Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.in vivo in mice.In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status. However, other subtypes namely H7, H9, and H5 which primarily cause infections and death among avian species have crossed the species barrier and caused mild to severe or fatal disease in humans [Annual influenza epidemics caused by influenza A and B viruses result in three to five million cases of severe illness with about 250,000 to 500,000 deaths globally every year. In the United States, complications from influenza infections result in approximately 250,000 hospitalizations and 36,000 deaths in an average year, with majority of the fatalities occurring among the elderly population . Influenn humans . Since 2n humans . In factn humans .Vaccination is the primary strategy for reducing the morbidity and mortality associated with human influenza -15. HoweThe immune system has evolved to recognize and eliminate pathogens. A number of pathogen recognition receptor (PRRs) families are involved in pathogen sensing and can be present in the host as soluble molecules in tissue fluids and serum or as molecules on cell membranes, localized in various cellular compartments, or in the cytosol -31. RecoThe 1918 pandemic resulted in 20-50 million deaths worldwide. To test if the activation of RIG-I with 5'PPP-RNA could suppress the replication of the reconstructed 1918 virus, we treated A549 cells with 5'PPP-RNA or CIAP-RNA for 24 hr, and then infected them with 1918 virus at an MOI of 0.01. Supernatants were collected 24 hr post-infection and assayed for viral titer. Figure Next we evaluated the prophylactic antiviral potential of 5'PPP-RNA against avian influenza H5N1 viruses with pandemic potential. A549 cells were transfected with 5'PPP-RNA or CIAP-RNA for 0, 24 or 48 hr, and infected with A/Vietnam/1203, an H5N1virus. Viral titers were determined 24 hr post-infection. As shown in Figure Stockpiling of the antiviral drug, oseltamivir is one strategy for pandemic preparedness, but the emergence of oseltamivir-resistant H5N1 viruses would seriously impede these efforts. Hence, we also tested the ability of 5'PPP-RNA to inhibit the replication of drug-resistant A/Vietnam/1203/2004 viruses that include zanamivir- and oseltamivir-sensitive and adamantanes-resistant and adamantanes- and oseltamivir-resistant virus variants, A/VN/30408/2005 (H274Y) and A/VN/HN/30408/2005 (N294S). As shown in Figure in vivo administration of 5'PPP-RNA will reduce viral titers, we delivered 5'PPP-RNA or PBS formulated in in vivojet-PEI daily for four days and infected mice on day 1 with A/Mexico/4482/09. Daily administration of 5'PPP-RNA alone had no side effects on body weight gain and activity (data not shown). On day 4 post-challenge, lungs from all animals were collected to determine viral titers. Control group had significantly higher viral load in lungs when compared to those that received 5'PPP-RNA  that circumvent host immune responses. Several studies, including our own have shown independently that non-structural protein (NS1) of influenza A viruses inhibits RIG-I function. We, therefore, investigated the expression of IFNβ and RIG-I as well as NS1 by real-time RT-PCR in 5'PPP-RNA or CIAP-RNA transfected A549 cells that were either uninfected or infected with H5N1 virus at an MOI of 0.1 for 24 hr following transfection. As shown in Figure RIG-I activation has been reported to stimulate type 1 interferon expression that induces an antiviral state. To study the direct role of 5'PPP-RNA induced RIG-I and type 1 interferon induction we knockdown RIG-I or IFNαβ-receptor using gene specific siRNA. As shown in Figure We also investigated the therapeutic potential of 5'PPP-RNA in A549 cells infected with seasonal influenza virus A/New York/02/2001 and its oseltamivir-resistant H274Y mutant. As shown in Figure Three influenza pandemics occurred during the 20th century, with varying degrees of severity; outcomes ranged from the high levels of illness and death observed during the 1918 Spanish flu pandemic (estimates of deaths range from 20 to 100 million) to the much lower levels observed during the pandemics of 1957 and 1968 (approximately one million deaths each) ,55. Furtin vitro transcribed 5'PPP-RNA, a ligand for cytoplasmic RNA sensor, RIG-I, in antiviral innate immune response against a panel of influenza viruses including drug-resistant, potential pandemic as well as pandemic strains in human lung epithelial cells. The broad-spectrum prophylactic potential of 5'PPP-RNA against influenza viruses is clearly evident from our studies. 5'PPP-RNA significantly inhibited in vitro growth of not only wild-type drug-sensitive and resistant H5N1 strains with comparable efficacy but also highly pathogenic 1918 pandemic strain. We also included 2009 pandemic H1N1 viruses in our studies to see the effect of 5'PPP-RNA both in vitro and in vivo. Data shown Figure in vitro suppression level of A/California/08/09 was similar to that of other viruses used in this study. Furthermore, 5'PPP-RNA also inhibited A/California/08/09 viral replication in vivo in Balb/c mice. These data suggest that irrespective of type or subtypes, drug-susceptibility status, or in vivo virulence, the replication of influenza viruses was inhibited by 5'PPP-RNA. The dose and frequency of administration of 5'PPP-RNA may influence the extent of inhibition of viral replication.In the present study, we explored the role of Treatment with 5'PPP-RNA not only induced the expression of IFN-I but also upregulated RIG-I expression. 5'PPP-RNA induced expression of IFN-I and RIG-I was not affected by subsequent infection with H5N1 Figure  or 1918 Viruses have acquired mechanism(s) to escape host immune surveillance by inhibiting IFN induction pathways. The NS1 protein of Influenza A virus inhibits host antiviral defenses by direct interaction with RIG-I -44. 5'PPTo evaluate the therapeutic potential of 5'PPP-RNA, we treated pre-infected (with wild-type and drug-resistant A/New York/02/2001) A549 cells with 5'PPP-RNA at different time-points. We observed relatively weak antiviral effect of 5'PPP-RNA, when A549 cells were transfected with 5'PPP-RNA post 8 hr infection. Interestingly, at this time point we also observed significantly low RIG-I and IFNβ expression  treatment was performed to remove tri-phosphate groups from in vitro synthesized RNA. Briefly, 100 μg of in vitro transcribed RNA was treated with 150U of CIAP for 3 hr at 37°C in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA and 50U of RNase inhibitor. RNA was purified as described above. Chemically synthesized RNA with same sequence and length that contains an OH-group at its 5'-end was purchased from Dharmacon .5'PPP-RNAs were synthesized using T7 RNA polymerase. Using annealed complimentary oligo nucleotides, a DNA template was constructed that contains a T7 RNA polymerase promoter followed by the sequence of interest (AGCUUAACCUGUCCUUCAA) to be transcribed ,61. Twenin vitro transcribed 5'-PPP-RNA has been shown to contain dsRNA as well that activates RIG-I [in vitro transcribe 5'PPP-RNA is single stranded RNA (ssRNA) and/or double stranded RNA,(dsRNA) we used RNase A treatment under the conditions that degrades ssRNA, but not dsRNA. This confirmed that in vitro transcribed RNA used in our study was RNA only  supplemented with 10% fetal bovine serum (FBS), 100? U/ml penicillin and 100 μg/ml streptomycin. Normal human bronchial epithelial (NHBE) cells  were maintained under the conditions and media specified by the supplier.Seasonal viruses used in this study include the laboratory H1N1 strain A/Puerto Rico/8/34, wild-type H1N1 , H3N2 , and B viruses (wild-type B/Memphis/20/96 and its R152K mutant resistant to NA inhibitors zanamivir and oseltamivir), HPAI H5N1 viruses , and A/Vietnam/HN30408/2005 (N294S),, 2009 pandemic H1N1 viruses, A/California/08/09 and A/Mexico/4482/09, and the reconstructed 1918 Spanish influenza pandemic virus (H1N1) generated by plasmid-based reverse genetics . All humin vitro transcribed 5'PPP-RNA, chemically synthesized RNA or CIAP-treated RNA using Lipofectamine 2000. This dose was determined by dose-kinetics studies using 1, 2, 4 and 6 μg of RNA. After 24 hr, cells were infected with the different viruses used in this study in a 6-well plate. Unless specified, infection of cells was performed at a multiplicity of infection (MOI) of 0.1 with or without trypsin supplement. Each treatment was carried out in duplicate cultures. After 24 hr, cells were harvested for RNA and protein analysis and cell-culture supernatants were collected and stored at -80°C for determination of viral titer by plaque assay as described previously using MDCK cells[Cells were transfected with 2 μg of DCK cells. This tith of each reaction) were analyzed in using syber green Q-PCR reagents . PCR condition was kept as 94°C for 15 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s for a total of 45 cycles. The threshold cycle number for cDNA was normalized to that of β-actin mRNA, and the resulting value was converted to a linear scale. Data from three independent experiments were taken account for analysis. All data points fell into a normal distribution and there were no outliers.Total RNA was isolated from cells using the RNAeasy kit  and real time RT-PCR was conducted using a Stratagene Q3005 PCR machine for mRNA expression of RIG-I, IFNβ, NS1, and β-actin. For each sample, 2 μg of RNA was reverse transcribed using Superscript II Reverse Transcriptase  according to the manufacturer's directions. Parallel reactions without reverse transcriptase were included as negative controls. Reverse transcription reactions  for 20 minutes at 4°C. The protein content of different samples was determined using a protein assay reagent . Equal quantities of solubilized protein were resolved by 10% SDS-PAGE, blotted to nitrocellulose membrane and probed with the indicated primary antibodies. Anti-RIG-I was purchased from Axxora, LLC . Anti-β-actin antibody was purchased from Sigma, St. Louis, MO, USA. Antibody signals were detected by chemiluminescence using secondary antibodies conjugated to horseradish peroxidase and an ECL detection kit .A549 cells grown on cover-slips were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr. Cell monolayers were then washed with cold PBS and fixed with 2% paraformaldehyde for 15 min at room temperature. Cells were permeablized with 0.05% saponin in PBS for 30 min at room temperature, and then blocked with blocking buffer  at room temperature. Cells were incubated with primary antibodies (1 μg/ml) at 4°C overnight, and were washed 3 times with wash buffer (0.05% saponin in PBS). Cell monolayers were further incubated with secondary antibodies (Alexa 594 or 488) in blocking buffer for 1 hr at room temperature followed by three washes with buffer. To stain nuclei, cells were incubated with Hoechst dye (5 μM in PBS containing 0.05% saponin). Cells were further washed (3 times) with PBS, and were mounted on slides using Prolong antifade mounting media  and