New insights on neurodegeneration triggered by iron accumulation: Intersections with neutral lipid metabolism, ferroptosis, and motor impairment.

Brain iron accumulation constitutes a pathognomonic indicator in several neurodegenerative disorders. Metal accumulation associated with dopaminergic neuronal death has been documented in Parkinson's disease. Through the use of in vivo and in vitro models, we demonstrated that lipid dysregulation manifests as a neuronal and glial response during iron overload. In this study, we show that cholesterol content and triacylglycerol (TAG) hydrolysis were strongly elevated in mice midbrain. Lipid cacostasis was concomitant with the loss of dopaminergic neurons, astrogliosis and elevated expression of α-synuclein. Exacerbated lipid peroxidation and markers of ferroptosis were evident in the midbrain from mice challenged with iron overload. An imbalance in the activity of lipolytic and acylation enzymes was identified, favoring neutral lipid hydrolysis, and consequently reducing TAG and cholesteryl ester levels. Notably, these observed alterations were accompanied by motor impairment in iron-treated mice. In addition, neuronal and glial cultures along with their secretomes were used to gain further insight into the mechanism underlying TAG hydrolysis and cholesterol accumulation as cellular responses to iron accumulation. We demonstrated that TAG hydrolysis in neurons is triggered by astrocyte secretomes. Moreover, we found that the ferroptosis inhibitor, ferrostatin-1, effectively prevents cholesterol accumulation both in neurons and astrocytes. Taken together, these results indicate that lipid disturbances occur in iron-overloaded mice as a consequence of iron-induced oxidative stress and depend on neuron-glia crosstalk. Our findings suggest that developing therapies aimed at restoring lipid homeostasis may lead to specific treatment for neurodegeneration associated with ferroptosis and brain iron accumulation.

Circadian Regulation and Clock-Controlled Mechanisms of Glycerophospholipid Metabolism from Neuronal Cells and Tissues to Fibroblasts.

Along evolution, living organisms developed a precise timekeeping system, circadian clocks, to adapt life to the 24-h light/dark cycle and temporally regulate physiology and behavior. The transcriptional molecular circadian clock and metabolic/redox oscillator conforming these clocks are present in organs, tissues, and even in individual cells, where they exert circadian control over cellular metabolism. Disruption of the molecular clock may cause metabolic disorders and higher cancer risk. The synthesis and degradation of glycerophospholipids (GPLs) is one of the most highly regulated metabolisms across the 24-h cycle in terms of total lipid content and enzyme expression and activity in the nervous system and individual cells. Lipids play a plethora of roles (membrane biogenesis, energy sourcing, signaling, and the regulation of protein-chromatin interaction, among others), making control of their metabolism a vital checkpoint in the cellular organization of physiology. An increasing body of evidence clearly demonstrates an orchestrated and sequential series of events occurring in GPL metabolism across the 24-h day in diverse retinal cell layers, immortalized fibroblasts, and glioma cells. Moreover, the clock gene Per1 and other circadian-related genes are tightly involved in the regulation of GPL synthesis in quiescent cells. However, under proliferation, the metabolic oscillator continues to control GPL metabolism of brain cancer cells even after molecular circadian clock disruption, reflecting the crucial role of the temporal metabolism organization in cell preservation. The aim of this review is to examine the control exerted by circadian clocks over GPL metabolism, their synthesizing enzyme expression and activities in normal and tumorous cells of the nervous system and in immortalized fibroblasts.

Neutral lipids as early biomarkers of cellular fate: the case of α-synuclein overexpression.

