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2007 Awarded Research Projects
Basic Research Imaging myelination and demyelination During brain development, specialized cells, the oligodendrocytes, contact and wrap around axons, the long cable-like extensions that are connecting neurons together. This developmental process is called myelination. It is also known that in some demyelinating diseases such as multiple sclerosis, oligodendrocytes can initially remyelinate demyelinated areas within the brain. Many genes and proteins expressed by oligodendrocytes and in myelin have been identified and some classic anatomical methods allow one to visualize oligodendrocytes during myelination on brain sections. However many important questions are still unanswered: How the number of oligodendrocytes that contact a single axon evolves over time? Are oligodendrocytes that myelinate the same axons arranged in a topographic manner forming oligodendroglial networks? Do oligodendrocytes interact and communicate during myelination? Do myelination and remyelination proceed similarly? This imprecision prevents establishing a realistic and comprehensive diagram of axon/oligodendrocyte interactions which is a prerequisite for a precise modelling of myelination and remyelination. Answering these question requires to study oligodendrocytes in real time, using in vivo and in vitro imaging methods. The aim of our project is to apply a novel and powerful imaging technology, the “Brainbow”, to the analysis of the oligodendroglial network, as well as myelination and remyelination. With this technique many oligodendrocytes will express random fluorescent proteins thereby allowing to “label” live oligodendrocytes with distinct colors. Our project will combine powerful in vivo studies using transgenic mice and in vitro myelination assays to cutting-edge imaging techniques. In conclusion our project should considerably improve our understanding of myelination and remyelination. A role for Sox17 in oligodendrocyte development and regeneration Oligodendrocytes, the myelinating cells of the central nervous system, play a major role in the synthesis of myelin sheaths and axonal protection. Unravelling the cellular and molecular mechanisms of proliferation, differentiation and maturation of oligodendrocytes is essential for a better understanding of the pathophysiology of myelin diseases such as leukodystrophies and multiple sclerosis. Recently, several genes regulating the different steps for oligodendrocyte development have been characterized but their functions in diseases where myelin and oligodendrocytes are affected remain poorly understood. The study of genes in demyelinating diseases shows that their altered expression could be one of the causes explaining failure of endogenous remyelination in the central nervous system. The role of oligodendroglial exosome secretion in glia We have learned in the past years that myelin is not just a mere insulator to enable fast nerve conduction, but furthermore, myelinating oligodendrocytes send signals mediating trophic support to axons. In agreement with this, axonal degeneration is observed in many myelin diseases. The molecular nature of the glial signal protecting axons from degeneration not known. We have discovered recently, that cultured oligodendrocytes secrete membrane vesicles, called exosomes, that in addition to myelin proteins contain a group of substances with potential neuroprotective functions. In our study, we follow up the hypothesis that exosomes carry trophic substances from the oligodenroglial cells to the axon. We will make use of PLP and CNP deletion mutant mice, which are models for axonal degeneration, and compare the composition of exosomes isolated from these mice with those from normal mice. Furthermore, we will analyse the role of exosomes in glia to axon transfer and test the potential neuroprotective function of the molecules present in exosomes in cultures of isolated neurons.
Understanding glutamate and ATP signalling pathways involved in oligodendrocyte death Neurotransmitter signalling in the white matter has received relatively little attention, but it is likely to play a major role in both the life and death of oligodendrocytes. Novel therapeutic strategies for treating white matter diseases may be possible based on the knowledge obtained about how GluR and ATP receptor- and ischemia-mediated oligodendrocyte death occurs. The current project proposal builds up in previous experimental observations obtained by our group and other laboratories, and aims at deciphering signalling pathways relevant to experimental oligodendrocyte excitotoxicity and ischemia, as well as to acute and chronic diseases of the white matter, including stroke and multiple sclerosis. The information obtained in this project will serve to design therapeutic strategies to protect oligodendrocytes from lethal insults and to reduce the extent of white matter lesions underlying those diseases. PPAR-γ agonists as potential therapeutic agents to protect oligodendrocytes and promote myelination Myelin formation is highly dependent on the appropriate energy supply, mainly deriving from metabolic reactions taking place in cellular organelles named mitochondria. Mitochondria are also the main cellular source of reactive oxygen species (ROS), highly reactive and potentially deleterious to the integrity of cells. ROS production increases massively during myelination. Substances capable to activate receptors called peroxisome proliferator-activated receptor-_ (PPAR-_) may control brain inflammation and be of potential therapeutic use in human brain diseases. The activation of PPAR-_ receptors could play a protective role on mitochondrial integrity, which might be instrumental for the recovery from demyelination. Such protective function could be accomplished through multiple mechanisms aimed to protect the organelles from the deleterious reactions due to ROS. The aim of the proposed project is to unravel such protective mechanisms in order to fully exploit the therapeutic potential of PPAR-_ activators, some of which are currently used in clinical practice (type 2 diabetes) or ongoing clinical trials. We intend to use cultures of oligodendrocytes to evaluate: a) the effect of PPAR-_ activation on the synthesis of proteins capable to protect from ROS or capable to decrease mitochondrial production of ROS (uncoupling protein 2: UCP2); b) the role of UCP-2 in the protection of mitochondrial functions from inflammatory/oxidative insults; c) the effects on oligodendrocyte maturation. These goals will be pursued by using biochemical and molecular biology techniques, together with single-cell fluorescence video-imaging by which the direct recording of some peculiar mitochondrial activities will be accomplished. Molecular characterization, in a model system, of the genetic relationship between pABC1 (ALDP ortholog) and the peroxisomal RING-finger complex Peroxisomes are present in nearly all eukaryotic cells. They contain a wide variety of enzymes which perform numerous metabolic functions. Their deficiencies are responsible for severe human diseases. At present, the relationship between peroxisomal dysfunction and the highly pleiotropic phenotype of the patients remains obscure. Several model organisms have been employed to understand the basis of these diseases. Mice have provided an invaluable tool to understand their pathophysiological aspects and organisms like fungi have been particularly important in the understanding of cellular and molecular basis of the peroxisomal disorders. A large number of genes involved in peroxisome biogenesis and function has been identified thanks to yeasts. On the other hand, filamentous fungi have proof to be relevant since the developmental outcome of the peroxisomal dysfunction can be easily evaluated and might reflect key cellular processes affected in some peroxisomal diseases. The filamentous fungus Podospora anserina is a suitable model system to study the role of peroxisomes in development. Like in humans, functional peroxisomes are required to perform a proper life cycle. In P. anserina one of the results of lacking functional peroxisomes is the inability to form sexual spores. This has been observed when cells lack PEX2, a protein that takes part of a multiprotein complex (RING-finger complex) involved in peroxisome biogenesis. In humans, defects in each protein of this complex generate the Zellweger syndrome.
