| Ciliary neurotrophic factor (CNTF) is a cytokine with neurotrophic and differentiation-inducing effects across a broad spectrum of peripheral and central nervous system cells. CNTF enhances the survival of sensory neurons, motor neurons, cerebral neurons and hippocampal neurons. It is promising for the treatment of neurodegenerative diseases. However, the blood-brain barrier hinders the systemic delivery of CNTF while its therapeutic potential for central nervous system diseases has been clear for sometime. Poor permeability of most proteins across cell membranes compromises their potential as therapeutic reagents. Invasive application strategies like intracerebroventricular infusion and intrathecal implantation of encapsulated cells with CNTF was used to treat the Huntington's disease and amyotrophic lateral sclerosis. The traumatic and complicated usages severely limited its clinical application. Recently, it has been shown that the protein transduction domains (PTD) have the ability to be internalized into the cell from the external environment even carrying a variety of large, biologically-active molecules. TAT (47–57) is an efficient transduction domain without the neurotoxicity of the full-length TAT of 86 amino acids. TAT has been shown to deliver various macromolecules passing the cell membranes and blood-brain barrier of mammals.In this paper, the conjugate (TAT-tCNTF) of TAT (47-57) of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed successfully in E.Coli. A highly purified soluble and biologically active TAT-tCNTF was obtained by denaturation and renaturation methods.It was demonstrated that TAT-tCNTF could promote the survival of TF-1 cells, induce the differentiation of SH-SY5Y cells in vitro, and protect SH-SY5Y cells from death after Aβ25-35 exposure. Cell permeation at different temperature of 37℃or 4℃indicated that a little of rhCNTF penetrated across the cell membrane via receptor-mediated endocytosis mechanism, however, TAT-tCNTF can all pass through the cell membrane by an additional way (e.g. lipid-craft pathway) besides endocytosis. The immunofluorescence assay clearly demonstrated that TAT-tCNTF did successfully penatrate into the neurons and reached the deeper parenchyma of the dentate gyrus while CNTF per se could not just because of the limitation of the blood-brain barrier. It is confirmed that the administered fusion protein has penetrated into the SH-SY5Y cells and crossed blood-brain barrier into the brains as well as the skin and rectum barriers. So TAT-tCNTF with high activities and effective transmembrane ability was obtained for the first time.Alzheimer's disease (AD) is the most common neurodegenerative dementia in the elder, affecting cognition, behavior and functioning, because of the loss of neurons. Neurodegeneration may be due to a decrease in expression of neurotrophic factors. The neuroprotective effect of neurotrophic factor therapy appears to be one of the most promising approaches toward effective treatment of AD. CNTF is a pleiotropic cytokine with neurotrophic properties for a number of neurons in vitro and in vivo and is the most active of the neurotrophic factors studied in promoting neurogenesis. However, little was done on research or application of CNTF for the treatment of AD. The present animal study aimed to evaluate whether TAT-tCNTF is protective against the Aβ25-35-induced dementia in mice since TAT-tCNTF inhibited amyloid fibrils formation in vitro. The learning and memory impairments of AD mice were substantially rescued by supplement with the TAT-tCNTF. Furthermore, mRNAs of enzymes involved in the Aβmetabolism e.g. endothelin-converting enzyme 1 (ECE-1) and insulin degrading enzyme (IDE) increased in the TAT-tCNTF -treated dementia mice, accompanied by the proliferation of nestin- and ChAT-positive cells in hippocampus. It implies that the delivery of TAT-tCNTF may be a novel treatment for Alzheimer's disease.In addition to promoting cell proliferation and differentiation, CNTF also has the ability to cause fat-loss in animals and humans. TAT-tCNTF was found to possess the ability of reducing the fat weight in the diet-induced obese (DIO) mice by means of a non-invasive administration route (per rectum). Significant reductions of the fat weight and Lee's index after treatment of TAT-tCNTF were observed in DIO mice. The adipocytes in the DIO mice became enlarged compared with the normal mice fed on the basic fodder. After treatment of TAT-tCNTF, the diameter of the enlarged adipocytes nearly recovered to that of the normal control. The level of serum glucose was also reduced. However, TAT-tCNTF has no effect on the levels of serum TG and TCHO. High dose of TAT-tCNTF reduced the mRNA expression of NPY compared with that of DIO control whereas it had no effect on the expression of leptin.Notably, the therapeutical effects of TAT-tCNTF on AD or obesity were alike no matter what kind of the administration route (i.p. or p.r.) was used. The rectal route, a non-invasive and practical choice, has an acceptable safety profile with a tiny first-pass effect. It produced less damage to the body for long term administration than the route of i.p. or i.v. which was commonly used for the protein drugs. Therefore, TAT-tCNTF provides a new, non-traumatic delivery into the brain for central therapeutic drugs. TAT-tCNTF may be an effective, non-invasive bio-drug in the therapy of AD, obesity and other neurodegeneration diseases.In summary, TAT-tCNTF is a pleiotropic factor, with actions which involve the protection of cells from apoptosis, inhibition of the Aβdeposit, promotion of the degradation and removal Aβ, improvement of the neurogenesis and the ChAT expression. And TAT-tCNTF also caused the fat-loss in DIO mice as well as the decrease of blood glucose. The outstanding characteristics of non-invasive administration of TAT-tCNTF make it a novel therapeutic strategy for Alzheimer's disease, obesity and other neurodegeneration diseases. |
