Polyphosphoinositides are enriched in plant membrane rafts and form microdomains in the plasma membrane.

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-beta-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.

Docosahexaenoic acid prevents lipopolysaccharide-induced cytokine production in microglial cells by inhibiting lipopolysaccharide receptor presentation but not its membrane subdomain localization.

Recognition of lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, by microglia occurs through its binding to specific receptors, cluster of differentiation 14 and toll-like receptor-4. LPS binding to these receptors triggers the synthesis of proinflammatory cytokines that coordinate the brain innate immune response to protect the CNS of the infection. Docosahexaenoic acid (DHA), a n-3 polyunsaturated fatty acid highly incorporated in the brain, is a potent immunomodulator. In this study, we investigated whether DHA modulates LPS receptor localization and, as a consequence, LPS-induced signaling pathway and proinflammatory cytokine production. We demonstrated that DHA, when added exogenously, is specifically enriched in membrane phospholipids, but not in raft lipids of microglial cells. DHA incorporation in membrane impaired surface presentation of LPS receptors cluster of differentiation 14 and toll-like receptor-4, but not their membrane subdomain localization. LPS-induced nuclear factor kappa B activation was inhibited by DHA, hence, LPS-induced proinflammatory cytokine synthesis of interleukin-1beta and tumor necrosis factor alpha was strongly attenuated. We suggest that DHA is highly anti-inflammatory by targeting LPS receptor surface location, therefore reducing LPS action on microglia. This effect represents a new insight by which DHA modulates in the brain the expression of proinflammatory cytokines in response to bacterial product.

Acetyl-CoA carboxylase and SREBP expression during peripheral nervous system myelination.

The expression of acetyl-CoA carboxylase (ACC) in mouse peripheral nervous system (PNS) was investigated. Both ACC 265 and ACC 280 isoforms were expressed in the sciatic nerve, although ACC 265 was predominant. ACC 265 transcripts originating from promoters P1 and P2 could be detected in the developing nerve, as well as the two splice products, which are characterized by the presence or the absence of a 24-base sequence before the codon serine-1200. The mRNA levels for ACC 265 parallel those of other lipogenic genes whose expression is linked to the myelination process. In addition, ACC 265 mRNA and protein levels in the nerves of the trembler mutant, which is a mouse model of PNS dysmyelination, represented around 30% of the normal values. The expression of the sterol regulatory element-binding proteins (SREBPs) was also studied. SREBP 1 mRNAs were expressed at a constant level during nerve development, and their quantities were normal in trembler. On the contrary, SREBP 2 mRNA quantities varied during the myelination period similarly to the lipogenic gene mRNAs, and the levels measured in trembler represented only 10% of the normal values. Taken together, these results suggest that the coordinate expression of several lipogenic genes, which occurs during PNS myelination, could possibly be regulated by SREBP 2.

Fatty acid synthase expression during peripheral nervous system myelination.

The expression of fatty acid synthase (FAS) in rat and mouse sciatic nerves during postnatal development was investigated. FAS activity was not sensitive to the nutritional status of the animals. During development, the specific activity of FAS was low in rat and mouse nerves immediately after birth. Then, there was a steady increase in the activity (8- to 10-fold) which reached a maximal level around postnatal day 11, plateaued till day 32, and decreased to reach 30% of the maximum at day 80. A similar developmental profile was obtained when the amount of FAS protein was quantified, thus suggesting that the variations in activity observed during sciatic nerve development are mainly due to variations in FAS protein content. Northern blot analysis showed that the mRNA levels for FAS parallels those of the ceramide galactosyl transferase (CGT) during mouse sciatic nerve development and in a rat demyelination-nerve regeneration model. In addition, we measured FAS expression in the sciatic nerves of the trembler mutant, which is a mouse model of PNS dysmyelination. In 20-day-old trembler nerves, FAS specific activity, protein amount and mRNA levels represented only 25% of the normal values. Altogether, our data indicate that FAS expression is linked to the PNS myelination process, and that the main regulation occurs at the level of the gene expression.

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus.

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Trembler as a Mouse Model of CMT1A?

The Trembler mouse suffers from a dominantly inherited autosomal mutation that results in an abnormal myelination of the peripheral nervous system. Biochemical studies have shown that dysmyelination is the primary event, demyelination being a late-occurring process. The expression of myelin protein genes has been studied. The steady-state levels for PMP22 mRNA represent 10 and 5% of normal values in the nerves of heterozygous and homozygous Trembler, respectively. This is due to a reduced expression of the specific transcript driven by the promoter 1 of the PMP22 gene. Collective results indicate that Trembler dysmyelination is not necessarily the consequence of a large accumulation of the mutated PMP22 protein. Moreover, it appears that the situation in the Trembler is different from that encountered in most CMT1A patients, where an increased PMP22 gene dosage is responsible for the disease. Therefore, the Trembler mutant is perhaps not an ideal model for this human neuropathy.