Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with bad prognosis, which predominates in males with advanced age. However, studies of the endocannabinoid system and how it affects tumour behaviour provides the basis for designing innovative therapeutic strategies that could open new opportunities for treatment of patient with MCL. It has earlier been shown that the cannabinoid receptor ligand (R)-(+)-methanandamide (R-MA) induce cell death in MCL by accumulation of ceramide. Ceramide has a pro-apoptotic effect on the cell but could be metabolized by the enzymes glucosylceramide synthase (GCS) and sphingosine kinase 1 (SphK1) to molecules with pro-proliferative effect. Therefore, treatments with R-MA on Jeko-1 MCL cell line were performed in this study to determine interference in the proliferative behaviour as well as in the gene expression of the enzymes GCS and SphK1. In addition, treatments with chemotherapeutic substances, such as doxorubicin or cytarabine (Ara-C), and combinations of R-MA and chemotherapeutic substance, were performed for the same reason. Results showed that the proliferation behaviour of Jeko cells remained unaffected when treated with R-MA, in contrast to the decreased proliferative effects shown when treated with cytostatics or combinations of R-MA and cytostatics. Furthermore, a tendency for up-regulation of GCS and SphK1 expression was recognized when cells were treated with cytostatics or combination of cytostatics and R-MA, in contrast to cells treated with R-MA alone. Although, R-MA alone had a tendency for a small down-regulation of GCS expression, it contributed to a potential elevation of GCS expression when combined with Ara-C or doxorubicin. It is believed that the effect from upregulated levels of the metabolizing enzymes GCS and SphK1 is balanced by, earlier observed, up-regulations of the ceramide synthesis enzymes.

Le vieillissement normal s’accompagne d’une diminution du contenu du cholestérol cérébral. Au contraire, une accumulation de cholestérol est associée aux processus toxiques dans plusieurs pathologies dégénératives (maladie d’Huntington, maladie de Parkinson, épilepsie, maladie de Niemann Pick de type C, maladie d’Alzheimer). De plus, les parallèles étroits existent entre la physiopathologie moléculaire de la maladie d’Alzheimer et celle de la maladie de Niemann Pick de type C, maladie de l’homéostasie du cholestérol. Ainsi on retrouve dans ces deux pathologies une hyperphosphorylation de la protéine Tau, associée à une augmentation des endosomes élargis et à la production de peptides A. L’ensemble de ces éléments évoque le rôle potentiel de la surcharge en cholestérol cérébral comme facteur favorisant le développement de la maladie d’Alzheimer. L’objectif de mon travail de doctorat a été de déterminer si une surcharge en cholestérol in vivo dans les neurones de l’hippocampe, région précocement touchée par la maladie d’Alzheimer, pouvait être à l’origine de processus neurotoxiques et de modifications biochimiques et neuropathologiques proches de ceux qui sont observés dans cette pathologie. La quasi-totalité du cholestérol cérébral est synthétisée in situ, la barrière hémato-encéphalique ne permettant qu’un apport minime du cholestérol périphérique. L’excès de cholestérol est exporté de la circulation sanguine sous la forme du 24-hydroxycholestérol, un métabolite produit exclusivement dans les neurones par la cholestérol-24-hydroxylase codée par le gène Cyp46a1.La surcharge en cholestérol a été induite in vivo par inhibition de la cholestérol-24-hydroxylase, dans l’hippocampe par une stratégie d’ARN interférence délivré par une injection stéréotaxique d’un vecteur AAV5. Nous avons étudié, d’une part la capacité d’une accumulation de cholestérol à induire chez la souris normale, un phénotype clinique et neuropathologique proche de la maladie d’Alzheimer et d’autre part si cette même accumulation de cholestérol neuronal pouvait aggraver ou compléter le phénotype Alzheimer d’un modèle murin de la maladie, la souris APP23. L’injection du vecteur AAV5-shCYP46A1 dans la stratum lacunosum moleculare de l’hippocampe conduit à une inhibition significative de l'expression du gène Cyp46a1, associée à une diminution de la concentration du 24-hydroxycholestérol et une augmentation du contenu en cholestérol dans les neurones de l’hippocampe, 3 semaines après l’injection. En réponse à cet excès de cholestérol, des mécanismes régulateurs permettent de diminuer, d’une part l’import et d’augmenter l’export du cholestérol de la cellule et d’autre part d’augmenter le contenu en phosphatidylcholine afin de rétablir un ratio phospholipide/cholestérol physiologique. Cependant, l'accumulation majeure de cholestérol intracellulaire conduit, 3 semaines après l’injection, à une activation de la réponse