Study Of Transmembrane Mechanism Of Transducible TAT-tCNTF And Effect Of TAT-tCNTF On Mice Injured By Aβ

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, and with thepopulation aging, people who suffer from AD are increasing, so these bring heavy burden tothe family and the society. Now drugs for the treatment of AD like anticholinesterase drugsbelong to the symptomatic treatment, couldn’t fundamentally repair neurons and preventdisease development processes. So it’s very necessary to seek for an effective treatment forAD to prevent and delay disease development. Amyloid plaques forming from β-amyloidprotein (Aβ) deposition and neurofibrillary tangles (NFTs) from tau hyperphosphorylation arethe two main pathological features, and the former is an initiating factor of AD and may causeneuronal degeneration in patients with AD.Previous studies and the domestic and foreign literatures have reported that ciliaryneurotrophic factor (CNTF) as one of the factors in the neurotrophin family not onlypromoted the growth of neurons and maintain cell survival but also significantly reduced Aβaggregation and deposition which just need to be treated for AD. So CNTF has a goodprospect for AD therapy.However, CNTF is a macromolecule protein, too difficult to cross theblood brain barrier (BBB) and so cannot reach the target area and its receptor, which limits itsclinical application. Trans-activator transcription (TAT) of human immunodeficiency virus-1(HIV-1), an effective protein transduction domain (PTD), can carry exogenousmacromolecules transferred to the cell, which provide a good modified means for drugs withthe large molecular weight difficult across the membrane. So our research group adoptedgenetic recombination technology with TAT and truncated CNTF and expressed in E. coli, andthen obtained the fusion protein, named TAT-tCNTF after separation and purification. Thisissue mainly investigated transmembrane properties and the mechanism of transmembrane ofTAT-tCNTF, and the effect and mechanism of TAT-tCNTF to cells and mice injured byAβ.Developing the BBB model with two layers of cells in double rooms showedTAT-tCNTF crossed the BBB model, and the fluorescence intensity of CF488A labeledTAT-tCNTF in the lower room increased as time went on, which suggested that TAT-tCNTFwas able to pass through the BBB cell model. In cultured SH-SY5Y cells, TAT-tCNTFsmoothly crossed the cell membrane and entered the cells, but the fluorescence signal ofrecombinant human CNTF （rhCNTF）due to its features of difficult transmembrane werehardly detected in SH-SY5Y. In that the transmembrane of TAT-tCNTF is similar to TAT and TAT crossed the membrane mainly via endocytosis, the transmembrane features ofTAT-tCNTF were studied with endocytosis inhibitors. As a result, the giant pinocytosisinhibitors cytochalasin D (CD) and amiloride Hydrochloride (AMI) suppressed TAT-tCNTFto enter cells, that’s to say, TAT-tCNTF entered cells mainly by way of giant pinocytosis.However, CD did not completely inhibit the effect of transmembrane of TAT-tCNTF, whichmight be associated with the structure of intracellular F-actin and the content of calcium ion.In mice, hippocampal tissue sections were obtained after TAT-tCNTF by intraperitonealinjection and proceeded immunofluorescence experiments for TAT, with the result thatTAT-tCNTF crossed the BBB and distributed in hippocampus more than rhCNTF.In vitro, TAT-tCNTF relieved the injury caused by Aβ and improved cell viability andreduced early apoptosis induced by Aβ, and even previous studies indicated that TAT-tCNTFlowered intracellular peroxide by increasing the content of glutathione (GSH) and the level ofsuperoxide dismutase (SOD). This topic also explored that the effect of TAT-tCNTF to cellsinjured by Aβ. Hippocampal neurons were separated and cultured in vitro. The cell viabilitylowered and the early apoptosis increased after Aβ treated hippocampal neurons, whileTAT-tCNTF increased the cell viability and decreased the early apoptosis. Mice with i.c.v. Aβappeared amyloid deposits, which was significantly reduced after i.p. TAT-tCNTF.TAT-tCNTF had the effect on Aβ injured mice both in histopathology and behaviorstics.In Morris water maze test, the escape latency of Aβ injured mice was significant longer andthe average swimming velocity was slower than the control group of mice. Meanwhile, in theshuttle box of passive escape test, the first electric shock time of Aβ injured mice was shorterand times suffered shock in limited time was more than control. These reflected that Aβ led toweaken the ability of learning and memory in mice. TAT-tCNTF ameliorated Aβ injured micein the above aspects. Brain tissues were collected and made sections, and then detected byimmunohistochemistry for Ki67, which suggested that Aβ reduced the Ki67-positive cells inDG, CA1and CA3of the hippocampus, that’s to say Aβ inhibited cell proliferations in micehippocampus. Western blot showed a similar result when detecting the cyclinD1proteinexpression, while both Ki67+cells and the cyclinD1protein expression increased in mice afteri.p. TAT-tCNTF, which indicated TAT-tCNTF improved cell inhibition produced by Aβ. Notonly is this conclusion to the total number of cells in the brain, but also to the NeuN-positivecells in the hippocampus of mice, namely Aβ decreased the number of NeuN+neurons whileTAT-tCNTF improved NeuN+neurons growth through the immunohistochemistry for NeuN.BrdU+GFAP double marked immunofluorescence test showed that Aβ caused reactiveastrogliosis the DG, CA1and CA3of hippocampus in mice brains, where BrdU positive cellsincreased that suggested BrdU was detained much and cell proliferation was slow. These results were accordance with the Ki67cells test and the cyclinD1protein expression test. Butin the brains of mice via i.p. TAT-tCNTF BrdU+and GFAP+cells were hardly observed,which illustrated TAT-tCNTF promoted cell proliferation in mice brains and relieved thereactive astrogliosis induce by Aβ. Further on, TAT-tCNTF significantly reduced tauhyperphosphorylation led by Aβ and yet rarely affected the Aβ precursor protein (APP) afterwestern blot detected Aβ precursor protein (APP), tau and p-tau protein expression. The allshowed TAT-tCNTF decreased NFTs formed from p-tau induced by Aβ but did not affect theupstream channel ofAβ.Due to biological effects of TAT-tCNTF nearly from CNTF, TAT-tCNTF played a roleunder conditions that it needed to bind with CNTF receptor CNTFRα and activated thedownstream of CNTF. This trail investigated that the Akt, Erk and Stat3protein expressionsof CNTF downstream channels, as a result that TAT-tCNTF increased the phosphorylation ofErk and Akt while not to Stat3in the DG, Cx, SVZ and other areas of the hippocampus areaon the day26and/or day45of the animal experiments, which showed TAT-tCNTF activatedthe Erk and Akt pathway while not do Stat3pathways. In brief, TAT-tCNTF produced cascadeeffects after binding with CNTFRα and then improved the Aβ injured mice in pathology andphenotype.The issue constructed and expressed the TAT-tCNTF fusion protein by generecombination of CNTF with the feature of difficult transmembrane, which had been provedthat TAT-tCNTF was relatively easy to pass trough biomembrane and BBB. And TAT-tCNTFimproved both cells and mice injured by Aβ. Meanwhile, it’s explained that in vivoTAT-tCNTF crossed the BBB and entered the mice brains mainly via the macropinocytosisand then TAT-tCNTF bind to CNTFRα and activated the Erk and Akt channels so thatimproved the ability of learning and memory of mice injured by Aβ, and promoted cellproliferation and survivals and relieved reactive astrogliosis. The topic solved the problem ofTAT-tCNTF unable to be widely applied in clinical medicine, which provided the basis ofTAT-tCNTF clinical practice and good prospects for AD therapy.