The 4L/4L mice have ∼10% WT GCase activity and no apparent CNS abnormalities (22). Saposin C−/− mice showed ∼60% of WT GCase activity and protein in several tissues and developed a CNS phenotype around 1 year of age (23).

The 4L;C* mice were born in normal sized litters with expected Mendelian ratios for the offspring. They began showing phenotypic abnormalities and neurological deficits ∼30 days and died by ∼48 days (Fig. 1) due to inability to eat and drink. At ∼35 days, the 4L;C* mice had outwardly splayed hind limbs and their stomachs dragged when walking. The gait of 4L;C* mice was a duck-like waddling due to the splaying of their hindlimbs and apparent stiffness and progressive bradykinesia, relative to WT, that progressed until death when the 4L;C* mice were nearly immobile (Fig. 1). Also, grip weakness of the 4L;C* mice was progressive. By age 43–48 days, they weighed 25% less than the control groups (C+/−, 4L/4L, 4L/4L;C+/− and wt/wt; wt/wt) with an average of 17.2 g compared with 23.0 g in controls. All control mice had weights and lifespans similar to WT.

The neuronal cells of hippocampal sections in the brain were recorded as excitatory postsynaptic potential (EPSP) slope potentiation (LTP, long-term potentiations) on a MED64 array chamber in the warm artificial cerebral spinal fluid. After accounting for a baseline of activity, the hippocampi from 4L;C* mice at 35 days showed significant (P < 0.05) decreases of LTP, compared with the WT mice (Fig. 2), suggesting an overall reduction of hippocampal plasticity in 4L;C* mice.

In the hematoxylin and eosin (H&E) stained sections, axonal degeneration (Fig. 3 arrow) was evident in the brain stem, spinal cord and white matter of cerebellum of 4L;C* mice at 46 days. The anti-CD68 positive microglial cells were numerous in the brainstem, basal ganglion, thalamus, hypothalamus, cerebral cortex and spinal cord (Fig. 4A). Moderate astrogliosis as evidenced by anti-GFAP positivity was observed in the midbrain, brainstem, multiform layer of cerebral cortex, thalamus, corpus collosum, cerebellum and spinal cord (Fig. 4B). The enhanced microglial cell activation and astrogliosis indicates proinflammation in 4L;C* CNS. Lamp2 is a membrane protein located in the late endosome and lysosome (24). Lamp2 immunoreactivity was increased in thalamus (Fig. 5), brain stem, basal ganglion and white matter of cerebellum. P62/sequestosome 1 is involved in autophagic clearance of ubiquitinated proteins (25). Enhanced p62 signals were intense in the thalamus (Fig. 5) and scattered p62 positive cells were in brain stem, basal ganglion and cortex. Accumulation of p62 was found in the neurons and astrocytes, but not in the microglial cells (Fig. 5B). These results suggest impairment of autophagosome and lysosome apparatus in 4L;C* brain. Tunnel assays in 4L;C* CNS tissues were negative. No histological abnormalities were found in the visceral tissues of 4L;C* mice. The brain and spinal cord morphology in saposin C−/− and 4L/4L mice were normal at 46 days.

Ultrastructural studies of CNS regions revealed the inclusion materials in the neuronal cell processes from brain stem, spinal cord, white matter of cerebellum and midbrain regions of 43-day-old mice (Fig. 6). The inclusions were visualized as electron dense and lucent materials in axonal processes. The storage materials were heterogeneous and coarsely granular. The ultrastructure shows axonal cell processes expanded by numerous tightly packed single-membrane bound bodies consistent with phagolysosomes (Fig. 6C, E and F). The sequestered materials range from dense osmiophilic undigested material to partially digested electron-lucent material. In the hippocampus, dendritic processes contained membrane-like material. Separation of myelin sheath was evident in spinal cord (Fig. 6C), brainstem and midbrain regions of 43-day 4L;C* mice. Degenerating cells and swollen cell processes were identified in cortex and hippocampal regions. In many processes, the subcellular organelles were compressed to the periphery by the accumulating storage materials. The general appearance of the soma of neurons was normal in 4L;C* mice (Fig. 6D).