UPR (Unfolded Protein Response ou stress du réticulum endoplasmique) caractérisée par l'expression des gènes codant les facteurs XBP1s, ATF6, GRP78 associée à celles des protéines PERK phosphorylée, CHOP et caspase 12, entraînant l'activation des caspases 9 et 3. Elle est associée à la phosphorylation des protéines GSK3 (Tyr216) et Tau (Thr231). En parallèle, l’augmentation du cholestérol induit, 3 semaines après l’injection, une augmentation de l’expression de la protéine Rab5 (marqueur des endosomes précoces) et une relocalisation de la protéine APP dans les fractions de radeaux lipidiques associées à l'activation de la voie amyloïdogénique (production des fragments-CTF et des peptides A42). L’étude lipidomique met en évidence, 4 semaines après l’injection, une augmentation du contenu en céramide à longues chaînes et à une augmentation des gangliosides. Tous ces éléments aboutissent à un processus de perte neuronale associée à un recrutement des astrocytes dès la quatrième semaine après l’injection.... / An increasing number of arguments suggest a close and complex link between cholesterol metabolism and neurodegenerative diseases, particularly with Alzheimer’s disease. Normal ageing is associated with a decrease of brain cholesterol content. Conversly, accumulation of brain cholesterol is associated with several neurodegenerative diseases (Huntington disease, Parkinson disease, epilepsy, Niemann Pick C disease, Alzheimer’s disease). Moreover, close connections exist between molecular physiopathology of AD and that of Niemann Pick, a disease of cholesterol homeostasis. Altogether, these results suggest that cholesterol overload might play a role, as an initiating factor for the development of AD.In the brain, cholesterol metabolism is tightly controlled. In adults, cholesterol is mainly synthetized by astrocytes, then shuttles to neurons where it is used. All cholesterol excess must be eliminated. Cholesterol cannot cross freely the blood-brain-barrier. To be metabolized, brain cholesterol must be converted in 24-hydroxy-cholesterol by the cholesterol-24 hydroxylase enzyme, coded by CYP46A1 gene. The objective of my PhD project was to determine if cholesterol accumulation in vivo in hippocampal neurons, a region early involved in AD pathology, could trigger neurotoxic processes with biochemical and neuropathological modifications close to what is observed in AD. Cholesterol overload in vivo was induced by inhibiting cholesterol 24-hydroxylase enzyme activity, using an RNA interference strategy. Stereotactic injection of an AAV5- shCYP46A1 vector in the stratum lacunosum moleculare of the hippocampus led to significant and rapid (as soon as 3 weeks after injection) inhibition of the Cyp46a1 gene in the hippocampus with an absence of RNA interference off-target effect. This inhibition was associated with a decrease of 24-hydroxycholesterol content and an increase of the cholesterol content. In response to this cholesterol excess, cell control mechanisms were initiated leading to decrease import and increase export of cholesterol, accompanied with an increase of phosphatidylcholine content to restore a physiological ratio of phospholipide/cholesterol. However, major accumulation of cholesterol led to neuronal death with activation of caspases 9 et 3, suggesting an apoptotic process. The cholesterol overload drives to an endoplasmic reticulum stress, with activation of the unfolded protein response (UPR) and expression of spliced XBP1, ATF6, GRP78, phosphorylated PERK, CHOP and caspase 12. These modifications were associated with phosphorylation of GSK3 (Tyr 216) and tau (Thr 231) proteins. In parallel, cholesterol accumulation led to increased expression of Rab5 (early endosome marker) and relocalization of APP in rafts domains associated to activation of amyloid pathway (production of -CTF fragments and A42 peptides). Lipidomic analysis showed an increase of ceramides and gangliosides content. All these modifications were associated with neuronal death 4 weeks after injection and astrocytosis, leading to an EEG theta rhythm accelerated to beta frequencies, memory deficits and hippocampal atrophy. In a mouse model of Alzheimer disease, the APP23 mouse, cholesterol accumulation led to major aggravation of the phenotype, with increased production of A peptides, occurring of tau phosphorylation and UPR response, leading to accelerated neuronal death. Altogether, these results suggest a direct link between cholesterol accumulation in the brain and Alzheimer’s disease. Brain cholesterol accumulation could seed the sows to the development of Alzheimer’s pathology. Reducing cerebral cholesterol could thus be a relevant therapeutic strategy to prevent the development, or at least slow down the evolution of the pathology in Alzheimer’s disease.