α-synuclein (α-syn) accumulation and aggregation is a common pathological factor found in synucleinopathies, a group of neurodegenerative disorders that includes Parkinson´s disease (PD). It has been proposed that lipid dyshomeostasis is responsible for the occurrence of PD-related processes, however, the precise role of lipids in the onset and progression of neurodegenerative disorders remains unclear. Our aim was to investigate the effect of α-syn overexpression on neutral lipid metabolism and how this impacts on neuronal fate. We found lipid droplet (LD) accumulation in cells overexpressing α-syn to be associated with a rise in triacylglycerol (TAG) and cholesteryl ester (CE) levels. α-syn overexpression promoted diacylglycerol acyltransferase 2 upregulation and acyl-CoA synthetase activation, triggering TAG buildup, that was accompanied by an increase in diacylglycerol acylation. Moreover, the CE increment was associated with higher activity of acyl-CoA:cholesterol acyltransferase. Interestingly, α-syn overexpression increased cholesterol lysosomal accumulation. We observed that sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 were differentially regulated by α-syn overexpression. The latter gave rise to a reduction in SREBP-1 nuclear translocation and consequently in fatty acid synthase expression, whereas it produced an increase in SREBP-2 nuclear localization. Surprisingly, and despite increased cholesterol levels, SREBP-2 downstream genes related to cholesterolgenesis were not upregulated as expected. Notably, phospholipid (PL) levels were diminished in cells overexpressing α-syn. This decrease was related to the activation of phospholipase A2 (PLA2) with a concomitant imbalance of the PL deacylation-acylation cycle. Fatty acids released from PLs by iPLA2 and cPLA2 action were esterified into TAGs, thus promoting a biological response to α-syn overexpression with uncompromised cell viability. When the described steady-state was disturbed under conditions favoring higher levels of α-syn, the response was an enhanced LD accumulation, this imbalance ultimately leading to neuronal death.

Crosstalk between sterol and neutral lipid metabolism in the alga Haematococcus pluvialis exposed to light stress.

The presence, biosynthesis and functional role of sterols in the green microalga Haematococcus pluvialis remain poorly understood. In this work we studied the effect of high-light (HL) stress on sterol synthesis in H. pluvialis UTEX 2505 cells. HL stress induced the synthesis of sterols in parallel with that of triacylglycerides (TAG), giving rise to the synthesis of cholesterol over that of phytosterols. Blockage of the carotenogenic 1-deoxy-D-xylulose 5-phosphate (MEP) pathway is shown to be involved in HL-induced sterol synthesis. In addition, high irradiance exposure induced MEP- and fatty acid (FA)-biosynthetic transcripts. The pharmacological inhibition of these pathways suggests a possible feedback regulation of sterol and FA homeostasis. Finally, both lipid classes proved crucial to the adequate photosynthetic performance of H. pluvialis grown under HL intensity stress. Our findings reveal new insights into H. pluvialis lipid metabolism that contribute to the development of value-added bioproducts from microalgae.

In vitro 6-hydroxydopamine-induced neurotoxicity: New insights on NFκB modulation.

Neuronal exposure to 6-hydroxydopamine (6-OHDA), a hydroxylated analog of dopamine, constitutes a very useful strategy for studying the molecular events associated with neuronal death in Parkinson's disease. 6-OHDA increases oxidant levels and impairs mitochondrial respiratory chain, thus promoting neuronal injury and death. Despite the extensive use of 6-OHDA in animal models, the exact molecular events triggered by this neurotoxicant at the neuronal level have not been yet fully understood. Human IMR-32 neuroblastoma cells exposed to increasing concentrations of 6-OHDA displayed high levels of reactive oxygen species and increased plasma membrane permeability with concomitant cell viability diminution. As part of the neuronal response to 6-OHDA exposure, the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) p65 subunit was observed. NFκB nuclear localization was also accompanied by an increase of IκB phosphorylation as well as a rise in cyclooxygenase-2 (COX-2) and the prostaglandin receptor, EP4, mRNA levels. Even though the canonical pathways participating in the modulation of NFκB have been extensively described, here we tested the hypothesis that 6-OHDA-induced injury can activate lipid signaling and, in turn, modulate the transcriptional response. 6-OHDA challenge triggered the activation of lipid signaling pathways and increased phosphatidic acid (PA), diacylglycerol and free fatty acid levels in human neuroblastoma cells. The inhibition of PA production was able to prevent the decrease in cell viability triggered by 6-OHDA, the nuclear translocation of NFκB p65 subunit and the rise in COX-2 mRNA expression. Our results indicate that the onset of the inflammatory process triggered by 6-OHDA involves the activation of PA signaling that, in turn, governs NFκB subcellular localization and COX-2 expression.

