Temporal gene expression profiling reveals CEBPD as a candidate regulator of brain disease in prosaposin deficient mice

<p>Abstract</p> <p>Background</p> <p>Prosaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipid (GSL) hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian prosaposin deficiencies.</p> <p>Results</p> <p>Our hypomorphic prosaposin deficient mouse, PS-NA, exhibited 45% WT levels of brain saposins and showed neuropathology that included neuronal GSL storage and Purkinje cell loss. Impairment of neuronal function was observed as early as 6 wks as demonstrated by the narrow bridges tests. Temporal transcriptome microarray analyses of brain tissues were conducted with mRNA from three prosaposin deficient mouse models: PS-NA, prosaposin null (PS-/-) and a V394L/V394L glucocerebrosidase mutation combined with PS-NA (4L/PS-NA). Gene expression alterations in cerebrum and cerebellum were detectable at birth preceding the neuronal deficits. Differentially expressed genes encompassed a broad spectrum of cellular functions. The number of down-regulated genes was constant, but up-regulated gene numbers increased with age. CCAAT/enhancer-binding protein delta (CEBPD) was the only up-regulated transcription factor in these two brain regions of all three models. Network analyses revealed that CEBPD has functional relationships with genes in transcription, pro-inflammation, cell death, binding, myelin and transport.</p> <p>Conclusion</p> <p>These results show that: 1) Regionally specific gene expression abnormalities precede the brain histological and neuronal function changes, 2) Temporal gene expression profiles provide insights into the molecular mechanism during the GSL storage disease course, and 3) CEBPD is a candidate regulator of brain disease in prosaposin deficiency to participate in modulating disease acceleration or progression.</p

Isofagomine In Vivo Effects in a Neuronopathic Gaucher Disease Mouse

The pharmacological chaperone, isofagomine (IFG), enhances acid β-glucosidase (GCase) function by altering folding, trafficking, and activity in wild-type and Gaucher disease fibroblasts. The in vivo effects of IFG on GCase activity, its substrate levels, and phenotype were evaluated using a neuronopathic Gaucher disease mouse model, 4L;C* (V394L/V394L + saposin C-/-) that has CNS accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) as well as progressive neurological deterioration. IFG administration to 4L;C* mice at 20 or 600 mg/kg/day resulted in life span extensions of 10 or 20 days, respectively, and increases in GCase activity and protein levels in the brain and visceral tissues. Cerebral cortical GC and GS levels showed no significant reductions with IFG treatment. Increases of GC or GS levels were detected in the visceral tissues of IFG treated (600 mg/kg/day) mice. The attenuations of brain proinflammatory responses in the treated mice were evidenced by reductions in astrogliosis and microglial cell activation, and decreased p38 phosphorylation and TNFα levels. Terminally, axonal degeneration was present in the brain and spinal cord from untreated and treated 4L;C* mice. These data demonstrate that IFG exerts in vivo effects by enhancing V394L GCase protein and activity levels, and in mediating suppression of proinflammation, which led to delayed onset of neurological disease and extension of the life span of 4L;C* mice. However, this was not correlated with a reduction in the accumulation of lipid substrates

Analyses of GC and GS levels in the spleen and lung by LC/MS.

<p>(A) Spleen GC level in 4L;C* mice was not different from that in the WT. GS level was accumulated in 4L;C* spleen. By 600 mg/kg/d IFG treatment, GC or GS were increased by 2-fold in 4L;C* spleen at 14 day or 28 day, respectively. (B) GC and GS were accumulated in 4L;C* lung. IFG treatment did not reduce the substrate level in 4L;C* lung. Increased GC level was in 600 mg/kg/d IFG treated lung at terminal stage (Age was same as spleen in Fig. 5A). The data represent the mean±S.E. and analyzed by Student's t-test. *, p<0.05 (n = 3 mice). The GC and GS levels were normalized by tissue weight.</p

Analyses of GC and GS levels in the cortex and liver by LC/MS.

<p>(A) Cortex GC and GS levels in IFG treated 4L;C* did not show significant reduction relative to the untreated mice. GC and GS levels remained accumulated at terminal stage in 600 mg/kg/d IFG treated 4L;C* brain. (B) Increased liver GC were detected in 600 mg/kg/d IFG treated 4L;C* mice at 14 and 44 days relative to untreated 4L;C* liver. GS level decreased in 20 mg/kg/d IFG treated 4L;C* liver at 28 days. GS level in 600 mg/kg/d IFG treated 28 day liver were higher than untreated 4L;C* liver. The data represent the mean±S.E. and analyzed by Student's t-test. *, p<0.05 (n = 3 mice). The GC and GS levels were normalized by protein level in the tissues lysate.</p

Life span and body weight.

