Specialized microenvironments, called niches, control adult stem cell proliferation and differentiation. The brain lateral ventricular (LV) neurogenic niche is generated from distinct postnatal ...radial glial progenitors (pRGPs), giving rise to adult neural stem cells (NSCs) and niche ependymal cells (ECs). Cellular-intrinsic programs govern stem versus supporting cell maturation during adult niche assembly, but how they are differentially initiated within a similar microenvironment remains unknown. Using chemical approaches, we discovered that EGFR signaling powerfully inhibits EC differentiation by suppressing multiciliogenesis. We found that EC pRGPs actively terminated EGF activation through receptor redistribution away from CSF-contacting apical domains and that randomized EGFR membrane targeting blocked EC differentiation. Mechanistically, we uncovered spatiotemporal interactions between EGFR and endocytic adaptor protein Numb. Ca2+-dependent basolateral targeting of Numb is necessary and sufficient for proper EGFR redistribution. These results reveal a previously unknown cellular mechanism for neighboring progenitors to differentially engage environmental signals, initiating adult stem cell niche assembly.
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•EGFR downregulation promotes postnatal radial glial differentiation into ependyma•Apical EGFR redistributes to ependymal basolateral domains, limiting its activation•Endocytic adaptor Numb traffics ependymal EGFR during adult neurogenic niche assembly•Progenitors differentially act on the same extracellular cues via receptor redistribution
Constructing adult stem niches from postnatal progenitors requires differential cellular interpretations of similar microenvironmental signals. Abdi et al. show that in an EGF-rich environment needed for adult neural stem cell proliferation, a subpopulation of postnatal progenitors downregulates EGFR signaling via targeted receptor trafficking, promoting multiciliated ependymal niche cell differentiation.
The choroid plexus (ChP) secretes a half-liter/day of cerebrospinal fluid (CSF) and is the blood-CSF barrier with critical immune functions. Hydrocephalus, commonly caused by infection or hemorrhage, ...lacks non-surgical treatments due to obscure pathobiology. We hypothesize that in acquired hydrocephalus, the dual ChP functions of CSF secretion and blood-CSF barrier maintenance become pathologically entangled in a maladaptive disease state. To test this hypothesis, we created novel rat models of post-infectious (PIH) and posthemorrhagic hydrocephalus (PHH) and conducted a multi-omics investigation of these animals to dissect the cellular, molecular, and physiological pathology underlying PIH and PHH . Our systems approach combined functional genomics to characterize the ChP immune response, including the first-ever single-cell transcriptomic profile of the ChP in a pathological condition, as well as in vivo assessments of ChP secretory capacity and CSF dynamics to systematically characterize fluid homeostasis. Integrated multi-omic analysis of diverse post-infectious (PIH) and post-hemorrhagic (PHH) hydrocephalus models show lipopolysaccharide and blood products, respectively, trigger highly similar Tlr4-dependent immune-secretory responses at the ChP-CSF interface. Activated ChP macrophages elicit a CSF “cytokine storm” that stimulates CSF production from ChP epithelial cells via a kinase-regulated multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents acute PIH and PHH by antagonizing ChP-mediated CSF hypersecretion. These data expand our understanding of ChP immune-epithelial cell crosstalk, reframe PIH and PHH as related neuroinflammatory disorders, and identify a druggable target of ChP immune-secretory function.
INTRODUCTION:
Radiation necrosis (RN), a common and morbid side effect of radiosurgical treatment of brain metastasis, is becoming more prevalent as immunotherapy options and patient survival ...improve. Thought to be immunologically driven, the pathophysiology of RN remains poorly defined. Further, RN is often indistinguishable from regrowing metastases after stereotactic radiosurgery (SRS), requiring invasive brain biopsy for diagnosis.
METHODS:
We collected 20 intraoperative samples from patients undergoing craniotomy for RN and/or metastatic tumor, as well as time-matched blood and CSF. We created a tissue bank, processed samples, and using fluorescence-activated cell sorting (FACS) and 10x Genomics single cell RNA sequencing, analyzed the cellular immune profile of lesional tissue, blood, and CSF.
RESULTS:
RN tissue demonstrates a robust, detectable expression of immune cells, including CD8+ and CD4+ T cells, natural killer (NK) and myeloid cells. Importantly, clustering of RN-derived immune cells (including T cells, myeloid cells, and NK cells) is reflected in the blood and CSF, with similar immune profiles. We demonstrate a subpopulation of myeloid cells with a microglia signature (CD14+, LYZ-, MS4A7+), and a separate population expressing interferon-stimulated gene (ISG). Supporting these findings, pathway analysis of top blood markers reveals pathways involved in innate and inflammatory immune responses in the blood of RN patients.
