Björn Scheffler

Microglia Instruct Subventricular Zone Neurogenesis

Microglia are increasingly implicated as a source of non‐neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self‐renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia‐conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self‐renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain.

Focal Cortical Dysplasia of Taylor's Balloon Cell Type: A Clinicopathological Entity with Characteristic Neuroimaging and Histopathological Features, and Favorable Postsurgical Outcome

Summary: Background and Purpose: Focal cortical dysplasia of Taylors balloon‐cell type (FCD‐BC) are a frequent cause of pharmacoresistant epilepsy in young patients. In order to characterize FCD‐BC, we coupled MRI and histopathology, and analyzed the clinical outcome following epilepsy surgery.

c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype

The tyrosine kinase c-Met promotes the formation and malignant progression of multiple cancers. It is well known that c-Met hyperactivation increases tumorigenicity and tumor cell resistance to DNA damaging agents, properties associated with tumor-initiating stem cells. However, a link between c-Met signaling and the formation and/or maintenance of neoplastic stem cells has not been previously identified. Here, we show that c-Met is activated and functional in glioblastoma (GBM) neurospheres enriched for glioblastoma tumor-initiating stem cells and that c-Met expression/function correlates with stem cell marker expression and the neoplastic stem cell phenotype in glioblastoma neurospheres and clinical glioblastoma specimens. c-Met activation was found to induce the expression of reprogramming transcription factors (RFs) known to support embryonic stem cells and induce differentiated cells to form pluripotent stem (iPS) cells, and c-Met activation counteracted the effects of forced differentiation in glioblastoma neurospheres. Expression of the reprogramming transcription factor Nanog by glioblastoma cells is shown to mediate the ability of c-Met to induce the stem cell characteristics of neurosphere formation and neurosphere cell self-renewal. These findings show that c-Met enhances the population of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met–responsive reprogramming transcription factors.

Focal cortical dysplasia of Taylor's balloon cell type: Mutational analysis of the TSC1 gene indicates a pathogenic relationship to tuberous sclerosis

Focal cortical dysplasia (FCD) is characterized by a localized malformation of the neocortex and underlying white matter. Balloon cells, similar to those observed in tuberous sclerosis, are present in many cases (FCDbc). In these patients, a hyperintense funnel‐shaped subcortical lesion tapering toward the lateral ventricle was the characteristic finding on fluid‐attenuated inversion recovery magnetic resonance imaging scans. Surgical lesionectomy results in complete seizure relief. Although the pathogenesis of FCDbc remains uncertain, histopathological similarities indicate that FCDbc may be related pathogenetically to tuberous sclerosis. Here, we studied alterations of the TSC1 and TSC2 genes in a cohort of patients with chronic, focal epilepsy and histologically documented FCDbc (n = 48). DNA was obtained after microdissection and laser‐assisted isolation of balloon cells, dysplastic neurons, and nonlesional cells from adjacent normal brain tissue. Sequence alterations resulting in amino acid exchange of the TSC1 gene product affecting exons 5 and 17 and silent base exchanges in exons 14 and 22 were increased in patients with FCDbc compared with 200 control individuals (exon 5, 2.3% FCDbc vs 0% C; exon 17, 35% FCDbc vs 1.0% C; exon 14, 37.8% FCDbc vs 15% C; exon 22, 45% FCDbc vs 23.8% C). Sequence alterations could be detected in FCDbc and in adjacent normal cells. In 24 patients, DNA was suitable to study loss of heterozygosity at the TSC1 gene locus in microdissected FCDbc samples compared with control tissue. Eleven FCDbc cases exhibited loss of heterozygosity. In the TSC2 gene, only silent polymorphisms were detected at similar frequencies as in controls. Our findings indicate that FCDbc constitutes a clinicopathological entity with distinct neuroradiological, neuropathological, and molecular genetic features. These data also suggest a role of the TSC1 gene in the development of FCDbc and point toward a pathogenic relationship between FCDbc and the tuberous sclerosis complex.

Derivation and large-scale expansion of multipotent astroglial neural progenitors from adult human brain.

The isolation and expansion of human neural cell types has become increasingly relevant in restorative neurobiology. Although embryonic and fetal tissue are frequently envisaged as providing sufficiently primordial cells for such applications, the developmental plasticity of endogenous adult neural cells remains largely unclear. To examine the developmental potential of adult human brain cells, we applied conditions favoring the growth of neural stem cells to multiple cortical regions, resulting in the identification and selection of a population of adult human neural progenitors (AHNPs). These nestin+ progenitors may be derived from multiple forebrain regions, are maintainable in adherent conditions, co-express multiple glial and immature markers, and are highly expandable, allowing a single progenitor to theoretically form sufficient cells for∼ 4×107 adult brains. AHNPs longitudinally maintain the ability to generate both glial and neuronal cell types in vivo and in vitro, and are amenable to genetic modification and transplantation. These findings suggest an unprecedented degree of inducible plasticity is retained by cells of the adult central nervous system.

Residual tumor cells are unique cellular targets in glioblastoma.

