NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes.
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ABSTRACT:
SUBMITTER: Brulet R
PROVIDER: S-EPMC5470076 | biostudies-literature | 2017 Jun
REPOSITORIES: biostudies-literature
Publications
NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes.
Brulet Rebecca R Matsuda Taito T Zhang Ling L Miranda Carlos C Giacca Mauro M Kaspar Brian K BK Nakashima Kinichi K Hsieh Jenny J
Stem cell reports 20170511 6
Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses th ...[more]
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Project description:BackgroundReprogramming resident glial cells to convert them into neurons in vivo represents a potential therapeutic strategy that could replenish lost neurons, repair damaged neural circuits, and restore function. AAV (adeno-associated virus)-based expression systems are powerful tools for in vivo gene delivery in glia-to-neuron reprogramming, however, recent studies show that AAV-based gene delivery of Neurod1 into the mouse brain can cause severe leaky expression into endogenous neurons leading to misinterpretation of glia-to-neuron conversion.MethodsAAV-based delivery systems were modified for improved in vivo delivery of Neurod1, Math5, Ascl1, and Neurog2 in the adult mouse retina and brain. To examine whether bona fide glia-to-neuron conversion occurs, stringent fate mapping experiments were performed to trace the lineage of glial cells.FindingsThe neuronal leakage is prevalent after AAV-GFAP-mediated delivery of Neurod1, Math5, Ascl1, and Neurog2. The transgene-dependent leakage cannot be corrected after lowering the AAV doses, using alterative AAV serotypes or injection routes. Importantly, we report the development of two new AAV-based tools that can significantly reduce neuronal leakage. Using the new AAV-based tools, we provide evidence that Neurod1 gene transfer fails to convert lineage traced glial cells into neurons.InterpretationStringent fate mapping techniques independently of an AAV-based expression system are the golden standard for tracing the fate of glia cells during neuronal reprogramming. The newly developed AAV-based systems are invaluable tools for glia-to-neuron reprogramming in vivo.FundingThe work in Chen lab was supported by National Institutes of Health (NIH) grants R01 EY024986 and R01 EY028921, an unrestricted challenge grant from Research to Prevent Blindness, the New York Eye and Ear Infirmary Foundation, and The Harold W. McGraw, Jr. Family Foundation for Vision Research. The work in Zhang lab was supported by NIH (R01 NS127375 and R01 NS117065) and The Decherd Foundation.
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Project description:Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and resembles aberrant immunoglobulins as observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in astrocytes adult rats, whereas in primary astrocytes from human fetal astrocytes we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 IgG2B constant heavy chain (IgH6), overexpression, proximal labeling of rat astrocytes IgH6 and targets identification through 2D gels were performed. In CRISPR-Cas9 IgG2B KO, overexpression of factors involved in hematopoietic cells, neural stem cells and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis whereas CRISPR-Cas9 IgG2B KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex; and behaves partially like CD98hc through its association with LAT1. Proximity-dependent biotin labeling experiments suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypotheses that aberrant neural IgG chains may act as gatekeepers of astrocytes fate.
2023-05-10 | PXD040562 | Pride
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Project description: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 (Ascl1 SA 6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1 SA 6 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-Ascl1 SA 6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1 SA 6 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 Ascl1 SA 6)-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), Ascl1 SA 6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1 SA 6 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 Ascl1 SA 6 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 Ascl1 SA 6 vs. Ascl1, and position Ascl1 SA 6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.
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