CONCLUSIONS:
These findings demonstrate the feasibility of extracting live cells from RN tissue for scRNAseq analysis and suggest that there are unique signatures of RN that can be detected in the blood and CSF of patients. This work has the potential to open new avenues for improved diagnostic and therapeutic approaches to allow earlier detection and more effective treatment of RN in patients with metastatic brain tumors.
INTRODUCTION:
Craniosynostosis (CS) is the premature fusion of the calvarial sutures and has long been considered mostly an aesthetic disorder, despite many affected children experiencing ...neurocognitive deficiencies and seizures. The two types of CS, syndromic and non-syndromic, present with significant genetic clinical heterogeneity, suggesting a complex interplay of genetic and environmental causes. Given the neurodevelopmental symptoms observed in CS patients, we hypothesized the genes involved in the pathophysiology of CS may play a key role in the functional development of the brain.
METHODS:
We applied a multimodal integrative genomic approach to examine the functional relationships as well as the spatiotemporal and cellular interactions of all currently known craniosynostosis-associated genes (CSGs) and also explored whether they are linked to other neurodevelopmental disorders (NDD). To this end, we constructed gene co-expression modules using a bulk-RNA expression dataset with 27 human brains encompassing 16 human brain regions across fetal development into early childhood. We further used scRNA-seq datasets derived from embryonic mouse meninges, calvarial sutures and developmental human brain to identify cell-types enriched with CSGs.
RESULTS:
The set of co-expression modules significantly enriched with CSGs were highly expressed during mid-gestation and in primary motor, somatosensory and prefrontal cortices, and the ganglionic eminence. Moreover, some of these modules were also significantly enriched with autism-spectrum disorder (ASD) and NDD genes. The most significant modules were associated with histone-3 lysine-4 methylation, neural crest differentiation, endochondral ossification and reactive astrogliosis. The mouse meninges scRNAseq analysis showed enrichment in pial and dural fibroblasts. The whole-brain scRNA-seq analysis showed CSGs were enriched in medial ganglionic eminence (MGE)-derived cortical interneurons, astrocytes and oligodendrocyte precursors during mid-fetal brain development.
CONCLUSION:
CSGs may contribute to pathogenesis through epigenetic dysregulation, disrupted neural crest differentiation facilitated by neuroglial cells and MGE interneuron migration very early during development. These processes are in confluence with premature suture fusion mediated by dural and pial fibroblasts.
Abstract
INTRODUCTION
The incidence of radiation necrosis (RN), a common and morbid side effect of radiosurgical management of brain metastases, is growing as patient survival and use of ...immunotherapy increases. RN is thought to be immunologically driven; however, its pathophysiology remains poorly understood. Further, difficulty distinguishing it radiographically from recurrent metastatic disease often necessitates invasive brain biopsies for diagnosis. We sought to define the neuroimmune profile of RN to better understand the underlying pathophysiological mechanisms and improve patient diagnosis.
METHODS
Over 12 months, we collected intraoperative samples from patients undergoing craniotomy for RN and/or metastatic tumor. We created a bank of these 16 patient samples and processed them for scRNAseq analysis. Using fluorescence-activated cell sorting (FACS) and 10x Genomics single cell RNA sequencing, we analyzed the cellular immune profile of lesional tissue and time-matched blood from each patient.
RESULTS
Compared to metastases, RN demonstrates decreased expression of FOXP3, NKG7, and GZMB, markers of regulatory and cytotoxic T, and natural killer (NK) cells, and increased expression of interleukin-7 receptor (IL7R), a marker of naïve and memory T cells – profiles reflective of bystander transcriptional signatures. In contrast, metastatic tissue demonstrates an antigen-reactive profile, including commonly observed exhaustion signatures. Importantly, blood samples from patients also reflect these differences. Interestingly, increased IL7R is implicated in autoimmune disorders, suggesting an iatrogenically-induced autoreactive process underlying RN.
DISCUSSION
These findings demonstrate, firstly, the feasibility of extracting live cells from RN tissue, and, secondly, suggest RN and metastases have unique immune profiles that can be detected in patient blood. Therefore, in addition to beginning to define the immune landscape of this poorly understood pathology, this work has the potential to open new avenues for the development of improved diagnostic and therapeutic approaches to allow for earlier detection and more effective treatment of RN in metastatic brain tumors patients.
