Cross-Talk between Oxysterols and Glucocorticoids: Differential Regulation of Secreted Phopholipase A2 and Impact on Oligodendrocyte Death

BACKGROUND: Oxysterols are oxidized forms of cholesterol. They have been shown to be implicated in cholesterol turnover, inflammation and in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis. Glial cells are targets of oxysterols: they inhibit astrocyte proliferation after brain injury, and we have previously shown that 25-hydroxycholesterol (25OH) provokes oligodendrocyte apoptosis and stimulates the expression of sPLA2 type IIA (sPLA2-IIA), which has a protective effect. METHODOLOGY/PRINCIPAL FINDINGS: As glucocorticoids are well-known for their anti-inflammatory effects, our aim was to understand their direct effects on oxysterol-induced responses in oligodendrocytes (sPLA2-IIA stimulation and apoptosis). We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) abolishes the stimulation of sPLA2-IIA by 25-hydroxycholesterol (25-OH). This inhibition is mediated by the glucocorticoid receptor (GR), which decreases the expression of the oxysterol receptor Pregnane X Receptor (PXR) and interferes with oxysterol signaling by recruiting a common limiting coactivator PGC1alpha. Consistent with the finding that sPLA2-IIA can partially protect oligodendrocytes against oxysterol-triggered apoptosis, we demonstrate here that the inhibition of sPLA2-IIA by Dex accelerates the apoptotic phenomenon, leading to a shift towards necrosis. We have shown by atomic force microscopy and electron microscopy that 25-OH and Dex alters oligodendrocyte shape and disorganizes the cytoplasm. CONCLUSIONS/SIGNIFICANCE: Our results provide a new understanding of the cross-talk between oxysterol and glucocorticoid signaling pathways and their respective roles in apoptosis and oligodendrocyte functions

Sex-specific Gene Expression in Flupirtine-Treated Cln3Δex7/8 Mouse Brain

Gene expression is a powerful tool to understand structure-function relationships in the nervous system. This study reports global gene expression changes induced by flupirtine in brain of male and female Cln3Δex7/8 mice, exposing potential flupirtine targets at the molecular level. Gene expression analysis of male and female Cln3Δex7/8 mouse brain was determined following oral administration of flupirtine for 14 weeks, using Mouse Genome 430 2.0 array Chips and an Affymetrix platform. Fifty-six genes in males and 79 in females were differentially expressed in flupirtine- versus vehicle-treated Cln3Δex7/8 mouse brain. Flupirtine altered several pathways in Cln3Δex7/8 mouse brain: apoptosis, the complement cascade, NF-kB, and p38α MAPK signaling pathways. Gene-gene network analysis highlighted networks and processes functionally pertinent to flupirtine treatment. These encompassed neurodegeneration, neuro-inflammation, and implicated neurological disorders such as Alzheimer and Parkinson disease. Flupirtine mediates its action in males and females through distinctive actionable targets in the same pathways. This work consolidates the groundwork for considering flupirtine as a treatment option in human CLN3 disease

Exogenous Flupirtine as Potential Treatment for CLN3 Disease

CLN3 disease is a fatal neurodegenerative disorder affecting children. Hallmarks include brain atrophy, accelerated neuronal apoptosis, and ceramide elevation. Treatment regimens are supportive, highlighting the importance of novel, disease-modifying drugs. Flupirtine and its new allyl carbamate derivative (compound 6) confer neuroprotective effects in CLN3-deficient cells. This study lays the groundwork for investigating beneficial effects in Cln3Δex7/8 mice. WT/Cln3Δex7/8 mice received flupirtine/compound 6/vehicle for 14 weeks. Short-term effect of flupirtine or compound 6 was tested using a battery of behavioral testing. For flupirtine, gene expression profiles, astrogliosis, and neuronal cell counts were determined. Flupirtine improved neurobehavioral parameters in open field, pole climbing, and Morris water maze tests in Cln3Δex7/8 mice. Several anti-apoptotic markers and ceramide synthesis/degradation enzymes expression was dysregulated in Cln3Δex7/8 mice. Flupirtine reduced astrogliosis in hippocampus and motor cortex of male and female Cln3Δex7/8 mice. Flupirtine increased neuronal cell counts in male mice. The newly synthesized compound 6 showed promising results in open field and pole climbing. In conclusion, flupirtine improved behavioral, neuropathological and biochemical parameters in Cln3Δex7/8 mice, paving the way for potential therapies for CLN3 disease

Flupirtine Derivatives as Potential Treatment for the Neuronal Ceroid Lipofuscinoses