Analysis of VLCFA (very long chain fatty acid)-induced cell-toxic mechanisms triggering myelin degeneration in X-ALD Accumulation of very long chain fatty acids (VLCFA) is a prominent biochemical characteristic of X-linked adrenoleukodystrophy (X-ALD), a severe hereditary disease. The genetic background of X-ALD has been thoroughly investigated. However, the question how the per se non-toxic fatty acids are linked with demyelination and neuronal cell death remains unanswered. We plan to clarify events, which cause the death of the myelin-forming cells, oligodendrocytes. Therefore, we will focus on the consequences of VLCFA accumulation on this cell type. In an ongoing study we already found a high susceptibility of oligodendrocytes to acute application of VLCFA regarding Ca2+ deregulation. Ca2+ within the cell is essential for information transfer from cell plasma to the nucleus. Cellular Ca2+ has many other regulatory functions, it is involved in modulation of energy generation and induction of cellular suicide. The X-ALD- induced changes in cellular functions will be mimicked by long-term application of VLCFA to oligodendrocytes in a cell culture system. Our special interest is the generation of reactive oxygen species due to VLCFA accumulation. Free radicals are known to induce cellular damage and to be involved in various neurodegenerative disorders. In addition, we will focus on immune factors, cytokines, which can induce inflammatory reactions. The cytokine tumor necrosis factor _ (TNF_) was found to be elevated in lesions of X-ALD brain in astrocytes. Therefore, we will analyze the release of cytokines from astrocytes due to VLCFA accumulation. To complete our studies we will investigate cells from X-ALD-mouse to mimic conditions, which are comparable to brain cells of X-ALD patients. We aim to contribute to the understanding of the events leading to the death of oligodendrocyte cells and therefore to the loss of myelin, which is observed in patients suffering from X-ALD. This understanding will indicate on which cell type drug targets could be used to develop remedies to stop myelin degeneration.
Is PICK1 involved in the pathogenesis of excitotoxic injury to oligodendrocytes in PVL? Approximately 55,000 babies are born prematurely (less than 32 gestational weeks) each year in the United States. Despite major advances in the intensive care of premature infants, 10% of these infants develop cerebral palsy. The major brain abnormality underlying cerebral palsy is a lesion characterized by injury to developing oligodendrocytes (OLs). Injury to these cells causes the motor and cognitive deficits that are prominent features of cerebral palsy. To understand why the white matter is prone to this age-specific lesion requires careful evaluation of the variables involved in predisposing developing OLs to injury. Clinical, anatomical, and neuropathological studies as well as work with animal and cell culture models strongly suggest that hypoxia/ischemia to the developing brain is important in the pathogenesis of this disorder. Hypoxia-ischemia is deprivation of the blood supply to the brain, starving the brain of oxygen and glucose, and is a common problem in premature babies. During hypoxia-ischemia an increase in the concentration of glutamate occurs in the brain that activates glutamate receptors on OLs leading to cell death, a process called excitotoxicity. The source of this glutamate is not well understood; however, it is known that glutamate transporters, which normally remove excessive glutamate, can operate in reverse and release glutamate. Therapeutic approaches to block glutamate receptors and glutamate transporters may not be feasible since these same proteins are involved in all aspects of neurological function because glutamate is a major brain signalling molecule. We have discovered that a protein, PICK1, which interacts with glutamate receptors in neurons, also interacts with a glutamate transporter called GLT1. We hypothesize that this protein could be involved in regulating glutamate receptor and transporter function making OLs very vulnerable to excitotoxicity. Understanding how PICK1 is involved in regulating glutamate receptors and transporters may uncover a potentially significant therapeutic target for this devastating disorder.
New PLP1 gene mRNAs isoforms: translation study and expression in a “PLP1 humanized” transgenic mouse model Pelizaeus-Merzbacher disease (PMD) and the X linked spastic paraplegia type 2 (SPG2) are two different diseases due to mutations in the human PLP1 gene. This gene is responsible of the production of proteins called proteolipoproteins PLP and DM20 in the myelinating cells of the brain: oligodendrocytes (OLs). These proteins are involved in the compaction and the stabilization of the myelin sheath. Three new coding regions called exons (containing DNA message) have been identified in our lab in the intron 1 non coding region of the human PLP1 gene. These exons are responsible of the formation of new PLP RNAs isoforms which have been identified in neurons whereas classic PLP is expressed in OLs. These RNAs are human specific and are expressed as soon as foetal development. These preliminary results suggest that the PLP1 gene could have other potential functions in the human brain, particularly in the communication between neurons and OLs.