INTRODUCTION:
Postinfectious hydrocephalus (PIH) is the most common cause of acquired hydrocephalus worldwide. Treatment is typically through CSF diversion via ventriculoperitoneal shunting, which ...has high rates of morbidity and is often unavailable in developing countries.
METHODS:
SPAK knockout (KO) rats were generated using Crispr/CAS9. Control and SPAK KO male rats were implanted with an infusion catheter to introduce E. coli (genetically engineered +/-LPS) or LPS into the lateral ventricles over 72 hours. CSF secretion was measured by catheter placement into the lateral ventricle, and ventricular size by MRI. ChP were harvested for immunohistochemistry (IHC), western blot (WB), genomic/proteomic analysis, and single-cell RNA sequencing (scRNAseq). To investigate SPAK-associated ChP targets, we purified the SPAK-protein complex, identified physical interactors using liquid chromatography/dual mass spectroscopy (LC-MS/MS), and validated top candidates by WB/IHC.
RESULTS:
Robust bumetanide-sensitive, SPAK-dependent, CSF hypersecretion and ventriculomegaly was observed and dependent on the presence of LPS. LPS triggered significant up-regulation of activating SPAK phosphorylation (Thr233/185), and the bumetanide-sensitive cation-chloride cotransporter NKCC1 (Thr203/207/212). LC-MS/MS pull-down assay additionally revealed three other important SPAK-bound ion transporters, alpha-1 subunit of Na/K ATPase, K+ channel KCNJ13/Kir7.1, and Cl- channel CLIC6. All SPAK-bound targets co-localized with pSPAK at the ChP apical membrane in LPS conditions. This interaction, as well as CSF hypersecretion and ventriculomegaly, were abrogated in LPS-treated SPAK KO animals.
CONCLUSION:
Multi-omic analysis identified a SPAK-regulated ensemble of ion transport proteins relevant for PIH at the ChP apical membrane. Given the recent development of SPAK kinase inhibitors, our findings uncover a targetable mechanism for the pharmacological treatment of PIH and potentially other forms of inflammation-associated acquired hydrocephalus.
INTRODUCTION:
Arachnoid cysts (ACs) are the most common congenital intracranial lesions. While familial cases implicate genetic factors, their ultimate cause remains unknown. Herein, we describe a ...multi-omics approach to disease state characterization including traditional whole exome sequencing, integrative genomics with analytics of large bulk-RNA and single cell RNA sequencing (scRNAseq) data, and a novel phenomics technique using natural language processing and Human Phenotypic Ontological (HPO) language clustering.
METHODS:
Whole exome sequencing was performed on 1560 individuals (520 parent-offspring "trios"). Genes with likely pathogenic mutations were defined as those with >1 de novo variant (DNV) reaching threshold for exome-wide significance (p<2.5 x 10-6) and having high pLI score (>0.9). This high-confidence pathogenic gene set and a secondary probable pathogenic gene set (>1 DNV, regardless of exome-wide significance or pLI score) were analyzed in bulk-RNA and scRNAseq datasets to assess spatiotemporal and cell-type relationships of gene expression. Phenomic analysis was conducted by subjecting patient medical records to a natural-language-processing (NLP) algorithm. Phenotypes were catalogued with the Human Phenotype Ontology (HPO) language tree and mapped with uniform manifold approximation and projection (UMAP) clustering.
RESULTS:
7 genes reached our pathogenic threshold, ADNP, ARIDB1, KDM5C, PURA, FOXP1, MAP2K1, and SCN2A. Strikingly, all but SCN2A play critical roles in epigenetic regulation of gene expression in the developing brain. Bulk-RNA datasets reveal that these genes converge in networks essential for chromatin remodeling and mRNA processing. scRNAseq datasets revealed an association with excitatory and inter-neurons known to play a role in developmental delay. HPO clustering revealed four major phenotypic clusters defined by neurodevelopmental delay (NDD), seizures, both or asymptomatic. In a subgroup of traditionally phenotyped patients, there was statistically significant enrichment of neuraxial phenotypes - including NDD - among those harboring DNVs.
CONCLUSION:
This novel association between ACs and multiple epigenetic regulators of gene expression provides the first insight into intracranial AC pathobiology. HPO clustering reveals clinically relevant subsets of ACs and implies that traditionally symptomatic or asymptomatic ACs may be harbingers of genetically-driven aberrant neurodevelopment.
Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason ...for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.
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