Isolectin B4 (IB4)-conjugated streptavidin for the selective knockdown of proteins in IB4-positive (+) nociceptors

Animals

All experiments were performed on 260–320 g adult male Sprague–Dawley rats (Charles River Laboratories, Hollister, CA, USA). Animals were housed in a controlled environment (21–23°C, 12-h day-night cycle, food and water were available ad libitum) in the animal care facility at the University of California, San Francisco. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California at San Francisco and in compliance with the National Institutes of Health Guide for the care and use of laboratory animals. Every effort was made to minimize the number of animals used in experiments and their suffering.

Randall-Selitto paw-pressure test

Mechanical nociceptive threshold was quantified using an Ugo Basile Analgesymeter^® (Stoelting, Chicago, IL, USA), a device that uses a cone-shaped pusher with a rounded tip, to apply a linearly increasing mechanical force to the dorsum of a rat’s hind paw, as previously described.^25,26 Rats were lightly restrained in a cylindrical acrylic tube that has lateral ports at one end, to allow easy access to the hind paw for mechanical nociceptive threshold testing. Rats were acclimatized to the testing procedure by placing them in the restrainers and training them with the Analgesymeter for ∼2 h per day for three consecutive days before measuring baseline mechanical nociceptive threshold. Nociceptive threshold was defined as the force in grams at which a rat withdrew its paw. Baseline mechanical nociceptive threshold was defined as the mean of the three readings taken before test agents were injected.

Drugs and their administration

The selective PKCε agonist, psi ε receptor for activated C kinase (ψεRACK) ²⁷ was purchased from Biomatik (Wilmington, DE, USA), and the proinflammatory cytokine prostaglandin-E₂ (PGE₂) from Sigma-Aldrich (St Louis, MO, USA). The selection of drug doses was based on dose-response relationships determined during earlier studies.^15,28 Stock solutions of PGE₂ (1 µg/µl) in 100% ethanol (EtOH) were diluted (1:50) with physiological saline (0.9% NaCl) immediately before intradermal injection. The EtOH concentration of the final PGE₂ solution was 2% (w/v) and the injection volume 5 µl. ψεRACK was dissolved and diluted in physiological saline (c_final = 0.4 µg/µl). ψεRACK (1 µg) was administered by hypotonic shock as previously described. ¹² All drugs were administered via a 30-gauge beveled hypodermic needle connected to a Hamilton micro syringe with polyethylene tubing (PE-10).

Covalent conjugation of streptavidin to IB4

The streptavidin conjugation kit (cat. #: ab 102921) was purchased from Abcam (Waltham, MA, USA) and IB4 (cat. #: L-1104-1) was purchased from Vector Laboratories (Burlingame, CA, USA). All reagents were equilibrated to room temperature (RT) before use. 100 μL of IB4 (c = 2.15 mg/mL), dissolved in sterile phosphate buffered saline (PBS), was activated by the addition of 20 μL of modifier reagent of the streptavidin conjugation kit, and then pipetted onto 100 μg of streptavidin (stochiometric ratio between IB4 and streptavidin = 1:1). This solution was gently mixed and incubated at room temperature for 24 h in the dark. Free excess amine groups on IB4 and streptavidin were inactivated by adding 20 μL of quenching reagent of the streptavidin conjugation kit to the reaction mixture. IB4-streptavidin conjugate was stored at 4°C until use.

Intrathecal injection of antisense oligonucleotides

The antisense oligodeoxynucleotide (ODN) for PKCε, Biotin-GCO AGC TOG ATO TTG OGC OC (O = C-phosphorthioate) directed against a unique region of the rat mRNA was synthesized by Invitrogen (Waltham, MA, USA). The corresponding GenBank accession number and ODN position within the mRNA sequence are NM_017171.2 and 420-439, respectively. We have previously proven that this antisense ODN for PKCε can be used to downregulate PKCε expression in rat DRG.^9,26 The corresponding sense ODN sequence for PKCε, Biotin-GGE CGC AFG ATO GAG OTG EC (E = G-phosphorthioate, F = A-phosphorthioate), was used as the control. ODNs were dissolved in ultrapure water to a final concentration of 100 pmol/µl, aliquoted and stored at −20°C, until use.

