abnormal

Novel model of secreted human tau protein reveals the impact of the abnormal N-glycosylation of tau on its aggregation propensity

Construction of pcDNA4 vector carrying htau fused to a signal peptide

The sequence encoding signal peptide originated by PCR from pCMV/myc/ER (V820-23, Thermo Fisher Scientific), using the following primers: F: 5′-GGCCGCGAATTCATGGGATGGAGCTGTATCATCCTC and R: 5′-CTGGCGGGGCTCAGCCATGGAGTGCGCGCCTGTGGA (the bold sequence represents restriction site of EcoR1; the single-underlined sequence represents part of the signal peptide; the double-underlined sequence contains part of the sequence of the longest htau isoform).

The htau-encoding sequence was amplified from the pNG2 plasmid (courtesy of Prof. Eckhard Mandelkow, DZNE, Bonn, Germany)40, using the following primers: F: product of the above mentioned PCR, and R: 5′-GCCTCGAGTCACAAACCCTGCTTGGCCAGG (the bold sequence represents Xho1 restriction site; the double-underlined sequence contains part of htau sequence. These two PCR reactions produced a sequence of signal peptide, followed by a wild type sequence of htau. Finally, the construct was cloned in to a pcDNA4 mammalian expression vector (Invitrogen) using the EcoR1 and Xho1 restriction sites. The sequence of the construct, termed hereafter SP-htau, was verified prior using.

Maintenance of cultured cells and stable transfection

SH-SY5Y (ATCC CRL-2266) cells were cultured in Dulbecco’s Modified Eagle’s Medium /Nutrient Mixture F-12 (DMEM-F12) (Biological Industries) supplemented with 10% fetal bovine serum (Biological Industries), 1% L-glutamine (Biological Industries), 1% Pen-Strep Nystain (Biological Industries), and 1% non-essential amino acids (Biological Industries). Cells were incubated under 5% CO2 at 37 °C. Cells were stably transfected with the pcDNA4 plasmid containing SP-htau, using Lipofectamine LTX (Invitrogen). Cells were subcloned and one colony was chosen for subsequent experiments.

For selection, Zeocin antibiotic (Tamar laboratories) at a concentration of 150 μg/mL was added from day three post transfection throughout the culture period.

Immunofluorescence staining

Monolayer culture cells grown on cover slips coated with Poly-D-Lysine (EMD Millipore) were fixed for 15 min in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) and washed three times for 5 min each in PBS. The cells were incubated with 0.25% Triton X-100 in PBS for 10 min at room temperature, followed by three times washing in PBS, 5 min each. The cells were then blocked for 30 min in blocking solution (1% BSA, diluted in PBS, 0.1% Tween-20 (PBS-T)) and incubated overnight at 4 °C with blocking solution which contained primary antibody recognizing total tau (5A6, Hybridoma bank, 1:200) and primary antibody recognizing Calnexin (sc-11397, Santa Cruz Biotechnology, 1:200). On the next day, the cells were washed three times in PBS, 5 min each, followed by 2 h of incubation with the secondary antibodies: goat Anti-Mouse IgG Cy3 (115-165-003, Jackson ImmunoResearch, 1:200) and goat Anti-Rabbit IgG Cy5 (sc-45101, Santa Cruz Biotechnology, 1:200). Finally, cells were washed three times for 5 min each in PBS and mounted using VECTASHILED medium containing DAPI (H-1200, Vector Laboratories). Images were taken with LSM510 confocal microscope (Zeiss).

We performed immunofluorescence staining with an additional ER marker against GRP78/BIP, using the following protocol: The cells grown on cover slips coated with Poly-D-Lysine (EMD Millipore) were fixed for 5 min with 100% Methanol at −20 °C and additional 5 min with Methanol:Acetone (1:1) at −20 °C, followed by two washes in PBS 5 min each and one wash with 2% PBS/BSA. The cells then were blocked for 30 min in blocking solution (normal goat IgG in 2% PBS/BSA 1:100), washed with 0.5 ml of 2% PBS/BSA and incubated with primary antibody recognizing total tau (5A6, Hybridoma bank, 1:200) together with primary antibody recognizing GRP78/BIP (G9043, Sigma-Aldrich, 1:200) for 2 h. Next, cells were washed three times with 2% PBS/BSA followed by 1 h incubation with the secondary antibodies: goat Anti-Mouse IgG Cy3 (ab97035, 1:100, abcam) and goat Anti-Rabbit IgG Cy5 (111-175-144, Jackson ImmunoResearch, 1:100). Finally, cells were washed three times for 5 min each in PBS and mounted using VECTASHILED medium containing DAPI (H-1200, Vector Laboratories). Images were taken with LSM510 confocal microscope (Zeiss).

