Rebecca A. Ihrie

Corridors of migrating neurons in the human brain and their decline during infancy

The subventricular zone of many adult non-human mammals generates large numbers of new neurons destined for the olfactory bulb. Along the walls of the lateral ventricles, immature neuronal progeny migrate in tangentially oriented chains that coalesce into a rostral migratory stream (RMS) connecting the subventricular zone to the olfactory bulb. The adult human subventricular zone, in contrast, contains a hypocellular gap layer separating the ependymal lining from a periventricular ribbon of astrocytes. Some of these subventricular zone astrocytes can function as neural stem cells in vitro, but their function in vivo remains controversial. An initial report found few subventricular zone proliferating cells and rare migrating immature neurons in the RMS of adult humans. In contrast, a subsequent study indicated robust proliferation and migration in the human subventricular zone and RMS. Here we find that the infant human subventricular zone and RMS contain an extensive corridor of migrating immature neurons before 18 months of age but, contrary to previous reports, this germinal activity subsides in older children and is nearly extinct by adulthood. Surprisingly, during this limited window of neurogenesis, not all new neurons in the human subventricular zone are destined for the olfactory bulb—we describe a major migratory pathway that targets the prefrontal cortex in humans. Together, these findings reveal robust streams of tangentially migrating immature neurons in human early postnatal subventricular zone and cortex. These pathways represent potential targets of neurological injuries affecting neonates.

Perp is a p63-regulated gene essential for epithelial integrity

p63 is a master regulator of stratified epithelial development that is both necessary and sufficient for specifying this multifaceted program. We show here that Perp, a tetraspan membrane protein originally identified as an apoptosis-associated target of the p53 tumor suppressor, is the first direct target of p63 clearly involved in mediating this developmental program in vivo. During embryogenesis, Perp is expressed in an epithelial pattern, and its expression depends on p63. Perp-/- mice die postnatally, with dramatic blistering in stratified epithelia symptomatic of compromised adhesion. Perp localizes specifically to desmosomes, adhesion junctions important for tissue integrity, and numerous structural defects in desmosomes are observed in Perp-deficient skin, suggesting a role for Perp in promoting the stable assembly of desmosomal adhesive complexes. These findings demonstrate that Perp is a key effector in the p63 developmental program, playing an essential role in an adhesion subprogram central to epithelial integrity and homeostasis.

Persistent Sonic Hedgehog Signaling in Adult Brain Determines Neural Stem Cell Positional Identity

Neural stem cells (NSCs) persist in the subventricular zone (SVZ) of the adult brain. Location within this germinal region determines the type of neuronal progeny NSCs generate, but the mechanism of adult NSC positional specification remains unknown. We show that sonic hedgehog (Shh) signaling, resulting in high gli1 levels, occurs in the ventral SVZ and is associated with the genesis of specific neuronal progeny. Shh is selectively produced by a small group of ventral forebrain neurons. Ablation of Shh decreases production of ventrally derived neuron types, while ectopic activation of this pathway in dorsal NSCs respecifies their progeny to deep granule interneurons and calbindin-positive periglomerular cells. These results show that Shh is necessary and sufficient for the specification of adult ventral NSCs.

Cells in the astroglial lineage are neural stem cells

A common assumption of classical neuroscience was that neurons and glial cells were derived from separate pools of progenitor cells and that, once development was completed, no new neurons were produced. The subsequent disproving of the “no new neuron” dogma suggested that ongoing adult neurogenesis was supported by a population of multipotent neural stem cells. Two germinal regions within the adult mammalian brain were shown to contain neural progenitor cells: the subventricular zone (SVZ) along the walls of the lateral ventricles, and the subgranular zone (SGZ) within the dentate gyrus of the hippocampus. Surprisingly, when the primary progenitors (stem cells) of the new neurons in these regions were identified, they exhibited structural and biological markers of astrocytes. The architecture of these germinal regions and the pattern of division of neural stem cells have raised fundamental questions about the mechanism of adult neurogenesis. This review describes studies on the origin of adult neural stem cells, the features distinguishing them from astrocytes in non-germinal regions, and the control mechanisms of the proliferation and differentiation of these cells. Astrocytic adult neural stem cells are part of a developmental lineage extending from the neuroepithelium to radial glia to germinal astrocytes. Adult neural stem cells appear to be strongly influenced by their local microenvironment, while also contributing significantly to the architecture of these germinal zones. However, environment alone does not seem to be sufficient to induce non-germinal astrocytes to behave as neural stem cells. Although emerging evidence suggests that significant heterogeneity exists within populations of germinal zone astrocytes, the way that these differences are encoded remains unclear. The further characterization of these cells should eventually provide a body of knowledge central to the understanding of brain development and disease.