Im Rahmen dieser Arbeit wurden fluoreszierende Sphingomyelin-Analoga zu Studium der sauren Sphingomyelinase (ASM) synthetisiert. Ausgehend von L-Serin wurde ein Sphingosin-Derivat mit natürlicher Stereochemie dargestellt. Anschließend wurde mittels Phosphorodichloridat-Chemie eine Aminoethylphosphat-Gruppe installiert. Zweifache Fluoreszenzmarkierung ergab Sonden mit der Fähigkeit zu Förster-Resonanzenergietransfer (FRET). Diese wurden als Substrate der ASM akzeptiert und erlaubten die Verfolgung der Enzymaktivität in vitro. Durch die Analyse der photophysikalischen Eigenschaften der Fluorophore wurde das allgemeine Konzept der Phasentrennungs-gestützten Signalverstärkung (PS) abgeleitet. Dieses Konzept wurde erfolgreich bestätigt durch die Synthese einer 30-mal leistungsfähigeren zweiten Generation der FRET-Sonde. Ein homogener Assay wurde entwickelt, der die Quantifizierung der ASM-Aktivität erlaubte. Unter Verwendung von gereinigter rekombinanter humaner ASM, HeLa-Zelllysaten oder Lysaten von murinen embryonalen Fibroblasten (MEFs) als Enzymquelle wurde ausschließlich unter den von der ASM bevorzugten Bedingungen eine vollständige und spezifische Hydrolyse der Sonde beobachtet. Des Weiteren erlaubte die Sonde die Detektion relativer Unterschiede der Aktivität der ASM in kultivierten MEFs mittels Fluoreszenzmikroskopie mit Zweiphotonenanregung (2PE). / Fluorescent sphingomyelin analogues have been synthesized to probe the acid sphingomyelinase (ASM). Starting from L-serine, a sphingosine with natural stereochemistry was synthesized. Subsequently, phosphorodichloridate chemistry was used to install an aminoethyl phosphate moiety. Dual fluorescent labeling afforded probes capable of Förster resonance energy transfer (FRET). They were recognized as substrates of ASM and allowed for monitoring of the enzyme’s activity in vitro. Through analysis of the fluorophores’ photophysical properties, the general concept of partition aided amplification of a FRET probe’s signal (PS) was developed. This concept was successfully confirmed by the synthesis of a second-generation probe with 30-fold improved response. A homogenous assay was developed, which allowed for a quantitation of ASM activity. Using either purified recombinant human ASM, or lysates of HeLa cells or mouse embryonic fibroblasts (MEFs) as an enzyme source, complete and specific cleavage was observed exclusively under conditions preferred by ASM. Furthermore, the probe enabled the detection of relative levels of ASM activity in cultivated MEFs using fluorescence microscopy with two-photon excitation (2PE).

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text

Indiana University-Purdue University Indianapolis (IUPUI) / Autophagy is a conserved cellular process that involves sequestration and degradation of cytosolic contents. The cell can engulf autophagic cargo (lipids, long-lived proteins, protein aggregates, and pathogens) through a double bound membrane called an autophagosome that fuses with a lysosome where hydrolases then degrade these contents. This process is one of the main defenses against starvation and is imperative for newborns at birth. Research on this process has increased exponentially in the last decade since its discovery almost a half a century ago. It has been found that autophagy is an important process in many diseases, continues to be at the forefront of research, and is clearly not fully understood. Our preliminary cell culture data in endothelial and epithelial cells show that a blockade of the de novo ceramide synthesis pathway, during treatment with an autophagy stimulus (cigarette smoke extract exposure), does not result in any reduction in autophagy or autophagic flux. Conversely, when acid sphingomyelinase (ASM) is pharmacologically inhibited, which prevents the generation of ceramide from sphingomyelin in an acidic environment, a profound increase in autophagy is observed. In this work, we hypothesize that (ASM) is an endogenous inhibitor of autophagy. ASM has two forms, a secreted form and a lysosomal form. N-terminal processing in the Golgi determines its cellular fate. In the lysosomal form, the phosphodiesterase is bound in the lysosomal membrane. The pharmacological inhibition mechanism is to release ASM from the membrane and allow other hydrolases to actively degrade the enzyme which, in turn, decreases the activity of ASM. This suggests that either the activity of ASM is a regulator of autophagy or that the presence of ASM, activity aside, is required for the lysosomal nutrient sensing machinery (LYNUS) to function properly. Here, we show that ASM is, in fact, an endogenous inhibitor of autophagy in vitro. The phosphorylation status of P70 S6k, a downstream effector of mammalian target of rapamycin (mTOR), which is part of the LYNUS, shows that dissociation of ASM from the membrane regulates mTOR and disturbs the LYNUS in such a manner as to signal autophagy.