OBJECTIVE: Neuronal Ceroid Lipofuscinoses (NCL) are fatal inherited neurodegenerative diseases with established neuronal cell death and increased ceramide levels in brain, hence, a need for disease-modifying drug candidates, with potential to enhance growth, reduce apoptosis and lower ceramide in neuronal precursor PC12 cells and human NCL cell lines using enhanced flupirtine aromatic carbamate derivatives in vitro. METHODS: Aromatic carbamate derivatives were tested by establishing growth curves under pro-apoptotic conditions and activity evaluated by trypan blue and JC-1 staining, as well as a drop in pro-apoptotic ceramide in neuronal precursor PC12 cells following siRNA knockdown of the RESULTS: Retigabine, the benzyl-derivatized carbamate and an allyl carbamate derivative were neuroprotective in CLN3-defective PC12 cells and rescued CLN1-/CLN2-/CLN3-/CLN6-/CLN8 patient-derived lymphoblasts from diminished growth and accelerated apoptosis. All drugs decreased ceramide in CLN1-/CLN2-/CLN3-/CLN6-/CLN8 patient-derived lymphoblasts. Increased INTERPRETATION: These findings establish that compounds analogous to flupirtine demonstrate anti-apoptotic activity with potential for treatment of NCL disease and use of ceramide as a marker for these diseases

Developmental Comparison of Ceramide in Wild-Type and Cln3Δex7/8 Mouse Brains and Sera

CLN3 disease is a neurodevelopmental disease leading to early visual failure, motor decline, and death. CLN3 pathogenesis has been linked to dysregulation of ceramide, a key intracellular messenger impacting various biological functions. Ceramide is upregulated in brains of CLN3 patients and activates apoptosis. Ceramide levels over the lifespan of WT and Cln3Δex7/8 mice were measured using the DGK assay. Ceramide subspecies were determined by LC-MS. Ceramide synthesis enzymes and pre- and post-synaptic mRNA expression was measured in Cln3Δex7/8 and normal mouse brains. Neuronal cell death was established by PARP cleavage and Caspases 3/6/9 and cytochrome C mRNA expression in Cln3Δex7/8 and normal mouse brains. In WT mouse, a ceramide peak was noted at 3 weeks of age. The absence of this peak in Cln3Δex7/8 mice might be related to early disease pathogenesis. Increase of ceramide in Cln3Δex7/8 mouse brain at 24 weeks of age precedes neuronal apoptosis. The correlation between serum and brain ceramide in WT mice, and dysregulation of ceramide in serum and brain of Cln3Δex7/8 mice, and the significant increase in ceramide in Cln3Δex7/8 mouse brains and sera argue for use of easily accessible serum ceramide levels to track response to novel therapies in human CLN3 disease

Shift from extracellular signal-regulated kinase to AKT/cAMP response element-binding protein pathway increases survival-motor-neuron expression in spinal-muscular-atrophy-like mice and patient cells

Spinal muscular atrophy (SMA), a recessive neurodegenerative disease, is characterized by the selective loss of spinal motor neurons. No available therapy exists for SMA, which represents one of the leading genetic causes of death in childhood. SMA is caused by a mutation of the survival-of-motor-neuron 1 (SMN1) gene, leading to a quantitative defect in the survival-motor-neuron (SMN) protein expression. All patients retain one or more copies of the SMN2 gene, which modulates the disease severity by producing a small amount of stable SMN protein. We reported recently that NMDA receptor activation, directly in the spinal cord, significantly enhanced the transcription rate of the SMN2 genes in a mouse model of very severe SMA (referred as type 1) by a mechanism that involved AKT/CREB pathway activation. Here, we provide the first compelling evidence for a competition between the MEK/ERK/Elk-1 and the phosphatidylinositol 3-kinase/AKT/CREB signaling pathways for SMN2 gene regulation in the spinal cord of type 1 SMA-like mice. The inhibition of the MEK/ERK/Elk-1 pathway promotes the AKT/CREB pathway activation, leading to (1) an enhanced SMN expression in the spinal cord of SMA-like mice and in human SMA myotubes and (2) a 2.8-fold lifespan extension in SMA-like mice. Furthermore, we identified a crosstalk between ERK and AKT signaling pathways that involves the calcium-dependent modulation of CaMKII activity. Together, all these data open new perspectives to the therapeutic strategy for SMA patients.This project was supported by the Association Française contre les Myopathies. J.B. is the recipients of a fellowship from the Ministry of Research and Technology, and F.Chal. is the recipient of a fellowship from AXA Research Fund/Garches Foundation.Peer reviewe

Wnt/β-Catenin Signaling Pathway Is a Direct Enhancer of Thyroid Transcription Factor-1 in Human Papillary Thyroid Carcinoma Cells