Sample preparation for Western blot and aggregation analysis

Cells (3 × 106) from each cell line (SH-SY5Y and SP-htau expressing SH-SY5Y) were plated in growth medium and incubated in a 5% CO2 at 37 °C for 24 h. Then the medium was replaced with fresh same medium lacking fetal bovine serum and incubation was continued for 60 h under 5% CO2 at 37 °C. Next, the media from the various cell lines were collected separately and centrifuged at 700 rpm to remove dead cells. The supernatant was collected and concentrated from 10 mL to 100 µL using centrifugal filters (50 kDa, Amicon Ultra, Merck). The concentrated media were then used for Western blotting or diluted with PBS pH 7.4 (Dulbecco’s phosphate buffer saline) for biophysical studies.

PNGase-F treatment

The concentrated media, collected from SH-SY5Y and SP-htau expressing SH-SY5Y were treated with the glycosidase, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase-F, Sigma-Aldrich) to cleave off N-linked glycosidic linkages, according to manufacturer’s protocol with few modifications. Each concentrated medium (100 µL) was first treated with 3.4 µL Na2HPO4 (1 M), and 1.0 µL of NaH2PO4 (1 M). Then the samples were boiled for 10 min at 100 °C, allowed to cool down in ice bath and 3.5 units of PNGase-F were added. The samples were incubated for 3 h at 37 °C, followed by centrifugation using centrifugal filters (50 kDa, Amicon Ultra, Merck) in an attempt to remove the PNGase-F, excess salt and the detached glycans. Removal of glycans from the SP-htau protein was verified by Western blot analysis.

Dot blot analysis

Cells (2 × 106) from each cell line (SH-SY5Y and SP-htau expressing SH-SY5Y) were plated in growth medium and incubated under 5% CO2 at 37 °C for 24 h. Then the medium was replaced with fresh same medium lacking fetal bovine serum and incubation continued for 60 h in a 5% CO2 at 37 °C. Next the media from the various cell lines were collected separately and centrifuged at 5000 rpm to remove dead cells. The supernatant was collected and concentrated from 6 mL to 500 µL using centrifugal filters (50 kDa, Sartorius). The concentrated media were transferred to PVDF membrane (GE healthcare) using a Bio-Dot apparatus (Bio-Rad). The membrane was subsequently fixed in 4% paraformaldehyde (PFA), washed two times for 5 min each in Tris Buffered Saline (TBS), 0.1% Tween-20 (TBS-T), blocked for 1 h in blocking solution (5% milk, diluted in TBS-T) and incubated overnight at 4 °C with primary antibody (5A6, Hybridoma Bank, 1:1000) recognizing total tau, diluted in blocking solution. Next, the membrane was washed three times for 5 min each in TBS-T, incubated 1 h with goat anti mouse secondary antibody (sc-2060, Santa Cruz Biotechnology, 1:10,000) and washed again three times for 5 min each in TBS-T. The membrane was developed using Luminata Forte Western HRP Substrate (Millipore), according to the manufacturer’s instructions, and developed using Amersham Imager 600 (GE healthcare).