were observed under a Zeiss LSM 510 fluorescence microscope.in vivo-jetPEI according to manufacturer's protocol  in a volume of 200 μl for 4 days. On day 1, mice were challenged intranasaly with 1000 MID50 of 2009 pandemic virus (A/Mexico/4482/09). A separate group of animals that received only 5'PPP-RNA complexed with in vivo-jetPEI without viral challenge were observed to determine changes in body weight and activity. Lungs from control and treated challenged animals were collected on day 4 to determine viral titers using MDCK cells as described earlier.Female Balb/c mice 6-12 weeks old purchased from Jackson Laboratories were intravenously injected 100 μg of 5'PPP-RNA, capped 5'-RNA or PBS complexed with P < 0.05 was considered significant. All data points were included in the analysis and there were no outliers.To determine the statistical significance among the 5'PPP-RNA or CIAP-RNA treated and untreated groups, we used Analysis of variance and a value of The authors declare that they have no competing interests.PR participated in the planning and execution of the experiments, analyses of the results, and manuscript writing. LJ, VD, and MAH performed studies with human and avian viruses. VJ, WGD, JBB, MEW and MBP assisted in making 5'PPP-RNA and tested mRNA samples for the expression of various host innate immune response genes with cytokine arrays. SG provided assistance in assessing the functionality of cytokines and chemokines. HZ and TMT performed studies with the 1918 pandemic virus. LG, JMK, AG, and TF provided advice in the planning and execution of the studies. SS participated in the planning, coordination, supervision, and execution of the experiments that led to the present manuscript. All the authors saw and approved the final version of the manuscript.Figure S1. 5'PPP-RNA inhibits replication of drug-resistant human viruses. A459 cells (1 × 106 cells/well) in a 6-well tissue culture plate were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr and then infected with (a&b) wild-type and drug-resistant human H1N1 viruses; (b&c) drug-resistant H3N2 viruses and (d&e) wild-type and drug-resistant B viruses. Supernatants collected were assayed for viral titers as indicated in material and methods. Results shown are mean ± SD from three independent experiments and are expressed as viral titer (pfu/ml).Click here for fileFigure S2. Testing viral colonies if they are interferon escape mutant. A/NewYork/02/2001 colonies from 5'PPP-RNA treated A549 cells grown on MDCK cell were isolated and viral stocks were made by growing them in MDCK cells. Subsequently, these viruses were tested for 5'PPP-RNA sensitivity in A549 cells transfected 24hr earlier with either CIAP-RNA or 5'PPP-RNA. Culture supernatants were collected 24hr post-infection to determine viral titers in MDCK cells as described in materials and methods.Click here for fileFigure S3. Analysis of size, integrity and single or double-strandness of RNA. In vitro transcribed RNA (1 μg) generated by T7 polymerase was digested with 0.1 μg/ml RNase A (Ambion) or DNase I (10U/ml) (Ambion) at 37C for 1 hr, separated on agarose gel, and visualized by ethidium bromide staining.Click here for file


Parkinson's disease (PD) is the most common movement disorder. While neuronal deposition of α-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, α-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of α-synuclein, however, is unclear.Here we show that PD-related A53T mutant α-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of α-synuclein aggregates was found in presymptomatic and symptomatic mouse brains and correlated with the expansion of reactive astrogliosis. The normal function of astrocytes was compromised as evidenced by cerebral microhemorrhage and down-regulation of astrocytic glutamate transporters, which also led to increased inflammatory responses and microglial activation. Interestingly, the activation of microglia was mainly detected in the midbrain, brainstem and spinal cord, where a significant loss of dopaminergic and motor neurons was observed. Consistent with the activation of microglia, the expression level of cyclooxygenase 1 (COX-1) was significantly up-regulated in the brain of symptomatic mice and in cultured microglia treated with conditioned medium derived from astrocytes over-expressing A53T α-synuclein. Consequently, the suppression of COX-1 activities extended the survival of mutant mice, suggesting that excess inflammatory responses elicited by reactive astrocytes may contribute to the degeneration of neurons.Our findings demonstrate a critical involvement of astrocytic α-synuclein in initiating the non-cell autonomous killing of neurons, suggesting the viability of reactive astrocytes and microglia as potential therapeutic targets for PD and other neurodegenerative diseases. GenetNeuronal expression of either human wild-type or PD-related mutant α-syn induces neurodegeneration associated with pathological accumulations of α-syn and reactive astrogliosis -13. In aHere we generated a new line of α-syn inducible transgenic mice in which the PD-related A53T α-syn was selectively expressed by astrocytes. Interestingly, the mutant mice developed rapidly progressed paralysis, likely resulting from widespread astrocytosis, severe microglial activation, and especially midbrain dopaminergic and spinal cord motor neuron degeneration. These results demonstrate that α-syn-mediated cytotoxicity to astrocytes is critical for inducing the non-cell autonomous degeneration of neurons, suggesting that the maintenance of normal function of astrocytes is important in ameliorating the progression of neurodegeneration in PD and other neurodegenerative diseases.syn is most abundant in neurons [syn in astrocytes has not been rigorously examined. Here we prepared the astrocytic and neuronal cell cultures from postnatal day 1 pups and compared the expression of α-syn in these cells. Consistent with previous findings [α- neurons . Howeverfindings , α-syn wsyn in the pathogenesis of PD, we generated new lines of α-syn inducible transgenic mice that selectively expressed the human wild-type (WT) or PD-related A53T α-syn in astrocytes. As outlined in Fig. GFAP-tTA) transgenic mice [syn transgenic mice (tetO-WT or tetO-A53T) to generate the GFAP-tTA/tetO-WT (WT) and GFAP-tTA/tetO-A53T double transgenic (A53T) mice. The expression of exogenous α-syn in the double transgenic mice was demonstrated by Western blot using an antibody specifically against human α-syn. We have obtained one WT and two A53T lines of transgenic mice  littermate controls , a tetracycline derivative, to block the expression of A53T α-syn from embryonic stages to postnatal day 21 (P21). The DOX-exposed A53T mice behaved normally at one and two months of age  and symptomatic mice (A53T*) were examined for GFAP staining   Figs. . Taken tA53T mice, we examined the expression and subcellular distribution of aquaporin 4, glucose transporter 1 (Glut1) and von Willebrand factor (vWF) in astrocyte and vascular endothelial cells. As shown in the brainstem of nTg mice, aquaporin 4 was normally confined to the end-foot of astrocytes that outlined the blood vessels in the brain  G was observed in the brainstem of A53T symptomatic mice -positive dopaminergic neurons in SNpc and ventral tegmental area (VTA) were not altered in the A53T mice at 1 month of age , a selective inhibitor of COX-1 [A53T mice was significantly extended  and plays an important role in the neuroinflammatory process . To evalof COX-1  once a dA53T mice. We first checked the presence of α-syn aggregates in the brain, which appeared as α-syn-positive high molecular weight bands in Western blot analysis and resistant to Triton-X100 (TX) extraction in tissue fractionation studies. The aggregation of α-syn was significantly increased in the brain of symptomatic A53T mice compared with nTg controls and asymptomatic mice . Compared to the early onset paralysis exhibited in A53T-E2 mice (the high expression line), no obvious behavior abnormalities were observed in A53T-A8 mice up to 12 months of age (data now shown). However, astrogliosis and modest microglial activation were detected in the brainstem and SNpc area of A53T-A8 mice at 12 months of age  [A53T mice may attribute to the activation of nearby microglia. This hypothesis is in line with recent reports that lipopolysaccharide, an activator of microglia, causes SNpc dopaminergic neuron degeneration in vivo [A53T mice.The degeneration of midbrain dopaminergic neurons and spinal motor neurons may account for the paralysis of symptomatic sporters , which wes (ROS) -45. Micres (ROS) . Thus th in vivo -49. NoneOur findings firmly support the non-cell autonomous toxicity which is well documented in neurodegenerative disease including PD, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and Alzheimer's disease (AD) . α-syn esyn transgenic mice not only provide strong evidences for the critical involvement of astrocytes in the pathogenesis of PD and motor neuron diseases, but also provide a very useful in vivo system to test novel therapeutics for preventing the loss of dopaminergic and motor neurons.Our studies from this new line of A53T α-α-synuclein (α-syn) inducible transgenic mice, a DNA fragment containing human wild-type (WT) or A53T α-syn coding sequence was inserted into a tetracycline operator-regulated gene expression vector (tet-O) to generate the WT or A53T inducible expression construct. The insert was then purified and microinjected into fertilized oocytes derived from C57BL6/J mice. One WT and two A53T founders were obtained, which were used to cross with GFAP-tTA mice [GFAP-tTA/tetO-α-syn double transgenic mice. The mice were housed in a pathogen-free climate-controlled facility with ad libitum access to regular diet and water. Genotypes were determined by PCR analysis of tail DNA extracted by DirectPCR Reagents . PCR primers for genotyping GFAP-tTA mice are: tTA-F (5'CCCTTGGAATTGACGAGTAC GGTG3') and MP1R (5'TGGTGTATGAGCGGCGGCGACGGCAG3'); and for genotyping tetO-α-syn mice are: PrpEx2-F (5'TACTGCTCCATTTTGCGTGA3') and SNCA-R (5'TCCAGAATTCCTTCCTGTGG3'). All mouse work follows the guidelines approved by the Institutional Animal Care and Use Committees of the National Institute of Child Health and Human Development.To generate human A53T tTA mice  to generRotarod test: as described previously [eviously , mice weOpen-field test: as described previously [eviously , the ambGrip strength measurement: as described previously [eviously , mice wez-series stack scans at 0.8 μM intervals from individual field. The numbers of microglia and microglia cluster in the images taken from 25 × objective (368 μm × 368 μm) were counted.As described previously , mice weth section, Bregma -2.06-1.54 mm), SNpc , as well as cervical (approximate T1-T6) and lumbar spinal cord  were stained with NeuN plus TH and NeuN, respectively, and visualized using the Vectastain ABC kit . The number of NeuN or TH-positive cells was assessed using Stereo Investigator 8, an unbiased