Suppression of brain cholesterol synthesis in male Mecp2-deficient mice is age dependent and not accompanied by a concurrent change in the rate of fatty acid synthesis

Earlier studies in 56-day-old Jaenisch mice showed that, unlike the brain, other organs including the liver did not manifest a lower rate of cholesterol synthesis.

“The earlier studies in 56-day-old Jaenisch mice showed that, unlike the brain, other organs including the liver did not manifest a lower rate of cholesterol synthesis (Buchovecky et al., 2013).”

• cites Buchovecky et al., 2013 sourcePartly supported

  The Results section (page unavailable) reports decreased cholesterol synthesis in the brain of adult Jaenisch mice, but a modest increase—not a decrease—in their liver at P56. That supports the brain–liver contrast. Our copy does not establish the same result for other organs generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Studies using hemizygous Mecp2 mouse models have revealed disruptions to some aspects of lipid metabolism, including a partial suppression of cholesterol synthesis in the brains of mature Mecp2 mutants.

“Studies using hemizygous Mecp2 mouse models have revealed disruptions to some aspects of their lipid metabolism including a partial suppression of cholesterol synthesis in the brains of mature Mecp2 mutants.”

• cites Buchovecky et al., 2013 sourceSupported

An earlier publication using gas chromatography and mass spectrometry in Mecp2−/y mice and wildtype controls at 56 and 70 days showed significantly lower brain desmosterol concentrations in the mutants.

“An earlier publication utilizing this technique in Mecp2−/y mice and their wildtype controls at 56 and 70 days showed significantly lower brain desmosterol concentrations in the mutants.”

• cites Buchovecky et al., 2013 sourcePartly supported

  The Results section reports lower brain desmosterol in Mecp2 mutant males at P56 (p=.01) and a further decrease at P70, but gives no significance value for the P70 comparison. The Online Methods section says cholesterol intermediates were measured by tandem mass spectrometry; gas-liquid chromatography was used to measure tissue cholesterol, not desmosterol. No page numbers are available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The dissociation between cholesterol and fatty acid synthesis partly reflects the extraordinarily high demand for newly generated cholesterol for myelin formation in early development.

“This dissociation partly reflects the extraordinarily high demand for newly generated cholesterol for use in myelin formation in the early stages of development (Dietschy and Turley, 2004).”

• cites Dietschy and Turley, 2004 sourcePartly supported

  In “Cholesterol synthesis in the CNS” (page unavailable), the review says that most early sterol synthesis is associated with myelin production. The Conclusions likewise describe very high synthesis rates during perinatal development as primarily reflecting construction of membranes and compact myelin. These passages support the high demand for newly synthesized cholesterol, but do not establish the claimed dissociation between cholesterol and fatty acid synthesis. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The earlier finding of lower brain desmosterol concentrations in older Mecp2 mutants was consistent with the lower rates of brain cholesterol synthesis in older Mecp2 mutant mice.

“That finding was consistent with the lower rates of brain cholesterol synthesis in older Mecp2 mutant mice (Fig. 2A) (Buchovecky et al., 2013).”

• cites Buchovecky et al., 2013 sourceSupported

In 70-day-old Mecp2−/y mice (Bird allele) on a 129S6/SvEv background, plasma total cholesterols were about 50% higher than in wildtype controls.

“In 70-day-old Mecp2−/y mice (Bird allele) on a 129S6/SvEv background, plasma total cholesterols were about 50% higher than in their wildtype controls (Buchovecky et al., 2013).”

• cites Buchovecky et al., 2013 sourcePartly supported

  The Results section (‘Mecp2 null males have abnormal cholesterol metabolism’; page not provided) reports significantly elevated total serum cholesterol in 129-background Bird-allele mutant males relative to controls at postnatal day 56. It does not establish the claimed approximately 50% increase at day 70; the Online Methods specify serum-cholesterol assessments at days 28 and 56. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The present data confirm and extend earlier published findings that specific aspects of cholesterol metabolism in the CNS, particularly the biosynthetic pathway, are disrupted in mouse models with loss-of-function mutations in Mecp2.

“Together, the present data confirm and extend earlier published findings that specific aspects of cholesterol metabolism in the CNS, particularly the biosynthetic pathway, are disrupted in mouse models with loss-of-function mutations in Mecp2 (Buchovecky et al., 2013).”

• cites Buchovecky et al., 2013 sourceSupported

The present finding of lower rates of brain cholesterol synthesis in Mecp2 mutant mice is incongruous with earlier data showing a favorable response of this model to statin treatment.

