Neural stem/precursor cells dynamically change their epigenetic landscape to differentially respond to BMP signaling for fate switching during brain development

During neocortical development, tight regulation of neurogenesis-to-astrogenesis switching of neural precursor cells (NPCs) is critical to generate a balanced number of each neural cell type for proper brain functions. Accumulating evidence indicates that a complex array of epigenetic modifications and the availability of extracellular factors control the timing of neuronal and astrocytic differentiation. However, our understanding of NPC fate regulation is still far from complete. Bone morphogenetic proteins (BMPs) are renowned as cytokines that induce astrogenesis of gliogenic late-gestational NPCs. They also promote neurogenesis of mid-gestational NPCs, although the underlying mechanisms remain elusive. By performing multiple genome-wide analyses, we demonstrate that Smads, transcription factors that act downstream from BMP signaling, target dramatically different genomic regions in neurogenic and gliogenic NPCs. We found that histone H3K27 trimethylation and DNA methylation around Smad-binding sites change rapidly as gestation proceeds, strongly associated with the alteration of accessibility of Smads to their target binding sites. Furthermore, we identified two lineage-specific Smad-interacting partners—Sox11 for neurogenic and Sox8 for astrocytic differentiation—that further ensure Smad-regulated fate-specific gene induction. Our findings illuminate an exquisite regulation of NPC property change mediated by the interplay between cell-extrinsic cues and -intrinsic epigenetic programs during cortical development.

Soft surfaces promote astrocytic differentiation of mouse embryonic neural stem cells via dephosphorylation of MRLC in the absence of serum

Astrocytes, which can be obtained from neural stem cells (NSCs) by adding serum and/or recombinant proteins in culture media or by passaging NSCs repeatedly, are expected to be applicable in regenerative medicine for the treatment of neurodegenerative diseases. However, astrocytes obtained using existing methods are costly and have poor quality. The stiffness of culture surfaces has been reported to affect astrocytic differentiation of adult NSCs. However, the influence of surface stiffness on astrocytic differentiation of embryonic NSCs has not yet been reported. In this study, we showed that astrocytic differentiation of embryonic NSCs was increased on soft surfaces (1 kPa and 12 kPa) compared with the NSCs on stiff surfaces (2.8 GPa) in serum-free condition. Furthermore, di-phosphorylated myosin regulatory light chain (PP-MRLC) was decreased in embryonic NSCs cultured on the soft surfaces than the cells on the stiff surfaces. Additionally, astrocytic differentiation of embryonic NSCs was induced by a Ras homolog associated kinase (ROCK) inhibitor, which decreased PP-MRLC in NSCs. These results suggest that decreasing the PP-MRLC of embryonic NSCs on soft surfaces or treating NSCs with a ROCK inhibitor is a good method to prepare astrocytes for application in regenerative medicine.

Endothelial cells induce proliferation and bone morphogenic protein mediated differentiation of PDGFRα+ human oligodendrocyte precursor cells to astrocytes in trans-well co-culture

