Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys. Chikungunya virus (CHIKV) is a re-emerging Alphavirus that has caused recent massive outbreaks in the Indian Ocean region. In addition, outbreaks have been documented in Europe and elsewhere in the world, initiated by infected travelers returning to their homelands. The recent outbreak strains possess extended vector range and as such, raise the potential of CHIKV outbreaks in the Southeastern parts of the United States. In this study, we examined CHIKV immunity in adult and aged Rhesus macaques following infection with two different CHIKV strains (recent outbreak strain CHIKV-LR and a West African Strain CHIKV-37997). CHIKV-LR causes persistent infection in the aged animals and replicates, on average, to higher levels than CHIKV-37997. Irrespective of the viral strain used, aged animals had delayed and/or reduced immunity compared to adult animals. Our data support the clinical findings of CHIKV susceptibility in vulnerable populations including the aged and provide mechanistic evidence that an effective immune response directed against the virus is required for preventing persistent CHIKV infection. Aedes (Ae.) albopictus and Ae. aegypti mosquitos, which are widely distributed throughout the world Ae. albopictus mosquitoes Chikungunya virus (CHIKV) is a re-emerging member of the Alphavirus genus within the Togaviridae family. CHIKV was first isolated in the 1950's from the serum of a febrile patient in Tanzania during a dengue fever-like outbreak Clinical symptoms of CHIKV infection in humans include acute fever lasting up to two weeks and severe poly-arthralgia of the peripheral joints that can be very debilitating and last for several months. Additional symptoms include nausea, headache, rash and lymphadenopathy. Although CHIKV infection is usually a self-limiting disease, the outbreaks occurring after 2005 were explosive and exhibited complicated clinical-pathological manifestations including neurological involvement. Aged individuals and adults with underlying immunological conditions experienced severe morbidity and sometimes mortality The immune response to CHIKV infection has been relatively understudied. A limited number of studies characterized changes in plasma cytokine/chemokine levels following CHIKV infection in order to identify predictors of poor disease prognosis. These analyses revealed that the acute phase of infection (4 days post onset of illness) is associated with the production of robust Type-1 interferon (IFN) Opy-1). We detail the innate and adaptive immune antiviral responses following infection. Rhesus macaques infected with CHIKV become viremic within 24 hours of inoculation and this was typically resolved by 5 days post-infection (dpi). All animals generated robust T and B cell responses that peaked by 14 dpi. However, our study demonstrates that aged animals persistent viral RNA in the spleen. Interestingly, this inability to clear the virus in the aged animals correlated with reduced innate and adaptive immune responses compared to adult animals. Our findings suggest that defects in the aged immune system play a critical role promoting CHIKV persistence.The adaptive immune responses to CHIKV infection have not been as widely investigated. In one study, the acute phase of CHIKV infection in a human cohort from a 2007 CHIKV outbreak in Gabon was found to be associated with CD8+ T cell activation. However, the antiviral specificity of the CD8 T cell response and its contribution to disease resolution were not investigated All Rhesus macaques were handled in accordance with good animal practice as defined by relevant national and/or local animal welfare bodies. The use of non-human primates was approved by the Oregon National Primate Research Center (ONPRC) Institutional Animal Care and Use Committee (IACUC #0826). The ONPRC is fully accredited by the Assessment and Accrediatation of Laboratory Animal Care-International. For blood collection monkeys were anesthetized with ketamine by intramuscular injection. Monkeys were humanely euthanized by the veterinary staff at ONPRC in accordance with endpoint policies. Euthanasia was conducted under anesthesia with ketamine followed by overdose with sodium pentobarbital. This method is consistent with the recommendation of the American Veterinary Medical Association.The full-length infectious clones for the West African  strain CHIKV-39779 and the recent outbreak strain La Réunion  were obtained from Dr. Stephen Higgs University of Texas Medical Branch-Galveston. The viruses produced from the infectious clones retain their original viral phenotypes of CHIKV in both cell culture and in mosquitoes. RNA was transcribed using the T7 mMessage mMachine kit (Ambion). The transcribed RNA was transfected into BHK cells and the resultant virus was propagated in C6/36-insect cells to produce low passage, high titer CHIKV stocks by pelleting through a 20% sucrose cushion by ultracentrifugation . Plaque titration assays on Vero cells using a carboxy-methylcellulose overlay were performed to determine titers (plaque forming units) of the stock virus.RNA transcribed from the infectious clones of CHIKV-LR and CHIKV-37997 was transfected into BHK cells and passaged one time in insect cells to generate the virus stock used to infect the animals.7, 1×108 or 1×109 plaque forming units (pfu) CHIKV-LR or CHIKV-37997 strain in 1 ml of phosphate buffered saline (PBS), and a sample of all viral inoculums was back-titered to confirm the delivery of correct dosage. Blood samples were obtained following ketamine sedation (10 mg/Kg) at 4 and 2 weeks prior to infection, on the day of infection (day 0), 1, 2, 3, 5, 7, 10 and14 days post infection and on a weekly basis thereafter. Complete blood cell counts were obtained at every time point to monitor changes in immune cell numbers/µl of blood. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by centrifugation over Histopaque gradient .Adult male and female rhesus macaques (ranging from 6–13 years old) and aged female rhesus macaques (>17 years old) were utilized for infection studies as described in GGAACGAGCAGCAACCTTTG, rev: ATGGTAAGAGTCTCAGACAGTTGCA, and probe: GGAATAAGGGCTTGT; CHIKV-37997 fwd: CGGAGAGCCGTAAGCTTCTTAA, rev: TTCACACGAAACCACTGTATCACA, and probe: CTTCAGTGTTCCATCTAAA. Total RNA was prepared from 100 µl of animal sera using the ZR Viral RNA kit  or from 5×106 total PBMC using Trizol. RNA was prepared from tissue specimens by the Trizol method. The isolated RNA was quantified using a Nanodrop spectrophotometer. RNA was first treated with RNAse-free DNAse and then single stranded cDNA was generated using random hexamers and Superscript III RT . Gene amplicons served as quantification standards (limit of detection is 10–100 copies). Quantiative RT-PCR results were analyzed using ABI StepOne Plus Real-Time PCR system .Viral load in peripheral blood (plasma and PBMC) as well as tissues at necropsy of the rhesus macaques was quantified using real-time RT-PCR. Primers and probes include: CHIKV-LR fwd: CHIKV peptides were designed to be 18 mers that overlapped by 12 amino acids covering the entire CHIKV proteome (peptides were purchased from Thermo Fischer Scientific). Peptides were resuspended in DMSO at 1 mg/ml. Separate peptides pools were made for each of the nine viral protein products.PBMC were stained with surface antibodies against CD4, CD8β, CD28, and CD95 to delineate naïve, central memory (CM), and effector memory (EM) T cell subsets. All antibodies with the exception of CD8β (Beckman Coulter) were purchased from Biolegend . Cells were then fixed and the nuclear membrane permeabilized as per manufacturer's recommendation  before staining with anti-Ki67 . Samples were acquired using the LSRII instrument  and data analyzed by FlowJo .5 rhesus monkey PBMC (n = 3). Positive controls  and Ionomycin (7 mM stock)) and negative controls  were also performed for each monkey PBMC sample. Plates were incubated for 18 hrs at 37°C. After incubation the plates were washed thoroughly with PBS and then incubated with a biotin-conjugated anti-IFN-γ antibody (1 µg/ml in PBS plus 0.5% FCS) for 2 hrs at room temperature. Plates were washed five times with PBS and then incubated with Streptavidin-ALP in PBS plus 0.5% FCS for 1 hour. Plates were washed five times and then developed with filtered BCIP/NBT substrate solution. Flushing the wells with water stopped color development. The plates were air-dried and the number of spot forming cells was determined by subtracting the mean number of spots counted in negative controls wells (n = 3) from the spots counted in each well. The magnitude of the response was determined as the sum of the spots counted per 6×105 PBMC.Gamma interferon (IFN-γ) enzyme linked immunospot (ELISPOT) assays were performed using the ELISpotPlus for Monkey IFN-γ kit (MabTech). Plates were washed one time with 200 µl/well of PBS and then blocked for 30 minutes in 200 µl/well of RPMI containing 10% fetal calf serum (FCS) and penicillin, streptomycin and glutamine (RPMI-10). Overlapping CHIKV peptide pools (1 µg/well) for each of the nine CHIKV proteins were added to wells containing 2×10PBMC were stained with surface antibodies directed against CD20 (Beckman Coulter), IgD (Southern Biotech) and CD27  to delineate naive, marginal zone-like and memory B cells. The cells were then fixed and the nuclear membrane permeabilized as per manufacturer's recommendation before addition of Ki67  specific antibodies. Samples were acquired using the LSRII instrument  and data analyzed by FlowJo .Antiviral IgG levels were measured in circulating plasma using a standard ELISA assay using plates coated with CHIKV lysate or CHIKV particle preparations, which reacts with CHIKV Abs. In these experiments, serial three-fold dilutions of plasma were incubated in triplicates CHIKV lysate or virus-coated ELISA plates for 1 hr prior to washing, staining with detection reagents (HRP-anti-IgG) and addition of chromogen substrate to allow for detection and quantitation of bound antibody molecules. Log-log transformation of the linear portion of the curve was then performed, 0.1 OD units was the cut-off point to calculate end point titers. Each plate contained a positive control sample to normalize ELISA titers between assays, and a negative control sample to ensure assay specificity.PBMC were stained with surface antibodies directed against CD3, CD11c, CD14, CD20, CD123 and DR to delineate plasmacytoid dendritic cells, myeloid dendritic cells, DR+ lineage negative other DCs and monocyte/macrophages. Samples were acquired using the LSRII instrument  and data analyzed by FlowJo .Blood plasma (100 µl) obtained from adult and aged CHIKV infected