Semi-quantitative scale developed to assess morphological changes following treatment of zebrafish embryos with an amino-terminal fragment of nApoE4_1-151.

Rubric for developmental abnormalities was accomplished in 48 hpf zebrafish following 24-hour treatment with 25 ?g/ml of nApoE41-151. This scale was established to quantify the effects of nApoE4_1-151 as compared to untreated, control embryos. Identified hallmark defects that appeared consistently following treatment with nApoE4_1-151 included inflation of pericardial cavity, enlarged hearts, pigmentation alterations, and delays or lack of development in ear and brain structures. Data are representative of 10 embryos treated with 25 ?g/ml nApoE4_1-151 per trial for a total of 30 embryos.

A sublethal concentration of nApoE4_1-151 leads to morphological abnormalities in zebrafish embryos at the hatching phase.

Representative light phase contrast microscopic images following live imaging of embryos at 48 hpf following a 24-hour period with respective treatments (Control, 25 ?g/ml of nApoE3_1-151, or 25 ?g/ml of nApoE4_1-151. A: Arrows point to consistent morphological changes as compared to untreated controls that are healthy and categorized as having a developmental abnormality score of <1.0 (Panel A). The blue arrows designate pigmentation changes; orange arrows designate cerebellar primordium junction differences in the hindbrain. B: Exogenous treatment with nApoE3_1-151 impacted pigmentation pattern in otherwise healthy embryos. Embryos in this category were most likely to receive a score of <1. C: 24-hour incubation of a sublethal concentration of nApoE4_1-151 resulted in developmental abnormality scores >3. Embryos in this category were typically observed to be delayed in development with limited hindbrain folding (orange arrow), limited or lacking pigmentation (blue arrow), as well as enlargement of the cardiac cavity (red arrow). D. Quantitative developmental abnormality scores for each treatment group following treatment of embryos for 24 hours with respective fragments at low E3 (orange bar), E4 (pink bar) concentrations (25 ?g/ml) or at high concentrations (50 ?g/ml) E3 (gray bar), E4 (yellow bar). The nApoE4_1-151 25 ?g/ml-treated groups were significantly different from controls (H(4) = -2.43, p = 0.0074). At 50 ?g/ml both nApoE4_1-151 (H(4) = -3.32, p = 0.0004) and nApoE3_1-151-treatment groups (H(4) = -1.77, p = 0.037) were significantly different from controls. Errors bars represent ± S.E.M. *p<0.05, **p<0.01, ***p<0.001 E. Heart rate data obtained from live microscope analyses in 25 ?g/ml and 50 ?g/ml treatment groups nApoE3_1-151 and nApoE4_1-151 compared to non-treated controls. nApoE4_1-151 (pink bar, 25 ?g/ml) was significantly different from controls (H(4) = 1.77, p = 0.038). nApoE4_1-151 (yellow bar, 50 ?g/ml) was significantly different from nApoE3_1-151 25 ?g/ml (H(4) = 1.94, p = 0.026) and controls (H(4) = 2.665, p = 0.0036). Errors bars represent ±S.E.M. All other comparisons were insignificant. *p<0.05, **p<0.01, ***p<0.001.

Survivability is decreased in zebrafish embryos following exogenous treatment with an amino-terminal fragment of nApoE4_1-151.

A. Embryos at 24 hpf (prim-9 stage) were segregated into three groups: controls (untreated), 25 ?g/ml or 50 ?g/ml nApoE3_1-151, and 25 ?g/ml or 50 ?g/ml nApoE4_1-151. Embryos that lacked a heartbeat for 10 seconds were stimulated to induce movement. If no movement or heartbeat was detected, embryos were considered to be non-viable. Significant mortality was observed at both concentrations of nApoE4_1-151 by 48 hpf (orange and green dotted lines) compared to non-treated controls (orange dotted line) or nApoE31-151 (blue dashed line and black dotted lines). N = 3 independent trials, N = 5 fish/treatment. B. The Mosaic Plot depicts mortality based on a lack of heartbeat and response to physical stimuli following exogenous treatment of 48 hpf zebrafish embryos with either 25 ?g/ml nApoE3_1-151 or nApoE4_1-151 for 24 hours. The blue filled region of the bar graph designates 25 ?g/ml treatment of nApoE4_1-151 which led to a significant portion of the embryos being designated as dead. The red dotted portion of the bar graphs indicates less than expected were alive (p = 1.17e-13). All blank cells indicate the sample group followed the estimated trend. Data indicated significant morality for only the nApoE4_1-151 group. N = 3 independent experiments, 15 embryos/treatment.