High glucose-induced phospholipase D activity in retinal pigment epithelium cells: New insights into the molecular mechanisms of diabetic retinopathy.

Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of diabetic retinopathy (DR). In this work we study the role of phospholipase D (PLD) pathway in an in vitro model of high glucose (HG)-induced damage. To this end, we exposed human retinal pigment epithelium (RPE) cell lines (ARPE-19 and D407) to HG concentrations (16.5 or 33 mM) or to normal glucose concentration (NG, 5.5 mM) for 4, 24 or 72 h. Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72 h of incubation. In addition, short term HG exposure (4 h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NFκB) nuclear translocation and IκB phosphorylation. The increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24 h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NFκB activation were downstream events of both PLD isoforms. The increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. Furthermore, the inhibition of PLD1, PLD2 and MEK/ERK pathway prevented the loss of cell viability and the activation of caspase-3 induced by HG. In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response triggered by HG in RPE cells, pointing to their potential use as a therapeutic target for DR treatment.

Lipids at the Crossroad of α-Synuclein Function and Dysfunction: Biological and Pathological Implications.

Since its discovery, the study of the biological role of α-synuclein and its pathological implications has been the subject of increasing interest. The propensity to adopt different conformational states governing its aggregation and fibrillation makes this small 14-kDa cytosolic protein one of the main etiologic factors associated with degenerative disorders known as synucleinopathies. The structure, function, and toxicity of α-synuclein and the possibility of different therapeutic approaches to target the protein have been extensively investigated and reviewed. One intriguing characteristic of α-synuclein is the different ways in which it interacts with lipids. Though in-depth studies have been carried out in this field, the information they have produced is puzzling and the precise role of lipids in α-synuclein biology and pathology and vice versa is still largely unknown. Here we provide an overview and discussion of the main findings relating to α-synuclein/lipid interaction and its involvement in the modulation of lipid metabolism and signaling.

Lipid metabolism alterations in the neuronal response to A53T α-synuclein and Fe-induced injury.

Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) ß-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties. Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content. We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease.

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.

Cativic acid-caffeic acid hybrid exerts cytotoxic effects and induces apoptotic death in human neuroblastoma cells.

The development of hybrids from natural products is a promising strategy for drug discovery. In cancer therapy, there is a need to discover novel agents that can induce apoptosis in cancer cells. To contribute to this field of interest, we investigated the effect of a synthetic hybrid from cativic acid and caffeic acid (5) on viability, proliferation, and apoptosis in human neuroblastoma cells (IMR-32). Three hybrids were prepared via Mitsunobu esterification from 17-hydroxycativic acid (1) and natural phenols. Cell viability was analyzed by MTT assay. SYTOX green and LDH leakage were used to determine the cytotoxic effect. Caspase-3 activity, cell cycle phases, and proliferation were analyzed in order to characterize the biological effects of hybrid 5. The mitogen-activated protein kinase (MAPK) status was evaluated for elucidating the potential mechanisms involved in hybrid 5 effect. Hybrid 5 reduced the viability of IMR-32 cells in a time- and concentration-dependent manner (IC50 = 18.0 ± 1.3 µM) as a result of its antiproliferative effect through changes in the cell cycle distribution and induction of apoptosis associated with activation of caspase-3. Exposure to 5 triggered ERK1/2 activation and nuclear translocation. Hybrid 5 also promoted an increase in nuclear localization of the transcription factor c-Jun. Inhibition of ERK1/2 and JNK potentiated 5-induced inhibition of IMR-32 viability. Hybrid 5 displays cell growth inhibition by promoting cell cycle arrest and apoptosis, through ERK1/2 and JNK participation.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/ßII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and ß protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, ß and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage.

Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -PLC.

This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.

The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it is involved in many ocular inflammatory diseases that may end in loss of vision and blindness. In this work the role of phospholipase D (PLD) classical isoforms, PLD1 and PLD2, in the inflammatory response of human RPE cells (ARPE-19) was studied. ARPE-19 cells exposed to lipopolysaccharide (LPS, 10 µg/ml) displayed increased levels of NO production and diminished mitochondrial function after 48 h of incubation. Furthermore, 24h LPS treatment strongly induced cyclooxygenase-2 (COX-2) expression and activation of extracellular signal-regulated kinase (ERK1/2). EGFP-PLDs showed the typical subcellular localization, perinuclear for PLD1 and plasma membrane for PLD2. LPS increased PLD activity by 90% with respect to the control. The presence of PLD1 inhibitor (EVJ 0.15 µM) or PLD2 inhibitor (APV 0.5 µM) reduced LPS-induced COX-2 induction but only PLD2 inhibition reduced ERK1/2 activation. Mitochondrial function was restored after inhibition of PLD2 and ERK1/2. These findings evidence the participation of PLD2 as a promoter of RPE inflammatory response through ERK1/2 and COX-2 regulation. Our results demonstrate for the first time distinctive roles of PLD isoforms in pathological conditions in RPE.

Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions.

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.

Decreased zinc availability affects glutathione metabolism in neuronal cells and in the developing brain.

A deficit in zinc (Zn) availability can increase cell oxidant production, affect the antioxidant defense system, and trigger oxidant-sensitive signals in neuronal cells. This work tested the hypothesis that a decreased Zn availability can affect glutathione (GSH) metabolism in the developing rat brain and in neuronal cells in culture, as well as the capacity of human neuroblastoma IMR-32 cells to upregulate GSH when challenged with dopamine (DA). GSH levels were low in the brain of gestation day 19 (GD19) fetuses from dams fed marginal Zn diets throughout gestation and in Zn-deficient IMR-32 cells. γ-Glutamylcysteine synthetase (GCL), the first enzyme in the GSH synthetic pathway, was altered by Zn deficiency (ZD). The protein and mRNA levels of the GCL modifier (GCLM) and catalytic (GCLC) subunits were lower in the Zn-deficient GD19 fetal brain and in IMR-32 cells compared with controls. The nuclear translocation of transcription factor nuclear factor (erythroid-derived 2)-like 2, which controls GCL transcription, was impaired by ZD. Posttranslationally, the caspase-3-dependent GCLC cleavage was high in Zn-deficient IMR-32 cells. Cells challenged with DA showed an increase in GCLM and GCLC protein and mRNA levels and a consequent increase in GSH concentration. Although Zn-deficient cells partially upregulated GCL subunits after exposure to DA, GSH content remained low. In summary, results show that a low Zn availability affects the GSH synthetic pathway in neuronal cells and fetal brain both at transcriptional and posttranslational levels. This can in part underlie the GSH depletion associated with ZD and the high sensitivity of Zn-deficient neurons to pro-oxidative stressors.

Distinctive roles of PLD signaling elicited by oxidative stress in synaptic endings from adult and aged rats.

