Figure 2. Impact of α-synucleinopathy on APE1 expression in mice and humans.

Three-month-old male and female mice were infused in the right OB/AON with sonicated α-synuclein fibrils (5 μg) or an equal volume of PBS (1 μL). Sagittal brain sections were collected and immunostained for APE1 after a six-month survival period. A blinded observer analyzed APE1 expression and the area of select brain regions by tracing the region of interest in the right hemisphere on (A) low-resolution, high-sensitivity scans captured with a 16 bit-depth imager. (B) Manual sketches of sagittal brain sections with anatomical regions of interest (red shading = AON). (C-D) Heatmaps of average APE1 signal per unit area. APE1 expression levels per unit area in traces of the mouse (E) whole brain, (I) AON, and (K) OB. Differential expression of APE1 elicited by fibril infusions within the mouse (F) whole brain, (J) AON, and (L) OB (see Fig. S8). (M) Average size of the traced outline of the mouse OB. (G-H) Eight-month-old male and female mice were bilaterally infused in the OB/AON with α-synuclein fibrils or PBS. Three months later, APE1 mRNA levels were assessed by RT-qPCR in whole-brain extracts (G). (H) Global APE1 mRNA levels expressed as the difference between fibril and PBS-infused animals. (N-R) The UCLA and University of Miami Brain Banks provided human OB samples from deceased male and female control subjects and subjects diagnosed with Lewy body disorders (see demographics in Table S3 in (Bhatia et al., 2021)). Bulk RNA Smart-Sequencing was performed on the UCLA samples, and transcripts per million (TPM) for the APEX1 gene are shown in N. (O) Differential expression of APEX1 TPM in the OB of subjects with Lewy body disorders compared to unaffected controls. (P) APE1 expression in the human OB from UCLA and Miami cohorts was determined by Western immunoblotting and expressed as a fraction of total protein levels (REVERT stain from LI-COR). (Q) Differential APE1 protein expression levels in the human OB of subjects with Lewy body disorders compared to unaffected controls. (R) Full-length immunoblots depicting APE1 and total protein expression. The entire lane of the Total Protein REVERT stain was quantified (for assay validation see Fig. S9 and for brighter images of blots see Fig. S10). Data in panels E-Q are shown as the mean ± S.D. (except box plots with interquartile ranges in H and J). Two-way ANOVAs in E, G, I, K, M, N, and P were followed by the Bonferroni post hoc, and statistical interactions between biological sex and disease are noted below respective graphs. For F, L, O, and Q, two-tailed Student’s t tests were performed. The Mann-Whitney U test was performed in H and J. *p≤0.05, **p≤0.01, ***p≤0.001 male vs. female; +p≤0.05, ++p≤0.01 PBS vs. fibrils.