Western blot analysis for total cellular proteins

The same cells (SH-SY5Y and SP-htau expressing SH-SY5Y) which were used for Dot blot analysis, were harvested and lysed, immediately after the media was removed, in 1 mL extraction buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mM MgCl2, 0.5% NP-40) containing a protease inhibitor cocktail (Roche) and 100 mM phenylmethanesulfonyl fluoride (PMSF). Next, lysates were incubated on ice for 30 min then were centrifuged at 10,300 rpm for 30 min at 4 °C. The supernatants were collected and boiled at 100 °C for 10 min in sample buffer containing β-mercaptoethanol. Samples were resolved in 4–20% (w/v) polyacrylamide gels (GenScript) using a Mini-PROTEAN Tetra Vertical Electrophoresis Cell apparatus (Bio-Rad), and were transferred onto polyvinylidene difluoride (PVDF) membrane by a semi-dry blot technique using eBlot Protein Transfer Device (GenScript). Next, the membrane was blocked for 1 h in blocking solution (5% milk, diluted in Tris Buffered Saline (TBS), 0.1% Tween-20 (TBS-T)) and incubated overnight at 4 °C with the primary antibody recognizing actin (ab8224, abcam, 1:10,000) diluted in blocking solution. On the following day, the membrane was washed three times for 5 min each in TBS-T and incubated 1 h with goat anti mouse secondary antibody (sc-2060, Santa Cruz Biotechnology, 1:10,000). The membrane was developed using Luminata Crescendo Western HRP Substrate (Millipore), according to the manufacturer’s instructions, and developed using Amersham Imager 600 (GE healthcare).

Western blot analysis for secreted htau

The proteins in concentrated media (see PNGase-F treatment section) were resolved in 4–20% (w/v) polyacrylamide gels (GenScript) and were transferred onto PVDF membrane, as described in the previous section. The membrane was then blocked for 1 h in blocking solution (5% milk, diluted in Tris Buffered Saline (TBS), 0.1% Tween-20 (TBS-T)) and incubated overnight at 4 °C with the primary antibody recognizing total tau (5A6, Hybridoma Bank, 1:1000) diluted in blocking solution. Next day, the membrane was washed three times for 10 min each in TBS-T and incubated 1 h with goat anti mouse secondary antibody (sc-2060, Santa Cruz Biotechnology, 1:10,000). The membrane was developed using EZ-ECL (Biological Industries), according to the manufacturer’s instructions, and exposed to Fuji Medical X-Ray Film which were developed using Kodak X-OMAT.

Mass spectrometry

For identification, tau digest peptides were loaded onto a bespoke column (15 cm 75 mm, fused silica) packed with beads (Jupiter C-18, 300 mm, 5 mm; Phenomenex, Torrance, CA) and connected to an Ekspert nano LC system (Eksigent, Dublin, CA). Elution was performed with Buffer A (acetonitrile (2%) with formic acid (0.1%)) and Buffer B (acetonitrile (80%) with formic acid (0.1%)), with a linear gradient (5–65% Buffer B, 45 min). MS peptide analysis and tandem MS fragmentation were performed with an LTQ-Orbitrap spectrometer (Thermo Scientific), operated in the data-dependent mode to enable switching between MS and collision-induced dissociation tandem MS analyses of the top five ions. Collision-induced dissociation fragmentation was performed at 35% collision energy with a 30 ms activation time.

Tau was identified and validated by using the SEQUEST algorithm in Proteome Discoverer software (Thermo Scientific) and the Uniprot- Swissprot FASTA database. Mass tolerance for precursor and fragmentations were set to 10 ppm and 0.8 Da, respectively. Only high-confidence peptides with the best XCorr score as obtained by the standard Percolator node parameters were chosen.

Thioflavin T/S fluorescence assay

Stock solutions of Thioflavin T or Thioflavin S (ThT/ThS, 200 µM, Sigma-Aldrich) and heparin (100 µM, Sigma-Aldrich) were prepared in PBS (pH 7.4). For ThT/ThS experiments, the stock solution was diluted to 200 µL in each well of a 96-well black plate so that the final mixture contained 80 µL of the concentrated medium (untreated or PNGase-F treated) and 100 µL of ThT/ThS. Prior to the experiment, heparin was added (20 µL in each well) to initiate protein aggregation. Kinetic fluorescence data were collected for 5610 min at 37 °C in triplicate using Infinite M200 microplate fluorescence reader (Tecan, Switzerland), with measurements taken at 15 min intervals. Excitation and emission wavelengths were 440 nm and 490 nm, respectively. All of the experiments were repeated 3–4 times to ensure reproducibility.

