Ganglioside and related-sphingolipid profiles are altered in a cellular model of Alzheimer's disease.

Sphingolipid-related issues are increasingly discussed to contribute to the neuropathological process of Alzheimer's disease (AD). In this study, gangliosides and related-sphingolipids (ceramides, neutral glycosphingolipids and sphingomyelins) were analyzed in neuroglioma (H4) cells expressing the Swedish mutation of the human amyloid precursor protein (H4APPsw) and compared with those of wild-type control H4 cells. These cells were chosen since H4APPsw cells were previously reported to reproduce well some essential features of AD. We found that H4APPsw cells exhibited a striking elevation of the simplest ganglioside GM3, an abnormality that was consistently reported in AD patients and animal models of AD. Concomitantly, the levels of both lactosylceramide (the immediate metabolic precursor of GM3) and ganglioside GD1a increased, suggesting a deregulation in the biosynthesis of gangliosides in the H4APPsw cells. Moreover, while the total ceramide level remained unaltered in H4APPsw cells, a shift in ceramide composition from long chain - to very long chain fatty acid-ceramide species was recorded. Because sphingolipid alterations occurring in H4APPsw cells were similar to those observed in transgenic mice and in human brains, this cellular model might be useful to further explore the complex role of sphingolipids in AD pathogenesis.

Anti-amyloidogenic effects of glycosphingolipid synthesis inhibitors occur independently of ganglioside alterations.

Evidence has suggested that ganglioside abnormalities may be linked to the proteolytic processing of amyloid precursor protein (APP) in Alzheimer's disease (AD) and that pharmacological inhibition of ganglioside synthesis may reduce amyloid ß-peptide (Aß) production. In this study, we assessed the usefulness of two well-established glycosphingolipid (GSL) synthesis inhibitors, the synthetic ceramide analog D-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol) and the iminosugar N-butyldeoxynojirimycin (NB-DNJ or miglustat), as anti-amyloidogenic drugs in a human cellular model of AD. We found that both GSL inhibitors were able to markedly inhibit Aß production, although affecting differently the APP cleavage. Surprisingly, the L-enantiomer of PDMP, which promotes ganglioside accumulation, acted similarly to D-PDMP to inhibit Aß production. Concurrently, both D- and L-PDMP strongly and equally reduced the levels of long-chain ceramides. Altogether, our data suggested that the anti-amyloidogenic effects of PDMP agents are independent of the altered cellular ganglioside composition, but may result, at least in part, from their ability to reduce ceramide levels. Moreover, our current study established for the first time that NB-DNJ, a drug already used as a therapeutic for Gaucher disease (a lysosomal storage disorder), was also able to reduce Aß production in our cellular model. Therefore, our study provides novel information regarding the possibilities to target amyloidogenic processing of APP through modulation of sphingolipid metabolism and emphasizes the potential of the iminosugar NB-DNJ as a disease modifying therapy for AD.

The Tyr216 phosphorylated form of GSK3ß contributes to tau phosphorylation at PHF-1 epitope in response to Aß in the nucleus of SH-SY5Y cells.

AIMS: GSK3ß activation in Aß conditions leading to tau phosphorylation at pathological sites is a well-known phenomenon. However, the serine/tyrosine phosphorylation processes implied in Aß-induced GSK3ß activation and responsible for tau phosphorylation, especially at the GSK3ß specific Ser396/Ser404 (PHF-1) site, are still debated. MAIN METHODS: Experiments were performed on SH-SY5Y cells exposed to 20µM Aß1-42 in a time ranging from 5min to 8h. The phophorylated forms (Ser9 and Tyr216) of GSK3ß and pTau at PHF-1 epitope were measured by immunoblotting in nuclear extracts. KEY FINDINGS: We showed a superimposable time-dependent increase of nuclear pGSK3ßTyr216 and nuclear pTau at PHF-1 site, both reaching their maximal level after 8h of Aß1-42 exposure. In addition, nuclear accumulation of pTau is accompanied by its cytoplasmic decrease suggesting that pTau is translocated in response to Aß treatment. Besides, our experiments showed that specific pGSK3ßTyr216 inhibition is required to drop nuclear pTau, ensuring the involvement of Tyr216 phosphorylation in Aß-mediated tau phosphorylation at PHF-1 epitope. SIGNIFICANCE: These data suggested that in response to Aß exposure in SH-SY5Y cells, GSK3ß activation is performed through Tyr216 phosphorylation and resulted in tau phosphorylation at PHF-1 epitope and in its translocation.

Inhibition of GSK3ß by pharmacological modulation of sphingolipid metabolism occurs independently of ganglioside disturbance in a cellular model of Alzheimer's disease.

