Helga Jurevics

Cholesterol for synthesis of myelin is made locally, not imported into brain.

Abstract: We examined whether cholesterol needed for myelin formation is locally synthesized or whether it comes from the circulation. The experimental design was to inject [3H]water and to use incorporation of label into brain cholesterol as a measure of the rate of accumulation of newly synthesized cholesterol in brain. The contribution of the circulation to this labeled cholesterol pool was minimized by repressing liver synthesis of cholesterol with a high cholesterol diet. The rate of accumulation of total cholesterol was calculated from the increasing amounts of sterol in brain regions at successive time intervals during development. Thus, accumulating cholesterol not explained as being newly synthesized (radioactive) could be assumed to have come from the circulation. Long‐Evans rats, ranging in age from birth to 35 days, were injected intraperitoneally with [3H]water (0.3–1.0 mCi/g of body weight) and killed 2 h later. The brain was dissected into brainstem, cerebellum, and cerebral hemispheres, and total lipids were extracted. Cholesterol and its precursors were quantified by HPLC. The radioactivity associated with the sterol fractions and the specific activity of body water determined from serum were used to calculate the absolute amount of newly synthesized sterol. The rates of cholesterol synthesis were compared with the rates of accumulation of total cholesterol in each brain region. The rate of accumulation of total sterol (cholesterol and desmosterol) closely followed that of newly synthesized total sterol in all brain regions from the second through the fifth postnatal weeks. Thus, all sterol accumulation in brain during the period of rapid myelination can be explained by local synthesis; neither diet nor production of cholesterol by other organs plays a direct role in supplying cholesterol for myelination in brain.

Origin of cholesterol in myelin

We review some of the older literature concerning metabolic turnover of cholesterol in the nervous system. The overall picture is that incorporation of radioactive precursors into brain cholesterol is roughly proportional to the rate of myelination and that, once incorporated, radioactive cholesterol is relatively stable metabolically. We outline a strategy for demonstrating the source (local synthesis or uptake from the circulation) of cholesterol in brain. The experimental design involves determining the rate of accumulation of cholesterol this is calculated as the increasing amounts of sterol in brain at successive time intervals during development. The rate of appearance of newly synthesized cholesterol is determined from incorporation of radioactivity from3H2O (injected i.p. several hours prior to sacrifice) into cholesterol. The radioactivity associated with the sterol fractions and the specific activity of body water determined from the serum can be used to calculate the absolute amount of sterol newly synthesized during the time when3H2O was present. The results obtained demonstrated that all of the bulk cholesterol accumulating in brain can be accounted for by newly synthesized cholesterol. None of the radioactive cholesterol came from the circulation, since cholesterol feeding suppressed cholesterol biosynthesis in the liver and specific radioactivity of circulating cholesterol was negligible. Thus, almost all cholesterol accumulating in brain during development is locally synthesized.

Parameters related to lipid metabolism as markers of myelination in mouse brain.

Myelination, during both normal development and with respect to disorders of myelination, is commonly studied by morphological and/or biochemical techniques that assay as their end‐points the extent of myelination. The rate of myelination is potentially a more useful parameter, but it is difficult and time‐consuming to establish, requiring a complete developmental study with labor‐intensive methodology. We report herein development of methodology to assay the absolute rate of myelination at any desired time during development. This involves intraperitoneal injection of 3H2O to label body water pools, followed by determination of label in the myelin‐specific lipid, cerebroside. The absolute amount of cerebroside synthesized can then be calculated from the specific radioactivity of body water and knowledge of the number of hydrogens from water incorporated into cerebroside. During development, the rate of cerebroside synthesis correlated well with the rate of accumulation of the myelin‐specific components, myelin basic protein and cerebroside. For purposes of control, we also tested other putative, albeit less quantitative, indices of the rate of myelination. Levels of mRNA for ceramide galactosyltransferase (rate‐limiting enzyme in cerebroside synthesis) and for myelin basic protein did not closely correlate with myelination at all times. Cholesterol synthesis closely matched the rate of cholesterol accumulation but did not track well with myelination. Synthesis of fatty acids did not correlate well with accumulation of either fatty acids (phospholipids) or myelin markers. We conclude that measurement of cerebroside synthesis rates provides a good measure of the rate of myelination. This approach may be useful as an additional parameter for examining the effects of environmental or genetic alterations on the rate of myelination.

Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination.

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6u2003weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down‐regulated during demyelination and up‐regulated during remyelination. Genes related to microglia/macrophages appeared in a time‐course (peaking at 6u2003weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4u2003weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.

Cerebroside synthesis as a measure of the rate of remyelination following cuprizone-induced demyelination in brain.

