Fig 1: Improved behavior and markers of brain atrophy in M83+/+ mice carrying STI1 hypomorphic allele. a Grip force (N) analysis at 8–10 months of age for WT control mice (n = 8), M83+/+:STI1WT mice (n = 8) and M83+/+:?HET mice (n = 9) (genotype: F(2,22) = 2.761, p = 0.0851, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT p = 0.0754, WT vs. M83+/+:?HET p =0.2828, M83+/+:STI1WT vs. M83+/+:?HET p = 0.6983). b. Grip force analysis at 12 months of age for WT control mice (n = 8), M83+/+:STI1WT mice (n = 8) and M83+/+:?HET mice (n = 9) (genotype: F(2,22) = 3.31, p = 0.0554, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT, p = 0.0455, WT vs. M83+/+:?HET p = 0.2834, M83+/+:STI1WT vs. M83+/+:?HET p = 0.5418). c Grip force analysis performed at 13–14 months old for one-way ANOVA (genotype: F(2,21) = 5.583, p = 0.0114, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT p = 0.0106, WT vs. M83+/+:?HET p = 0.6579, M83+/+:STI1WT vs. M83+/+:?HET, p = 0.0551). d Number of sessions to reach acquisition criteria in the PVD-R test using Marble (S-) and Fan (S+) stimuli during the acquisition phase. One-way ANOVA (genotype: F(2,55) = 1.238, p = 0.2978, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT p = 0.8587, WT vs. M83+/+:?HET, p = 0.2751, M83+/+:STI1WT vs. M83+/+:?HET, p = 0.5555). e Reversal learning phase for the PVD-R test. Mice were baselined for 2 days (B1 and B2) and tested on reversal learning. Repeated-measures two-way ANOVA (Session: F(6.820, 373.9) = 154.2, p < 0.0001; Genotype: F(2, 55) = 2.821, p = 0.0682; Interaction: F(22, 603) = 3.744, p < 0.0001, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT p = 0.0025, WT vs. M83+/+:?HET, p = 0.7172, M83+/+:STI1WT vs. M83+/+:?HET p = 0.0311)). f Number of correction trials during reversal learning. Repeated-measures two-way ANOVA (Session: F(4.312, 237.1) = 84.47, p < 0.0001; Genotype: F(2, 55) = 7.170, p = 0.0017; Interaction: F(22, 605) = 2.866, p < 0.0001, Adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT, p < 0.0001, WT vs. M83+/+:?HET p = 0.5810, M83+/+:STI1WT vs. M83+/+:?HET, p < 0.0001). WT control mice (n = 20), M83+/+:STI1WT mice (n = 20), and M83+/+:?HET mice (n = 18). Parameters were measured across baseline days 1 and 2 (B1, B2) and reversal days 1 to 10 (R1–R10). g Coronal slices of the group average MRI brain from posterior (left) to anterior (right) slices. h Volumetric differences between M83+/+:STI1WT and WT. i Volumetric differences between M83+/+:?HET and M83+/+:STI1WT. In terms of t-statistics thresholded at FDR, 5% are displayed on top of the group average brain, such that the color maps denote the significance of the difference observed; cooler colors describe volumetric decreases whereas warmer colors described volumetric increases for the group of interest. We observed global volumetric decreases throughout the brain in M83+/+ mice harboring two copies of the human A53T SNCA transgene compared to WT mice (images in h) and volume increases in the M83 mice with one copy of the TPR1 hypomorphic allele (M83+/+:?HET) (compared to M83+/+:STI1WT mice with no ?TPR1 copy, images in i. j Examination of both patterns of volumetric difference by overlaying the two. Green regions denoting smaller volumes for M83+/+:STI1WT compared to WT, red regions denoting larger volumes for the M83+/+:?HET compared to M83+/+:STI1WT, and yellow denoting the regions observed in both comparisons). We observed, for several of the regions, that the volumetric decreases for the M83+/+:STI1WT mice (compared to WT) get reverted with the introduction of the ?TPR1 hypomorphic allele, such that we observe volumetric increases in M83+/+:?HET mice compared to M83+/+:STI1WT controls. k–m Quantitative analysis