Rhesus macaques (3 dpi) was UV inactivated (3× for 30 seconds at 600 µJ). Plasma was added to rhesus macaques fibroblasts cultured in 24-well plates for 24 hr. Recombinant universal Type-1 IFN  treated cells were used as positive controls and untreated cells were used as a negative control. Total RNA was isolated from the cells using the Trizol method (Life Technologies). Quantitiative RT-PCR was used to measure Interferon-stimulated gene (ISG) expression using primers and probes specific for Mx-1 and ISG-56. cDNA was generated using Superscript III (Life Technologies) and analyzed on an ABI StepOne Real-Time PCR system and normalized to L32. Gene amplicons served as quantification standards (limit of detection ≤100 copies/gene). ISG expression levels for each sample were expressed as fold-change by comparison to untreated cells. Data were analyzed by Prism Graph Pad software.Data were analyzed using Prism Graph Pad software using Two-way ANOVA, no post-test corrections were carried out due to the small sample size.Ae. albopictus (Asian Tiger) mosquitoes as well as increased virulence. We sought to compare CHIKV-LR virus disease progression and host immune responses infection with that of a West African reference strain (37997) to determine whether CHIKV-LR was also more pathogenic in non-human primates. We also explored the impact of aging on virus replication and immune response to both of these strains. We first performed a titration experiment to determine the effects of virus strain and inoculation dosage on in vivo CHIKV replication and pathogenesis. Two cohorts of adult rhesus macaques were infected intravenously with either CHIKV-LR or CHIKV-37997. Each cohort consisted of 3 groups of 2 animals each that received 1×107, 1×108 or 1×109 plaque forming units of either strain and animals were observed daily for signs of disease. Animals developed lymphadenopathy 7–14 dpi as well as fever between 3–7 dpi. A rash was observed on the chest of some of the animals at 7 dpi (data not shown). We compared viral loads in peripheral blood mononuclear cells (PBMC) and plasma at 0, 1, 2, 3, 5, and 10 dpi using qRT-PCR . CHIKV RNA levels were very low in PBMC but as described for plasma were slightly higher in CHIKV-LR infected animals  or CHIKV-37997 (n = 2). We compared peak viral loads and the kinetics of viral replication between the aged and adult cohorts  with 1×10 cohorts . The kin cohorts .All animals were euthanized at 35–42 dpi, at which point, blood, spleen, lymph nodes and joint tissues were harvested to determine viral loads. At the time of necropsy, we observed no gross joint inflammation in any of the animals. In addition, we detected no virus in tissues harvested from adult animals at 35 dpi. However, viral RNA was present in spleen of aged CHIKV-LR infected animals but not in aged animals infected with CHIKV-37997 . InteresNext, we investigated the impact of age on the adaptive immune response to CHIKV. One of the hallmarks of the immune response against a viral infection is rapid and robust proliferation of antigen specific lymphocytes. We therefore compared the kinetics and magnitude of the T cell proliferative burst in adult and aged animals by determining the frequency of T cells expressing the nuclear protein Ki67, which is induced during cell cycle Proliferation of CD4 CM cells was evident by 7 dpi in both CHIKV-LR and CHIKV-37997-infected adult animals . The freSimilar patterns of proliferation were observed in the CD8 T cell subsets. Proliferation within the CD8 CM subset was detected in all LR-infected animals at 7 dpi . Prolifein vivo, we used IFN-γ ELISPOT assays to determine the frequency of CHIKV-specific T cells in PBMC. A complete overlapping peptide library to CHIKV was subdivided into peptides for each of the 9 proteins . PBMC collected at 35 dpi were incubated with peptides and the mean numbers of spot forming units 5 PBMC were determined for four adult and four aged animals. A representative example of the data is presented in To further characterize the impact of age on T cell response to CHIKV infection We next assessed the B cell response by measuring the kinetics and magnitude of the B cell proliferative response as well as IgG production following infection. B cells can be subdivided into three subsets based on the expression of CD27 and IgD as shown in To further characterize B cell response, we measure IgG end point titers by ELISA using 96-well plates coated with either purified virus or cellular lysates produced from infected Vero cells. When using purified virions as the antigen source, anti-CHIKV IgGs were detected at 14 dpi and peaked near 21 dpi in LR-infected adult animals . The IgGGiven the proposed role for monocyte/macrophages in CHIKV pathogenesis To determine whether the innate immune response was also affected by age following CHIKV infection, we measured plasma levels of bioactive IFN. For this experiment, rhesus macaque fibroblasts were treated with UV inactivated plasma from CHIKV infected aged or adult animals at 3 dpi. Interferon stimulated gene (ISG) expression was determined by quantitative RT-PCR using specific primers. We detected a trend towards increased ISG expression in fibroblasts treated with plasma from the adult animals indicating that the levels of bioactive IFN was higher in those samples but this difference did not reach statistical significance .Chikungunya virus is a re-emerging Alphavirus that causes debilitating arthralgia. The 2006 outbreak on the island of La Reunion involved a newly emerged CHIKV strain. This strain exhibits both an increased vector range making more widespread transmission highly likely but also more severe virus-induced disease. Ultimately these phenomena demonstrate that CHIKV is an adaptable pathogen capable of rapidly expanding its ecological niche and potentially emerging globally. Moreover, this new strain was associated with increased mortality in the elderly and newborns. Unfortunately, the host response to CHIKV infection and associated determinants of disease remain poorly understood. Characterization of the immune response following CHIKV infection in humans is hampered by the occurrence of symptoms at least a week after infection as well as severity of disease that can differ dramatically between patients, which both influence the time of presentation in the clinic. Thus, novel models that recapitulate human CHIKV disease are needed to facilitate the identification of the immune correlates of protection against CHIKV infection, and for evaluation of therapeutics and vaccines. Recently studies in Cynomolgus monkeys have shown that infection of non-human primates results in similar pathophysiology as that described for humans with the involvement of several joints, but the host immune responses were not evaluated This is the first report describing the impact of age on CHIKV disease and immune response in non-human primates. Our results show that although viremia is detected within 24 hpi in both adult and aged animals, LR infection resulted in slightly higher peak viremia. Interestingly, at necropsy (35 dpi) we were able to detect persistent viral RNA in spleens harvested from aged but not adult animals infected with the LR strain suggesting that aged animals were unable to fully clear viral infection. One possible explanation for persistent viral RNA in aged animals is a reduced immune response to CHIKV. Therefore, we characterized the innate and adaptive immune responses following CHIKV infection. Following infection with CHIKV-LR, adult and aged rhesus macaques generated comparable T and B cell proliferative responses, similar IgG antibodies to viral lysate but lower IgG antibodies to whole virions. In contrast, aged animals infected with 37997 generated weaker T cell and B cell proliferative responses (delayed and reduced) compared to adult animals, but the IgG responses were comparable. We also measured frequency of CHIKV-specific T cells using an overlapping peptide library for the entire CHIKV proteome. Overall CHIKV-specific T cell responses were lower in aged animals and the immunogenicity profiles differed. Aged animals also failed to exhibit an increase in the frequency of myeloid innate immune cells. This failure could be mediated by a defect in innate immune cell mobilization or proliferation. Lastly, production of bioactive Type 1 IFN was lower in the plasma of the aged animals at 3 dpi. Taken together, our findings suggest that aged animals have impaired ability to mount an effective immune response against CHIKV infection.Aging results in a general decline in immune function, commonly known as immune senescence, which contributes to the increased susceptibility to pathogens and cancer. Defects associated with immune senescence have been shown in both innate and adaptive processes. In the current report we found a reduction in innate immune responses in the aged animals infected with CHIKV compared to adult animals. Importantly, these responses were observed in animals infected with either the LR or 37997 strains. Of note, aged animals do not exhibit an increase in the frequency of pDC. Given that pDCs play a critical role in the antiviral response through the production of Type 1 IFN In line with decreased magnitude of the T cell proliferative burst, we found reduced frequency of CHIKV-specific T cells in the aged animals by IFN-γ ELISPOT. Interestingly, the immunodominance profile also differed between adult and aged animals. Similarly, we observed a decreased IgG antibody response to whole virions. However, IgG titers measured using plates coated with viral lysate were higher than those measured using purified virions suggesting that some of the antibody response is directed against viral antigens that are not included in the virion. Collectively, these findings indicate that the aged immune system is incapable of mounting a broad and effective adaptive immune response to CHIKV.In summary, we present data indicating that the recent outbreak strain CHIKV-LR replicated, on average, to higher levels compared to the CHIKV strain 37997. While we did not observe CHIKV-associated joint inflammation or disease for either virus in these animals at 35 dpi, we did identify the spleen as a potential site of CHIKV persistence in aged animals. This effect was limited to CHIKV-LR and did not occur in monkeys infected with CHIKV-37997, which is in line with the increased severity of CHIKV-LR disease. We have also identified a number of differences in the ability of adult vs. aged animals to respond to CHIKV infection, with a general decline in both the innate and adaptive immune responses that could explain the increased disease severity observed in older individuals.


