Fig 2: IHC localization of ETV:IDS demonstrates more effective distribution into the brain parenchyma than IgG:IDS across multiple brain regions. The distribution of ETV:IDS and IgG:IDS was assessed using sagittal brain sections from TfRmu/hu KI mice 0.5, 4, and 24 h after an IV dose of 10 mg/kg. TfRmu/hu KI mice are on a C57BL/6 background. (A) Schematic indicating approximate location of sagittal brain regions of interest (ROIs) for images and quantification in Fig. 4, A and B (cortex), and Fig. S2 (hindbrain, hippocampus, and cerebellum; adapted from the Allen Adult Mouse Brain Atlas; original image credit: Allen Institute; Lein et al. [2007]). (B–D) Sections were immunostained with antibodies against huIgG, imaged, and post-stitched using a wide-field fluorescence slide scanner in the hindbrain (B), hippocampus (C), and cerebellum (D). Scale bars = 200 µm. Dashed boxes indicate regions shown at higher magnification displayed in the far-right panel; scale bars = 50 µm. Arrows indicate huIgG staining localized to vascular profiles; arrowheads indicate cellular internalization of huIgG staining. (E–G) Quantification of huIgG staining in the parenchyma of the hindbrain (E), hippocampus (F), and cerebellum (G) was calculated based on the total sum intensity of all parenchymal staining in the ROI divided by the total parenchymal area in the ROI. A custom macro script was used to identify blood vessels present in the tissue and masked out of subsequent image analyses. (H–J) The ratio of normalized huIgG signal in the brain parenchyma to brain vasculature was also quantified in the hindbrain (H), hippocampus (I), and cerebellum (J). Data presented are from a single study with n = 4–5 mice per group. Each symbol represents a biological replicate. Graphs display mean ± SEM and P values: two-way ANOVA with Sidak’s multiple comparison test; *, P = 0.05; **, P = 0.01; ***, P = 0.001; ****, P = 0.0001.

Fig 3: ETV:IDS normalizes neurobehavioral deficits in Ids-KO TfRmu/hu mice.Vehicle-treated TfRmu/hu (n = 22; black), vehicle-treated Ids-KO TfRmu/hu mice (n = 20; gray), and ETV:IDS-treated Ids-KO TfRmu/hu mice (n = 19; orange) underwent a battery of behavioral tests from week 11 to week 15 of dosing. (A) The proportion of mice that successfully completed the running trials at each treadmill speed was assessed: Fisher’s exact or ?2 test. (B) The latency to descend the pole was averaged across 3 trials: linear mixed effects model. (C) The level of pre-pulse inhibition of startle was assessed for each pre-pulse intensity: linear model. (D) The whole-body startle response (N) was assessed for each acoustic stimulus: linear mixed effects model. (E) In the active place avoidance assay (APA), mice were trained to avoid an unmarked aversive zone (orange) where a mild foot shock was presented upon entrance in the zone. The number of entrances in the aversive zone were measured during each phase of testing: linear mixed effects model to assess treatment effect in Ids-KO TfRmu/hu mice. (F and G) The entrances in the aversive zone were graphed for the third and reinstatement trials: Wilcoxon’s rank sum test. (H) The classification of performance in the APA was based on the number of entrances in the aversive zone during the reinstatement trial; poor (>1), good (1), top (0): Fisher’s exact test. (I) Hind paw withdrawal was measured in the hot plate test: Wilcoxon’s rank sum test, not significant. (J) Visual function was assessed by evaluating an animal’s preference toward the shallow versus the deep zones in the visual cliff test: ?2 test, not significant. All graphs display mean ± SEM except A and H. Comparison of vehicle-treated TfRmu/hu mice and Ids-KO TfRmu/hu mice, or as indicated by brackets; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 4: IHC localization of ETV:IDS demonstrates enhanced distribution into the brain parenchyma compared with IgG:IDS. The distribution of ETV:IDS and IgG:IDS was assessed using sagittal brain sections from TfRmu/hu KI mice 0.5, 4, and 24 h after an IV dose of 10 mg/kg. TfRmu/hu KI mice are on a C57BL/6 background. Schematic indicating approximate location of sagittal brain regions of interest (ROIs) for images and quantification is in Fig. S2 A. (A) Sections were immunostained with antibodies against huIgG, imaged, and post-stitched using a wide-field fluorescence slide scanner. Scale bars = 200 µm. Dashed boxes indicate cortical regions shown at higher magnification displayed in the far-right panel; scale bars = 50 µm. Arrows indicate huIgG staining localized to vascular profiles; arrowheads indicate cellular internalization of huIgG staining. (B) Quantification of huIgG staining in the parenchyma of the cortex was calculated based on the total sum intensity of all parenchymal staining in the ROI divided by the total parenchymal area in the ROI. A custom macro script was used to identify blood vessels present in the tissue and masked out of subsequent image analyses. The ratio of normalized huIgG signal in the brain parenchyma to brain vasculature was also quantified. Data presented are from a single study with n = 4–5 mice per group. Each symbol represents a biological replicate. Graphs display mean ± SEM and P values: two-way ANOVA with Sidak’s multiple comparison test; **, P = 0.01; ****, P = 0.0001. (C and D) Sagittal brain sections from TfRmu/hu KI mice 24 h after an IV dose of 10 mg/kg were immunostained with antibodies against huIgG and the neuronal marker NeuN (C) or huIgG and the endolysosomal marker LAMP2 (D). Confocal Z-stacks were acquired using SRCM. Scale bars = 10 µm. Dashed boxes indicate cortical regions shown at higher magnification displayed in the far-right panel; scale bars = 5 µm. For the analysis, huIgG-positive signal was segmented into vascular (magenta) and parenchymal (grayscale) components. Intraneuronal huIgG (yellow, shown with surface rendering; C) and intralysosomal huIgG (yellow, shown with surface rendering; D) was further segmented using either NeuN or LAMP2 (cyan) as a mask. In D, the merged panel shows both LAMP2 signal non-colocalized (cyan) and colocalized (yellow, shown with surface rendering) with huIgG signal. For better visualization, the subsequent intralysosomal panels show only the parenchymal LAMP2 signal that colocalized with huIgG (yellow). The intraneuronal (C) and intralysosomal (D) huIgG signal was quantified and normalized to the total neuronal volume or total lysosomal volume, respectively. Data presented are from a single study with n = 5 mice per group. Graphs display superimposed summary statistics from n = 5 mice (solid shapes) consisting of two to three different image volumes from each animal (open shapes). Each animal is coded by different shapes. The five means were then used to calculate the mean ± SEM and P values: unpaired t test analysis; **, P = 0.01; ***, P = 0.001. a.u., arbitrary unit.

Fig 5: ETV:IDS has improved brain uptake compared with IgG:IDS in TfRmu/hu KI mice. (A–C) Serum (A), liver (B), and brain (C) concentrations of ETV:IDS or IgG:IDS from TfRmu/hu KI mice were measured 0.5, 4, 8, and 24 h after an IV dose of 1, 3, or 10 mg/kg and determined using an IDS capture/IDS detect immunoassay. TfRmu/hu KI mice are on a C57BL/6 background. Data presented are from a single study. ELISA data represent the average of n = 2 technical replicates per mouse; each symbol represents an average of n = 3–5 mice per group per time point; graphs display mean ± SD.