Given the fundamental roles of histone deacetylases (HDACs) in the regulation of DNA repair, replication, transcription and chromatin structure, it is fitting that therapies targeting HDAC activities are now being explored as anti-cancer agents. In fact, two histone deacetylase inhibitors (HDIs), SAHA and Depsipeptide, are FDA approved for single-agent treatment of refractory cutaneous T cell lymphoma (CTCL). An important target of these HDIs, histone deacetylase 3 (HDAC3), regulates processes such as DNA repair, metabolism, and tumorigenesis through the regulation of chromatin structure and gene expression. Here we show that HDAC3 inhibition using a first in class selective inhibitor, RGFP966, resulted in decreased cell growth in CTCL cell lines due to increased apoptosis that was associated with DNA damage and impaired S phase progression. Through isolation of proteins on nascent DNA (iPOND), we found that HDAC3 was associated with chromatin and is present at and around DNA replication forks. DNA fiber labeling analysis showed that inhibition of HDAC3 resulted in a significant reduction in DNA replication fork velocity within the first hour of drug treatment. These results suggest that selective inhibition of HDAC3 could be useful in treatment of CTCL by disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death. HDAC3 (a class I HDAC) is involved in the regulation of chromatin structure and gene expression, which controls DNA repair, metabolism, and even tumorigenesis o-aminophenyl) carboxamides, can show 10-fold or higher selectivity for HDAC3, over HDACs 1 and 2  (acetyl K5) (ab51997), Histone H3 (acetyl K27) (ab4729), HDAC 1 (ab19845), HDAC 2 [Y461] (ab32117), HDAC 3 (ab16047) and Histone H2B (ab1790). Histone H3 [96C10] (3638S) and Histone H4 [L64C1] (2935S) were used as loading controls and purchased from Cell Signaling. Anti-acetyl histone H3 (or H3K9K14ac) (06–599) and Anti-phospho-Histone H2A.X (Ser 139) clone JBW301 (05–636) were purchased from Millipore. Histone H3 (acetyl K56) (2134-1) was purchased from Epitomics, and anti-actin (A2066) was purchased from Sigma Aldrich. PCNA [FL261] was purchased from Santa Cruz (SC7907).50 values for HDAC1, HDAC2, and HDAC3: RGFP966: >15, >15, 0.08 µM; RGFP233∶0.034, 0.059, 3.33 µM; and RGFP136∶5.2, 3.0, 0.4 µM. Bexarotene (SML0282), Methotrexate (M8407), and ATRA (R2625) were purchased from Sigma Aldrich.Depsipeptide  was kindly provided by Celgene. The HDIs RGFP233, RGFP136, and RGFP966 were synthesized and kindly given to us by Repligen Corporation. These compounds are analogs of previously published compounds For preparation of whole cell protein lysates, cell pellets were washed with PBS and then sonicated in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Roche). For preparation of liver lysates, livers were minced in RIPA buffer with protease inhibitors with a razor blade and then homogenized using a dounce homogenizer. Samples were sonicated and then cleared by centrifugation. Then samples were diluted 1∶2 in Laemmli’s sample buffer (Bio-Rad) and subjected to 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analyses were performed using primary antibodies listed above and for histone modification or γH2ax westerns, fluorophore conjugated secondary antibodies and the Odyssey system (LiCor) were used. For the iPOND experiment, a HRP secondary antibody and Western Lightning Plus enhanced chemiluminescence substrate  was used.For protein separation, soluble chromatin obtained from Hela cells was fractionated using a Superose 6 10/300 GL  gel filtration column. Fractions (0.5 ml) were collected, concentrated using trichloroacetic acid precipitation, and analyzed by western blotting using the antibodies indicated in the figure legends. Molecular weight standards were added to the sample as controls. Their elution fractions are indicated at top of the figure.5 cells/mL. Cells were then treated with DMSO, or HDIs once at hour 0. 100 µl aliquots were taken in triplicate from each flask at 0 hr, 24 hrs, 48 hrs, and 72 hrs after treatment, distributed into a flat bottom 96-well plate, and 10 µl of alamar blue added to each well. After a 4 hr incubation, fluorescence was measured using the Biotek Synergy MX Microplate Reader. For the dual treatment curves, the same protocol was followed except ATRA was re-administered at 48 hrs after the initial treatment.Alamar blue was purchased from Invitrogen (DAL1100). Cells were counted and split into T25 (Corning) flasks at 2×10Annexin V analysis of HH and Hut78 cells was performed using annexin V-fluorescein isothiocyanate (annexin V-FITC) apoptosis detection kit I (BD Pharmingen - 556547) per the manufacturer’s instructions. Briefly, cells were treated with DMSO, Depsi, or HDIs for 24 hrs, pelleted, washed with PBS, and counted. Cells were then resuspended in annexin V binding buffer, labeled with annexin V-FITC and propidium iodide (PI), and then analyzed by flow cytometry using the 5-laser BD LSRII instrument in the Vanderbilt Flow Cytometry Core. Here propidium iodide (PI) is used as a vital dye.6 cells per sample were pelleted, resuspended in 200 µl cold PBS and 5 mls of cold 100% ethanol, covered with foil, and stored at 4°C overnight. The next day cells were pelleted, resuspended in 1 mL 2N HCL supplemented with 0.5 mg/mL pepsin, and then incubated for exactly 30 mins at 37°C. Samples were then neutralized with 3 mL 0.1M Sodium Tetraborate (pH 8.5) and pelleted for 7 mins. Then samples were washed 1× with 1 mL of PBS +0.5% BSA, pelleted, washed 1× with PBS +0.5% BSA +0.5% Tween 20, and pelleted again. Samples were then resuspended in FITC-Conjugated anti-BrdU and incubated for 45 mins at room temperature in the dark. Samples were washed one more with PBS +0.5% BSA +0.5% Tween 20 and resuspended in 400 µL of PBS. Propidium iodide and RNase A were added to each sample and then analyzed by flow cytometry using the 5-laser BD LSRII instrument in the Vanderbilt Flow Cytometry Core.Cell cycle status was analyzed using the FITC Mouse Anti-BrdU set (BD Pharmingen-556028). Cells were treated with DMSO, Depsi, or HDIs for 24 hrs and then BrdU  was added to each flask one and a half hours before harvesting. The cells were then pelleted, washed with PBS, and counted. 1×10Analysis of proteins associated with DNA replication forks was performed using the iPOND (isolated proteins on nascent DNA) method described previously 6 cells/ml, and mixed with non-labeled cells for better spreading results (20 µL labeled cells +60 µL non-labeled cells). 2 µL of cell suspension and 10 µL of spreading buffer  was added to each slide, let sit for 6 mins at RT and then tilted to 15 degrees to allow the DNA to run slowly down the slide. 5 slides were made for each sample. Slides were then air dried for at least 40 mins, fixed in 3∶1 methanol:acetic acid for 2 mins, air dried again for 20 mins, and then stored at 4°C overnight.DNA fiber labeling analysis was used to assess DNA replication fork progression The next day, slides were submerged in 2.5M HCl for 30 mins, rinsed 3× in PBS and then incubated in 10% goat serum/0.1%Triton in PBS for 1 hr. Then slides were incubated in the dark for 1 hr in rat monoclonal anti-CldU  and mouse anti-IdU (Becton Dickinson 347580) diluted 1/100 in 10% goat serum/0.1% Triton in PBS. Slides were then rinsed 3× in PBS and incubated 30 min with secondary antibodies (Invitrogen Alexa Fluor 568 goat anti-rat-IgG A-11077 and Alexa Fluor 488 goat anti-mouse-IgG A-11029) in 10% goat serum/0.1% Triton in PBS in the dark. Slides were then rinsed 3× in PBS, air dried in the dark, mounted with 110 µL of Prolong Gold with no Dapi (Invitrogen P36930) using whole slide coverslips, let dry overnight at RT and then stored at 4°C. Samples were imaged at 1000× and 100 fibers were measured for each sample.Fork velocity was determined by the total length of fibers (IdU plus CldU) divided by 40 min. The above listed protocol was followed for all experiments except for changes in the labeling scheme as listed below: For experiments where DMSO or HDIs were added after labeling with IdU followed by CldU, cells were labeled with IdU for 20 mins followed by 20 mins of CldU, washed, and then either immediately treated with DMSO or HDIs for 25 mins or incubated in fresh medium for 4 hrs and then treated with DMSO or HDIs for 25 mins. Fork Velocity was determined by the total length of fibers (IdU plus CldU) divided by 40 min pulse or by the length of either the IdU label or CldU label divided by 20 min pulse.Hdac3 caused increases in the acetylation of H4K5, H4K8, H4K12, H4K16, H3K9K14, and H3K27 Hdac3 deletion did not cause the accumulation of the modification recognized by the rabbit monoclonal antibody to H3K56ac . In contrast, inhibitors of class I HDACs ), caused a more dramatic accumulation of H3K56ac than nicotinamide, which impairs the Sirtuins  and Hdac3 selective