A recent development has produced the K14 lnl/lnl mouse model of type 2 Gaucher disease (hereinafter referred to as K14); this mousemodel closely recapitulates the human disease, showing ataxia, seizures, spasticity and a median lifespan of only 14 days. [3]. As in patients with nGD, several mouse models of the disease have increased levels of GluCer and GluSph in the brain due to the deficiency in GC activity [4,5]. A homozygous GC knockout mouse presents with an ,100-fold elevation of GluSph in the brain as early as day 13 of gestation, and these levels increase until early neonatal death from disruption of the epithelial barrier of the skin [3,6]. An increase in GluSph has also been observed in human fetuses with type 2 Gaucher disease, thus confirming the similarity of the pathologic processes in mice and humans [7]. Restricting GC expression to the skin with a keratin-14 promoter helped overcome the early mortality observed in previous mouse models of the disease. The resulting “K14” mice display a neuropathic phenotype that shares many pathologic features with type 2 Gaucher disease, such as neurodegeneration, astrogliosis, microglial proliferation, and increased levels of GluCer and GluSph in specific brain regions [3].
Clinical management of patients affected by nGD poses a challenge for treating physicians both because of the severity of type 2 disease and the inability of the current therapies to cross the blood brain barrier (BBB). In type 3 Gaucher disease, treatment using high doses of intravenous recombinant human glucocerebrosidase (rhGC) has been evaluated [8]. Even though this treatment is useful for reducing visceral disease, it has not provided convincing evidence of its ability to reduce the rate of progression of the neurological symptoms [9,10]. Recent studies have explored the possibility of directly administering lysosomal enzymes to the brain to circumvent the BBB, and have shown efficient biodistribution, clearance of substrate, amelioration of pathology and behavioral improvements in mouse models of Neuronal Ceroid Lipofuscinosis, Niemann-Pick disease and Gaucher disease [11,12]. Indeed, we have shown recently that brain pathology and survival in the K14 Gaucher mouse model could be enhanced by intracerebroventricular injection of GC in neonatal animals [13]. Miglustat, a marketed, non specific glucosylceramide synthase inhibitor (GSI) which crosses the BBB does not appear to address the neuropathic symptoms of nGD. To this point, we have shown in a mouse model of Sandhoff disease [14] that NB-DNJ (active ingredient in miglustat) counterintuitively increases brain GluCer levels, while GZ 112638 a specific GSI in clinical trials, which does not cross the BBB, has no effect as expected. To identify a glucocerebroside synthase (GCS) inhibitor with the ability to cross the BBB, we screened a series of novel synthetic compounds, resulting in the nomination of GZ 161 as a potential therapeutic. An evaluation of GZ 161 in the K14 mouse model of type 2 Gaucher disease demonstrated that it could indeed reduce brain GluCer and GluSph. It also reduced brain neuropathology and extended the lifespan of this model. Although this substrate reduction approach may have promise for type 3 Gaucher disease, in the K14 model inhibiting GCS in this way did not appear to be as efficacious as supplying GC directly to the murine brain. Thus a combined approach using both enzyme replacement and small molecule substrate reduction may, at least in principle, represent a more effective approach for neuropathic Gaucher disease.did not appear to have an impact on brain weight (relative to that of WT mice). Given the known toxicity of GluSph, therapeutic strategies geared towards reducing the accumulation of these substrates in the K14 mouse brain might be expected to have an impact on the pathologic features of the disease and the lifespan of the animals.
Intraperitoneal Administration of GZ 161 Reduces GluCer and GluSph Levels in the Brains of K14 mice
Figure 2 shows that compared to vehicle-treated K14 mice at the humane endpoint (14?5 days of age), daily intraperitoneal (IP) administration of GZ 161 reduced brain levels of both GluCer and GluSph by .60%. K14 mice treated with GZ 161 were asymptomatic at this time point. Even though GZ 161 administration significantly reduced the levels of these glycosphingolipids, Figure 2 shows that they nonetheless remained elevated severalfold over those of age-matched WT mice; GluSph was not detected in samples analyzed from WT or heterozygote littermates. The reduction of brain glycosphingolipids as a consequence of systemic drug administration strongly suggests that GZ 161 is both capable of crossing the blood brain barrier and inhibiting its target enzyme, GCS.
Intraperitoneal Administration of GZ 161 Reduces Microglial/macrophage Staining throughout the Brain of K14 mice
Cells of the myeloid lineage can be detected in the murine brain using antibodies to antigens such as F4/80 and CD68. F4/80 is a transmembrane glycoprotein found on ramified (quiescent) microglia [16] and macrophages, while CD68 is a lysosomal protein expressed at relatively high levels in macrophages and activated (reactive) microglia and at lower levels in ramified microglia [17,18,19]. Increased F4/80 and CD68 staining in the brain may occur through recruitment of monocytes or microglial proliferation [20] and is a normal response to injury and inflammation. Figure 3 shows qualitatively and quantitatively that compared to wild type mice at 10 days of age (P10), the K14 mouse brain has increased numbers of CD68+ cells in multiple locations (hippocampus, thalamus, brainstem, cerebellum). The greatest concentration of CD68+ cells was seen in the thalamus and brainstem, two sites that also show pathology in type 2 Gaucher patients [21,22,23]. Figure 3 also shows that systemic administration of GZ 161 reduces the numbers of CD68+ cells in all of these locations; treatment also reduced CD68+ cells in the olfactory bulb and frontal cortex (data not shown). Consistent with the CD68 histopathology, Figure 4 shows increased F4/80 staining relative to WT animals in vehicle treated K14 mice at P10. Daily IP injections of GZ 161 reduced the numbers of F4/80+ cells in the thalamus and brainstem, but had marginal effects in other brain regions. Taken together with the CD68 data, these results suggest that systemic treatment of the K14 mouse with GZ 161 results in decreased numbers of macrophages/microglia in multiple brain regions. Whether these decreases are stable relative to vehicle treated K14 mice or simply indicate a delay in the accumulation of these cells in the brain is not clear.