Effects of plasmalogens deficiency and very-long-chain fatty acid accumulation in myelin: a structural and biochemical study The Boston College portion of the project is focused on the role of a special class of lipids called plasmalogens, which are enriched in the white matter (or myelin) of the nervous system, including the brain, optic nerve, spinal cord, and peripheral nerves. We are testing the hypothesis that these lipids are the “first line of defence” against oxidative damage in myelin. By comparing results between normal mice and the plasmalogen-deficient mouse mutant PEX7 knockout, we hope to determine whether plasmalogen protects the myelin more effectively in normal than in the lipid-deficient animal. Myelin Repair Exploring the requirement of Cdk2 for normal white matter development In diseases affecting the white matter, defect of myelination involves errors in the complex sequence of events occurring during the development of myelinating cells, the oligodendrocyte progenitor cells (OPC). Here, we focused on early events, proliferation of OPC and arrest of this proliferation which are crucial steps for an effective differentiation in mature cells.
Determining the neural population with the greatest capacity for myelin repair Multiple sclerosis (MS) affects nearly 1 million people world wide. The most common type of MS is the relapsing/remitting form which results from periodic loss of the nerve cell insulation known as myelin. Following episodic myelin loss, some of the myelin is replaced however the extent of repair does not completely offset the initial damage. Over time the cumulative loss of myelin results in nerve cell loss, which appear to be responsible for the irreversible motor and cognitive deficits of MS. Currently there is no cure for MS but one promising avenue for effective MS treatment involves facilitating spontaneous myelin repair in order to stem nerve cell loss. Within the brain, three mitotically active populations that serve as potential sources for the production of mature myelin forming cells known as oligodendrocytes (OLs) have been identified. These populations include cells of the subventricular zone (SVZ) of the 3rd and 4th ventricle, the subgranular zone (SGZ) of the dentate gyrus and the subcortical white matter. Based on their proliferative response following injury, all three populations are candidates to mediate spontaneous myelin repair; however, the individual myelin repair capacity of these populations has not been demonstrated. Elucidating the myelin formation potential of each of these populations is fundamental for therapeutic manipulations designed to maximize remyelination; thus, it is imperative to identify the cell population that provides the greatest potential for endogenous myelin repair. This concept defines the goal of this proposal— to provide unequivocal evidence that identifies the cellular population that provides the greatest potential for spontaneous CNS myelin repair.
Enhancing remyelination in the ageing CNS Myelin is an important substance which wraps around nerve fibres in the brain and spinal cord allowing them to conduct electrical impulses efficiently. When myelin is lost in demyelinating diseases such as the leukodystrophies and multiple sclerosis it is important to replace the lost myelin so that the brain and spinal cord can work properly again. This process is called ‘remyelination’. Remyelination can occur as a spontaneous healing event after myelin loss. However, the efficiency of this healing or regenerative process declines rapidly with age – as do all types of healing. Recently it has been shown that exposing old animals to the blood circulation of young animals can significantly improve the ability of damaged muscle to regenerate. In this application two internationally-recognized experts in remyelination and in reversing the age-associated decline in regeneration of muscle combine their expertise to find out whether the poor remyelination in older individuals can be improved using a similar strategy to that which has proven successful for damaged muscle. If this does occur it will be a very significant advance in the development of remyelination treatments. The role of the adhesion molecule TAG-1 in glial cell function We are interested in exploring the properties of a protein that appears to play a role in the interaction of nerve fibers and their myelin coating. Myelin insulation is not continuous but is separated by unmyelinated spots, called the nodes of Ranvier. The proteins surrounding the nodes are essential for maintaining communication between nerve fibers and myelin. One of these molecules, TAG-1, is shown to be important in this process since mice engineered to be deficient in TAG-1 exhibit alterations in motor function and in the organization of nerve fibers. Deciphering the function of this myelin protein may be helpful in understanding more about myelin/nerve interactions and the myelin damage that occurs in demyelinating disorders. Myelin repair : The role of gender and androgens Myelin is an important insulating material that surrounds nerve fibres in the brain and spinal cord. When it is lost, as occurs in diseases such as the leukodystrophies and multiple sclerosis, the brain and spinal cord do not work properly with devastating consequences for the patient. If it were possible to replace the lost myelin, a process which can occur as a natural healing event but does so with frustrating inconsistency, there would be immense benefits for patients. At present, however, there are no effective therapies for helping the body to replace myelin. This represents a very significant gap in the ability of clinicians to treat these diseases that needs to be urgently addressed by focused research. In order to develop treatments that promote myelin regeneration (a process called remyelination) it is necessary to have a thorough understanding of this process works. The sex of an individual (whether they are male or female) is one of the most profound factors determining how our bodies function. Surprisingly, very few studies have looked at how whether being male or female affects the processes of myelin formation and regeneration. However, recent work, partly carried out by the two applicants of this project, indicate that these effects are likely to be very important. In this project, the laboratories of the two applicants will combine their effects in a comprehensive and collaborative effort to unravel the complex relationships between an individual’s sex and how myelin is made and repaired. This will involve studying the role of substances important in determining biological differences between males and females (steroid hormones). This is important because the effects of these substances can be relatively easily mimicked by synthetic compounds which easily cross the barriers of the nervous system. Thus, this project will address fundamental questions, the answers to which may potentially open up the possibility of treatments that can be used to help repair myelin. The prospects are exciting but unless the necessary studies are undertaken will remain only possibilities Imaging CNS demyelination and myelin repair in Multiple Sclerosis: a PET study with 11C-2-(4’-methyl-aminophenyl)-6-hydroxybenzothiazole (11C-PIB) Myelin is a ubiquitous structure within the nervous system, which is essential for the normal conduction of neural impulses along axons. The lack or loss of myelin resulting from an inherited or acquired disease produces a delay or failure of conduction in affected nerve fibers, with concomitant neurological dysfunction. Promoting myelin repair is one of the most promising therapeutic avenues for such diseases. To facilitate the development of clinical trials in this field, we need an imaging technique, suitable for humans, aimed at visualizing and measuring myelin loss and repair. In this project, we plan to assess such an imaging method, using Positron Emission Tomography (PET), among healthy volunteers and patients with a chronic demyelinating disease, multiple sclerosis (MS). We have previously shown that a chemical compound, named PIB, could selectively bind to myelin, and could be used for myelin imaging by PET. As this biomarker is safe and already used for PET imaging in humans, we will perform two consecutive PET examinations in 10 volunteers and 20 MS patients. Results will be compared with current available MRI sequences previously proposed to detect demyelination and remyelination. This study will improve our knowledge about the pathophysiology and kinetic of endogenous remyelination in MS patients, and may provide a new way to image and quantify repair in clinical trials. Therapeutic approach of central nervous system remyelination: development of a pre-clinical model of demyelination Myelin diseases of the central nervous system (CNS) such as Multiple Sclerosis (MS) can result in severe neurological deficits. During this process myelin and oligodendrocytes, the myelin-forming cells, are slowly lost at defined lesion sites. There is no currently satisfactory treatment for MS. Approaches aiming at promoting myelin repair could benefit patients and improve their quality of life. These are investigated in rodent models of demyelination but rarely in species closer to man. Our goal is to develop a preclinical model of demyelination pertinent of MS, resulting in a quantifiable clinical deficit. We are in the process of developing the model in the macaque spinal cord. During this first year of the program, we succeeded in training animals to perform a food retrieval task, which will allow evaluation of the functional motor deficit. We also developed a surgical approach to achieve demyelination of a large white matter area. Finally, we gained evidence that macaque Schwann cells can be labelled with diphosphonate (HEDP) coated biocompatible organic iron particles for their MRI tracking in vivo. For the coming year, we propose to analyse whether demyelination of this large area is sufficient to alter the capacity of the animal to perform their food retrieval task. If not successful, we will attempt to induce focal inflammatory demyelination, a condition, which is closer to MS and more likely to alter myelin and axon integrity. Analyze myelinating or remyelinating potentiality of adult bone marrow and umbilical cord stem cells in different models of leukodystrophy A fundamental problem in our understanding of demyelinating processes and therapy approaches for myelin neurodegenerative diseases is to know what are the molecular factors that play a trophic role in normal and affected brains during disease progression, as well as in the reactive mechanisms that the affected brain activates against cellular death and myelin degeneration. In our previous work we have demonstrated that adult bone marrow stem cells can generate oligodendroglial cells (the cells that generate the myelin) in animal models of demyelination. In addition we have showed that bone marrow stem cells also drive trophic effects to neural cells in the brain and spinal cord (providing (protective factors that promote their survival). During this first year of ELA grant we have developed cellular and molecular tools to make experiments and reproduce the observed regenerative and trophic effects using mouse cells, now grafting human umbilical cord stem cells in demyelinating mouse models. Initial experiments have demonstrated the neural potential in human cells and the existence of substrate factors in the adult and embryo brain that modify the production of oligodendrocytes and their maturation from these grafted stem cells. The study in progress will analyze these molecular factors and how could be improved the efficiency of myelin trophism and regeneration of adult human stem cells, in order to propose cell therapy approaches in demyelinating diseases.
Identification of new genes Localization and identification of the gene responsible of leukodystrophy and oligodontia in a large family We previously described a new syndrome: Leukodystrophy with oligodontia (OMIM 607694) in a large inbred Syrian pedigree. It is an autosomal recessive neurodegenerative disorder. The clinical picture of the six affected children is oligodontia, and a degenerative neurological condition with onset around age 12, characterized by progressive ataxia and pyramidal syndrome.
Pathophysiopathology Identification of modifier genes in X-ALD Adrenoleukodystrophy (ALD) is characterized by a variability of clinical expression (children cerebral form, adrenomyeloneuropathy (AMN), patients with AMN developing a cerebral injury), often within the same family. Neither the type of mutation on the ALD gene nor the plasmatic levels of VLCFA can predict this variability which considerably complicates family care from a medical and a psychological point of view. This variation in clinical expression certainly depends on genetic factors, i.e. is influenced by tiny variations in all the other normal genes of the ALD patient. The project aims to identify the genes and their variants (known as “modifier genes”) inducing a risk to develop a form of ALD and implicated in the severity of the disease. The methodological approach used is twofold: 1/ to test candidate genes; 2/ to test by a global approach all the genes of the individual. A potential modifier gene has already been identified, and this project aims to validate, using a whole set of studies, this variant which could be the first predictive marker of ALD phenotype. Two other candidate genes will be studied. The first results of the global approach ("to test all the genes”) should be available by July 2007. Role of peroxisomes in the formation and maintenance of myelinated axons Peroxisomes were the latest discovered subcellular compartments. During the last 30 years, much has been discovered about their metabolic function in the liver, which is primarily related to lipids and about the way they arise and multiply. A major step forward was the identification of patients with either fully inactive peroxisomes due to a biogenesis defect or partially inactive peroxisomes due to a single enzyme defect. It is very striking that neuropathological abnormalities are a major hallmark of all these peroxisomal diseases. These include neurodevelopmental defects causing malformations of the cortex and cerebellum, defects in the formation or maintenance of myelin and axonal or neuronal degenerations. Until now, the relationship between the metabolic abnormalities and pathologies have remained obscure.
