Generation of Alzheimer's Disease Transgenic Zebrafish Expressing Human APP Mutation Under Control of Zebrafish appb Promotor.

BACKGROUND: Amyloid peptide precursor (APP) as the precursor protein of peptide betaamyloid (ß-amyloid, Aß), which is thought to play a central role in the pathogenesis of Alzheimer's disease (AD), also has an important effect on the development and progression of AD. Through knocking-in APP gene in animals, numerous transgenic AD models have been set up for the investigation of the mechanisms behind AD pathogenesis and the screening of anti-AD drugs. However, there are some limitations to these models and here is a need for such an AD model that is economic as well as has satisfactory genetic homology with human. METHODS: We generated a new AD transgenic model by knocking a mutant human APP gene (APPsw) in zebrafish with appb promoter of zebrafish to drive the expression of APPsw. RESULTS: Fluorescent image and immunochemistry stain showed and RT-PCR and western blot assay confirmed that APPsw was successfully expressed in the brain, heart, eyes and vasculature of the transgenic zebrafish. Behavioral observation demonstrated that the transgenic zebrafish had AD-like symptoms. Histopathological observation found that there were cerebral ß-amyloidosis and angiopathy (CAA), which induced neuron loss and enlarged pervascular space. CONCLUSION: These results suggest that APPsw transgenic zebrafish well simulate the pathological characters of AD and can be used as an economic AD transgenic model. Furthermore, the new model suggested that APP can express in microvasculatures and cause the Aß generation and deposition in cerebral vessel which further destroys cerebral vascular structure resulting in the development and/or the progress of AD.

Amelioration of dementia induced by Aß 22-35 through rectal delivery of undecapeptide-hEGF to mouse brain.

A group of growth factors have been shown to play important roles in amelioration of the malfunction of the neurodegenerative diseases. However, the proteins or polypeptides passing across the blood-brain barrier (BBB) to access the brain parenchyma are relatively few so that it hinders the therapies in clinic. Here a genetically reconstructed fusion peptide of human epidermal growth factor (hEGF) with an undecapeptide YGRKKRRQRRR (P11) was used to investigate the permeability between the cell membrane and the BBB via rectal administration. The efficiency to rescue the Aß 22-35-induced dementia in mice was assessed after administration of P11-hEGF per rectal. Our results showed that P11-hEGF permeates across not only the 3T3 cell membrane in vitro, but also the endothelia of vessels after intravenous injection (IV), and the mucosa of the rectum after per rectal administration. Further results showed that the circulating P11-hEGF allowed penetrating through the blood-brain barrier and then getting into the brain manifesting biological responses. In the animal experiments, treatment with P11-hEGF not only ameliorated the dementia induced by Aß 22-35 but also rescued the dementia of the aged mice, no matter how it was administrated (IV or per rectal). These results suggest that the rectal non-invasive delivery of the P11 polypeptide-conjugated growth factor is an efficient way for BBB transduction, thus raises the hope of real therapeutic progress against neurodegenerative diseases.

Changes of autologous neural stem cells in the hippocampi of aging mice.

Although there is possibility of cognitive disturbance in aging people, many of them live for long life and enjoy well-functioning brain during the whole life-span. The biological basis of longevity is unknown. In this study, we investigated the influence of aging on hippocampal neural stem cells (NSCs), and the correlations between hippocampal neurogenesis and cognitive function. The result showed that the protein production and mRNA expression of nestin, and the number of BrdU(+) cells in dentate gyrus (DG) of the aged non-dementia mice were clearly higher than that in the aged dementia mice and the young adult mice. We also found that the number of NeuN(+) (neuron-specific nuclear antigen) cells in DG and CA1, choline O-acetyltransferase (ChAT, EC 2.3.1.6) production and mRNA expression in hippocampi of the aged-dementia mice were significantly reduced as compared to that of the young adult mice and the aged non-dementia mice, whereas the number of NeuN(+) cells, ChAT production and mRNA expression of the aged non-dementia mice has no difference with that of the young adult mice. Glial fibrillary acidic protein (GFAP) expression in the hippocampi of aged dementia mice significantly higher than that of the young adult mice and the aged non-dementia mice. Our results suggest that aging sometimes does not cause changing of the number of neurons and the hippocampal neurogenesis. Increment of DNA replication and neuron replacement, promotion of differentiation of neural stem cells, enhancement of neuronal proliferation, facilitation of synaptic plasticity of neurons may all benefit to the maintenance of the normal cognitive ability in the aged mice.