The Wnt/β-catenin signaling pathway is involved in the normal development of thyroid gland, but its disregulation provokes the appearance of several types of cancers, including papillary thyroid carcinomas (PTC) which are the most common thyroid tumours. The follow-up of PTC patients is based on the monitoring of serum thyroglobulin levels which is regulated by the thyroid transcription factor 1 (TTF-1): a tissue-specific transcription factor essential for the differentiation of the thyroid. We investigated whether the Wnt/β-catenin pathway might regulate TTF-1 expression in a human PTC model and examined the molecular mechanisms underlying this regulation. Immunofluorescence analysis, real time RT-PCR and Western blot studies revealed that TTF-1 as well as the major Wnt pathway components are co-expressed in TPC-1 cells and human PTC tumours. Knocking-down the Wnt/β-catenin components by siRNAs inhibited both TTF-1 transcript and protein expression, while mimicking the activation of Wnt signaling by lithium chloride induced TTF-1 gene and protein expression. Functional promoter studies and ChIP analysis showed that the Wnt/β-catenin pathway exerts its effect by means of the binding of β-catenin to TCF/LEF transcription factors on the level of an active TCF/LEF response element at [−798, −792 bp] in TTF-1 promoter. In conclusion, we demonstrated that the Wnt/β-catenin pathway is a direct and forward driver of the TTF-1 expression. The localization of TCF-4 and TTF-1 in the same area of PTC tissues might be of clinical relevance, and justifies further examination of these factors in the papillary thyroid cancers follow-up

Rôle des stéroïdes et de la signalisation Wnt dans les cellules de Schwann (implications dans la régénération et les neuropathies périphériques)

La formation de la gaine de myéline est un processus très complexe et finement régulé. En effet, une altération de l expression des gènes codant deux protéines structurales et majeures de la myéline périphérique (MPZ et PMP22) entraîne des neuropathies graves comme la maladie de Charcot-Marie-Tooth (CMT). Notre objectif était donc d identifier des voies de signalisation capables de moduler l expression de ces gènes. Tout d abord, nous nous sommes intéressés à la voie Wnt/b-caténine qui joue un rôle essentiel dans le développement du système nerveux. Nous avons montré que les composants de la voie Wnt sont essentiels pour l expression des gènes de la myéline in vitro et in vivo. L activation de cette voie joue un rôle très important dans le processus de myélinisation. Pour ces raisons, nous avons réalisé des tentatives thérapeutiques en utilisant le lithium qui mime l activation de la voie Wnt. Le traitement par le lithium d animaux, dont les nerfs sciatiques ou faciaux sont lésés par compression, permet la remyélinisation des nerfs et une récupération fonctionnelle. De plus, nous avons montré que la b-caténine est un coactivateur jouant un rôle de carrefour entre la voie Wnt et celle des glucocorticoïdes. Ceci permettrait donc une potentialisation des effets de la voie Wnt par les glucocorticoïdes au niveau de l activation des gènes de la myéline. La deuxième partie de ma thèse à pour objet l étude de l impact des oxystérols dans la régulation des gènes de la myéline périphérique. Les oxystérols sont connus pour leur rôle dans le maintien de l homéostasie du cholestérol et dans la progression des maladies neurodégénératives du SNC. Nos résultats suggèrent que les oxystérols exercent un effet inhibiteur vis-à-vis de l expression des gènes de la myéline périphérique MPZ et PMP22 via les récepteurs XR. Cet effet est indirect puisqu il passe par le recrutement du corégulateur Rip140 et par une régulation négative de la voie Wnt/b-caténine. Enfin, notre travail a d importantes perspectives thérapeutiques soit pour le traitement d une démyélinisation due à un écrasement du nerf mimant un traumatisme ou pour le traitement des maladies neurodégénératives telle que la CMT1A, surtout pendant la phase précoce de la maladie, qui est caractérisée par une surexpression du gène PMP22PARIS-BIUP (751062107) / SudocSudocFranceF

Sex differences in gene expression with galactosylceramide treatment in Cln3Δex7/8 mice.

BackgroundCLN3 disease is caused by mutations in the CLN3 gene. The purpose of this study is to discern global expression patterns reflecting therapeutic targets in CLN3 disease.MethodsDifferential gene expression in vehicle-exposed mouse brain was determined after intraperitoneal vehicle/Galactosylceramide (GalCer) injections for 40 weeks with GeneChip Mouse Genome 430 2.0 arrays.ResultsAnalysis identified 66 genes in male and 30 in female brains differentially expressed in GalCer-treated versus vehicle-exposed Cln3Δex7/8 mice. Gene ontology revealed aberrations of biological function including developmental, cellular, and behavioral processes. GalCer treatment altered pathways of long-term potentiation/depression, estrogen signaling, synaptic vesicle cycle, ErbB signaling, and prion diseases in males, but prolactin signaling, selenium compound metabolism and steroid biosynthesis in females. Gene-gene network analysis highlighted networks functionally pertinent to GalCer treatment encompassing motor dysfunction, neurodegeneration, memory disorder, inflammation and astrogliosis in males, and, cataracts, inflammation, astrogliosis, and anxiety in females.ConclusionsThis study sheds light on global expression patterns following GalCer treatment of Cln3Δex7/8 mice. Understanding molecular effects of GalCer on mouse brain gene expression, paves the way for personalized strategies for treating this debilitating disease in humans