Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

BRG1 programs PRC2-complex repression and controls oligodendrocyte differentiation and remyelination.

Chromatin-remodeling protein BRG1/SMARCA4 is pivotal for establishing oligodendrocyte (OL) lineage identity. However, its functions for oligodendrocyte-precursor cell (OPC) differentiation within the postnatal brain and during remyelination remain elusive. Here, we demonstrate that Brg1 loss profoundly impairs OPC differentiation in the brain with a comparatively lesser effect in the spinal cord. Moreover, BRG1 is critical for OPC remyelination after injury. Integrative transcriptomic/genomic profiling reveals that BRG1 exhibits a dual role by promoting OPC differentiation networks while repressing OL-inhibitory cues and proneuronal programs. Furthermore, we find that BRG1 interacts with EED/PRC2 polycomb-repressive-complexes to enhance H3K27me3-mediated repression at gene loci associated with OL-differentiation inhibition and neurogenesis. Notably, BRG1 depletion decreases H3K27me3 deposition, leading to the upregulation of BMP/WNT signaling and proneurogenic genes, which suppresses OL programs. Thus, our findings reveal a hitherto unexplored spatiotemporal-specific role of BRG1 for OPC differentiation in the developing CNS and underscore a new insight into BRG1/PRC2-mediated epigenetic regulation that promotes and safeguards OL lineage commitment and differentiation.

Nuclear condensates of YAP fusion proteins alter transcription to drive ependymoma tumourigenesis.

Nuclear localization of HIPPO-YAP fusion proteins has been implicated in supratentorial ependymoma development. Here, unexpectedly, we find that liquid-liquid phase separation, rather than nuclear localization, of recurrent patient-derived YAP fusions, YAP-MAMLD1 and C11ORF95-YAP, underlies ependymoma tumourigenesis from neural progenitor cells. Mutagenesis and chimaera assays demonstrate that an intrinsically disordered region promotes oligomerization of the YAP fusions into nuclear, puncta-like, membrane-less condensates. Oligomerization and nuclear condensates induced by YAP fusion with a coiled-coil domain of transcriptional activator GCN4 also promote ependymoma formation. YAP-MAMLD1 concentrates transcription factors and co-activators, including BRD4, MED1 and TEAD, in condensates while excluding transcriptional repressive PRC2, and induces long-range enhancer-promoter interactions that promote transcription and oncogenic programmes. Blocking condensate-mediated transcriptional co-activator activity inhibits tumourigenesis, indicating a critical role of liquid phase separation for YAP fusion oncogenic activity in ependymoma. YAP fusions containing the intrinsically disordered region features are common in human tumours, suggesting that nuclear condensates could be targeted to treat YAP-fusion-induced cancers.

Human fetal cerebellar cell atlas informs medulloblastoma origin and oncogenesis.

Medulloblastoma (MB) is the most common malignant childhood brain tumour1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3-5 have not fully defined the compositional heterogeneity of MBs. Here we undertook single-cell profiling of freshly isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group 3 and metastatic tumours. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group 3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group 3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group 3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs show potential cell populations predisposed to transformation and regulatory circuitries underlying tumour cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

Single-cell multiomics identifies clinically relevant mesenchymal stem-like cells and key regulators for MPNST malignancy.

Malignant peripheral nerve sheath tumor (MPNST), a highly aggressive Schwann cell (SC)-derived soft tissue sarcoma, arises from benign neurofibroma (NF); however, the identity, heterogeneity and origins of tumor populations remain elusive. Nestin+ cells have been implicated as tumor stem cells in MPNST; unexpectedly, single-cell profiling of human NF and MPNST and their animal models reveal a broad range of nestin-expressing SC lineage cells and dynamic acquisition of discrete cancer states during malignant transformation. We uncover a nestin-negative mesenchymal neural crest-like subpopulation as a previously unknown malignant stem-like state common to murine and human MPNSTs, which correlates with clinical severity. Integrative multiomics profiling further identifies unique regulatory networks and druggable targets against the malignant subpopulations in MPNST. Targeting key epithelial-mesenchymal transition and stemness regulators including ZEB1 and ALDH1A1 impedes MPNST growth. Together, our studies reveal the underlying principles of tumor cell-state evolution and their regulatory circuitries during NF-to-MPNST transformation, highlighting a hitherto unrecognized mesenchymal stem-like subpopulation in MPNST disease progression.