stereological procedure with an optical fractionator . The sampling scheme was designed to have coefficient of error (CE) less than 10% in order to get reliable results. All stereological analyses were performed under the 100 × objective of a Zeiss Axio microscope (Imager A1).According to stereotaxic coordinates of mouse brains , a series of coronal sections across the striatum . The cells were pre-incubated at 37°C in a humidified atmosphere of air/5% CO2 and the medium was changed first 24 h later and on alternate 3-days thereafter. After a pre-culture period of 8-11 days the cellular debris, microglia were lifted from astrocytes layer by shaking the culture flasks at 190 rpm for 3 h at 30°C. The cells attached to the flask were passed and grown in six-well plates for 3 days until harvest for astrocytes culture. The cells floating in the medium were collected by centrifuge and plated on 6-well plates in DMEM supplemented with 10% FBS for microglial culture. After 24 h incubation, the medium of microglial cultures was switched to conditioned medium from cultured astrocytes.Primary cortical neuron cultures were conducted as described previously  by using3, 1 mM MgCl2, and 0.5 mM CaCl2, plus protease and phosphatase inhibitor cocktails). Lysates were centrifuged at 1, 000 g for 10 min to separate supernatant (S1) and pellet (P1). Protein concentrations in S1 were measured by BCA . S1 contains total α-synuclein protein, representing the sucrose fraction. An aliquot of S1 was diluted in the same volume of Triton extraction buffer , homogenized by sonication, and centrifuged at 20, 000 × g for 30 min to obtain the Triton X-100-soluble (TX-sol) supernatant (S2) and Triton X-100-insoluble (TX-insol) pellet (P2). P2 was washed 4 times by 1% Triton X-100 buffer and centrifuged at 20, 000 × g for 10 min. The pellet fraction was further extracted in 2% SDS buffer  by sonication and centrifuged at 20, 800 × g for 5 min. The supernatant (S3) were present as Triton X-100-insoluble (TX-insol) or SDS-soluble fraction.As previously described , brain tissues  were weighed and homogenized with 10 volumes of sucrose buffer  using MOPS running buffer (Invitrogen), and transferred to polyvinylidene difluoride (PVDF) or Nitrocellulose membranes. Antibodies specific to human/mouse α-synuclein   and β-acFor COX-1 inhibitor treatment, mutant mice and control littermates at 2 months of age were administrated with SC-560  or vehicle  through intraperitoneal (IP) injection once a day for 7 days .2 First Strand Kit (SuperArray Bioscience Corporation). Quantitative real-time PCR was performed using an ABI Prism 7900HT Fast Detection System (Applied Biosystems).RNA was harvested using Qiagen RNeasy mini kit and converted into first-strand cDNA using RTStatistical analysis was performed using the Graphpad Prism 5 . Data are presented as Means ± SEM. Statistical significances were determined by comparing means of different groups using t-test or ANOVA followed by Post Hoc Tukey HSD test, and presented as *p < 0.05, **p < 0.01, ***p < 0.001.The authors declare that they have no competing interests.XLG and HC designed the experiments and wrote the manuscript. XLG, CXL, CSX and LS performed the experiments. XL provided some materials. All authors have read and approved the final manuscript.α-synuclein was selectively expressed in the astrocytes of A53T transgenic micePD-related human A53T . (A) Western blot analysis reveals the expression level of exogenous α-syn in asymptomatic A53T mice. Protein extracts (5 μg) from the hippocampus were diluted by 1, 2, 4, 8, 16, 32 and 64-fold and equal volume of diluted samples was subjected to Western blot with a human/mouse specific α-syn antibody, α-syn (C20). (B-E) To determine the expression pattern of tTA under the GFAP promoter, GFAP-tTA mice were crossbred with tetO-HIST1H2BJ/GFP to yield GFAP-tTA/tetO-GFP mice. HIST1H2BJ/GFP is located in the nucleus. Brain sections of GFAP-tTA/tetO-GFP mice were stained with NeuN , Iba1 , OSP , and GFAP . Scale bars: 20 μm.Click here for fileA53T miceProgressive reduction of body weight and spontaneous locomotor activities in . (A) Representative photos of A53T mice and age-matched littermate controls under anesthetized condition. (B) Bar graph shows the body weight of A53T mice and control littermates at 1 and 2 months of age. ***p < 0.001. (C-D) Bar graphs depict the fine movement (C) and rearing activities (D) of A53T mice and control littermates at 1 and 2 months of age. *p < 0.05, ***p < 0.001. (E) Bar graph displays results from Rotarod test of A53T mice and littermate controls. Latency to fall was recorded at 1 and 2 months of age. (F) Body weight curves of A53T mice and littermates from 1 to 3 months of age. The body weight of A53T mice and littermates was measured twice a week. Concurrent with the abnormal motor behavior symptoms, the body weight of A53T mice was dropped continuously. Once the mice were unable to feed themselves , they were sacrificed for histology and biochemistry study.Click here for fileA53T mice.The TreadScan Gait Analysis System  was used to record the gait information of  Each mouse was placed on the belt of treadmill unit. The movement of each paw was recorded with the treadmill running at 17 cm/s, 5 cm/s and 2 cm/s, respectively [nTg mouse was running on the treadmill with the speed set at 17 cm/s. The later part of movie was replayed four times slower than the first part to better visualize the placement of each paw on the belt.ectively . A repreClick here for fileA53T mice were only able to run at the speed of 5 cm/s.Symptomatic  The first mouse showed paralysis in both forelimbs. The second mouse displayed paralysis in the left forelimb and in both hindlimbs.Click here for fileA53T mice developed rapid progression of paralysis in four limbs. This mouse was able to run at the speed of 5 cm/s on postnatal day 65 (P65), but 2 cm/s on P70. The right forelimb started to show paralysis on P71 and then the left forelimb was affected on P73.Click here for fileA53T mice treated with doxycycline (DOX) from embryonic stages to postnatal day 21 (P21)Behavior analysis of . (A) Diagram outlines the treatment of A53T mice with DOX. DOX-containing mouse feed  was provided to breeding pairs and young pups till the pups were weaned at postnatal day 21. The new weanlings were then switched to regular feed. (B) Dot plot shows the change of body weight of A53T mice and littermate controls after the stop of DOX treatment at P21. (C-E) Dot plots show the results of Open-field test of A53T and control mice after the stop of DOX treatment at P21. The spontaneous ambulatory (C), fine movement (D), and rearing activities (E) were quantified. (F) Dot plot depicts the performance of A53T and control mice on Rotarod test after the stop of DOX-treatment at P21. (G) Line graph shows the onset of paralysis of A53T mice after the stop of DOX-treatment at P21.Click here for fileA53T miceIncrease of GFAP and Iba1 expression in . (A-B) Bar graph shows quantitative RT-PCR analysis of Gfap and Iba1 transcripts expressed in the brainstem and cortex of A53T mice and age-matched GFAP-tTA mice (n = 3 per genotype). BS, brainstem; Ctx, cortex; 1 M, 1 month of age; 2.5 M, 2.5 months of age; A53T*, symptomatic A53T mice. *p < 0.05, and ***p < 0.001. (C) Western blots analysis of GFAP and Iba1 protein expression in the brainstem of A53T mice and littermate controls. (D-E) Bar graphs show the quantification GFAP (D) and Iba1 (E) expression in the brainstem, spinal cord, and cerebral cortex (n = 3 per genotype) of A53T and control mice. *p < 0.05, **p < 0.01 and ***p < 0.001.Click here for fileActivated microglia surrounded dopaminergic and spinal motor neurons. (A-F) Representative images show double immunofluorescent labeling of Iba1 (green) with TH (red) and SMI32 (red) in the SNpc (A-C) and spinal cord (SC) (D-F) of symptomatic A53T mice. Scale bar: 20 μm.Click here for fileA53T miceQuantification of cortical and striatal neurons remained in symptomatic . (A), Bar graph depicts the numbers of TH-positive dopaminergic neurons in the substantia nigra pars compacta (SNpc) and ventral tegmentum area (VTA) in nTg and A53T mice at 1 month of age. N = 3 per genotype.(B), Bar graph depicts the numbers of NeuN-positive neurons in the cerebral cortex and striatum of symptomatic A53T mice and age-matched nTg littermates estimated by unbiased stereological methods. N = 3 per genotype.Click here for fileA53T lower expresser mice (A53T-A8)Reactive astrocytes in the brainstem and substantia nigra pars compacta of .  Representative images of GFAP (red) and Iba1 (green) staining show mild astrocytosis in the brainstem and SNpc but not in the cerebral cortex of 12-month old A53T-A8 mice compared to littermate nTg mice.  High magnification views of  reveal the morphology of astrocyte and microglia in control nTg and A53T mice at 12 month of age. Scale bars: 50 μm; 20 μm .Click here for file


In Alzheimer's disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions.Transgenic mouse models do not resolve this problem conclusively, i.e. the relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here, we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers.We present novel AAV-based models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies, including Alzheimer's disease. MAPT gene encoding protein tau, are dominantly associated with various tauopathies Aggregation of hyper-phosphorylated protein tau into filaments and eventually neurofibrillary tangles (NFT) is characteristic for tauopathies, a large and diverse group of neurodegenerative disorders, including Alzheimer's disease (AD) Alzheimer's disease (AD) is the most prominent secondary tauopathy, wherein intracellular tau inclusions combine with amyloid deposits Transgenic mice have been invaluable for understanding molecular mechanisms underlying amyloid peptide generation, but amyloid mice lack two major pathological features of AD, i.e. tauopathy and neuro-degeneration Here we expressed Tau or APP, both wild-type and mutants, by adeno-associated viral vectors (AAV) injected directly into the hippocampus of wild-type mice. The observed dramatic pyramidal neuro-degeneration inflicted by wild-type Tau4R and by mutant Tau-P301L within weeks, contrasted with mutant APP that provoked amyloid pathology after 6 months but with only minor neurodegeneration. Importantly, tau-mediated neurodegeneration was not caused by fibrillar tau-aggregates. Most prominent were cell-cycle markers, indicating that degenerating neurons were attempting to re-entry the cell-cycle. The in vivo AAV-based models firmly support the unifying hypothesis that protein tau mediates neurodegeneration by forcing post-mitotic neurons to re-enter the cell-cycle in primary and secondary tauopathies.Initial experiments were performed with triple mutant APP.SLA, described in the next paragraph, and mutant Tau.P301L, both packaged in AAV-vectors with hybrid serotype-1/2 The generated triple mutant APP.SLA construct contained the Swedish, London and Austrian