“The final point of emphasis centers on the incongruity between the present finding of lower rates of brain cholesterol synthesis in the Mecp2 mutant mice and earlier data detailing the favorable response of this model to the subcutaneous administration of fluvastatin or lovastatin, starting at the beginning of the fifth or sixth week after birth (Buchovecky et al., 2013).”

• cites Buchovecky et al., 2013 sourceSupported

Data presented here and earlier show that the fall in brain cholesterol synthesis in Mecp2 mutants is not seen in organs like the liver and lung.

“Although the technique has yet to be applied to obtain rates of cholesterol synthesis in all the major organs in Mecp2−/y mice and their controls at different ages, data presented here (Fig. 3), and earlier (Buchovecky et al., 2013), show that the fall in brain cholesterol synthesis in the mutants is not seen in organs like the liver and lung.”

• cites Buchovecky et al., 2013 sourcePartly supported

  The Results section reports decreased cholesterol synthesis in the adult Mecp2 mutant brain, while liver synthesis showed a modest increase in one mutant line rather than the same decrease. Our copy does not report lung cholesterol-synthesis measurements, so the claim’s extension to the lung is unsupported (pages unavailable). (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The beneficial effects of statins in Mecp2-deficient mice presumably reflect mechanisms of action other than inhibition of cholesterol synthesis, given that brain cholesterol synthesis is already moderately suppressed in these mice.

“As statins were designed to act primarily by inhibiting cholesterol synthesis, their beneficial effects in the Mecp2-deficient mouse presumably reflect other mechanisms of action given that brain cholesterol synthesis is already moderately suppressed in these mice (Orth and Bellosta, 2012; Martin et al., 2014).”

• cites Orth and Bellosta, 2012 sourcePartly supported

  Section 7, “Cholesterol Synthesis Inhibitors” (page not provided), says statins primarily inhibit Hmgcr and may affect brain function through pathways other than cholesterol synthesis. It does not address Mecp2-deficient mice, whether their brain cholesterol synthesis is suppressed, or why statins benefit those mice. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Martin et al., 2014 sourcePartly supported

  The ‘Cholesterol in brain disease: hereditary causes’ section (page unavailable) reports decreased brain sterol synthesis in adult Mecp2-mutant mice and improved behavior and lifespan after fluvastatin treatment. The ‘Therapeutic approaches to cholesterol-related brain diseases’ section (page unavailable) describes other possible beneficial actions of statins. The paper does not conclude that those other actions explain fluvastatin’s benefits in the Mecp2-deficient mice. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: Statins act primarily by inhibiting cholesterol synthesis.

The values for brain cholesterol concentration in 7-day-old animals, and to some extent in 14-day-old mice, include desmosterol, an immediate precursor of cholesterol.

“Another point regarding the brain cholesterol concentration measurements is that the values for the 7-day-old animals in particular, and to some extent for the 14-day-old mice, include desmosterol, an immediate precursor of cholesterol (Paoletti et al., 1965; Sharpe and Brown, 2013).”

• cites Paoletti et al., 1965 sourceNot checked — no full text of the source
• cites Sharpe and Brown, 2013 sourcePartly supported

  The paper’s discussion of DHCR24 identifies desmosterol as its substrate in the cholesterol-synthesis pathway and cites work on desmosterol accumulation in the developing brain (indexed as Abstract; page unavailable). It does not say that cholesterol concentration measurements in 7-day-old or 14-day-old mice included desmosterol, so the measurement-specific claim is unsupported by this source. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

In 56-day-old mice, fatty acid synthesis rates in the brain and lungs did not vary with genotype.

“The fatty acid synthesis rates in the brain (Fig. 4G) and lungs (Fig. 4I) also did not vary with genotype”

Cholesterol synthesis rates in the liver and lungs of 56-day-old Mecp2−/y mice were normal.

“Cholesterol synthesis rates in the liver and lungs of 56-day-old Mecp2−/y mice were normal.”

• Also stated in: § 2.2 Suppression of brain cholesterol synthesis in Mecp2−/y mice was not evident until the third week after birth · plain

  “In the case of the liver (Fig. 4E) and lungs (Fig. 4F), cholesterol synthesis rates did not show a genotypic difference.”

The age-related decline in lipogenesis was much more gradual than the age-related decline in sterol synthesis.

“and second, that the age-related decline in lipogenesis was much more gradual than was the case for sterol synthesis.”

In 7- and 14-day-old mice there was no genotypic difference in the rate of brain cholesterol synthesis.

“In the 7- and 14-day-old mice there was no genotypic difference in the rate of brain cholesterol synthesis”

• Also stated in: § 2.2 Suppression of brain cholesterol synthesis in Mecp2−/y mice was not evident until the third week after birth · plain

  “No genotypic difference in the rate of cholesterol synthesis was discernible at 7 or 14 days postpartum”

Plasma total cholesterol levels in 43-day-old Mecp2−/y mice (98.9 ± 0.8 mg/dl, n=9) were not significantly different from those in Mecp2+/y controls (89.2 ± 11.2 mg/dl, n=8).

