Long (ObRb) and short (ObRa) leptin receptor isoforms are thought to play essential roles in mediating leptin signaling and the transport and degradation of leptin, respectively. Although the capacity of these cloned receptor species to mediate signal transduction has been reported, there is no information on the ability of individual receptor species to mediate leptin internalization and degradation or to undergo ligand-induced downregulation. We therefore studied these parameters in Chinese hamster ovary (CHO) cells stably expressing either ObRa or ObRb isoforms of the leptin receptor. We determined that both ObRa and ObRb mediated internalization of 125I-labeled leptin by a temperature- and coated pit-dependent mechanism. Both ObRa and ObRb also mediated degradation of 125I-leptin by a lysosomal mechanism, and this was more efficiently mediated by ObRa in these cells. Neither leptin internalization nor degradation by ObRa was affected by mutation of the conserved Box 1 motif. By studying deletion mutants of ObRa, we found that efficient internalization was dependent on a motif located between amino acids 8 and 29 of the intracellular domain of ObRa. Exposure of cells expressing ObRa or ObRb to unlabeled leptin for 90 min at 37 degrees C produced downregulation of available surface receptors, and this effect was of greater magnitude in cells expressing ObRb. Whereas CHO cells expressing the growth hormone receptor showed marked downregulation of ligand binding after exposure to dexamethasone (DEX) or phorbol myristic acid (PMA), PMA had no effect on expression of ObRa or ObRb, and DEX reduced binding to cells expressing ObRb by 15%. Thus, the two leptin receptor isoforms, ObRa and ObRb, mediate leptin internalization by a coated pit-dependent mechanism, leptin degradation by a lysosomal pathway, and ligand-induced receptor downregulation. The differential capacity of the two receptor isoforms may relate to the different roles of the receptor isoforms in the biology of leptin.

With refinement, the ECB technology is positioned to become an important therapeutic platform in restorative neurosurgery and, in combination with other therapeutic factors, may be relevant for the treatment of a variety of neurological disorders. Clinical trial registration no.: NCT01163825.

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

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