Effects of a behaviorally active antibody on the brain uptake and clearance of amyloid beta proteins

Antibodies directed against amyloid beta protein (AssP) have been suggested to be effective in the treatment of Alzheimer's disease (AD). Here, we used in vivo and in vitro models to test some of the mechanisms by which antibodies may produce their effects. We found that the blood-to-brain uptake of murine AssP1-42 was significantly reduced when co-injected peripherally with an antibody known to reverse cognitive defects in the SAMP8, an mouse model of AD. This antibody was not effective when tested against the more slowly transported human AssP1-42. Antibody given by intracerebroventricular (icv) injection did not improve the clearance of murine AssP1-42 from the brains of young healthy mice, which already rapidly clear AssP by saturable and non-saturable mechanisms. Antibody given icv also did not improve the clearance of human AssP1-42 from the brains of aged SAMP8 mice, a combination in which the AssP is only poorly cleared from brain. IV antibody also did not affect retention of murine AssP in young mice. In vitro transwell studies with monolayers of mouse brain endothelial cells (MBEC) found no evidence that antibody in the vascular chamber would retard the reuptake of AssP which had been effluxed from the brain-side chamber. A statistical trend suggested that antibody might decrease the association of AssP with brain vasculature. In conclusion, we found that icv administration of antibody was not effective in aiding clearance of AssP already in brain, but that blood-borne antibody can inhibit the entry of AssP into brain and might prevent AssP from associating with the brain vasculature.     

Effect of semax on the temporary dynamics of brain-derived neurotrophic factor and nerve growth factor gene expression in the rat hippocampus and frontal cortex

Semax is a synthetic peptide, which consists of the N-terminal adrenocorticotropic hormone fragment (4-7) (ACTH4-7) and C-terminal Pro-Gly-Pro peptide. Semax promotes neuron survival in hypoxia, increases selective attention and memory storage. It was shown that this synthetic peptide exerted a number of gene expressions, especially brain derived neurotrophic factor gene (Bdnf) and nerve growth factor gene (Ngf). Temporary dynamics of Bdnf and Ngf ex- pression in rat hippocampus and frontal cortex under Semax action (50 mg/kg, single intranasal administration) was studied in this work. It was shown that the studied gene expression levels changed significantly both in the hippocampus and the frontal cortex tissues 20 minutes after the peptide preparation application. The expression levels decreased in the hippocampus and increased in the frontal cortex. Forty minutes after Semax administration both gene expression levels returned to the level typical of control tissues. After that they increased significantly by 90 minutes after experiment start. Bdnf and Ngf expression levels decreased up to the control levels by 8 hours after medicine applying maximum gene expression levels were attained. Thus, Semax administration results in rapid, long-term, and specific activation of Bdnf and Ngf expression changes in different rat brain departments.     

Analysis of inducers of the E.coli lac repressor system in mammalian cells and whole animals.

Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system. These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers. In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours. Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration. Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG. In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes. HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor. These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.     

CD4 expression decrease by antisense oligonucleotides: inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.     

胶质细胞谷氨酸转运体-1反义寡核苷酸对脑缺血预处理神经保护效应的抑制作用

本研究应用胶质细胞谷氨酸转运体-1(glial glutamate transporter-1,GLT-1)的反义寡核苷酸(antisense oligo-deoxynucleotides,AS-ODNs)抑制Wistar大鼠GLT-1蛋白的表达,观察其对脑缺血预处理(cerebral ischemic preconditioning.CIP)增强脑缺血耐受作用的影响,探讨GLT-1在CIP诱导的脑缺血耐受中的作用.将凝闭双侧椎动脉的Wistar大鼠随机分为7组:(1)Sham组:只暴露双侧颈总动脉,不阻断血流;(2)CIP组:夹闭双侧颈总动脉3 min;(3)脑缺血打击组:夹闭双侧颈总动脉8 min;(4)CIP 脑缺血打击组:夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后,夹闭双侧颈总动脉8min;(5)双蒸水组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射双蒸水,每次5 μL,其它同sham组;(6)AS-ODNs组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同sham组,再根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组;(7)AS-ODNs CIP 脑缺血打击组:于CIP前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同CIP 脑缺血打击组,根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组.Western blot分析法观察GLT-1蛋白的表达,硫堇染色观察海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)情况.Western blot分析显示,侧脑室注射GLT-1 AS-ODNs可剂量依赖性地抑制大鼠海马CA1区GLT-1蛋白表达.硫堇染色显示,sham组和CIP组海马CA1区未见明显的DND;脑缺血打击组海马CA1区有明显的DND:预先给予CIP可显著对抗脑缺血打击引起的DND,表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗脑缺血打击引起的DND;而在GLT-1 AS-ODNs CIP 脑缺血打击组,侧脑室注射GLT-1 AS-ODNs后,大鼠海马CA1区出现了明显的DND,表明GLT-1 AS-ODNs通过抑制大鼠GLT-1蛋白表达从而减弱CIP对抗脑缺血打击的神经保护作用.以上结果进一步证实了GLT-1参与CIP诱导的脑缺血耐受.     