Exogenous treatment of zebrafish embryos with nApoE4_1-151 leads to nuclear localization.

A-C. Representative images from confocal immunofluorescence in 5 mm paraffin-embedded sections that were stained with DAPI (A), anti-His antibody (green, B), or anti-His together with NeuN (C). There was no detection of nApoE4_1-151 fragments in untreated control neuronal cells as indicated by the lack of labeling in Panel A. Nuclear localization of the nApoE4 fragment was evident (Panels B and C) following exogenous treatment. For the E4 fragment, staining appears punctate and co-localized with NeuN and DAPI (B and C). All images were captured within the area of the cerebellum and fourth ventricle. D. Identical to Panels B-C with the exception that whole embryo mounts were triple labeled in order to display overall labeling in the entire organism at low magnification. Labeling of head, eye and tail is presented for orientation. The intense orange fluorescence area (arrows) represents regions with strong overlap between the E4 fragment and NeuN. In this case, labeling of the E4 fragment that co-localized with DAPI and NeuN was apparent in the hindbrain brain region. Data are representative of five independent experiments.

Tau pathology present after treatment with exogenous nApoE4_1-151 fragment in 72 hpf zebrafish brain.

Representative 40X images from confocal immunofluorescence in 5 mm paraffin embedded sections of non-treated control 72 hpf zebrafish (A-D), nApoE3_1-151-treated at 25 ?g/ml (E-H), or nApoE4_1-151-treated at 25 ?g/ml (I-L). Strong PHF-1 labeling was only observed following treatment with nApoE4_1-151 (I-L). Panel K depicts a separate, representative merged image following treatment with nApoE4_1-151. In this case, at high magnification the fibrillar nature of PHF-1 labeling was apparent. All scale bars represent 50 ?m. Data are representative of three independent experiments.

Negative trends in motor behavior in zebrafish following treatment with nApoE4_1-151.

A. Groups for non-treated controls (green bar), nApoE3_1-151 25 ?g/ml (orange bar), and ApoE4_1-151 25 ?g/ml (blue bar)) were assessed via video monitoring to determine number of spontaneous tail flicks per minute that were then averaged per group for each trial. Data are representative of N = 5 trials, for a total of 25 embryos per group, ±SEM. Data depicted show limited spontaneous tail flick activation from every group with no difference detectable between groups (F(2,27) = 1.24, p = 0.305). B. Results from the touch-evoked response motor behavior experiment. Non-treated controls had a 90% response rate to the evoked, tactile stimulus, whereas for nApoE4_1-151-treated groups responded to fewer than 50% of stimuli. No significant difference was observed (F(12) = 1.482, p = 0.266). C and D. Results from TEMR analyses similar to Panels A and B with the exception that in this case, total distance traveled (C) or the total time swimming (D) were recorded via video monitoring and using Noldus tracking software. For Panel C, representative heat maps of individual larvae representing either wild-type controls (left Panel), or a low-performing nApoE4_1-151-treated zebrafish (right Panel). Each bar represents the average total distance traveled or averaged cumulative duration during 5 independent trials, for a total of 15 embryos per group, (±SEM). No significant differences were observed, with for example the Ctl group vs. nApoE4_1-151 having a p value = 0.08 in Panel C. P-values for Panel D were Ctl vs. E3 fragment = 0.86 and Ctl vs. E4 fragment = 0.06.

The presence of nApoE4_1-151 within tail regions of zebrafish embryos.

A-C: Representative images from confocal immunofluorescence in 5 mm paraffin-embedded sections of non-treated control 48 hpf zebrafish embryos that were stained with DAPI (A), anti-His antibody (1:500) (B), and the merged image together with PHF-1 (1:250) in Panel (C). There was no detection of any nApoE4_1-151 fragments in untreated control sections as indicated by the lack of labeling in Panel B. D-F: Identical to Panels A-D with the exception that embryos were exogenously treated for 24 hours with 25 ?g/ml of nApoE4_1-151. In this case punctate staining of the fragment was observed that appeared cytoplasmic. PHF-1 labeling was identified in muscle cells that exhibited abnormal morphology (arrows, Panel F). All scale bars represent 20 ?m.