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All experiments were carried out with proper oversight by institutional review boards for animal care. Comparisons of i.v. and i.c.v. routes of administration were completed using wild-type (WT) C57BL/6J mice (Jackson Laboratory, stock number 000664). Proof-of-concept experiments for GBA-PD were completed using homozygous Gba^D409V/D409V mutant mice (Jackson Laboratory, C57BL/6N-Gba^tm1.1Mjff/J, stock number 019106)³⁹ (link) and WT C57BL/6NJ mice (Jackson Laboratory, stock number 005304) as controls. Proof-of-concept experiments for GRN-FTD were completed using heterozygous and homozygous Grn^R493X mutant mice on the C57BL/6J background.⁴¹ (link) Donor and recipient animals for all experiments were 8–12 weeks of age. Terminal collection for all animals occurred 14–16 weeks after cell administration except for the long-term cohort for scRNA-seq experiments (12–13 months post-transplantation) and the long-term cohort for the GRN-FTD proof-of-concept study (32–36 weeks post-transplantation). At necropsy, animals first underwent whole-body transcardial perfusion with either heparinized saline or PBS followed by tissue harvesting. Samples for biochemistry analysis were flash frozen and stored at -80°C. Samples for immunohistochemistry (IHC) analysis were fixed in 4% PFA in PBS overnight at 4°C.

Heterozygous GBA1 D409V mice (C57BL/6N-Gba tm1.1 Mjff/J, stock number: 019106, Jackson Laboratory, Bar Harbor, ME) were rederived, bred, and maintained at Taconic. Animals were acclimated to housing for at least one week from delivery and kept on a normal 12 h/12 h light/dark cycle for a week or longer during which time access to food and water were provided ad libitum. Mice were divided into separate groups and fed chow formulated with a potent GCS inhibitor BZ1 or matching control chow for four days or four weeks. BZ1 content in chow (Rodent diet with 10 kcal% Fat and 220 mg of BZ1 API/kg; Research Diets, Inc., New Brunswick, NJ) was selected to target chronic exposure levels of unbound BZ1 approximating two times the reported GCS enzyme IC50 value of 18 nM (20 (link)). Weight of chow consumed was measured daily across individuals to estimate BZ1 daily doses and to verify similar feeding rates between mice-fed control versus BZ1-formulated diets. At the conclusion of treatment, animals were anesthetized with isoflurane (1–4% in oxygen at 2 l/minute) and blood was collected via cardiac puncture immediately prior to euthanasia via decapitation. Plasma was prepared and stored at −70°C until lipid extraction and analyses. All animal studies were performed under the approval of the Merck & Co., Inc., Kenilworth, NJ, Institutional Animal Care and Use Committee.