Olig2 Ablation in Immature Oligodendrocytes Does Not Enhance CNS Myelination and Remyelination.

The oligodendrocyte (OL) lineage transcription factor Olig2 is expressed throughout oligodendroglial development and is essential for oligodendroglial progenitor specification and differentiation. It was previously reported that deletion of Olig2 enhanced the maturation and myelination of immature OLs and accelerated the remyelination process. However, by analyzing multiple Olig2 conditional KO mouse lines (male and female), we conclude that Olig2 has the opposite effect and is required for OL maturation and remyelination. We found that deletion of Olig2 in immature OLs driven by an immature OL-expressing Plp1 promoter resulted in defects in OL maturation and myelination, and did not enhance remyelination after demyelination. Similarly, Olig2 deletion during premyelinating stages in immature OLs using Mobp or Mog promoter-driven Cre lines also did not enhance OL maturation in the CNS. Further, we found that Olig2 was not required for myelin maintenance in mature OLs but was critical for remyelination after lysolecithin-induced demyelinating injury. Analysis of genomic occupancy in immature and mature OLs revealed that Olig2 targets the enhancers of key myelination-related genes for OL maturation from immature OLs. Together, by leveraging multiple immature OL-expressing Cre lines, these studies indicate that Olig2 is essential for differentiation and myelination of immature OLs and myelin repair. Our findings raise fundamental questions about the previously proposed role of Olig2 in opposing OL myelination and highlight the importance of using Cre-dependent reporter(s) for lineage tracing in studying cell state progression.SIGNIFICANCE STATEMENT Identification of the regulators that promote oligodendrocyte (OL) myelination and remyelination is important for promoting myelin repair in devastating demyelinating diseases. Olig2 is expressed throughout OL lineage development. Ablation of Olig2 was reported to induce maturation, myelination, and remyelination from immature OLs. However, lineage-mapping analysis of Olig2-ablated cells was not conducted. Here, by leveraging multiple immature OL-expressing Cre lines, we observed no evidence that Olig2 ablation promotes maturation or remyelination of immature OLs. Instead, we find that Olig2 is required for immature OL maturation, myelination, and myelin repair. These data raise fundamental questions about the proposed inhibitory role of Olig2 against OL maturation and remyelination. Our findings highlight the importance of validating genetic manipulation with cell lineage tracing in studying myelination.

Reduction of WDR81 impairs autophagic clearance of aggregated proteins and cell viability in neurodegenerative phenotypes.

Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington's disease (HD), Parkinson's disease (PD) and Alzheimer's disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

WDR81 regulates adult hippocampal neurogenesis through endosomal SARA-TGFß signaling.

Adult hippocampal neurogenesis, a process considered important for hippocampal function, is regulated at multiple molecular levels. Mutations in the gene encoding the WD40 repeat-containing protein WDR81 are associated with neurological disorders, including cerebellar ataxia, mental retardation, quadrupedal locomotion syndrome (CAMRQ2), and microcephaly. In this study, we show that ablation of WDR81 in adult neural progenitor cells (aNPCs) markedly reduced adult hippocampal neurogenesis and impaired hippocampus-dependent learning. WDR81 suppresses endosomal PtdIns3P synthesis, likely by inhibiting the assembly of the PI3K-III complex. In the absence of WDR81, endosomal PtdIns3P levels are greatly elevated, leading to endosomal persistence of the PtdIns3P-binding protein SARA and consequently hyperactivation of SARA-dependent TGFß signaling. Inhibition of PI3K-III activity or suppression of SARA-dependent TGFß signaling markedly ameliorated the defective adult neurogenesis in WDR81-deficient mice. Taken together, these findings not only uncover the requirement for the WDR81-SARA-TGFß axis in adult hippocampal neurogenesis, but also suggest that defective adult hippocampal neurogenesis contributes to the etiology of WDR81-related neurological diseases.

Single-Cell Transcriptomics in Medulloblastoma Reveals Tumor-Initiating Progenitors and Oncogenic Cascades during Tumorigenesis and Relapse.