inhibitors (RGFP136 and RGFP966) for specificity. RGFP233 (233) showed 100- and 50-fold selectivity respectively towards HDAC1 and HDAC2 over HDAC3, and RGFP136 (136) and RGFP966 (966) were 10- and >100-fold respectively more selective for HDAC3 in To determine how treatment with selective HDIs affects CTCL cell lines, we first performed cell proliferation assays using alamar blue to measure cell growth and viability in the presence of different HDIs. HH and Hut78 cells were treated at hour 0 with either DMSO, Depsipeptide, 233, or 966 and then analyzed at hours 0, 24, 48, and 72 for changes in cell proliferation as measured by changes in alamar blue-dependent fluorescence. Both cell lines were sensitive to treatment with 10 µM 233 or 966, as demonstrated by decreases in cell growth over time . HoweverDose curves were performed on each cell line to determine the optimal dose for dual treatment with drugs that are used or have been used to treat CTCL . Cells w50 were chosen , Methotrexate (inhibitor of dihydrofolate reductase), or ATRA  e chosen . Both HHe chosen , while oe chosen .We next determined whether the decreased cell growth seen when HH and Hut78 cells were treated with selective HDIs  and 3 waHdac3 caused increased DNA damage and cell cycle delays in an S phase dependent manner in fibroblasts Deletion of HDACs 1 and 2 regulate deacetylation of histones deposited on newly synthesized DNA during S phase and are enriched at replication forks The gel filtration analysis suggested that HDAC3 might be associated with histone deposition machinery, yet not directly bound to the DNA replication machinery. Therefore, isolation of proteins on nascent DNA (iPOND) was used to further probe HDAC3 localization to DNA replication forks. A similar analysis in HEK293T cells suggested that, not only were HDAC1 and HDAC2 present at DNA replication forks, but HDAC3 was also detected Although HDAC3 did not appear to move with replication forks using iPOND, loss of HDAC3 activity using siRNA or gene deletion showed a requirement for this deacetylase for optimal DNA replication fork velocity [Next, DNA fiber labeling analysis was used to visualize individual DNA fibers by sequential labeling of cells with IdU and CldU followed by immunofluorescence to detect the incorporation of these analogs Finally, to determine if this replication defect was due to a localized effect, we treated Hut78 cells for 5 min with either Depsi or 966 before labeling with IdU followed by CldU. Remarkably, even treatment within this short timeframe caused a shortening of DNA fiber track lengths and slower fork velocity . These dCutaneous T cell lymphoma (CTCL) diagnosed during early stage disease generally has an indolent course and good outcome Mechanistically, the apoptosis observed was associated with the accumulation of DNA damage in HDI treated cells  and S3. The rapid effects of 966 on DNA replication suggest an important role for HDAC3 in DNA replication. In addition, by inhibiting HDAC3 at various times before DNA fiber labeling, we were able to narrow the possible mechanisms by which this might occur to localized effects at or around the DNA replication fork, as it took greater than 30 min before global changes in histone acetylation were observed . HoweverHdac3 is not compensated for by higher expression of other class 1 Hdacs. Thus, we conclude that HDAC3 plays a distinct role from HDAC1 and HDAC2 during chromatin maturation , or different concentrations of either 233 or 966 at hour 0. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment using alamar blue. A representative curve is shown from experiments performed in triplicate that are consistent with other biological replicates.(TIFF)Click here for additional data file.Figure S2CTCL cell lines exhibit sensitivity to multiple doses of 233 and high dose 136. Dose curves of HH cells (left) or Hut78 cells (right) treated with 10 µM 233 (A) or 966 (B). Cells were treated once with DMSO, 10 nM Depsipeptide (Depsi), or different concentrations of either 233 or 136 at hour 0. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment using alamar blue. For both (A) and (B), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 233 2 µM: p = 0.005, 233 5 µM: p = 0.005, and 233 10 µM: p = 0.004. For the Hut78 cells (right), Depsi: p = 0.002, 233 2 µM: p = 0.01, 233 5 µM: p = 0.005, and 233 10 µM: p = 0.006. (B) HH cells (left), Depsi: p = 0.001, 136 1 µM: p = 0.1, 136 5 µM: p = 0.1, and 136 10 µM: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.001, 136 1 µM: p = 0.08, 136 5 µM: p = 0.02, and 136 10 µM: p = 0.005.(TIFF)Click here for additional data file.Figure S3Dose curves for Bexarotene, Methotrexate, and ATRA reveal optimal concentrations for combination treatments. Dose curves of Bexarotene (A), Methotrexate (B), and ATRA (C) treated HH cells or Hut78 cells. Cells were treated at hour 0 with DMSO, 10 nM Depsipeptide (Depsi), or varying concentrations of Bexarotene, Methotrexate, or ATRA. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. In all studies except for (A), the HH and Hut78 cells were treated with the same varying concentrations of CTCL drugs. HH cells were treated with 10, 20, or 50 µM of Bexarotene while Hut78 cells were treated with 50,75, or 100 µM of Bexarotene. In (B) DMSO and a solution containing Na2CO3 served as vehicle controls. (C) ATRA was administered at hour 0 and re-dosed at 48 hours after treatment. For (A–C), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI or CTCL drug treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0007; Bexarotene 10 µM: p = 0.001; Bexarotene 20 µM: p = 0.004; Bexarotene 50 µM: p = 0.001. Hut78 cells (right), Depsi: p = 0.002; Bexarotene 50 µM: p = 0.8; Bexarotene 75 µM: p = 0.1; and Bexarotene 100 µM: p = 0.04. (B) HH cells (left), Depsi: p = 0.001; Methotrexate 0.1 µM: p = 0.007; Methotrexate 1 µM: p = 0.01; Methotrexate 10 µM: p = 0.01; Methotrexate 100 µM: p = 0.006. Hut78 cells (right) Depsi: p = 0.001; Methotrexate 0.1 µM: p = 0.005; Methotrexate 1 µM: p = 0.006; Methotrexate 10 µM: p = 0.004; Methotrexate 100 µM: p = 0.004. (C) HH cells (left), Depsi: p = 0.001; ATRA 500 nM: p = 0.008; ATRA 1 µM: p = 0.002; ATRA 2 µM: p = 0.003. Hut78 cells (right) Depsi: p = 0.001; ATRA 500 nM: p = 0.02; ATRA 1 µM: p = 0.005; ATRA 2 µM: p = 0.006.(TIFF)Click here for additional data file.Figure S4HDIs increased in apoptosis, DNA damage, and cell cycle defects in HH cells. (A) HH cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and apoptosis levels were assessed by Annexin V/PI staining and flow cytometry. Untreated (UT) and DMSO treated cells were used as controls. Shown is a representative graph from an experiment performed in duplicate that is consistent with other biological replicates. (B) Western blot analysis of γH2aX levels in HH cells treated with DMSO, 10 nM Depsi, or 10 µM 966 for 8 hrs. Untreated and DMSO treated cells were used as controls. (C) Cell cycle status was analyzed using BrdU/PI and flow cytometry. HH cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and pulsed for an hour and a half with BrdU prior to cell harvest and analysis. Shown are representative flow cytometry plots from an experiment performed in duplicate that is consistent with other biological replicates. (D) Graphical representation of BrdU incorporation from the experiment described in (C). (E) Graphical representation of the percent of S phase cells that did not incorporate BrdU (shown by box in panel (C)). Statistical analysis for both the Annexin V and BrdU experiments was performed using a two-tail T-test and comparing the HDI treated cells to the DMSO treated cells resulting in the following p-values: (A) Depsi: p = 0.02, 233: p = 0.01, and 966: p = 0.06. (D) Depsi: p = 0.002, 233: p = 0.05, and 966: p = 0.3. (E) Depsi: p = 0.03, 233: p = 0.07, and 966: p = 0.8.(TIFF)Click here for additional data file.Figure S5HDI treatment after labeling with IdU and CldU shows no changes in DNA fiber length. (A) DNA fiber labeling analysis was used to assess DNA fiber length in Hut78 cells treated with either DMSO, 10 nM Depsipeptide (left) or 10 µM 966 (right) 4 hrs after labeling the cells with IdU for 20 mins (green) followed by 20 mins of CldU (red). (A) Graphical representation of fork velocity as determined by the total length of fibers (IdU plus CldU) divided by 40 min pulse is shown. Representative measured fibers are shown at the right for DMSO, Depsi, and 966. 100 fibers were measured for each sample. Statistical analysis was performed using Mann-Whitney test and standard deviations were calculated. HDI treated cells were compared to the DMSO treated cells resulting in the following p-values: Depsi: p = 0.5 and 966: p = 0.4. The average velocities for both Depsi and 966 were within 1 standard deviation of the average velocity for DMSO.(TIFF)Click here for additional data file.


Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature. Human papillomaviruses (HPVs) contain a single, circular dsDNA genome of approximately 8 kb which associates with cellular histones to form a minichromosome. This minichromosome is encapsidated within a nonenveloped, icosahedral capsid composed of the late proteins, L1 and L2. Similar to polyomaviruses, 360 monomers of the major capsid protein L1 assemble into 72 pentameric capsomeres which are geometrically arranged on a T = 7 icosahedral lattice Due to the relative inefficiency of organotypic culture in the production of native HPVs, structural analyses of HPV capsids has been largely limited to the utilization of more efficient monolayer culture-derived papillomavirus particles such as virus-like particles (VLPs), and PsV Since previous studies into the disulfide bonding patterns of HPV16 capsids utilized particles produced in monolayer culture, or from fully developed BPV-containing warts, disulfide interactions were not placed in the temporal context of stratified and differentiating human tissue. Here we extend upon our previous observation, that HPV16 capsids become stabilized over time To develop producer cell lines which can synthesize organotypic culture-derived native virions from differentiating epithelia, primary human foreskin keratinocytes (HFKs) were electroporated with linearized wild-type and site-directed mutagenized HPV16(114/B) DNA Previous literature suggests that HPV16 and BPV1 PsV can mature within 293TT cell lysates if incubated overnight at 37°C Previous studies indicate that C428S, C175S, and C185S substitutions in HPV16 VLPs and PsV dramatically alters either initial assembly of viral particles, or their stabilities after purification HPV titers are measured similar to other viruses such as the Kaposi's sarcoma virus (KSHV), quantifying viral genome equivalents While significant reductions in titers were detected in 10 and 20-day mutant virus preparations, all preparations contained quantifiable titers . These rWhen we compared to 10-day wild-type virus, the infectivity relative to L1 protein of 10-day C428S, C175S, C185S, and C175,185S viruses was 41%±28, 0.3%±0.2, 148%±60, and 49%±26, respectively. Compared to 20-day wild-type, the infectivityrelative to L1 protein of 20-day C428S, C175S, C185S, and C175,185S viruses was 5%±3, 0.1%±0.1, 20%±3, and 10%±9, respectively.In We have previously demonstrated that highly infectious wild-type virus migrates into Optiprep fractions #7 and #8 The instability of mutant capsids was compared to wild-type capsids by assessing the relative stability of virus within the Optiprep gradient . The relTo determine if Optiprep-fractionated mutant virions retained full infectivity, we performed the RT-qPCR-based infectivity assay on each Optiprep fraction . Using eInterestingly, when we compared the total amount of endonuclease-resistant genomes within each Optiprep fraction and normalized for total L1 protein concentration, we observed a dramatic defect in DNA encapsidation efficiency of C428S and C175S virions . In contPrevious studies have shown that treatment of HPV PsV or HPV-infected organotypic raft cultures with 5 mM GSSG enhances capsid maturation. However, work with PsV suggests that GSSG treatment also results in an increase in misfolded capsids since they are more susceptible to partial tryptic proteolysis than untreated capsids In this study, we sought to determine individual temporal roles for the highly conserved HPV16 L1 cysteine residues: C428, C175, and C185 in capsid assembly within the context of stratifying and differentiating human epithelial tissue, HPV's natural host environment. Previous studies have depicted that C428S and C427S mutations in HPV16 and HPV33 VLPs, respectively, lead to the complete loss of capsid integrity, being disrupted into their respective capsomeres Thought to reside on an external loop neighboring C175, it has been suggested by others that C185 might form similar interpentameric contacts with C428 with only minor structural rearrangements of this loop Our data suggests that all three conserved cysteine residues, C428, C175, and C185 are critical for proper assembly and maturation of native HPV16 virions. While no individual substitution led to the complete disruption of particles as was previously seen with C428S mutations in HPV16 VLPs and PsV, each substitution undermined particle formation efficiency and stability. These virions were more susceptible to intrusion of endonucleases into the capsid lumen, and less able to withstand the forces of ultracentrifugation Contrary to work with VLPs and PsV, in the context of organotypic culture-derived native virions, the C428S mutation led to approximately 50% of wild-type titers within 10-day viral preparations suggesting that hydrophobic interactions and/or cellular factors are involved in reinforcing the integrity of C428S virions In Analysis of 20-day viruses reveals the temporal importance of C175, C428 and C185. Titers remain low in 20-day C175S viral preparations as they had in 10-day viral preparations. Titers for C428S and C185S mutations were significantly down within 20-day viral preparations . This obFurther supporting the role of a complex interplay of disulfide bonding between C428, C175, and C185, is our finding that C428S, C175S, C185S, and C175,185S 20-day viral preparations have a significant decrease in titers compared to their 10-day counterparts. This suggests that each of these residues is important in the efficient accumulation of mature capsids . This reE. coli cells , we controlled for polymerase fidelity and/or random mutations that may arise during PCR amplification by extracting plasmid DNA from many isolated bacterial clones and sequencing the L1 ORF of each clone to verify the correct incorporation of nucleotide substitutions and the absence of spurious mutations elsewhere in the L1 ORF. Multiple mutant viral genomic clones containing correctly mutagenized sequences were isolated per each mutation and utilized in subsequent experimentation. To create a full-length, HPV16(114/B) genome with a Cys175Ser substitution, the two complimentary oligonucleotides: forward 5′CTGGGGCAAAGGATCCCCATCTACCAATGTTGCAGTAAATC3′ and reverse 5′GATTTACTGCAACATTGGTAGATGGGGATCCTTTGCCCCAG3′, were used with the change of G to C at nucleotide 6161. To create a full-length, HPV16(114/B) genome with a Cys185Ser substitution, the two complimentary oligonucleotides: forward 5′GCAGTAAATCCAGGTGATTCTCCACCATTAGAG3′ and reverse 5′CTCTAATGGTGGAGATCACCTGGATTTACTGC3′, were used with the change of G to C at nucleotide 6191. To create a full-length, HPV16(114/B) genome with a Cys428Ser substitution, the two complimentary oligonucleotides: forward 5′GTAACCCAGGCAATTGCTTCTCAAAAACATACACCTCC3′ and reverse 5′GGAGGTGTATGTTTTTGAGAAGCAATTGCCTGGGTTAC3′, were used with the change of G to C at nucleotide 6917. To create a full-length, HPV16(114/B) genome with Cys175,185Ser substitutions, the two complimentary oligonucleotides: forward 5′GCAGTAAATCCAGGTGATTCTCCACCATTAGAG3′ and reverse 5′CTCTAATGGTGGAGATCACCTGGATTTACTGC3′, were used with the change of G to C at nucleotides 6161 and 6191, respectively. The Cys175,185Ser amplimers were made utilizing the Cys175Ser template. In all instances, the most prevalent serine codon in the HPV16(114/B) L1 ORF, TCT, was utilized.pBSHPV16(114/B) DNA, a generous gift from M. Dürst  was utilized as the template for site-directed mutagenesis using Strategene's Quikchange II XL Site-Directed Mutagenesis Kit. Following transformation and selection of the mutant amplimers into XL10 Ultracompetent Immortalized HFK lines which stably maintained episomal HPV16 DNA were grown in monolayer culture using E medium in the presence of mitomycin C-treated J2 3T3 feeder cells BamHI, linearizing the viral DNA at nucleotide 6151 in L1 and separating it from the vector sequence. HFKs were electroporated with the prepared DNA as described previously Primary human foreskin keratinocytes (HFKs) were isolated and grown from newborn circumcision as described previously BamHI, which linearizes the HPV16 genome. Samples were separated by 0.8% agarose gel electrophoresis and transferred onto a GeneScreen Plus membrane (New England Nuclear Research Products) as previously described Total cellular DNA was isolated as previously described Tissues were fixed in 10% neutral buffered formalin, and embedded in paraffin. Four-micrometer sections were cut and stained with hematoxylin and eosin as previously described 2). Homogenizers were rinsed with 250 µl phosphate buffer. 1.5 µl (375 units) Benzonase (Sigma) was added to 750 µl of CVPs and incubated at 37°C for 1 hour. Samples were brought to 1 M NaCl by adding 130 µl ice cold 5 M NaCL. Then, samples were vortexed and centrifuged at 4°C for 10 minutes at 10,500 rpm in a microcentrifuge. Supernatants were stored at −20°C.For Optiprep fractionation, RT-PCR, RT-qPCR, and qPCR-based DNA encapsidation assays, 3-raft tissues viral preparation or VLP and PsV lysates were prepared by dounce homogenization in 500 µl phosphate buffer  and reverse 5′GGATTGGAGCACTGTCCACTGAG 3′ (nt 3535–3557) at final concentrations of 4 µM. A fluorogenic, dual-labeled, HPV16 E1∧E4 probe of 5′ 6-FAMCACCGGAAACCCCTGCCACACCACTAAGBHQ-1 3′ (nt 3493–3520) was utilized at a final concentration of 0.2 µM to detect E1∧E4 cDNA. Primers and probe were developed using Gene Link Software: OligoAnalyzer 1.2, and OligoExplorer 1.2. TATA-binding protein (TBP) amplicons were created using primers 5′ CACGGCACTGATTTTCAGTTCT 3′ (nt 627–648) and 5′ TTCTTGCTGCCAGTCTGGACT 3′ (nt 706-686) at final concentrations of 0.125 µM. TBP amplicons were detected by the fluorogenic TaqManTM probe 5′HEX TGTGCACAGGAGCCAAGAGTGAAGA BHQ-13′ used at 0.2 µM. TBP primer sequences were obtained from those previously described HaCaT cells were grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin and seeded 50,000 cells/well in 24-well plates. 