Effects of Chinese herbal medicine fuzhisan on aged rats.

Fuzhisan (FZS), a Chinese herbal complex prescription, has been used in the treatment of Alzheimer's disease (AD) for more than 15 years. Previous studies showed that FZS enhanced the cognitive ability in AD patients and AD model rats. FZS modulated the impaired cellular functions, and attenuated the damage caused by beta-amyloid protein, dose-dependently regulated and ameliorated the cholinergic functions of the Abeta(25-35)-induced AD-model mice. The SPECT imaging revealed that FZS improved the blood flow of the frontal and temporal lobes and the callosal gyrus in AD patients. However, little investigation of the effects of FZS on the naturally aged rats was reported. The underlying mechanism also remains to be explored. Recently we investigated the effects of the aqueous extract of FZS on the cognitive functions of the aged rats and the pharmacological basis for its therapeutic efficacy. The results showed a significant improvement made by FZS (0.3, 0.6, and 1.2 g/kg/d) for impaired cognitive functions of the aged rats. The rats manifested a shortened latency in Morris water maze test after intra-gavage administration (ig) of FZS for 30 consecutive days. The micro-positron emission tomography (microPET) using (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) as the tracer demonstrated that FZS promoted the glucose metabolism in the whole brains especially the temporal and parietal regions in the aged rats. The spectrophotometry and Western blot showed that FZS obviously increased the activity and the production of choline O-acetyltransferase (ChAT, EC 2.3.1.6) and the acetylcholine (ACh) contents in the hippocampus, thus regulated and ameliorated the impaired cholinergic functions of the aged rats. The therapeutical effects of FZS on the learning and memory of the aged rats were dose-dependent. The mechanism of action of FZS in ameliorating the memory dysfunction of the aged rats is ascribed to the reinforcement of the function of the cholinergic system and the enhancement of the glucose metabolism in the brains. The results of this study, together with a survey of the findings in the clinical treatment with FZS suggest that FZS may not merely alleviate the symptoms of the dementia, but may also augment the production of the neurotransmitter ACh and the energy supply in the brain to build up fitness of the patients. FZS may be beneficial for the treatment of Alzheimer's disease or cognitive impairment in old people.

Transducible P11-CNTF rescues the learning and memory impairments induced by amyloid-beta peptide in mice.

Alzheimer's disease is a progressive brain disorder with the loss of memory and other intellectual abilities. Amyloid species and neurofibrillary tangles are the prime suspects in damaging and killing nerve cells. Abnormal accumulation of Amyloid-beta peptide (Abeta) may cause synaptic dysfunction and degeneration of neurons. Drugs that can prevent its formation and accumulation or stimulate its clearance might ultimately be of therapeutic benefit. Ciliary neurotrophic factor (CNTF), a neurotrophic cytokine, promotes the survival of various neurons in brain. However, the blood-brain barrier hinders the systemic delivery of CNTF to brain. Recently the 11-amino acid of protein transduction domain TAT has successfully assisted the delivery of many macromolecules to treat preclinical models of human disease. The present study aimed to evaluate whether P11-CNTF fusion protein (P11-CNTF) is protective against the Abeta25-35-induced dementia in mice. Immunofluorescence experiments showed that P11 effectively carried CNTF to the SH-SY5Y cells in vitro, and to the brains of mice in vivo. The learning and memory impairments of mice induced by Abeta were substantially rescued by supplement with the P11-CNTF. Furthermore, mRNAs of enzymes involved in the Abeta metabolism, e.g. neprilysin (NEP), endothelin-converting enzyme 1 (ECE-1) and insulin degrading enzyme (IDE), increased in the P11-CNTF treated dementia mice, accompanied by the proliferation of nestin- and choline acetyltransferase (ChAT)-positive cells in hippocampus. It implies that the delivery of P11-CNTF may be a novel treatment for Alzheimer's disease.

