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Novel fluorescent probes to resolve the role of oxygen radicals in peroxisomal disorders: from single cell to knockout mice and patients.

Projectomschrijving

Stoornissen in peroxisomen, die zijn betrokken bij de afbraak van vetten in de cellen, leiden tot een ophoping van vetzuren en stapelingsziekten. Die kan onder andere het zenuwstelsel aantasten. Met behulp van genetisch veranderde muizen en gedetailleerd biochemisch en beeldvormend onderzoek in cellen is achterhaald welke mechanismen ten grondslag liggen aan de stoornis en welke onderdelen van de cellen erbij zijn betrokken. Daarvoor zijn ook huidcellen van patiënten met stapelingsziekten als het Zellweger syndroom bestudeerd. De nadruk lag op oxidatieve processen die de onderzoekers in de cellen hebben kunnen volgen. Daartoe hebben ze speciale fluorescerende merkstoffen ontwikkeld die specifiek zijn voor vetzuren. Deze reageren op oxidatieve veranderingen in de cel, waardoor deze zichtbaar gemaakt worden.

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Samenvatting van de aanvraag

One of the major challenges faced by neuroscience is to understand which neuronal circuitries are essential for memory formation, and which molecular mechanisms in these neurons are responsible for the control of this process. In the present program we propose to investigate the role of the olivocerebellar system in the control of motor learning by changing single parameters via a genetic approach. This multidisciplinary effort follows large research programs like the Human Genome Project that are moving towards the use of modern genetics to investigate developmental and physiological processes in the brain. To date, several processes may underly memory formation in the cerebellar cortex; these include postsynaptic LTD, postsynaptic long-term potentiation (LTP), and presynaptic LTP. Postsynaptic LTD of synaptic transmission at the excitatory parallel fiber - Purkinje cell synapses is thought to be one of the major cellular substrates of cerebellar motor learning. Scientists agree that LTD induction requires a calcium influx into a Purkinje cell together with activation of ionotropic AMPA and probably metabotropic subtypes of glutamate receptors, and that this influx and activation can be evoked by conjunctive activation of the two major inputs of the Purkinje cell, the climbing fibers and parallel fibers. The debate is now focused on which second-messenger pathways contribute to LTD and to what extent they do so; in particular the investigations are aimed at determining the roles of PKC and NO-PKG pathways. Postsynaptic LTP at the inhibitory stellate cell / basket cell - Purkinje cell synapse may act in concert with postsynaptic LTD induced at the parallel fiber - Purkinje cell synapse, while presynaptic LTP at the parallel fiber - Purkinje cell synapse, while presynaptic LTP at the parallel fiber - Purkinje cell synapse may counteract LTD. Interestingly, both postsynaptic and presynaptic LTP are supposed to be dependent on the activation of PKA. It is the long-term objective of this proposal to elucidate the contribution of each of these molecular pathways and cellular mechanisms to cerebellar motor learning and to determine to what extent these processes are impaired in cerebellar diseases such as paraneoplastic ataxia. So far, most if not all of the experiments aimed at unravelling the molecular basis of motor learning have either been limited to in vitro studies or they have been hampered by the general caveats of global knock out mice (e.g. no cell specificity, upregulation of iso-enzymes, developmental aberrations). For the present proposal, we will only create and test cell specific, conditional mutant mice. These include two conditional mutant mice in which postsynaptic LTD is affected, one for the PKC pathway and one for the NO-PKG pathway; one conditional mutant in which postsynaptic LTP is affected; and one conditional mutant in which presynaptic LTP is affected. To obtain cell specificity and to compensate for upregulation of iso-enzymes we will follow the strategy we recently presented (for details, see De Zeeuw et al., 1998, Neuron). For all mutants we will insert specific inhibitory peptides of one of the protein knases (for LTD PKCi or PKGi, and for LTP PKAi) into a cell specific promotor (for the postsynaptic processes we will use the Purkinje cell specific L7 promotor and for the presynaptic process we will use the granule cell specific GABA-A-asubunit promotor). Thus, the beauty of the experimental design is that each cellular process can be selectively attacked in a cell specific and dosage dependent manner so that different abnormalities in the learning behaviour can be correlated to a specific molecular and cellular deficit. Moreover, once we have mapped these