For each injection 2.3 μL IB4-streptavidin (30 pmol of conjugate with 120 pmol of biotin binding sites), 1.2 μL biotinylated ODN (c = 100 pmol/μl) and 16.5 μL PBS were mixed (injection volume = 20 μL), incubated at RT for 5 min and transferred into a 3/10 cc insulin syringe with a 29-gauge ultra-fine 1/2-inch fixed hypodermic needle (Walgreens, Deerfield, Il, USA). Rats were briefly anesthetized with 2.5% isoflurane (Phoenix Pharmaceuticals, St Joseph, MO, USA) to facilitate intrathecal injection, and the hypodermic needle inserted into the subarachnoid space on the midline, between the L4 and L5 vertebrae. ²⁹ The intrathecal site of injection was confirmed by the elicitation of a tail flick produced by the insertion of the needle into the intrathecal space and injection of the solution. ³⁰ Intrathecal injections were performed on day 1, 4 and 7 and DRG harvested on day 10, to be used for either Western blotting, quantitative real-time PCR (Polymerase Chain Reaction) or immunohistochemical analysis.

Tissue harvesting

Rats treated with antisense or sense ODN were euthanized by exsanguination while under deep isoflurane anesthesia. Then L4 and L5 DRG were surgically removed, snap frozen on dry ice and stored at −80°C until further processing.

Protein extraction

DRG were transferred into homogenization buffer [150 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA, 2% sodium dodecyl sulfate (SDS)] supplemented with complete protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA) and manually homogenized with a plastic pestle in an Eppendorf tube. Proteins were extracted by incubating the homogenate for 2 h at RT in an Eppendorf thermomixer at 1400 r/min and separated from insoluble components by centrifugation for 15 min at 14,000 r/min in a tabletop centrifuge (Eppendorf, Hamburg, Germany). Protein concentration of the samples was determined using the micro-BCA protein assay kit (Pierce, Rockford, Il, USA) with BSA as a standard.

Western blotting

Changes in PKCε expression were evaluated by Western blotting. Briefly, 20 μg of protein per sample were mixed with 4 x sample buffer (62.5 mM Tris-HCl pH 6.8, 3% SDS, 10% glycerol, 0.025% bromophenol blue), denatured by shaking for 10 min at 500 r/min and 90°C in an Eppendorf thermomixer, and electrophoresed on a 4%–15% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) in 25 mM Tris buffer containing 192 mM glycine and 0.1% SDS. Proteins were transferred onto a nitrocellulose membrane (NC) using the semidry blotting method (transfer time 1 h at 10 V with 47.9 mM Tris, 38.9 mM glycine, 0.038% SDS and 20% methanol). The blotting membrane was saturated by shaking in Tris buffered saline (TBS), pH 7.4, containing 5% BSA and 0.1% Tween 20 (antibody dilution buffer) for 1 h at RT. The NC membrane was cut in half at ∼64 kDa and the upper half of the membrane was probed at 4°C overnight with a 1:500 dilution of a rabbit anti-PKCε antibody (sc-214, Santa Cruz Biotechnology, Dallas, Texas, USA) in antibody dilution buffer, while the lower half was probed at 4°C overnight with a 1:1000 dilution of a rabbit anti-β actin antibody (ab8227, Abcam, Waltham, MA, USA), in antibody dilution buffer. Blots were rinsed with TBS containing 0.1% Tween 20 (3 times for 15 min under shaking at RT) and probed with an anti-rabbit HRP conjugated antibody (NA934V, GE healthcare, Piscataway, NJ, USA) for 2 h at RT. Blots were rinsed with TBS containing 0.1% Tween 20 (3 times for 15 min under shaking at RT) and PKCε and β-actin immunoreactivities were visualized with the enhanced chemiluminescence detection kit (Pierce) and analyzed by computer-assisted densitometry. ³¹

Immunohistochemistry

Rats received three intrathecal injections of either IB4-streptavidin complexed with biotinylated antisense or sense oligonucleotides against PKCε mRNA on day 1, 4 and 7. On day 10 rats were deeply anesthetized with isoflurane and perfused through the left ventricle with 100 mL cold PBS containing 10 U heparin/ml PBS followed by 300 mL methanol-buffered 10% formalin (Thermo Fisher Scientific, Waltham, MA, USA). L4 and L5 DRG were dissected out and transferred to PBS with 30% sucrose and stored overnight at 4°C. The DRGs were then embedded in Tissue Tek (OCT compound, Electron Microscopy Sciences, Hatfield, PA, USA), sectioned in a Leica cryostat at 10 µm and mounted on gelatinized microscope slides (Southern Biotech, Birmingham, AL, USA).