50 µl of HPV16 viral stock was diluted with cell culture medium to a total volume of 0.5 ml. Medium was aspirated from HaCaT cells and 0.5 ml of diluted virus was added per well. One well on each plate received 0.5 ml of medium without virus as a negative control. The cells were incubated with the virus for 48 h at 37°C. mRNA was harvested with the SurePrep TrueTotal RNA Purification Kit (Fisher Scientific). DNA contamination of columns was insignificant in that the optional on-column DNase-I treatment of extracted mRNA had no effect on downstream signal. Amplification of both the viral target and endogenous cellular control target was performed using a duplex format in 0.2 ml, 96-well PCR plates (BIO-RAD) with a total reaction volume of 25 µl. All reactions containing RNAs from virus-infected cells were performed in duplicate or triplicate. Reverse transcription and quantitative PCR were performed in the same closed tube with approximately 250 ng of total RNA per reaction using the Quantitect Probe RT-PCR Kit (Qiagen). HPV16 E1∧E4 primers used were the splice-site straddling, forward 5′CCATATAGACTATTGGAAACACATGCGCC3′ as the forward primer (nt 2839–2868) and 0.3 µM 5′CGTTAGTTGCAGTTCAATTGCTTGTAATGC3′ as the reverse primer (nt 2960–2989). Amplification of human repetitive Alu DNA was performed using 0.3 µM 5′GTCAGGAGATCGAGACCATCCC3′ as the forward primer and 0.3 µM 5′TCCTGCCTCAGCCTCCCAAG3′ as the reverse primer 2 values for standard curves were at or above 0.99. A Bio-Rad iQ5 Multicolor Real-Time qPCR machine and software were utilized for PCR amplifications and subsequent data analysis. The PCR thermocycling profile was as follows: 15 min. hot-start at 95°C, followed by 40 cycles at 15 sec. at 94°C, 30 sec. at 52°C, and 30 sec. at 72°C. Data analysis commenced during the extension phase.To detect endonuclease-resistant genomes in viral preparations or Optiprep fractions, only benzonase-treated samples were used so that all non-encapsidated genomes were digested. To release all encapsidated viral genomes, 10 µl sonicated virus preparation or 20 µl Optiprep fraction was added to 2 µl Proteinase K, 10 µl 10% SDS, 2 µl pCMV-GFP (140 ng/µl) carrier DNA, and brought up to 200 µl with Hirt buffer. Tubes were rotated at 37°C for 2 hours. Immediately, an equal amount of phenol-chloroform-isoamyl alcohol (25∶24∶1) was added and the mixture was extracted for the aqueous phase. An equal amount of chloroform was added and again extracted for the aqueous phase. DNA was EtOH precipitated overnight at −20°C. After centrifugation, the DNA pellet was washed with 70% EtOH and resuspended in 20 µl TE overnight. To detect viral genomes or cellular DNA, a Qiagen Quantitect SYBR Green PCR kit was utilized. Amplification of the viral target was performed in 0.2 ml, 96-well PCR plates (BIO-RAD) with a total reaction volume of 25 µl. l µl of each endonuclease-resistant viral genome prep was analyzed in triplicate for each independent experiment. Amplification of HPV16 genomes was performed using 0.3 µM 40 µl aliquots from Optiprep fractions were boiled for 10 minutes in 6% 2-ME loading buffer and loaded on 8% polyacrylamide gels. To detect HPV16 L1, anti-HPV16 L1 monoclonal antibody  was utilized at a dilution of 1∶4,000 per manufacturer's recommendations. For nonreducing gels, loading buffer without 2-ME was utilized. Samples were boiled for 10 minutes at 70°C and loaded on 6% polyacrylamide gels. L1 was detected using the anti-HPV16 L1 monoclonal antibody, H16.J4


The polypyrimidine tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which—along with structure-based mutagenesis—reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB. ► First crystal structures of PTB reveals how it binds Raver1 via PRI motifs ► Observed mode of binding is general for PRIs and PTB paralogues ► Results place constraints on models of splicing repression by PTB-Raver1 complexes Over 95% of human multiexon genes have multiple splice isoforms . The pro introns . The temOne of the most intensively studied regulators of alternative splicing is the polypyrimidine tract-binding protein (PTB). PTB is a versatile protein. In addition to its nuclear splicing activity, PTB is also found in the cytoplasm, where it has roles in the stabilization and localization of mRNA . It is aPTB is expressed in a variety of tissues  and reprThough best known as a negative regulator of splicing that acts to exclude specific exons, PTB has recently been found to determine exon inclusion in some cases . In commSeveral different mechanisms have been proposed for the repressive splicing activity of PTB, including direct binding to pre-mRNA in order to block binding of U2AF65—a component of the spliceosome—to the polypyrimidine tract  or oligoNevertheless, it is clear that PTB binds to both RNA and proteins during splicing. The protein binds pyrimidine-rich motifs within RNA , which are found in regulatory elements  that mayTpm1). PTB acts to exclude the mutually exclusive exon 3 of Tpm1 in smooth muscle cells but not in other cells where PTB is expressed -restrained docking model because the affinity of purified PTB RRM2 for synthetic PRI3 peptides was too low for a full structure determination . AlthougAPPSLLGKD—the conserved core residues, which we number 1-7, are underlined) followed by residues 156-285 of PTB RRM2. Residues 156-179 are from the polypeptide that links RRM1 to RRM2 in the full-length protein; residues 180-285 correspond to the structured RRM2 domain  also crystallized.The expression levels in  6 mg/ml . Althoug 6 mg/ml D. All thAPPSLLG; PRI4: SEGLLGL) , Leu 2 and Leu 3 both project in the same direction into the binding pocket on PTB RRM2 because of a pinched backbone conformation that is stabilized by an internal hydrogen bond from the side-chain hydroxyl of Ser 1 to the backbone amide of Gly 4 A. In thi3LGL5 sequence to wrap around Tyr 247.In contrast, PRI4 (core sequence: GLLGLGP) makes only three intermolecular hydrogen bonds. The absence of Ser at position 1 eliminates the intrapeptide hydrogen bond and results in a more open backbone conformation. The loss of this internal interaction allows the peptide bond between Leu 3 and Gly 4 to flip with respect to PRI3, a conformational change that eliminates a hydrogen bond to RRM2 , was previously shown to be fully active as a splicing repressor domain when fused to MS2 coat protein and tethered to ing site . We thering site A, lane 4ing site B and 2D.ing site A and 4B.ing site D and to ing site C. As exping site , and coning site , the str1 exon 3  may be dTpm1. Though the structures contain only a single domain of PTB, this is the major structured portion of PTB-2L, the minimal fragment required to recapitulate the activity of the full-length protein in exon exclusion , despite observations of PTB-binding partners that have accumulated over the past 10 years and now include not only Raver1 and Raver2 , but alsThe crystal structures of PTB RRM2 in complex with Raver1 reveals a mode of peptide binding that has been observed in other RRM-peptide complexes such as U2AF35/U2AF65 , SPF45/SNevertheless, the PTB-Raver1 complex marks an interesting variation on this theme. Although in the three examples of perpendicular binding mentioned above peptide binding is anchored by insertion of a Trp side chain from the peptide into a deep apolar pocket between the helices on the RRM, in the Raver1 peptide the role of this Trp is taken by a pair of Leu side chains that insert into a shallower hydrophobic depression in a similar location on the RRM. This pair of Leu side chains make important hydrophobic contacts that contribute to binding of Raver1 PRIs to PTB RRM2; however, they are not sufficient for high-affinity binding, because this sequence feature is also found in PRI2 and PRI4, which have lower affinity . AdditioGiven the close sequence similarity between the PRI1 and PRI3 motifs from Raver1, the structural and binding data presented here offer a plausible explanation for the high affinity of PRI1 and suggest strongly that it binds to PTB RRM2 in the same way. The same can probably also be said of the two high-affinity PRI motifs in Raver2, which are similar to Raver1 PRI1 and PRI3 D.We have also shown that despite a cluster of sequence differences between PTB and its neuronal paralogue near the Raver1-binding site on RRM2, nPTB exhibits a very similar pattern of variation of affinity for the Raver1 PRIs . The RavFurthermore, the results allow us to refine the definition of what constitutes a high-affinity PRI sequence from [S/G][I/L]LGxxP to [S/G][I/L]LGxΦP, where the Φ at position 6 indicates a preference for a small hydrophobic residue . Using ScanProsite , 36 humaTpm1. Against this, however, it is has been observed that mutation of a single PRI3 motif in Raver1—and of an identical PRI in Raver2  to mutate MS2-Raver1 fusion proteins that were made previously . These iE. coli BL21 (DE3). Cell pellets were lysed by sonication in Buffer A , 0.1% (v/v) Triton X-100, and 0.5 mM phenylmethanesulfonylfluoride containing 1 mg/ml lysozyme. 