Developmental Context Determines Latency of MYC-Induced Tumorigenesis

One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYCs ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis.

Perp is a mediator of p53-dependent apoptosis in diverse cell types

The induction of apoptosis by the p53 protein is critical for its activity as a tumor suppressor. Although it is clear that p53 induces apoptosis at least in part by direct transcriptional activation of target genes, the set of p53 target genes that mediate p53 function in apoptosis in vivo remains to be well defined. The Perp (p53 apoptosis effector related to PMP-22) gene is highly expressed in cells undergoing p53-dependent apoptosis as compared to cells undergoing p53-dependent G1 arrest. Perp is a direct p53 target, and its overexpression is sufficient to induce cell death in fibroblasts, implicating it as an important component of p53 apoptotic function. Here, through the generation of Perp-deficient mice, we analyze the role of Perp in the p53 apoptosis pathway in multiple primary cell types by comparing the cell death responses of Perp null cells to those of wild-type and p53 null cells. These experiments demonstrate the involvement of Perp in p53-mediated cell death in thymocytes and neurons but not in E1A-expressing MEFs, indicating a cell type-specific role for Perp in the p53 cell death pathway. In addition, we show that Perp is not required for proliferation-associated functions of p53. Thus, Perp selectively mediates the p53 apoptotic response, and the requirement for Perp is dictated by cellular context.

Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy.

Although malignant astrocytomas are a leading cause of cancer-related death in children, rational therapeutic strategies are lacking. We previously identified activating mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) (BRAFT1799A encoding BRAFV600E) in association with homozygous cyclin-dependent kinase inhibitor 2A (CDKN2A, encoding p14ARF and p16Ink4a) deletions in pediatric infiltrative astrocytomas. Here we report that BRAFV600E expression in neural progenitors (NPs) is insufficient for tumorigenesis and increases NP cellular differentiation as well as apoptosis. In contrast, astrocytomas are readily generated from NPs with additional Ink4a-Arf deletion. The BRAFV600E inhibitor PLX4720 significantly increased survival of mice after intracranial transplant of genetically relevant murine or human astrocytoma cells. Moreover, combination therapy using PLX4720 plus the Cyclin-dependent kinase (CDK) 4/6-specific inhibitor PD0332991 further extended survival relative to either monotherapy. Our findings indicate a rational therapeutic strategy for treating a subset of pediatric astrocytomas with BRAFV600E mutation and CDKN2A deficiency.

Perp-etrating p53-Dependent Apoptosis

The induction of apoptosis is a fundamental mechanism by which the p53 transcriptional activator protein suppresses tumor development. Recently, the roles of several p53 target genes in mediating the p53 apoptotic response have been queried through loss-of-function analysis with knockout mouse models. These studies have demonstrated that the p53 targets Noxa, Puma, and Perp play cell type-specific roles in p53-mediated apoptosis. Perp, a tetraspan protein localizing to the plasma membrane, rather than to mitochondria, is a novel type of p53 effector that may stimulate apoptosis through a different mechanism from the BH3-containing proteins Noxa, Puma, and Bax.

Single cell analysis of human tissues and solid tumors with mass cytometry

Mass cytometry measures 36 or more markers per cell and is an appealing platform for comprehensive phenotyping of cells in human tissue and tumor biopsies. While tissue disaggregation and fluorescence cytometry protocols were pioneered decades ago, it is not known whether established protocols will be effective for mass cytometry and maintain cancer and stromal cell diversity.

A New Perp in the Lineup: Linking p63 and Desmosomal Adhesion

The process of stratified epithelial development depends upon a transcriptional program directed by the p53-related transcription factor p63. p63 is required for the commitment of the ectoderm to stratification and for the completion of terminal differentiation in stratified epithelia, and mutations in p63 have been identified in multiple developmental disorders affecting ectoderm-derived tissues. Recent work from our laboratory has determined that the p53 target gene Perp is required for the integrity of the stratified epithelia specified by p63, and that expression of Perp in these structures depends on the presence of p63. In these tissues, Perp is a critical component of the desmosome, a cell-cell adhesion complex whose constituents are frequently mutated in human diseases affecting the skin and hair. Perp’s position downstream of p63 and p53, as well as its essential role in normal desmosome function, suggest that it, like other adhesion proteins, may be a target for mutation in human blistering diseases or cancer.