Background CD140a /PDGFRα + human oligodendrocyte precursor cells (OPCs) are a lineage of OPCs with proven potential for use in cell therapy against demyelinating diseases. However, little is known about the contribution of human endothelial cells in the biology of PDGFRα + human OPCs in the stem cell niche. Methods Transwell co-culture technique with human umbilical vein endothelial cells (HUVECs) was adopted under proliferative or differentiating conditions to understand the role of endothelial cells in these processes within OPCs. Proliferation was followed by measuring OPC sphere size, count, sphere dissociation followed by cell count and 3H-methyl thymidine incorporation. Differentiation was followed by immunocytochemistry. Taqman gene expression assay for selective soluble factors was performed for the two co-culture partner cells to determine the expression of these factors on the biology of the OPCs in presence of the endothelial cells. Results In co-culture with HUVECs, under proliferative conditions, OPCs show increased proliferation and sphere formation. In contrast, under differentiating conditions, OPCs show increased differentiation to astrocytes, with a concomitant decrease in differentiation to oligodendrocytes, compared to no co-culture controls. Transcript assay for selected humoral factors in the OPCs and HUVECs revealed bone morphogenic proteins (BMPs), endothelin1, growth arrest specific 6 (GAS6), and interleukin 6 (IL6) to be in higher abundance in HUVECs than OPCs. Whereas the OPCs show higher expression for pleiotrophin (PTN), fibroblast growth factor 9 (FGF9), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF) compared to the endothelial cells. Among the transcripts analyzed, BMP4 transcripts were the highest in relative abundance in the endothelial cells indicating possibilities of BMPs being the critical mediator of endothelial cell-mediated effects. In agreement to this, Noggin effectively attenuated HUVEC mediated astrocytic differentiation of CD140a /PDGFRα + fetal human OPCs. Conclusion Based on the above results, the study concludes that human endothelial cells can significantly alter the biology of PDGFαR + fetal human OPCs mediated by humoral factors to induce increased proliferation and BMP mediated astrocytic differentiation. It can be secondarily inferred from these conclusions that using pharmacological inhibitors of BMP signaling along with the PDGFRα + fetal OPC transplantation may make these cells more effective in remyelination therapy.

Diffuse Midline Glioma with H3-K27M Mutation: A Rare Case with GFAP-positive Anucleate Whorled Patterns

Background: Diffuse midline glioma with H3-K27M mutation is a new tumor type in the 2016 WHO classification of tumors of the central nervous system, with predominantly astrocytic differentiation and is characterized by K27M mutation in either H3F3A/-HIST1H3B/C. It occurs at the midline of the CNS. It is mainly found in children, and adult cases are relatively rare . Although its histopathological features have been widely described, GFAP-positive anucleate whorled patterns have not been reported to the best of our knowledge. Hence, we have reported this unusual case.Case presentation：A woman presented with complaints of walking instability. MRI showed a mass shadow of isometric T1 and slightly longer T2 with mild mixed signals in the third ventricle of the suprasellar region, about 3.4 x 2.5 x 3.4 cm in size. The enhanced scan showed that the lesion was irregular and annular, with a clear edge. The lesion had spread to the right lateral ventricle, which dilated the ventricular system, especially the right lateral ventricle. Furthermore, the lamellar long T2 signal was seen in bilateral paraventricular white matter, the midline structure had shifted to the left, and the corpus callosum was thinner. Radiological findings suggested ependymoma. Subtotal resection craniotomy was performed. After surgery, the patient was routinely treated with temozolomide for chemotherapy and synchronous radiotherapy (Dt=60 Gy/30f). It has been 11 months now, and the patient is living well.Result: We described an extremely rare case of diffuse midline glioma, H3-K27M mutation with GFAP-positive anucleate whorled patterns. Conclusions: This case report provides information on the microscopic morphological features of diffuse midline glioma with H3K27M mutation, which can help pathologists to make a definitive diagnosis of this tumor.

NF-κB and TNF Affect the Astrocytic Differentiation from Neural Stem Cells

The NF-κB signaling pathway is crucial during development and inflammatory processes. We have previously shown that NF-κB activation induces dedifferentiation of astrocytes into neural progenitor cells (NPCs). Here, we provide evidence that the NF-κB pathway plays also a fundamental role during the differentiation of NPCs into astrocytes. First, we show that the NF-κB pathway is essential to initiate astrocytic differentiation as its early inhibition induces NPC apoptosis and impedes their differentiation. Second, we demonstrate that persistent NF-κB activation affects NPC-derived astrocyte differentiation. Tumor necrosis factor (TNF)-treated NPCs show NF-κB activation, maintain their multipotential and proliferation properties, display persistent expression of immature markers and inhibit astrocyte markers. Third, we analyze the effect of NF-κB activation on the main known astrocytic differentiation pathways, such as NOTCH and JAK-STAT. Our findings suggest that the NF-κB pathway plays a dual fundamental role during NPC differentiation into astrocytes: it promotes astrocyte specification, but its persistent activation impedes their differentiation.