“Plasma total cholesterol levels were determined in 43-day-old mice. The concentrations in the Mecp2−/y mice (98.9 ± 0.8 mg/dl, n=9) were not significantly different than in their Mecp2+/y controls (89.2 ± 11.2 mg/dl, n=8).”

At most age points, the body weights of Mecp2−/y mice were less than those of their Mecp2+/y controls.

“At most age points, the body weights of the Mecp2−/y mice were less than those of their Mecp2+/y controls (Fig. 1B).”

For Sqle, the mRNA level was lower in Mecp2−/y mutants only at 43 days.

“The mRNA level for Sqle (Fig. 5C) was lower in the mutants only at 43 days.”

From 21 days and later, the rate of brain cholesterol synthesis was marginally lower in Mecp2−/y mice than in age-matched Mecp2+/y littermates.

“but, from 21 days and later, it was always marginally lower in the Mecp2−/y mice than in age-matched Mecp2+/y littermates.”

• Also stated in: § 2.2 Suppression of brain cholesterol synthesis in Mecp2−/y mice was not evident until the third week after birth · boosted

  “but clearly by 21 days, the rate was lower in the mutants, and this remained the case at 28, 42 and 56 days of age.”

Brain weight was consistently lower in Mecp2−/y mice than in their Mecp2+/y controls except at 7 days of age.

“Brain weight was consistently lower in the Mecp2−/y mice than in their Mecp2+/y controls except at 7 days of age.”

• Also stated in: § 2.1 Mecp2−/y mice exhibited prototypical shortened lifespan and reduced brain weight · plain

  “As illustrated in Fig. 1C, from the second week after birth, the weight of the brain in the Mecp2−/y mice was consistently lower than in their Mecp2+/y controls.”

At no age was a genotypic difference detected in cholesterol concentration in the brain.

“At no age was a genotypic difference detected in either the rate of fatty acid synthesis or cholesterol concentration in the brain.”

• Also stated in: § 2.2 Suppression of brain cholesterol synthesis in Mecp2−/y mice was not evident until the third week after birth · plain

  “Despite the lower rate of cholesterol synthesis in the brains of Mecp2−/y mice after 14 days, the brain cholesterol concentration did not show a genotypic difference at any age (Fig. 2C).”

In 56-day-old mice, irrespective of Mecp2 genotype, total cholesterol concentration in the liver and lungs averaged 2.1 and 4.7 mg/g, respectively.

“In 56-day-old mice, irrespective of their Mecp2 genotype, the total cholesterol concentration in the liver and lungs averaged 2.1 and 4.7 mg/g, respectively (data not shown).”

In mice of both genotypes, the rate of cholesterol synthesis declined markedly after 21 days.

“In mice of both genotypes, the rate of cholesterol synthesis declined markedly after 21 days, in keeping with what has been well documented for the developing brain in several species (Jurevics and Morell, 1995; Quan et al., 2003).”

• cites Jurevics and Morell, 1995 sourceNot checked — no full text of the source
• cites Quan et al., 2003 sourceNot checked — no full text of the source

The rate of fatty acid synthesis at any age was much higher than the rate of cholesterol synthesis.

“Furthermore, when compared to the data in Fig. 2A, it is seen first, that the rate of fatty acid synthesis at any age was much higher than the rate of cholesterol synthesis”

At no age was a genotypic difference detected in the rate of fatty acid synthesis in the brain.

“At no age was a genotypic difference detected in either the rate of fatty acid synthesis or cholesterol concentration in the brain.”

• Also stated in: § 2.2 Suppression of brain cholesterol synthesis in Mecp2−/y mice was not evident until the third week after birth · plain

  “The next important finding was that, at no age, did a genotypic difference in the rate of fatty acid synthesis in these same brains become manifest (Fig. 2B).”

Apoe mRNA expression level showed no genotypic difference at any of the three ages.

“The mRNA expression level for both Apoe (Fig. 5E), and Cyp46a1 (Fig. 5F), showed no genotypic difference at any of the three ages.”

At 56 days, the brain cholesterol synthesis rate in Mecp2−/y mice (81 ± 8) was lower than in Mecp2+/y controls (105 ± 5).

“A comparable genotypic difference was also found at 56 days (81 ± 8 in the Mecp2−/y mice vs. 105 ± 5 in the Mecp2+/y controls).”

In 56-day-old mice, the fatty acid synthesis rate in the liver was clearly elevated in the Mecp2−/y mutants.