mutations that are associated with early-onset familial AD Initially, brains were analyzed 12 weeks after intracerebral injection of AAV-vectors in wild-type mice (age 3–6 months). Expression of APP.SLA was pronounced in pyramidal neurons in CA and cortex . AntibodIn sharp contrast, the intracerebral injection of AAV-Tau.P301L presented at first a very surprising outcome at 12 weeks p.i., because expression of human tau was detected in cortex but was hardly detectable in the hippocampus, i.e. where the virus was injected . CounterHuman protein tau was more clearly expressed in cortical neurons, as well as in apparently intact hippocampal neurons that were located adjacent to the degenerated CA regions . These rLower doses of AAV-Tau-P301L resulted in less neurodegeneration with marked thinning of CA1/2 in injected mice , coincidTemporal progression of neurodegeneration, analyzed at 1.5, 3, 6, 9 and 12 weeks p.i. of AAV-Tau.P301L was evident in CA2 already at 1.5 weeks p.i., progressing to CA1 at 3 weeks p.i. and evolving into nearly complete pyramidal neuron-loss at 6–12 weeks p.i. . At the FluoroJadeB (FJB) Inflammation was confirmed by IHC for activated microglia and astroglia . MicroglWe conclude that intracerebral injection of AAV-Tau.P301L, but not of AAV-APP.SLA or of AAV.EGFP, caused pyramidal neurons to degenerate in CA and cortex. Moreover, microgliosis is closely linked to Tau-mediated neurodegeneration, but not to amyloid production, nor to reaction to viral particles or proteins.Biochemical analysis was performed in parallel, on four separate cohorts of wild-type mice injected intracerebrally with AAV-APP.SLA or AAV-Tau.P301L, analyzed at 1.5 weeks p.i. which marks the beginning intense neurodegeneration in AAV-Tau.P301L mice . We analWestern blotting of hippocampal protein extracts demonstrated relative levels of APP.SLA or Tau.P301L to be about two-fold higher than endogenous murine APP or murine protein tau, respectively .The hypothesis that microtubule binding of tau was involved in the observed neurodegeneration was supported by AAV-Tau255 that we generated to express C-terminally truncated Tau4R lacking the microtubule binding domain . At 3 weThe unequivocal demonstration that wild-type and mutant protein tau are equally neurotoxic for hippocampal and cortical pyramidal neurons in vivo, instigated a search for molecular markers in these novel mouse models for tau-mediated neurodegeneration. We analyzed a broad panel of markers and characteristics of degenerating neurons , S2 baseApoptotic markers were evident in AAV-Tau injected mice, albeit outside the actual degenerating regions, i.e. particularly in dentate gyrus granular cells and sparingly in CA1 adjacent to the subiculum .Beclin and Atg8/LC3 are essential regulators of autophagy that mark early and mature steps of autophagosomes Ultrastructurally, degenerating CA neurons presented with nuclear and cytoplasmic condensation and vacuolization, clumped chromatin and indentated or blebbing nuclear membranes . These fTruncated AAV-Tau255, lacking the microtubule binding domain and failing to provoke neurodegeneration, favored the hypothesis that microtubular networks, and associated synaptic architecture, are affected which was supported by marked changes in tubulin and actin, as well as in synaptophysin . These fExpression of cell cycle-related proteins fueled the hypothesis that post-mitotic neurons attempting to reactivate cell-cycling contribute to neurodegeneration in AD Among cell-cycle and related markers, cyclinD2 and cyclinB1 were strongly up-regulated 3 weeks p.i. in degenerating neurons in AAV-Tau mice  corroborRas-dependent mitogen-activated protein (MAP) kinases control cell-proliferation and stress responses. Expression of activated, phosphorylated forms of three members of the MAPK family by IHC revealed no signal for phospho-SAPK/JNK, whereas phospho-p44/42 MAPK was elevated mainly in glia cells. Only phospho-p38 MAPK was observed in a subset of degenerating neurons .The strong expression of cyclinD2, i.e. the only D-type cyclin expressed in dividing cells derived from neuronal precursors in adult hippocampus Here we provide direct in vivo experimental evidence for protein tau-mediated hippocampal neuro-degeneration using intracerebral injection of specified adeno-associated viral vectors. The salient features of the model, based on extensive characterization, qualify them as innovative and unique in several aspects: protein tau at near-physiological protein levels invokes rapid and specific degeneration of pyramidal neurons in limbic regions. Moreover, wild-type Tau4R is as effective as mutant Tau.P301L which is unique, to our knowledge. Thereby AAV-tau model recapitulates, and is informative, for the majority of tauopathies that are caused by wild-type Tau4R, including all AD cases.We discuss three important aspects that are directly concerned by the AAV-models: (i) the mechanism by which protein tau destroys pyramidal neurons; (ii) the information content of the experimental models for human disease, i.e. primary tauopathies and AD; (iii) the marked difference with classical transgenic mice that have amyloid deposits and/or tangles with minimal neurodegeneration.Importantly, the mechanism whereby AAV-Tau inflicts neurodegeneration does not depend on the formation of large, fibrillar tau-aggregates. Conversely, surviving pyramidal neurons flanking the degenerated neurons in CA1/2, contained hyper-phosphorylated tau aggregates, but nevertheless appeared morphologically intact. This remarkable outcome corroborates our previous observation in bigenic mice with massive forebrain tauopathy, i.e. fibrillar tau is not neurotoxic per se We analyzed the phosphorylation status of tau and collected extensive biochemical and immuno-histochemical data-sets on tau phospho-epitopes in the AAV models, as in our transgenic mice We first and foremost consider important the only known physiological function of protein tau, i.e. binding to microtubules. If phosphorylated protein tau fails to stabilize microtubules, or affects microtubule dynamics, a dysfunctional cytoskeleton with axonal, dendritic and synaptic defects as results. The observed changes in cytoarchitecture in degenerating neurons, support the hypothesis that they contribute to the overall process. The experimental data obtained with truncated Tau255 most strongly imply that microtubule binding of protein tau is essential in inflicting neurodegeneration and involve the microtubular network as a structural and transport scaffold. The alternative explanation, i.e. that wild-type and mutant full-length Tau but not truncated Tau255 interact with cellular proteins other than microtubuli, remains open for experimental verification.We went on to define the underlying mechanism of tau-mediated neurodegeneration by analysis of a large and wide panel of molecular targets and pathways, conform the hypothesis that neurons do not die by a single mechanism In AD and unrelated tauopathies, cell-cycle events are suggested to correlate with, if not cause neuro-degeneration. Specifically, cell-cycle activation caused neurodegeneration in a Drosophila tauopathy model Cyclins B1 and D2 were most prominent in degenerating CA1 neurons of AAV-Tau mice. Cyclins D1, D2, D3 with cdk4 and cdk6, regulate G1/S transition by releasing E2F transcription factors via increased phosphorylation of retinoblastoma protein. The latter is here also demonstrated in degenerating pyramidal neurons expressing protein Tau. Upregulation of cyclinB1 also points to the G2/M checkpoint, which depends on cdk2/cyclinB1 activity. Both cyclinD2 and cyclinB1 have been shown to mark cell-cycle events in AD brain The fact that cyclinD2 deficient mice that have problems with adult neurogenesis From the many markers analyzed , S2 we cThe rapid and dramatic neurodegeneration inflicted by protein tau contrasts with the minimal neurotoxic effects sorted by the mutant APP.SLA under the same experimental conditions. Only marginal neuro-degeneration resulted despite important accumulation of amyloid peptides that led even at 6 months p.i. to amyloid plaques in hippocampus and cortex. This marked dissociation in outcome by the same experimental approach, corroborates the growing awareness that neurodegeneration in AD is not mediated primarily or directly by amyloid. The essential contribution of protein tau to pyramidal cell-death is thereby joined seamlessly to the primary tauopathies. Consequently, tau pathology is essential and decisive, together with amyloid, in the overall pathogenesis of AD, notwithstanding its pathological classification as a ‘secondary tauopathy’.We adhere the hypothesis that in AD the accumulation of amyloid is the trigger, but that protein tau executes specified neurons. The transgenic models for amyloid and tau pathology have not, however, substantiated this hypothesis to the fullest, although they have been invaluable for understanding molecular mechanisms of amyloid peptide generation, amyloid pathology, repercussions on cognition and behavior Remarkably, transgenic mouse models now even support the reverse hypothesis, i.e. that tangle deposition is protective, similar to amyloid plaques in amyloid pathology. Both are sinks for mis-folded, aggregated, indigestible peptides or proteins. Whereas such detoxification by aggregation and deposition can be effective as a short-term strategy, it eventually becomes futile and then aggravates the pathology, particularly in humans with their long life-span. Of note, the view on tauopathy is evolving very analogous as that on amyloid pathology in AD: the emphasis shifts from large visible deposits, i.e. amyloid plaques and neurofibrillary tangles, to smaller molecular entities, i.e. amyloid- and tau-oligomers. A similar evolution is evident in other neurodegenerative disorders.The combined transgenic and AAV-models allow us to propose confidently that the link between amyloid, tau pathology and neurodegeneration does not involve directly the formation or repercussions of amyloid plaques and neurofibrillary tangles. Rather, these are the products of dead-end escape-routes whereby cells, in the case of AD pyramidal neurons in limbic brain regions, attempt to limit the negative impact of accumulating misfolded and aggregated proteins that cannot be properly handled by normal cellular proteolytic degradation mechanisms, i.e. endosomal, lysosomal, auto-phagosomal, proteasomal, … It is then not surprising to see many of these involved in, or attained by, the amyloid or tau pathology.Tau mutations in familial primary tauopathies has promoted tauopathy to be mechanistically relevant and important. Experimental evidence in vivo that was largely lacking is convincingly provided by the current AAV-models.Although tauopathy with tangles is and remains the post-mortem pathological hallmark that is co-diagnostic for AD, the genetic evidence of A final important point concerns the question why AAV-based models are more powerful in producing neuro-degeneration than transgenic models