Steroid-responsive meningitis-arteritis (SRMA) is a systemic inflammatory disease affecting young adult dogs and a potential large animal model for neutrophilic meningitis. Similarities between SRMA and infectious central nervous system (CNS) diseases in lymphocyte subsets suggest an infectious origin.Toll-like receptors (TLRs) are pattern recognition receptors playing an important role in innate immunity. Due to their ability to recognize both self and non-self antigens, we hypothesize that TLRs are among the key factors for the induction of the inflammatory process in SRMA and provide an indirect hint on the etiology of the disease.The expression profile of cell surface TLRs  and intracellular TLRs (TLR3 and TLR9) of canine leukocytes was analyzed by immunophenotyping and subsequent flow cytometric measurements. Experiments were performed on cerebrospinal fluid (CSF) and peripheral blood (PB) samples of dogs affected with SRMA during the acute phase (n = 14) as well as during treatment (n = 23) and compared with those of dogs with bacterial meningitis (n = 3), meningoencephalitis of unknown etiology (n = 6), neoplasia of the central nervous system (n = 6) and a group of dogs with miscellaneous neurological diseases (n = 9). Two additional control groups consisted of dogs with pyogenic infections (n = 13) and of healthy dogs (n = 6).All examined groups showed a high percentage of TLR2, TLR4 and TLR5 positive PB polymorphonuclear cells (PMNs) in comparison to healthy dogs. Very high values of TLR9 positive PB PMNs were detected in acute SRMA. Only a few similarities were found between SRMA patients and dogs with pyogenic infections, both groups were characterized by high expression of TLR4 positive PB monocytes. Glucocorticosteroid therapy reduced TLR2, TLR4 and TLR9 expression in PB monocytes.A relatively high expression of TLR4 and TLR9 in acute SRMA suggests that these two receptors might be involved in the inflammatory process in SRMA, enhancing the autoimmune reaction. Systematic CSF cell analysis for TLRs can be performed in future treatment studies in larger animals, such as dogs. Steroid-responsive meningitis-arteritis (SRMA) is a systemic inflammatory disease affecting young adult dogs. It is the most common cause of meningitis  and the Toll-like receptors (TLRs) are pattern recognition receptors which recognize both invading pathogens  and endogenous molecules produced by injured tissue  .This recognition process plays a role in innate immunity and in the development of the adaptive immune response ,9. AdditTo date, SRMA is believed to be characterized by a Th2-mediated immune response , but it Due to their ability to recognize both self (DAMPs) and non-self (PAMPS) molecules, TLRs are suspected to be involved in the inflammatory process in SRMA. To confirm the hypothesis that SRMA is triggered by an environmental factor, such as a bacterial infection, which is specifically changing the TLR pattern, the expression profile of cell surface TLRs  and intracellular TLRs (TLR3 and TLR9) were examined on canine leukocytes. An indirect hint on the etiology of SRMA was expected.The study population consisted of 80 dogs referred to the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany between May 2009 and April 2011. The studies were conducted according to the ethical guidelines of the University for Veterinary Medicine Hannover. Depending on the clinical diagnosis, the dogs were assigned to one of the following groups (see Table SRMA Acute (SRMA A): The diagnosis of SRMA was supported by the detection of typical findings during physical and neurological examinations, complete blood and CSF examinations, cervical radiographs, elevated IgA levels in CSF and serum and the absence of other conditions causing cervical pain . Dogs wiSRMA Therapy (SRMA Th): dogs from the former group under glucocorticosteroid treatment that did not show clinical signs at the time of sampling. Dogs under treatment for SRMA received prednisolone, with dosages ranging from 1 mg/kg/24 h to 0.5 mg/kg/48 h.The other groups were: bacterial meningitis or meningoencephalitis (BM); meningoencephalitis of unknown etiology (MUE); CNS neoplasia (Neopl) and a group of dogs with miscellaneous neurological diseases (Mix)  for collection of peripheral blood (PB) leukocytes.Cisternal cerebrospinal fluid (CSF) collection under general anesthesia was part of the work-up in all dogs for collection of CSF leukocytes with the exception of healthy animals and patients with pyogenic infections.After collection, 1 mL of EDTA PB was used for staining of cell surface TLRs  and 0.5 mL for intracellular staining (TLR3 and TLR9). Leukocytes were fixed, in order to preserve their marker expression.For the staining of cell surface TLRs, the cells were fixed using a previously described method of preparing blood leukocytes for flow cytometric analysis . The metTM Lysing Solution, twice its volume, diluted 1:10 , and incubated for 10 minutes. After a washing step at 500 x g for five minutes using PBS containing 1.25% pooled dog serum, BD FACSTM Permeabilizing Solution 2 was added for 10 minutes according to the description of the manufacturer (BD Biosciences). Ultimately, an additional washing step was performed.For intracellular staining the blood was mixed with BD FACS5 leukocytes/50 μL using PBS containing 1.25% pooled dog serum.The number of leukocytes was determined in both procedures using a hemocytometer and the cell suspension was adjusted to 2.5 10et al.[Immediately after tapping, CSF was aliquoted in two tubes and centrifuged at 200 x g for 10 minutes, as described by Schwartz et al..As described above for blood leukocytes, one CSF aliquot underwent fixation (cell surface TLRs) and the other CSF aliquot underwent permeabilization (intracellular TLRs). Both procedures were performed as described for PB with the exception of the lysing steps.et al.[+ or CD21+), polymorphonuclear cells (CD11a+/CD11b+) and monocytes (CD14+),  (1:200 dilution).A study from Burgener et al. demonstrNegative controls consisted of isotype-matched primary antibodies .Leukocyte populations Figure  were gatAll events in CSF samples and a minimum of 10,000 events in blood samples were collected.P <0.05).The percentage of positive cells and the mean fluorescence intensity (MFI) of each group were used for statistical analysis using a commercial statistical program . The Wilcoxon rank sum test and the Kruskal–Wallis one-way analysis of variance were applied for comparison of the results deriving from the different groups. Statistical significance was set at the 5% level  was noted towards a decreased expression of TLR2 on PMNs  in comparison to dogs with the acute form of SRMA   of TLR4 positive PB PMNs  in comparison to healthy controls  and to dogs affected with miscellaneous diseases of the nervous system  (P = 0.0095)  to cases with encephalitides of unknown etiology  and with CNS neoplasia   in comparison to healthy animals   was found in SRMA A  in comparison with MUE  and Neopl   in comparison to dogs with pyogenic infections . SRMA Th and Pyo dogs had a significant lower (P <0.05) percentage of TLR3 positive PMNs in comparison to healthy dogs , SRMA Th , Neopl  and Mix  groups showed a higher percentage of PMNs positive for TLR3 (P <0.05), with the exception of MUE and Pyo  in comparison to all other groups (P = 0.0149) and Healthy (P = 0.0077).In general, the fluorescence expression intensity did not differ significantly among the groups examined. An exception was the expression intensity of TLR3 on PB PMNs, SRMA A had a lower TLR3 MFI in comparison to Pyo , Neopl  and Mix   and untreated SRMA dogs , being significantly higher than in SRMA Th  and in dogs with pyogenic infections  in comparison to control group . SRMA dogs under therapy showed a decrease in TLR4 positive PB monocytes  in comparison to the untreated dogs  in comparison to healthy dogs . The expression of TLR5 positive monocytes in CSF did not statistically differ among the groups.Similar to SRMA Th, MUE and Pyo, untreated SRMA dogs showed higher percentage of TLR5 positive PB monocytes  in comparison to dogs affected with pyogenic infections . Similar percentages have been found in the remaining groups, with the exception of the healthy controls  percentage of TLR9 positive PB monocytes  in comparison to healthy dogs . Similar values were found also in the remaining groups. In dogs with SRMA the expression of TLR9 positive PB monocytes statistically decreased (P = 0.0499) under therapy   of TLR2 positive PB lymphocytes  in comparison to healthy dogs . Similar findings were seen in the other groups, with the exception of MUE and Neopl. The lowest values were found in dogs with bacterial meningoencephalitis , being statistically lower than those found in SRMA A (P = 0.0197) . SRMA A had a higher percentage of TLR2 positive lymphocytes in CSF  than BM   and the remaining groups (except MUE and Pyo) had a lower percentage of TLR4 positive PB lymphocytes than Healthy   and Healthy (P = 0.004). Dogs with untreated SRMA displayed statistically lower (P <0.01) percentage of TLR4 positive lymphocytes in CSF  compared to SRMA dogs under therapy  and dogs with miscellaneous diseases of the nervous system   in comparison to SRMA Th.SRMA A . Additionally, there was a tendency for SRMA A towards lower MFI values in comparison to healthy dogs, but this difference was statistically relevant only for SRMA Th (P = 0.0056).The percentage of TLR5 positive lymphocytes in PB and CSF did not differ among the groups. However, significant differences among the groups were found in the fluorescence expression intensity of TLR5 in PB lymphocytes. Dogs from both the SRMA A and SRMA Th groups showed lower MFI values in comparison to dogs affected with pyogenic diseases  of TLR9 positive PB lymphocytes in untreated SRMA dogs  and under treatment  were found in comparison to dogs with pyogenic diseases  and healthy dogs  , fibronectin, hyaluronic acid, fibrinogen and heparan sulfate . In the The role of TLR4 in human patients with sepsis, but also in non-infectious diseases, such as inflammatory bowel disease and rheumatoid arthritis, were studied . In the P = 0.019). These findings strongly suggest that TLR4 plays an important role in triggering the described pathological findings in SRMA. Additionally, it opens the discussion for new treatment modalities, such as anti-TLR4 antibodies and TLR4 antagonist; some compounds from the latter class are already under clinical trials for treatment of sepsis in human patients [The role of TLR4 for neutrophil recruitment into the CNS was demonstrated in a murine model of systemic inflammation . Similarpatients ,42.TLR9 seems to be constantly increased on PB leukocytes in almost every disease examined in the current study. However, patients with SRMA and dogs affected with meningoencephalitides of unknown etiology showed the highest expression of TLR9, suggesting a potential role of this TLR in inflammatory CNS diseases with a possible autoimmune component. TLR9 is primarily involved in the recognition of bacterial DNA . In huma+ T cells. The last are known to shift towards Th17-differentiation under the effect of IL-6 and transforming growth factor beta 1 (TGFβ1) [Dendritic cells, once activated by TLR4 and TLR9, produce interleukin-23 (IL-23), which subsequently activate CD4 (TGFβ1) ,44. This (TGFβ1) ,46. A re (TGFβ1) , suggestAlthough most TLR responses lead to inflammation, there are studies suggesting an important role of TLRs in homeostasis ,48. The Limitations of this study include the absence of true controls, the number of patients in some groups and the variation of the time of sampling in the course of the disease. We tried to overcome the limitations comparing a broad spectrum of different neurological diseases. Including only a few cases of a relatively uncommon disease in dogs, bacterial meningoencephalitis, might be the reason TLRs in CSF samples were not statistically comparable. We tried to overcome this limitation by including dogs with pyogenic infections not affecting the nervous system, since SRMA is considered to be a systemic inflammatory disorder .We suggest that TLRs are involved in different aspects of the pathogenesis of SRMA. This study supports the hypothesis that an infectious agent can only trigger the disease. SRMA itself seems to be maintained by multiple alterations of the immune system resulting in an autoimmune disease, TLRs, such as TLR4 and TLR9, might act as receptors maintaining the inflammation.BM: Bacterial meningitis; CD: Cluster of differentiation; CNS: Central nervous system; CSF: Cerebrospinal fluid; DAMPs: Damage associated molecular pattern molecules; EDTA: Ethylene diamine tetraacetic acid; HSP: Heat shock proteins; IL: Interleukin; mAb: Monoclonal antibody; MFI: Mean fluorescence intensity; Mix: Miscellaneous non-inflammatory neurological diseases; MRI: Magnetic resonance imaging; MUE: Meningoencephalitis of unknown etiology; Neopl: CNS neoplasia; PAMPs: Pathogen-associated molecular patterns; PB: Peripheral blood; PBS: Phosphate-buffered saline; PMNs: Polymorphonuclear cells; Pyo: Pyogenic infection; rSpear: Spearman’s rank correlation coefficient; SRMA: Steroid-responsive meningitis-arteritis; TGF: Transforming growth factor; Th17: T helper 17; TLR: Toll-like receptor.The authors declare that they have no competing interests.AT designed and supervised the study. AM performed the experiments and analyzed the data. RC gave substantial contributions to acquisition, analysis and interpretation of the data. AM drafted the manuscript and all authors contributed to the critical revision of the manuscript for important intellectual content and have read and approved the final version.


