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1 MO reports Phenotype of 1T human tau transgenic mice Sumihiro Maeda et al xpanded View igures Western lotting LIS HT7/GPH 3 1 HT7/ctin 3 1 HT7-77G7P htau Level (ng/mg) 1 1 htau-wt (L1) htau-1t (L3) htau Level (ng/mg) 1 1 htau-wt (L1) htau-1t (L3) ragments/ull Length ragments/ull Length G7-TauP G m+htau Level (ng/mg) 1 1 htau-wt (L1) m+htau Level (ng/mg) H 1 1 htau-wt (L1) igure V1. Tau levels in brain tissues of htau-wt and htau-1t mice determined by Western blotting or LIS. H Tau levels in the cortex (,,, G) and hippocampus (,,, H) of mice were determined at 1 months of age by Western blotting ( ) or LIS ( H). ( ) omparable levels of full-length tau (, ) and higher levels of htau fragments (, ) in htau-wt than htau-1t mice were confirmed with the HT7 antibody in a cohort independent from the cohort described in ig 1. ( H) htau levels determined by htau-specific LIS (HT7 for capture and 77G7 conjugated with alkaline phosphatase for detection) (, ) or pan-tau LIS (77G7 for capture and Tau conjugated with alkaline phosphatase for detection) (G, H). n=3 mice per group. P <., P <.1, P <.1 by two-tailed unpaired t-test (, ), two-tailed Welch s t-test (, ), or Welch s t-tests and Holm adjustment ( H). Values are means SM. V1 MO reports ª 1 The uthors

2 Sumihiro Maeda et al Phenotype of 1T human tau transgenic mice MO reports Percent of Survival 1 TR- 1 3 ge (Months) Percent of Survival 1 TR- 1 1 ge (Months) Male emale Months of ge ody Weight (g) 3 1 ody Weight (g) Months of ge ody Weight (g) 3 1 ody Weight (g) 3 1 igure V. Normal survival curves and subtle weight reduction in htau-wt and htau-1t mice., Survival curves of () and () mice were compared with those of littermates. No significant differences were detected by logrank test. ody weights of male (, ) and female (, ) mice from and were compared with those of and singly transgenic littermates at 7 (, ) or 1 1 (, ) months of age. ata information: n = 1 (), 17 (), 8 (), 7 (), (), or 3 () ; 9 () TR-; 87 () TR-; 1 (), 88 (), 19 (), (), or 1 (, ) ; 91 (), 8 (, ), 1 (), or 8 () ; and 8 (), 1 (), 7 (), 13 (), or 11 () mice. P <., P <.1, P <.1 versus by one-way NOV and Tukey test ( ). Values are means SM. ª 1 The uthors MO reports V

3 MO reports Phenotype of 1T human tau transgenic mice Sumihiro Maeda et al TR- TR- G H I J K L M N PH1/PY O P 1. PH1/PY.8... Iba1 Positive ells 3 1 htau-wt(l1) Q R S igure V3. dditional immunohistochemical and biochemical characterization of htau transgenic mice. oronal brain sections from -month-old mice were immunostained with the htau-specific antibody HT7. (), TR- (), and TR-hTau-1T (L1) () sections showed similar levels of faint background staining, confirming the lack of htau expression in both TR-hTau singly transgenic lines. M Photomicrographs of HT7-stained coronal 1 sections from 3- to-month-old mice highlight differences in the accumulation of htau in the stratum radiatum in mice (more string-like) versus mice (more dispersed). igures V3 and J are magnified images of igure 3I and H, respectively. N, O p-tau/total tau ratios in cortical (N) and hippocampal (O) homogenates were determined by Western blot analysis with the PH1 (p39, ) antibody and the PY pan-tau antibody. n = 3 mice per group. P <.1 by Welch s t-test with Holm adjustment. P oronal brain sections from -to8-month-old and mice were immunostained for the microglial marker Iba1. The quantitation of Iba1- positive cells in the hippocampus revealed no significant difference between the groups (two-tailed unpaired t-test). n= 8mice per group. Q S oronal brain sections from - to-month-old mice were immunostained with the conformation-dependent tau antibody M1. ata information: Scale bars: 1 mm ( ), 1 lm ( M), 3 lm (Q S). Values in (N P) are means SM. V3 MO reports ª 1 The uthors