垂体在刺激脑内渗透压感受器引起的肾脏反应中的作用

实验在α氯醛糖和氨基甲酸乙酯混合麻醉的大鼠中进行。脑室内注射高张盐水(icv.HS)后,肾血浆流量、肾小球滤过率、尿量、尿钠排出量、尿钾排出量和渗透物质清除率均增加,游离水清除率下降。去除垂体后,icv.HS不再能引起上述肾脏反应。另外给大鼠静脉注射血管升压素(VP)拮抗剂(V_1和V_2受体拮抗剂),并不能削弱上述icv.HS引起的肾脏反应。脑室内注射高张盐水后,尿中多巴胺(DA)排出量无显著增多;给予多巴脱羧酶抑制剂苄丝肼也不能削弱icv.HS引起的肾脏反应。上述实验结果表明,在本实验条件下刺激脑内渗透压感受器引起的肾脏反应依赖于垂体的完整性,但看来并不依赖于外周的VP和DA,故垂体通过何种机制介导icv.HS引起上述肾脏反应,有待于进一步的研究。     

Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay. Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression.

Antisense oligodeoxynucleotides (AS-ODNs) are frequently used for the down-regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness, fast screening methods for the selection of effective AS-ODNs are needed. We describe a new cellular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of the gene of interest and the gene encoding the enhanced green fluorescent protein (EGFP) are cotransfected with AS-ODNs to COS-7 cells. Subsequently, cells are analysed for expression of the EGFP fusion protein by flow cytometry. With the assay, we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S-transferase Mu1 (GSTM1) and/or Mu2 (GSTM2). We found several AS-ODNs that demonstrated potent, sequence-specific, and concentration-dependent inhibition of fusion protein expression. At 0.5 microm, AS-6 and AS-8 inhibited EGFP-GSTM1 expression by 95 +/- 4% and 81 +/- 6%, respectively. AS-5 and AS-10 were selective for GSTM2 (82 +/- 4% and 85 +/- 0.4% decrease, respectively). AS-2 and AS-3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77-95% decrease). Other AS-ODNs were not effective or displayed non-target-specific inhibition of protein expression. The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity. As isoform-selective, chemical inhibitors of GSTM and GSTM knock-out mice are presently unavailable, the selected AS-ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protection against chemical-induced carcinogenesis.     

Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration

Peptide nucleic acids (PNAs) are DNA analogs that hybridize to complementary nucleic sequences with high affinity and stability. In our previous work, we showed that a PNA complementary to a 12-base pair (bp) sequence of the coding region of the rat neurotensin receptor (rNTR1) mRNA is effective in significantly blocking a rat's central responses to neurotensin (NT), even when the PNA is injected intraperitoneally (i.p.). Using a novel gel shift detection assay to detect PNA, we have now used this same PNA sequence to derive its pharmacokinetic variables and its tissue distribution in the rat. The PNA has a distribution half-life of 3 +/- 3 minutes and an elimination half-life of 17 +/- 3 minutes. The total plasma clearance and volume of distribution of this PNA were 3.4 +/- 0.9 ml/min x kg and 60 +/- 30 ml/kg. Two hours after dosing, the PNA was found at detectable but low levels in all organs examined-in order of decreasing concentration: kidney, liver, heart, brain, and spleen. Approximately 90% of the PNA dose was recovered as unchanged parent compound in the urine 24 hours after administration.     

Brain Gene Expression of a Sporadic (icv-STZ Mouse) and a Familial Mouse Model (3xTg-AD Mouse) of Alzheimer’s Disease

