Dependence of reversibility and progression of mouse neuronopathic Gaucher disease on acid beta-glucosidase residual activity levels

Abstract

Genetic and chemically induced neuronopathic mouse models of Gaucher disease were developed to facilitate understanding of the reversibility and/or progression of CNS involvement. The lethality of the skin permeability barrier defect of the complete gene knock out [gba, (glucocerebrosidase) GCase] was avoided by conditional reactivation of a low activity allele (D409H) in keratinocytes (kn-9H). In kn-9H mice, progressive CNS disease and massive glucosylceramide storage in tissues led to death from CNS involvement by the age of 14 days. Conduritol B epoxide (CBE, a covalent inhibitor of GCase) treatment (for 8-12 days) of wild type, D409H, D409V or V394L homozygotes recapitulated the CNS phenotype of the kn-9H mice with seizures, tail arching, shaking, tremor, quadriparesis, extensive neuronal degeneration loss and apoptosis, and death by the age of 14 days. Minor CNS abnormalities occurred after daily CBE injections of 100 mg/kg/day for 6 doses, but neuronal degeneration was progressive and glucosylceramide storage persisted in D409V homozygotes in the 2 to 5 months after CBE cessation; wild type and D409H mice had persistent neurological damage without progression. The persistent CNS deterioration, histologic abnormalities, and glucosylceramide storage in the CBE-treated D409V mice revealed a threshold level of GCase activity necessary for the prevention of progression of CNS involvement.

Figure 1. Schematic of mice containing k14-Cre/neo-9h gene constructs and PCR genotyping

(A) The gba structure of k14-Cre/neo-D409H (kn-9H) mice containing the loxP-neo in intron 8 gba with a D409H encoding point mutation in exon 9(*) (top line). The neo was excised in skin by k14-Cre (second line). The resultant recombinant D409H allele (*) contained one loxP (◊) site only in skin; the other tissues retain neo. WT gba is shown in the lower diagram. The B and C panels show the PCR genotyping for Cre transgenes. (B) A 400 bp PCR Cre containing fragment was in tissues of kn-9H (kn) mice, but not in WT (+) mice. (C) PCR genotyping shows that the mutant D409H allele (9H) was only re-activated in skin, but not in other tissues. The 391 bp PCR fragment (bottom band) is from WT (+) and the 485 bp PCR fragment (upper band) is from the skin of kn-9H (391+loxP, D409H) gba mice. Sk: skin; Lv: liver; and Sp: spleen. The (−) lanes are no template DNA controls.

Figure 2. kn–9H mouse phenotype

(A) Homozygote that had the neogene in intron 8 (neo-9h/neo-9h) (Fig. 1) pups was similar to the gba null mouse [13] with red-wrinkled dry skin and death after birth. (B) kn-9H pups (i.e., neo in intron 8 was present in all tissues but skin) were smaller than WT mice and had nearly normal skin. (C and D) These mice survived to the age of 14 days, but were small and severely neurologically impaired. They often flipped on their side and displayed tail arching, tremor, myoclonus and tonic/clonic seizures.

Figure 3. Immunofluorescence analyses of skin from kn-9H or WT mice

Biopsies were analyzed using anti-mouse GCase (mGCase) or anti-Cre, or -K14, antibodies. (A–C) mGCase signals (A, FITC) and Cre (B, Alexa-610) were present in skin cells of kn-9H mice. The merged images (C) show co-localization of Cre and mGCase (i.e., the D409H protein). K14 (D, FITC) and Cre signals (E, Alexa-610) were detected in skin of K14-Cre mice. Panel F merges D & E. In WT mice, only K14 (G), but no Cre (H) signals were detected. The magnifications are 400X ; scale bar, 50 µm.

Figure 4. Expression of mGCase in kn-9H and WT mice

Lung, liver, cerebellum and cerebrum sections from kn-9H (A–D) or WT (E–H) were treated with rabbit anti-mGCase antiserum for immunofluorescence, respectively. The mGCase positive signals (red) were detected in tissues from WT (E–H), but not from kn-9H (A–D). Cell nuclei were counterstained with DAPI (blue). The magnifications are 400X; scale bar, 50 µm.

Figure 5. CNS phenotypes of CBE-injected mice

After 6 daily injections (CBE; 100 mg/kg/day) beginning at post-partum day 5, WT and 9V mice displayed similar CNS signs to those of kn-9H mice. The CBE-4L mouse only showed paresis of the hind limbs after 24 injections.

Figure 6. Histological and immunochemical analyses of kn-9H mouse visceral tissues

Storage cells (arrows) were in liver (A), lung (B), thymus (C) and (D) bone marrow (BM) from 13 day old kn-9H mice, but not in littermate WT control tissues (E–H). The magnifications of H&E stained sections (A–H) are 1000X (Scale bar, 50 µm). CD68 positive macrophages (stained as dark brown) were detected in liver (I), lung (J), thymus (K), or BM (L) of kn-9H mice, but not in matched littermate WT control tissues (M–P). The magnifications of CD68 stained sections are 400X for liver, lung and thymus, and 1000X for BM (Scale bar, 50 µm).

