Animal use overview

The GBA1 mouse colony C57BL/6N-Gba^tm1.1Mjff/J (strain No. 019106) was bred and housed at The Jackson Laboratory mouse repository in a standard barrier mouse pathogen-free vivarium (designated as Area MP23; http://jaxmice.jax.org/health/mp23.pdf) in individually ventilated cages with aspen bedding. The mice had ad libitum access to water and food and were fed a standard LabDiet® 5K52 formulation with 6% fat; water was acidified. The mouse colony was maintained under a 12 hr light/12 hr dark cycle. From the heterozygous x heterozygous breeding colony, the mice were pooled at weaning based on date of birth into N = 20 males per cage. Once genotyped prior to 8 wks of age (using the standard PCR genotype procedure described above) the mice were rehoused based on genotype at N = 10/genotype/weaning cage. As the mice aged or signs of hair loss were observed, cage density was dropped to N = 5 per cage for males. If signs of aggression were noted, males were separated and individually housed as needed.

For this study 66 homozygous (hom) GBA1 D409V KI and 66 wild type (WT) littermate male mice were allocated. The 66 mice per genotype were divided into three equal groups and aged to different timepoints– 4, 8, and 12 months of age (n = 22 mice/genotype/age) with dates of birth ranging no more than plus/minus 3 days for the 4 month cohort and no more than plus/minus 1 week for the 8 and 12 month old cohorts. Within each age group, mice were assigned to one of three groups: Group 1 for biochemistry (n = 7/genotype), Group 2 for neurochemistry (n = 6/genotype), and Group 3 for neurohistology (n = 9/genotype). Assignment to these groups was performed at random for each genotype using a computer program based on stratification of body weights. Group sizes were selected based on previous studies phenotyping models with other PD-related mutations [32].

When each cohort of n = 22 mice/genotype reached the target age, the mice were shipped live to Charles River Labs (formerly WIL Research). At Charles River Labs, viability observations for pain, moribundity, and mortality were performed twice daily and body weight was assessed twice weekly and on the day of scheduled euthanasia. No animals were noted as experiencing pain, significant weight loss, or measures of moribundity during the study. Following a brief period of acclimation (at least one week) mice were examined weekly in a functional observational battery (fully described below) and underwent behavioral testing (see details below) prior to euthanasia and necropsy for phenotyping analyses.

A separate cohort of heterozygous (het) GBA1 D409V KI mice and WT controls (n = 10/genotype) were obtained from the het x het breeding colony at The Jackson Labs. The het and WT mice were aged to 5 months at which point they were shipped to Amicus Therapeutics for analyses of GCase activity and GSL levels. Finally, an independent cohort of 4 month old WT (n = 8), het (n = 8), and hom (n = 7) mice were obtained from a het x het breeding colony at Merck & Co, Inc (with breeders obtained from the founding Jackson Labs colony) for analysis of GBA1 mRNA and GCase protein expression.

All animal monitoring and procedures performed at The Jackson Laboratory, WIL Research (now Charles River Laboratory), and Merck & Co met guidelines and regulations of federal, state, and local agencies, as well as the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All work was approved by the Institutional Animal Care and Use Committee (IACUC) of that institution. Specifically, the behavioral testing and sacrifice of WT and hom mice for biochemistry, neurochemistry, and neurohistology was approved by the Charles River Ashland IACUC on December 1, 2014 as protocol WIL-784012.