“but in the liver, the rate was clearly elevated in the mutants (Fig. 4H).”

At 42 days, the brain cholesterol synthesis rate in Mecp2−/y mice (133 ± 5) was 21% lower than in Mecp2+/y littermates (168 ± 2).

“However, at 42 days, the rate in the Mecp2−/y mice (133 ± 5) was 21% lower than in their Mecp2+/y littermates (168 ± 2).”

Cyp46a1 mRNA expression level showed no genotypic difference at any of the three ages.

“The mRNA expression level for both Apoe (Fig. 5E), and Cyp46a1 (Fig. 5F), showed no genotypic difference at any of the three ages.”

There was no genotypic difference in brain desmosterol concentration at 7 or 14 days.

“As shown in Fig. 3, there was no genotypic difference in the brain desmosterol concentration at 7 or 14 days, just as there was no difference in the rate of brain cholesterol synthesis at these two ages (Fig. 2A).”

The changes in cholesterol synthesis rates in the CNS from early neonatal life to adulthood seen here and in earlier studies far exceed the magnitude of the difference in brain cholesterol synthesis rates between Mecp2−/y and Mecp2+/y mice.

“In the case of the mouse, the changes in cholesterol synthesis rates in the CNS from early neonatal life to adulthood seen here (Fig. 2A), and in earlier studies (Quan et al., 2003), far exceed the magnitude of the difference in brain cholesterol synthesis rates between Mecp2−/y and Mecp2+/y mice.”

• cites Quan et al., 2003 sourceNot checked — no full text of the source

The effect of Mecp2 dysfunction on brain cholesterol synthesis rates is modest.

“Given that the effect of Mecp2 dysfunction on brain cholesterol synthesis rates is modest, it could be that this is also the case with any genotypic differences in oxysterol concentrations that are detected.”

The rate of brain cholesterol synthesis in Mecp2 mutant mice falls below normal beginning in the third week after birth.

“the brain cholesterol concentration in the mutant mice is unchanged from that seen in their matching wildtypes at all ages even though their rate of cholesterol synthesis falls below normal beginning in the third week after birth.”

In the present studies, no genotypic difference in brain Cyp46a1 mRNA levels was detected over the age span of 14 to 56 days.

“In the present studies, no genotypic difference in brain Cyp46a1 mRNA levels was detected over the age span of 14 to 56 days.”

Brain cholesterol concentration in Mecp2 mutant mice is unchanged from that seen in matching wildtypes at all ages.

“the brain cholesterol concentration in the mutant mice is unchanged from that seen in their matching wildtypes at all ages even though their rate of cholesterol synthesis falls below normal beginning in the third week after birth.”

By 43 days of age, there was a clear trend towards lower levels of mRNA for the three key cholesterol biosynthetic enzymes.

“By 43-days of age, there was however a clear trend towards lower levels of mRNA for these enzymes (Fig. 5).”

The conventional gas chromatographic method used was not sufficiently sensitive to determine desmosterol levels beyond 14 days of age.

“Our conventional gas chromatographic method was not sufficiently sensitive to determine desmosterol levels beyond 14 days of age.”

The absence of Mecp2 in the mutants at each age was confirmed.

“As shown in Fig. 5A, the absence of Mecp2 in the mutants at each age was confirmed.”

Once myelin formation begins to reach completion, the requirement for locally synthesized cholesterol falls dramatically.

“Once this process begins to reach completion, the requirement for locally synthesized cholesterol falls dramatically (Jurevics and Morell, 1995; Quan et al., 2003).”

• cites Jurevics and Morell, 1995 sourceNot checked — no full text of the source
• cites Quan et al., 2003 sourceNot checked — no full text of the source

The finding that at no stage of development was a genotypic difference in brain fatty acid synthesis detected points to a selective, albeit modest, downregulation of sterol biosynthesis starting in the third postnatal week.

“The finding that at no stage of development was a genotypic difference in brain fatty acid synthesis detected points to a selective, albeit modest, downregulation of sterol biosynthesis, starting in the third postnatal week.”

The lack of a detectable impact of the lower sterol synthesis rate on whole-brain cholesterol concentration suggests there was no disruption to the supply of cholesterol for myelin formation.

“This suggests there was no disruption to the supply of cholesterol for myelin formation”

Taken together, the findings point to a selective suppression in cholesterol synthesis in the developing brain starting at about the time that closure of the blood brain barrier is purported to occur in the mouse.

“Taken together then, all these findings point to a selective suppression in cholesterol synthesis in the developing brain starting at about the time that closure of the blood brain barrier is purported to occur in the mouse (Vorbrodt et al., 2001).”

• cites Vorbrodt et al., 2001 sourceNot checked — no full text of the source