The receptor tyrosine phosphatase CRYPalpha promotes intraretinal axon growth.

Retinal ganglion cell axons grow towards the optic fissure in close contact with the basal membrane, an excellent growth substratum. One of the ligands of receptor tyrosine phosphatase CRYPalpha is located on the retinal and tectal basal membranes. To analyze the role of this RPTP and its ligand in intraretinal growth and guidance of ganglion cell axons, we disrupted ligand- receptor interactions on the retinal basal membrane in culture. Antibodies against CRYPalpha strongly reduced retinal axon growth on the basal membrane, and induced a dramatic change in morphology of retinal growth cones, reducing the size of growth cone lamellipodia. A similar effect was observed by blocking the ligand with a CRYPalpha ectodomain fusion protein. These effects did not occur, or were much reduced, when axons were grown either on laminin-1, on matrigel or on basal membranes with glial endfeet removed. This indicates that a ligand for CRYPalpha is located on glial endfeet. These results show for the first time in vertebrates that the interaction of a receptor tyrosine phosphatase with its ligand is crucial not only for promotion of retinal axon growth but also for maintenance of retinal growth cone lamellipodia on basal membranes.     

CRYP-2: A receptor-type tyrosine phosphatase selectively expressed by developing vertebrate neurons

Axonal growth and guidance, like other aspects of neuronal differentiation, can be regulated by changes in tyrosine phosphorylation. Although much is known concerning the role of tyrosine kinases in these processes, relatively little is known about the nature and function of protein tyrosine phosphatases (PTPs) that may be involved. To identify the PTPs expressed in the embryonic chicken CNS at the time of axon growth, we performed a polymerase chain reaction based “screen” using degenerate primers directed against conserved regions of the PTP catalytic domain. We obtained five distinct PTP-related cDNAs, two of which code for novel PTPs. One, designated CRYP-2, is selectively expressed in the CNS. Full-length cloning of CRYP-2 revealed that it is a receptor-type PTP with an adhesion molecule-like extracellular region comprising fibronectin (FN) type III repeats and a single catalytic domain in the intracellular region. It is alternatively spliced in the juxtamembrane region, similar to other PTPs recently cloned. CRYP-2 mRNA is strongly expressed in the brain during the time of axon growth; it is downregulated toward the end of embryo-genesis. Western blot analysis identifies a 330-kDa glycoprotein as CRYP-2 and confirms that the protein is downregulated after hatching. Immunostaining of cerebellar neurons in vitro reveals that CRYP-2 is expressed on neuronal cell bodies and processes, but not on glia. The CAM-like structure, developmental pattern of expression, and neuron-specific localization of the CRYP-2 PTP suggest that it is involved in neuronal differentiation, particularly axon growth. © 1996 John Wiley & Sons, Inc.     

HIV-1 utilizes the CXCR4 chemokine receptor to infect multipotent hematopoietic stem and progenitor cells

HIV infection is characterized by gradual immune system collapse and hematopoietic dysfunction. We recently showed that HIV enters multipotent hematopoietic progenitor cells and establishes both active cytotoxic and latent infections that can be reactivated by myeloid differentiation. However, whether these multipotent progenitors include long-lived hematopoietic stem cells (HSCs) that could establish viral reservoirs for the life of the infected person remains unknown. Here we provide direct evidence that HIV targets long-lived HSCs and show that infected HSCs yield stable, multilineage engraftment in a xenograft model. Furthermore, we establish that the capacity to use the chemokine receptor CXCR4 for entry determines whether a virus will enter multipotent versus differentiated progenitor cells. Because HSCs live for the life span of the infected person and are crucial for hematopoietic health, these data may explain the poor prognosis associated with CXCR4-tropic HIV infection and suggest HSCs as long-lived cellular reservoirs of latent HIV.     

A comparison of the anti-luteolytic activities of recombinant ovine interferon-alpha and -tau in sheep

Interferon tau (IFNT) is secreted by the trophectoderm of ruminant conceptuses during the peri-implantation period and serves an anti-luteolytic function. The question as to whether IFNT is superior as an anti-luteolytic agent to closely related Type I IFNs, such as IFN alpha (IFNA), which have a different function, remains unanswered. Thus, the aim of this study was to determine whether equivalent antiviral (AV) units of ovIFNA and ovIFNT are equipotent in extending estrous cycle length. Four distinct ovIFNA mRNA (ovIFNA1-4) were cloned from ovine lymphocytes. Recombinant ovine IFNs (ovIFNT4 and ovIFNA1) were prepared in the yeast Pichia pastoris. The AV activity of the purified IFNs was determined on a bovine cell line (MDBK) and on transformed ovine luminal uterine epithelial cells. Indwelling uterine catheters were fitted into crossbred ewes on Day 3 postestrus (Day 0 = estrus). Between Days 10 and 18 postestrus, ewes received twice-daily infusions of 0.7 x 10(7) IU of either ovIFNA1 or T4, plus serum albumin. Control ewes received serum albumin only. Daily blood samples were collected for progesterone determination, and ewes were monitored twice daily for estrus. Both ovIFNA (P = 0.04) and ovIFNT (P = 0.01) caused estrous cycle extension in nonpregnant ewes compared to controls when administered at equivalent AV doses. In conclusion, the uniqueness of IFNT as an anti-luteolytic agent most likely resides in its unique expression pattern rather than its special biopotency.     

CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro

Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.