Progenitor heterogeneity and identities underlying tumor initiation and relapse in medulloblastomas remain elusive. Utilizing single-cell transcriptomic analysis, we demonstrated a developmental hierarchy of progenitor pools in Sonic Hedgehog (SHH) medulloblastomas, and identified OLIG2-expressing glial progenitors as transit-amplifying cells at the tumorigenic onset. Although OLIG2+ progenitors become quiescent stem-like cells in full-blown tumors, they are highly enriched in therapy-resistant and recurrent medulloblastomas. Depletion of mitotic Olig2+ progenitors or Olig2 ablation impeded tumor initiation. Genomic profiling revealed that OLIG2 modulates chromatin landscapes and activates oncogenic networks including HIPPO-YAP/TAZ and AURORA-A/MYCN pathways. Co-targeting these oncogenic pathways induced tumor growth arrest. Together, our results indicate that glial lineage-associated OLIG2+ progenitors are tumor-initiating cells during medulloblastoma tumorigenesis and relapse, suggesting OLIG2-driven oncogenic networks as potential therapeutic targets.

C. elegans-based screen identifies lysosome-damaging alkaloids that induce STAT3-dependent lysosomal cell death.

Lysosomes are degradation and signaling centers within the cell, and their dysfunction impairs a wide variety of cellular processes. To understand the cellular effect of lysosome damage, we screened natural small-molecule compounds that induce lysosomal abnormality using Caenorhabditis elegans (C. elegans) as a model system. A group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A-D) were identified that caused lysosome enlargement in C. elegans macrophage-like cells. Intriguingly, these compounds triggered cell death in the germ line independently of the canonical apoptosis pathway. In mammalian cells, ervachinines A-D induced lysosomal enlargement and damage, leading to leakage of cathepsin proteases, inhibition of autophagosome degradation and necrotic cell death. Further analysis revealed that this ervachinine-induced lysosome damage and lysosomal cell death depended on STAT3 signaling, but not RIP1 or RIP3 signaling. These findings suggest that lysosome-damaging compounds are promising reagents for dissecting signaling mechanisms underlying lysosome homeostasis and lysosome-related human disorders.

A histone deacetylase 3-dependent pathway delimits peripheral myelin growth and functional regeneration.

Deficits in Schwann cell-mediated remyelination impair functional restoration after nerve damage, contributing to peripheral neuropathies. The mechanisms mediating block of remyelination remain elusive. Here, through small-molecule screening focusing on epigenetic modulators, we identified histone deacetylase 3 (HDAC3; a histone-modifying enzyme) as a potent inhibitor of peripheral myelinogenesis. Inhibition of HDAC3 enhanced myelin growth and regeneration and improved functional recovery after peripheral nerve injury in mice. HDAC3 antagonizes the myelinogenic neuregulin-PI3K-AKT signaling axis. Moreover, genome-wide profiling analyses revealed that HDAC3 represses promyelinating programs through epigenetic silencing while coordinating with p300 histone acetyltransferase to activate myelination-inhibitory programs that include the HIPPO signaling effector TEAD4 to inhibit myelin growth. Schwann cell-specific deletion of either Hdac3 or Tead4 in mice resulted in an elevation of myelin thickness in sciatic nerves. Thus, our findings identify the HDAC3-TEAD4 network as a dual-function switch of cell-intrinsic inhibitory machinery that counters myelinogenic signals and maintains peripheral myelin homeostasis, highlighting the therapeutic potential of transient HDAC3 inhibition for improving peripheral myelin repair.

The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy.

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak-Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder.

Protein kinase C controls lysosome biogenesis independently of mTORC1.

Lysosomes respond to environmental cues by controlling their own biogenesis, but the underlying mechanisms are poorly understood. Here we describe a protein kinase C (PKC)-dependent and mTORC1-independent mechanism for regulating lysosome biogenesis, which provides insights into previously reported effects of PKC on lysosomes. By identifying lysosome-inducing compounds we show that PKC couples activation of the TFEB transcription factor with inactivation of the ZKSCAN3 transcriptional repressor through two parallel signalling cascades. Activated PKC inactivates GSK3ß, leading to reduced phosphorylation, nuclear translocation and activation of TFEB, while PKC activates JNK and p38 MAPK, which phosphorylate ZKSCAN3, leading to its inactivation by translocation out of the nucleus. PKC activation may therefore mediate lysosomal adaptation to many extracellular cues. PKC activators facilitate clearance of aggregated proteins and lipid droplets in cell models and ameliorate amyloid ß plaque formation in APP/PS1 mouse brains. Thus, PKC activators are viable treatment options for lysosome-related disorders.

Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion.

Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.

Clathrin and AP2 are required for phagocytic receptor-mediated apoptotic cell clearance in Caenorhabditis elegans.

Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.