The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis  for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice. Discovered in 1993, these endogenous non-coding transcripts, represents approximately 1–2% known genes in eukaryotes and function to negatively regulate gene expression by repression or degradation through base-pairing to target mRNAs In blood samples, it is well recognized that circulating miRNAs are either packaged in microparticles  or associated with RNA-binding proteins [Argonaute 2 (Ago2)] or lipoprotein complexes (high-density lipoprotein (HDL)) To address this issue, several studies have tried to develop different approaches Other problems related to the very short length and low levels of microRNAs in such fluidic samples make problematic to obtain efficient and reproducible microRNA recovery. In the qPCR approach, it is common to test only a few numbers of molecules. The short structure of such PCR targets may affect the efficiency of the amplification process, including adding bias to the quantification of miRNA expression. In addition, it is reported that RNA purified from plasma can contain inhibitors that may also affect qPCR efficiency.Since the first reports revealing the presence of miRNAs in plasma and serum Twenty consecutive peripheral blood samples from newly diagnosed of inflammatory bowel disease patients at the Gastroenterology Department of the Hospital Universitari i Politècnic La Fe  and healthy volunteer controls were collected for the study. The control group comprised patients who were undergoing an ileocolonoscopy but who did not met a clinicopathological suspicion of inflammation, such as polyps, and resulted in normal colonoscopy. Blood samples were collected prior to any therapeutic procedure. Biopsies from bowel mucosa were obtained at the time of the ileocolonoscopy. All the subjects gave their written informed consent on the day of recruitment. The study was approved by the Ethical Committee on Clinical Research of the Hospital Universitari i Politècnic La Fe (ref: 2010/0468) in compliance with the Declaration of Helsinki.Anticoagulated blood (K3-EDTA) was obtained after 12 hours of fasting. Two different approximations were followed to obtain plasma samples: 1) Blood was carefully layered onto Histopaque 1077  at RT and centrifuged at 213 g 30 min (without brakes). The upper-layer phases containing the white cell-rich plasma were removed and cells and platelets were subsequently separated from the plasma by spinning down at 2375 g 10 min. At this point, plasma aliquots were either processed immediately for total RNA isolation or stored at −80°C for later extractions. 2) Other protocols Three different commercial kits/reagents were employed to test which was most suitable for obtaining RNA from human plasma samples: mirVana PARIS kit , TRIzol-LS  and miRNeasy Serum/Plasma Kit . Two different approximations were applied in mirVana kit: one following the exact manufacturer’s instructions and the other, a modified version of the centrifugation step to 30 min at 4°C after the phenol/chloroform step in order to increase the final RNA yields. In both cases, a starting volume of 400 µL of thawed plasma or freshly isolated plasma was used. Total RNA was eluted into 100 µL of pre-heated (95°C) RNase-free water.RNA was also extracted from plasma using the TRIzol-LS reagent. From frozen plasma, 4 aliquots of 400 µL each were used and 3,5 mL from freshly isolated plasma. In all cases, RNA extraction was done according to the commercial protocol, including glycogen addition before isopropanol and an overnight precipitation step. In addition, a combined version of TRIzol-LS extraction followed by purification with mirVana column kit was employed Finally, for the Qiagen kit assay, two 200 µL of plasma aliquots were used to get enough final RNA volume to perform all quality tests and miRNA expression determinations. RNA was eluted with 14 µL of RNase-free water according the manufacturer’s instructions and the elution’s volumes from the same sample were pooled to obtain a final volume of 28 µL.In addition, RNA extracted from biopsies using the mirVana kit was employed as a control of the whole RNA recovery process and microarray detection. All biopsy samples selected for further microarray tests were obtained from the same plasma patients or donors, thereby being paired.Due to the difficulty in quantifying RNA in plasma samples, and with the aim of RNA yield increase, purified Torulla yeast RNA carrier  was included in trial processes at two different concentrations: 10 µg/mL (as previously reported) RNA purity and concentration were evaluated by spectrophotometry using NanoDrop ND-2000 (ThermoFisher). Initially, only the A260/230 and A260/280 ratios were used to assess the presence of contaminants: peptides, phenols, aromatic compounds, or carbohydrates and proteins. In the end, however, the full spectrum was considered because absorbance was also detected in other wavelengths.Quality and the related size of total and small RNA was assessed by the Agilent 2100 Bioanalyzer microfluidics-based platform  with two chips: Agilent RNA 6000 Nano Kit for total RNA and Agilent Small RNA kit for low molecular weight RNA. Electropherograms were visualized using the Agilent 2100 Expert software which included data collection, peak detection and interpretation of the different profiles.All plasma RNA preparations assessed by microarray technology were those obtained from duplicate starting material (200 µL and 200 µL of plasma) using the miRNeasy kit to control for variation in the RNA preparation steps, loading the aqueous phase onto a single affinity column and mixing the final elution volume (14 µL each) in a single tube.A total of 14 samples from patients and donors were hybridized following the manufacturer protocols: 4 plasma samples (PL), 2 plasma samples with carrier at standard previously reported 10 µg/ml concentration (PLsc) and 4 plasma samples with carrier at ten-fold lower 1 µg/ml concentration (PLlc) and 4 biopsies (BIO). For biopsies, 500 ng of total RNA were added to the labelling mix. Despite the previously determined concentration, 8 µL of plasma and plasma with carrier RNA were labeled. FlashTag miRNA direct labelling (Genisphere) was used following the manufacturer’s procedures. The GeneChip® Scanner 3000 7G System and reagents from Affymetrix were used to hybridize, wash, stain and scan the arrays.The latest version of the miRNA Affymetrix platform miRNA 3.0 based on mirBase version 17 Expression console free software from Affymetrix was used for quality control and assessment of probe set normalized values. Partek Genomic Suite 6.6 version  was used for microarray statistical analysis, PCA and HC generation. Following the miRNA expression workflow, normalization of data included RMA background correction, quartile normalization, log2 transformation values and median polish according to Genisphere indications for the FlashTag Biotin labelling kit. ANOVA test at the different categories was performed and FDR and p-values were applied to generate the list of more significant probe values. GraphPad software  was employed for t-test statistical analysis.http://www.ncbi.nlm.nih.gov/geo/). The accession number at GEO is GSE51375.All microRNA data described in this manuscript have been deposited in NCBI’s Gene Expression Omnibus Database  for plasma study were extracted with mirVana protocol, employing them as an example of good quality RNA for microarray experiments and to compare expressed miRNAs that could be related afterwards to its presence in the plasma from those individuals. BIO samples were very useful as expression controls of some characteristic miRNA molecules being homogeneously expressed in all but the PLsc sample , S3.Human plasma has been a rich source of biomarker discovery In this study we have tested different commercial kits or combinations of them to define the most suitable microRNA isolation protocol for human plasma samples . To idenBesides the detailed validation of the pre-analytical steps affecting miRNA purification, addition of different carrier amounts has also been tested. Our observations support the hypothesis that a lower concentration of carrier than the previously one published for total RNA extraction in plasma and serum can be more helpful in miRNA purification, increasing purity and quantity . It is iPlasma RNA isolation procedure is important for improving yield but is also determining level of inhibitors. As we have seen, some current methods or combinations of them increase the final concentration of RNA, leading to higher levels of contaminants, therefore reducing applicability or efficiency of miRNA detection protocols. Such contaminants may be derived from biofluids or introduced by reagent carry-over during sample preparation Some papers reinforce the idea that PCR is the best way for microRNA detection and quantification In conclusion, we have demonstrated that microRNA from RNA plasma samples obtained using the miRNeasy serum/Plasma kit and adding 10 fold lower doses of carrier than previously published gives rise to reproducible and efficient RNA extractions for later analysis by microarrays. By detecting broadly and highly expressed miRNA molecules we confirmed the efficiency and performance of those extraction conditions. Highly expressed molecules were detected at very similar levels in biopsies, plasma and plasma including a low amount of carrier samples. Plasma extracted using commonly reported carrier concentration reported very different in microarray assays so miRNA target selection must be still well validated according to different protocols for RNA extraction and detection techniques.Figure S1Quality samples control of microarrays results. A) Log expression signals after of robust multi-array average RMA, detected above background or DABG normalization. B) Relative log expression signals.(TIF)Click here for additional data file.Figure S2PCA scatter plot of data for all four RNA methodology extraction categories: Biopsies (BIO), plasma with standard carrier concentration (PLsc), plasma with low carrier concentration (PLlc) and plasma without carrier (PL).(TIFF)Click here for additional data file.Figure S3Hierarchical clustering showing microRNA expression profiles of differentially expressed in the four categories.(TIFF)Click here for additional data file.Table S1Quality control results: List of intensity values generated by Expression console software, showing normalized array probes values for all plasma samples under RMA and DABG algorithms.(XLS)Click here for additional data file.Table S2Summary of quality control (QC) Affymetrix values for all array tested samples, including: Biopsies (BIO), plasma with standard carrier concentration (PLsc), plasma with low carrier concentration (PLlc) and plasma without carrier (PL).(XLSX)Click here for additional data file.Table S3List of more significant miRNA  differentially expressed in all four categories.(XLS)Click here for additional data file.Table S4ANOVA contrast of each category two by two. Lists are generated by p<0.05 and Fold Changes ≥2. Those lists were used for Venn diagram generation and overlapping lists.(XLS)Click here for additional data file.