The GCase activity in 4L;C* was reduced to <10% of WT in liver, lung, spleen, brain and cultured skin fibroblasts. These levels were 40–60% of those in the respective tissues of 4L/4L mice (Fig. 7A). The GCase activity (WT enzyme) in saposin C−/− mice was ∼60% of those in saposin C+/+ mice (Fig. 7A). Liver GCase protein was determined by immunoblot using anti-mouse GCase antibody. Consistent with the activity data, 4L;C* mice had ∼40% of 4L/4L levels GCase protein levels (Fig. 7B). These results indicate that the deficiency of saposin C led to a concordant reduction in mutant V394L GCase protein and activity.

Relative to WT mice, marked increases of GS were present in cerebral cortex (∼20-fold) and midbrain (∼30-fold) from 43-day-old 4L;C* mice. Levels of GS also were massively increased in 43-day-old 4L;C* mouse lung and liver (Fig. 8A). GC levels were increased ∼3-fold in the liver and lung, and by ∼1.5-fold in the cortex and midbrain relative to WT. Increases of ceramide (1.6-fold) and sphingosine (3.4-fold) were found only in the liver of 4L;C*. Lactosylceramide levels was not significantly altered in 4L;C* mice. No excess accumulations of GC and GS were detected in 10 weeks 4L/4L and 13 months C−/− mouse brains (data not shown). Elevated GC (1.3-fold) and GS (6.5-fold) were detected in 4L;C* skin at 14 days.

GSL was analyzed in hippocampal tissue from 44-day-old mice. Both GC (3.4-fold) and GS (132-fold) were elevated in 4L;C* mice (Fig. 8C). Galactosylceramide (GalCer) levels in 4L;C* were at WT levels, indicating normal GalCer metabolism in 4L;C* mice. This result suggests a correlation of substrate accumulation with abnormal LTP in 4L;C* mice.

The following were from commercial sources: NuPAGE 4–12% Bis-Tris gel, NuPAGE MES SDS running buffer, Anti-GFAP mouse monoclonal antibody conjugated Alexa Fluor 488 (Invitrogen, Carlsband, CA, USA). 4-Methyl-umbelliferyl-β-d-glucopyranoside (4MU-Glc; Biosynth AG, Switzerland). Sodium taurocholate (Calbiochem, La Jolla, CA, USA). Rat anti-mouse CD68 monoclonal antibodies (Serotec, Oxford, UK). M-PER Mammalian Protein Extraction Reagent, ImmunoPure immobilized Protein G and BCA Protein Assay reagent (Pierce, Rockford, IL, USA). Hybond™-ECL™ nitrocellulose membrane and ECL detection reagent (Amersham Biosciences, Piscataway, NJ, USA). Anti-fade/DAPI, Methyl green, ABC Vectastain and Alkaline Phosphatase Kit II (Black) (Vector Laboratory, Burlingame, CA, USA). Anti-pig GFAP mouse monoclonal antibody (Sigma, St Louis, MO, USA). Anti-recombinant p62/SQSTM1 mouse monoclonal antibody (Abnova, Taipei, Taiwan). Anti-mouse NeuN mouse monoclonal antibody conjugated Alex Fluor 488 (Millipore, Temecula, CA, USA). Anti-mouse Lamp2 rat monoclonal antibody (Abcam, Cambridge, MA, USA).

Saposin C deficient (C−/−) mice (23) were generated by introducing a Cys → Pro substitution in the saposin C region of prosaposin. Substitution of this conserved cysteine (Cys) breaks one of the three disulfide bridges of saposin C and resulted in the specific deficiency of saposin C. This strategy has been used to produce several single and double saposin deficient mice (36,37). Saposin C−/− were cross-bred with point mutated GCase V394L homozygotes (4L/4L) (22) to generate C−/−;4L/wt and C+/−;4L/4L. 4L;C* mice (C−/−;4L/4L) were produced by intercrossing C−/−; 4L/wt and C+/−;4L/4L. The strain background of 4L;C* was C57BL/6J/129SvEV. The mice were maintained in microisolators in accordance with institutional guidelines under IACUC approval at Cincinnati Children's Hospital Research Foundation.

