Enzyme blockade: a nonradioactive method to determine the absolute rate of cholesterol synthesis in the brain

Abstract

The standard in vivo method to determine rates of brain cholesterol synthesis involves systemic injection of 3 H 2 O and measurement of incorporated radioactivity in sterols. Herein, we describe an alternative method ("enzyme blockade") that obviates the use of radioactivity. The method relies on the ability of AY9944, a potent and relatively selective inhibitor of cholesterol synthesis, to cause the time-dependent accumulation of 7-dehydrocholesterol (DHC), a cholesterol precursor detected with sensitivity and specificity by reverse-phase HPLC-coupled spectrophotometry at 282 nm. To validate the method, adult AY9944-treated and control mice were injected with [ 3 H]acetate. After 24 h, most of the radioactivity in brain sterols from treated mice accumulated in DHC, without significantly perturbing overall sterol pathway activity, compared with controls (where cholesterol was the dominant radiolabeled sterol, with no label found in DHC). When adult mice were treated continuously with AY9944, the time-dependent accumulation of DHC in brain was linear (after ‫ف‬ 8 h) for 3 days. The rate of brain cholesterol synthesis determined by this method ( ‫ف‬ 30 g/g/day) closely agrees with that determined by the radioactive method. We also determined the cholesterol synthesis rate in different regions of adult mouse brain, with frontal cortex having the highest rate and cerebellum having the lowest rate. There is growing interest in, and experimental documentation of, the relationship between central nervous system (CNS) cholesterol metabolism and neurodegeneration, particularly in age-related diseases such as Alzheimer's disease (AD) (1-3) and age-related macular degeneration (ARMD) (4, 5). For instance, retrospective studies have suggested that statins, which act on the rate-limiting enzyme of cholesterol synthesis (HMG-CoA reductase), decrease the risk of developing AD (6), and expression of specific allelic isoforms of apolipoprotein E, a cholesterol transport protein, is associated with an increased risk of developing AD (7). The relationship between statin use, apolipoprotein E isoform expression, and ARMD development or progression, although suggestive, is somewhat more speculative (8-11). These findings provide the impetus for developing techniques to investigate cholesterol metabolism in the CNS. In general, the rate of de novo cholesterol synthesis in the mammalian brain is relatively high in the fetus and newborn, where it is synthesized de novo, with little or no contribution from maternal sources (12, 13). Brain cholesterol synthesis decreases precipitously after weaning (14), as the rate of myelination dramatically declines [for review, see ref. (15)]. The fate of cholesterol in the adult CNS is uncertain, but all indications are that the turnover is relatively slow, with a half-life on the order of months (16). A portion of brain cholesterol is metabolized to 24 S -hydroxycholesterol, which then exits across the blood-brain barrier into the bloodstream (17). More than 50% of cholesterol release from...

Survival of adult neurons lacking cholesterol synthesis in vivo

“…Since these two very different populations of neurons show a similar tolerance for loss of the fdft1 gene, this might be a general feature of adult neurons. However, the adult cerebellum has only about half the steady state level of cholesterol production compared to the forebrain, indicating that the cholesterol requirements might differ between different brain regions [24]. A further indication for diversity comes from culture experiments where hippocampal neurons have different cholesterol requirements compared to cortical neurons [25].…”

A critical role for the cholesterol‐associated proteolipids PLP and M6B in myelination of the central nervous system

“…6C), a method suitable for identifying qualitative differences (Lappe-Siefke et al, 2003;Saher et al, 2005). When HPLC was used to quantify cholesterol levels (Keller et al, 2004), cholesterol was reduced by about 40% in PLP null and PLP null *M6B null myelin compared with M6B null and WT myelin (Fig. 6D).…”

“…Quantification of myelin sterols (n 5 3 animals per genotype) was as described (Keller et al, 2004). Briefly, lipids were extracted with choloroform/methanol (1:1, v/v).…”

The ins and outs of cholesterol in the vertebrate retina

“…The brain has the highest cholesterol content ( ‫ف‬ 14 mg/g wet weight) of all organs in the body compared with only ‫ف‬ 2-3 mg/g in most tissues ( 7,8 ), including the neural retina and RPE ( 9 ). When compared with the major cholesterogenic tissues (i.e., liver and intestine), the rate of cholesterol synthesis in the mature brain and retina is relatively slow ( 10,11 ). Despite this, however, essentially all of the cholesterol in the brain is derived from endogenous de novo synthesis, most of which is utilized to support the primates) spectral types according to their wavelength maxima.…”