Transduced PTD-BDNF fusion protein protects against beta amyloid peptide-induced learning and memory deficits in mice.

Brain-derived neurotrophic factor (BDNF) promotes the survival and differentiation of hippocampal, cortical, and basal forebrain cholinergic neurons. However, the efficacy of BDNF via peripheral (i.v.) administration is limited by the lack of transport of the neurotrophin through the blood-brain barrier (BBB) in vivo. The present study describes that the i.v. administered recombinant human BDNF (rhBDNF) conjugated with a protein transduction domain (PTD ) is able to survive and promote the growth of hippocampal neurons impaired by Abeta25-35 (10 microM) in vitro and transport through the BBB in vivo. The Morris water maze test indicated that the i.v. PTD-rhBDNF improved the spatial learning and memory of mice impaired by the aggregated Abeta25-35. The peripherally administered PTD-rhBDNF exhibited neuroprotective effects in brain and raise the possibility of delivery of the exogenous rhBDNF in treatment of the brain diseases.

[PTD-hEGF fusion protein--gene expression and function analysis].

To produce membrane-permeable human epidermal growth factor (hEGF), a pPTD-hEGF prokaryocyte expression vector was constructed and transformed into E. coli BL 21 (DE3). The PTD-hEGF fusion protein was induced by 0.3 mmol/L of IPTG expressed in the form of inclusion body with an yield of 40% of the total protein in the cells, and then purified by Ni2+ -NTA affinity chromatography. The SDS-PAGE analysis showed a single fusion protein band with a molecular weight of 16 kD. The amino acid sequence was checked by MALDI-TOF-MS analysis. The genetic engineering PTD-hEGF fusion protein obviously promoted the proliferation and growth of the HEK-293 cells in vitro.

A novel therapeutic approach to depression via supplement with tyrosine hydroxylase.

Tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2, TH) is the rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, dopamine (DA), noradrenaline (NE), and adrenaline, in the neurons. The regulated activity of TH is thought to play a critical role in modulating the functional activity of catecholaminergic neuronal systems in the brain. It is well known that the catecholaminergic neuronal systems are associated with depression. Here we showed that TH, delivered by protein transduction domain (PTD), passed through the blood-brain barrier and entered the neurons. Systemic TH treatment improved the behavioral despair in the forced swim test (FST) and the tail suspension test (TST), the two models widely used to screen the potential anti-depressant efficacy. The results indicated a novel and potential therapeutic use of TH in the depression disorder.

Protective effect of N-acetyl-L-cysteine on amyloid beta-peptide-induced learning and memory deficits in mice.

This study aimed to examine the effects of N-acetyl-L-cysteine (NAC) on protecting neurons function and improving learning and memory deficits in mice. Mice were intracerebroventricularly (icv) injected with the aggregated amyloid beta-peptide (Abeta) to produce Alzheimer's disease (AD). Learning and memory functions in mice were examined by the step through test and the water maze performance. The results showed that the mice pretreated with NAC had significantly greater retention in the step through test and shorter latencies in the water maze performance. Biochemical studies showed the potential role of free radical toxicity and the damage of cholinergic neurons in the Abeta-treated mice. There was an increased lipid peroxidation as indicated by elevated malondehyde (MDA) and decrease of glutathione (GSH) levels. There was also an increase in acetylcholinesterase (AChE) activity and a reduction in the choline acetyltransferase (ChAT) activity and acetylcholine (ACh) levels. NAC pretreatment significantly reversed the elevated MDA, AChE and the reduced GSH, ChAT and ACh in the Abeta-model mice. The results of the present study suggest the potential usage of the neuroprotective action of NAC on AD.

A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase.

The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 microM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD.

Antisense inhibition of acetylcholinesterase gene expression for treating cognition deficit in Alzheimer's disease model mice.

To examine whether the selected antisense oligodeoxynucleotides (AS-ODN) targeting against human brain acetylcholinesterase (AChE) mRNA could improve the cognitive deficit in the Alzheimer's disease (AD) model mice induced by amyloid-beta peptide (Abeta), we determined the time-effect relationship of AChE activity and the learning and memory after AS-ODN delivery. The results showed that the AChE activity decreased gradually along with time, initiating at 8 h and lasting 42 h. The time-effect curves of acetylcholine (ACh) behaved consistency with that of AChE activity. The animal cognition studies showed that in step-through test, the error number of the AS-ODN-treated AD model mice was significantly decreased, and the memory retention was increased. In the water maze performance, the swimming time obviously shortened. Our results indicated that antisense therapy is of potential use in the treatment of cognitive deficit in the Abeta model mice.

