EPEN-21. IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION IN PEDIATRIC EPENDYMOMA REVEALED BY SINGLE-CELL RNA-SEQ

Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

Age-related loss of neural stem cell O-GlcNAc promotes a glial fate switch through STAT3 activation

Increased neural stem cell (NSC) quiescence is a major determinant of age-related regenerative decline in the adult hippocampus. However, a coextensive model has been proposed in which division-coupled conversion of NSCs into differentiated astrocytes restrict the stem cell pool with age. Here we report that age-related loss of the posttranslational modification, O-linked β-N-acetylglucosamine (O-GlcNAc), in NSCs promotes a glial fate switch. We detect an age-dependent decrease in NSC O-GlcNAc levels coincident with decreased neurogenesis and increased gliogenesis in the mature hippocampus. Mimicking an age-related loss of NSC O-GlcNAcylation in young mice reduces neurogenesis, increases astrocyte differentiation, and impairs associated cognitive function. Using RNA-sequencing of primary NSCs following decreased O-GlcNAcylation, we detected changes in the STAT3 signaling pathway indicative of glial differentiation. Moreover, using O-GlcNAc–specific mass spectrometry analysis of the aging hippocampus, together with an in vitro site-directed mutagenesis approach, we identify loss of STAT3 O-GlcNAc at Threonine 717 as a driver of astrocyte differentiation. Our data identify the posttranslational modification, O-GlcNAc, as a key molecular regulator of regenerative decline underlying an age-related NSC fate switch.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10–Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Tectal stem cells display diverse regenerative capacities

How diverse adult stem and progenitor populations regenerate tissue following damage to the CNS remains unknown across most neurogenic domains. To understand the role of quiescent radial-glial (qRG) stem cells during regeneration, we tested the hypothesis that qRG could be induced to proliferate and produce newborn neurons. We designed a stab lesion assay in the midbrain tectum of the adult zebrafish to target an isolated population of qRG, and investigated their proliferative behaviour, differentiation potential, and requirement of Wnt/β-catenin signalling for the regenerative response. EdU-labelling showed that a small proportion of qRG transit to a proliferative state (pRG), but that progeny of pRG are restricted to a radial-glial fate. Lesion promoted upregulation of proliferation and neurogenesis from neuro-epithelial-like amplifying progenitors (NE-Ap) of the tectal marginal zone (TMZ). Homeostatic levels of Wnt/β-catenin signalling persisted under lesioned conditions in the qRG/pRG population, whereby increased β-catenin staining and axin2 expression was present in the NE-Ap progenitor zone. Attenuation of Wnt signalling using Dickkopf-1, demonstrated that proliferative responses post-injury appeared to be Wnt-independent. Our results align with the emerging view that adult stem/progenitor phenotypes are characterized by discrete, rather than mutual, regenerative programs and that different stem cell domains employ different modes of regeneration.

Transient hypothyroidism favors oligodendrocyte generation providing functional remyelination in the adult mouse brain

In the adult brain, both neurons and oligodendrocytes can be generated from neural stem cells located within the Sub-Ventricular Zone (SVZ). Physiological signals regulating neuronal versus glial fate are largely unknown. Here we report that a thyroid hormone (T3)-free window, with or without a demyelinating insult, provides a favorable environment for SVZ-derived oligodendrocyte progenitor generation. After demyelination, oligodendrocytes derived from these newly-formed progenitors provide functional remyelination, restoring normal conduction. The cellular basis for neuronal versus glial determination in progenitors involves asymmetric partitioning of EGFR and TRα1, expression of which favor glio- and neuro-genesis, respectively. Moreover, EGFR+ oligodendrocyte progenitors, but not neuroblasts, express high levels of a T3-inactivating deiodinase, Dio3. Thus, TRα absence with high levels of Dio3 provides double-pronged blockage of T3 action during glial lineage commitment. These findings not only transform our understanding of how T3 orchestrates adult brain lineage decisions, but also provide potential insight into demyelinating disorders.

Regulation of neuronal-glial fate specification by long non-coding RNAs

Neural stem cell transplantation is becoming a promising and attractive cell-based treatment modality for repairing the damaged central nervous system. One of the limitations of this approach is that the proportion of functional cells differentiated from stem cells still remains at a low level. In recent years, novel long non-coding RNAs (lncRNAs) are being discovered at a growing pace, suggesting that this class of molecules may act as novel regulators in neuronal-glial fate specification. In this review, we first describe the general features of lncRNAs that are more likely to be relevant to reveal their function. By this, we aim to point out the specific roles of a number of lncRNAs whose function has been described during neuronal and glial cell differentiation. There is no doubt that investigation of the lncRNAs will open a new window in studying neuronal-glial fate specification.