Connexin43 Region 266–283, via Src Inhibition, Reduces Neural Progenitor Cell Proliferation Promoted by EGF and FGF-2 and Increases Astrocytic Differentiation

Neural progenitor cells (NPCs) are self-renewing cells that give rise to the major cells in the nervous system and are considered to be the possible cell of origin of glioblastoma. The gap junction protein connexin43 (Cx43) is expressed by NPCs, exerting channel-dependent and -independent roles. We focused on one property of Cx43—its ability to inhibit Src, a key protein in brain development and oncogenesis. Because Src inhibition is carried out by the sequence 266–283 of the intracellular C terminus in Cx43, we used a cell-penetrating peptide containing this sequence, TAT-Cx43266–283, to explore its effects on postnatal subventricular zone NPCs. Our results show that TAT-Cx43266–283 inhibited Src activity and reduced NPC proliferation and survival promoted by epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). In differentiation conditions, TAT-Cx43266–283 increased astrocyte differentiation at the expense of neuronal differentiation, which coincided with a reduction in Src activity and β-catenin expression. We propose that Cx43, through the region 266–283, reduces Src activity, leading to disruption of EGF and FGF-2 signaling and to down-regulation of β-catenin with effects on proliferation and differentiation. Our data indicate that the inhibition of Src might contribute to the complex role of Cx43 in NPCs and open new opportunities for further research in gliomagenesis.

Parp3 promotes astrocytic differentiation through a tight regulation of Nox4-induced ROS and mTorc2 activation

Parp3 is a member of the Poly(ADP-ribose) polymerase (Parp) family that has been characterized for its functions in strand break repair, chromosomal rearrangements, mitotic segregation and tumor aggressiveness. Yet its physiological implications remain unknown. Here we report a central function of Parp3 in the regulation of redox homeostasis in continuous neurogenesis in mice. We show that the absence of Parp3 provokes Nox4-induced oxidative stress and defective mTorc2 activation leading to inefficient differentiation of post-natal neural stem/progenitor cells to astrocytes. The accumulation of ROS contributes to the decreased activity of mTorc2 as a result of an oxidation-induced and Fbxw7-mediated ubiquitination and degradation of Rictor. In vivo, mTorc2 signaling is compromised in the striatum of naïve post-natal Parp3-deficient mice and 6 h after acute hypoxia-ischemia. These findings reveal a physiological function of Parp3 in the tight regulation of striatal oxidative stress and mTorc2 during astrocytic differentiation and in the acute phase of hypoxia-ischemia.

Diffuse Midline Glioma (H3K27M mutant) in Adult: A Diagnostic Challenge

Introduction: Diffuse midline glioma (DMG), H3K27M mutant is an infiltrative midline high grade glioma with predominantly astrocytic differentiation and K27M mutation in either H3F3A or HIST1H3B/C. Case Report: A 45-year-old female presented with complaints of headache and memory loss for 3 months. MRI was suggestive of an infiltrative mass lesion in the quadrigeminal plate cistern suggestive of pineal neoplasm. Squash and histomorphology showed a low-grade astrocytic tumour with infiltrative growth pattern. Microvascular proliferation and necrosis were absent. Immunohistochemistry showed loss of ATRX protein, focal positivity for p53 proteinand IDH1R132H negativity. On molecular analysis, H3K27M mutation was noted and the case was labelled as DMG H3K27Mmutant (WHO IV) Conclusion: DMG (H3K27M) is a newly added entity in the WHO 4th revised editionof 2016. It presents with a diagnostic challenge as it has varied histomorphology, not requiring atypia, mitosis, endothelial hyperplasia and necrosis for diagnosis as Grade IV.