6xHis-tagged proteins were purified from clarified lysates using TALON resin (Clontech). Purified proteins were eluted in Buffer A containing 100 mM imidazole. The 6xHis tag was removed by overnight incubation at 4°C with 1 mg of his-tagged TEVpro per 30 mg of PRI-RRM2 during dialysis against Buffer A with 5 mM β-mercaptoethanol. The cleaved tag and TEVpro were removed in a second round of TALON purification. For crystallization, proteins were further purified by gel filtration on a Superdex 75 16/60 column  in 100 mM NaCl, 25 mM Tris (pH 7.8), and 1 mM dithiothreitol (DTT).PTB proteins were expressed at 37°C in E. coli in a similar manner and applied a glutathione sepharose 4B column . The column was washed with Buffer A and proteins were eluted in Buffer A + 5 mM DTT and 20 mM glutathione (pH 9.0). Purified proteins were concentrated by centrifugal filtration to 22 mg/ml, 28 mg/ml, 5.5 mg/ml, and 4.3 mg/ml for PRI3-RRM2, PRI4-RRM2, GST-PTB, and GST-nPTB, respectively and stored at −80°C.For pull-down assays to monitor Raver1 binding, glutathione S-transferase (GST) tagged PTB proteins were extracted from 2PO4, 1 mM DTT, 3 mM PMSF, EDTA-free protease cocktail [Roche]). Soluble and insoluble fractions were separated by centrifugation at 8,000 g for 10 min. GST fusion proteins were purified through a glutathione sepharose 4B column , washed with MTPBS + 1% Triton X-100, and step-eluted using reduced glutathione. Protein-containing fractions were dialyzed in Buffer E . The GST-PTB and GST-SXL proteins used as a control in GST-PTB RRM2 fusion proteins used in 2. X-ray diffraction data were collected at beamlines X13 at DESY, Hamburg, Germany, and I02 at Diamond Light Source, Didcot, UK. The data were processed with iMosfilm and scaled using SCALA from the CCP4 suite , and 20% polyethylene glycol 3350. Crystals were soaked for 1 min in mother liquor with 20% glycerol before flash freezing in liquid NP4 suite . PRI3-RRP4 suite  using thP4 suite . PRI4-RRP4 suite  and refiP4 suite .35S-Met-labeled MS2-Raver1 proteins produced by in vitro transcription-translation reactions  were incubated for 3 hr at 4°C with GST, GST-PTB, or GST-nPTB in wash buffer  in the presence of 5 μl glutathione sepharose 4B. Beads were then washed three times with wash buffer, and bound protein was eluted using SDS loading buffer for SDS-PAGE analysis. Protein band intensities on dried gels were recorded using a Fuji FLA-5000 phosphor imager; quantitative densitometry was performed using AIDA software (Raytest).In vitro GST pull-down assays were performed essentially as described in . BrieflyTpm1 exon 3 polypyrimidine tract, which is enriched in PTB binding sites  for 30 min at 30°C and loaded directly onto a 5% polyacrylamide (40:1) gel.GST-PTB RRM2 proteins (0.2-3.2 μM) were incubated with 10 fmol of an RNA probe spanning the ng sites  in 10 mMTpm1 exon 3, which leads to enhanced exon skipping in HeLa cells (Splicing transfection assays were carried out as described previously . BrieflyLa cells .


However, the involvement of zinc in amyloid precursor protein (APP) processing and Aβ deposition has not been well established in AD animal models in vitro.In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD. The presence of extracellular β-amyloid (Aβ) plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). Mounting evidence has demonstrated that aberrant zinc homeostasis is involved in the pathogenesis of AD in vitro studies have shown that both APP and its proteolytic product Aβ contain zinc binding domains. However, the involvement of zinc in APP processing and Aβ deposition has not been well established in AD transgenic models in vivo.The disruption of zinc homeostasis in the AD brain is associated with the aberrant distribution and altered expression of zinc-regulating metalloproteins, such as metallothionein, zinc transporters (ZnT) and divalent metal transporter 1 (DMT1). We have reported that high levels of ZnT1, 3-7 and DMT1 proteins are located in the degenerating neurites in or around the Aβ-positive plaques associated with human AD and the APP/presenilin 1 (PS1) transgenic mouse brain in vivo and in vitro. Our data support the possibility that dietary zinc overload has the potential to be a contributing factor to the pathophysiology of AD.In the present study, we extended our experiments to examine whether chronic intake of water containing a high level of zinc accelerates Aβ deposition and APP cleavage in APP/PS1 mouse brain. We found that a high level of dietary zinc could cause cognition dysfunction and enhance the aggregation of Aβ. Furthermore, we found that a high level of zinc also enhanced Aβ generation through altering the expression levels of APP and APP cleavage enzymes 4 (20 mg/ml). Morris water maze tests were performed to evaluate whether high dietary zinc treatment affects learning and memory in APP/PS1 mice at the age of 9 months. These included 2 days of visible platform training, 5 days of hidden platform tests, and a probe trial 1 day after the last hidden platform test , compared with the control (0.43±0.12 µg/ml) p<0.01; . We alsop<0.01; p<0.01; The autometallography (AMG) procedure allows demonstration of a striking condensation of ionic zinc within plaques in the human postmortem AD brain and the APP/PS1 transgenic mouse brain p<0.01; p<0.01; To determine whether chronic high intake of dietary zinc potentiates Aβ deposition, brain sections of APP/PS1 mice were subjected to Aβ immunohistochemical analysis. As shown in To further test whether a high dose of dietary zinc affects APP expression, the levels of APP mRNA and APP695 protein were measured by RT-PCR and Western blotting, respectively. As can be seen in p<0.05) and Aβ1-42 (p<0.01) in the brain, compared with controls. The levels of Aβ1-40 and Aβ1-42 were increased by 122.19±20.60%  and cleavage fragments of APP  in the brain samples of APP/PS1 mice were subjected to Western blot analyses. As shown in p<0.01; p<0.01; p<0.01; We then examined the levels of α-secretase-generated sAPPα/C83 and β-secretase-generated sAPPβ/C99 fragments in the transgenic mouse brain. Zinc treatment significantly reduced the level of sAPPα by 53.55±3.32% p<0.01; , and incTaken together, these results indicate that the α-secretase cleavage activity is markedly reduced while β- and γ-secretase are increased in the brain of zinc-treated transgenic mice.in vitro model p<0.01; p<0.01; To further verify that high zinc exposure might be involved in APP processing and Aβ secretion, a human neuroblastoma SHSY-5Y cell line stably transfected with APPsw was used as an in vivo data from APP/PS1 transgenic mice, high zinc (70 µM) exposure significantly increased the APP cleavage enzyme levels of BACE1 by 135.50±17.05% and PS1 by 132.79±13.11% (p<0.01), and reduced the levels of ADAM 10 by 80.11±5.04% (p<0.05), respectively, in APPsw cells (p<0.01), while the levels of α-secretase-generated sAPPα and C83 were decreased by 79.89±4.63% (p<0.01) and 76.94±5.61% (p<0.05), respectively, following 70 µM zinc treatment  treatment guided APP processing to the non-amyloidogenic pathway in our APPsw overexpressing cells. Following 1 µM zinc treatment, the levels of AMAM10, sAPPα and C83 were increased by 142.21±12.04% p<0.05; , 116.71±Both APP and its proteolytic byproduct Aβ, which play central roles in senile plaque formation in the pathogenesis of AD, are zinc-containing metalloproteins that contain zinc-binding domains We and others have reported that zinc is highly concentrated in amyloid plaques in human postmortem brain samples Zinc is toxic and, besides its physiological roles, it is involved in neuronal and glial death through activation of multiple intracellular pathways leading to necrotic, apoptotic and autophagic neuronal death in vitro studies that tested the effects of zinc on APP processing in vivo data, high zinc (70 µM) exposure suppresses α-secretase cleavage, but enhances β- and γ-secretase cleavage of APP and Aβ generation in APPsw overexpressing cells. Thus, our in vivo and in vitro studies clearly show that high-dose zinc treatment enhances the amyloidogenic APP cleavage pathway and Aβ secretion. Interestingly, a recent study involving APP/PS1 mice fed a zinc-deficient diet has shown that such a diet increases the plaque volume but does not alter the total plaque number in the brain APP protein contains a novel zinc binding motif which is located between the cysteine-rich and negatively charged ectodomains In summary, the present study provides evidence that chronic exposure to high zinc levels in drinking water leads to an increase in APP expression, amyloidogenic APP cleavage and Aβ deposition in the APP/PS1 transgenic mouse brain. The present data, together with previous reports, suggest that excess zinc exposure could be a risk factor for AD pathological processes, and corrections of metal abnormalities in the brain are beneficial strategies for AD prevention and therapy.The experimental procedures were carried out in accordance with the regulations of the animal protection laws of China and approved by the animal ethics committee