The 4L;C* and strain-matched WT mice were subjected to CO₂ narcosis and then, perfused with saline followed by 4% paraformaldehyde before tissue dissection. The tissues were fixed in 10% formalin or 4% paraformaldehyde and processed for paraffin or frozen blocks, respectively. Paraffin sections of brain were stained with H&E. Karnovsky's fixative was used for ultrastructural studies. CD68 monoclonal antibody staining was as described (14). Frozen sections were stained with mouse anti-pig glial fibrillary acidic protein (GFAP) that detects astrocytes, mouse anti-recombinant p62 that is an autophagic receptor and rat anti-mouse Lamp2 that is a late endosome and lysosomal membrane protein. Biotinylated goat anti-mouse followed by streptavidin-conjugated fluorescent 488 (green) and goat anti-rat conjugated to FITC were applied to the sections. Both mouse anti-pig GFAP and mouse anti-recombinant p62 antibodies have reactivities in the mouse tissues (38,39). Colocalization of p62 in neuron, astrocyte or microglial cells was carried out on frozen sections using mouse anti-recombinant p62 (1:100) followed by biotinylated goat anti-mouse and streptavidin-conjugated fluorescent 610 (red). Mouse anti-NeuN conjugated to Alexa Fluor 488 (1:100), mouse anti-GFAP conjugated to Alexa Fluor 488 (1:100) or rat anti-CD68 (1:100) were applied as marker for neuron, astrocyte or microglial cells, respectively. Rat anti-CD68 was detected by biotinylated goat anti-rat and streptavidin-conjugated fluorescent 488 (green). DAPI (blue) in anti-fade were used for cell nuclei staining.

GSLs in the tissues lysate (cortex, midbrain, liver and lung) were extracted and analyzed by LC/MS at the Lipidomic Core in Medical University of South Carolina. The GSL content was normalized to phosphate content in the same sample after lipid extraction. (GC and GalCer were not separated by LC/MS analysis.) Because 4L;C* mice had normal levels of saposin A and galactosylceramidase, 4L;C* and WT mice would have the same levels of GalCer. The alterations of GC and GalCer in the analysis will reflect the changes for GC. The data are presented as fold-change of GC in 4L;C* relative to WT after being normalized to phosphate content in the same sample. Hippocampal tissues were analyzed by a modified method in order to separate GC from GalCer at the Lipidomic Core in University of South Carolina. GC, GS and GalCer content in hippocampus were normalized to mg protein in the same samples. Three animals for each genotype were included in the analyses. Data were analyzed by Student's t-test.

Mouse tissues were homogenized and their GCase activities were determined fluorometrically with 4MU-Glc in 0.25% Na taurocholate and 0.25% Triton X-100 in the presence and absence of CBE (40). Assay mixtures were incubated for 60 min (37°C). WT control tissues were analyzed in parallel. Mouse liver GCase protein detection by immunoblot was as described (14). The amounts of GCase protein were quantitated by ImageQuant software relative to the amount of β-actin in the same sample. Data were analyzed by Student's t-test.

LTP was measured using the MED64 multielectrode array (Alpha Med Sciences, Kadoma, Japan) (41) as described previously (39). Animals were anesthetized using isofluorane and decapitated. Brains were removed and placed in an ice cold solution of aCSF. Parasagittal sections (350 µm) of the hippocampus were collected using a vibrotome and transferred to a warm (32°C), constantly oxygenated aCSF bath for a minimum of 1 h. Slices were transferred to the recording chamber, which consists of an 8 × 8 electrode array. Electrodes are 50 µm and are spaced 150 µm apart. Slices were placed so that the CA1 region of the hippocampus was placed across the grid, with recordings coming from the Schaffer collateral pathway. The section was constantly perfused with warmed (32°C) aCSF at a flow rate of 2 mL/min and supplied with humidified 95% O₂/5% CO₂. A stimulation and recording channel were selected out of the 64 possible electrodes. Single-pulse stimuli were delivered from the stimulation channel and the slope of the line from the resulting EPSPs was measured from the defined recording channel. Stimulus amplitude was selected by determining maximum response slope and then selecting a stimulus amplitude that produced a response that was ∼40% of the maximal response. Baseline recordings were collected for a minimum of 10 min. Following baseline, a theta burst stimulus (TBS; 5 Hz for 2 s) was applied to the slice from the stimulation channel. Single-pulse stimuli were again delivered and the resulting EPSPs were collected 1/min for 90 min following TBS. LTP was calculated by taking the percent increase in slope following TBS. Data were analyzed using Performer 2.0 software (Alpha Med Sciences). Six WT and eight 4L;C* mice were used in this study with two slices used per animal. The average of the two slices per animal was used in the subsequent analysis. Data were analyzed using a general linear model ANOVA with gene as a between subject factor and time as a repeated factor.