An RNA-seq study in Friedreich ataxia patients identified hsa-miR-148a-3p as a putative prognostic biomarker of the disease.

Friedreich ataxia (FRDA) is a life-threatening hereditary ataxia; its incidence is 1:50,000 individuals in the Caucasian population. A unique therapeutic drug for FRDA, the antioxidant Omaveloxolone, has been recently approved by the US Food and Drug Administration (FDA). FRDA is a multi-systemic neurodegenerative disease; in addition to a progressive neurodegeneration, FRDA is characterized by hypertrophic cardiomyopathy, diabetes mellitus and musculoskeletal deformities. Cardiomyopathy is the predominant cause of premature death. The onset of FRDA typically occurs between the ages of 5 and 15. Given the complexity and heterogeneity of clinical features and the variability of their onset, the identification of biomarkers capable of assessing disease progression and monitoring the efficacy of treatments is essential to facilitate decision making in clinical practice. We conducted an RNA-seq analysis in peripheral blood mononuclear cells from FRDA patients and healthy donors, identifying a signature of small non-coding RNAs (sncRNAs) capable of distinguishing healthy individuals from the majority of FRDA patients. Among the differentially expressed sncRNAs, microRNAs are a class of small non-coding endogenous RNAs that regulate posttranscriptional silencing of target genes. In FRDA plasma samples, hsa-miR-148a-3p resulted significantly upregulated. The analysis of the Receiver Operating Characteristic (ROC) curve, combining the circulating expression levels of hsa-miR-148a-3p and hsa-miR-223-3p (previously identified by our group), revealed an Area Under the Curve (AUC) of 0.86 (95%, Confidence Interval 0.77-0.95; p-value < 0.0001). An in silico prediction analysis indicated that the IL6ST gene, an interesting marker of neuroinflammation in FRDA, is a common target gene of both miRNAs. Our findings support the evaluation of combined expression levels of different circulating miRNAs as potent epi-biomarkers in FRDA. Moreover, we found hsa-miR-148a-3p significantly over-expressed in Intermediate and Late-Onset Friedreich Ataxia patients' group (IOG and LOG, respectively) compared to healthy individuals, indicating it as a putative prognostic biomarker in this pathology.

Interferon Gamma Enhances Cytoprotective Pathways via Nrf2 and MnSOD Induction in Friedreich's Ataxia Cells.

Friedreich's ataxia (FRDA) is a rare monogenic disease characterized by multisystem, slowly progressive degeneration. Because of the genetic defect in a non-coding region of FXN gene, FRDA cells exhibit severe deficit of frataxin protein levels. Hence, FRDA pathophysiology is characterized by a plethora of metabolic disruptions related to iron metabolism, mitochondrial homeostasis and oxidative stress. Importantly, an impairment of the antioxidant defences exacerbates the oxidative damage. This appears closely associated with the disablement of key antioxidant proteins, such as the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the mitochondrial superoxide dismutase (MnSOD). The cytokine interferon gamma (IFN-γ) has been shown to increase frataxin expression in FRDA cells and to improve functional deficits in FRDA mice. Currently, IFN-γ represents a potential therapy under clinical evaluation in FRDA patients. Here, we show that IFN-γ induces a rapid expression of Nrf2 and MnSOD in different cell types, including FRDA patient-derived fibroblasts. Our data indicate that IFN-γ signals two separate pathways to enhance Nrf2 and MnSOD levels in FRDA fibroblasts. MnSOD expression increased through an early transcriptional regulation, whereas the levels of Nrf2 are induced by a post-transcriptional mechanism. We demonstrate that the treatment of FRDA fibroblasts with IFN-γ stimulates a non-canonical Nrf2 activation pathway through p21 and potentiates antioxidant responses under exposure to hydrogen peroxide. Moreover, IFN-γ significantly reduced the sensitivity to hydrogen peroxide-induced cell death in FRDA fibroblasts. Collectively, these results indicate the presence of multiple pathways triggered by IFN-γ with therapeutic relevance to FRDA.

Drug Repositioning in Friedreich Ataxia.