of China Medical University (JYT-20060948). All efforts were made to minimize animal suffering and the number of animals used.ad libitum. (2) Zinc group: mice were given a standard diet and deionized water containing ZnSO4 (20 mg/ml). The doses of zinc chosen for this study were based on previous reports showing that it produced no serious toxicity in C57BL/6 mice after chronic treatment Male APP/PS1 double transgenic mice  85Dbo/J mice) were used in the present study . They were kept in cages in a controlled environment . Mice at the age of 3 months were randomly assigned to one of two groups (n = 12 in each group). (1) Control group: mice were given a standard diet (30 ppm zinc) and deionized water Morris water maze tests were carried out as previously described with few modifications The day after the Morris water maze tests, mice were anaesthetized with sodium pentobarbital  and sacrificed by decapitation. The brains were removed immediately and split sagittally into halves. The left hemisphere was kept at −80°C for Western blotting, RT-PCR or ELISA analyses. The right hemisphere was further cut into two slabs. One was immersed in 3% glutaraldehyde for zinc autometallographic analysis while the other was placed in 4% paraformaldehyde for immunohistochemical staining.AMG was performed to analyze the distribution of zinc in the transgenic mouse brain according to our previous reports The stereological assessment of zinc load in the AMG-stained brain sections was performed as reported previously 2O2) in PB for 10 min to reduce endogenous peroxidase activity. After rinsing, the sections were treated with 5% bovine serum albumin and 3% goat serum in TBS for 1 h. They were then incubated with mouse anti-Aβ  overnight at 4°C. After rinsing in TBS, the sections were incubated with 1∶200 diluted biotinylated goat anti-mouse IgG for 1 h at room temperature (RT). The sections were then rinsed and treated with an ABC kit for 1 h at RT. The sections were rinsed in TBS and incubated with 0.025% 3, 3-diaminobenzidine (DAB) plus 0.0033% H2O2 in TBS for 10 min. After rinsing, the sections were dehydrated, covered with neutral balsam, and examined with a light microscope. Control sections were incubated with normal serum instead of Aβ antibody followed by all subsequent incubations as described above. The stereological assessment of Aβ load in the Aβ-immunostained brain sections was performed according to the above described procedure.Routine free floating ABC procedures were applied. Cryostat sections (30 µm) were treated with 3% hydrogen peroxide , TPEN (1 µM) or ZnSO4 (70 µM) plus TPEN (1 µM) for 8 h. The concentrations of zinc and TPEN were selected based on routine MTT assay. To analyze the zinc entry into the cells, the staining with a zinc-specific fluorescent dye, Zinquin, was carried out by incubating the cells with 0.24 µM Zinquin ethyl ester  for 30 min, and then examining them under a fluorescent microscope equipped with an image analysis system.Human neuroblastoma SHSY-5Y cells stably transfected with human APPsw The preparation of lysates from the transgenic mouse brain samples and culture cells, and the Western blots were performed as described previously 5′-GACTGACCACTCGACCAGGTTCTG-3′ (upstream), 5′-CTTGAAGTTGGATTCTCATACCG-3′ (downstream); GAPDH: 5′-ACGGATTTGGTCGTATTGGG-3′ (upstream), 5′-CGCTCCTGGAAGATGGTGAT-3′ (downstream). Amplification was performed as follows: APP: 35 cycles of 95°C for 30 s, 62°C for 30 s and 72°C for 30 s; GAPDH: 30 cycles of 95°C for 45 s, 58°C for 45 s, and 72°C for 60 s. The PCR products were normalized in relation to standards of GAPDH mRNA. The results were determined and quantified with ChemDoc XRS Quantity One software.Total RNA was isolated using Trizol reagent (Invitrogen) after homogenizing the brain tissue samples. The isolated RNA was examined by UV-spectroscopy at 260 nm. Total RNA (2 µg) of each sample was first transcribed to cDNA using a Reverse Transcription System kit . PCR amplification was performed with reagents from Promega. The cDNA solution was amplified with primers based on the human APP sequences. The primer sequences were: APP: Aβ levels were determined using ELISA assay as described previously t test, and one-way ANOVA Post hoc Fisher's PLSD for cultured cells. All data were analyzed using SPSS 13.0 software, and statistical significance was assumed if p<0.05.Data are expressed as means ± SEM. For water maze analysis of latencies and path length, repeated measures analysis of variance (ANOVA) were performed, and differences among means were evaluated with multivariable ANOVA. Other comparisons were analyzed by Student's


Repeated visual processing of an unfamiliar face suppresses neural activity in face-specific areas of the occipito-temporal cortex. This "repetition suppression" (RS) is a primitive mechanism involved in learning of unfamiliar faces, which can be detected through amplitude reduction of the N170 event-related potential (ERP). The dorsolateral prefrontal cortex (DLPFC) exerts top-down influence on early visual processing. However, its contribution to N170 RS and learning of unfamiliar faces remains unclear. Transcranial direct current stimulation (tDCS) transiently increases or decreases cortical excitability, as a function of polarity. We hypothesized that DLPFC excitability modulation by tDCS would cause polarity-dependent modulations of N170 RS during encoding of unfamiliar faces. tDCS-induced N170 RS enhancement would improve long-term recognition reaction time (RT) and/or accuracy rates, whereas N170 RS impairment would compromise recognition ability. Participants underwent three tDCS conditions in random order at ∼72 hour intervals: right anodal/left cathodal, right cathodal/left anodal and sham. Immediately following tDCS conditions, an EEG was recorded during encoding of unfamiliar faces for assessment of P100 and N170 visual ERPs. The P3a component was analyzed to detect prefrontal function modulation. Recognition tasks were administered ∼72 hours following encoding. Results indicate the right anodal/left cathodal condition facilitated N170 RS and induced larger P3a amplitudes, leading to faster recognition RT. Conversely, the right cathodal/left anodal condition caused N170 amplitude and RTs to increase, and a delay in P3a latency. These data demonstrate that DLPFC excitability modulation can influence early visual encoding of unfamiliar faces, highlighting the importance of DLPFC in basic learning mechanisms. Repetition of a visually presented stimulus leads to suppression of neural activity in the cortical areas responsible for processing the stimulus RS is a fundamental sensory process, but is also embedded in declarative memory and higher associative forms of learning. For instance, stronger RS in the temporal cortex at retrieval, as detected by blood-oxygen level dependent (BOLD) signal reduction, is associated with higher perceived familiarity for previously encoded visual stimuli, which may provide a basis for declarative memory formation These neuropsychological signs suggest that the capacity to learn new faces is highly dependent on the presence of RS in face-specific cortical areas. Investigations into the RS properties of the N170 event-related potential (ERP) component in normal subjects provide further insight into the reason why. The N170 component is a robust finding in ERP research and is described as a negative deflection peaking over occipito-temporal sites around 170ms post-stimulus, maximal for face stimuli and thought to reflect early structural detection and perceptual processing of faces The dorsolateral prefrontal cortex (DLPFC) is a source of top-down control that has been shown to influence the course of bottom-up visual processing through increases in extrastriate neural activity enhancing attention to elements of the visual field Although many neural models for RS have been put forth Transcranial direct current stimulation (tDCS) is a non-invasive form of electrical brain stimulation which allows transient and superficial cortical excitability modulation through application of a weak electrical current via electrodes placed on the surface of the scalp. Cortical excitability modulation is polarity-dependent: anodal stimulation increases excitability of the underlying cortex whereas cathodal stimulation decreases it Based on the body of evidence reviewed, we hypothesized that application of tDCS over the DLPFC would modulate N170 RS over occipito-temporal sites in response to repeated presentation of unfamiliar faces. Differential effects would arise in a polarity-dependent fashion whereby anodal tDCS would have an effect opposite to that induced by cathodal stimulation. Similarly, the P3a component would also show polarity-dependent changes in RS and amplitude, reflecting top-down influences including attention. As tDCS-induced cortical excitability modulation is transient Comité d’éthique de la recherche de la Faculté des arts et des sciences (CÉRFAS) of the University of Montreal and was conform to the Declaration of Helsinki. All participants gave written informed consent prior to testing. Subjects in The project was reviewed and approved by the Participants were 14 healthy young adults , all undergraduate and graduate students of the University of Montreal. All participants had normal or corrected-to-normal vision and reported