Fig 2: Effects of Txnip gene silencing in vitro. 3T3-CrT-HA cells were transfected with a small-interfering RNA (siRNA) oligonucleotide to inhibit mouse Txnip open-reading frame (siTxnip). A: basal Txnip mRNA levels were reduced to 50.74% (SE ±2.236) in the presence of siTxnip, and the Cr-mediated increase in Txnip transcript was abolished (52 ± 5; P = 0.018). B: there was a trend for reduced Txnip protein expression after siTxnip (70 ± 5), whereas the synthesis of new Txnip protein in response to 5 mM Cr was significantly attenuated (117 ± 9 vs. 145 ± 107 in the absence of siRNA; P = 0.018). C: Cr uptake is reduced following exposure to saturating levels of [Cr] for 3 h (3.007 ± 0.13), and this substrate inhibition is efficiently abolished by silencing Txnip (5.848 ± 0.34; P = 0.001). D: this effect was associated with preservation of CrT gene expression (1-way ANOVA, Dunnett's posttest vs. untreated; P = 0.0619). C: conversely, under basal conditions, siTxnip reduced Cr uptake via mechanisms unrelated to CrT gene expression (72 ± 1); n = 3/treatment was included in all experiments. E: intracellular Cr was raised by 65% (from 213 ± 15 to 383 ± 20 nmol/well) after exposure to 5 mM Cr for 3 h in vitro. When Txnip mRNA was reduced by siTxnip, the increasing effects of Cr were enhanced by a further 38% (to 470 ± 18 nmol/well; P = 0.0064). ns, Nonsignificant P > 0.05, *P < 0.05, **P < 0.01, and ****P < 0.001.

Fig 3: A: verification of gene array result by qRT-PCR. Cells were exposed to either 250 µM or 5 mM Cr. While 250 µM Cr did not change thioredoxin-interacting protein (Txnip) mRNA (107 ± 3.7; P = 0.449), saturating Cr caused a rise in Txnip mRNA levels in agreement to the gene array observation (n = 3 from 3 independent experiments; 161 ± 2; P = 0.0016). Moreover, the induction of Txnip expression was specific for Cr and did not occur at 5 mM taurine (n = 3; 106; P = 0.5481). B: Txnip protein levels were elevated by 5 mM Cr, at 3 h (n = 3/treatment; 145 ± 11; P = 0.0139), whereas neither 250 µM Cr nor taurine changed protein expression. ns, Nonsignificant P > 0.05, *P < 0.05, and **P < 0.005.

Fig 4: Relevance to myocardial tissue. A: LV Cr levels in mice overexpressing the Cr transporter (CrT-OE) were significantly higher than wild type (mean 124 nmol/mg protein vs. 74 nmol/mg, respectively; P = 0.0012), whereas Cr was confirmed to be undetectable in hearts from guanidinoacetate methyltransferase knockout (GAMT-/-) mice. B: GAMT-/- had lower Txnip transcript levels than wild-type littermates (60 ± 7; P = 0.021). In contrast, CrT-OE showed higher levels of Txnip transcript compared with the wild type (158 ± 15; P = 0.0397). C: protein expression of Txnip was not statistically different in GAMT-/- compared with the wild type, whereas CrT-OE had a 29% increase in protein (P = 0.012). Wild-type n = 10; GAMT-/- n = 5; CrT-OE n = 8. Statistical symbols on graphs correspond to nonsignificant (ns) P > 0.05, *P < 0.05, and **P < 0.005.

Fig 5: Knockdown of TXNIP Rescues a-Synuclein Oligomers in LRRK2-G2019S Knockin 3D Organoids(A) Validation of microarray and real-time qPCR gene expression data.(B) Real-time qPCR analysis of TXNIP expression in LRRK2-G2019S knockin 3D organoids and midbrain 3D organoids. Data represent the mean ± SEM. **p < 0.01 by ANOVA (n = 3 per sample).(C) Real-time qPCR analysis of TXNIP expression in control and LRRK2-G2019S 3D organoids compared with control and LRRK2-G2019S 2D cultures. Data represent the mean ± SEM. **p < 0.01 by ANOVA (n = 3 per sample).(D) Validation of TXNIP gene expression in LRRK2-G2019S knockin 3D organoids treated with TXNIP-short hairpin RNA (TXNIP-shRNA) (target sequence: agt gga ggt gtg tga agt tac tcg tgt ca, i026466a) via real-time qPCR. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 3 per sample).(E and F) Western blot analysis (E) and quantification (F) of pS129-a-synuclein reveals a reduction in pS129-a-synuclein oligomers in LRRK2-G2019S knockin 3D organoids treated with TXNIP-shRNA. Data represent the mean ± SEM. *p < 0.05 by ANOVA (n = 3 per sample).(G) Representative immunofluorescence images of LAMP1 and pS129-a-synuclein puncta in LRRK2-G2019S knockin 3D organoids. Scale bar, 10 µm.(H and I) Quantification (H) of LAMP1+ and pS129-a-synuclein+ puncta showing a reduction (I) of pS129-a-synuclein inclusions upon treatment with TXNIP-shRNA. Data represent the mean ± SEM. *p < 0.05 by ANOVA (n = 5 per sample).