Accumulating evidence implicates ganglioside and/or related-sphingolipid disturbance in the pathogenesis of Alzheimer's disease (AD). However, it is not known whether these lipidic alterations are connected with other important features of AD, such as deregulated insulin/Akt/GSK3 signaling. In this study, we have treated neuroglioma cells expressing the double Swedish mutation of human amyloid precursor protein (H4APPsw) with several glycosphingolipid (GSL)-modulating agents, and we have analyzed the impact of the aberrant ganglioside composition on the GSK3 activation state. We found that both ceramide analogs D- and L-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol), which have opposite effects on ganglioside synthesis, selectively inhibited GSK3ß via Ser9 phosphorylation independently of the upstream insulin/Akt pathway. Conversely, the iminosugar N-butyldeoxynojirimycin (NB-DNJ) which displayed similar reduction of gangliosides as D-PDMP, did not affect the phosphorylation state of GSK3ß. Concurrently, while NB-DNJ did not modify the cellular ceramide content, both PDMP enantiomers strongly and equally reduced the levels of long-chain ceramide species. Altogether, our findings led us to hypothesize that the PDMP-induced altered ganglioside composition is not the principal mechanism involved in the inhibition of GSK3ß, but seems to implicate, at least in part, their ability to reduce ceramide levels. Nevertheless, this study provides new information regarding the possibilities to target GSK3ß through modulation of sphingolipid metabolism.

Lithium chloride and staurosporine potentiate the accumulation of phosphorylated glycogen synthase kinase 3ß/Tyr216, resulting in glycogen synthase kinase 3ß activation in SH-SY5Y human neuroblastoma cell lines.

Glycogen synthase kinase 3ß (GSK3ß) activity is regulated by phosphorylation processes and regulates in turn through phosphorylation several proteins, including eukaryotic initiation factor 2B (eIF2B). Serine 9 phosphorylation of GSK3ß (pGSK3ßSer9), usually promoted by activation of the PI3K/Akt survival pathway, triggers GSK3ß inhibition. By contrast, tyrosine 216 phosphorylation of GSK3ß (pGSK3ßTyr216) increases under apoptotic conditions, leading to GSK3ß activation. Lithium chloride (LiCl) is usually described to increase pGSK3ßSer9 through the PI3K/Akt pathway, resulting in GSK3ß inhibition. The purpose of this study is to demonstrate that in some cases LiCl is also able to increase pGSK3ßTyr216, resulting in GSK3ß activation. For this, we used SH-SY5Y cells and primary neuronal cultures and investigated the effects of LiCl on the two phosphorylated forms of GSK3ß under staurosporine (STS)-intoxicated conditions. The ratios between the phosphorylated and total forms of GSK3ß and eIF2B were determined by Western blotting. Our results revealed that, besides its ability to increase pGSK3ßSer9, LiCl is also able to increase pGSK3ßTyr216 greatly in STS-intoxicated SH-SY5Y cells but not in STS-intoxicated primary neuronal cultures. This accumulation of both Ser9 and Tyr216 phosphorylation results in GSK3ß activation in STS-intoxicated SH-SY5Y cells in spite of the presence of LiCl. These findings indicate that LiCl treatment is not necessarily correlated with GSK3ß inhibition even though it generates Ser9 phosphorylation. Consequently, the ratio pGSK3ßSer9/pGSK3ßTyr216, which takes into account the balance between the two inactive (Ser9) and active (Tyr216) forms of GSK3ß, could be more useful for predicting GSK3ß inhibition.

Ceramide and Related-Sphingolipid Levels Are Not Altered in Disease-Associated Brain Regions of APP and APP/PS1 Mouse Models of Alzheimer's Disease: Relationship with the Lack of Neurodegeneration?

There is evidence linking sphingolipid abnormalities, APP processing, and neuronal death in Alzheimer's disease (AD). We previously reported a strong elevation of ceramide levels in the brain of the APP(SL)/PS1Ki mouse model of AD, preceding the neuronal death. To extend these findings, we analyzed ceramide and related-sphingolipid contents in brain from two other mouse models (i.e., APP(SL) and APP(SL)/PS1(M146L)) in which the time-course of pathology is closer to that seen in most currently available models. Conversely to our previous work, ceramides did not accumulate in disease-associated brain regions (cortex and hippocampus) from both models. However, the APP(SL)/PS1Ki model is unique for its drastic neuronal loss coinciding with strong accumulation of neurotoxic Aß isoforms, not observed in other animal models of AD. Since there are neither neuronal loss nor toxic Aß species accumulation in APP(SL) mice, we hypothesized that it might explain the lack of ceramide accumulation, at least in this model.