Friedreich ataxia is a rare neurodegenerative disorder caused by insufficient levels of the essential mitochondrial protein frataxin. It is a severely debilitating disease that significantly impacts the quality of life of affected patients and reduces their life expectancy, however, an adequate cure is not yet available for patients. Frataxin function, although not thoroughly elucidated, is associated with assembly of iron-sulfur cluster and iron metabolism, therefore insufficient frataxin levels lead to reduced activity of many mitochondrial enzymes involved in the electron transport chain, impaired mitochondrial metabolism, reduced ATP production and inefficient anti-oxidant response. As a consequence, neurons progressively die and patients progressively lose their ability to coordinate movement and perform daily activities. Therapeutic strategies aim at restoring sufficient frataxin levels or at correcting some of the downstream consequences of frataxin deficiency. However, the classical pathways of drug discovery are challenging, require a significant amount of resources and time to reach the final approval, and present a high failure rate. Drug repositioning represents a viable alternative to boost the identification of a therapy, particularly for rare diseases where resources are often limited. In this review we will describe recent efforts aimed at the identification of a therapy for Friedreich ataxia through drug repositioning, and discuss the limitation of such strategies.

Hsa-miR223-3p circulating level is upregulated in Friedreich's ataxia and inversely associated with HCLS1 associated protein X-1, HAX-1.

Frataxin (FXN) deficiency is responsible for Friedreich's ataxia (FRDA) in which, besides the characteristic features of spinocerebellar ataxia, two thirds of patients develop hypertrophic cardiomyopathy that often progresses to heart failure and premature death. Different mechanisms might underlie FRDA pathogenesis. Among them, the role of miRNAs deserves investigations. We carried out an miRNA PCR-array analysis of plasma samples of early-, intermediate- and late-onset FRDA groups, defining a set of 30 differentially expressed miRNAs. Hsa-miR223-3p is the only miRNA shared between the three patient groups and appears upregulated in all of them. The up-regulation of hsa-miR223-3p was further validated in all enrolled patients (n = 37, Fc = +2.3; P < 0.0001). Using a receiver operating characteristic curve analysis, we quantified the predictive value of circulating hsa-miR223-3p for FRDA, obtaining an area under the ROC curve value of 0.835 (P < 0.0001) for all patients. Interestingly, we found a significant positive correlation between hsa-miR223-3p expression and cardiac parameters in typical FRDA patients (onset < 25 years). Moreover, a significant negative correlation between hsa-miR223-3p expression and HAX-1 (HCLS1-associated protein X-1) at mRNA and protein level was observed in all FRDA patients. In silico analyses suggested HAX-1 as a target gene of hsa-miR223-3p. Accordingly, we report that HAX-1 is negatively regulated by hsa-miR223-3p in cardiomyocytes (AC16) and neurons (SH-SY5Y), which are critically affected cell types in FRDA. This study describes for the first time the association between hsa-miR223-3p and HAX-1 expression in FRDA, thus supporting a potential role of this microRNA as non-invasive epigenetic biomarker for FRDA.

Frataxin deficiency in Friedreich's ataxia is associated with reduced levels of HAX-1, a regulator of cardiomyocyte death and survival.

Frataxin deficiency, responsible for Friedreich's ataxia (FRDA), is crucial for cell survival since it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. In FRDA, the heart is frequently affected with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and cause premature death. A microarray analysis performed on FRDA patient's lymphoblastoid cells stably reconstituted with frataxin, indicated HS-1-associated protein X-1 (HAX-1) as the most significantly upregulated transcript (FC = +2, P < 0.0006). quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and western blot analysis performed on (I) HEK293 stably transfected with empty vector compared to wild-type frataxin and (II) lymphoblasts from FRDA patients show that low frataxin mRNA and protein expression correspond to reduced levels of HAX-1. Frataxin overexpression and silencing were also performed in the AC16 human cardiomyocyte cell line. HAX-1 protein levels are indeed regulated through frataxin modulation. Moreover, correlation between frataxin and HAX-1 was further evaluated in peripheral blood mononuclear cells (PBMCs) from FRDA patients and from non-related healthy controls. A regression model for frataxin which included HAX-1, group membership and group* HAX-1 interaction revealed that frataxin and HAX-1 are associated both at mRNA and protein levels. Additionally, a linked expression of FXN, HAX-1 and antioxidant defence proteins MnSOD and Nrf2 was observed both in PBMCs and AC16 cardiomyocytes. Our results suggest that HAX-1 could be considered as a potential biomarker of cardiac disease in FRDA and the evaluation of its expression might provide insights into its pathogenesis as well as improving risk stratification strategies.