The role of iron in oxidative injury in the nervous system has been extensively described. However, little is known about the role of lipid signal transduction in neurodegeneration processes triggered by iron overload. The purpose of this work was to characterize the regulation and the crosstalk between phosphatidylcholine (PC)-derived diacylglycerol (DAG) and cannonical signaling pathways during iron-induced oxidative stress in cerebral cortex synaptic endings (Syn) obtained from adult (4 months old) and aged (28 months old) rats. DAG production was increased in Syn exposed to iron. This rise in DAG formation was due to phospholipase D1 (PLD1) and PLD2 activations. In adult rats, PKD1, ERK1/2 and PKCα/ßII activations were PLD1 and PLD2 dependent. In contrast, in senile rats, DAG formation catalyzed by PLDs did not participate in PKD1, ERK1/2 and PKCα/ßII regulations, but it was dependent on ERK and PKC activities. Iron-induced oxidative stress promoted an increased localization of PLD1 in membrane rafts, whereas PLD2 was excluded from these domains and appeared to be involved in glutamate transporter function. Our results show a differential regulation and synaptic function of DAG generated by PLDs during iron-induced oxidative stress as a consequence of aging.

Lactobacillus reuteri CRL1098 soluble factors modulate tumor necrosis factor alpha production in peripheral blood mononuclear cells: involvement of lipid rafts.

The aim of the present study was to evaluate the capacity of Lactobacillus reuteri CRL1098 soluble factors (Lr-S) to modulate TNF-α production in peripheral blood mononuclear cells (PBMC) and to study lipid rafts participation in this response. PBMC treated with Lr-S showed a reduced production of TNF-α. In addition, Lr-S treatment activated ERK and p38 MAPK pathways in PBMC. Lipid rafts participation in the reduced production of TNF-α by PBMC induced by Lr-S was verified by lipid rafts disruption with methyl-ß-cyclodextrin and the reduction of the Src-tyrosine kinase Lck localization in rafts. Moreover, PBMC pre-treatment with Lck inhibitors blocked the effect of Lr-S on TNF-α production suggesting that activation and mobilization of Lck from lipid rafts would be involved in the modulatory effect of L. reuteri CRL1098. A secreted peptide of 5785 Da would be responsible of the modulatory effect of CRL1098 strain. This study demonstrated for the first time the lipid rafts participation in a response induced by a beneficial bacterium. Also, these results open new possibilities for investigating the molecular mechanisms involved in the interaction of probiotic bacterial extracellular compounds with immune cells.

A deficit in zinc availability can cause alterations in tubulin thiol redox status in cultured neurons and in the developing fetal rat brain.

Zinc (Zn) deficiency during early development can result in multiple brain abnormalities and altered neuronal functions. In rats, a gestational deficit of Zn can affect the fetal brain cytoskeleton and signaling cascades involved in cellular processes that are central to brain development. In this paper, we tested the hypothesis that oxidative stress is involved in Zn deficiency-induced altered tubulin dynamics and the associated dysregulation of transcription factor NF-κB. For this purpose, we used two cell culture models (rat cortical neurons, human IMR-32 neuroblastoma cells) and an animal model of Zn deficiency. A low rate of in vitro tubulin polymerization, an increase in tubulin oligomers, and a higher protein cysteine oxidation were observed in the Zn-deficient neuronal cells and in gestation day 19 fetal brains obtained from dams fed marginal-Zn diets throughout pregnancy. These alterations could be prevented by treating the Zn-deficient cells with the reducing agent tris(2-carboxyethyl)phosphine or by the presence of N-acetylcysteine (NAC) and α-lipoic acid (LA). Consistent with the above, Zn deficiency-induced tubulin-mediated alterations in transcription factor NF-κB nuclear translocation were prevented by treating IMR-32 cells with LA and NAC. Binding of the NF-κB protein p50, dynein, and karyopherin α (components of the NF-κB transport complex) to ß-tubulin as well as the expression of NF-κB-dependent genes (Bcl-2, cyclin D1, and c-myc) was also restored by the addition of LA and NAC to Zn-deficient cells. In conclusion, a deficit in Zn viability could affect early brain development through: (1) an induction of oxidative stress, (2) tubulin oxidation, (3) altered tubulin dynamics, and (4) deregulation of signals (e.g., NF-κB) involved in critical developmental events.