Biochemical and genetical analyses of the MLC1 interactome Mutations in MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), characterized clinically by an abnormally large head, loss of motor functions, epilepsy and mild mental decline. Although we know where the MLC1 protein is located, we still don’t know anything about the normal function of MLC1 in the brain, and how the loss of MLC1 function causes the disease. The second problem is that in some MLC patients, no MLC1 mutations can be found. Genetic studies have indicated that there is at least one other as yet unknown gene involved in MLC. The low number of patients without mutations in MLC1 and the possibility that there are multiple other genes for MLC hamper a conventional genetic linkage study as a way to find new MLC disease genes. Mitochondrial dysfunction and oxidative stress underlying X-linked adrenoleukodystrophy (X-ALD) physiopathogenesis Our ultimate goal is to develop new therapies for X-linked adrenoleukodystrophy (X-ALD), the most frequent inherited monogenic demyelinating disease (minimal incidence 1:17,000), often lethal and currently lacking of satisfactory therapy. The disease is caused by loss of function of the ALD gene, a peroxisomal ATP-binding cassette transporter, which function is related to the import of VLCFA (very long-chain fatty acids) for their further degradation through _-oxidation in the peroxisome. Impact of inflammatory cytokines on developing oligodendrocytes in a mouse model of white matter diseases of the premature infant Among the most cited explanations for occurrence of cerebral palsy in premature babies, maternal and/or fetal infections happening during the pregnancy are of a great concern for public health. These infectious conditions trigger general activation of immune system and local inflammation.
The role of VLCFA in the pathogenesis of X-ALD: generation of an ELOVL1 over-expressing X-ALD mouse X-linked adrenoleukodystrophy (X-ALD) is a serious progressive, genetic disorder that affects the adrenal glands and the white matter (myelin) of the nervous system. X-ALD is characterized by an accumulation of very long-chain fatty acids (VLCFA) in all tissues of the body, especially the brain and the adrenal glands.
The role of mitochondrial respiration on myelination and axon-glia interaction Axons, the vital neuronal processes that relay information from one neuron to the next, are very vulnerable structures. They consume lots of energy to sustain electrical stimulus conduction and can reach dozens of inches away from the cell body. Building blocks for maintenance and repair of axons thus need to be transported over very long distances. Most axons in the brain and the spinal cord don’t lay “naked”, but are tightly wrapped by specialized glial cells: oligodendrocytes and astrocytes. Alexander disease: Pathophysiological mechanisms Alexander's disease (AXD) affects myelin in the central nervous system (CNS). It is caused by mutations in the gene coding for the glial fibrillary acidic protein (GFAP). With other proteins, GFAP produces filaments that form the cellular frame.
Pathophysiological role of MLC1, a protein involved in megalencephalic leukoencephalopathy with subcortical cysts The aim of this project is to shed light into the functional role of a protein, named MLC1, which is expressed in the central nervous system, particularly in astrocytes. Mutations in the gene encoding for MCL1 have been associated with a rare inherited form of childhood-onset spongiform leukodystrophy, named megalencephalic leukoencephalopathy with subcortical cysts (MLC). This disease is characterized by myelin alterations and progressive neurodegeneration with deterioration of motor functions, spasticity, epilepsy and mental decline. To date, the role of MLC1 is still unknown and there is no specific therapy to cure MLC. The experimental strategy used to study MLC1 function involves the combined use of molecular biology, biochemistry, electrophysiology and proteomics techniques in cultured human astrocytes. Through this approach, we shall try to unravel the specific function of MLC1 and to study its interactions with other cellular proteins. During this year of support we obtained results indicating that MLC1 protein is expressed in specific functional compartments of the cell membranes where it associates to the dystrophin-glycoprotein complex, a well characterized multiprotein complex involved in cellular interactions and fluid homeostasis in the CNS. Data obtained in this part of the project constitute an essential step in the comprehension of the pathological role of MLC1 as they allow to start more focused investigations on MLC1 physiological function in astrocytes and to identify components functionally associated with MLC1. Once the physiological role of MLC1 will be established and a functional assay will be developed, we shall investigate how mutations in the MLC1 gene, identified in patients with MLC, alter the function of the encoded protein. It is expected that a better understanding of the physiological and pathological role of MLC1 might provide information relevant for the development of a specific therapy to cure children with MLC. MLC disease: elucidating the role of MLC1 protein in brain physiology and physiopathogenesis; research for MLC genes We have been particularly concerned with the collection and analysis of Turkish patients affected by megalencephalic leukodystrophy with cysts (MLC), a disabling and progressive disorder with onset in childhood, and inherited by an autosomal recessive trait. The patients were screened for mutations in the MLC1 gene, and the purpose was twofold: a) to have an array of the mutations in Turkey were the disease is known to be more frequent that in the other countries; b) to