth inhibitory effects in oligodendrogliomas, higher expression levels of several extracellular vesicle biogenesis genes (SMPD3, TSG101, STAM1) correlates with longer survival in oligodendroglioma patients. Furthermore, SMPD3 overexpression slows oligodendroglioma cell growth in culture. Conversely, SMPD3 knockdown enhances oligodendroglioma proliferation in vitro, in murine xenografts, and in human cerebral organoid co-cultures. Oligodendroglioma-derived extracellular vesicles thus mediate tumor cell microenvironmental interactions that contribute to low aggressivity.