4 Sumihiro Maeda et al Phenotype of 1T human tau transgenic mice MO reports 8 Months 3 Months 1 Months rtg1 G H I J K Total raction L Sarcosyl Insoluble raction TR-hTau htau-1t htau-wt htau-1t htau-wt HT7 Tubulin HT7 Tubulin HT7 M N O P HT7/Tubulin HT7/Tubulin Relative Human Tau Levels (HT7) (a.u.) Relative Human Tau Levels (HT7) (a.u.) 1 1 igure V. bsence of dense Gallyas silver-positive tau inclusions and insoluble -ka tau aggregates in htau-1t mice. J oronal brain sections from mice of the indicated genotypes and ages were silver-stained by the Gallyas method. Representative images of the cortex ( ) and the hippocampal /3 regions ( J) from one mouse per genotype and age are shown. Note the dense labeling of cortical and hippocampal neurons in the rtg1 mouse (a positive control for this type of tau pathology), the absence of such labeling in mice at both ages, and the faint and diffuse labeling of cortical but not hippocampal neurons in old mice. Scale bar: lm. K P ortical or hippocampal tissues from - to3-month-old mice of the indicated genotypes were lysed in RIP buffer and saved as total fractions (K, M, N). Sarkosylinsoluble fractions (L, O, P) were extracted from the total fractions. htau levels were determined by Western blot analysis with the HT7 antibody. P19 mice (age: 11 1 months expressing aggregation-prone P31S-mutant htau served as a positive control for the detection of insoluble tau. Representative Western blots are shown in (K, L) and quantitations of Western blot signals in (M P). n = 3 7 mice per genotype. P <., P <.1 by one-way NOV and Tukey test. Values are means SM. ª 1 The uthors MO reports V

5 MO reports Phenotype of 1T human tau transgenic mice Sumihiro Maeda et al Nesting Score Months h h h rossings Months 18 3 Months ay: 1 3 ay: Target Non-target G rossings igure V. Nesting behavior of old htau-1t mice and MWM deficits in htau-1t mice from cohort. Nesting behaviors of mice and littermates at 19 3 months of age were scored 7 by an investigator blinded to genotype. mice showed a trend toward a deficit. Unadjusted P=.1981 and Holm-adjusted P=.9 by nonparametric Wilcoxon rank-sum test. G,, and mice from the same cohort were tested in the MWM at the indicated ages. (, ) Learning curves during hidden platform training at () or 18 3 months. ata at day are from the first trial on day 1. or each age range, we used ox proportional hazards model analysis with mixed effects and Holm adjustment to compare with mice and with mice. Only mice had significant learning impairments at 18 3 months of age (P <.1). ( G) uring probe trials (platform removed) h after the last training trial, we recorded latency to cross the original platform location (, ) and the number of times mice crossed the original platform location in the target quadrant (black bars) versus the average of crossings of the corresponding locations in the three other quadrants (white bars) (, G). ata information: n = 1 (,,, G) or 17 (,, ) ; 1 (,,, G) or 1 (,, ) ; and 1 (,,, G) or 19 (,, ) mice. P <. by oneway NOV and Tukey test (, ). Values are means SM. V MO reports ª 1 The uthors

6 Sumihiro Maeda et al Phenotype of 1T human tau transgenic mice MO reports Open ield Pole Test alance eam Rotarod levated Plus Maze 3 Months Months Months 7 Months 3 Months Locomotion Locomotion TR Months G 8 TR Months Latency to ross (sec) Latency to ross (sec) H TR Months Latency to all (sec) Latency to all (sec) I 1 1 TR Months Open/losed rm Ratio J Open/closed rm Ratio TR Months igure V. htau transgenic mice did not have robust alterations in locomotor activity or anxiety. J Mice of the indicated genotypes and ages were tested in the open field (, ), pole test (, G), balance beam (, H), rotarod (, I), and elevated plus maze (, J) at young ( ) and old ( J) ages. (, ) Total number of beam breaks during 1 min of exploration. (, G) Latency to climb down from the top of the pole. (, H) verage latency to cross the beam on day 3. (, I) verage latency to fall from the rotarod on day 3. (, J) Ratio of time spent in open arm versus closed arm of the elevated plus maze. n = 1 ( ) or 17 ( J) ; 13 ( ) TR-; 1 (, ), 11 ( ), 13 (, J), or 1 (G I) ; 13 (, J) or 1 (G I) htau-wt (L3); and 13 (, ), 1 ( ), or 1 ( J) mice. P <. by one-way NOV and Tukey test. Otherwise, no significant differences between genotypes were detected by one-way NOV with post hoc Tukey test (, I) or by two-tailed Welch s t-test with Holm adjustment (, J). ª 1 The uthors MO reports V