Rationally designed DNA therapeutics can modulate human TH expression by controlling specific GQ formation in its promoter

Abstract

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the catecholamine (CA) biosynthesis pathway, making TH a molecular target for controlling CA production, specifically dopamine. Dysregulation of dopamine is correlated with neurological diseases such as Parkinson's disease (PD) and post-traumatic stress disorder (PTSD), among others. Previously, we showed that a 49-nucleotide guanine (G)-rich sequence within the human TH promoter adopts two different sets of G-quadruplex (GQ) structures (5'GQ and 3'GQ), where the 5'GQ uses G-stretches I, II, IV, and VI in TH49, which enhances TH transcription, while the 3'GQ utilizes G-stretches II, IV, VI, and VII, which represses transcription. Herein, we demonstrated targeted switching of these GQs to their active state using rationally designed DNA GQ Clips (5'GQ and 3'GQ Clips) to modulate endogenous TH gene expression and dopamine production. As a translational approach, we synthesized a targeted nanoparticle delivery system to effectively deliver the 5'GQ Clip in vivo. We believe this strategy could potentially be an improved approach for controlling dopamine production in a multitude of neurological disorders, including PD.

Keywords: G-quadruplex; Parkinson’s disease; dopamine; nanoparticles; nucleic acids therapeutics; targeted drug delivery; tyrosine hydroxylase.

Graphical abstract

Figure 1

Schematic representation of the position of TH49 within the tyrosine hydroxylase (TH) promoter and our strategy of blocking of a specific GQ formation in the TH promoter using GQ Clips

Figure 2

GQ Clips bind to TH49 to elicit structural changes (A and B) DNA Clips bind to TH49 in a dose-dependent fashion. Native PAGE of TH49 GQ (1 μM) in the absence and presence of increasing concentrations of 5′GQ Clip or 3′GQ Clip (0, 1, 10, and 50 μM). (C) The 5′GQ Clip blocks the formation of 3′GQ structure. DMS structural mapping of wtTH49 in the presence of systematically increasing the concentration of 5′GQ Clip was performed. Lane designations are as follows: lane 1, DMS probing at 0 mM K⁺; lane 2, DMS probing at 100 mM K⁺; lanes 3–5, DMS structural mapping in the presence of increasing concentrations of 3′GQ Clip at 100 mM K⁺. The TH49 sequence is shown on the left side and the G-stretches in the sequence and corresponding bands in the gel are labeled in red letters. (D) The 3′GQ Clip blocks the formation of 5′GQ structure. DMS structural mapping was repeated with the 3′GQ DNA Clip. The TH49 sequence is shown on the left side and the G-stretches in the sequence and corresponding bands in the gel are labeled in red letters.

Figure 3

GQ Clips can modulate TH expression and dopamine production in dopaminergic neurons (A) The rationally designed GQ Clips modulate the endogenous TH transcription in human SH-SY5Y cells. Histograms representing the ratio of endogenous TH to GAPDH mRNA levels in SH-SY5Y cells. The mRNA level in the cells treated with scramble Clip was set to 1, and mRNA from GQ Clip-treated cells were normalized accordingly (data represent mean values ± the standard error of the mean (SEM); n = 9; ∗∗∗p < 0.001 from a Student’s t test). (B) GQ Clips modulate the endogenous TH protein expression. TH protein levels in SH-SY5Y cells were treated with 5′GQ Clip, 3′GQ Clip, and scrambled Clip as the control oligonucleotide. Western blot of the TH protein expression levels when treated with various oligonucleotides. Histogram representing densitometry analysis of the western blot image was performed using ImageJ software. The TH bands were normalized with corresponding GAPDH bands to correct for experimental loading errors. (C) Targeted control of endogenous dopamine production in SH-SY5Y cells by GQ Clips. Cellular dopamine levels in SH-SY5Y cells treated with 5′GQ Clip, 3′GQ Clip, and scrambled Clip sequences. The dopamine level of 2 × 10⁶ cells was measured using HPLC equipped with ECD. The histogram represents a quantitative analysis of the cellular dopamine level. Data represented as mean ± SEM, and the significance of the data was determined by t test analysis (∗∗∗p < 0.001).