Figure 7. Macrophage presence in visceral tissues of CBE-injected D409V mice

Liver, lung, thymus, or BM from CBE-injected 9V mice with 6 doses (middle row, E–H) or 2-month after the 6 doses (bottom row, I–L) were analyzed with anti-CD68 antibody. The CD68 positive cells were stained as dark brown. No-CBE controls showed absence of CD68 positivity (top row, A–D). The magnifications are 400X for liver, lung and thymus, and 1000X for BM. Scale bar, 50 µm.

Figure 8. Histological and immunochemical analyses of kn-9H mouse CNS

Degenerated neuron cells (arrows) were observed in cerebrum (A) or spinal cord (G) of day-13 kn-9H mice, but not in WT mice (D or J). CD68 positive microglial cells were present in cerebrum (B) and spinal cord (H) of kn-9H mice (dark brown cells), but not in WT control tissues (E or K). Many TUNEL positive cells (dark brown cells) were in the spinal cord (I), but only a few in cerebrum (C) of kn-9H mice. In WT (F or L) control mice, no neuronal degeneration, microglial response or apoptosis was observed in the cerebrum and spinal cord.

Figure 9. Neuronal lesions in CBE-9V mouse CNS

Spinal cord and cerebral sections from CBE-9V mice after 6 injections (middle column, B, E, H and K) or 2 month after the initial 6 injections of CBE (right column, C, F, I and L). Degenerated neuronal cells are shown by arrows and TUNEL positive cells are shown as dark brown. The corresponding no-CBE controls are in left column (A, D, G and J). The magnifications are 400X; scale bar, 50 µm.

Figure 10. Expression of CCL3 and CD68 in CNS of kn-9H mice

Spinal cord sections from kn-9H (bottom, D–F) and WT (top, A–C) mice were stained for CCL3 (FITC green, D, arrows) or anti-CD68 (Alexa-610 red, E, arrows). The co-localization of CD68 and CCL3 is shown in merged image (yellow, F, arrows). The magnifications are 400X; scale bar, 50 µm.

Figure 11. Glycosphingolipid (GSL) TLC profiles from kn-9H and CBE-treated mouse tissues

(A) GSLs of brain, lung, and kidney tissues from 13 day kn-9H (−/−) or littermates (+/+ or +/−) mice. Glucosylceramide was in many fold excess in kn-9H tissues compared to the unaffected counterpart (almost undetectable). (B) GSLs in brains from CBE-9V or CBE-WT mice. Mice were sacrificed after 6 injections (6Inj) of CBE or sacrificed 2 months after CBE injections (6InjR2m). CBE+ or − indicates injected or un-injected age and genotype matched mice. CBE-WT and CBE-9V show greatly and similarly increased GC after 6Inj (left panel). Large amounts of GC persisted in the brain of three 9V-6InjR2m mice (middle panel), whereas, little, if any, GC was present in matched WT-6InjR2m brains (right panel). GC in lung of 9V-6InjR2m was increased compared the untreated 9V mutant mice (middle panel). The 9V-6InjR2m mice that did not receive CBE show no GC accumulation in brain (middle panel). Arrows indicate the glucosylceramide in tissues. Migration standards are made from visceral organ spleen. These are GlcCer, glucosylceramide; GalCer, galactosylceramide; LacCer, lactosylceramide; Tri-cer, ceramide trihexoside; Sph, sphingomyelin.

Figure 11. Glycosphingolipid (GSL) TLC profiles from kn-9H and CBE-treated mouse tissues

(A) GSLs of brain, lung, and kidney tissues from 13 day kn-9H (−/−) or littermates (+/+ or +/−) mice. Glucosylceramide was in many fold excess in kn-9H tissues compared to the unaffected counterpart (almost undetectable). (B) GSLs in brains from CBE-9V or CBE-WT mice. Mice were sacrificed after 6 injections (6Inj) of CBE or sacrificed 2 months after CBE injections (6InjR2m). CBE+ or − indicates injected or un-injected age and genotype matched mice. CBE-WT and CBE-9V show greatly and similarly increased GC after 6Inj (left panel). Large amounts of GC persisted in the brain of three 9V-6InjR2m mice (middle panel), whereas, little, if any, GC was present in matched WT-6InjR2m brains (right panel). GC in lung of 9V-6InjR2m was increased compared the untreated 9V mutant mice (middle panel). The 9V-6InjR2m mice that did not receive CBE show no GC accumulation in brain (middle panel). Arrows indicate the glucosylceramide in tissues. Migration standards are made from visceral organ spleen. These are GlcCer, glucosylceramide; GalCer, galactosylceramide; LacCer, lactosylceramide; Tri-cer, ceramide trihexoside; Sph, sphingomyelin.