Seong-Seng Tan

Separate Progenitors for Radial and Tangential Cell Dispersion during Development of the Cerebral Neocortex

Cell lineage analyses suggest that cortical neuroblasts are capable of undertaking both radial and tangential modes of cell movement. However, it is unclear whether distinct progenitors are committed to generating neuroblasts that disperse exclusively in either radial or tangential directions. Using highly unbalanced mouse stem cell chimeras, we have identified certain progenitors that are committed to one mode of cell dispersion only. Radially dispersed neurons expressed glutamate, the neurochemical signature of excitatory pyramidal cells. In contrast, tangential progenitors gave rise to widely scattered neurons that are predominantly GABAergic. These results suggest lineage-based mechanisms for early specification of certain progenitors to distinct dispersion pathways and neuronal phenotypes.

The Tumor Suppressor PTEN Is Exported in Exosomes and Has Phosphatase Activity in Recipient Cells

A tumor suppressor protein with lipid phosphatase activity is carried to target cells in microvesicles. PTEN Goes Traveling The tumor suppressor protein and phosphatase PTEN antagonizes the kinase PI3K, resulting in inhibition of the kinase Akt downstream of PI3K and reduced cellular proliferation. Mutations in PTEN are found in many different tumors. PTEN functions at the cytosolic side of the plasma membrane and in the nucleus, but Putz et al. extend its sphere of influence by showing that it can be secreted from cells in exosomes and taken up by recipient cells, where its functional activity is intact. PTEN-deficient cells that received exosomes bearing PTEN exhibited Akt inhibition and reduced proliferation. Secretion of PTEN from cells required components of the Nedd4 ubiquitination system. Given the role of PTEN in tumor suppression, these results suggest that the delivery of PTEN to tumor cells may provide an effective therapeutic strategy. Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non–cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease.

Clonal analysis of Patterns of growth, stem cell activity, and cell movement during the development and maintenance of the murine corneal epithelium

Patterns of growth and cell movement in the developing and adult corneal epithelium were investigated by analysing clonal patches of LacZ‐expressing cells in chimeric and X‐inactivation mosaic mice. It was found that cell proliferation throughout the basal corneal epithelium during embryogenesis and early postnatal life creates a disordered mosaic pattern of LacZ+ clones that contrasts with patterns of proliferation and striping produced during the later embryonic stages of retinal pigmented epithelium development. The early mosaic pattern in the corneal epithelium is replaced in the first 12 postnatal weeks by an ordered pattern of radial stripes or sectors that reflects migration without mixing of the progeny of clones of limbal stem cells. In contrast to previous assumptions, it was found that maturation of the activity of limbal stem cells and the pattern of migration of their progeny are delayed for several weeks postnatally. No evidence was found for immigration of the progeny of stem cells until the 5th postnatal week. There are approximately 100 clones of limbal stem cells initially, and clones are lost during postnatal life. Our studies provide a new assay for limbal and corneal defects in mutant mice.

Clonal expansion and cell dispersion in the developing mouse retina.

The present study has used two different approaches for labelling progenitor cells at the optic vesicle stage in order to examine patterns of clonal expansion and cellular dispersion within the developing retina. X‐inactivation transgenic mice and chimeric mice expressing the lacZ reporter transgene were examined during development and in adulthood to study the radial and tangential dispersion of proliferating neuroepithelial cells and postmitotic retinal cells of known identities. Chimeric retinas were used to measure tangential dispersion distances, while transgenic retinas were used to assess the frequency of tangential dispersion for individual populations of retinal neurons. Tangential dispersion is shown to be a universal feature of particular retinal cell types, being contrasted with the strictly radial dispersion of other cells. Tangential dispersion is a relatively short‐distance phenomenon, with distinct dispersion distances characteristic for cone, horizontal, amacrine and ganglion cells. Embryonic and postnatal retinas show that tangential dispersion occurs at different times for these distinct cell types, associated with their times of differentiation rather than their neurogenetic periods. These developmental results rule out the possibility that tangential dispersion is due to a passive displacement produced by the proliferation of later‐born cells, or to the lateral dispersion of a dividing sibling; rather, they are consistent with the hypothesis that tangential dispersion plays a role in the establishment of the orderly spatial distribution of retinal mosaics.

Growth and migration markers of rat C6 glioma cells identified by serial analysis of gene expression

Tumors derived from rat C6 cell implants into rat brain exhibit similar morphological characteristics and degree of vascularization to human glioblastomas. To establish a molecular basis for C6 gliosarcoma malignancy, we have constructed a molecular profile of the most abundantly expressed genes, using serial analysis of gene expression (SAGE). Sequence tags (1168) representing 738 individual transcripts were collected and tag‐to‐gene mapping was carried out using the UniGene data set for rat. Differentially expressed C6 transcripts were identified by comparison of tags collected for C6 cells with a similar number (1002) of tags from a rat primary astrocyte library. Genes found to be expressed at increased levels in C6 cells are associated with cell surface interactions, migration, or metastasis formation and proliferation. These include the receptor for hyaluronan‐mediated motility (RHAMM), S‐100 related protein 42A, galectin I, preproenkephalin, osteopontin, autocrine motility factor, α‐tubulin, ad1 antigen, and cofilin. In addition, a tag with no database match probably representing a previously uncharacterized transcript was differentially expressed in C6 cells. Transcripts showing reduced expression in C6 cells relative to astrocytes included the extracellular matrix glycoprotein osteonectin/SPARC (secreted protein, acidic, rich in cysteine), actin‐binding proteins thymosins β‐4 and β‐10, the cysteine protease inhibitor cystatin C, the actin‐gelling protein SM22/transgelin, and ferritin‐H. SAGE results were confirmed by Northern blot for all transcripts tested, reaffirming the value of the SAGE technique for expression profiling in cancer biology. GLIA 32:146–154, 2000.

Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2

Many ion channels and transporters are regulated by ubiquitination mediated by the Nedd4 family of HECT-type ubiquitin ligases (E3s). These E3s commonly interact with substrates via their WW domains that bind to specific motifs in target proteins. However, not all potential targets of these E3s contain WW-binding motifs. Therefore, accessory proteins may mediate the interaction between Nedd4 family members and their targets. Here we report that the divalent metal ion transporter DMT1, the primary nonheme iron transporter in mammals, is regulated by ubiquitination mediated by the Nedd4 family member WWP2. DMT1 interacts with 2 WW domain-interacting proteins, Ndfip1 and Ndfip2, previously proposed to have roles in protein trafficking. This promotes DMT1 ubiquitination and degradation by WWP2. Consistent with these observations, Ndfip1(-/-) mice show increased DMT1 activity and a concomitant increase in hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. This novel mechanism of regulating iron homeostasis suggests that Ndfips and WWP2 may contribute to diseases involving aberrant iron transport.

Statistical modeling of sequencing errors in SAGE libraries

MOTIVATION In the study of many systems, cells are first synchronized so that a large population of cells exhibit similar behavior. While synchronization can usually be achieved for a short duration, after a while cells begin to lose their synchronization. Synchronization loss is a continuous process and so the observed value in a population of cells for a gene at time t is actually a convolution of its values in an interval around t. Deconvolving the observed values from a mixed population will allow us to obtain better models for these systems and to accurately detect the genes that participate in these systems. RESULTS We present an algorithm which combines budding index and gene expression data to deconvolve expression profiles. Using the budding index data we first fit a synchronization loss model for the cell cycle system. Our deconvolution algorithm uses this loss model and can also use information from co-expressed genes, making it more robust against noise and missing values. Using expression and budding data for yeast we show that our algorithm is able to reconstruct a more accurate representation when compared with the observed values. In addition, using the deconvolved profiles we are able to correctly identify 15% more cycling genes when compared to a set identified using the observed values. AVAILABILITY Matlab implementation can be downloaded from the supporting website http://www.cs.cmu.edu/~zivbj/decon/decon.html

Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons

The regulation of metal ion transport within neurons is critical for normal brain function. Of particular importance is the regulation of redox metals such as iron (Fe), where excess levels can contribute to oxidative stress and protein aggregation, leading to neuronal death. The divalent metal transporter 1 (DMT1) plays a central role in the regulation of Fe as well as other metals; hence, failure of DMT1 regulation is linked to human brain pathology. However, it remains unclear how DMT1 is regulated in the brain. Here, we show that DMT1 is regulated by Ndfip1 (Nedd4 family-interacting protein 1), an adaptor protein that recruits E3 ligases to ubiquitinate target proteins. Using human neurons we show the Ndfip1 is upregulated and binds to DMT1 in response to Fe and cobalt (Co) exposure. This interaction results in the ubiquitination and degradation of DMT1, resulting in reduced metal entry. Induction of Ndfip1 expression protects neurons from metal toxicity, and removal of Ndfip1 by shRNAi results in hypersensitivity to metals. We identify Nedd4–2 as an E3 ligase recruited by Ndfip1 for the ubiquitination of DMT1 within human neurons. Comparison of brains from Ndfip1−/− with Ndfip1+/+ mice exposed to Fe reveals that Ndfip1−/− brains accumulate Fe within neurons. Together, this evidence suggests a critical role for Ndfip1 in regulating metal transport in human neurons.

Nedd4 Family-interacting Protein 1 (Ndfip1) Is Required for the Exosomal Secretion of Nedd4 Family Proteins

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50–90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.

Leukaemia inhibitory factor or related factors promote the differentiation of neuronal and astrocytic precursors within the developing murine spinal cord

Previously we have shown that leukaemia inhibitory factor (LIF) potentiates the development of murine spinal cord neurons in vitro, suggesting that it, or related factors, may play an important regulatory role in neuronal development. We have further investigated this role and show here that the generation of neurons in cultures of embryonic day 10 spinal cord cells is inhibited by antibodies to the β subunit of the LIF receptor. Since there are more undifferentiated precursors in antibody‐treated cultures than in control and LIF‐treated cultures, it is concluded that the primary action of LIF, or related molecules, is to promote neuronal differentiation, not precursor survival. In addition, the failure of LIF to support neuronal survival in the period immediately following differentiation suggests that the increased numbers of neurons generated with LIF are not attributable to its neurotrophic action. By selecting neuronal precursors on the basis of their inability to express class I major histocompatibility complex molecules, it was shown that LIF acted directly upon these cells and not via an intermediary cell. LIF also appears to be involved in regulating the differentiation of astrocytes, since it increases the number of glial fibrillary protein (GFAP)‐positive cells present in the cultures and since the spontaneous production of GFAP‐positive cells is blocked by antibodies to the LIF β receptor. These findings suggest that LIF or related factors promote the differentiation of neural precursors in the spinal cord, but that they are not involved in preferentially promoting precursors down a specific differentiation pathway.