Complementary remedy of aged-related learning and memory deficits via exogenous choline acetyltransferase.

The present study aimed to examine whether the aged mice with naturally occurring cognitive deficits in learning and memory would benefit from supplementation of choline acetyltransferase (ChAT), the biosynthetic enzyme for neurotransmitter acetylcholine. Delivered by protein transduction domain (PTD), ChAT could pass through the blood-brain barrier, enter the neurons, interact with heat shock protein 70kDa, and retain enzyme activity. In behavior tests, PTD-ChAT given to the aged and memory-deficient mice almost completely reversed the behavioral changes, such as impairment of memory retention in the step-through test (an index of long-term memory) and prolonged swimming time in water maze test (an index of spatial recognition memory). The results suggest a novel and potential therapeutic use of PTD-ChAT in the age-related cognitive deficits.

Purification and characterization of recombinant human liver prolidase expressed in Saccharomyces cerevisiae.

The recombinant human liver prolidase (rh-prolidase, EC 3.4.13.9) from the lysate supernatant of engineering yeast Saccharomyces cerevisiae was purified in two steps employing anion-exchange gradient chromatography (DEAE-Sepharose fast flow) and gel filtration chromatography (Sephacryl S-200 high resolution). The purified recombinant protein furnished a single band with a molecular weight of 56 kD. Intensity scanning of the SDS-PAGE gel revealed that the prolidase accounted for more than 90% of total protein. The optimum pH of the catalytic reaction was 8.0. The enzyme was stimulated by Mn2+, but strongly inhibited by Cu2+ and Zn2+. The rh-prolidase expressed in S. cerevisiae had both dipeptidase and organophosphorus acid anhydrolase activity. It catalyzed the hydrolysis of soman and the dipeptide Gly -Pro. In a detoxification test in vitro, purified rh-prolidase was remarkably efficient at eliminating the toxicity of a lethal dose of soman, with the result that mice survived injection of such a dose.

Naked DNA prevents soman intoxication.

Paraoxonase (Q isoenzyme, PON1) can effectively hydrolyze chlorpyrifos-oxon (CPO), soman, sarin, and other organophosphates. Previous studies had indicated that the levels of serum PON1 in gene therapy with adenoviral vector could decrease the toxicity of CPO. In our study, plasmid pcDNA/PON1 injected into the tail vein of mice gave excellent expression at 24h after delivery, and PON1 activity decreased gradually along with days. The PON1 activities of mice treated with different doses of the plasmid (150, 300, and 600 microg/mouse) indicated a very good dose-effect relationship. Toxicity study has been performed using one lethal dose of soman (200 microg/kg). The mean death latency of mice pre-treated with 150, 300, 600, and 1200 microg pcDNA/PON1 extended and the mortality decreased vs control mice received the null pcDNA. These results demonstrate that increasing serum PON1 by naked DNA can offer protection toward the acute toxicity of soman.

Alternative therapy of Alzheimer's disease via supplementation with choline acetyltransferase.

Much evidence indicates that the memory and cognitive deficits of patients with Alzheimer's disease are closely associated with dysfunction of central cholinergic system. The degree of reduction of choline acetyltransferase activity in cerebral cholinergic neurons is significantly correlated with the severity of dementia or cognitive impairments observed in Alzheimer's disease. Therefore, Alzheimer's disease may be slowed by supplementation of exogenous choline acetyltransferase. Here we show that choline acetyltransferase mediated by TAT protein transduction domain passes through the blood-brain barrier and enters the neurons in mice, increasing choline acetyltransferase and neurotransmitter acetylcholine contents. The recombination TAT-choline acetyltransferase fusion protein injected intravenously improves the memory and cognitive dysfunction in Alzheimer's disease model mice induced by amyloid-beta peptide. Our results imply a novel and potentially effective way for Alzheimer's disease therapy.