Transmission electron microscopy (TEM)

Aliquots (10 μL) from the aggregated concentrated media were applied onto the dark side of 400-mesh copper grids covered with carbon-stabilized Formvar film (Electron Microscopy Sciences) and allowed to float for 2 min. Excess solution was removed using blotting paper. Then, 2% uranyl acetate solution (10 μL) was added to the grid and allowed to float for 2 min. Excess solution was removed using blotting paper. The grid was dried at room temperature and was kept in a desiccator before taking TEM analysis on JEOL (Model: JEM 1400) instrument at 80 kV.

Congo red birefringence

Aliquots (10 μL) from PNGase F-treated or untreated aggregated samples was placed over a glass slide followed by 10 μL of the saturated Congo red solution (in 80% aqueous ethanol). Excess solution was removed using a blotting paper. The sample was dried at room temperature and analyzed under a Nikon HD polarizable microscope under cross polarized light.

Immuno-Gold Assay

A 2 μL aliquot from the PNGase F-treated or untreated aggregated concentrated media was applied onto the dark side of 400-mesh copper grid covered with carbon-stabilized Formvar film (Electron Microscopy Sciences) and allowed to float for 2 min. Excess solution was removed using blotting paper and the grid was allowed to dry for 2 min28. Then, the grid was blocked with SuperBlock blocking buffer (Thermo Scientific) for 30 min. Samples were incubated with the primary antibody recognizing total tau (ab64193, abcam, 1:100) in blocking buffer for 30 min, washed five times with the same buffer solution, and then incubated with secondary goat anti-rabbit antibody conjugated with 18-nm gold (111-215-144, Jackson ImmunoResearch, 1:20) for 30 min and similarly washed. Samples were viewed using a JEM-1400Plus electron microscope operating at 80 kV.

ANS fluorescence assay

A 20 μL aliquot from the PNGase F-treated or untreated aggregated concentrated media was mixed with 100 μM ANS in PBS (Sigma-Aldrich). ANS fluorescence intensities were measured with excitation at 380 nm and emission between 400 and 750 nm using Infinite M200 microplate fluorescence reader (Tecan, Switzerland).

Life/Dead cell counting

Cells (1 × 106) from each cell line (SH-SY5Y and SP-htau expressing SH-SY5Y) were plated in growth medium and incubated in a 5% CO2 at 37 °C for 24 h. Then the medium was replaced with fresh same medium FBS and incubation was continued for 60 h under 5% CO2 at 37 °C. For the control experiment, the medium was replaced with fresh regular media with FBS and incubated in a similar manner. After 60 h, the media from the various cell lines were collected separately and mixed with the respective trypsinized cells followed by centrifugation at 700 rpm. Then the supernatant was discarded and 1 mL of regular media was added to each of the cell pellets and total number of cell vs percentage of live/dead cells were counted using Countess® II Automated Cell Counter from Thermo Fisher Scientific.

Fluorescent live/dead staining assay

Cells (1 × 105) from each cell line (SH-SY5Y and SP-htau expressing SH-SY5Y) were seeded in 24 well plate in growth medium and incubated in a 5% CO2 at 37 °C for 24 h. Then the medium was replaced with fresh same medium lacking FBS and incubation was continued for 60 h under 5% CO2 at 37 °C. For the control experiment, the medium was replaced with fresh regular media with FBS and incubated in a similar manner. After 60 h, we performed fluorescent live/dead staining assay (SigmaAldrich) containing fluorescein diacetate (6.6 μg/mL) and propidium iodide (5 μg/mL) to visualize the proportion of viable versus nonviable cells. The dye solutions were added directly to the cells containing no FBS. Whereas, for control experiment, the FBS containing media was replaced by fresh media containing no FBS followed by addition of dye solution. Then, the stained cells were immediately viewed under Nikon Eclipse Tifluorescent microscope with ZylascMOS camera using Nikon Intensilight C-HGFI fluorescent lamp.