Tissue Tek was removed by rinsing the DRG tissue sections twice with Tris-buffered saline (TBS) containing 0.1% Triton X-100 (TBST). Tissue sections were incubated for 1 h at room temperature (RT) in TBST, with 10% normal goat serum (Jackson Immunoresearch, West Grove, PA, USA) (antibody dilution buffer). Sections were incubated overnight at 4°C with a 1:500 dilution of a Rabbit (Rb) anti-PKCε antibody (sc-214, Santa Cruz Biotechnology) in antibody dilution buffer, washed three times for 10 min each at RT with TBST and probed for 2 h at RT with a 1:500 dilution of an Alexa Fluor conjugated Goat anti-Rb antibody (Invitrogen cat. #: A11012) and a 1:1000 dilution of IB4-FITC (Invitrogen, cat. #: I21411) in antibody dilution buffer supplemented with 0.1 mM CaCl₂, 0.1 mM MnCl₂, 0.1 mM MgCl₂ for 2h at RT. ⁶ Sections were washed three times for 10 min at RT with TBST supplemented with 0.1 mM CaCl₂, 0.1 mM MnCl₂, 0.1 mM MgCl₂ and mounted with DAPI containing mounting media (Invitrogen, cat. #: P36935).

RNA extraction and quantitative real-time PCR

Total RNA from L4 and L5 DRG was extracted using Trizol (Invitrogen) and the “PureLink^TM RNA Mini Kit” (Invitrogen) according to the manufacturer’s instruction. The RNA concentration in each sample was determined using a spectrophotometer (Shimadzu, Santa Clara, CA, USA). RNA was transcribed into cDNA using the “iScript^TM Advanced cDNA Synthesis Kit for RT-qPCR” (Bio-Rad, Hercules, CA, USA). The real-time PCR was performed with the “SsoAdvanced^TM Universal SYBR Green Supermix” (Bio-Rad) and specific primers for PKCε (Bio-Rad, assay ID: qRnoCID0008431) on the CFX 96^TM real-time PCR detection system (Bio-Rad). The PCR program was run under the following conditions: 2 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 30 s at 60°C. Each sample was run in triplicate. Change in gene expression was determined by the 2^−ΔΔCT method and expressed as relative change compared to control level. ³² Glycerinaldehyde-3-phosphate dehydrogenase (GAPDH) RNA was used as reference gene (Bio-Rad, assay ID: qRnoCID0057018).

Statistical analysis

(a) Western blot and quantitative real-time PCR: Statistical comparisons were made with GraphPad Prism 10 statistical software (GraphPad Software In., La Jolla, CA, USA). Comparison between treatments were performed using Student’s t-test and a p-value less than 0.05 was considered statistically significant. (b) Immunohistochemistry: Statistical comparisons were made with GraphPad Prism 10 statistical software. Comparisons between treatments were performed using contingency testing and Fisher’s exact test. A p-value of less than 0.05 was considered statistically significant. (c) Behavior: Statistical comparisons were made with GraphPad Prism 10 statistical software. Comparisons between treatments were performed using two-way repeated measures ANOVA with Post hoc analysis. A p-value of less than 0.05 was considered statistically significant.

Data availability

The data generated in this study are available upon request from the corresponding author.

Transfection with IB4-streptavidin

To evaluate whether IB4-streptavidin can be used as a transfection reagent, rats were treated with a complex of IB4-streptavidin and biotinylated antisense ODN (or for the control biotinylated sense ODN) directed against PKCε mRNA. Two groups of three rats each received three intrathecal injections on days 1, 4 and 7 before their L4 and L5 DRG were harvested on day 10. Western blot analysis of protein extracts from DRG from the group that had been subjected to the antisense ODN treatment showed a significant decrease (∼40%) in the PKCε expression (9.5 ± 1.2 a.u., [N = 3]) compared to the group that was treated with the sense ODN sequence (15.9 +/− 1.3 a.u., [N = 3], Student’s t-test, p = .01, Figure 1(a) and (b)) indicating successful knockdown of PKCε.

