Studies on cultured Schwann cells: the induction of myelin synthesis, and the control of their proliferation by a new growth factor

We have recently described the use of immunological methods to identify and purify rat Schwann cells. In dissociated cultures of neonatal sciatic nerve, all of the cells can be identified by antigenic criteria as either Schwann cells or fibroblasts. The fibroblasts may be removed by treatment with antiserum to the Thy-1 antigen and complement. The purified Schwann cells have been used to study the regulation of the expression of myelin components, and the stimulation of Schwann cell division by a soluble growth factor. Among the components of myelin, we have concentrated on the peripheral myelin glycoprotein P_0, which constitutes 50–60% of the protein in peripheral myelin. We have studied the distribution of P_0 in vitro and in vivo by immunofluorescence, immuno-autoradiography on SDS gels, and solid-phase radioimmunoassay. Our results support the hypothesis that P_0 is induced specifically as a consequence of the interaction between the Schwann cell and the myelinated type of axon. The level of P_0 in the myelin membrane is at least 1000-fold higher than in the Schwann cell membrane. Purified Schwann cells divide very slowly in a conventional tissue culture medium. This has allowed us to purify a new growth factor from extracts of brain and pituitary, tentatively named Glial Growth Factor (GGF). The activity resides in a basic protein with a native molecular weight of 6 × 10^4 daltons and a subunit molecular weight of 3 × 10^4 daltons, which is active at levels comparable to those of epidermal growth factor. GGF is mitogenic for Schwann cells, astrocytes and muscle fibroblasts

Identification of the orphan gene Prod 1 in basal and other salamander families.

The urodele amphibians (salamanders) are the only adult tetrapods able to regenerate the limb. It is unclear if this is an ancestral property that is retained in salamanders but lost in other tetrapods or if it evolved in salamanders. The three-finger protein Prod 1 is implicated in the mechanism of newt limb regeneration, and no orthologs have been found in other vertebrates, thus providing evidence for the second viewpoint. It has also been suggested that this protein could play a role in salamander-specific aspects of limb development. There are ten families of extant salamanders, and Prod 1 has only been identified in two of them to date. It is important to determine if it is present in other families and, particularly, the basal group of two families which diverged approximately 200 MYA

Mechanism of Action of Secreted Newt Anterior Gradient Protein

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family

Protection against glucose-induced neuronal death by NAAG and GCP II inhibition is regulated by mGluR3

Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective against long-term neuropathy in diabetic animals. In the current study, we have determined that the GCP II inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) is protective against glucose-induced programmed cell death (PCD) and neurite degeneration in dorsal root ganglion (DRG) neurons in a cell culture model of diabetic neuropathy. In this model, inhibition of caspase activation is mediated through the group II metabotropic glutamate receptor, mGluR3. 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist (S)-α-ethylglutamic acid (EGLU). In contrast, group I and III mGluR inhibitors have no effect on 2-PMPA neuroprotection. Furthermore, we show that two mGluR3 agonists, the direct agonist (2 R ,4 R )-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) and N -acetyl-aspartyl-glutamate (NAAG) provide protection to neurons exposed to high glucose conditions, consistent with the concept that 2-PMPA neuroprotection is mediated by increased NAAG activity. Inhibition of GCP II or mGluR3 may represent a novel mechanism to treat neuronal degeneration under high-glucose conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65724/1/j.1471-4159.2003.02321.x.pd

Regeneration of Cryoinjury Induced Necrotic Heart Lesions in Zebrafish Is Associated with Epicardial Activation and Cardiomyocyte Proliferation

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death

Hair organ regeneration via the bioengineered hair follicular unit transplantation

Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia

Role of Sox-9, ER81 and VE-Cadherin in Retinoic Acid-Mediated Trans-Differentiation of Breast Cancer Cells

Many aspects of development, tumor growth and metastasis depend upon the provision of an adequate vasculature. This can be a result of regulated angiogenesis, recruitment of circulating endothelial progenitors and/or vascular trans-differentiation. The present study demonstrates that treatment of SKBR-3 breast cancer cells with retinoic acid (RA), an important regulator of embryogenesis, cancer and other diseases, stimulates the formation of networks in Matrigel. RA-treatment of SKBR-3 cells co-cultured with human umbilical vein endothelial cells resulted in the formation of mixed structures. RA induces expression of many endothelial genes including vascular endothelial (VE) cadherin. VE-cadherin was also induced by RA in a number of other breast cancer cells. We show that RA-induced VE-cadherin is responsible for the RA-induced morphological changes. RA rapidly induced the expression of Sox-9 and ER81, which in turn form a complex on the VE-cadherin promoter and are required to mediate the transcriptional regulation of VE-cadherin by RA. These data indicate that RA may promote the expression of endothelial genes resulting in endothelial-like differentiation, or provide a mechanism whereby circulating endothelial progenitor cells could be incorporated into a growing organ or tumor

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro . Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47466/1/11068_2005_Article_3332.pd