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. 2024 Jan-Dec:20:17448069241230419.
doi: 10.1177/17448069241230419.

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

Oliver Bogen  1 , Dionéia Araldi  1 , Anatol Sucher  2 , Kord Kober  2 , Peter T Ohara  3 , Jon D Levine  1   4
Affiliations

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

Oliver Bogen et al. Mol Pain. 2024 Jan-Dec.

Abstract

In vivo analysis of protein function in nociceptor subpopulations using antisense oligonucleotides and short interfering RNAs is limited by their non-selective cellular uptake. To address the need for selective transfection methods, we covalently linked isolectin B4 (IB4) to streptavidin and analyzed whether it could be used to study protein function in IB4(+)-nociceptors. Rats treated intrathecally with IB4-conjugated streptavidin complexed with biotinylated antisense oligonucleotides for protein kinase C epsilon (PKCε) mRNA were found to have: (a) less PKCε in dorsal root ganglia (DRG), (b) reduced PKCε expression in IB4(+) but not IB4(-) DRG neurons, and (c) fewer transcripts of the PKCε gene in the DRG. This knockdown in PKCε expression in IB4(+) DRG neurons is sufficient to reverse hyperalgesic priming, a rodent model of chronic pain that is dependent on PKCε in IB4(+)-nociceptors. These results establish that IB4-streptavidin can be used to study protein function in a defined subpopulation of nociceptive C-fiber afferents.

Keywords: Antisense oligonucleotides; Isolectin B4; hyperalgesic priming; ligand-conjugated antisense; nociceptors; pain.

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Conflict of interest statement

Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Effect of intrathecal PKCε antisense oligonucleotides on PKCε expression in rat lumbar dorsal root ganglia. (a) Western blot image of protein extracts from L4/L5 dorsal root ganglia of rats treated with either biotinylated PKCε sense (SE) or antisense (AS) oligonucleotides (bound to IB4 streptavidin). The calculated molecular weight of PKCε is ∼83.5 kDa (according to UniProt database entry P09216). β-Actin was used as a loading control. Its calculated molecular weight is ∼42 kDa (according to UniProt database entry P60711). (b) Column bar graph showing normalized PKCε immunoreactivity in both groups. Data are presented as mean ± SEM with N = 3 in each group (p < .05).
Figure 2.
Figure 2.
Knockdown of PKCε in IB4(+) C-fiber afferents. Representative images from L4/L5 DRG from rats treated with either IB4-streptavidin biotinylated PKCε sense ODN (upper image panels) or IB4-streptavidin biotinylated PKCε antisense ODN (lower image panels). Color coding: Red = PKCε immunoreactivity, Blue = DAPI, Green = IB4 immunoreactivity, Yellow = PKCε/IB4 colocalization. Note that up to 40% of all DRG neurons in the rat are IB4 positive (+) and the vast majority are small diameter C-fiber DRG neurons. PKCε immunoreactivity can be observed in small-, medium-, and large-diameter DRG neurons. Interestingly, the vast majority of PKCε expressing DRG (∼90%) are IB4 positive (+). Scale bar = 50 µm.
Figure 3.
Figure 3.
Effect of intrathecal PKCε antisense oligonucleotides on PKCε transcripts in rat L4 and L5 DRG. The histogram depicts the level of PKCε expression in rats treated with either a complex of IB4streptavidin/biotinylated PKCε sense ODN (left) or IB4 streptavidin/biotinylated antisense ODN (right). Gene expression was determined with SYBR green RT-PCR and values (mean ± SEM) represent expression relative to GAPDH. Rats treated with IB4 streptavidin/biotinylated PKCε antisense ODN show a significant decrease in their PKCε mRNA levels compared to those treated with a mixture of IB4 streptavidin/biotinylated PKCε sense ODN (Student’s t-test, p = .0001, N = 6 for each group).
Figure 4.
Figure 4.
Reversal of hyperalgesic priming: Rats were primed by the intradermal administration of ψεRACK. Starting 3 days after the injection of ψεRACK, rats received intrathecal injections on day 1, 4, and 7 of IB4-streptavidin complexed with either biotinylated sense or antisense ODN for PKCε mRNA. Three days after the last injection PGE2 was administered into the same site as ψεRACK before. To test for hyperalgesic priming nociceptive paw withdrawal thresholds were measured prior to and 30 min and 4 h post intradermal administration of PGE2. PGE2-induced hyperalgesia remained elevated at 4h in sense-treated rats, indicating the presence of priming. However, hyperalgesia was not present at the 4th h in antisense-treated rats, showing its ability to reverse priming. A two-way repeated measures ANOVA for the baseline readings showed no significant effect of treatment (F1,10 = 0, p > .999), time (F2,20 = 1.25, p = .308) and treatment by time interaction (F2,20 = 1.67, p = .225). These results show that the two ODN groups did not differ significantly until the PGE2 injection. A second two-way repeated measures ANOVA that included the two post-PGE2 injection time points showed significant effects for treatment (F1,10 = 159, p < .0001), time (F1,10 = 426.2, p < .0001) and treatment by time interaction (F1,10 = 277.9, p < .0001). Post hoc analysis revealed significant differences between sense and antisense treated rats only 4 h post PGE2 administration (t20 = 19.74, p < .0001).
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