APOE4-induced patterned behavioral decline and neurodegeneration requires endogenous tau in a C. elegans model of Alzheimer's disease

Abstract

Alzheimer's disease (AD) causes a characteristic spatiotemporal pattern of neurodegeneration, resulting in the loss of associated faculties such as cognition. The factors which account for this pattern of degeneration are unclear, as AD risk genes are numerous and often broadly expressed. Previously, we generated a model of AD using the nematode Caenorhabditis elegans in which the AD risk variant of apolipoprotein E, APOE4, is pan-neuronally expressed. We showed that HSN class motor neurons degenerate in early adult. Here, we expand on our past work by performing behavioral analyses to deduce the effect of APOE4 on the function of distinct neuronal circuits. We found evidence that APOE4 induces dysfunction of other neurons; this spatiotemporal pattern of degeneration roughly correlates with endogenous levels of PTL-1, the C. elegans homolog of human MAPT also known as tau. Moreover, deletion of ptl-1 suppressed defects in multiple behaviors, suggesting broad protective effects across the nervous system including the HSN neurons. Lastly, we show that PTL-1 in the touch receptor neurons, where PTL-1 is most abundant, is required cell nonautonomously for degeneration of the HSN neurons. Our results suggest that C. elegans may provide a useful in vivo system to study how endogenous Tau acts downstream of APOE4 to cause progressive, patterned neurodegeneration.

Figure 1.. ptl-1 is required for widespread, progressive neuronal dysfunction induced by APOE4.

(A left) Timeline from egg lay to the adult age of onset at which defective or abnormal performance in APOE4 versus control animals are first detected. (A right) Endogenous ptl-1 locus with all known isoforms and the portion deleted in the ok621 allele as indicated below. (B-H) Results of behavioral assays, used to proxy neuronal dysfunction, which are arranged by the adult age of onset for APOE4-induced defective or abnormal performance. (B-E) D1 adult onset. (B) Subtle defect in gentle touch sensitivity which is fully suppressed by ptl-1 deletion. N=47 for control, N=30 for Δptl-1, N=33 for APOE4, and N=35 for APOE4;Δptl-1. (C) Subtle defect in pharyngeal pumping which is fully suppressed by ptl-1 deletion. N=94 for control, N=37 for Δptl-1, N=61 for APOE4, and N=91 for APOE4;Δptl-1. (D) Severe defect in swim-to-crawl gait transition which is partially suppressed by ptl-1 deletion. N=164 control, N=136 for Δptl-1, N=162 for APOE4, N=165 for APOE4;Δptl-1. (E) Subtle defect in short-term locomotion which is not suppressed by ptl-1 deletion. N=47 for control, N=38 for Δptl-1, N=44 for APOE4, and N=44 for APOE4;Δptl-1). (F) D2 adult onset. Subtle defect in long-term locomotion which is not suppressed by ptl-1 deletion. N=31 control, N=32 for Δptl-1, N=28 for APOE4, and N=29 for APOE4;Δptl-1. (G,H) D3 adult onset. (G) Severe egg laying defect which is partially suppressed by ptl-1 deletion. N=196 for control, N=134 for Δptl-1, N=214 for APOE4, N=243 for APOE4;Δptl-1. (H) Subtle, abnormal increase in harsh touch sensitivity. N=25 for control, N=17 for Δptl-1, N=26 for APOE4, and N=31 for APOE4;Δptl-1. All data are represented as mean ± SEM. N is the total sample size representing all animals across replicates. Statistical comparisons were made using χ² tests of independence for D,G and Student’s t-test for B,C,E,F,G. *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 2.. ptl-1 is required for APOE4-induced HSN axon degeneration.

