CJ LI LRRK2 MOUSE COMPARISON STUDY. Phenotyping Data Results

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Esmond Tyler
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1 CJ LI MOUSE COMPARISON STUDY Phenotyping Data Results

2 NOTE FOR USE This comparison study was run as an opportunistic look at the phenotype of multiple CJ Li-generated mouse lines. The primary purpose of these data are to display common phenotypes of PD preclinical models and provide investigators with an idea of how the different lines broadly compare and perform in multiple outcome measures. This could aid in choosing a model for one s studies. The secondary purpose of these data are to provide investigators an idea of the phenotype of models previously unpublished or an idea of robustness of previously reported phenotypes. The mice used in this study were acquired at different times and were not necessarily analyzed in the same run. Data should be interpreted with caution. 2

3 DESCRIPTION OF MICE Genotype Transgene/Promoter/Zygosity Background JAX # C57Bl/6 WT N/A Control FVB N/A WT G219S R1441G Human wildtype driven by the human promoter on the BAC transgene. (Hemi) Human G219S driven by the human promoter on the BAC transgene. (Hemi) Human R1441G driven by the human promoter on the BAC transgene. (Hemi) FVB 961 FVB 969 FVB 964 3

4 DESCRIPTION OF METHODS Outcome Measure Body Weight Rotarod Forelimb/Hindlimb Grip Strength Hindlimb Footsplay Stereology Protein Levels Statistics for Behavior, Stereology, and Protein Description Individual body weights were recorded twice weekly, beginning the day of animal receipt and ending the day of euthanasia. Body weights graphed are averages of the recorded weight for each animal at the given age. Animals were tested once daily for 5 days on an accelerating rotarod. Rotation speed was set to increase at a constant rate to 4 RPM over 6 seconds (testing period was 6 seconds in duration). As no statistically significant effect of testing day was observed for any group, averages for each animal were taken and graphed as a single point. The animal is allowed to grip a T-shaped grip bar with its forepaws and is pulled back gently along a platform until its grip is broken. As the backward locomotion continues, the animal s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Digital force gauges record the maximum strength required to break grip. The average of three valid measurements is taken as and reported as an average score. Heel pads of the hindfeet of each animal are painted with a non-toxic paint. The animals are gently dropped onto a Hindlimb Foot Splay test sheet for three trials, with the first designated as practice. The closest distance between the inner edge of the ink blots made by each foot is measured. The measurements from the two test trials are used to calculate an average footsplay distance. Brains were embedded and sectioned at 4um in the coronal plane and stored in antigen preserve solution. Tyrosine hydroxylase staining was performed on every 6th free-floating section. Stereological counts were performed in the substantia nigra pars compacta in one hemisphere of each animal. Counter was blinded to animal age and genotype. Human protein was quantified using western blots and the Protein Simple WES system using the Antibodies Inc anti- antibody (N231B/34, Catalog #: ) and Abcam anti-vinculin antibody (Catalog #:ab15512) as the control in the striatum, and cortex. Absolute amounts of protein were detected and normalized to vinculin. A two way ANOVA for the effect of genotype and the effect of age was run with Bonferroni post hoc tests for differences between ages/genotypes. Outliers were removed uniformly across groups. All analyses and graphing were performed using GraphPad Prism software. 4

5 BEHAVIOR DATA BODY WEIGHT ROTAROD TIME TO FALL FORELIMB GRIP STRENGTH HINDLIMB GRIP STRENGTH HINDLIMB FOOTSPLAY [n = 15 mice per group]

6 B o d y W e ig h t B o d y W e ig h t B o d y W e ig h t B o d y W e ig h t BODY WEIGHT 5 4 M o n t h s 5 8 M o n t h s M o n t h s B o d y W e ig h t O v e r v ie w Body Weight Change Across Age FVB WT G219S R1441G 4-8 Mo *** = = ** 8-12 Mo *** ** *** * 4-12 Mo *** *** *** *** Significance: ***= p<.1; **= p<.1; *= p<.5 A g e o f m ic e (m o n th s ) 6