Figure 4

Synthesis of a 5′GQ Clip nanoparticle system for the targeted delivery of 5ʹGQ Clip (A) Schematic of targeted clip nanoparticle for treatment of dopaminergic neurons. (B) TEM of AuNP. The black bar represents 10 nm. (C and D) UV-vis spectroscopy was used to determine the loading and stability of the 5ʹGQ Clip nanoparticle. The absorbance of DNA at 260 nm was measured to quantitate conjugation onto the nanoparticle complex, as after conjugation and purification the increase in intensity was used to measure loading efficiency (~85%). The 5ʹGQ Clip nanoparticle was incubated in increasing concentrations of NaCl to assess the stability of the complex in salt. Through the addition of 500 mM NaCl, no redshift or broadening of the 515 nm AuNP-specific peak was observed, which both resemble nanoparticle aggregation. (E and F) In vitro release of Clip DNA from 5ʹGQ Clip nanoparticle via increased glutathione level. Glutathione has an affinity for the gold surface (ligand-metal interactions between thiol and gold), replacing the thiolated DNA. The DNA was radiolabeled, and the higher band resembled bound DNA to the nanoparticle complex, while the lower band was released DNA after being incubated in 10 mM glutathione for various time points. The bands were quantified using ImageJ software and the data plotted.

Figure 5

5ʹGQ Clip nanoparticle improves cellular efficacy (A) Neuronal uptake of 5ʹGQ Clip nanoparticle is dependent on the Trk-B aptamer for successful cellular recognition. SH-SY5Y cells were incubated with 270 nM free fluorescently labeled 5′GQ Clip, no-aptamer 5′GQ Clip nanoparticle, or 5ʹGQ Clip nanoparticles (with TrkB). After 24 h, the TrkB aptamer was shown to be essential for cellular uptake presumably through receptor-mediated endocytosis and eventually reaching the nucleus. Scale bar: 10 μM. (B and C) 5ʹGQ Clip nanoparticle dramatically increases the expression of TH. SH-SH5Y cells were treated with 270 nM 5′GQ Clip nanoparticle and 5′GQ Clip for 24 h. Cellular RNA and protein were extracted, and the TH expression was normalized to GAPDH showing the nanoparticle complex increased expression by 9-fold and 6-fold, respectively. Data represented as mean ± SEM (∗∗p < 0.01). (D and E) 5ʹGQ Clip nanoparticle treatment can improve TH expression in disease-state cells. SH-SH5Y cells were treated with 270 nM 5ʹGQ Clip nanoparticle in the presence of 5 μM 6OHDA for 24 h. Similar to (A) and (B), qRT-PCR and western blot were used to verify the nanoparticle complex can increase mRNA and protein expression of TH 3-fold in the presence of high oxidative stress mimicking PD disease state. Data represented as mean ± SEM (∗∗p < 0.01).

Figure 6

5′GQ Clip nanoparticle is taken up by neurons and can increase the expression of GFP in TH-GFP transgenic rat model (A) Confocal fluorescent images display successful cellular recognition and uptake of the 5′GQ Clip nanoparticle complex in rat brains. Rats were treated with 10 μg of total 5′GQ Clip DNA (conjugated to the 5ʹGQ Clip nanoparticle). After 24 h, cellular uptake in the substantia nigra of treated rats. (B) Confocal fluorescent images show successful upregulation of GFP through targeting of the TH promoter by the 5′GQ Clip nanoparticle complex in rat brains. Transgenic TH-GFP rats were treated with 10 μg 5′GQ Clip or scrambled nanoparticles. After 24 h, GFP expression was assessed in the substantia nigra of treated rats. The activity of the TH promoter is shown to greatly increase in the presence of the 5′GQ Clip compared to the scramble sequence (or endogenous GFP expression). Scale bar: 100 μM. (C) 5′GQ Clip nanoparticle treatment in TH-GFP rats shows a 5-fold increase in GFP mRNA expression. Cellular RNA was extracted, and qRT-PCR was used to quantify GFP mRNA concentrations compared to GAPDH. (D) GFP protein expression is increased with 5′GQ Clip nanoparticle treatment in TH-GFP rats. Quantification of GFP within rats (n = 3) of the substantia nigra shows an average of a 5-fold increase in fluorescence per cell. Data represented as mean ± SEM (∗p < 0.05).