Hyaluronic Acid (HA), Platelet-Rich Plasm and Extracorporeal Shock Wave Therapy (ESWT) promote human chondrocyte regeneration in vitro and ESWT-mediated increase of CD44 expression enhances their susceptibility to HA treatment.

Novel strategies have been proposed for articular cartilage damage occurring during osteoarthritis (OA) and -among these- Extracorporeal Shock Wave Therapy (ESWT), intra-articular injections of Platelet-Rich Plasma (PRP) or Hyaluronic Acid (HA) revealed encouraging results. To investigate the possible mechanisms responsible for those clinical benefits, we established primary cultures of human chondrocytes derived from cartilage explants and measured the in vitro effects of ESW, PRP and HA therapies. After molecular/morphological cell characterization, we assessed those effects on the functional activities of the chondrocyte cell cultures, at the protein and molecular levels. ESWT significantly prevented the progressive dedifferentiation that spontaneously occurs during prolonged chondrocyte culture. We then attested the efficiency of all such treatments to stimulate the expression of markers of chondrogenic potential such as SOX9 and COL2A, to increase the Ki67 proliferation index as well as to antagonize the traditional marker of chondrosenescence p16INK4a (known as Cdkn2a). Furthermore, all our samples showed an ESW- and HA-mediated enhancement of migratory and anti-inflammatory activity onto the cytokine-rich environment characterizing OA. Taken together, those results suggest a regenerative effect of such therapies on primary human chondrocytes in vitro. Moreover, we also show for the first time that ESW treatment induces the surface expression of major hyaluronan cell receptor CD44 allowing the increase of COL2A/COL1A ratio upon HA administration. Therefore, this work suggests that ESW-induced CD44 overexpression enhances the in vitro cell susceptibility of human chondrocytes to HA, presumably favouring the repair of degenerated cartilage.

Drug repositioning screening identifies etravirine as a potential therapeutic for friedreich's ataxia.

BACKGROUND: Friedreich's ataxia is an autosomal-recessive cerebellar ataxia caused by mutation of the frataxin gene, resulting in decreased frataxin expression, mitochondrial dysfunction, and oxidative stress. Currently, no treatment is available for Friedreich's ataxia patients. Given that levels of residual frataxin critically affect disease severity, the main goal of a specific therapy for Friedreich's ataxia is to increase frataxin levels. OBJECTIVES: With the aim to accelerate the development of a new therapy for Friedreich's ataxia, we took a drug repositioning approach to identify market-available drugs able to increase frataxin levels. METHODS: Using a cell-based reporter assay to monitor variation in frataxin amount, we performed a high-throughput screening of a library containing 853 U.S. Food and Drug Administration-approved drugs. RESULTS: Among the potentially interesting candidates isolated from the screening, we focused our attention on etravirine, an antiviral drug currently in use as an anti-human immunodeficiency virus therapy. Here, we show that etravirine can promote a significant increase in frataxin levels in cells derived from Friedreich's ataxia patients, by enhancing frataxin messenger RNA translation. Importantly, frataxin accumulation in treated patient cell lines is comparable to frataxin levels in unaffected carrier cells, suggesting that etravirine could be therapeutically relevant. Indeed, etravirine treatment restores the activity of the iron-sulphur cluster containing enzyme aconitase and confers resistance to oxidative stress in cells derived from Friedreich's ataxia patients. CONCLUSIONS: Considering its excellent safety profile along with its ability to increase frataxin levels and correct some of the disease-related defects, etravirine represents a promising candidate as a therapeutic for Friedreich's ataxia. © 2019 International Parkinson and Movement Disorder Society.

E3 Ligase RNF126 Directly Ubiquitinates Frataxin, Promoting Its Degradation: Identification of a Potential Therapeutic Target for Friedreich Ataxia.