Gender-dependent accumulation of ceramides in the cerebral cortex of the APP(SL)/PS1Ki mouse model of Alzheimer's disease.

Altered sphingolipid metabolism plays an emergent role in the etiology of Alzheimer's disease (AD). In this study, we determined the levels of ceramides and other related-sphingolipids (sphingomyelins, sulfatides and galactosylceramides) in the cerebral cortex of an APP(SL)/PS1Ki mouse model of AD. The results demonstrate that ceramides accumulated in the cortex of APP(SL)/PS1Ki mice, but not in PS1Ki mice, whereas all others major sphingolipids (except galactosylceramides) were not altered in comparison with those from age-matched wild-type mice. Furthermore, as early as 3 months of age, female mice but not males, exhibit a strong increase in 2-hydroxy fatty acid-containing ceramides, whereas males display a significant elevation of non-hydroxy fatty acid ceramide species. Therefore, the gender differences in ceramide accumulation in the brain of mice expressing APP(SL) suggest that additional factors like modified ceramide metabolism may contribute to the increased propensity of females to develop AD.

The oxindole/imidazole derivative C16 reduces in vivo brain PKR activation.

Inhibition of double-stranded RNA-dependent protein kinase (PKR) represents an interesting strategy for neuroprotection. However, inhibiting this kinase which triggers the apoptotic process could favour in counterpart cell proliferation and tumorigenesis. Here, we use an in vivo model of 7-day-old rat displaying a high activation of brain PKR to investigate the effects of a new PKR inhibitor identified as an oxindole/imidazole derivative (C16). We show for the first time that acute systemic injection of C16 specifically inhibits the apoptotic PKR/eIF2alpha signaling pathway without stimulating the proliferative mTOR/p70S6K signaling mechanism.

Regulation of initiation factors controlling protein synthesis on cultured astrocytes in lactic acid-induced stress.

The goals of this work were first to assess whether the lactic acidosis observed in vivo in ischemia may by itself explain the inhibition of protein synthesis described in the literature and second to study the factors controlling the initiation of protein synthesis under lactic acid stress. Primary rat astrocyte cultures exposed to pH 5.25 underwent cell death and a strong inhibition of protein synthesis assessed by [3H]methionine incorporation, which was solely due to acidity of the extracellular medium and was not related to lactate concentrations. This result was associated with a weak phosphorylation of eukaryotic initiation factor (eIF)4E and a rapid phosphorylation of eIF2alpha via the kinases PKR and PKR-like endoplasmic reticulum kinase. The inhibition of PKR by PRI led first to a significant but not complete dephosphorylation of eIF2alpha that probably contributed to maintain the inhibition of the protein synthesis and second to surprising phosphorylations of extracellular signal-regulated protein kinase, p70S6K and eIF4E, suggesting a possible cross-link between the two pathways. Conversely, cell death was weak at pH 5.5. Protein synthesis was decreased to a lesser extent, the phosphorylation of eIF2alpha was limited, extracellular signal-regulated protein kinase 1/2 was activated and its downstream targets, p70S6K and eIF4E, were phosphorylated. However, the strong phosphorylation of eIF4E was not associated with an activation of the eIF4F complex. This last result may explain why protein synthesis was not stimulated at pH 5.5. However, when astrocytes were exposed at pH 6.2, corresponding to the lower pH observed in hyperglycemic ischemia, no modification in protein synthesis was observed. Consequently, lactic acidosis cannot, by itself, provide an explanation for the decrease in protein synthesis previously reported in vivo in ischemia.

Genotype-related changes of ganglioside composition in brain regions of transgenic mouse models of Alzheimer's disease.

In this study, brain gangliosides of different transgenic mouse models of Alzheimer's disease (AD) were analyzed and compared with age-matched wild-type mice. Gangliosides were analyzed in cerebral cortex, a region with extensive A beta plaques, and cerebellum, a non-vulnerable region with no A beta containing plaques. There was a marked increase in simple gangliosides GM2 and GM3 only within the cortex of all mice expressing APP(SL). Additionally, loss of complex "a" gangliosides (GT1a, GD1a and GM1) was recorded in APP/PS1Ki model, whereas in APP(SL) and APP/PS1 mice, the complex "b" gangliosides (GQ1b, GT1b and GD1b) moderately decreased. Surprisingly, expression of either mutant PS1(M146L) or PS1 mutant FAD (Ki model) alone tended to lower the levels of both GM2 and GM3 within the cortex. Conversely, only slight changes of the ganglioside pattern were found in the cerebellum. Because ganglioside alterations occurring in APP transgenic mice were similar to those observed in human AD brain, these transgenic models would represent valuable tools to further investigate the role of altered ganglioside metabolism in the pathogenesis of AD.