Alzheimer’s disease (AD) can be divided into sporadic AD (SAD) and familial AD (FAD). Most AD cases are sporadic and may result from multiple etiologic factors, including environmental, genetic and metabolic factors, whereas FAD is caused by mutations of presenilins or amyloid-β (Aβ) precursor protein (APP). A commonly used mouse model for AD is 3xTg-AD mouse, which is generated by over-expression of mutated presenilin 1, APP and tau in the brain and thus represents a mouse model of FAD. A mouse model generated by intracerebroventricular (icv) administration of streptozocin (STZ), icv-STZ mouse, shows many aspects of SAD. Despite the wide use of these two models for AD research, differences in gene expression between them are not known. Here, we compared the expression of 84 AD-related genes in the hippocampus and the cerebral cortex between icv-STZ mice and 3xTg-AD mice using a custom-designed qPCR array. These genes are involved in APP processing, tau/cytoskeleton, synapse function, apoptosis and autophagy, AD-related protein kinases, glucose metabolism, insulin signaling, and mTOR pathway. We found altered expression of around 20 genes in both mouse models, which affected each of above categories. Many of these gene alterations were consistent with what was observed in AD brain previously. The expression of most of these altered genes was decreased or tended to be decreased in the hippocampus of both mouse models. Significant diversity in gene expression was found in the cerebral cortex between these two AD mouse models. More genes related to synaptic function were dysregulated in the 3xTg-AD mice, whereas more genes related to insulin signaling and glucose metabolism were down-regulated in the icv-STZ mice. The present study provides important fundamental knowledge of these two AD mouse models and will help guide future studies using these two mouse models for the development of AD drugs.     

Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells

The deletion mutation in the HPRT-deficient mouse embryonic stem (ES) cell line E14TG2a has been corrected by gene targeting. The presence of plasmid sequences in the correcting vector DNA did not affect the frequency of correction. We have characterized three different HPRT gene structures in correctants. Cells from one corrected clone have been introduced into mouse blastocysts, and germ line transmission of the ES cell-derived corrected gene has been achieved. The corrected gene has the same pattern of expression as the wild-type gene, with the characteristic elevated level of expression in brain tissue. Hence, we have demonstrated the feasibility of introducing targeted modifications into the mouse germ line by homologous recombination in ES cells.     

Evaluation of the neurorestorative effects of the murine beta-nerve growth factor infusions in old rat with cognitive deficit

The nerve growth factor (NGF) is known to participate in the regulation of the expression levels and activity of the choline acetyltransferase (ChAT) in the nervous system. This enzyme is sensitive to the degenerative changes found in Alzheimer's disease (AD). We compared the effectiveness of intraparenchymal (ip) and intracerebroventricular (icv) administration of the murine beta-NGF (beta-NGFm) produced in our laboratories, through the determination of the expression levels and activity of the ChAT, and the evaluation of behavioral recovery in aged rat with cognitive deficit. Our results indicated that icv infusion of beta-NGFm stimulates the expression levels of ChAT gene in the striatum of old rats. Remarkable losses in the ChAT activity were observed in the septum and striatum of old rats. Exogenous administration of beta-NGFm produced a significant increase of ChAT activity in these brain regions differentially according to the administration pathway. The behavioral studies demonstrated that the administration pathway is an important factor in order to obtain the best results for a neurorestorative treatment.     

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice

BACKGROUND: The tissue-specific expression of an exogenous gene, under the influence of a tissue-specific promoter, has been examined in the past with pro-nuclear injections of the transgene and the development of transgenic mouse models. 'Adult transgenics' is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration. METHODS: The organ specificity of exogenous gene expression in adult mice was examined with a bacterial beta-galactosidase (LacZ) expression plasmid under the influence of the bovine rhodopsin gene promoter. The 8-kb plasmid DNA was delivered to organs following an intravenous administration with the pegylated immunoliposome (PIL) non-viral gene transfer technology. The PIL carrying the gene was targeted to organs with the rat 8D3 monoclonal antibody (MAb) to the mouse transferrin receptor (TfR). RESULTS: The rhodopsin/beta-galactosidase gene was expressed widely in both the eye and the brain of adult mice, but was not expressed in peripheral tissues, including liver, spleen, lung, or heart. Ocular expression included the retinal-pigmented epithelium, the iris, and ciliary body, and brain expression was observed in neuronal structures throughout the cerebrum and cerebellum. CONCLUSIONS: The expression of trans-genes in adult animals is possible with the PIL non-viral gene transfer method. The opsin promoter enables tissue-specific gene expression in the eye, as well as the brain of adult mice, whereas gene expression in peripheral tissues, such as liver or spleen, is not observed.     

Action sites of adrenomedullin in the rat brain: functional mapping by Fos expression.