Immunohistochemistry

To analyze whether the downregulation of PKCε expression is restricted to IB4-positive DRG neurons, an immunohistochemical analysis was performed. Two groups of three rats each received three intrathecal injections of either biotinylated PKCε sense oligonucleotides bound to IB4-streptavidin or biotinylated PKCε antisense oligonucleotides bound to IB4-streptavidin on days 1, 4, and 7. Three days later, rats were transcardially perfused with 4% PFA, their L4 and L5 DRG excised and analyzed for PKCε immunoreactivity (Figure 2). A contingency test was performed to compare the proportion of IB4(+)/PKCε(+) neurons between DRGs from rats treated with the IB4-streptavidin biotinylated-PKCε sense ODN complex and rats treated with the IB4-streptavidin biotinylated PKCε antisense ODN complex (IB4(+)/PKCε(+) in PKCε sense ODN-treated rats [31%, 191/600 neurons, 95% CI = (28-35)%], in PKCε antisense ODN-treated rats [19%, 117/600 neurons, 95% CI = (16-22)%]). Fisher’s exact test shows a significant difference between IB4(+)/PKCε(+) neurons in DRG of rats treated with IB4-streptavidin biotinylated PKCε sense ODN and those treated with IB4-streptavidin biotinylated PKCε antisense ODN [p < .0001, N = 600/group (3 rats per group with 200 neurons/DRG per rat; 95% CI were calculated using the Wilson/Brown method)]. A second contingency test was performed to compare the proportion of IB4(−)/PKCε(+) neurons between DRGs from rats treated with the biotinylated PKCε sense ODN IB4-streptavidin complex and rats that were treated with the biotinylated PKCε antisense ODN IB4-streptavidin complex (IB4(−)/PKCε(+) in PKCε sense ODN-treated rats [10%, 65/600 neurons, 95% CI = (8–13)%], in PKCε antisense ODN-treated rats [11%, 70/600 neurons, 95% CI = (9-14)%]). Fisher’s exact test shows no significant difference between IB4(−)/PKCε(+) neurons in DRG of rats treated with IB4-streptavidin bound to biotinylated PKCε sense ODN and those treated with IB4-streptavidin bound to biotinylated PKCε antisense ODN [p = .7, N = 600/group (3 rats per group with 200 neurons/DRG per rat; 95% CI were calculated using the Wilson/Brown method). These results demonstrate that the knockdown in PKCε expression is limited to IB4-positive (+) neurons, most of which are known to be non-peptidergic C-fiber nociceptors.^33,34

Quantitative real-time PCR

To determine whether the decrease in PKCε protein levels in rat DRG is due to antisense ODN-stimulated degradation of transcripts of the PKCε gene or to a reduction in the frequency with which the PKCε mRNAs are translated into the corresponding amino acid sequence we performed a real-time PCR. Rats received three intrathecal injections with either IB4-streptavidin complexed with biotinylated antisense oligonucleotides to PKCε, or IB4-streptavidin complexed with biotinylated sense oligonucleotides to PKCε, on days 1, 4 and 7 before their L4 and L5 DRG were harvested on day 10. Quantitative real-time PCR analysis of RNA extracts from DRG from rats subjected to the antisense treatment showed a significant decrease in the number of PKCε transcripts (24% ± 2.3%, [N = 6], p < .0001, Figure 3) compared to the group of rats treated with the sense sequence, suggesting that the reduction in cellular PKCε protein levels is caused by a reduction in the number of transcripts of the PKCε gene in rat DRGs.

Reversal of hyperalgesic priming in IB4(+)-nociceptors

Finally, to assess the potential therapeutic utility of our transfection method, we tested the efficacy of the knockdown on hyperalgesic priming, a well-characterized preclinical model of chronic pain ¹³ known to be dependent on PKCε activity in IB4(+)-nociceptors.^10,11 Rats were first primed with the selective PKCε activator ψεRACK, ¹⁵ then treated with the complex of IB4-streptavidin and biotinylated PKCε antisense ODN (or biotinylated PKCε sense ODN for the control), and finally tested for the presence of hyperalgesic priming. Rats treated with the IB4-streptavidin biotinylated PKCε sense ODN complex demonstrate prolonged PGE₂ hyperalgesia, a behavioral phenotype that characterizes the presence of hyperalgesic priming (Figure 4).^9,13 Rats treated with the IB4-streptavidin biotinylated PKCε antisense ODN complex only show the short lasting PGE₂ hyperalgesia, a behavioral phenotype that characterizes the wild-type nociceptor.^9,13 These results demonstrate that the knockdown of PKCε in IB4(+)-DRG neurons can reverse the neuroplastic changes associated with the primed IB4(+)-nociceptor.