of some of the volumetric changes observed (albeit with differing t values). k White matter tracts such as the corpus callosum. l Right primary motor areas. m Subcortical structures (right caudoputamen). n Thalamus. We observed reversal of volumetric decreases in the corpus callosum, motor cortex, right caudateputamen, and thalamus. Notably, we also observed regions where atrophy is not recovered with the presence of ?TPR1 allele. Namely, the (left caudoputamen), we did not observe a reversal of the volumetric decrease in M83+/+ . In fact, in the left caudoputamen, we observed further volumetric increase in the M83+/+:?HET mice (compared to M83+/+ mice). Similarly, we observed regions that show volume increases only in the M83+/+:?HET mice. Namely, the right primary motor area. Individual unfilled black circles (WT), dark blue circles (M83+/+:STI1WT), and half-filled pink triangles (M83+/+:?HET) represent individual animals
Fig 2: SNCA fibril treatment increases the colocalization of TRIM16 and ATG16L1 with mCherry LGALS3 and SNCA fibrils. (A-C) Representative images from mCherry LGALS3 SH-SY5Y cells treated with SNCA fibrils and stained for TRIM16 and LC3B (B), or ATTO 647 labeled SNCA Fibrils and stained for TRIM16 (A) or ATG16L1 (C). The white arrows point to instances of triple colocalization. (D-F) an Imaris masking algorithm built around TRIM16 (A-B, D, and F) or ATG16L1 puncta (C, E). Each data point represents the averaged maximum intensity from the total masked puncta in an individual image. The same algorithm was applied to all experiments and treatment conditions. Intensity data are expressed as M ± SE (n = 3 independent experiments, 7–10 images per experiment). Statistical significance was determined by unpaired t-tests. For all statistical tests *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively.
Fig 3: A summary diagram of the hypothesized cellular mechanism. This diagram illustrates a hypothetical mechanism where endocytosed pathological SNCA enters the cell and induces lysosomal rupture. Lysosomal rupture results in MTOR inactivation subsequently increasing ULK1 and RPS6KB1 activity. Concurrently, lysosomal rupture exposures its inner-luminal glycans, which are recognized by LGALS3. Bound LGALS3 recruits TRIM16 and ULK1, and subsequently ATG16L1 to mediate further autophagic machinery assembly, increase cellular autophagy, and promote autophagosome development. This response, ultimately, increases SNCA secretion. (Bottom) LGALS3 recruitment to lysosomes during early membrane stress facilitate membrane repair.
Fig 4: BF-PLA specifically detects pS129 α-synuclein in mouse brain sections. A-P) BF-PLA staining of pS129 asyn was performed on coronal mouse brain sections (30μm). R13 (1:1,000) and syn-1 (1:2,000) monoclonal antibodies were used together (A–H). The BF-PLA was also performed using either syn-1 (I–L) or R13 (M–P) alone as controls. WT (A-D,I-P) or SNCA KO sections (E–H) containing neocortex (A,E,I,M), striatum (B,F,J,N), hippocampus (C,G,K,O), and substantia nigra (D,H,L,P) were used.
Fig 5: Fluorescent PLA specifically detects pS129 α-synuclein in primary cortical neuron cultures and HeLa cells. A–D) Schematic illustration of the principle experimental steps in the PLA protocol. E–H) The F-PLA specifically stains for pS129 asyn in WT (F) but not in SNCA KO (E) primary cortical neuron cultures. AAV overexpression of mouse asyn (AAV masyn) (G) and human asyn (AAV hasyn) (H) in WT cultures were used to further exemplify the utility of the F-PLA. In HeLa cells, the F-PLA also staines pS129 asyn (I) while cells treated with the PLK inhibitor BI2536 show a reduced staining signal (J).
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