Arrest of my parents 'abnormal' - Renzi

(see related story on M5S vote on Salvini's immunity).    (ANSA) - Rome, February 19 - Ex-premier and former Democratic Party (PD) leader Matteo Renzi on Tuesday described a decision by magistrates to put his parents under house arrest over alleged fraudulent bankruptcy and false invoices as "abnormal".    He added that, while he would not say his family was the victim of a plot, the news was "media masterpiece" as it had overshadowed the vote by 5-Star Movement members against M5S lawmakers backing Deputy Premier and Interior Minister Matteo Salvini's parliamentary immunity being lifted in the Diciotti case.    "Those who read the documents (for the arrest of Renzi's parents) and have a minimum of legal knowledge know that depriving someone of their personal liberty for something like this is abnormal," Renzi, a PD Senator, said on enews newsletter. "What's more, those who know how things really are know that the documents do not say the truth. But that will be for the trial. "Everyone is equal before the law. My parents, like everyone, have the right to a just trial and I hope a rapid one.    "I'm not shouting 'plot': I ask for trials to be conducted in courtrooms, not on the web or in newspaper editorial desk rooms.    "We await the sentences, but sentences are announced in court, not on the streets of populism. "There's no need to say that the case of my parents has totally overshadowed everything that happened yesterday in the world of politics. "All you have to do is read the newspapers to realise. A media masterpiece, hats off".   

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Movie Review – Abnormal Attraction (2018)

Abnormal Attraction, 2018.

Directed by Michael Leavy.Starring Bruce Davison, Nathan Reid, Nicole Balsam, Leslie Easterbrook, Malcolm McDowell, and Jason Leavy.

SYNOPSIS:

While humans and monsters work to put aside their differences, Nick (Nathan Reid) tries to keep his impending nuptials on track in this monster comedy.

In a world where monsters and humans live side by side, no one bats an eye at an abominable snowman (Michael Barra) selling snow cones from an ice cream truck, or a fish man in the water at a pool party, but that doesn’t mean everyone’s happy about it. There are plenty of people who’d like to see monsters out of the picture, and vice versa. Can the two species learn to give tolerance a chance?

One of Abnormal Attraction’s most attractive features is how liberal the film is with the term “monster”. The film’s biggest name is probably Frank Stein (Jim Hanks), Frankenstein’s Jewish monster, but there are other storybook characters, the muffin man who lives on Drury Lane (Nicholas Siggia), who you might not classically think of as monsters that are included as well. Then there are the monsters you don’t see often enough, like the Purple People Eater (Craig Loydgren), if his is one of the less successful make-ups.

Divided into three chapters, Nick (Nathan Reid) is having relationship problems with his fiancée and is determined to make it home for dinner on time but when his car breaks down, Nick gets kidnapped and brought to Camp Morning Wood, a camp for monsters. Run by Hildie (Leslie Easterbrook), a human-hating witch, and her associate, Boogeyman (Malcolm McDowell), Nick becomes one of her prisoners but what Hildie doesn’t know is Nick is a counselor and might just be the one person who can start a dialog between the species.

Abnormal Attraction is a comedy where, due to the high concentration of jokes, you expect a certain number won’t land but, while viewers mileage will vary on Finbar (Jason Leavy), a character you’re not entirely certain is human until he declares himself anti-monster, the movie falls impressively in the plus column. One of my favorite gags takes place in the title sequence. Medusa’s talking to a guy on a bench and the next time you see them the guy’s been turned into stone while Medusa’s casually folding her sunglasses away. All of the actors invest their characters with heart, and it’s the details that make the film (sleep masks for the eyes on Boogeyman’s hands). Even when you know a joke is coming, Pig (Gilbert Gottfried) not being swayed into buying bricks instead of straw), the punchline can be unexpected.

The one time the movie stumbles is with abusive relationships. This mostly comes up in the first chapter when Nick asks a colleague, Dr. Stanley Cole (Bruce Davison), to cover an AA meeting for him. The catch is that the AA meeting isn’t for Alcoholics Anonymous but Abnormal Attraction, but Dr. Cole doesn’t know that. Without being told what Abnormal Attraction means, it’s clear from the stories shared that many of the humans are forcing monsters to be with them but the name of the group implies interspecies relationships are the problem, which is inconsistent with the film’s message. While the section is well-acted, there’s something conceptually uncomfortable about it and, other than establishing how an AA meeting runs, the chapter could be dropped and not be missed.