Brain Cholesterol Biosynthetic Pathway Is Altered in a Preclinical Model of Fragile X Syndrome

Abstract

Fragile X Syndrome (FXS) is the most frequent form of inherited X-linked pathology, associated with an intellectual and developmental disability, and currently considered the first monogenic cause of autism spectrum disorder (ASD). Low levels of total cholesterol reported in the serum of FXS patients, and evidence that FMRP targets a subset of mRNAs encoding proteins of lipid synthesis and transport suggests that the cholesterol metabolism impairments could be involved in FXS. Thus, the aim of the presented work was to investigate the modulations of the cholesterol biosynthetic pathway and its end-products in a recently developed Fmr1-Δexon 8 rat model of FXS. Here, we show that this experimental model mimics what is found in FXS patients, exhibiting a lower serum cholesterol content, accompanied by a reduction in food intake and body weight compared to WT animals. Moreover, alterations of proteins committed to cholesterol synthesis and uptake have been observed in the amygdala, prefrontal cortex and nucleus accumbens. Interestingly, the end-products show a brain region-dependent modulation in Fmr1-Δexon 8 rats. Overall, our results demonstrate that the cholesterol biosynthetic pathway is altered in some brain regions of this preclinical model of FXS. This finding has relevance for future studies to delve deeper into the involvement of this metabolic process in FXS, and thus its possible role as a therapeutic target.

Keywords: 3-Hydroxy 3-methylglutaryl Coenzyme A reductase; Fmr1-Δexon 8 rat; Fragile X Syndrome; brain; cholesterol; liver; low-density lipoprotein receptor; plasma; prenylated proteins.

Figure 1

A schematic illustration of the mevalonate pathway.

Figure 2

HMGCR and LDLR protein level in the liver of Fmr1-^Δexon 8 and WT male adolescent (PND35) and adult (PND90) rats. (A) illustrates a typical Western blot (top) and the densitometric analysis of HMGCR and LDLR in PND35 rats. (B) illustrates a typical Western blot (top) and the densitometric analysis of HMGCR and LDLR in PND90 rats. Values represent mean ± SD obtained from 6 animals performed in duplicate. Tubulin served as a housekeeping protein to normalize protein loading. Statistical analysis was carried out by using the unpaired Student’s t-test.

Figure 3

Body weight and food intake measurements of the Fmr1-^Δexon 8 and WT male rats. (A) shows the weight of the Fmr1-^Δexon 8 and WT adolescent (PND35) and adult (PND90) rats at the moment of euthanasia. (B) illustrates the grams of food intake of Fmr1-^Δexon 8 and WT PND90 rats, measured during the last 6 days before euthanasia. Values represent mean ± SD obtained from 6 animals performed in duplicate. Statistical analysis was carried out by using the unpaired Student’s t-test. °°° = p < 0.001 vs. WT PND90; **** = p < 0.00001 vs. PND35; ** = p < 0.01 vs. WT.

Figure 4

HMGCR and LDLR protein level in brain areas of Fmr1-^Δexon 8 and WT male adolescent (PND35) and adult (PND90) rats. (A) illustrates a typical Western blot (top) and the densitometric analysis of HMGCR and LDLR in the amygdala of PND35 and PND90 rats. (B) illustrates a typical Western blot (top) and the densitometric analyses of HMGCR and LDLR in the prefrontal cortex (CORTEX) of PND35 and PND90 rats. (C) illustrates a typical Western blot (top) and the densitometric analyses of HMGCR and LDLR in nucleus accumbens of PND35 and PND90 rats. Tubulin served as a housekeeping protein to normalize protein loading. Values represent mean ± SD obtained from 6 animals performed in duplicate. Statistical analysis was carried out by using the unpaired Student’s t-test. * = p < 0.05 vs. WT; ** = p < 0.01 vs. WT; *** = p < 0.001 vs. WT; **** = p < 0.0001 vs. WT.

Figure 5

CYP46A1 and nSREBP2 protein level in amygdala of Fmr1-^Δexon 8 and WT adult (PND90) male rats. (A) illustrates a typical Western blot (top) and the densitometric analyses of CYP46A1 in the amygdala of PND90 rats. (B) illustrates a typical Western blot (top) and the densitometric analyses of nSREBP2 in the amygdala of PND90 rats. Vinculin served as a housekeeping protein to normalize protein loading. Values represent mean ± SD obtained from 6 animals performed in duplicate. Statistical analysis was carried out by using the unpaired Student’s t-test. * = p < 0.05 vs. WT; *** = p < 0.001 vs. WT.

Figure 6

Membrane-bound and total level ratio of HRas and RhoA protein content in brain areas of Fmr1-^Δexon 8 and WT adult (PND90) male rats. The figure illustrates a typical Western blot and a graph representing the ratio of the densitometric analysis between membrane-bound and total levels of HRas (top) and RhoA (bottom) in PND90 rats. The loading control was performed by using caveolin 1 (Figure S3). (A): amygdala; (B): prefrontal cortex (cortex); (C): nucleus accumbens. Values represent mean ± SD obtained from 6 animals performed in duplicate. Statistical analysis was carried out by using the unpaired Student’s t-test. ** = p < 0.01 vs. WT.