select the patients/families who were negative for mutations in order to include them in the linkage study for the ch2q35-q36.1 locus and in the Genome wide analysis for further loci. We have collected altogether 40 new families/46 patients. We analyzed in Rome (Italy) 31 families (12 mutated families having other healthy or affected sibs + 5 mutated families with single affected sibs + 14 families with no mutations in the MLC1 gene), and 9 families (7 were mutated and 2 were negative) were analyzed in Ankara (Turkey). We found mutations in 24 families, that make up 52 mutated alleles, indicating marked heterogeneity of mutated alleles. Only 2 mutations were recurrent in a few Turkish families. The families negative for mutations have been included in the linkage/Genome wide analysis. Role of phytanic acid in nervous system pathogenesis using mouse models Refsum disease is caused by the accumulation of a branched-chain fatty acid, named phytanic acid. Patients with
Molecular mechanisms in the pathogenesis of metachromatic leukodystrophy Metachromatic leukodystrophy is caused by the inability of the patient to degrade a lipid, sulfatide, which is an essential component of myelin. If sulfatide cannot be degraded it is stored and accumulates mainly in oligodendrocytes and Schwann cells, the myelin producing cells. It is not known why the storage of sulfatide causes functional impairment of these cells. In addition, sulfatide accumulates also in other cell types, for example neurons, though it is not clear if sulfatide accumulation in these cells contributes to the pathology of the disease. This project tries to identify genes that are turned on or off in sulfatide storing cells. This is done by a so called transcriptome profiling, which can measure many thousand components in a single experiment. The correct function of a cell also depends on signals received from outside and on correct sorting of proteins and lipids. External signals frequently regulate the survival but also the death of a cell. It will therefore be examined whether the storage of the lipid alters the cell’s reception of external signals. This could provide a direct explanation for the demyelination occurring in metachromatic leukodystrophy. Elongation of very long-chain fatty acids in X-linked adrenoleukodystrophy: X-linked adrenoleukodystrophy (X-ALD) is a serious progressive, genetic disorder that affects the white matter (myelin) of the nervous system and the adrenal glands. It has an incidence of 1 in 17.000 life births. All male patients and most female patients have higher than normal levels of very long-chain fatty acids (VLCFA) in all tissues of the body, especially the brain and the adrenal glands. While some of these VLCFAs come from the diet, most of them are derived from production within the body itself. Normally, there is a balance in the amount of VLCFAs and excess amounts of VLCFAs are degraded in small compartments in the cell which are named peroxisomes. The ultimate aim of this study is to identify the enzyme that is responsible for this phenomenon and to determine whether inhibition of this enzyme or the overall synthesis of VLCFAs can prevent further elongation of VLCFAs and be a possible therapeutic target for X-ALD. Physiopathogenesis of X-linked adrenoleukodystrophy in the mouse. X-ALD is a fatal demyelinating, neurodegenerative disease, with a prevalence of 1:17.000. Patients accumulate very long-chain fatty acids, such as C26:0 in organs and plasma, due to mutations in the ABCD1 gene, whose function is the import of very long-chain fatty acids into the peroxisomes for degradation. ABCD1 null mice develop a late-onset neurodegenerative phenotype, starting at around 15 months of age, comparable to the most common X-ALD phenotype in patients, adrenomyeloneuropathy. To gain insight into physiopathogenesis of the disease, we have chosen to explore transcriptome profiles in spinal cords of these mice. We have identified oxidative stress and mitochondria depletion as early events in the pathogenesis. Also, we have demonstrated that exposure to C26:0 of fibroblasts and spinal cord slices induces free radical generation, oxidative damage to proteins and decrease of mitochondria membrane potential. We have also generated an ex-vivo spinal cord slice cultures system that recapitulate closely the pathogenic events seen in spinal cords, and will constitute a powerful screening tool for therapeutic agents, and for deciphering molecular cues underlying neurodegeneration in X-ALD. We have also generated mouse mutants for the Abcd4 transporter, whose expression has been positively correlated to a milder phenotype in X-ALD. Once available, they will be analysed and crossed to double Abcd1/Abcd2, in an attempt to create an earlier onset and/or more severe phenotype of the disease.
A combined magnetic resonance imaging and histological approach to understanding and treating white matter disease of prematurity We have been optimizing magnetic resonance imaging techniques to obtain high-resolution high signal to noise images of the immature brain postmortem. We have used a high field 3 Tesla MR Scanner. Optimisation requires changing parameters such as the number of signal averages collected. This improves the signal to noise but at the expense of the time it takes to acquire the images. In ante mortem imaging, increasing the length in time of the acquisition risk the images being contaminated by motion even in the sedated infant. This is not a problem in the postmortem examination where we are able to spend several hours imaging the brain. Basic images (T2 weighted) are relatively easy to optimize but more advanced imaging sequences which allow us to interrogate tissue microstructure such as diffusion tensor imaging present more of a challenge.