Friedreich ataxia (FRDA) is a severe genetic neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. To date, there is no therapy to treat this condition. The amount of residual frataxin critically affects the severity of the disease; thus, attempts to restore physiological frataxin levels are considered therapeutically relevant. Frataxin levels are controlled by the ubiquitin-proteasome system; therefore, inhibition of the frataxin E3 ligase may represent a strategy to achieve an increase in frataxin levels. Here, we report the identification of the RING E3 ligase RNF126 as the enzyme that specifically mediates frataxin ubiquitination and targets it for degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner, both in vivo and in vitro. Most importantly, RNF126 depletion results in frataxin accumulation in cells derived from FRDA patients, highlighting the relevance of RNF126 as a new therapeutic target for Friedreich ataxia.

Extracorporeal Shock Wave Treatment (ESWT) enhances the in vitro-induced differentiation of human tendon-derived stem/progenitor cells (hTSPCs).

Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury.

Src inhibitors modulate frataxin protein levels.

Defective expression of frataxin is responsible for the inherited, progressive degenerative disease Friedreich's Ataxia (FRDA). There is currently no effective approved treatment for FRDA and patients die prematurely. Defective frataxin expression causes critical metabolic changes, including redox imbalance and ATP deficiency. As these alterations are known to regulate the tyrosine kinase Src, we investigated whether Src might in turn affect frataxin expression. We found that frataxin can be phosphorylated by Src. Phosphorylation occurs primarily on Y118 and promotes frataxin ubiquitination, a signal for degradation. Accordingly, Src inhibitors induce accumulation of frataxin but are ineffective on a non-phosphorylatable frataxin-Y118F mutant. Importantly, all the Src inhibitors tested, some of them already in the clinic, increase frataxin expression and rescue the aconitase defect in frataxin-deficient cells derived from FRDA patients. Thus, Src inhibitors emerge as a new class of drugs able to promote frataxin accumulation, suggesting their possible use as therapeutics in FRDA.

Highly specific ubiquitin-competing molecules effectively promote frataxin accumulation and partially rescue the aconitase defect in Friedreich ataxia cells.

Friedreich ataxia is an inherited neurodegenerative disease that leads to progressive disability. There is currently no effective treatment and patients die prematurely. The underlying genetic defect leads to reduced expression of the mitochondrial protein frataxin. Frataxin insufficiency causes mitochondrial dysfunction and ultimately cell death, particularly in peripheral sensory ganglia. There is an inverse correlation between the amount of residual frataxin and the severity of disease progression; therefore, therapeutic approaches aiming at increasing frataxin levels are expected to improve patients' conditions. We previously discovered that a significant amount of frataxin precursor is degraded by the ubiquitin/proteasome system before its functional mitochondrial maturation. We also provided evidence for the therapeutic potential of small molecules that increase frataxin levels by docking on the frataxin ubiquitination site, thus preventing frataxin ubiquitination and degradation. We called these compounds ubiquitin-competing molecules (UCM). By extending our search for effective UCM, we identified a set of new and more potent compounds that more efficiently promote frataxin accumulation. Here we show that these compounds directly interact with frataxin and prevent its ubiquitination. Interestingly, these UCM are not effective on the ubiquitin-resistant frataxin mutant, indicating their specific action on preventing frataxin ubiquitination. Most importantly, these compounds are able to promote frataxin accumulation and aconitase rescue in cells derived from patients, strongly supporting their therapeutic potential.

Recovery of immunological homeostasis positively correlates both with early stages of right-colorectal cancer and laparoscopic surgery.

Differences in postoperative outcome and recovery between patients subjected to laparoscopic-assisted versus open surgery for colorectal cancer (CRC) resection have been widely documented, though not specifically for right-sided tumors. We investigated the immunological responses to the different surgical approaches, by comparing postoperative data simultaneously obtained at systemic, local and cellular levels. A total of 25 right-sided CRC patients and controls were managed, assessing -in the immediate followup- the conventional perioperative parameters and a large panel of cytokines on plasma, peritoneal fluids and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) tissue cultures. A general better recovery for patients operated with laparoscopy compared to conventional procedure, as indicated by the analysis of typical pre- and post-surgical parameters, was observed. The synchronous evaluation of 12 cytokines showed that preoperative plasma levels of the proinflammatory cytokines IL-6, IL-8, IL-1ß, TNFα were significantly lower in healthy donors versus CRC patients and that such differences progressively increase with tumor stage. After surgery, the IL-6 and IL-8 increases were significantly higher in open compared to laparoscopic approach only in CRC at early stages. The postsurgical whole panel of cytokine levels were significantly higher in peritoneal fluids compared to corresponding plasma, but with no significant differences depending on kind of surgery or stage of disease. Then we observed that, pre- compared to the corresponding post-surgery derived LPS-stimulated PBMC cultures, produced higher supernatant levels of the whole cytokine panel. In particular IL-6 in vitro production was significantly higher in PBMC derived from patients subjected to laparoscopic versus open intervention, but -again- only in CRC at early stages of disease. Our results thus show that laparoscopy compared to open right resection is associated with a shorter compromission of the immunological homeostasis, mainly in early stages of right-CRC patients.

Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans.

Severe mitochondria deficiency leads to a number of devastating degenerative disorders, yet, mild mitochondrial dysfunction in different species, including the nematode Caenorhabditis elegans, can have pro-longevity effects. This apparent paradox indicates that cellular adaptation to partial mitochondrial stress can induce beneficial responses, but how this is achieved is largely unknown. Complete absence of frataxin, the mitochondrial protein defective in patients with Friedreich's ataxia, is lethal in C. elegans, while its partial deficiency extends animal lifespan in a p53 dependent manner. In this paper we provide further insight into frataxin control of C. elegans longevity by showing that a substantial reduction of frataxin protein expression is required to extend lifespan, affect sensory neurons functionality, remodel lipid metabolism and trigger autophagy. We find that Beclin and p53 genes are required to induce autophagy and concurrently reduce lipid storages and extend animal lifespan in response to frataxin suppression. Reciprocally, frataxin expression modulates autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts also display increased autophagy, indicating an evolutionarily conserved response to reduced frataxin expression. In sum, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, thus providing the rationale for investigating autophagy in the pathogenesis and treatment of Friedreich's ataxia and possibly other human mitochondria-associated disorders.

Interferon gamma upregulates frataxin and corrects the functional deficits in a Friedreich ataxia model.

Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting ∼3 in 100 000 individuals in Caucasian populations. It is caused by intronic GAA repeat expansions that hinder the expression of the FXN gene, resulting in defective levels of the mitochondrial protein frataxin. Sensory neurons in dorsal root ganglia (DRG) are particularly damaged by frataxin deficiency. There is no specific therapy for FRDA. Here, we show that frataxin levels can be upregulated by interferon gamma (IFNγ) in a variety of cell types, including primary cells derived from FRDA patients. IFNγ appears to act largely through a transcriptional mechanism on the FXN gene. Importantly, in vivo treatment with IFNγ increases frataxin expression in DRG neurons, prevents their pathological changes and ameliorates the sensorimotor performance in FRDA mice. These results disclose new roles for IFNγ in cellular metabolism and have direct implications for the treatment of FRDA.

Preventing the ubiquitin-proteasome-dependent degradation of frataxin, the protein defective in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent frataxin ubiquitination and degradation, and increase frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the frataxin degradation pathway.

Molecular control of the cytosolic aconitase/IRP1 switch by extramitochondrial frataxin.

The inability to produce normal levels of the mitochondrial protein frataxin causes the hereditary degenerative disorder Friedreich's Ataxia (FRDA), a syndrome characterized by progressive gait instability, cardiomyopathy and high incidence of diabetes. Frataxin is an iron-binding protein involved in the biogenesis of iron-sulfur clusters (ISC), prosthetic groups allowing essential cellular functions such as oxidative phosphorylation, enzyme catalysis and gene regulation. Although several evidence suggest that frataxin acts as an iron-chaperone within the mitochondrial compartment, we have recently demonstrated the existence of a functional extramitochondrial pool of mature frataxin in various human cell types. Here, we show that a similar proteolytic process generates both mature mitochondrial and extramitochondrial frataxin. To address the physiological function of human extramitochondrial frataxin, we searched for ISC-dependent interaction partners. We demonstrate that the extramitochondrial form of frataxin directly interacts with cytosolic aconitase/iron regulatory protein-1 (IRP1), a bifunctional protein alternating between an enzymatic and a RNA-binding function through the 'iron-sulfur switch' mechanism. Importantly, we found that the cytosolic aconitase defect and consequent IRP1 activation occurring in FRDA cells are reversed by the action of extramitochondrial frataxin. These results provide new insight into the control of cytosolic aconitase/IRP1 switch and expand current knowledge about the molecular pathogenesis of FRDA.