no history of neurological or psychiatric disorders. Behavioural and ERP data of two participants were rejected because of concerns they did not perform the encoding task correctly (recognition accuracy ≤50%). An additional participant’s EEG data were rejected because of excessive artifacts. Behavioural data of this participant were also removed from analyses, to maximize comparability of results between behavioural and EEG analyses. Behavioural and EEG analyses therefore included 11 participants. These remaining participants were right-handed.http://www.mapageweb.umontreal.ca/gosselif/alignTools/) and a procedure similar to that described by Taschereau-Dumouchel and colleagues 2), contrast and spatial frequency were equated across stimuli using functions from the SHINE toolbox for Matlab , conceived by Willenbockel et al. Stimuli were 270 different unfamiliar male (177) and female (93) grayscale Caucasian faces. Images were selected from the FERET database on the criteria of their neutral expressions and absence of glasses, earrings or beards, to avoid any facilitation in learning or recognition based on these features. As this study aimed at characterizing the contribution of top-down processes in basic learning, it was essential to minimize any possible confounds arising from bottom-up processes by controlling for low-level image properties such as size, spatial location, luminance, contrast and spatial frequency. Firstly, to equate size and spatial location across stimuli, we aligned the internal attributes of faces using Matlab functions  anodal/left (F3) cathodal, right cathodal/left anodal and sham. For each active tDCS condition, the battery-driven stimulator was programmed to deliver a 1.5mA direct current for 15 minutes, with 10 seconds of linear fade in and fade out. These current intensity and stimulation duration parameters were chosen for their reported ability to limit undesirable side-effects while effectively inducing cortical excitability modulation tDCS was applied using a Magstim DC stimulator via two rubber electrodes  placed inside 35 cmFor each tDCS condition, an encoding task was administered, followed by a matching recognition task see . Each enEncoding task . The EEG cap was installed first with placement of tDCS electrodes underneath, ensuring precision and consistency of installation across conditions and participants. However, to avoid any electrical contamination of EEG signals, tDCS was administered prior to the start of recording such that the two systems were not simultaneously active. During tDCS, participants were instructed to close their eyes and relax while remaining awake. Both EEG recording and the encoding task started immediately following removal of tDCS electrodes and verification of impedances (∼5 minute delay). Each encoding task comprised 3 separate blocks. In each block, 15 different face identities were presented consecutively 15 times each. Each encoding task therefore contained 45 different face identities presented 15 times each for a total of 675 (45×15) trials per encoding task. In later analyses, blocks were added as a factor to assess the expected decay of tDCS effects over time. Each encoding task block lasted 10 min 13 sec. Participants were not instructed to use any particular encoding strategy but were asked to attentively observe the faces on which they would subsequently be tested. Stimuli were presented for 2000ms with a 600ms intertrial interval showing a central fixation cross.Recognition task . The 45 encoded faces were presented amongst 45 novel distractor faces in 90 randomly ordered trials. In each trial, a face appeared for 600ms followed by a fixation cross at which point participants had to perform a forced choice old/new discrimination decision using the left mouse button for known (i.e.: previously encoded) faces and the right button for unknowns. Participants were instructed to respond as fast as possible without making mistakes. Reaction time (RT) and accuracy were recorded at every trial.To assess the long-term repercussions of DLPFC activity modulation during encoding and avert any cross-over effects from one tDCS stimulation condition to the next, tasks and conditions were administered at three-day (∼72 hours) intervals. This led to the four-session experimental design depicted in During encoding tasks, continuous EEG was recorded from 32 Ag/AgCl sintered electrodes (10/20 system) mounted in a Quik-cap (Compumedics). Data were acquired at 500 Hz sampling rate and high-pass filtered at 0.1 Hz with NeuroScan 4.5. Linked mastoids were used as reference and impedances were kept below 5 kΩ. Vertical and horizontal eye movements were monitored by EOG with four additional bipolar electrodes positioned on the outer canthus of each eye as well as above and under the orbit of the left eye.Offline signal processing and averaging was performed  separately for each condition, participant and channel. EEG raw data were first segmented into 675 trials lasting 2200ms each (200ms baseline) based on stimulus onset marker positions. A high-pass digital filter was applied at 0.5 Hz with an additional low-pass filter set to 50 Hz (24dB/octave) and 60 Hz notch filter. Eye movement artifacts were corrected by algorithm All analyses were conducted using SPSS Statistics 20 software. Correct answers in the recognition tasks were defined as correct identification of known and novel faces. Accuracy was then calculated as percentages of correct answers for every participant and condition. RT and accuracy data were first entered in two-way repeated-measures ANOVAs with tDCS condition (3) and stimulus familiarity (previously encoded vs. novel distractors) as within-subjects factors. To assess the specificity of tDCS effects, RT and accuracy data were subsequently divided into those of faces encoded while under the influence of tDCS and those of novel faces (i.e.: first presented ∼72 hours following active tDCS). Encoded faces RTs and accuracy were entered in two-way repeated-measures ANOVAs with tDCS condition (3) and block (3) as within-subjects factors. Novel faces RTs and accuracy were entered in one-way repeated-measures ANOVAs with tDCS condition (3) as within-subjects factor (no block factor can be assigned to novel faces).ERP analyses were conducted for latency and amplitude values of the P100, N170 and P3a components. Peaks were individually detected for all trial averages  in a ±30ms time window around the grand-average means at Oz, O1 and O2 sites for P100, TP8 and TP7 for N170 and along the midline  for P3a. A four-way repeated-measures ANOVA was conducted on P100 data, using electrode (3), tDCS condition (3), block (3) and trial average (3) as within-subjects factors. For the N170 component, a four-way repeated-measures ANOVA was carried out with hemisphere (2), stimulation condition (3), block (3) and trial average (3) as within-subjects factors. The P3a component was analyzed using a four-way, repeated-measures ANOVA with electrode (5), stimulation condition (3), block (3) and trial average (3) as within-subjects factors. When obtained, main effects were further analyzed at specific factor levels. Interaction effects were analyzed with interaction-graph guided ANOVAs at specific levels of the independent variables. All statistical analyses involved Greenhouse-Geisser adjusted degrees of freedom when necessary and Bonferroni corrections for multiple comparisons.PSQI. Descriptive statistics indicate a mean global score of 3.86±0.29 (range: 2–5) which is within recommended "good" sleeping quality values (≤5) tDCS questionnaire. Participants reported some adverse side-effects due to tDCS. During stimulation, 93% of participants reported an "itching" sensation under the electrodes and 78.5% reported "burning". Such side-effects were anticipated as they are relatively common in tDCS studies Recognition task accuracy. Recognition task difficulty was set to yield accuracies lower than ceiling to allow for improvement and decrement of performance. The range of accuracy rates was 51-80% (mean ± SD: 61%±7%), all conditions, participants and types of stimuli (encoded vs. novel) included. No effect of tDCS condition  = 1.13, P>0.1, ηp2 = 0.1), stimulus familiarity  = 2.23, P>0.1, ηp2 = 0.18) or interaction  = 0.76, P>0.1, ηp2 = 0.07) were found on recognition accuracy.Recognition task RTs. A marginally significant main effect of tDCS condition was found on overall RTs at recognition  = 3.2, P = 0.06, ηp2 = 0.24), but not of stimulus familiarity  = 1.5, P>0.1, ηp2 = 0.13) or interaction  = 0.64, P>0.1, ηp2 = 0.06). For encoded stimuli, a main effect of tDCS condition was found  = 4.2, P<0.05, ηp2 = 0.29), but not of block  = 1.54, P>0.1, ηp2 = 0.13) or interaction  = 0.6, P>0.1, ηp2 = 0.06). Indeed, Bonferroni-adjusted pairwise comparisons indicate RTs following the right anodal/left cathodal condition were faster than both the right cathodal/left anodal (P<0.05) and sham conditions (P<0.01). For novel faces of the recognition task, no tDCS condition effect was found on RTs  = 2.2, P>0.1, ηp2 = 0.18), indicating that the effect of tDCS condition on RTs is confined to stimuli presented following active tDCS (ive tDCS . Single P100. All interactions were non-significant (P>0.1) and no main effect of electrode  = 1.63, P>0.1, ηp2 = 0.14), stimulation condition  = 2.3, P>0.1, ηp2 = 0.2), block  = 0.4, P>0.1, ηp2 = 0.04) or trial average  = 0.3, P>0.1, ηp2 = 0.01) was found for amplitude values of the P100 at posterior sites. For latency values, no electrode  = 1.1, P>0.1, ηp2 = 0.1), tDCS condition  = 0.23, P>0.1, ηp2 = 0.02), block  = 2.15, P>0.1, ηp2 = 0.2), trial average  = 0.46, P>0.1, ηp2 = 0.02) effects or interactions (P>0.1) were significant.N170. For amplitude values, an interaction effect of hemisphere x tDCS condition x block x trial average was found  = 2.12, P<0.05, ηp2 = 0.18). When hemispheres were analyzed separately, no main or interaction effects were found at left hemisphere electrode TP7 (P>0.1) however, right occipito-temporal site TP8  = 2.62, P = 0.01, ηp2 = 0.21). In turn, analyzing encoding blocks separately at TP8, revealed a significant tDCS condition x trial average effect  = 4.73, P<0.01, ηp2 = 0.32) specific to the first encoding block  = 11.2, P = 0.01, ηp2 = 0.53) was found to be circumscribed to the first five trials (Av1) within the first encoding block at TP8. Indeed, pairwise comparisons indicate that N170 amplitude is significantly suppressed for the right anodal/left cathodal condition relative to the right cathodal/left anodal condition . All other blocks and trial averages showed non-significant tDCS condition effects (P>0.1).site TP8  showed ang block . A signiF = 4.73, P<0.01, ηp2 = 0.32). As expected, a trial average effect was revealed  = 3.46, P<0.05, ηp2 = 0.26) in the sham condition where N170 amplitude reduced from Av1 to Av2 . Trial average effects were non-significant in the right anodal/left cathodal  = 2.62, P = 0.1, ηp2 = 0.21) and right cathodal/left anodal  = 2.37, P>0.1, ηp2 = 0.19) conditions. Secondly, to confirm that the tDCS effect suppresses activity rather than creating a non specific change in brain activity, the amplitude values of the very first presentations of each face of the first encoding block would have to be equal across conditions. This was in fact the case as no significant difference was found in a supplementary one-way repeated-measures ANOVA with tDCS condition as within-subjects factor and grand-averaged N170 amplitude values of the first stimulus presentation as outcome variable  = 0.32, P>0.1, ηp2 = 0.03). It follows from this analysis, that the tDCS condition effect at Av1 of the first block would be caused by a difference in the last four repetitions of Av1. Indeed, the tDCS condition effect remains when grand-averaged amplitude values of only the last four repetitions of Av1 are included  = 7.49, P<0.01, ηp2 = 0.43). Together, these results demonstrate that the tDCS condition effect on N170 amplitude reflects differential RS between the active tDCS conditions during the first five repetitions of stimuli. Furthermore, the fact that the tDCS condition effect was specific to the right hemisphere where face stimuli are preferentially processed Of special interest here, the tDCS condition effect on N170 amplitude at Av1 of the first encoding block suggests that differential right occipito-temporal RS took place between active stimulation conditions within the first five stimulus repetitions of the first block, when active tDCS modulatory influence on DLPFC excitability was presumably strongest. This supports the hypothesis that tDCS over the DLPFC would modulate N170 RS over occipito-temporal sites in response to repeated presentation of unfamiliar faces. However, as this is only inferred from observing an overall difference between resulting averages (i.e.: Av1), additional analyses were conducted to ensure the tDCS condition effect can be interpreted in the context of RS. First, to ascertain that the encoding task effectively induced RS in the absence of DLPFC activity modulation, the amplitude values of N170 should decrease as a function of trial average within the first block of the sham condition. In an analysis with tDCS condition (3) and trial average (3) as within-subjects factors, a significant interaction was found (P>0.1). No main effects of hemisphere  = 1.9, P>0.1, ηp2 = 0.16), stimulation condition  = 2.67, P = 0.1, ηp2 = 0.21), block  = 1.9, P>0.1, ηp2 = 0.16) or trial average  = 1.1, P>0.1, ηp2 = 0.1) were found.For latency values of the N170 component, all interactions were non-significant , main effects of stimulation condition  = 3.85, P<0.05, ηp2 = 0.29) and trial average  = 4.87, P<0.05, ηp2 = 0.52) were found on amplitude values of the P3a component. The tDCS condition effect was caused by overall higher P3a amplitude in the right anodal/left cathodal condition relative to the inversed polarity and sham conditions , indicating suppression of activity throughout repetitions (1–15) of single stimuli  = 4.33, P<0.05, ηp2 = 0.3), FCz  = 4.41, P<0.05, ηp2 = 0.31) and CPz  = 4.38, P<0.05, ηp2 = 0.49). These stimulation condition effects were driven by the second block at these sites  = 6.99, P<0.01, ηp2 = 0.41; FCz: F = 7.66, P<0.01, ηp2 = 0.43; CPz: F = 5.83, P = 0.01, ηp2 = 0.37). Indeed, the first and third blocks showed no tDCS condition effect (P>0.05). Trial average or RS effects also remained significant at Fz  = 5.58, P<0.05, ηp2 = 0.55), FCz  = 6.88, P<0.05, ηp2 = 0.6) and CPz  = 4.81, P<0.05, ηp2 = 0.52). P<0.05, . Also, A stimuli . When siF = 6.33, P<0.05, ηp2 = 0.58). This trial average effect was significant at both F3  = 4.54, P<0.05, ηp2 = 0.5) and F4  = 5.96, P<0.05, ηp2 = 0.57), where an additional interaction of tDCS condition x trial average was found  = 1.91, P<0.05, ηp2 = 0.52). This interaction further revealed that a significant RS effect was present only in the right anodal/left cathodal condition at this site  = 4.1, P<0.05, ηp2 = 0.3) between Av1 and Av2, as well as Av1 and Av3 , tDCS condition (3), block (3) and trial average (3) as within-subjects factors. In this analysis, a significant main effect of trial average was found  = 3.76, P<0.05, ηp2 = 0.27), specifically between the right cathodal/left anodal and sham conditions at the second block . This suggests that neural activity to repeated face identity processing in right occipito-temporal cortex was modulated through prefrontal activity disruption and enhancement. tDCS did modulate prefrontal activity, as concurrent effects on both amplitude and latency values of the P3a component reflected variations in prefrontal function. Notably, P3a RS interacted with tDCS condition over the right hemisphere, further supporting the DLPFC’s influence on RS. DLPFC stimulation also influenced behaviour, as RTs in subsequent recognition tasks followed a polarity-dependent pattern in line with ERP results. These data support the hypothesis that top-down connections may causally influence early visual encoding and extend it by showing a specific link between DLPFC activity modulation by tDCS and right-hemisphere N170 RS, having long-term repercussions on recognition.Our main behavioural finding was that participants made faster decisions about the familiarity of face stimuli encoded under the right anodal/left cathodal condition than under the right cathodal/left anodal. This effect held for all three encoding blocs and only for encoded faces, which enforces the notion that this variation is indeed tDCS-induced. There is strong evidence from studies on priming that leads us to expect facilitation of processing such as faster RT for previously encoded stimuli In the absence of tDCS influence, the N170 showed RS, as Av2 trial amplitude was significantly reduced relative to Av1. Moreover, although amplitude was equal across conditions for the first presentation of individual face identities, N170 average amplitude of the first five repetitions (Av1) varied as a function of tDCS polarity, indicating that different activity patterns took place within these first repetitions. Indeed, although our analyses do not provide a trial-by-trial resolution, it seems the right anodal/left cathodal condition induced a progressive suppression of activity in response to repeated face stimuli, whereas the right cathodal/left anodal condition increased activity, as reflected by its larger amplitude. Additionally, effects on N170 RS were significant at the first encoding block and fell thereafter, which suggests tDCS was responsible for early face identity processing effects over occipito-temporal cortex: as DLPFC activity modulation due to electrical current presumably faded, so did N170 RS modulation. This is comparable to results obtained by Zanto and colleagues Heightened P3a amplitude, found in the right anodal/left cathodal condition, can reflect high availability and focus of attentional resources, which has generally been linked to enhanced encoding and subsequent retrieval ability What remains uncertain is the exact mechanism through which tDCS has induced effects in this study. Improvement of many aspects of learning have been demonstrated using anodal stimulation Although N170 peak latencies of up to 240ms have been confidently reported e.g.: , the oneIn conclusion, results presented here demonstrate that tDCS of the DLPFC can influence early visual encoding of individual face identities indexed by N170 RS. The fact that reversal of tDCS polarity over the DLPFC yielded differences in prefrontal function, N170 repetition effects and long-term recognition RT, suggests that visual encoding efficacy has the potential to be either enhanced or impaired through DLPFC stimulation.