Lactic acidosis stimulates ganglioside and ceramide generation without sphingomyelin hydrolysis in rat cortical astrocytes.

Acidosis is a ubiquitous feature of cerebral ischemia, and triggers a cascade of biochemical events that results in neuronal injury. The purpose of this study was to evaluate the effects of lactic acidosis on the ganglioside composition, the ceramide and sphingomyelin (SM) levels in rat cortical astrocytes. Primary astrocyte cultures were exposed to lactic acid (pH 5.5) for 2, 5 and 17 h, and cell death was evaluated at each time point. Gangliosides, ceramides and SM were analyzed by high-performance thin layer chromatography. Lactic acidosis caused a progressive increase of both GM3 and GD3 gangliosides up to 5 h of treatment. However, at 17 h of acidosis, GM3 tented to return to the normal level whereas GD3 accumulated. Additionally, ceramides were gradually generated, whereas no significant decrease of SM occured for 17 h of acidosis. These results suggest that ceramides were not produced by the breakdown of SM and may be served as metabolic precursor for the biosynthesis of GM3 and GD3. Since these lipids are important messengers of the adaptative responses to stress, accumulation of sphingolipids triggered by lactic acid exposure of astrocytes might play an important role in determining the outcomes of injurious processes.

Evidence for the reactivity of fatty aldehydes released from oxidized plasmalogens with phosphatidylethanolamine to form Schiff base adducts in rat brain homogenates.

The vinyl ether bond of plasmalogens could be among the first target of free radicals attack. Consequently, because of their location in the membranes of cells, plasmalogens represent a first shield against oxidative damages by protecting other macromolecules and are often considered as antioxidant molecules. However, under oxidative conditions their disruption leads to the release of fatty aldehydes. In this paper, we showed using gas chromatography-mass spectrometry (GC-MS) analyses that fatty aldehydes released from plasmalogens after oxidation (UV irradiation and Fe2+/ascorbate) of cerebral cortex homogenates can generate covalent modifications of endogenous macromolecules such as phosphatidylethanolamine (PE), like the very reactive and toxic malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). These newly formed Schiff base adducts could be responsible for deleterious effects on cells thus making the protective role of plasmalogens potentially questionable.

Effect of ischemia on TBARS and lactate production in several cerebral regions of anaesthetised and awake rats.

The premise of neuroprotective therapy for acute ischemic stroke is based upon the possibility to interfere with the cellular ischemic cascade, so the understanding of the mechanisms and consequences of cerebral ischemia is necessary. The relationship between lipid peroxidation and acidosis was investigated in several regions of rat brain following ischemia without reperfusion. Male Wistar rats (280-300 g) were anaesthetised (Ketalar 33 mg/kg and Rompun 6.66 mg/kg) or not and underwent a four-vessel occlusion for 5 minutes. Then, thiobarbituric acid-reactive substances (TBARS) and lactate levels were measured in different brain regions (cerebellum, bulb, striatum, hippocampus, cortex). Induction of ischemia by ligation of two common carotid arteries and two vertebral arteries resulted in a production of TBARS (40-120%, p < 0.05) and lactate (20-60%, p < 0.05) in all cerebral regions of awake rats, especially in striatum, suggesting a potential link between lipid peroxidation and acidosis. When ischemia was realised on anaesthetised animals, an increase of lactate levels (30-50%, p < 0.05) was shown in all brain regions but TBARS were produced only in striatum (82%, p < 0.05). These data showed the particular vulnerability of striatum to ischemia and the possible opposite effects of an anaesthesia.

Lactic acidosis progressively impairs dopamine uptake in rat striatal synaptosomes by a mechanism partially independent of the Na+/K+-ATPase dysfunction.

Previous experiments reported that incubation of rat striatal synaptosomes with lactic acid (pH 5.5) resulted in an inhibition of dopamine (DA) uptake partially mediated by free radical damage. Since the DA uptake process is highly dependent on the functionality of Na+/K+-ATPase, the present study investigated whether this inhibition of DA uptake could be related to an alteration of the Na+/K+-ATPase activity. Striatal lactic acidosis was performed by direct addition of lactic acid in the incubation medium to obtain a pH as close as possible to that observed in ischemia. Acidosis (pH 5.5) induced a progressive decline in the specific DA uptake and a decrease of Na+/K+-ATPase activity in striatal synaptosomes. However, whereas loss of Na+/K+-ATPase activity was totally prevented by Trolox, a powerful antioxidant, DA uptake remained partially inhibited. Taken together, these data suggest that acidosis, in a degree encountered during ischemia, alters the high-affinity DA uptake in part by a mechanism that does not involve a Na+/K+ pump deficiency.