Functional analysis of the ALDRP protein X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is characterized by the accumulation of very-long-chain fatty acids (VLCFA). The disorder is associated with mutations in the ABCD1 gene, which encodes a half ABC transporter of the peroxisomal membrane supposed to allow saturated VLCFA to enter into the peroxisome. ABCD2 (ALDR), the closest homolog of this gene, displays a partial functional redundancy with ABCD1 since its overexpression in X-ALD fibroblasts or in ABCD1-deficient mice compensates for ABCD1 deficiency. Pharmacological induction of the ABCD2 gene could therefore constitute a novel therapeutic strategy. In order to better understand this phenomenon of functional redundancy, we intend to characterize the functional role of the ALDRP protein in its peroxisomal context and analyze its interactions with the surrounding proteins, interactions that we supposed to be tightly linked to the function. By using specific cell models allowing the dose-dependent control of the expression of ALDRP, we want to identify the specific substrate(s) transported by ALDRP and by extension the specific role of each of the peroxisomal ABC transporters. The identification of the substrates will be crucial to understand the function of ALDRP as a main target for a pharmacological therapy of X-ALD. Invariant NKT cells in the pathophysiology of adrenoleukodystrophy Our laboratory has studied for several years cells of the immune system with anti-inflammatory properties. These cells, the NKT lymphocytes, can inhibit various inflammatory responses and in particular can protect from the development of neurological diseases such as multiple sclerosis. Therefore, we would like to determine if these cells could also inhibit the development of adrenoleukodystrophy (ALD), another demyelinating neurological disease. This study is carried out on ALD patients and on two ALD animal models. . Proteomics for myelin (P4M) mass spectrometry platform dedicated to biomarker discovery and quantitative studies for leukodystrophies Leukodystrophies are orphan degenerative hereditary diseases that affect the white matter and the myelin and represent a very heterogeneous group of affections. Among different forms of leukodystrophies the most frequent are the adrenoleukodystrophy (X-ALD), the metachromatic leukodystrophy (MLD) and the childhood ataxia with central hypomyelination / leukoencephalopathy with vanishing white matter (CACH/VWM). Their clinical symptoms are very variable and no specific biological marker is available for their diagnosis and follow-up of their evolution during treatment. The purpose of the ‘‘P4M’’ Proteomics for Myelin Project is to discover biomarkers of leukodystrophies based on differential peptides and proteins mass fingerprinting (PPMF) studies, using the P4M platform installed at Atheris Laboratories (Geneva) which includes high technology mass spectrometers. In such studies, mass spectrometric (MS) profiles from individuals diagnosed with leukodystrophies are compared to age and sex matched controls. Due to the rarity of available samples for orphan disorders and to the complexity of the human plasma peptidome, the generation of high quality data sets was considered as a primary objective and required careful adjustments of experimental conditions. The optimized protocols and dedicated bio-computing tools were based on a label-free liquid chromatography followed by a 2D-MS approach (on-line LC-ESI-MS and offline MALDI-TOF-MS). The strategy included working on native sample (no chemical or enzymatic treatment), focusing on the low molecular weight proteome (400-30’000 Da) in human biofluids and brain tissues. This way, the sample complexity could be kept as low as possible and the molecular integrity was preserved in order to distinguish a protein from its endogenous degradation fragments. Furthermore, to improve accuracy and quantitative aspects, samples were spiked with a known amount of standard proteins that also served for quality control. Significantly over- or under-expressed biomolecules (n=56, of which 7 under-expressed identified so far) were identified in pathological samples from the X-ALD and CACH/VWM leukodystrophies investigated. Among the 48 over-expressed in X-ALD samples, 58% displayed p-values <0.01 and 46% were over-expressed with an estimated fold >100. The statistically most relevant candidates (n=3 for CACH/VWM and n=16 for X-ALD) were selected for further structural characterization (in process). The validation process to evaluate the specificity of the biomarker will be initiated thereafter. It is expected that the identification of specific biomarkers of different forms of these diseases will lead to the development of new clinical prognosis and diagnostic assays, which will help in predicting the phenotypic variability and the disease severity. In addition to new diagnostic tools, it will generate a map of protein metabolic pathways involved in leukodystrophies, which may provide clues for innovative therapeutic approaches. A second shutter of the project consists in developing efficient and reliable MS-based quantitative assays of sulfatides, a series of 29 important metabolites in MLD. Using palmitoyl sulfatide as internal standard, we have developed and optimized a simple assay based on a first “one-pot PP-SPE” extraction step valid for urine as well as serum and plasma samples. It is followed either by an ESI-MS/MS analysis to quantitate individual and total sulfatides, or by an ESI-MS and MS/MS analysis to generate a sulfatide landscape. Lower limits of detection (LLOD) for total sulfatides are now in the 30-60 nM range for urine, serum and plasma. The lower limits of quantification (LLOQ) have also been significantly improved down to the 100-130 nM range, which was our initial objective. Furthermore, the profiling of each individual native sulfatide has been made possible and our preliminary investigations on a murine model and on human clinical MLD samples revealed original sulfatide profiles that seem to be specific to the pathological state. We believe that such quantitative sulfatide landscapes have the potential to serve as a new diagnostic imaging system for diagnosis and to assist clinical decisions in the near future.. Therapeutic Approaches Gene therapy for X-ALD: First step for the production of the lentiviral vector
Arylsulfatase A over-expression in human hematopoietic cells for MLD gene therapy: pre-clinical and clinical assessment During the past years our group gained increasing experience in the field of gene therapy for Metachromatic Leukodystrophy (MLD), a severe Lysosomal Storage Disorder (LSD) due to inherited deficiency of Arylsulfatase A (ARSA). Based on our pre-clinical studies, we are currently working for the implementation of a gene therapy trial based on the transplantation of autologous Hematopoietic Stem and Progenitor Cells (HSPC – the mother cells of all blood elements), transduced with a Lentiviral Vector (LV – advanced generation vehicle for gene therapy) encoding the functional ARSA protein, which is expected to start by Q1-Q2 2008. Recently the European Medicinal Agency (EMEA) provided this approach with the Orphan Drug designation. Our preclinical work demonstrated that production of supra-normal levels of functional ARSA by blood elements is the fundamental therapeutic mechanism allowing the achievement of prevention of MLD manifestations and eventually correction of already established neurological disease by HSPC gene therapy in the mouse model. The demonstration of the feasibility and long-term safety of supra-normal ARSA expression in pre-clinical human samples and in hematopoietic cells of the patients enrolled in the trial will