Oxidative stress has been implicated in pathophysiology of aging and age-associated disease. Antioxidative medicine has become a practice for prevention of atherosclerosis. However, limited success in preventing cardiovascular disease (CVD) in individuals with atherosclerosis using general antioxidants has prompted us to develop a novel antioxidative strategy to prevent atherosclerosis. Reducing visceral adipose tissue by calorie restriction (CR) and regular endurance exercise represents a causative therapy for ameliorating oxidative stress. Some of the recently emerging drugs used for the treatment of CVD may be assigned as site-specific antioxidants. CR and exercise mimetic agents are the choice for individuals who are difficult to continue CR and exercise. Better understanding of molecular and cellular biology of redox signaling will pave the way for more effective antioxidative medicine for prevention of CVD and prolongation of healthy life span. Oxidative stress has been implicated in pathophysiology of aging and age-associated disease. Reactive oxygen species (ROS) have been considered as harmful molecules for organisms by destroying DNA and cell structures, thereby deteriorating multiple organ function, leading to aging. However, recently emerging paradigm reinforces a novel role of ROS as a messenger of redox signaling that modulates aging processes . Perturbα and interleukin-6 in individuals with abdominal obesity [The major origins of ROS in endothelial cells are mitochondrial electron transfer chain (ETC), NADPH oxidase (Nox), endothelial nitric oxide synthase (eNOS), and xanthine oxidase (XO). Mitochondrial ETC inevitably generates ROS associated with oxidative phosphorylation and energy production. The amount of ROS production by mitochondria increases with age and under certain pathophysiological conditions such as excessive food intake and sedative lifestyle , 3. Besi obesity , 6. Proi obesity , 8. Nox- obesity –11. Endo obesity , 13. ThuExtensive efforts have been exerted to ameliorate oxidative stress in the cardiovascular system especially to endothelial cells by general antioxidants. However, these antioxidants have conferred only limited success to prevent CVD. On the other hand, a growing body of evidence suggests that the site-specific blockade of ROS production might represent an alternative strategy to prevent atherosclerosis and CVD. The present review will discuss the issue as to why general antioxidants have failed to provide appreciable antiatherosclerotic effects, and how the site-specific antioxidative therapy exerts beneficial effects on the cardiovascular system. β-carotene failed to show any protective cardiovascular effects when smokers with acute myocardial infarction (MI) were treated for a long term with these agents [β-carotene or the combination of vitamin E and β-carotene. Some explanations have been offered to give an insight into the failure of clinical studies using general antioxidant therapies. It may be argued that more than one antioxidant is required for clinical effectiveness. This idea is based on the assumption that antioxidants exist as a “network,” wherein both lipid soluble (like vitamin E) and water soluble  molecules work in a network for the removal of oxidative stress in conjunction with the regeneration of oxidant defenses. To date clinical trials have generally not used synergistic combinations of lipid soluble and water soluble antioxidants despite the theoretical advantages and basic science demonstrations of effectiveness of “antioxidant cocktail.” Another explanation for the failure of clinical antioxidant studies is that antioxidants can act as prooxidants by retaining high energy electrons when scavenging ROS or reducing iron. It is also possible that antioxidants or by-products may impair mitochondrial oxidative phosphorylation and ATP production. For example, the β-carotene cleavage products have been shown to strongly inhibit state-3 respiration in isolated liver mitochondria [The use of general antioxidants has become a common practice for prevention of CVD and age-associated disease. However, there are as yet no clinical indications for the routine use of antioxidants for treatment of these diseases. This is because no appreciable benefits have been demonstrated in multiple clinical trials that employed general antioxidants. For example, a large trial of vitamin E and e agents . Converschondria . TherefoAstaxanthin, a xanthophyll carotenoid, which is contained in a variety of seafoods such as crabs, crayfish, krill, lobsters, salmon, shrimp, and trout, may represent a novel class of general antioxidant. Unlike other general antioxidants, astaxanthin is not converted into a prooxidant when scavenging ROS. Astaxanthin neutralizes ROS or other oxidants by either accepting or donating electrons, and without being destroyed or becoming a prooxidant in the process, because its linear, polar-nonpolar-polar molecular layout equips it to precisely insert into the membrane and span its entire width. In its position spanning the membrane, astaxanthin can intercept reactive molecular species within the membrane's hydrophobic interior and along its hydrophilic boundaries, thereby providing versatile antioxidant actions. Diverse benefits of astaxanthin in the clinical arena, with excellent safety and tolerability, have been extensively reviewed . It appeε and phosphatidylinositol-3 kinase which culminates in protection of mitochondria against ROS- and Ca2+-induced opening of mitochondrial permeability transition pore through activation of mitochondrial KATP channels and inhibition of glycogen synthase kinase-3β [ATP channels plays a crucial role in developing the memory of cardioprotection that converges on mitochondria. The use of general antioxidants may not be an ideal choice for treatment of CVD especially in subjects at high risk of MI or cerebral infarction. Indiscriminate removal of ROS may reduce the tolerance to hypoxic or ischemic stress, which may worsen the prognosis of heart attack. It is known that ROS is necessary for acquisition of tolerance to lethal ischemic insult. The most representative example of this phenomenon is ischemic preconditioning (IPC). IPC was first discovered by Murry and associates  who demoinase-3β . PositivROS generated during brief ischemia and reperfusion cycles have been consistently implicated in the trigger of IPC –22. In cphox was originally found in phagocytes such as neutrophils and macrophages. Nox2 plays a crucial role in host defense against microorganisms by releasing ROS upon the respiratory burst. The absence of Nox2 gene causes repetitive infections and premature death known as chronic granulomatous disease at an early stage of life [2O2-induced hyperpolarization of endothelial cells. These findings indicate that the use of nonselective Nox inhibitors is not suitable for ameliorating oxidative stress in the cardiovascular system and provides a basis for employing specific inhibitors that block only inducible Nox.Nox is a major ROS generating enzyme in the cardiovascular system. It has been demonstrated that Nox-derived ROS plays a physiological role in the regulation of endothelial function and vascular tone and a pathophysiological role in atherosclerosis and angiogenesis. There are seven homologs of Nox  . Nox2 al of life . Thus, i of life , 28. Nox of life . Convers of life  demonstrSome of the recently emerging drugs for CVD possess a property to inhibit inducible Nox as a preferable side effect. Angiotensin converting enzyme inhibitors (ACEs) and angiotensin II type 1 (AT1) receptor blockers (ARBs) are such a class of antihypertensive drugs. A growing body of evidence suggests that enhanced activation of the RAS is a key to promote endothelial dysfunction and hypertension , 32. Nox plays a crucial role in the RAS-mediated development of atherosclerosis. Nox-derived ROS oxidizes LDL that is incorporated into the intima of the blood vessel. Oxidized LDL is then taken up by macrophages via their scavenger receptors CD36 to form foam cells. Macrophage recruitment to the intima is likely to be regulated not only by a multiplicity of adhesion molecules, integrins, and selectins, but also by chemokines such as macrophage chemoattractant protein-1, which is constitutively synthesized and secreted by endothelial cells and SMCs migrated from the media , 34. Traβ signaling and promote interstitial fibrosis [Local activation of the RAS in the myocardium induced by hypertension and after myocardial infarction plays a crucial role in pathological remodeling of the heart. The RAS is activated in response to mechanical or hypoxic stress to cardiomyocytes under these conditions. AT1 receptor stimulation induces oxidative stress on cardiomyocytes by activating Nox, which triggers cardiomyocyte hypertrophy by stimulating hypertrophy signaling such as protein kinase C and ERK , 36. AT1fibrosis . CardiomThere is another reason for the preferential use of ACE inhibitors and ARBs for hypertensive subjects with visceral obesity. Adipocytes in the visceral adipose tissue are a suggested source of components of the RAS, with regulation of their production related to obesity-induced hypertension. Angiotensin II has been demonstrated to promote oxidative stress via overexpression of Nox in adipocytes. It has been demonstrated that blockade of AT1 receptors reduces oxidative stress in adipose tissue and ameliorates production of proinflammatory cytokines responsible for systemic inflammation and oxidative stress . Thus, ABecause atherosclerosis is facilitated by dyslipidemia in conjunction with oxidative stress, numerous studies have investigated relative contribution of dyslipidemia and oxidative stress to atherogenesis. It has been demonstrated that a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor lovastatin significantly reduced total cholesterol, selectively decreased non-HDL-cholesterol, and significantly reduced fatty streak lesion formation in the aortic arch of the diabetic hamsters fed the atherogenic diet . In thisThe pleiotropic effects of statins that prevent oxidative stress and atherosclerosis have been extensively investigated. Emerging evidence suggests that these cholesterol-independent effects are predominantly due to their ability to inhibit isoprenoid synthesis, particularly geranylgeranylpyrophosphate and farnesylpyrophosphate, which are important posttranslational lipid attachments of the Rho GTPases and activation of its downstream target, Rho-kinase (ROCK) . AngioteA number of studies show that increased physical activity reduces oxidative stress. The mechanism underlying reduced oxidative stress by exercise may be related to the decreased expression of Nox in the visceral adipose tissue associated with the prevention of dysregulated production of inflammation-related adipocytokines, suggesting that exercise is a fundamental approach to protect against cardiovascular risk. Szostak and Laurant  have pro A substantial body of experimental and epidemiological evidence suggests that serum uric acid is an important and independent risk factor for cardiovascular and renal disease, especially in patients with heart failure, DM, and hypertension . The levA large body of evidence supports an important role for XO-mediated ROS generation in atherosclerosis –51. Landα, which is upregulated after ischemia/reperfusion, and administration of anti-TNF-α antibodies at the onset of reperfusion partially restores nitric oxide- (NO-) mediated coronary arteriolar dilation and reduces superoxide production [α antibodies against ischemia/reperfusion injury remains controversial, presumably by the fact that TNF-α also mediates cardioprotective signal transduction as observed in IPC [XO mediates tissue injury during reperfusion typically encountered in the heart after percutaneous coronary interventions for acute MI or open-heart surgery. It has been demonstrated that XO in the heart is activated by tumor necrosis factor (TNF)-oduction , 56. Thed in IPC .Allopurinol and its active metabolite oxypurinol are the representative inhibitors of XO. Inhibition of XO by allopurinol is known to prevent atherosclerosis. It has been demonstrated that allopurinol reduces neointimal hyperplasia in the carotid artery ligation model in spontaneously hypertensive rats . In addiIt has been established that allopurinol