<p>(A) Both treated and untreated 4L;C* mice had normal growth of their body weight till 35 days. The body weight of untreated 4L;C* mice started to decline at ∼35 days. The mice on 20 mg/kg/d IFG showed decrease of body weight at ∼35 days. The body weight of 4L;C* mice treated by 600 mg/kg/d IFG started decreasing at ∼48 days. Their body weight grew slower than untreated 4L;C* mice. (B) The mice in both cohorts had significantly extended life span to average 54 days (p = 0.0002) by 20 mg/kg/d or 63 days (P<0.0001) by 600 mg/kg/d IFG treatment relative to the untreated mice (∼48 days). The Kaplan-Meier survival curves were analyzed using the Mantel-Cox Test.</p

Proinflammation.

<p>(A) The brain stem and spinal cord sections were stained with anti-CD68 antibody, a macrophage-like marker. Positive CD68 signals (brown) on microglial cells demonstrated proinflammation. IFG (20 or 600 mg/kg/d) treated 4L;C* showed decreases of proinflammation at 44 days and increases of proinflammation at 59 or 64 days (terminal stage). (B) Enhanced GFAP staining (green) was in untreated 44 days old 4L;C* brain stem. IFG (20 mg/kg/d) treated 4L;C* showed decreases of GFAP signal at 44 days. GFAP signal were increased at terminal stage at 57 days. (C) GFAP protein in 44 days midbrain tissues was determined by immunoblot. Compared to untreated 4L;C*, GFAP levels were decreased in 20 mg/kg/d IFG and further reduced in 600 mg/kg/d IFG treated midbrain. (D) Phospho-p38 in 44 days midbrain tissues was analyzed by immunoblot. Increased Phospho-p38 was in untreated 4L;C* midbrain relative to WT. Phospho-p38 level was decreased in 20 mg/kg/d IFG treated midbrain and nearly to WT level in 600 mg/kg/d IFG treated mice. (E) TNFα and IL-6 mRNA in midbrain was determined by qRT-PCR. TNFα and IL-6 mRNA levels were increased by 35- and 13-fold in the untreated 4L;C* midbrain, respectively. In IFG (20 or 600 mg/kg/d) treated midbrain, TNFα mRNA were reduced at 44 or 45 days and at terminal stage (51 or 57 days). IL-6 mRNA level was not altered by IFG treatment. The data represent the mean±S.E. and analyzed by Student's t-test (n = 3 mice, assayed in duplicate).</p

GCase activity and protein.

<p>(A) GCase activity was significantly increased in the treated 4L;C* liver by 3-fold in 20 mg/kg/d or 6-fold in 600 mg/kg/d IFG treatment groups, respectively. In the midbrain GCase was significantly increased by 1.4-fold in 4L;C* mice treated with 600 mg/kg/d IFG, but not changed with 20 mg/kg/d IFG compared to the untreated group. The GCase activity in the 4L;C* spleen was enhanced by 600 mg/kg/d IFG, whereas both 20 mg and 600 mg/kg/d IFG increased GCase activity in the lung. (B) GCase protein was increased in the liver of IFG (20 or 600 mg/kg/d) treated 4L;C* mice. GCase protein were determined by immunoblot analyses using anti-mouse GCase antibody and normalized to β-actin signal in the same sample. The levels were presented as percentage relative to GCase in WT mice. All the tissues used in GCase activity and protein analyses were from 28 days old mice. The data represent the mean±S.E. for three mice assayed in triplicate and analyzed by Student's t-test. ***, p<0.001.</p

Treatment and phenotypes.

<p>(A) IFG treatment regime. IFG was given to pregnant females 3 to 5 days prior birth at 20 mg/kg/d. The first cohort was continued on 20 mg/kg/d for entire course. The dose for the second cohort was increased to 600 mg/kg/d at postnatal 7days. (B) Phenotypes of IFG treated 4L;C* mice. The mice with 20 mg/kg/d IFG showed delayed duck-walk gait (arrow) as seen in the untreated 4L;C* mice. The mice on 600 mg/kg/d IFG showed spastic walk.</p