7 R o ta r o d T im e to F a ll (s e c o n d s ) R o ta r o d T im e to F a ll (s e c o n d s ) R o ta r o d T im e to F a ll (s e c o n d s ) R o ta r o d T im e to F a ll (s e c o n d s ) ROTAROD TIME TO FALL 4 M o n t h s 8 M o n t h s 1 2 M o n t h s * R o ta r o d T im e to F a ll O v e r v ie w Rotarod Time to Fall Change Across Age FVB WT G219S R1441G 4-8 Mo = = = = 8-12 Mo = = ** = 4-12 Mo = = ** = Significance: ***= p<.1; **= p<.1; *= p<.5 A g e o f m ic e (m o n th s ) 7

8 F o r e lim b G r ip S tr e n g th F o r e lim b G r ip S tr e n g th F o r e lim b G r ip S tr e n g th F o r e lim b G r ip S tr e n g th FORELIMB GRIP STRENGTH 4 M o n t h s 8 M o n t h s 1 2 M o n t h s 2 * * 2 2 * * * * F o r e lim b G r ip O v e r v ie w Forelimb Grip Strength Change Across Age FVB WT G219S R1441G A g e o f m ic e (m o n th s ) 4-8 Mo = = ** * 8-12 Mo * = *** = 4-12 Mo = * * * Significance: ***= p<.1; **= p<.1; *= p<.5 8

9 H in d lim b G rip S tr e n g th H in d lim b G rip S tr e n g th H in d lim b G rip S tr e n g th H in d lim b G rip S tr e n g th HINDLIMB GRIP STRENGTH M o n t h s * M o n t h s M o n t h s * * H in d lim b G r ip O v e rv ie w Hindlimb Grip Strength Change Across Age FVB WT G219S R1441G 4-8 Mo = = = = 8-12 Mo *** *** *** = 4-12 Mo ** ** *** = Significance: ***= p<.1; **= p<.1; *= p<.5 A g e o f m ic e (m o n th s ) 9

10 H in d lim b F o o ts p la y (m m ) H in d lim b F o o ts p la y (m m ) H in d lim b F o o ts p la y (m m ) H in d lim b F o o ts p la y (m m ) HINDLIMB FOOTSPLAY 6 4 M o n t h s 6 8 M o n t h s M o n t h s * F o o ts p la y O v e r v ie w Hindlimb Footsplay Change Across Age FVB WT G219S R1441G 4-8 Mo = ** = ** 8-12 Mo ** ** = = 4-12 Mo *** *** = = Significance: ***= p<.1; **= p<.1; *= p<.5 A g e o f m ic e (m o n th s ) 1

11 STEREOLOGY DATA TH+ NEURONS IN THE SUBSTANTIA NIGRA PARS COMPACTA [n = 9 mice per group]

12 # o f T H + N e u r o n s in S N p c # o f T H + N e u r o n s in S N p c # o f T H + N e u r o n s in S N p c # o f T H + N e u r o n s in S N p c TH-POSITIVE CELLS IN SN 4 M o n t h s 8 M o n t h s 1 2 M o n t h s 2 * * * T H + N e u r o n s in th e S N p c TH+ Cells in the SNpc Change Across Age 1 FVB WT G219S R1441G Mo = *** = = 8-12 Mo = = = = 4-12 Mo = ** = = Significance: ***= p<.1; **= p<.1; *= p<.5 A g e o f m ic e (m o n th s ) 12

13 EXPRESSION DATA HUMAN PROTEIN [n = 3 mice per group]

14 PROTEIN DATA COMING SOON! Striatal (STR) and Cortical (CTX) Protein Change Across Age FVB WT G219S R1441G STR CTX STR CTX STR CTX STR CTX 4-8 Mo 8-12 Mo 4-12 Mo Significance: ***= p<.1; **= p<.1; *= p<.5 14

MJFF KNOCKOUT RAT COMPARISON STUDY. Phenotyping Data Results

MJFF KNOCKOUT RAT COMPARISON STUDY Phenotyping Data Results NOTE FOR USE This comparison study was run as an opportunistic look at the phenotype of multiple MJFF-generated knockout rat lines. The primary