Increased 4R-Tau Induces Pathological Changes in a Human-Tau Mouse Model

Abstract

Pathological evidence for selective four-repeat (4R) tau deposition in certain dementias and exon 10-positioned MAPT mutations together suggest a 4R-specific role in causing disease. However, direct assessments of 4R toxicity have not yet been accomplished in vivo. Increasing 4R-tau expression without change to total tau in human tau-expressing mice induced more severe seizures and nesting behavior abnormality, increased tau phosphorylation, and produced a shift toward oligomeric tau. Exon 10 skipping could also be accomplished in vivo, providing support for a 4R-tau targeted approach to target 4R-tau toxicity and, in cases of primary MAPT mutation, eliminate the disease-causing mutation.

Figure 1. Total tau and tau isoform mRNA and protein expression in hTau mice following 3R to 4R MAPT mRNA splicing ASO treatment

A) Relative 4R human MAPT mRNA was significantly increased following 3R to 4R splicing ASO administration while B) total MAPT mRNA levels remained similar to controls (n=19 splicing ASO, n=9/saline and scrambled controls). MAPT mRNA data was normalized to GAPDH and the mean + SEM calculated relative to saline controls; **p<0.005 compared to saline and scrambled control values. C) 3R and 4R MAPT mRNA products visualized following RT-PCR confirm a shift in isoform composition following 3R to 4R splicing ASO treatment compared to mice treated with saline or scrambled ASO. D) Immunoblots for 3R- and 4R-tau show greater 3R-tau protein compared to 4R-tau in brain homogenates obtained from saline- and scrambled-treated mice, ipsilateral to the catheter placement. With 3R to 4R splicing ASO administration, the ratio of tau isoforms is altered, increasing the expression of 4R-tau and decreasing expression of 3R, E) without changing total tau protein (HT7). F) Total human tau protein expression quantified by human tau-specific ELISA closely paralleled total MAPT mRNA results, showing no change in total human tau protein after treatment. Data are expressed as mean + SEM relative to saline controls.

Figure 2. Detection of phosphorylated and aggregated tau species in ASO-treated hTau mice

Phosphorylated tau (AT8) was identified within the contralateral A) entorhinal cortex, D) amygdala, and G) cortex of 4 and 12-month old hTau mice following scrambled ASO treatment. Following 3R to 4R MAPT mRNA splicing ASO administration, AT8 cellular reactivity in these regions showed definitive increases (B, E, H). Non-transgenic (NonTg) mice displayed no AT8 pathology. Of note, one mouse treated with 3R to 4R splicing ASO did not meet our established criteria for ASO treatment (i.e. 80% increase in 4R-tau mRNA), and was eliminated from pathological analysis. Scale bars represent 250μm, insets 50μm. The percentage of AT8 immunoreactivity (IR) within a given area was quantified by densitometric analysis, revealing greater AT8 IR with 3R to 4R splicing ASO treatment (n=7) compared to NonTg (n=3) or scrambled ASO (n=5) treatment (C, F, I); mean + SEM; *p<0.05 scrambled vs. splicing treatment. J) Separation of monomeric tau from higher order tau species by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) revealed a shift toward more high molecular weight (HMW) tau forms with 3R to 4R splicing ASO treatment (n=4) compared to scrambled control (n=3) treatment in ipsilateral lysates obtained from 12-month-old hTau mice. When quantified by densitometric analysis, greater presence of HMW tau was identified with increased 4R-tau. K) Tau was also significantly more heavily phosphorylated (PHF-1) with a 3R to 4R splicing change. L) Ipsilateral cortical samples from treated hTau mice were fractionated and probed for human tau (HT7) to discern soluble vs. insoluble tau forms. Increased 4R-tau expression appeared to shift human tau from the RAB-soluble fraction toward the RIPA-soluble fraction (n=3 scrambled control, n=4 3R to 4R splicing ASO). No overt changes in insoluble tau in the formic acid fraction were detected between treatments. M) Quantification of RAB-, RIPA-, and formic acid-soluble tau fractions by human-specific ELISA was comparable to immunoblot findings, demonstrating a modest increase in RIPA-soluble tau following 3R to 4R splicing ASO treatment. Data are expressed as mean + SEM; * p<0.05. One lane of blots (black dashed lines) was cropped out to exclude the eliminated hTau sample within the 3R to 4R splicing ASO group.

Figure 3. Functional deficits measured by seizure and nesting activity in 3R to 4R MAPT mRNA splicing ASO-treated hTau mice

A) Following intraperitoneal PTZ injection, hTau mice treated with 3R to 4R MAPT mRNA splicing ASO exhibited a significant increase in seizure severity and B) a reduction in seizure latency compared to mice treated with saline or scrambled ASO (combined as controls) (n=18–19/treatment). Final seizure score data are plotted as mean stage + SEM and seizure latencies are mean time ± SEM across each seizure stage; *p<0.05 vs. controls. C) Representative images of nests constructed by hTau mice. Nesting activity was assessed using a modified scoring criteria to rate the quality of nest construction and amount of torn nestlet material. hTau mice treated with 3R to 4R splicing ASO (n=5) exhibited D) significantly lower nestlet scores and E) significantly greater untorn nestlet weights indicative of poor nesting activity compared to control (saline and scrambled ASO) treated hTau mice (n=5). Nestlet scores and weights are shown as mean + SEM; *p<0.05 vs. controls.

Figure 4. Validation of 4R to 3R human MAPT mRNA splicing ASOs in vivo

In both hTau and Tau N279K mice, a reverse MAPT splicing strategy was investigated that would induce exon 10 skipping and bias toward 3R-tau. A) In hTau mice, which exhibit tau isoform expression similar to humans, 4R to 3R MAPT splicing ASO induced a significant and widespread reduction of 4R mRNA levels in the cortex, hippocampus, and spinal cord compared to saline (n=3/treatment). B) No change in total tau mRNA was detected with either treatment. To further assess the potency of exon 10 exclusion, 4R to 3R splicing ASO was administered to Tau N279K mice and the ipsilateral temporoparietal cortex analyzed for MAPT isoform mRNA. C) Radiolabeled 4R and 3R PCR products demonstrate a clear shift toward 3R MAPT expression in mice treated with 4R to 3R splicing ASO (n=3 saline, n=5 ASO). When quantified, mRNA levels revealed that treatment with 4R to 3R splicing ASO afforded a significant decrease in 4R MAPT mRNA level and increase in 3R mRNA, in contrast to saline treatment, while D) total human MAPT mRNA levels remained unchanged. 4R mRNA levels were calculated as a percent of exon 10 inclusion normalized to total tau, and total tau mRNA levels to GAPDH. Data are expressed as the percent of exon 10 inclusion relative to saline treatment (mean + SEM); analysis by unpaired t-test (saline vs. ASO) within each region, *p<0.05, **p<0.005.