Protein Expression of TXNIP in the Dopaminergic Neurons of Subjects with Parkinson's Disease: Evidence from a Pilot Study

Abstract

Parkinson's disease (PD) is a progressive, multisystemic α-synucleinopathy, recognized as the second most prevalent neurodegenerative disorder globally. Its neuropathology is characterized by the degeneration of dopaminergic neurons, particularly in the substantia nigra pars compacta (SNpc), and the intraneuronal accumulation of α-synuclein-forming Lewy bodies. Oxidative stress is a key contributor to PD pathogenesis. Thioredoxin-interacting protein (TXNIP) is a crucial regulator of cellular redox balance, inhibiting the antioxidant function of thioredoxin. This pilot study aimed to investigate the protein expression and localization of TXNIP in the SNpc of PD patients compared to healthy controls. We performed immunohistochemical analyses on 12 post-mortem human brain sections (formalin-fixed, paraffin-embedded) from six subjects with PD and six healthy controls. The study was performed on PD subjects with Braak stage 6. Our findings revealed that in control samples, TXNIP protein was distinctly and closely associated with neuromelanin (NM) pigment within the cytoplasm of SNpc dopaminergic neurons. Conversely, in PD samples, there was a markedly weak cytoplasmic expression of TXNIP, and critically, this association with NM pigment was absent. Furthermore, PD samples exhibited a significant reduction in both dopaminergic neurons and NM content, consistent with advanced disease. These findings, which mirror previous transcriptomic data showing TXNIP gene under-expression in the same subjects, suggest that altered TXNIP expression and localization in SNpc dopaminergic neurons are features of late-stage PD, potentially reflecting neuronal dysfunction and loss.

Keywords: Parkinson’s disease; TXNIP protein; dopaminergic neurons; immunohistochemistry; substantia nigra.

Figure 1

Images acquired using cellSens of SN sections from the post-mortem brains of healthy subjects. Specifically, using the TXNIP monoclonal antibody that binds the corresponding epitope, positive immunostaining (magenta) is detected at the cytoplasmic level, as well as in the same areas where NM was present (brown), resulting in a glossy staining. The signal is present and clearly visible in all samples analyzed (panels (a,b), 20× and panels (c,d), 40×). In addition, the green arrows point to the NM signal (brown), while the blue arrows point to the positive signal (magenta).

Figure 2

Images acquired using cellSens of SN sections from the post-mortem brains of subjects with PD. A difference in the positive (magenta) signal is evident in all images compared with the panels in Figure 1, indicating a reduction in or absence of magenta coloration in most NM granules and, therefore, a loss of antigen–antibody complex formation (panels (a,b), 20× and panels (c,d), 40×). In addition, the arrows in green point to the NM signal (brown).

Figure 3

Images acquired using cellSens of SN sections from post-mortem brains. (a,b) The images show dopaminergic neurons, at 100× magnification, with positive immunostaining in proximity to the NM in CTRL subjects. (c,d) The images show dopaminergic neurons, at 100× magnification, with negative immunostaining in proximity to the NM in PD subjects. (e,f) The images show SNpc dopaminergic neurons in which the primary antibody was not added—a negative control; image (e) is from a CTRL subject while image (f) is from a PD subject. (panels (a–d), 100×; panel (e), 20× and panel (f), 40×). In addition, the arrows in green point to the NM signal (brown) while the arrows in blue point to the positive signal (magenta). Furthermore, the red asterisk in image (e) highlights a nonspecific pink coloration demonstrating that the magenta dye was introduced during the IHC experiment.

Figure 4

Images acquired using cellSens of SN sections from post-mortem brains. On the left, the panels of healthy subjects are shown, while on the right are the panels of subjects with PD. In all images, the following is evident: (1) a drastic decrease in dopaminergic neurons, part of the SNpc, between panels (a,d); (2) a drastic decrease in the amount of NM between panels (b,e); and, finally, (3) we hypothesize that all of the NM spots scattered in the PD panels are a consequence of the destruction of dopaminergic neurons and the release of the NM contained within them (panels (a,b), 4×; panels (c,d), 10×; panels (e,f), 20×). Panel (f) represents the enlargement of the part highlighted by the black box in panel (d).