be fundamental for predicting and monitoring clinical efficacy. Therefore, the main goal of the proposed project is the biochemical and molecular assessment of the efficacy and safety of ARSA over-expression in pre-clinical and clinical samples from MLD patients receiving HSPC gene therapy. Mitochondrial Impairment in Pelizaeus-Merzbacher Disease: Pelizaeus-Merzbacher (PM) Disease is caused by mutations in the CNS proteolipid protein (PLP1) gene. The PLP1 mutations fall into 3 broad classes: (1) missense mutations, (2) duplications of the native (wild-type) PLP1 gene, and (3) deletions of the PLP1 gene. Duplications and amplifications of the PLP1 gene account for 50% to 70% of human PLP1 mutations. In many cases, the duplications are lethal, with death ensuing as early as 2 years after birth. No treatments are available for PM patients. Development of therapy for PM patients involves understanding the molecular and cellular bases that cause hypo- and demyelination, and subsequent neurological sequelae. It is generally assumed that the underlying molecular and cellular defects in PM with missense mutations and duplications are similar because neurological signs in humans with PLP1 mutations in the three classes often overlap. We now show that different molecular mechanisms cause oligodendrocyte cell death in PLP1 mutants with missense mutations compared to PLP1 mutants with duplications of the native gene. We show that PLP1 mutants with duplications exhibit major oxidative phosphorylation defects whereas mice with PLP1 missense mutations do not. We are testing selected drugs to improve their neurological sequelae. Evaluation of mesenchymal stem cells and macrophage progenitors Transplantation of hematopoietic stem cells is the only effective treatment in ALD. When it is carried out at an early stage, it allows stopping demyelination. It acts by replacing the cerebral macrophages (or microglia) deficient for the ALD protein by normal macrophages from the donor. However this replacement is very slow, explaining the delay between the transplantation and the patient improvement (18 months). This is true for all the diseases of the CNS in which transplantation of hematopoietic cells is effective. It is thus crucial to develop strategies which will accelerate and increase this process of microglia replacement. Our objective is to test the capacity of mesenchymal stem cells (MSC) and macrophages progenitors to accelerate and increase this microglial replacement when they are injected in combination with hematopoietic stem cells. MSC are capable of accelerating hematopoietic reconstitution but their role on the microglia is unknown. For this project we use a human model of blood-brain barrier in culture and several mouse models (saposine A mouse with a demyelinating leukodystrophy, mouse with a localized demyelination induced by intracerebral injection of lysolecithin, mouse with a localized neuroinflammation induced by injection of lipopolysaccharide (LPS). Participation to a platform in Nantes producing viral vectors for clinical applications POPART’MUS: Prevention of Post Partum Relapses with Progestin and Estradiol in Multiple Sclerosis Multiple sclerosis (MS) affects 1 in 1000 people in western countries, mainly women in their childbearing years. It is an autoimmune disease of the central nervous system (CNS), which results in a chronic focal inflammatory response with subsequent demyelination and axonal loss. It usually begins with acute episodes of neurological dysfunction, the relapses, followed by periods of partial or complete remission. This relapsing-remitting phase is usually followed by a steady, continuous and irreversible worsening of the neurological dysfunction, which characterizes the progressive phase of the disease. Recent prospective studies reported a significant decline by two-third in the rate of relapses during the third trimester of pregnancy and a significant increase by two-third during the first three months post-partum by comparison to the relapse rate observed during the year prior to the pregnancy (Confavreux et al., 1998). These dramatic changes in the relapse rate occur at a time when impregnation of many substances, among which sexual steroids, is at its highest, before a dramatic decline to the pre-pregnancy levels, immediately following delivery. It may be hypothesized that sexual steroids could exert beneficial effects through a modulation of the immune state with a lowering of the pro-inflammatory lymphocyte responses and an enhancement of anti-inflammatory responses of the. Enzyme replacement therapy in an improved metachromatic leukodystrophy mouse model Metachromatic leukodystrophy (MLD) is caused by mutations of the arylsulfatase A gene encoding the enzyme ASA (also called ARSA). The disease is characterized by the accumulation of sulfatide, severe and deteriorating neurological symptoms and premature death. Bone marrow transplantation can retard the disease progression under certain circumstances, but transplantation is not applicable to all patients and does not prevent the fatal outcome of MLD. Different alternative therapeutic approaches are therefore under investigation. Due to ethical reasons experimental therapies have to be tested first in animal models before they can be applied to patients. The only animal model existing for MLD is a mouse strain which arylsulfatase A gene has been artificially destroyed so that the mice lack ASA. As a consequence, the mice develop a disease which resembles MLD. We have shown that treatment by repeated injection of active ASA into the circulation, a treatment strategy called enzyme replacement therapy, ameliorates the mouse disease. However, the mouse disease is comparably mild and does not proceed to the severe stages of the human disease. In view of the clinical application, we can therefore not predict therapeutic effects on the severe symptoms which dominate advanced stages of MLD. To fill this gap, we have artificially introduced two additional genes into the genome of conventional MLD mice. One gene encodes an enzyme which increases the bio-synthesis of sulfatide. Therefore, more sulfatide accumulates in the nervous system and the mice develop a more severe MLD-like disease. The other gene is a mutant arylsulfatase A gene which encodes an inactive ASA enzyme. Since the ASA is defective it does not influence the disease, but its mere presence prevents an immune response when thera-peutic active ASA is injected. Consequently, we have now a mouse which, in contrast to the conventional mouse model, allows the evaluation of long-term effects of enzyme replacement on severe MLD symptoms. We intend to treat the mice with ASA which has been optimized for enzyme replacement in a previous ELA-granted study. To differentiate between preventive and corrective effects mice will be treated before and after they have developed severe symptoms.
Newborn Screening for X-Linked Adrenoleukodystrophy and other Peroxisomal Disorders Adrenoleukodystrophy is a serious disorder affecting children and adults with an incidence of approximately 1:20,000. Therapies which have been developed rely extensively on presymptomatic diagnosis and monitoring which unfortunately occurs in only a limited percentage of individuals. We propose to develop a methodology which can occur with standard regional newborn screening and confirm the sensitivity and specificity of the technique. Broader use of this optimized method should allow quicker diagnosis for families and maximum translation of recent therapeutic interventions. Intracerebral administration of adeno-associated viral vector carrying the human ARSA cDNA in metachromatic leukodystrophy |
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