We studied markers of myelin content and of the rate of myelination in brains of mice between 8 and 20 weeks of age. During the 12‐week time‐course, control animals showed slight increases in the content of oligodendroglial‐specific cerebroside, as well as cholesterol (enriched in, but not specific to, myelin). In contrast, synthesis of these lipids, as assayed by in vivo incorporation of 3H2O, was substantial, indicating turnover of 0.4% and 0.7% of total brain cerebroside and cholesterol, respectively, each day. We also studied mice exposed to a diet containing 0.2% of the copper chelator, cuprizone. After 6 weeks 20%, and by 12 weeks, over 30% of brain cerebroside was gone. Demyelination was accompanied by down‐regulation of mRNA expression for enzymes controlling myelin lipid synthesis (ceramide galactosyl transferase for cerebroside; hydroxymethylglutaryl‐CoA reductase for cholesterol), and for myelin basic protein. Synthesis of myelin lipids was also greatly depressed. The 20% cerebroside deficit consequent to 6 weeks of cuprizone exposure was restored 6 weeks after return to a control diet. During remyelination, expression of myelin‐related mRNA species, as well as cerebroside and cholesterol synthesis were restored to normal. However, in contrast to the steady state metabolic turnover in the control situation, all the cerebroside and cholesterol made were accumulated. To the extent that accumulating cerebroside is targeted for eventual inclusion in myelin (discussed) the rate of its synthesis is proportional to remyelination. With our assay, in vivo rates of cerebroside synthesis can be determined for a time window of the order of hours. This offers greater temporal resolution and accuracy relative to classical methods assaying accumulation of myelin components at time intervals of several days. We propose this experimental design, and the reproducible cuprizone model, as appropriate for studies of how to promote remyelination.

Regenerating Sciatic Nerve Does Not Utilize Circulating Cholesterol

The rapid accumulation of myelin in the peripheral nervous system during the early postnatal period requires large amounts of cholesterol, a major myelin lipid. All of the cholesterol accumulating in the developing rat sciatic nerve is synthesized locally within the nerve, rather than being derived from the supply in lipoproteins in the systemic circulation (Jurevics and Morell, J. Lipid Res. 5:112–120; 1994). Since this lack of utilization of circulating cholesterol may relate to exclusion by the blood-nerve barrier, we examined the sources of cholesterol needed for regeneration following nerve injury, when the blood-nerve barrier is breached. One sciatic nerve was crushed or transected, and at various times later, the rate of cholesterol accumulation was compared with the rate of local in vivo synthesis of cholesterol within the nerve, utilizing intraperitoneally injected 3H2O as precursor. The accumulation of additional cholesterol in nerve during regeneration and remyelination could all be accounted for by that locally synthesized within the nerve. There was also an increase in cholesterol esters in injured nerve segments; in crushed nerves, these levels decreased during regeneration and remyelination, consistent with reutilization of cholesterol originally salvaged by phagocytic macrophages and Schwann cells. Thus, regeneration and remyelination following injury in sciatic nerve utilizes both salvaged cholesterol and cholesterol synthesized locally within the nerve, but not cholesterol from the circulation.

Normal metabolism but different physical properties of myelin from mice deficient in proteolipid protein

Proteolipid protein (PLP) is the primary protein component of CNS myelin, yet myelin from the PLPnull mouse has only minor ultrastructural abnormalities. Might compensation for a potentially unstable structure involve increased myelin synthesis and turnover? This was not the case; neither accumulation nor in vivo synthesis rates for the myelin‐specific lipid cerebroside was altered in PLPnull mice relative to wild‐type (wt) animals. However, the yield of myelin from PLPnull mice, assayed as levels of cerebroside, was only about 55% of wt control levels. Loss of myelin occurred during initial centrifugation of brain homogenate at 20,000g for 20 min, which is sufficient to sediment almost all myelin from wt mice. Cerebroside‐containing fragments from PLPnull mice remaining in the supernatant could be sedimented by more stringent centrifugation, 100,000g for 60 min. Both the rapidly and the more slowly sedimenting cerebroside‐containing membranes banded at the 0.85/0.32 M sucrose interface of a density gradient, as did myelin from wt mice. These results suggest at least some myelin from PLPnull mice differs from wt myelin with respect to physical stability (fragmented into smaller particles during dispersion) and/or density. Alternatively, slowly sedimenting cerebroside‐containing particles could be myelin precursor membranes that, lacking PLP, were retarded in their processing toward mature myelin and thus differ from mature myelin in physical properties. If this is so, recently synthesized cerebroside should be preferentially found in these “slower‐sedimenting” myelin precursor fragments. Metabolic tracer experiments showed this was not the case. We conclude that PLPnull myelin is physically less stable and/or less dense than wt myelin.