Production of human liver prolidase by Saccharomyces cerevisiae as host cells.

AIM: To clone and express the recombinant human liver prolidase in yeast and explore the activities of both dipeptidase and organophosphoric acid anhydrolase (OPAA). METHODS: The cDNA encoding human liver prolidase derived from healthy adult liver was cloned into the pYES2, an expression vector of S cerevisiae, and then transformed into S cerevisiae INVSc1 by electroporation. The transformant with the highest enzymatic activity was induced by galactose for expression. The optimal induction conditions (temperature, induction time, and the initial amount of inoculation cells) were estimated by orthogonal experimental design. The recombinant prolidase and OPAA activities were assayed by spectrocolorimetric methods. RESULTS: The recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions (20 h, 25 degree, initial OD(600)=0.4), the maximum activities of prolidase and OPAA came to 226.5 and 578 micromol.min(-1).g(-1) protein in cell lysate supernatants, respectively. SDS-PAGE of the recombinant enzyme in disrupted cell supernatants showed a molecular weight of 56 kDa. Intensity scanning of the SDS-PAGE gel revealed that the enzyme accounted for 3.16 % of the total protein in the supernatant. One liter incubation medium produced 7 g of wet yeast cell containing 4.56 mg of the recombination protein. CONCLUSION: The recombinant human liver prolidase produced by yeast cell (S cerevisiae) exhibited both dipeptidase and OPAA activities.

Anti-epitope antibody, a novel site-directed antibody against human acetylcholinesterase.

AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induction and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody. METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChE was chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE) polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

Specific oligobodies against human brain acetylcholinesterase.

In order to develop the specific oligobodies against human brain acetylcholinesterase (AChE) and distinguishes between human erythrocyte and brain AChEs, we applied the strategy of 'target switching' to obtain the specific polyclonal and monoclonal oligobodies. The specificity between human brain AChE and other ChEs was identified by Western blotting, dot blotting and enzyme protein binding assay (EPBA). The results showed that the oligobodies against the human brain AChE specifically immunoreacted with the human brain AChE and Torpedo AChE, not showing significant binding to AChE from human erythrocyte and butyrylcholinesterase (BChE) from human serum.

Screening of RNA molecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichment.

AIM: To acquire the specific RNA aptamers inhibiting human red blood cell (RBC) acetylcholinesterase (AChE). METHODS: Systematic evolution of ligands by exponential enrichment (SELEX) aptamer against human red blood cell membrane AChE was selected by microtiter plate method in vitro. The specifity binding to AChE was determined by gel mobility shift analysis. Microcolorispectrophotometric method was used to measure the activity of AChE. RESULTS: The aptamers to human RBC AChE were identified by 9 reiterative rounds. At the same concentration (2.25 micromol/L), the aptamers did not bind to the recombinant human butyrylcholinesterase (rhBChE) but specifically bound to human RBC AChE and inhibited the enzyme activity. CONCLUSION: It is an effective way to isolate the specific AChE inhibitor from the vast oligonucleotide combinatorial library by virtue of SELEX.

Molecular simulation of a single-chain antibody against AChE to explore molecular basis of inhibitory effect of 3F3 McAb on enzyme activity.

AIM: To explore the molecular basis of the inhibitory effect of 3F3, a monoclonal antibody against acetylcholinesterase (AChE), by computer-aided molecular simulation. METHODS: The single-chain 3F3 antibody (Sc3F3) was designed by joining VH and VL via a flexible linker (Gly4Ser)3. The amino acid sequence of the recombinant Sc3F3 was then subjected to computer-aided molecular modeling, and docking with the antigen molecule AChE to mimic the immunoactive interaction in a three-dimensional fashion. RESULTS: The modeled structure of Sc3F3 manifested the common features of a classical antibody. Both VH and VL were composed of two ?-sheets and connecting loops. The docking profile of the action between Sc3F3 with AChE demonstrated the formation of a stable structure. The van der Waals force played an important role suggesting that the complex was formed mainly via hydrophobic interactions between Sc3F3 and AChE molecules. CONCLUSION: The spatial structure of the complex of Sc3F3 and AChE showed that Sc3F3 overlaid the entrance of the active center gorge of AChE blocking the access of substrate.