The effects of intracerebroventricular (icv) administration of adrenomedullin (AM) and proadrenomedullin NH2-terminal 20 peptide (PAMP) on the expression of Fos in the central nervous system (CNS) were examined in conscious rats, using immunohistochemistry. Fos-like immunoreactivity (LI) was detected in various brain areas of the rats, including the supraoptic nucleus, the paraventricular nucleus, the locus coeruleus, the area postrema and the nucleus of the tractus solitarius 90 min after icv administration of AM. Few cells with Fos-LI were found in the CNS 90 min after icv administration of saline. Fos-LI was also detected in the various hypothalamic areas after icv administration of PAMP. These results suggest that centrally administered AM and PAMP may cause physiological responses through the activation of a neural network in the hypothalamus and the brainstem.     

Design and properties of hepatitis C virus antisense oligonucleotides for liver specific drug targeting

Different backbone modified antisense oligonucleotides (AS-ODNs) directed against the hepatitis C virus genome were 5'-conjugated to cholesterol, cholic acid or taurocholic acid to enhance liver specific drug targeting and hepatocellular uptake. The lipophilic character of modified AS-ODNs was determined from RP-HPLC retention times and duplex stability was correlated with Tm-values from UV melting curves.     

Intravenous neuromyelitis optica autoantibody in mice targets aquaporin-4 in peripheral organs and area postrema

The pathogenesis of neuromyelitis optica (NMO) involves binding of IgG autoantibodies (NMO-IgG) to aquaporin-4 (AQP4) on astrocytes in the central nervous system (CNS). We studied the in vivo processing in mice of a recombinant monoclonal human NMO-IgG that binds strongly to mouse AQP4. Following intravenous administration, serum [NMO-IgG] decreased with t_1/2 ∼18 hours in wildtype mice and ∼41 hours in AQP4 knockout mice. NMO-IgG was localized to AQP4-expressing cell membranes in kidney (collecting duct), skeletal muscle, trachea (epithelial cells) and stomach (parietal cells). NMO-IgG was seen on astrocytes in the area postrema in brain, but not elsewhere in brain, spinal cord, optic nerve or retina. Intravenously administered NMO-IgG was also seen in brain following mechanical disruption of the blood-brain barrier. Selective cellular localization was not found for control (non-NMO) IgG, or for NMO-IgG in AQP4 knockout mice. NMO-IgG injected directly into brain parenchyma diffused over an area of ∼5 mm² over 24 hours and targeted astrocyte foot-processes. Our data establish NMO-IgG pharmacokinetics and tissue distribution in mice. The rapid access of serum NMO-IgG to AQP4 in peripheral organs but not the CNS indicates that restricted antibody access cannot account for the absence of NMO pathology in peripheral organs.     

Cellular uptake,distribution, and stability of 10-23 deoxyribozymes

The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.     

Post-transcriptional silencing of Notch2 mRNA in chronic lymhocytic leukemic cells of B-CLL patients

Environmental and genomic stresses induce different pathological conditions and one of them is blood cancer. This escalating load of disease with a constant threat to life requires an intensive comprehensive response. For our understanding about the cancer treatment capabilities, novel medicinal platforms should be strived to explore among the existing conventional and molecular approaches that have already been proven to be successful in fighting against genetic diseases. Several DNA therapeutics previously studied are currently in clinical settings. RNA interfering antisense oligonucleotide (AS-ODN) is the most experimentally advanced molecular therapeutic which has the potential to modify the gene activity resulting in the down regulation of particular protein. In this study, we focused on the inhibition of Notch2 function in B-cell chronic lymphocytic leukemia (B-CLL) by AS-ODN (phosphorothioate oligomers) targeted to the initiation codon region of the Notch2 mRNA. We investigated the in vitro ability of four such oligomers to reduce the expression of Notch2 gene in peripheral blood mononuclear cells from B-CLL patients. Our findings implicate that AS-ODNs specifically designed for the region of 314–333 neucleotides (AS1) of Notch2 inhibits its gene expression better than other AS-ODNs designed for other regions and respond in a dose dependent manner. The results of cell proliferation assay for the evaluation of AS1 in gene silencing, infer that the number of cells were reduced to 80% (P < 0.001). Our results implicate that using the AS-ODNs against specific Notch2 nucleotide sequence can be used as future therapeutic agent with the ability of Notch2 down regulation, which is the root problem in the pathogenicity of B-CLL.     

DESIGN AND PROPERTIES OF HEPATITIS C VIRUS ANTISENSE OLIGONUCLEOTIDES FOR LIVER SPECIFIC DRUG TARGETING

Different backbone modified antisense oligonucleotides (AS-ODNs) directed against the hepatitis C virus genome were 5′-conjugated to cholesterol, cholic acid or taurocholic acid to enhance liver specific drug targeting and hepatocellular uptake. The lipophilic character of modified AS-ODNs was determined from RP-HPLC retentiontimes and duplex stability was correlated with T_m-values from UV melting curves.