expressing the same wild-type Tau4R isoform Although data to answer this problem do not abound, we consider as major difference the observed microgliosis that is much more intense in the AAV-Tau model than in the AAV-APP mice. Microgliosis is spatially and temporally most closely associated with degenerating neurons in the AAV-tau models. This is strongly reminiscent of our observations in inducible p25 mice that suffer a profound hippocampal and cortical sclerosis with pathological characteristics very similar to the AAV-Tau mice Aspects that are important to explain the apparently contradictory outcome in different models, relate to differences in time-scale and/or kinetics whereby neurons either degenerate or whereby tau aggregates and ‘sinks’ into tangles. It is evident that various post-translational modifications, e.g. phosphorylation, ubiquitinylation, glycation, O-glucosylation, … can and will contribute to either mechanism. The overall process is complex and implies enzymes, i.e. proteinases, kinases, phosphatases, … but also cellular factors like chaperones and heat-shock proteins. Their structural features and dynamic actions will tilt the balance to either slow death by progressive accumulation of aggregated, undigested or undigestible amyloid and/or protein tau, or to faster death by cell-cycle re-entry, accelerated by microglia derived pro-inflammatory neurotoxic factors Moreover, the tau-species that are responsible for aggregation and neurotoxicity are proposed to differ at the molecular level. We refer here also to a most recent report on the transmission and spreading of tauopathy in transgenic mouse brain, following intra-cerebral injection of tau-aggregates In conclusion, we present in vivo experimental evidence for a major problem in tauopathies: effective modeling of pyramidal neurodegeneration that is mediated by protein tau.4R, which is responsible for the majority of human tauopathies, including all Alzheimer patients. We further delineate two major mechanisms that contribute to the rapid neurodegeneration mediated by AAV-Tau: attempted cell-cycle re-entry by the post-mitotic neurons, and microgliosis. We are confident that these innovative models will contribute considerably to unravel the molecular factors and mechanistic details. Importantly, the ease whereby the AAV-vectors and the models can be implemented widely in research-projects on neurodegeneration is a further strong point of this report. The wider distribution of the model, and its application for analysis of mechanisms that will untangle tau-mediated neurodegeneration, is hoped to help meet the needs of patients suffering from primary tauopathies or Alzheimer's disease.Human cDNA of wild-type and mutant P301L in the longest tau isoform (Tau4R/2N) was used as described For cloning into the pAAV vectors 5′- AAAAAAGGATCCACCGGTCGCCACCATGGCTGAGCCCCGCCAGGAGTTCGAAGTGATGG – 3′5′- TTTTTTGCGGCCGCTCACAAACCCTGCTTGGCCAGGGAGGCAGACACCTCGTCAGC – 3′.and primers for APP5′- AAAAAAGCTAGCAAGGATCCACCGGTCGCCACCATGCTGCCCGGTTTGGCACTGCTCCTGCT - 3′5′- TTTTTTGCGGCCGCCTAGTTCTGCATCTGCTCAAAGAACTTGTAGGTTGG – 3′These primers contain suitable restriction sites for cloning into the pAAV-constructs. Amplified Tau cDNA was inserted into BamHI-NotI sites while amplified APP cDNA was inserted into NheI-NotI sites of pAAV. Integrity of the ITR was confirmed by restriction with SmaI and by sequencing. All molecular cloning procedures were performed in SURE bacteria  to minimize recombination events.Recombinant AAV vectors of hybrid serotype 1/2 were produced essentially as described Adult wild-type FVB/N mice were used for most studies, except for the cyclin D2 deficient mice All animal experiments were performed by certified researchers conforming to regional, national and European regulations concerning animal welfare and animal experimentation, authorized and supervised by the university animal welfare commission . We formally declare that we comply to the European FP7-Decision 1982/2006/EC, Article 6§1, i.e. all research activities is carried out in compliance with fundamental ethical principles and all experiments are approved and overlooked by the respective Animal Welfare Commissions.At the indicated time periods post-injection (p.i.) mice were anesthetized and perfused transcardiacally with ice-cold saline for 2 min. Brains were removed rapidly and fixed overnight in 4% paraformaldehyde for subsequent immunohistochemical analysis on free-floating coronal vibratome sections (40 µm). Primary antibodies were either affinity-purified polyclonal antibodies or mouse monoclonal antibodies that were biotinylated . Immune In separate experiments, the brain of injected mice was processed for biochemistry. Following anesthesia and perfusion as above, the brain was removed, dissected into hippocampus and cortex on ice and snap-frozen in liquid nitrogen. Hippocampi were homogenized in 6 volumes of homogenization buffer, i.e. 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM sodium fluoride, 1 nM okadaic acid, 0.2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail and stored at −20°C until analysis. For SDS-PAGE total homogenate was loaded onto 8% Tris-glycine gels . Following separation, proteins were transferred to nitrocellulose membranes at 180 mA for 2.5 hr and membranes were blocked for 1 hr with 5% non-fat milk-powder in TBST before incubation with primary antibodies  at 4°C oCompound X34 is a derivative of Congo Red binding to beta-pleated proteins Anesthetized mice  were perfused transcardiacally with ice-cold saline (2 min) followed by Karnovsky fixation for 10 min. Brains were removed and stored in Karnovsky solution at 4°C. Thick vibratome sections (300 µm) were cut and incubated with 1% osmium tetroxide (60 min) before dehydration through a graded series of ethanol and impregnation with Agar100 resin. Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate by standard procedures before analysis by transmission electron microscopy .Figure S1AAV1/2-mediated expression of EGFP. Intracerebral injection of 10E8 t.u. AAV-EGFP in wild-type mice analyzed 12 weeks months p.i. by fluorescence microscopy for EGFP in neurons in the hippocampal formation and in layers 5 and 6 of the cortex (upper panel). Confocal microscopy of CA1 (lower left panel) and CA2 (lower right panel) pyramidal neurons with EGFP expressed in soma and apical dendrites. Scale bar 40 µm.(8.88 MB TIF)Click here for additional data file.Figure S2Effect of APP-mutations on generation of amyloid peptides in transfected N2a. Mouse neuroblastoma N2a cells transiently transfected with pcDNA3 vectors encoding human APP695 containing no, Swedish (K670M/N671L), London (V717I) or Austrian (T714I) mutations, alone and in the combinations indicated. Panels A:. cellular growth media were collected after 48 hours of culture, immunoprecipitated with Mab 6E10 and protein G-agarose beads before Western blotting with Mab WO2, after microwave heating of the filters, as recommended for the WO2 antibody. Panel B: Cell extracts analyzed directly by Western blotting with WO2. Panel C: acid-urea SDS-PAGE to separate amyloid peptides with synthetic amyloid peptides as standards (Aβ-mix). Quantification by densitometric scanning using synthetic peptides as standards. Note the highest ratio Aβ42/Aβ40 for APP.SLA triple mutant, which was used in the AAV-construct.(1.40 MB TIF)Click here for additional data file.Figure S3Distribution of human tau following AAV-Tau.P301L injection. Compilation of 40 sections (each 40 µm) spaced each about 3–4 sections apart throughout the brain of a wildtype mouse injected with 10E8 t.u. AAV-TauP301L and analyzed at 1.5 weeks p.i. for human protein tau by IHC with Mab HT7.(10.05 MB TIF)Click here for additional data file.Figure S4Comparison of wild-type and mutant Tau. Intracerebral injection of 10E8 t.u. of the indicated AAV-vectors in wild-type mice analyzed 1.5 week (panel A) and 3 weeks p.i. (panel B) with indicated antibodies in Western blotting and IHC, respectively. Note some minor cross-reaction of the polyclonal antibody against Tau.P301L (ref. 46) with wild-type Tau in IHC (right upper panel).(10.17 MB TIF)Click here for additional data file.Figure S5Protein Tau255 lacking microtubuli binding domains is not neurotoxic. Intracerebral injection of 10E8 t.u. AAV-Tau255 vector in wild-type mice (n = 8) analyzed 3 weeks p.i. A: representation of Tau.255 and Tau4R constructs and representative IHC for human Tau with HT7, AT180, AT8, AT270. Note that Tau.255 lacks phosphorylation at AT8 and AT270 epitopes. B: IHC for NeuN (upper panels) and histological staining with FJB (lower panels) of injected (left) and noninjected (right) hemispheres. C: IHC for MHCII for microgliosis (upper panel) and for GFAP for astrogliosis (lower panel) in injected (left) and non-injected (right) hemispheres. D: IHC with HT7 for human tau in AAV-Tau.255 injected mice (left panel) compared to AAV-TauP301L injected mice (right panel). Note the lack of neurodegeneration inflicted by Tau255  and the different subcellular localization of Tau255  versus Tau.P301L . Scale bars: A, C 0.5 mm; B 0.5 mm (upper panel) and 50 µm (lower panel); D 40 µm(10.29 MB TIF)Click here for additional data file.Figure S6Morphological and pathological aspects of Tau-mediated neurodegeneration. Intracerebral injection of 10E8 t.u. AAV-Tau.P301L vector in wild-type mice analyzed 3 weeks p.i. A. IHC for active caspase-3 and quantification of apoptotic cells in ipsilateral and contralateral hemispheres . Note the distribution of presumed apoptotic neurons (arrowheads) in regions that do not correlate with degenerating neurons. B. IHC for LC3 and Beclin as mediators of autophagy. Scale bar 40 µm. C. Histological and ultra-structural analysis of brain sections stained with toluidin-blue a–d: shrunken dark neurons  absent at contralateral side . e: vacuolization of cytoplasm (green arrow) and condensed chromatin (blue arrow) f: indentations of nuclei (red arrowheads). Scale bars: a–d 20 µm, e–f 2 µm. D. IHC with AT8 and AT270 reveal sporadic tangles, spheroids and axonal dilatations.(9.31 MB TIF)Click here for additional data file.Figure S7Lipofucsin in degenerating neurons. Autofluorescent lipofucsin-like deposits in brain of mice injected with 10E8 t.u. of AAV-Tau.P301L analyzed at different periods p.i. as indicated, with enlarged view at higher magnification (utmost right panel). The efficient elimination with a proprietary reagent is illustrated (panel marked “Autofluo-eliminator”).(2.21 MB TIF)Click here for additional data file.Figure S8Defects in AAV-Tau.P301L injected mice. Intracerebral injection of 10E8 t.u. of AAV-TauP301L in wild-type mice 3 weeks p.i. analyzed by IHC for tubulin, actin and synaptophysin in injected (left panels) and non-injected (right panels) hemispheres.(9.50 MB TIF)Click here for additional data file.Table S1Antibodies used in this study(0.08 MB PDF)Click here for additional data file.Table S2Markers tested by IHC on AAV-Tau.P301L mice.(0.04 MB PDF)Click here for additional data file.


Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection.Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65 It is now well established that the memory subset of circulating T cells contribute to alloresponse, thus explaining that viral infections are associated with graft failure in human transplant recipients + T cell alloreactive repertoire was first described for an EBV T cell response specific to the EBNA3A325–333/B*0801 EBV epitope Though association between persistent viral infection and allograft rejection is well admitted, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. The influence of antiviral T cell responses on the CD8495–503/A*0201 HCMV epitope (NLVPMVATV) 54–64/B*3501: EPLPQGQLTAY 259–267/A*0201: GLCTLVAML To appraise the contribution of EBV- or HCMV-specific CD8 T cell responses to the allogeneic repertoire, we screened a number of CD8 T cell lines, that had been sorted with recombinant peptide/MHC class I (pMHC) multimeric complexes, on a large panel of HLA class I alleles expressed either by transfected COS cells or by EBV-transformed B lymphoblastoid cell lines (LCL) for cross-reactivity to allogeneic class I HLA molecules. Our study was focused on the pp65495–503/A*0201) or EBV (BMLF1259–267/A*0201 or BZLF154–64/B*3501) epitopes for cross-recognition of allogeneic MHC class I molecules, taking into account the immunodominance of those responses and the frequent expression of A*0201 and B*3501 alleles . Two unsorted T cell lines, derived from HLA-A*0201-negative donors were included in the screening. They showed allorecognition of HLA-A*0201, producing TNF-a selectively against COS cells expressing A*0201, and killing all A*0201+ LCL (data not shown). This indicates that results obtained from the two assays were fully consistent, thus validating the use of COS cells transfected with individual HLA allele to screen for allorecognition in the MHC class I context.Cross-reactivity to allogeneic HLA molecules was found for 3 out of 11 pp65/A2-sorted T cell lines, each one recognizing a different HLA-A allele. The T cell line derived from D03 killed only the LCL SBN, the only one of the panel to express A*3201. D01-derived T cell line killed LCL BOI and JHAF, sharing A*3101 expression, and D08-derived T cell line killed LCL BAR, the only one to express A*3001, but did not kill LCL DUC A*3002+ . The per+ . The cDNTo appraise the polyclonality of the pp65/A2-specific T cell lines, their TCR Vβ repertoire was analyzed by immunofluorescence, using Vβ-specific mAb . T cell + LCL and 36% of cells were stained by anti-IFN-γ mAb (not shown), thus confirming the result of the CD25 assay. In contrast, only 3% of T cells of the T cell line derived from D03 were stimulated by the LCL SBN, expressing A*3201. For the two cell lines, a fraction of CD25+ cells was stained by anti-Vβ13.1 mAb . Due to the down-modulation of the TCR-CD3 complex following activation by Ag, most probably the whole fraction of CD25+ T cells activated by the allogeneic HLA molecule is included in the TRBV6-9 subset. This suggests that alloreactive T cells contained in the pp65/A2-specific T cell lines from D01 and D03 used the same TCR Vβ (TRBV6-9), though they did not recognize the same allogeneic HLA allele.To assess the percentage of alloreactive T cells within a given pp65/A2-sorted T cell line and the TCR Vβ region used by the fraction of alloreactive T cells, we conducted a CD25 upregulation assay. T cell lines were stimulated overnight with LCL expressing, or not, the relevant allogeneic HLA allele and cells were triple stained by anti-CD3-APC, anti-CD25-FITC and by anti-Vβ-PE mAb specific to each one of the TCR Vβ used by this T cell line, as indicated in TRBV6-9; , while a+ LCL, but not with Cw*0602− LCL .Strong allorecognition of HLA-Cw*0602 was observed for the BZLF1/B*3501-sorted T cell line from D15. This T cell line killed selectively the four LCL of the panel , that shared Cw*0602 expression  and prod602− LCL , thus intetramer . All thrAnalysis of TCR Vβ repertoire of BZLF1/B*3501-sorted T cell lines showed a limited set of TCR Vβ regions, with a preferential usage of Vβ12 (TRBV10-3) for the three cell lines . RepertoEndothelial cell cultures were tested for their capacity to activate alloreactive CD8 T cell lines. As endothelial cell cultures, expressing A*31, A*32 or Cw*0602 were available, we tested their capacity to activate CD8 T cell lines that had shown alloreactivity toward these HLA alleles: T cell line from D01, alloreactive to A*3101, T cell line from D03, alloreactive to A*3201 and T cell line from D15, alloreactive to Cw*0602. While endothelial cell cultures alone did not produce TNF-α, they induced TNF-α responses from T cell lines specific for the relevant allogeneic MHC allele . MoreoveWe identified in this study the cross-reactivity of herpesvirus-specific CD8 T cell lines with allogeneic MHC class I molecules. The rationale for analyzing these particular HCMV and EBV epitopes was that their immunodominance has been well documented In agreement with a recent report by Amir et al. We could have missed alloreactive clonotypes poorly represented within the polyclonal T cell line. However, our data indicate that when as few as 3% of alloreactive T cells were present in the polyclonal population, alloresponse was detected, thus documenting the exquisite sensitivity of the functional assays herein used. In accordance with previous studies 54–64/B*3501-sorted T cell line, and this is, to our knowledge, the first example of cross-recognition of an HLA-Cw allele by HLA-B-restricted T cells. Cross-reactivity to Cw*0602 was observed in only one BZLF1/B*3501-specific T cell line out of three, thus indicating that alloreactivity is mediated by private T cell clonotype(s). It has been reported that a recurrent CDR3 sequence using TRBV10-3 is found in CTL recognizing BZLF154–64/B*3501 54–64/B*3501-sorted T cell lines but only one exhibit a cross-reactivity to Cw*0602, it would be of interest to characterize the CDR3 sequences in order to determine if a minor difference in TCR sequence influence the allo-cross-reactivity with HLA-Cw*0602.Though several HLA-C locus mismatch combinations have been described to be significant risk factors for severe acute graft-versus-host disease GVHD in vivo during acute GVHD and persistence of these T cells for up to one year has been reported Again, this underlies the difficulty to predict alloreactivity, given the huge number of HLA alleles and diversity of clonotypes directed against a particular p/MHC complex. Analysis of alloreactive T cell populations infiltrating human allografts undergoing rejection have shown highly biased TCR usage, associated in some cases with predominance of a single clone 325–333/B*0801 EBV epitope on the allogeneic repertoire has been well documented 325–333/B8 epitope and for HLA-B*14, B*44 or B*35 alleles, as alloantigens + renal transplant recipients A range of acute viral infections have been linked with initiation of complications that often follow transplantation, and most attention has focused on the role of herpesvirus infections in vitroin vitro are targets for alloreactive CD8+ T cells. Allograft rejection often involves injury of graft endothelium lining both large and small vessels. Human vascular endothelial cells display both class I and class II MHC molecules and are directly recognized in vitro by CD8+ and CD4+ alloreactive T cells, respectively While it has long been established that human vascular endothelium can activate alloreactive CD8 T cell PBL from healthy donors were obtained from the blood bank (EFS Pays de la loire), which informed the healthy donors about the final use of their blood. Based on their choice regarding the destination of their blood , healthy donors signed a consent statement. The approval of an ethical committee was thus not necessary. A signed convention was established between our institution (INSERM) and the blood bank (EFS Pays de la Loire) to have access to PBL from healthy donors for research purpose.T cell lines were derived from PBL, originating either from healthy donors  or from patients suffering from arthritis  that were recruited by the department of Rheumatology , as previously described + T lymphocytes were positively selected by magnetic cell sorting from PBL using anti-CD8 mAb, then expanded in vitro under nonspecific stimulation in IL-2/CM supplemented with leukoagglutinin at 1 mg/mL, irradiated allogeneic PBL and LCL as described T cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 1mM L-glutamine and 150 U/mL recombinant IL-2 (hereafter referred to as IL-2/CM). CD8A panel of thirty LCL was used. They were generated by exogenous transformation of peripheral B cells with EBV-containing supernatant from the virus-producing B95.8 marmoset cell line and were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS). Human arterial endothelial cells (HAEC) were isolated from renal artery patches collected at the time of kidney transplantation, harvested according to good medical practice and stored in the DIVAT Biocollection , and cultured as described previously 495–503/A*0201 from the HCMV pp65 protein; BZLF154–64/B*3501, BMLF1259–267/A*0201, from lytic EBV proteins (Genosys).The following antigenic peptides were used: pp65in vitro under non-specific stimulation, as described above, and maintained for 3 weeks without restimulation before analysis. The purity of Ag-specific T cells after one sorting was checked by tetramer staining. No major perturbation in the TCR Vβ repertoire is induced by the culture system Soluble pMHC monomers were synthesized as previously described 495–503/A*0201, BZLF154–64/B*3501 or BMLF1259–267/A*0201 tetramers was performed at 4°C with PE-labeled pMHC complexes at 10 mg/mL and anti-CD3-FITC mAb (Beckman Coulter) for 30 minutes at 4°C, as described previously T cell lines were phenotyped for 24 TCR Vβ expression (IOTest Beta Mark PN IM3497) and anti-CD3-PE-Cy5 mAb (Beckman Coulter). Staining with pp65Plasmids encoding the following HLA antigens were included in the analysis: A*0101, A*0201, A*0301, A*1101, A*2402, A*3201, A*3402, A*6801, B*0702, B*0801, B*1302, B*1401, B*1516, B*1801, B*2701, B*2702, B*2704, B*2705, B*2706, B*2709, B*3501, B*3508, B*4001, B*4002, B*4402, B*4403, Cw*0102, Cw*02021, Cw*0302, Cw*0303, Cw*0401, Cw*0501, Cw*0602, Cw*0701, Cw*0702, Cw*0802, Cw*1402, Cw*1501, Cw*1601. Plasmids encoding BMLF1, BZLF1, and pp65 were also used.3 cells from a CD8 T cell line were added to transfected COS-7 cells, culture supernatants were harvested 6 h later and their TNF-α content was determined by measuring their cytotoxicity on WEHI-164 clone 13 cells in a MTT colorimetric assay Transfection of COS-7 cells was performed by the DEAE-dextran-chloroquine method, as described 51Cr-release assay, as previously described 51Cr-labeled targets and incubated for 1 hour before excess unbound peptide was washed off. Results are expressed as percent specific 51Cr lysis = /(maximum release - spontaneous release)×100%. Maximum and spontaneous release were determined by respectively adding 1% Triton X-100 or medium to target cells in the absence of effector cells. Each test was performed in triplicate at the indicated E∶T ratio.Cytotoxicity of T cell lines against LCL or primary cultures of endothelial cells was evaluated in a standard 4h 3 T cells were incubated with cultured HAEC (3×104 cells/well) generated from different donors and the amount of TNF-a released in the supernatant was estimated after 6 hours by the WEHI-164 cytoxicity assay In TNF-α release assays, 5×105 cells/well) were stimulated overnight with allogeneic LCL cells expressing or not the relevant allogeneic HLA allele (E∶T = 1∶1). Sixteen hours later, cells were double stained by anti-CD25-FITC (BD Pharmingen) and anti-CD3-APC mAb (Beckman Coulter), and analyzed by flow cytometry using a BD FACSCalibur . To determine the TCR Vβ region used by the fraction of alloreactive T cells, the same protocol of stimulation with allogeneic LCL was followed and cells were triple stained by anti-CD3-APC, anti-CD25-FITC and by anti-Vβ-PE mAb specific to each one of the TCR Vβ used by those T cell lines, (Beckman Coulter).To assess the percentage of alloreactive T cells within a given T cell line, T cells (1.5×10


Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT2A receptor subtype, gene expression studies on 5-HT2A, 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters Bmax and Kd showed a significant decrease (p < 0.001) whereas 5-HT2A receptor binding parameters Bmax showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in Kd in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT2A, 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, Bmax , Kd of 5-HT, 5-HT2A and gene expression of 5-HT2A, 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT2A and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin,  Tubes were incubated at 37°C for 15 min. and filtered rapidly through GF/B filters (Whatman). The filters were washed quickly by three successive washing with 5.0 ml of ice cold 50 mM Tris buffer, pH 8.5. Bound radioactivity was counted with cocktail-T in a Wallac 1409 liquid scintillation counter.5-HT receptor assay was done using [ethod of . Crude m2A receptor assay was done using [3H] Ketanserin binding in crude synaptic membrane preparations of hippocampus by the modified method of [3H] Ketanserin was incubated with and without excess of unlabelled 10 μM Ketanserin. Tubes were incubated at 37°C for 15 minutes and filtered rapidly through GF/B filters (Whatman). The filters were washed quickly by three successive washing with 5.0 ml of ice cold 50 mM Tris buffer, pH 7.6. Bound radioactivity was counted with cocktail-T in a Wallac 1409 liquid scintillation counter. Protein was measured by the method of Lowry et al.  5-HT binding against 5-HT showed that the Bmax decreased significantly (p < 0.001) in the hippocampus of diabetic rats with significant increase (p < 0.001) in the affinity. Treatment with pyridoxine alone and in combination with Aegle marmelose and insulin in diabetic rats reversed the Bmax and Kd to near control compared to diabetic group  in the hippocampus of diabetic rats with significant increase (p < 0.001) in the affinity. Treatment groups reversed the Bmax of D+I (p < 0.001), DIP (p < 0.001), D+A (p < 0.001) and DAP (p < 0.001) to near control compared to diabetic group  in diabetic rats when compared to control and it was (p < 0.001) reversed to near control level on treatment with pyridoxine alone and in combination therapy with Aegle marmelose and insulin in diabetic rats  in diabetic rats when compared to control and it was (p < 0.001) reversed to near control level on treatment with pyridoxine alone and in combination therapy with s Figure-.(i) Behavioural response in streptozotocin induced diabetic Rats: Effect of insulin and pyridoxine treatment on open and closed arm entry in elevated plus- maze testThe experimental groups showed a significant increase in the attempt taken for open arm entry- D (p < 0.001) compared to C. D+I (p < 0.001), D+P (p < 0.01), DIP (p < 0.001), D+A(p < 0.001) and DAP (p < 0.001) treated groups showed the open arm entry to near control Figure-.There was a significant increase (p < 0.001) in the number of entries made into closed arm by D compared to C. D+I (p < 0.001), D+P (p < 0.01), DIP (p < 0.001), D+A (p < 0.001) and DAP (p < 0.001) treated groups showed the open arm entry to near control Figure-, 8.(ii) Behavioural response in streptozotocin induced diabetic Rats: Effects insulin and pyridoxine treatment on time spent in open and closed arms in Elevated plus-maze testThere was a significant decrease in time spent in open arm by D (p < 0.001) compared to C Figure-. Time sp2A receptor antibody staining in the hippocampus showed significant decrease (p < 0.001) in the 5-HT2A receptor in diabetic rats compared to control. There was significant reversal of 5-HT2A receptor to near control level in D+I (p < 0.001), D+P (p < 0.05), DIP (p < 0.001), D+A (p < 0.001) and DAP (p < 0.001) of 5-HT2A receptors on treatment with pyridoxine alone and in combination therapy with insulin and Aegle marmelose compared to diabetic rats  in the 5-HTT in diabetic rats compared to control. There was a significant reversal to near control level in expression of D+I (p < 0.001), DIP (p < 0.001), D+A (p < 0.001) and DAP (p < 0.001) of 5-HTT on treatment with insulin and t Figure-.Maintenance of euglycemia over a lifetime of diabetes cannot be accomplished safely with currently available treatment methods . The effvia its different receptors. Activation of 5-HT2A receptors found in the hippocampus has been suggested to induce depolarization in the dentate gyrus [2A receptor has been found to enhance Long term potentiation in the hippocampus [5-HT has both depolarising and hyperpolarizing effects in the hippocampus, te gyrus . 5-HT2A pocampus . The chapocampus .2A receptor binding with increase in affinity in hippocampus of diabetic rats. This decrease in the sympathetic activity thereby decreases the circulating 5-HT level. Treatment of pyridoxine along with Aegle marmelose and insulin, resulted in restoring the synthesis of 5-HT in hippocampus of diabetic rats. 5-HT levels reflect the intrasynaptic release indicated by the response of the Bmax of 5-HT receptor binding to its ligand. The results indicate that the pyridoxal phosphate content in hippocampus regulates the extent of decarboxylation of the 5-HTP, the precursor of 5-HT. Treatment of diabetic rats with pyridoxine reflected the synthesis and secretion into the synaptic cleft of the neurotransmitter 5-HT [Previous reports showed a decrease in 5-HT in brain regions during diabetes . 5-HIAA/ter 5-HT ,36. 5-HTter 5-HT  and thervia modulation of its expression and function. In brain, 5-HTT is situated both in presynaptic membranes of nerve terminals in proximity to serotonin-containing cell bodies [2A receptors. Synaptic 5-HT signaling are motivated by uptake of 5-HT2A from the synapse by 5-HTT.5-HTT regulates the entire serotonergic system and its receptors l bodies . 5-HTT ml bodies . 5-HT isAegle marmelose alone and in combination with pyridoxine. The distribution of insulin receptors in the brain and the presence of insulin-dependent glucose transporters suggest that brain insulin participate in several cognitive functions, including learning and memory [max of 5-HT2A receptors to near control level. Also, it is evident that pyridoxine along with insulin and Aegle marmelose leaf extract has neuroprotective action mediated through the 5-HTT at the transcription level.Recent evidence suggests that a dysfunction of the neuronal insulin receptor signalling cascade, with the subsequent abnormalities in glucose/energy metabolism, affect amyloid precursor protein metabolism and cause insulin dysfunction . In thisd memory . In animd memory . Treatmed memory . Cognitid memory . The rold memory . It was d memory . The revAegle marmelose was comparable to insulin in reversing blood glucose to normal levels. Anandharajan et al. [Aegle marmelose activate glucose transport in PI3 kinase-dependent fashion. Scopoletin (7-hydroxy-6-methoxy coumarin) isolated from leaves of Aegle marmelose was evaluated for its potential to regulate hyperglycemia in rats [Aegle marmelose is found to have anti-hyperglycemic activity by lowering the blood glucose [Aegle marmelose to diabetic rats caused a reversal in the Bmax of 5-HT, 5-HT2A receptors and gene expression to near control level. Also, it is evident that Aegle marmelose has a role in controlling INSR function. This study demonstrates the involvement of 5-HT2A receptor which has modulating effect on the diabetes stress. Administration of pyridoxine alone and in combination with Aegle marmelose and insulin significantly decreased the diabetic associated stress. Hence the treatment has pharmacological and neurobiological basis.n et al.  reported in rats . Alkaloi glucose . The lea glucose  the planThe change in brain 5-HT synthesis rate in diabetic animals is related to the various behavioural and psychological changes . AnxietyAegle marmelose leaf extract alone and in combination with pyridoxine has marked to normalize 5-HT, 5-HT2A receptor, gene expression studies along with the Elevated plus test implicate a role in reducing the stress associated with diabetic rats. Thus it is suggested that pyridoxine treated alone and in combination with insulin and Aegle marmelose have a functional role through serotonergic receptors regulation in the hippocampus. This represents a possibility for the better management of diabetic mediated neurological complications.The prevalence of diabetes among depressed and anxious patients is due to high sensitivity of diabetics to the adverse effects of stress, etiology and course of the disease. Serotonergic system and insulin function play key roles in the regulation of stress-related behaviours. The result of this study has demonstrated the effect of insulin, INSR: Insulin receptor; 5-HT: Serotonin; 5-HIAA: 5-hydroxy indole acetic acid; 5-HTT: 5-HT Transporter; EDTA: Ethylene Diamine Tetra Acetic Acid.The authors declare that they have no competing interests.PMA and CSP designed research. PMA, KPK, JM, AM and SJ carried out the experiments and drafted manuscript. All authors read and approved the final manuscript.


Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation. Mural cells, a peri-endothelial localized mesenchymal cell type including vascular smooth muscle cells and pericytes, exert a dominant effect on endothelial cell behavior, driving vessel maturation, regulating vascular function and influencing responsiveness to anti-angiogeneic therapeutics Prior to mural cell coverage, nascent vessels are susceptible to remodeling and VEGF-signaling inhibitors Heterotypic cell-cell contact at interdigitations between endothelial cells and mural cells provides a unique presentation context for paracrine factors such as VEGF and angiopoietins that regulate endothelial cellular responses In vitro organotypic angiogenesis models, such as the co-culture of primary endothelial  and mural cells , fibroblasts, mesenchymal stem cells) in two- and three-dimensional (imbedded in extracellular matrix) assays In spite of the central role that mural cells play in determining vascular maturation and anti-angiogenic therapeutic responses, our understanding of the molecular mechanisms mediating these effects is incomplete. in vitro model system comprising co-cultured primary human endothelial and vascular smooth muscle (EC-vSMC) cells To study how mural cells affect endothelial cell behavior we used a genetically and pharmacologically tractable Time-lapse fluorescence microscopy analysis of GFP-expressing EC in co-culture revealed no apparent cell division . AnalysiAdherens junctions represent the major class of homotypic endothelial cell-cell interactions Together these results indicate that while formation of a single interconnected network relies on the formation of EC-EC adherens junctions, these junctions are not required for the endothelial cells to undergo extensive and stable changes in cell morphology.Formation of the vascular basement membrane is a hallmark of vessel maturation To determine whether the formation of a collagen-rich vascular basement membrane-like structure was necessary to stabilize EC capillary-like networks, co-cultures were treated with a proline hydroxylase inhibitor (EDBH) or starved for ascorbic acid to inhibit collagen assembly and synthesis respectively . These tPrevious studies demonstrated the requirement for VEGF to drive endothelial network formation in different systems including EC-vSMC co-cultures 165 in vSMC (3238 pg/ml per 700 000 cells/day) by retroviral transduction and evaluated the effects on EC in co-culture. As shown in VEGF-High co-cultures displayed normal network formation. Interestingly, retroviral vector expression of VEGF165 in fibroblasts was insufficient to drive the EC capillary-like network formation observed for vSMC-EC co-cultures, instead inducing EC morphology changes characterized by extended filopodia (As co-cultures conducted in growth factor-rich or poor (i.e. 2% serum only) conditions both displayed network formation (data not shown), we reasoned that VEGF was provided by vSMCs. Indeed, RT-PCR  and ELISilopodia . These rMural cells exert a dominant effect on endothelial cell behavior that is necessary for vessel maturation and function, and that determines responses to anti-angiogenic therapeutics in vivoin vitro co-culture systems demonstrated that mesenchymal cells induce endothelial quiescence by direct cell-cell contact, mediated in part by activation of TGFβ Mural cell interactions attenuate endothelial proliferation, a prerequisite for maturation and stability. Reduced mural cell recruitment to sprouting endothelial cells is associated with vessel hypertrophy in vitro angiogenesis systems  have shown that VEGF is required for endothelial cell network formation Several studies using various in vitro model to study vascular basement membrane assembly.Assembly of the laminin and collagen IV-rich vascular basement membrane is a critical event in stabilizing newly formed blood vessel We demonstrate a singular requirement for mural cell-derived VEGF in generating a stable capillary-like network formation. RNAi knockdown of smooth muscle cell VEGF expression phenocopied the effect of VEGF signaling inhibitors. Surprisingly, neither the presence of VEGF, bFGF, EGF (EGM-2 medium) nor conditioned vSMC medium rescued the loss of SMC VEGF expression. This indicates that VEGF must be associated with the smooth muscle cells in a specific context in order to trigger EC morphogenesis. VEGF165, the major isoform expressed by vSMC, is retained at the cell surface by binding heparin sulfate proteoglycans (HSPG) in vivoMural cells have been shown to express VEGF In conclusion, our findings emphasize a unique role for mural cell-derived VEGF in driving blood vessel maturation and define an informative high throughput-high content imaging-compatible experimental system for studying blood vessel formation.2. The growth medium was changed every third day and cells were passaged prior to reaching confluence. The maximum passage number used for experiments was 8 (HUVEC), 12 (PaSMC), 8 (HUMVEC), 6 (hMSC). Primary human foreskin fibroblasts were provided by Dr. Ola Hammarsten, Sahlgrenska University Hospital, Gothenburg, Sweden. SV-40 immortalized mouse embryonic fibroblasts were provided by Prof. Donald Gullberg, University of Bergen, Norway.Human umbilical vein endothelial cells (HUVEC), pulmonary artery smooth muscle cells (Pa-vSMC), human adult dermal microvascular endothelial cells (HUMVEC) and human mesenchymal stem cells (hMSC) were purchased from Lonza . To simplify imaging, early passage HUVEC cells were infected with retrovirus carrying a GFP or RFP-expressing construct. Cells were maintained in culture in the supplier's recommended complete medium  at 37°C, 5% CO4 PaSMC, 10×103 HUVEC, 200 µl EGM-2; 6-well plates: 8×105 PaSMC, 1.5×105 HUVEC, 2 ml EGM-2; transwells: 1.75×105 PaSMC, 6×103 HUVEC, 1 ml EGM-2; 8-well chamber slides: 1.4×105 PaSMC, 3×104 HUVEC, 250 µl EGM-2. HUMVEC and hMSC experiments were conducted similarly using EGM-2MV as the culture medium.PaSMC and HUVEC were seeded together, centrifuged briefly at 200 g to achieve an even distribution of cells and cultured in EGM-2 for at least 72 hrs to allow network formation. For longer periods, culture medium was changed every third day. Cell numbers and culture volume were as follows (per well): 96-well plates: 5×10Tube total length is the total number of pixels comprising the network in the image field. Tube average length is the total number of pixels comprising the network in the image field divided by number of unconnected tube segments. Number of segments is the number of unconnected segments in the image field.Confocal images were acquired on a Zeiss LSM 510 Meta. A Zeiss Axiovert S100 fluorescence microscope with a Roper Quantix digital camera was used for conventional fluorescence and brightfield microscopy. For quantitative analysis of the co-cultures a BD Pathway 855 bioimaging system  was used for automated high throughput imaging. Statistical analysis of acquired images was done with BD Image Data Explorer software. Images were acquired as 3×3 montages using a 10×lens. Background subtraction, noise reduction  and image thresholding were performed using the AttoVision v1.6.1 software supplied by BD Biosciences. Statistics on tube branch lengths, and number of branch points per region of interest were obtained using the “Tube Formation” image analysis module of AttoVision v1.6.1. BrdU (10 µM) was added 4 hrs after seeding cells and incubated overnight. Cultures were trypsinated, fixed  and permeabilized . BrdU was visualized by staining with monoclonal anti-BrdU  followed by an allophycocyanin conjugated goat anti-mouse IgG secondary . BrdU staining was quantified by flow cytometry (10 000 events) on a FACSCalibur (BD Biosciences) and performed as three independent experiments.Cells in 8-well chamber slides were washed (PBS), fixed  and permeabilized and blocked . VE-cadherin was visualized with rabbit anti-VE-cadherin antibody . Cells were washed , treated with secondary antibody  and washed again .As for VE-cadherin staining using primary antibodies diluted 1∶200 in PBS/2% FBS and secondary antibodies diluted 1∶3000 in PBS/2% FBS . Primary antibodies were: Monoclonal mouse anti-Collagen type IV antibody , rabbit anti-Laminin , and monoclonal mouse anti-collagen type XVIII .Cells were fixed with 4% PFA as above. HUVEC in co-culture were selectively stained with FITC/TRITC-UAE 1 lectin  and washed .Mature day 8 co-cultures were incubated with Texas Red-conjugated 10,000 MW dextran .RNA interference was achieved using retrovirally expressed shRNAs. Complementary shRNA oligos  were annealed and ligated into the MMLV-derived retroviral shRNA expression vector L071 RRI-Green or L087 RRI-Red . These vectors also express a puromycin resistance marker and GFP or RFP respectively. Transduced cells were isolated by FACS utilizing fluorescent protein reporter expression. Final hairpin sequences were:GCAGATGTCTACAAATTACTTGTTCAGCTctggtcAGCTGAACAAGTAATTTGTAGACATCTGCGAACATTTGGGAAATCTCTTGCAAGCTAgaagcttgTAGCTTGCAAGAGATTTCCCAAATGTTCGTGGTGAAGTTCATGGATGTCTATCAGCGctggtcCGCTGATAGACATCCATGAACTTCACCACOverexpression of proteins in cells was performed by transfection of a 293T packaging cell line with a retroviral expression vector containing the DNA of interest. Virus was harvested 24–48 hrs after transfection in medium suited for the target cells. Infection was performed by filtering the virus containing medium, addition of proteamine sulphate, and transferring of the virus containing medium to the target cells which were incubated further for 24 hrs. Infection was ended by changing the medium. Protocol will be given out by request. GFP was expressed from pCGFP VEGF in cells and culture medium was quantified using an ELISA kit  and a HIDEX Plate Chameleon luminometer. Samples were prepared as follows: Culture medium from 24 hrs was collected, cell debris removed by centrifugation and samples were stored at −80°C after addition of protease inhibitors . Cells were treated with lysis buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM dithiothreitol, 1% deoxycholic acid, 0.5% sodium dodecylsulfate, 1% Nonidet P-40, and protease inhibitors. Prior to freezing of samples small aliquots were taken out to determine protein concentration.300 µM ethyl-3–4-dihydroxybenzoate  was added to the cell suspension before plating and treatment was continued for 72 hrs. Avastin Figure S1Retroviral shRNA knockdown in primary endothelial cells. (a) Cell lysates from HUVEC mono-cultures transduced with luciferase shRNA or α-catenin shRNA were analysed by 10% SDS-PAGE and show knock-down of the 102 kDa protein α-catenin. Actin, 42 kDa, was used as a loading control. Results are representative of 3 different shRNA sequences that silence human α-catenin expression. Each of these different α-catenin shRNA sequences engendered the non-branching phenotype shown in (0.09 MB PDF)Click here for additional data file.Figure S25′-GGGCAGAATCATCACGA-3′ (156–172) and 5′-CCGCCTCGGCTTGTCACA-3′ (629–612) VEGF inner: 5-′ATCGAGACCCTGGTGGACA-3′ (219–238) and 5′-CCGCCTCGGCTTGTCACA-3′ (629–612). The brackets indicate the positions of primers (Entrez: M32977). Expected sizes of cDNA fragments for the various vascular growth factor (VEGF) transcript alternative splice variants when amplified with outer/or the inner primer pairs: VEGF206 (597 bp/534bp); VEGF189 (546 bp/483 bp); VEGF165 (474bp/411bp); VEGF145 (414 bp/351bp); and VEGF121 (342 bp/279 bp).Dominant VEGF isoforms in PA-vSMC. RT-PCR analysis of mRNA isolated from monocultured PA-vSMC. Primers used for nested PCR, VEGF outer: (0.09 MB PDF)Click here for additional data file.Video S1Time-lapse microscopy analysis of endothelial-mural cell co-culture. Co-cultures (GFP-expressing HUVEC and PA-vSMC) were seeded in 96-well plates and images were acquired every 15th minute on a Zeiss LSM 510 Meta . Several time-lapse movies from different timepoints were merged and together span a time range from day 0 to day 9.(4.33 MB MOV)Click here for additional data file.Video S2Time-lapse microscopy analysis of a mature endothelial-mural cell co-culture Co-cultures (GFP-expressing HUVEC and PA-vSMC) were seeded in 96-well plates and images were acquired every 15th minute on a Zeiss LSM 510 Meta . Time-lapse is recorded from 216–240 hrs post-seeding of culture and shows a mature quiescent network.(0.33 MB MOV)Click here for additional data file.Video S3Time-lapse microscopy analysis of PTK787/ZK-treated endothelial-mural cell co-culture Co-cultures (GFP expressing HUVEC) were seeded in a 96-well plate and treated with 100 nM PTK787/ZK. A time-lapse was recorded overnight  by imaging the cells every 15th minute using a Zeiss LSM 510 Meta.(0.29 MB MOV)Click here for additional data file.


By covalent conjugation of amino acids to m-L-SL, a peptide complex consisting of six m-L-SL-based oligopeptides known as peptichemio (PTC) was developed previously. In the present study, the cytotoxic activity pattern of the different oligopeptides of PTC was investigated in ten human tumour cell lines representing different mechanisms of cytotoxic drug resistance using the fluorometric microculture cytotoxicity assay (FMCA). In the cell line panel, L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine (P2) was the most active oligopeptide, showing slightly lower mean IC50 values (2.6 vs 3.9 and 4.1 microg ml(-1)) than Mel and m-L-SL. The other five oligopeptides were less active than Mel. All active oligopeptides showed mechanistic similarity to Mel as judged by the correlation analysis of the cell line panel log IC50 values (R > or = 0.90), although P2 appeared to be less sensitive to GSH-mediated drug resistance. The relative activity of Mel and P2 was found to be related to degree of proliferation, P2 being more active towards low-proliferating cell lines. P2 and Mel were then further characterized in 49 fresh human tumour samples. In these samples P2 was considerably more active than Mel and showed a higher relative solid tumour activity (2.7 to 4.5-fold). However, the correlation of log IC50s between P2 and Mel in patient cells was high (R = 0.79), indicating a similar mechanism of action in this tumour model too. Cross-resistance with other standard drugs was lower for P2 than Mel. The results show that P2 is the most potent component of PTC and demonstrates a favourable activity profile compared with Mel. These data suggest that further investigation of P2 as a potential anti-tumour agent is warranted.