protects reperfusion injury of the heart. Manning et al.  for the NO plays a central role in the regulation of cardiovascular function. NO is one of gaseous signaling molecules, which were previously considered to be toxic. However, the identification of NO as the endothelium-derived relaxing factor combined with the discovery of NO generation by NOS primed an explosion of research in this area in the 1990s –65. It i4) which is a cofactor essential for the catalytic activity of all three NOS isoforms [2+/calmodulin orients the other domains to allow NADPH-derived electrons generated in the reductase domain to flow to the oxygenase domain [4 is bound in the dimer interface, where it interacts with amino acid residues from both monomers to stabilize NOS dimerization and participate in arginine oxidation through the N-hydroxyl-L-arginine intermediate and the subsequent generation of NO. BH4 depletion because of its oxidation and/or reduced synthesis can result in functional uncoupling of NOS. Uncoupled NOS generates more ROS and less NO, shifting the nitroso-redox balance and having adverse consequences on the cardiovascular system [4 may be beneficial for prevention of CVD.Biosynthesis of NO is dependent on enzymatic activity of NOS. NOS is a homodimeric oxidoreductase containing iron protoporphyrin IX (heme), flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin . Oxidative stress imposed on endothelial cells causes depletion of BH4 and eNOS uncoupling. The effect of eNOS uncoupling has been investigated in a wide variety of in vitro models, animal models of CVD, and human subjects with cardiovascular risk factors [ BH factors , 69, 70.4 under pathological conditions with oxidative stress has been shown to reverse eNOS dysfunction. However, true mechanistic relationship between endothelial BH4 levels and eNOS regulation in vivo by administration of BH4 remains controversial. High extracellular BH4 concentrations may result in nonspecific antioxidant effects that indirectly increase NO bioactivity by scavenging ROS rather than by modulating eNOS activity. Furthermore, the effects of supplementation with BH4 or biopterin analogues on NO bioactivity are unpredictable in vascular disease states in which oxidative stress is increased [4 levels in the endothelial cell, or whether the relative balance between reduced BH4 and oxidized BH2 may be more important [In many cases, supplementation with BHncreased , 72. Indmportant . 4 levels are regulated by the activity of the de novo biosynthetic pathway and the salvage pathway. In the de novo biosynthetic pathway guanosine triphosphate cyclohydrolase (GTPCH)-1 catalyzes GTP to dihydroneopterin triphosphate. BH4 is generated by further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. GTPCH-1 appears to be the rate-limiting enzyme in BH4 biosynthesis; transgenic overexpression of GTPCH-1 is sufficient to augment BH4 levels in endothelial cells and preserve NO-mediated endothelial function in diabetic mice. In the salvage pathway, BH4 is synthesized from BH2 by sepiapterin reductase and dihydrofolate reductase [4 is labile in physiological solution. It has been reported that in vivo half-life of BH4 is 3.3–5.1 hour in the plasma of healthy adult humans [4 is converted to BH2, which is further degraded to dihydroxanthopterin and excreted to urine [2 availability for the salvage pathway may be limited under oxidative stress even with BH4 supplementation. Thus, sepiapterin may serve as an effective substrate for BH4 via the salvage pathway. Folic acid and vitamin C are also able to restore eNOS functionality, most probably by enhancing BH4 levels through mechanisms yet to be clarified [Intracellular BHeductase . Exogenot humans . Becauseto urine , BH2 avalarified . 4 on CVD has been investigated in various animal models and in human subjects. BH4 ameliorated endothelial dysfunction and reversed hypoadiponectinemia as a result of oxidative stress in rats [4 and the treatment of endothelial dysfunction in insulin-resistant rats [4 has been examined in patients with hypertension, peripheral arterial disease, and coronary artery disease, and these studies consistently demonstrated the beneficial effect of BH4 on endothelial dysfunction [4 also improved endothelium-dependent vasodilation in chronic smokers [4 in patients with poorly controlled hypertension. This finding is not surprising, because chronic exposure to NO causes nitrate tolerance through S-nitrosylation and desensitization of soluble guanylyl cyclase, an enzyme that generates cGMP which is largely attributed to the vasodilating effect of NO [4 may not be effective in ameliorating hypertension through an NO/cGMP-dependent mechanism, recent studies suggest that an NO-dependent S-nitrosylation plays a key role in posttranscriptional modification of the variety of key proteins involved in cardiac contractile function and antiarrhythmias, angiogenesis, and protection against ischemia/reperfusion injury [4 may represent a promising therapeutic strategy for heart failure and ischemic heart disease. Further studies are warranted to address whether BH4 or its precursors truly exert salutary effects on endothelial dysfunction in a variety of CVD.The effect of BH in rats . In addiant rats , 78. Thefunction . BH4 als smokers . Howeverct of NO . Althougn injury –86. Thes Mitochondria are the critical component in control of aging. Mitochondrial dysfunction and increased generation of ROS are a prominent feature of aging and various age-related neurodegenerative diseases such as Parkinson's disease and Alzheimer disease . MitochoMitochondrial generation of ROS is increased with age due to a decrease in electron transport function especially at the site of complex I in the ETC . This leγ coactivator (PGC)-1α through deacetylation reaction. PGC-1α is an end target of events by which AMPK promotes mitochondrial biogenesis. Valle et al. [α show reduced accumulation of ROS, increased mitochondrial membrane potential, and reduced apoptotic cell death both in basal and oxidative stress conditions. Improvement of ETC function may be the most effective approach to reduce the production of ROS in mitochondria. An increase in mitochondrial biogenesis has been shown to improve ETC function and reduce ROS production. A growing body of evidence suggests that AMP-activated protein kinase (AMPK) plays a pivotal role in mitochondrial biogenesis . AMPK ace et al.  have demα appears to be triggered by mitochondrial generation of ROS. Irrcher et al. [2O2 increased PGC-1α promoter activity with concomitant increases in mRNA expression. This observation suggests that mitochondria-derived ROS is necessary for AMPK and SIRT1 to upregulate PGC-1α that feedbacks to inhibit mitochondrial generation of ROS by increasing mitochondrial biogenesis. Thus, AMPK upregulation of PGC-1α is another example for redox signaling-dependent protection of mitochondria.The mechanism by which AMPK upregulates PGC-1r et al.  demonstrα. NO induces mitochondrial biogenesis in skeletal muscle cells via upregulation of PGC-1α [α and mitochondrial biogenesis were also observed in fibroblasts and adipocytes [α.eNOS-derived NO and AMPK synergistically increase the activity of PGC-1f PGC-1α . Similaripocytes . Thus, Nα [α [α, and NRF under the redox regulation upon CR and endurance exercise.Nuclear respiratory factor (NRF) is another redox-sensitive transcriptional factor that is activated in the presence of ROS derived from mitochondria , 104. Miα . NRF is,α [α . Taken tα, thereby promoting mitochondrial biogenesis and ameliorating oxidative stress, may represent a promising pharmacological tool to prevent atherosclerosis and CVD. Because the long-term adoption of a CR lifestyle is not realistic in a significant proportion of the human population, the search for substances that can reproduce the beneficial physiologic responses of CR without a requisite calorie intake reduction, termed CR mimetic agents, have gained momentum. Of these, CR mimetics that activate NRF and PGC-1α, and NRF and has been suggested as a CR mimetic [Epidemiological studies suggest that the consumption of wine, particularly of red wine, reduces the incidence of mortality and morbidity from coronary heart disease. This has given rise to what is now popularly termed the “French paradox” . The car mimetic –111. α, and NRF [α and NRF, increased mitochondrial mass and mitochondrial DNA content, and upregulated protein expression of the ETC constituents in endothelial cells. Mitochondrial biogenesis induced by resveratrol treatment also has important impact on the liver and brain functions and seems to have significant consequences in the skeletal muscle, because resveratrol-treated mice exhibit an approximately twofold increase in endurance exercise [α, or NRF each alone or in combination can ameliorate age-associated diseases and prolong healthy life span in human subjects.Emerging evidence indicates that resveratrol increases mitochondrial biogenesis and reduces oxidative stress in a wide variety of age-associated disease models. It has been demonstrated that resveratrol induces mitochondrial biogenesis and ameliorates angiotensin II-induced cardiac remodeling through the activation of SIRT1, PGC-1 and NRF . Csiszar and NRF  demonstrexercise . Future Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. The term “uncoupling protein” was originally used for UCP1, which is uniquely present in mitochondria of brown adipocytes, the thermogenic cells that maintain body temperature . In thes The mechanism by which UCPs are activated appears to involve redox signaling through mild uncoupling and membrane depolarization of mitochondria that feedbacks to prevent robust ROS generation. It has been demonstrated that superoxide activates UCP3 in rat skeletal muscle mitochondria . Brand aγ) agonist thiazolidinedione (TZD) or PPAR-α agonist WY-14643 increased UCP gene expression in the brown adipose tissue [α induction of UCP2 protected against elevated ROS during drug-induced hepatotoxicity [α agonist WY-14643 in the rodent hearts [γ upregulated mitochondrial UCP2 expression, which decreased overproduction of ROS, improved mitochondrial ETC function at the site of complex I, and inhibited the mitochondrial apoptotic cascade leading to neuronal cell death in the hippocampus following status epilepticus [The physiological signals that modulate UCP3 gene expression include fasting , 124, ace tissue . PPAR-α toxicity . UCP3 ext hearts , 132. Itlepticus . γ and promote insulin sensitization and improve dyslipidemia in patients with type 2 DM, although it is unknown how PPAR agonists-induced upregulation of UCPs contributes to their beneficial effects on CVD. The PPAR-γ agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrosative stresses [PPAR signaling pathways are known to exert anti-inflammatory effects and attenuate atherosclerosis formation. The TZDs are the agonist for PPAR-stresses . Howeverstresses , 136. Cuα agonist that stimulates the oxidation of free fatty acids in the liver, diverting them away from triglyceride synthesis and thus reducing the hepatic synthesis of triglyceride-rich lipoproteins [γ by increasing the adiponectin level [γ and effectively treat insulin resistance and dyslipidemia without the toxicity sometimes associated with full PPAR-γ agonists [γ activation, which may at least in part be involved in their antioxidant or anti-inflammatory potential [Although CR is the fundamental approach to upregulate PPARs , a varieproteins . Fibratein level . The facin level , suggestagonists , 142. Stotential .  Oxidative stress to endothelial cells plays a central role in the development of atherosclerosis and the occurrence of CVD. CR and endurance regular exercise are the fundamental approach to prevent oxidative stress to endothelial cells and prolong healthy life span. In addition to the improvement of lifestyle, recently emerging drugs that are effective in treating CVD have a property to eliminate ROS with a site-specific manner without interrupting favorable redox signaling, thereby ameliorating oxidative stress to endothelial cells. CR and exercise mimetics will also be fascinating pharmacological tools that specifically mitigate oxidative stress to endothelial cells. The sources of ROS and their inhibitors as well as a strategy of the site-specific antioxidative therapy to prevent CVD are presented in


