Sabrina Ingrand

Activated double-stranded RNA-dependent protein kinase and neuronal death in models of Alzheimer’s disease

Neuronal death is a pathological hallmark of Alzheimers disease. We have shown previously that phosphorylated double-stranded RNA-dependent protein kinase is present in degenerating hippocampal neurons and in senile plaques of Alzheimers disease brains and that genetically down-regulating double-stranded RNA-dependent protein kinase activity protects against in vitro beta-amyloid peptide neurotoxicity. In this report, we showed that two double-stranded RNA-dependent protein kinase blockers attenuate, in human neuroblastoma cells, beta-amyloid peptide toxicity evaluated by caspase 3 assessment. In addition, we have used the newly engineered APP(SL)/presenilin 1 knock-in transgenic mice, which display a severe neuronal loss in hippocampal regions, to analyze the activation of double-stranded RNA-dependent protein kinase. Western blots revealed the increased levels of activated double-stranded RNA-dependent protein kinase and the inhibition of eukaryotic initiation factor 2 alpha activity in the brains of these double transgenic mice. Phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum-resident kinase was also increased in the brains of these mice. The levels of activated double-stranded RNA-dependent protein kinase were also increased in the brains of patients with Alzheimers disease. At 3, 6 and 12 months, hippocampal neurons display double stranded RNA-dependent protein kinase labelings in both the nucleus and the cytoplasm. Confocal microscopy showed that almost constantly activated double-stranded RNA-dependent protein kinase co-localized with DNA strand breaks in apoptotic nuclei of CA1 hippocampal neurons. Taken together these results demonstrate that double-stranded RNA-dependent protein kinase is associated with neurodegeneration in APP(SL)/presenilin 1 knock-in mice and could represent a new therapeutic target for neuroprotection.

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/eIF2α signaling pathway without stimulating the proliferative mTOR/p70S6K signaling mechanism.

Gender-dependent accumulation of ceramides in the cerebral cortex of the APPSL/PS1Ki mouse model of Alzheimer’s disease

Altered sphingolipid metabolism plays an emergent role in the etiology of Alzheimers 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.

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

Glycogen synthase kinase 3β (GSK3β) activity is regulated by phosphorylation processes and regulates in turn through phosphorylation several proteins, including eukaryotic initiation factor 2B (eIF2B). Serine 9 phosphorylation of GSK3β (pGSK3βSer9), usually promoted by activation of the PI3K/Akt survival pathway, triggers GSK3β inhibition. By contrast, tyrosine 216 phosphorylation of GSK3β (pGSK3βTyr216) increases under apoptotic conditions, leading to GSK3β activation. Lithium chloride (LiCl) is usually described to increase pGSK3βSer9 through the PI3K/Akt pathway, resulting in GSK3β inhibition. The purpose of this study is to demonstrate that in some cases LiCl is also able to increase pGSK3βTyr216, resulting in GSK3β activation. For this, we used SH‐SY5Y cells and primary neuronal cultures and investigated the effects of LiCl on the two phosphorylated forms of GSK3β under staurosporine (STS)‐intoxicated conditions. The ratios between the phosphorylated and total forms of GSK3β and eIF2B were determined by Western blotting. Our results revealed that, besides its ability to increase pGSK3βSer9, LiCl is also able to increase pGSK3βTyr216 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 GSK3β 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 GSK3β inhibition even though it generates Ser9 phosphorylation. Consequently, the ratio pGSK3βSer9/pGSK3βTyr216, which takes into account the balance between the two inactive (Ser9) and active (Tyr216) forms of GSK3β, could be more useful for predicting GSK3β inhibition.

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.

Ceramide and Related-Sphingolipid Levels Are Not Altered in Disease-Associated Brain Regions of APPSL and APPSL/PS1M146L Mouse Models of Alzheimer's Disease: Relationship with the Lack of Neurodegeneration?

There is evidence linking sphingolipid abnormalities, APP processing, and neuronal death in Alzheimers disease (AD). We previously reported a strong elevation of ceramide levels in the brain of the APPSL/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., APPSL and APPSL/PS1M146L) 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 APPSL/PS1Ki model is unique for its drastic neuronal loss coinciding with strong accumulation of neurotoxic Aβ isoforms, not observed in other animal models of AD. Since there are neither neuronal loss nor toxic Aβ species accumulation in APPSL mice, we hypothesized that it might explain the lack of ceramide accumulation, at least in this model.

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 eIF2α 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 eIF2α 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 eIF2α 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.

The Tyr216 phosphorylated form of GSK3β contributes to tau phosphorylation at PHF-1 epitope in response to Aβ in the nucleus of SH-SY5Y cells.

AIMS GSK3β activation in Aβ conditions leading to tau phosphorylation at pathological sites is a well-known phenomenon. However, the serine/tyrosine phosphorylation processes implied in Aβ-induced GSK3β activation and responsible for tau phosphorylation, especially at the GSK3β specific Ser396/Ser404 (PHF-1) site, are still debated. MAIN METHODS Experiments were performed on SH-SY5Y cells exposed to 20μM Aβ1-42 in a time ranging from 5min to 8h. The phophorylated forms (Ser9 and Tyr216) of GSK3β and pTau at PHF-1 epitope were measured by immunoblotting in nuclear extracts. KEY FINDINGS We showed a superimposable time-dependent increase of nuclear pGSK3βTyr216 and nuclear pTau at PHF-1 site, both reaching their maximal level after 8h of Aβ1-42 exposure. In addition, nuclear accumulation of pTau is accompanied by its cytoplasmic decrease suggesting that pTau is translocated in response to Aβ treatment. Besides, our experiments showed that specific pGSK3βTyr216 inhibition is required to drop nuclear pTau, ensuring the involvement of Tyr216 phosphorylation in Aβ-mediated tau phosphorylation at PHF-1 epitope. SIGNIFICANCE These data suggested that in response to Aβ exposure in SH-SY5Y cells, GSK3β activation is performed through Tyr216 phosphorylation and resulted in tau phosphorylation at PHF-1 epitope and in its translocation.

Inhibition of GSK3β 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 Alzheimers 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 GSK3β 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 GSK3β. 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 GSK3β, 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 GSK3β through modulation of sphingolipid metabolism.

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 Alzheimers disease (AD) and that pharmacological inhibition of ganglioside synthesis may reduce amyloid β-peptide (Aβ) 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 Aβ production, although affecting differently the APP cleavage. Surprisingly, the L-enantiomer of PDMP, which promotes ganglioside accumulation, acted similarly to D-PDMP to inhibit Aβ 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 Aβ 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.