In vivo maturation of human frataxin.

The defective expression of frataxin causes the hereditary neurodegenerative disorder Friedreich's ataxia (FRDA). Human frataxin is synthesized as a 210 amino acid precursor protein, which needs proteolytic processing into mitochondria to be converted into the functional mature form. In vitro processing of human frataxin was previously described to yield a 155 amino acid mature form, corresponding to residues 56-210 (frataxin(56-210)). Here, we studied the maturation of frataxin by in vivo overexpression in human cells. Our data show that the main form of mature frataxin is generated by a proteolytic cleavage between Lys80 and Ser81, yielding a 130 amino acid protein (frataxin(81-210)). This maturation product corresponds to the endogenous frataxin detected in human heart, peripheral blood lymphocytes or dermal fibroblasts. Moreover, we demonstrate that frataxin(81-210) is biologically functional, as it rescues aconitase defects in frataxin-deficient cells derived from FRDA patients. Importantly, our data indicate that frataxin(56-210) can be produced in vivo when the primary 80-81 maturation site is unavailable, suggesting the existence of proteolytic mechanisms that can actively control the size of the mature product, with possible functional implications.

A pool of extramitochondrial frataxin that promotes cell survival.

Frataxin is a mitochondrial protein involved in iron metabolism. Defective expression of frataxin causes Friedreich ataxia (FA), an inherited degenerative syndrome characterized by ataxia, cardiomyopathy, and high incidence of diabetes. Here we report that frataxin-deficient cells are more prone to undergo stress-induced mitochondrial damage and apoptosis, while the overexpression of frataxin confers protection to a variety of cell types. Moreover, we reveal the existence of an extramitochondrial pool of frataxin, which can efficiently prevent mitochondrial damage and apoptosis in different cellular systems. Remarkably, extramitochondrial frataxin can fully replace mitochondrial frataxin in promoting survival of FA cells.

9-O-acetyl GD3 protects tumor cells from apoptosis.

The ganglioside GD3 (Neu5Ac alpha8Neu5Ac alpha3Gal beta4GlcCer) is an intracellular lipid messenger that induces apoptosis by targeting mitochondria in various cell types. GD3 can also promote apoptosis when externally added to cells. Previous studies showed that the proapoptotic effects of GD3 can be counteracted by 9-O-acetylation. To determine whether 9-O-acetyl GD3 (acGD3) has a general antiapoptotic potential, the apoptosis-sensitive Jurkat cell line and an apoptosis-sensitive variant of the cell line Molt-4 were preincubated with micromolar concentrations of acGD3 and then treated with inducers of apoptosis. A reduced apoptotic index and an increased cell viability were observed. On the other hand, when the Jurkat cells were treated with GD3 for extended periods of time, a population was selected that was resistant to apoptosis induction by N-acetyl sphingosine as well as by the anti-leukemic drug daunorubicin. Comparative analysis of gangliosides revealed the formation of acGD3 in the resistant Jurkat cells that was not found in the apoptosis-sensitive cells. Conversely, exposing the acGD3 positive and apoptosis-resistant cell line Molt-4 to the O-deacetylating activity of salicylate resulted in a complete disappearance of acGD3 and an enhanced sensitivity to N-acetyl sphingosine-mediated apoptosis. Formation of acGD3 might thus represent a new mechanism how tumor cells can escape apoptosis.

Ceramide catabolism critically controls survival of human dendritic cells.

The regulation of dendritic cell (DC) survival is crucial for the modulation of adaptive immunity. Ceramide is a lipid mediator of the stress response, which accumulates intracellularly during DC differentiation. We found that ceramide levels are tightly regulated in human DCs and that the pharmacological inhibition of enzymes responsible for ceramide catabolism, such as ceramidases and sphingosine kinases, sensitizes DCs to ceramide-induced cell death. It is important that inhibition of sphingosine kinases, during lipopolysaccharide stimulation, causes extensive ceramide accumulation and death of DCs. These data indicate that ceramide catabolism regulates survival of human DCs and reveal novel potential targets for the pharmacological manipulation of the immune response.