Previous studies suggest that loss of γ-secretase activity in postnatal mouse brains causes age-dependent memory impairment and neurodegeneration. Due to the diverse array of γ-secretase substrates, it remains to be demonstrated whether loss of cleavage of any specific substrate(s) is responsible for these defects. The bulk of the phenotypes observed in mammals deficient for γ-secretase or exposed to γ-secretase inhibitors are caused by the loss of Notch receptor proteolysis. Accordingly, inhibition of Notch signaling is the main cause for untoward effects for γ-secretase inhibitors as therapeutics for Alzheimer’s disease. Therefore, we wished to determine if loss of canonical Notch signaling is responsible for the age-dependent neurodegeneration observed upon γ-secrectase deficiency in the mouse brain. We generated postnatal forebrain-specific RBPj conditional knockout (cKO) mice using the CamKII-Cre driver and examined behavior and brain pathology in 12–18 month old animals. Since all four mammalian Notch receptor homologues signal via this DNA binding protein, these mice lack canonical Notch signaling. We found that loss of RBPj in mature excitatory neurons was well tolerated, with no evidence for neurodegeneration or of learning and memory impairment in mice aged up to 18 months. The only phenotypic deficit we observed in the RBPj-deficient mice was a subtle abnormality in olfactory preferences, particularly in females. We conclude that the loss of canonical Notch signaling through the four receptors is not responsible for age-dependent neurodegeneration or learning and memory deficits seen in γ-secretase deficient mice. Alzheimer’s Disease (AD) is an incurable progressive neurodegenerative disease characterized by synaptic dysfunction and subsequent neurodegeneration, a hallmark of which are aggregates of amyloid β (Aβ) peptide  Notch receptors are one of the most intensively investigated γ-secretase substrates and a major source for untoward effects in clinical trials of GSI. Given the ambiguity discussed above and data demonstrating roles for Notch signaling in synaptic plasticity  from mutant RBPj  . QRT-PCRCre-mediated recombination of the floxed RBPj allele generates an RNA that could encode for a truncated RBPj protein . Similarly, there were no significant effects involving genotype for any of the measures within the sensorimotor battery, suggesting that these functions were intact in RBPcKO mice.To assess the effects of loss of RBPj in hippocampus-dependent spatial learning and memory, RBPcKO mice and control littermates at 12–18 months of age were evaluated on the Morris Water Maze (MWM) tasks. The mice were first tested on the cued (visible platform) trials to document that the RBPcKO mice did not have any non-associative  disturbances that would affect their subsequent spatial learning/memory performance. A rmANOVA conducted on the escape path length data from the cued condition  did not A rmANOVA conducted on the path length data from the place trials . Similarly, no significant effects involving genotype were revealed following analysis of the freezing data from the subsequent 8 min when the tone was presented during auditory cue testing (not shown). Firm conclusions are difficult to draw from the auditory cue data since mice may have hearing impairments during these ages A previous study using adenovirus Cre-mediated deletion of RBPj in the subventricular zone resulted in a reduction in the number of mature granule cells in the olfactory bulb To evaluate olfactory preferences, we conducted planned comparisons on the number of hole pokes made into the odorant-containing versus empty corner holes, as well as pokes into holes containing familiar or novel odorants. Results from these comparisons showed that none of the groups significantly preferred the odorant-containing versus the empty corner holes, although the WT control group tended to do so . However, an ANOVA conducted on these data revealed a significant genotype by sex by hole interaction, , suggesting sex-dependent effects across groups. Inspection of these data indicated that males and females of each genotype showed at least a tendency to prefer the odorant-containing versus the empty corner hole except for the female RBPcKO homozygous mice, which exhibited the opposite preference . SubsequPlanned comparisons conducted on poke frequencies into familiar and novel odorant-containing holes indicated that the WT control mice exhibited the expected robust preference for the familiar odorant, , while non-significant trends for this preference were shown by the RBPcKO heterozygous (p = 0.064) and RBPcKO homozygous groups . An ANOVPrevious studies identified age-dependent neurodegeneration when either PS or Nct were removed from excitatory neurons in postnatal forebrain Hints that Notch signaling may be implicated in this phenotype abound in the literature. Reduced Notch activity was linked to poor cognitive performance in mice heterozygous for Notch1 or RBPj We showed that aging RBPcKO mice lacking canonical Notch signaling in excitatory neurons of the forebrain were anatomically and behaviorally indistinguishable from their wild-type littermates. Although much larger cohorts may reveal subtle differences, this is in dramatic contrast to the severe deficits observed in similar size cohorts of aged PScDKO and NctcKO mice We propose three possible explanations for these observations. First, another substrate or a combination of substrates are responsible for the previously observed age- and γ-secretase-dependent neurodegeneration and memory impairments. Second, because loss of both Notch1 and Notch2 is tolerated with no evidence for neurodegeneration DrosophilaInterestingly, we observed that female RBPcKO mice at 12–18 months of age process certain olfactory stimuli differently compared to WT controls, suggesting that RBPj may mediate certain olfactory functions. This hypothesis is consistent with recent reports that Notch signaling may play a role in the generation of mature granule cells in the olfactory bulb In summary, Notch signaling deficiency in excitatory neurons is not responsible for the age-dependent neurodegeneration and memory impairment observed in the absence of γ-secretase. In addition, excitatory neurons in the neocortex maintain normal function in the absence of canonical Notch signaling in the adult mouse brain.Animal studies were approved by the Washington University Division of Comparative Medicine, protocol #20110027 and the mice were cared for following ICOC guidelines.All mouse lines used in this study have been previously described: RBPj f/f Mice were anesthetized and cardiac perfusion was conducted with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). For isolating different parts of the brain, after removing cerebellum and olfactory bulb, brain was cut sagittally in half and banana-shaped area encompassing the hippocampus was dissected out on ice. The frontal cortex was cut from the remaining tissue.mRNA was extracted from isolated cerebellum, hippocampus, frontal cortex, and olfactory bulb using RNeasy kit (Qiagen). Total RNA (1 ug) was treated with DNase I and reverse transcribed using the SuperScript First-Strand Synthesis kit (Invitrogen). PCR reactions in triplicate were performed in a total volume of 25 µl using SYBR Green PCR mastermix in a Step One Plus Real-time PCR system (Applied Biosystems) using 5 µl of diluted (1∶100) cDNA and gene-specific primers. Amplification was performed under the following conditions: 40 cycles of 95°C for 20 s and 60°C for 20 s. The forward primer for RBPj wt is located in exon 6 that is deleted in RBPcKO, and the reverse primer is located in exon 8. The forward primer for RBPj mut spans the junction between exon 4 and 5, and the reverse spans the novel junction between exons 5 and 8 that is generated upon Cre-mediated deletion of exon 6 and 7. We prepared mRNA from hippocampus, frontal cortex, cerebellum and olfactory bulb from 6 months old RBPcKO and control RBPj f/f mice. GAPDH was used as an internal control for PCR reaction, and the ratio of mRNA was normalized to that of cerebellum where deletion does not occur.Lysates were generated from various brain regions in RIPA-DOC, homogenized through sonication and equivalent amounts as determined by protein assays of the brain lysate and from mouse ES cells were loaded on reducing polyacrylamide gels. The lysates were probed with antibodies against total Notch1 ) and two independent antibodies for RBPj  and rat monoclonal T-6709 (Cosmo Bio)). Equivalent loading was confirmed by staining for Ponceau S.RBPj wt and RBPj mut sequences were cloned into the HindIII and BamHI sites in 3x Flag CMV-7 vector . TP-1 and Hes1 luciferase reporters were previously published A cohort of mice aged 12–18 months (RBPj f/f (control) n = 9 : CamKII-Cre RBPj f/+ (RBPcKO het) n = 7 : CamKII-Cre RBPj f/f (RBPcKO homo) n = 8 ) was evaluated through the use of several behavioral analyses, including a (1-h) locomotor activity/exploratory behavior test, a battery of sensorimotor measures, as well as the Morris water navigation, holeboard exploration/olfactory preference, and conditioned fear tests . Same coLocomotor activity was evaluated in all mice over a 1-h period using transparent (47.6×25.4×20.6 cm high) polystyrene enclosures and computerized photobeam instrumentation, as previously described Spatial learning and memory were evaluated in the Morris water maze using a computerized tracking system , as previously described Mice were evaluated for possible differences in exploratory behaviors and olfactory preference using a modified version of our previously published procedure FreezeFrame image analysis software , which allowed for simultaneous visualization of behavior while adjusting a “freezing threshold,” which categorized behavior as freezing or not freezing during 0.75 s intervals. Freezing was defined as no movement except for that associated with normal respiration, and the data were presented as percent of time spent freezing. Shock sensitivity was evaluated following completion of the conditioned fear testing, according to our previously described procedures Mice were evaluated on the conditioned fear test, as previously described Animals were anesthetized and tissues were fixed by intracardiac perfusion with PBS followed by 4% PFA and post-fixation in 4% PFA. Paraffin sections were prepared with standard procedures and sagittal brain sections (10 µm) were obtained. It was Nissl stained, or immunostained with antibodies reactive against MAP2 , GFAP , NeuN  or Iba1  and incubated with DyLight549-conjugated secondary antibodies  or stained with DAB. RBPj staining was performed as previously described Images were analyzed with Nikon Eclipse 80i, Apotome  or Nanozoomer (Alafi Neuroimaging Laboratory at Washington University).Cells were counted by the optical dissector method using Stereo Investigator software (MBF Bioscience). Cortex was outlined using 4x objective and cell quantification was performed using 10x objective. Section interval was 5–8. The counting frame size was 40 µm×40 µm. The grid size was 300 µm×300 µm. The thickness of the tissue was 50 µm. These conditions were optimized to obtain a Gundersen coefficient of error less than 0.1.Analysis of variance (ANOVA) models were used to analyze the behavioral data . Repeated measures (rm) ANOVA models containing two between-subjects variables (genotype and sex) and one within-subjects (repeated measures) variable  were typically used to analyze the learning and memory data. The Huynh-Feldt adjustment of alpha levels was utilized for all within-subjects effects containing more than two levels to protect against violations of sphericity/compound symmetry assumptions underlying rmANOVA models. One-way ANOVA models were also used to analyze other data . Planned comparisons were conducted within ANOVA models for certain holeboard test variables. In most other instances, pair-wise comparisons were conducted following relevant, significant overall ANOVA effects, which were subjected to Bonferroni correction when appropriate.Figure S1Experimental design. The same cohort of mice  were subjected to behavioral then biochemical/immunohistochemical analyses. A separate cohort of mice were used for the qPCR studies.(TIF)Click here for additional data file.


Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease,  Huntington's disease is an autosomal dominant neurodegenerative disorder in a family of CAG trinucleotide repeat disorders which includes Spinobulbar muscular atrophy and six types of Spinocerebellar ataxia Mutated huntingtin protein aggregates into insoluble, amyloid fibrils that have structural and cytotoxic similarities to aggregates associated with other neurodegenerative disorders such as Alzheimer's and Parkinson's disease in vitro shows that glutamine repeat length dependence strongly correlates with HD pathology and age of onset in vivo and in vitroin vitro characterization of the kinetics and thermodynamics of such systems can provide valuable insight into the mechanism of aggregation which would aid therapeutic inhibition strategies.Aggregation of polyglutamine (polyQ) peptides in vivoin vitroin vitro aggregation studies.While the potential to aggregate is a characteristic of proteins associated with neurodegenerative disorders, it is not clear that mature amyloid fibrils are the toxic species in these deposition diseases in vitro studies of amyloid aggregation kinetics.Many strategies have been employed to monitor polyglutamine peptide aggregation with the goal of elucidating fibril phase behavior, including analytical ultracentrifugation While ThT fluorescence is well suited to monitor β-sheet fibril formation, ThT binding assays cannot identify non-fibrilar species, interpret fibril hyperstructure, or distinguish many small fibrils from fewer large fibrils because the fluorescence associated with ThT binding only quantifies total β-sheet content. Equally, non-aggregated monomer quantification techniques like sedimentation assays cannot distinguish various species of aggregated protein. Therefore it is often informative to use multiple assays in concert, overlaying results from different techniques to reveal critical features of the aggregation pathway Dynamic light scattering (DLS) is a laser scattering technique capable of unbiased analysis of size distributions of diffusing particles in the nanometer to micron size range DLS is a useful complimentary technique to ThT fluorescence because in addition to detecting fibrils, it can detect diffusing species that ThT cannot, like non-fibrilar intermediates and large assemblies of fibrils. Meanwhile ThT can verify the fibrilar nature of the species which DLS resolves. The two methods are not orthogonal however as they both report some of the fundamental characteristics of polyQ aggregation, for example Q-length and concentration dependence as is shown in this work. In this work, an apparatus was built which can simultaneously perform dynamic light scattering measurements and measure ThT fluorescence in order to distinguish ThT-fibril binding events from other modes of aggregation in a single reaction . The difn = 15, 25, 35, or 45, was expressed in BL21DE3 . Following induction in the presence of 0.5 mM IPTG at 37°C for 2 h, the cells were sedimented and resuspended in 3 mM imidazole, 250 mM KCl, and 1 mM DTT. They were then lysed by sonication and the cell extract was spun at 30,000×g and 4°C for 30 minutes. The supernatant was filtered through 0.4 µm nitrocellulose membranes and loaded on a 10 ml Talon column and equilibrated in 3 mM Imidazole, 200 mM KCl, and 1 mM DTT. The column was washed with five bed volumes of equilibration buffer and the fusion protein was eluted using an imidazole gradient 3–200 mM. Purified [His-tag]-[MBP]-[TEVcleavage site]-[Qn] was flash frozen in liquid nitrogen and stored at −80°C. The concentration of [His-tag]-[MBP]-[TEVcleavage site]-[Qn] was determined spectrophotometrically using an extinction coefficient of 67840 M-1. cm-1 at 280 nm.The fusion polypeptide [His-tag]-[MBP]-[TEVcleavage site]-[Qn], where A dynamic light scattering apparatus was built in order to easily integrate DLS with the fluorescence excitation and detection system . The DLSThe SMF delivered scattered photons to an avalanche photo diode based photon counting module (PCM) . The PCM digitized photon arrival events and output a digital pulse train representing photon counts which were counted by a National Instruments PCI counter-card and a PC. Labview software  processed the raw data and computed the autocorrelation for dynamic light scattering analysis. The incident laser beam was gated by an electronic shutter so that the sample was only illuminated during data collection. In a typical experiment scattered photons were collected over sample time of 100 seconds in 50 µs bins.Autocorrelation functions were computed using a multi-tau scheme enabling a lag-time dynamic range spanning from tens of microseconds to minutes. This dynamic range is important for aggregation studies because of the large variation in diffusional timescales between polyQ monomers and large complexes of amyloid fibrils.D:q is the scattering vector, n is the index of refraction of the solvent and λ is the incident wavelength. For spherical particles, the hydrodynamic radius, hR, is related to the translational diffusion coefficient by the Stokes-Einstein relation.For a monodisperse suspension of Brownian particles the electric field autocorrelation function kB is the Boltzmann constant, T is the absolute temperature in Kelvin, and η is the solvent viscosity. In suspensions of biological samples, there is typically a polydisperse distribution of diffusional timescales associated with heterogeneity in hydrodynamic radius and particle shape. This is especially true for fibrilar systems. In these cases the autocorrelation function is better represented as continuous sum of exponential decays.S(Γ), like mean and variance, were computed using the method of cumulants hR>, of aggregating species was calculated from <Γ> using q.Properties of the decay distribution S(Γ) using constrained regularization.Cumulant analysis is typically appropriate for, monomodal distributions. When populations of diffusing particles are multimodal and have distinguishable peaks, it is informative to solve the distributions directly. CONTIN Thioflavin T  was dissolved in water to yield a 1 mM stock solution. This stock solution was added to polyQ aliquots before addition of TEV at a molar ratio of 10∶1 ThT:polyQ. Epi-mode fluorescence excitation and emission paths are depicted in To monitor fibril assembly with dynamic light scattering and Thioflavin T, 30 µl of a solution containing the [MBP]-[TEV cleavage site]-[Qn] fusion protein, ThT, and TEV protease was injected into the flow cell depicted in The nature of the oligomeric species was assessed using a Jeol 1400 transmission electron microscope  following adsorption of the samples onto carbon-coated 200-mesh grids and negative staining with 1% uranyl acetate. The images were recorded with a Gatan Orius CCD camera .Polyglutamine peptide aggregation was monitored while varying both glutamine repeat length and concentration. Polyglutamine peptides including tracts of about 35 or more continuous glutamines demonstrate relatively rapid assembly into amyloid fibrils and likewise, tracts of greater than 35 glutamines in the huntingtin exon 1 lead to HD pathogenesis. Aggregation was initiated by TEV-mediated release of the polyQ peptide from the MBP-TEV-polyQ fusion complex for Q45, Q35, Q25, and Q15 (70 µM). Thioflavin T fluorescence and dynamic light scattering both confirmed this critical Q-length reporting almost complete fibril assembly in a 12 hour period for Q45 and Q35 while reporting no ThT binding or any shift in mean diffusion coefficient in the same period for Q25 and Q15 . In figuhR> just after 100 minutes. Here mean hydrodynamic radius is calculated by use of the method of cumulants and normalized to the steady state Q45 values. Contributions to the amplitude of the static scattering signal from TEV protease and MBP-Q45 monomers are about 8×103 photons/sec and 104 photons/sec respectively. These magnitudes are 50 to 70 times smaller than the contributions from mature fibrils. As the polyQ peptides begin to aggregate the static, time-averaged scattering signal increases above the baseline scattering from these non-aggregating molecules . hR> on the order of 10 nm  confirming that fibril elongation is stunted at lower concentrations. ThT fluorescence alone only measures total β-sheet content, and is blind to the way in which β-sheets are distributed within fibrils. Thus without the complimentary DLS information it would be impossible to discern whether the lower relative ThT fluorescence measured in the less concentrated reactions was caused by shorter fibrils or fewer fibrils.This disparity between <aneously . ThT revaneously . EquallyhR> evolution  and extract particle size distributions from the light scattering data from aggregating Q45 and Q35 at 70 µM and 28 µM  and DLS measurements (B) of the beta-sheet content and mean hydrodynamic radius of a single Q45 (black filled circles) and Q35 (black open circles) assembly reaction at 70 μM compared with solutions containing only MBP-Q45 complex (blue stars) and TEV protease (red triangles). All measurements are normalized to their respective initial values.(EPS)Click here for additional data file.Figure S2Time-averaged static scattering of Q45 assembly. Absolute time-averaged scattering signal during the same experiments described in (EPS)Click here for additional data file.Figure S3TEV and MBP-complex particle size distributions. Intensity weighted particle size distributions measured by CONTIN of MBP-polyQ complex (blue stars) and TEV protease (red triangles) which add to the background in DLS measurements.(EPS)Click here for additional data file.