(A) Cartoon of the worm midbody, centered about the vulva area, depicting: the HSN proximal axon, the VC4,5 neurons, vulval musculature, and ventral nerve cord (VNC). (B) Representative live images of normal (1,2) and abnormal (3–10) HSN proximal axons visualized with GFP. Proximal axon segments are marked by i. and ii (with exception to image 2, where segment identity is less certain). The vulva area is also marked with a white arrowhead for anatomical reference. All scale bars are 20 μm. (C,D) Percentage of animals with abnormal HSN proximal axons in D1 and D3 adults. The baseline incidence of abnormal axons in control was similar between D1 and D3. (C) D1 adults. All other groups show non-significant, but slightly increased incidence of abnormal axons compared to control. N=37 for control, N=59 Δptl-1, N=51 for APOE4, N=22 for APOE4;Δptl-1 (left), and N=37 for APOE4;Δptl-1 (right). (D) The incidence of abnormal axons appears to correlate with defective egg laying behavior (See Fig. 1G) in D3 adults. N=101 for control, N=78 for Δptl-1, N=91 for APOE4, N=79 for APOE4;Δptl-1 (left), and N=86 for APOE4;Δptl-1 (right). (E,F) Quantification of HSN proximal axon lengths in D1 and D3 adults. The baseline axon length in control was similar between D1 and D3. (E) D1 adults. Axon length was similar across all groups. N=15 for control, N=13 Δptl-1, N=20 for APOE4, N=6 for APOE4;Δptl-1 (left), and N=10 for APOE4;Δptl-1 (right). (D) D3 adults. Axon length appears to correlate with the incidence of abnormal axons shown in (C) and defective egg laying behavior (See Fig. 1G). N=79 for control, N=66 for Δptl-1, N=72 for APOE4, N=54 for APOE4;Δptl-1 (left), and N=66 for APOE4;Δptl-1 (right). All data are represented as mean ± SEM. N is the total sample size representing all animals across replicates. Statistical comparisons were made using χ² tests of independence for C and D and Student’s t-test for (E) and (F). *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 3.. Targeted genetic ablation, but not abolished function, of the TRNs protects HSN against APOE4-induced dysfunction.

(A1) Egg-laying behavior across groups. Genetic ablation of the TRNs partially suppressed egg-laying defects in APOE4 animals as with the ptl-1 deletion (See Fig. 1G). N=104 for control, N=171 for TRNs::EGL-1, N=525 for APOE4, and N=190 for APOE4;TRNs::EGL-1. (A2 top) Cartoon depicting the head and tail areas wherein the nerve ring and PLM neuron, respectively, are located. (A2 bottom) Representative live images of nerve ring (arrowhead) and PLM neuron (within dashed circle if present; marked with asterisk if absent) visualized with PTL-1::mNG in control and TRNs::EGL-1 animals. Dashed borders indicate the worm body outline. All scale bars are 20 μm. (B) Egg-laying behavior across groups. In contrast to genetic ablation, abolishing TRN function did not suppress egg laying defects. N=104 for control, N=123 for mec-4(lf), N=525 for APOE4, and N=101 for APOE4;mec-4(lf) (left), and N=125 for APOE4;mec-4(lf) (right). All data are represented as mean ± SEM. N is the total sample size representing all animals across replicates. All statistical comparisons were made using χ² tests of independence. control and APOE4 data are re-plotted in (A1) and (B). *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 4.. Targeted depletion of PTL-1 in the TRNs protects HSN against APOE4-induced dysfunction.

(A1) Egg-laying behavior across groups. N=104 for control, N=180 for TRNs::ptl-1 KD, N=525 for APOE4, N=171 for APOE4;TRNs:: ptl-1 KD (Left), and N=108 for APOE4;TRNs:: ptl-1 KD (Right). (A2) Quantification of ptl-1 knockdown in the targeted TRNs and non-targeted VCs4,5 neurons between control and TRNs::ptl-1 KD animals using PTL-1::mNG fluorescence intensity in the soma. (B1) Egg-laying behavior across groups. N=104 for control, N=348 for TRNs::ptl-1 KO, N=525 for APOE4, N=423 for APOE4;TRNs:: ptl-1 KO (Left), and N=519 for APOE4;TRNs:: ptl-1 KO (Right). (B2) Representative live images of nerve ring (arrowhead) and PLM neuron (within dashed circle if present; area marked with asterisk if absent) visualized with PTL-1::mNG in control and TRNs::ptl-1 KO animals. Dashed borders indicate the worm body outline. All scale bars are 20 μm. All data are represented as mean ± SEM. N is the total sample size representing all animals across replicates. Statistical comparisons were made using χ² tests of independence for A1,B1 and Student’s t-test for A2. Control and APOE4 data are re-plotted in (A1) and (B1). *: p<0.05; **: p<0.01; ***: p<0.001.