Residual tumor cells remain beyond the margins of every glioblastoma (GBM) resection. Their resistance to postsurgical therapy is considered a major driving force of mortality, but their biology remains largely uncharacterized. In this study, residual tumor cells were derived via experimental biopsy of the resection margin after standard neurosurgery for direct comparison with samples from the routinely resected tumor tissue. In vitro analysis of proliferation, invasion, stem cell qualities, GBM‐typical antigens, genotypes, and in vitro drug and irradiation challenge studies revealed these cells as unique entities. Our findings suggest a need for characterization of residual tumor cells to optimize diagnosis and treatment of GBM. ANN NEUROL 2010;68:264–269

Preservation of Calretinin-immunoreactive Neurons in the Hippocampus of Epilepsy Patients with Ammon's Horn Sclerosis

Selective neuronal vulnerability and aberrant axonal reorganization in the hippocampus may play an important role for the pathogenesis of pharmaco-resistant temporal lobe epilepsy (TLE). Interneurons containing calcium-binding proteins (CaBPs) are candidates for pathogenetically relevant neurons in the hippocampus of patients with TLE. Here we have examined the cellular localization and distribution of calretinin (CR), a recently discovered CaBP, in the hippocampus of 35 patients with TLE. There was a striking preservation of CR-immunoreactive neurons in TLE patients with Ammons horn sclerosis (AHS). No significant differences in the distribution of CR-immunoreactive neurons were observed between patients with lesion-associated TLE and control patients without epilepsy. However, a subpopulation of CR-immunoreactive interneurons with morphological features of Cajal-Retzius-like cells, which are only transiently detectable in the normally developing hippocampus, was markedly increased in epilepsy patients with AHS. This increase did not correlate with the duration of the epileptic disorder. Another significant finding was a striking increase and reorganization of CR-immunoreactive neuropil throughout the entire molecular layer of the dentate gyrus (DG-ML) in patients with AHS as compared to patients with focal lesions and control specimens. Ultrastructural analysis identified the CR-immunoreactive axonal profiles as components of an inhibitory, intrinsic neuronal system. The presence of a CR-positive, aberrant cell population, in combination with sprouting of CR-positive axonal processes may significantly alter the gating function of the dentate gyrus and thereby increase hippocampal epileptogenicity in epilepsy patients with AHS

Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.

Chondroitin Sulfate Proteoglycans Potently Inhibit Invasion and Serve as a Central Organizer of the Brain Tumor Microenvironment

Glioblastoma (GBM) remains the most pervasive and lethal of all brain malignancies. One factor that contributes to this poor prognosis is the highly invasive character of the tumor. GBM is characterized by microscopic infiltration of tumor cells throughout the brain, whereas non-neural metastases, as well as select lower grade gliomas, develop as self-contained and clearly delineated lesions. Illustrated by rodent xenograft tumor models as well as pathological human patient specimens, we present evidence that one fundamental switch between these two distinct pathologies–invasion and noninvasion–is mediated through the tumor extracellular matrix. Specifically, noninvasive lesions are associated with a rich matrix containing substantial amounts of glycosylated chondroitin sulfate proteoglycans (CSPGs), whereas glycosylated CSPGs are essentially absent from diffusely infiltrating tumors. CSPGs, acting as central organizers of the tumor microenvironment, dramatically influence resident reactive astrocytes, inducing their exodus from the tumor mass and the resultant encapsulation of noninvasive lesions. Additionally, CSPGs induce activation of tumor-associated microglia. We demonstrate that the astrogliotic capsule can directly inhibit tumor invasion, and its absence from GBM presents an environment favorable to diffuse infiltration. We also identify the leukocyte common antigen-related phosphatase receptor (PTPRF) as a putative intermediary between extracellular glycosylated CSPGs and noninvasive tumor cells. In all, we present CSPGs as critical regulators of brain tumor histopathology and help to clarify the role of the tumor microenvironment in brain tumor invasion.

Altered distribution of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2(4) and the N-methyl-d-aspartate receptor subunit NMDAR1 in the hippocampus of patients with temporal lobe epilepsy

Abstract In patients with therapy-refractory temporal lobe epilepsy (TLE), alterations of glutamate receptors have been proposed as a mechanism for enhanced excitability. Using commercially available monoclonal antibodies specific for the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1 and for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2(4), we have examined the distribution of these polypeptides in human hippocampal tissue that was surgically removed from patients with intractable TLE. Surgical specimens were classified according to the presence of Ammon’s horn sclerosis (AHS) or a focal lesion in the temporal lobe. Cell counts and a densitometric analysis of the immunoreactivity patterns were carried out for all hippocampal subfields. NMDAR1 and GluR2(4) levels were markedly reduced in patients with AHS, primarily in those subfields with substantial neuronal cell loss (in particular CA1, CA4 and CA3), compared to those seen in patients with focal lesions and in control specimens obtained at autopsy. In contrast, the molecular layer of the dentate gyrus (DG-ML) showed significantly higher levels of GluR2(4) immunoreactivity in AHS compared to control tissue, while NMDAR1 showed no significant up-regulation in this sublayer. When the receptor staining intensity was normalized for alterations in neuronal density, no significant alterations could be detected except for an increase in GluR2(4) in the DG-ML of patients with AHS. These changes may reflect synaptic reorganization observed in the DG-ML of specimens from patients with chronic intractable TLE.