Glycosphingolipids (GSLs), composed of a polar saccharide head and a lipophilic ceramide tail, are ubiquitous components of the plasma membrane of eukaryotic cells. They serve in many regulatory capacities and have antigenic properties towards natural killer T (NKT) cells of the innate immune system. Critical to the recognition of glycosylceramides by NKT cells are antigen presenting cells (APC), such as dendritic cells, which are responsible for binding, processing, and delivery of ligands to these lymphocytes. This event is mediated by CD1d, a major histocompatibility complex-like protein expressed on the surface of APCs, which binds GSL antigens by the ceramide moiety and presents the polar group to the T cell receptors of CD1d-restricted cells. The subsequent immune response involves NKT cell proliferation and emission of numerous cytokines, such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), resulting in the stimulation of the innate and adaptive immune systems through maturation of APCs, activation of T cells, and secretion of antibodies by B cells. To understand the structure-activity relationship between GSLs and NKT cell activity and the requirements for intracellular processing of antigens, analogs of the model compound alphaGalCer (KRN-7000) have been synthesized. These include fluorophore-appended 6”-amino-α-galactosylceramides and N-alkenoyl GSLs, such as PBS-57, a potent alphaGalCer surrogate useful in NKT cell stimulation studies. A nonantigenic beta-C-galactosylceramide has also been prepared as an inhibitor of these innate lymphocytes. To probe the potential for using NKT cells to bias the immune system between the proinflammatory TH1 response or the immunomodulatory TH2 mode, versions of alphaGalCer with shortened ceramides have been created. One of these truncated analogs, PBS-25, has successfully been cocrystallized with CD1d and the binary complex structure solved by X-ray crystallography. Synthetic glycosphingolipids derived from Novosphingobium capsulatum and Sphingomonas paucimobilis have also been made. In assays with classical Valpha14i/Valpha24i NKT cell lines, these Gram-negative bacterial antigens were recognized directly and specifically by host immune systems through CD1d-restriction, unlike GSL-deficient microbes (e.g., Salmonella typhimurium). A search for other GSL-bearing alpha-proteobacteria led to the discovery of another natural glycosphingolipid, an N-alkenoylphytosphingoid-alpha-galactoside, isolated from the outer membrane of Ehrlichia muris.

<p dir="ltr">The recent global pandemic, COVID-19, has revealed to all the importance of understanding the complex relationship between viruses and hosts. Before COVID-19, I started my study of viral protein-host lipid interactions in the hemorrhagic fevers Ebola and Marburg viruses. These viruses contain a matrix protein that interacts with the plasma membrane to facilitate the formation of both authentic viruses and virus-like particles. My goal was to understand the limitations of their specific host lipid interactions. However, when the COVID-19 pandemic began, so to be our swift response in the development of a biosafety level 2 compatible model. This model can be used for studying severe acute respiratory distress syndrome 2 (SARS-CoV-2) assembly, egress, and entry. This model enabled exponentially greater access to more facilities to study the intricacies of SARS-CoV-2 assembly. With more access to studying the virus in a safe model, our goal is to push the understanding of viral assembly faster. I then began to take apart the individual pieces of the model and started to look at understanding the roles that they play independently. The membrane protein is the most abundant structural protein and I studied the specific lipid interactions of the soluble fraction of the protein. Physicians observed nucleocapsid protein mutations in the clinic with the increasing number of SARS-CoV-2 variants that are on the rise. The microscopy data collected can give us more insight into perhaps how the nucleocapsid protein induces the formation of filopodia structures at the plasma membrane. The envelope protein proved to be a challenge, but I determined a specific envelope and ceramide interaction in cells. The envelope protein was also causing the formation of microvesicles for an undefined function. I was able to determine the subcellular localization of the protein to the mitochondria. The localization to the mitochondria appears to induce depolarization of the mitochondria membrane action potential and induces the increase in mitochondria dysfunction signal, cytochrome c. Although the mitochondria were dysfunctional, there was no increase in apoptosis signal in the presence of the protein alone.</p>