individual correlations we can easily combine various investigations by crossbreeding the different mutants and retesting their deficits at both the cellular and system physiological level. The behavioural analysis will focus on two relatively simple reflexes that are controlled by different parts of the cerebellum: I. Compensatory eye movements controlled by the vestibulocerebellum; and II. Conditioning of eye blink responses controlled by the cerebellar hemispheres. Both approaches lend themselves well for testing both the amplitude and timing aspects of motor learning, and in addition, they complement one another in that each of them has particular features that provide unique experimental opportunities. In addition, we will subject slices of the cerebellar cortex of each of the mutants to investigations under the Multiphoton Laser Scanning Microscope (MPLSM) so that we can investigate in living tissue whether the induction of long term processes as LTD and LTP can be correlated with morphological changes of the spines of cerebellar Purkinje cells and their climbing fiber and/or parallel fiber input. Thus, the proposed program will allow us to elucidate with the use of modern gene technology and physiological methods the molecular mechanisms underlying learning processes in the cerebellum at both the cellular and system level. The present proposal is novel in that it combines new approaches both at the genetics engineering level and at the behavioural level to elucidate the molecular and cellular mechanisms underlying memory and learning, which is an emerging area of neuroscience research. Most of the advantages of our transgenic have been described above and they are partly due to the fact that specific promoters are available for the neurons in the system that we investigate. In this respect the cerebellum diverges from other memory systems such as the hippocampus for which cell specific promoters are difficult to find. The behavioural learning tests that we developed both for adaptation of the vestibulo-ocular reflex and eye blink conditioning are also unique in that they allow accurate measurements in the spatiotemporal domain at very high resolutions in milliseconds and tenths of degrees. In this respect, they are far more reliable than for example the water maze test, which is commonly used for learning and memory studies in the hippocampal field (see e.g. recent review on this test in Science by Crabbe et al., 1999). In addition, the cerebellar system and the reflexes controlled by it are far simpler than most other memory systems in mammals; they often include only three or four synapses and therefore allow a more robust interpretation of the behavioural data. Finally, we recently acquired a new MPLSM (Zeiss), which is designed to operate in parallel with cell physiological investigations. This combined approach is novel and will allow us to combine morphological and electrophysiological experiments at a high resolution in living tissue over prolonged periods of time. The outcomes of the investigations mentioned above will form the basis for our understanding of memory dysfunctions in the cerebellum (e.g. paraneoplastic syndromes), and since they will probably provide new insight on the general plasticity of pre- and postsynaptic processes, they may also be relevant as a model for memory and learning problems in other brain regions, which cannot be subjected to the same rigorous test. Exciting advances have been achieved in our understanding of many peroxisomal disorders including Zellweger syndrome and X-linked adrenoleukodystrophy. Detailed information has become available about the genetic defects that are associated with these disorders and the inherent biochemical and clinical consequences. In many instances, the impaired peroxisomal metabolism results in an accumulation of long chain and branched chain fatty acids. As a consequence various organ functions degenerate including those of the central nervous system. How the genetic and biochemical abnormalities cause this degenerative including those of the central nervous system. How the genetic and biochemical abnormalities cause this degenerative pathology is far from understood. There is a growing appreciation, however, that reactive oxygen plays a prominent role in the pathology of these diseases. Indeed it has been observed in rodents that ß-oxidation substrates which accumulate due to impaired peroxisomal function, activate receptors (PPARaRxR) that regulate the expression of peroxisomal enzymes. Sustained activation of these receptors results in an imbalanced expression of peroxisomal oxidases (hydrogen peroxide producing) and catalyse (hydrogen peroxide scavenging). The ensuing elevated levels of hydrogen peroxide are associated with increased oxidative damage to DNA and cause hepatic carcinomas. To resolve the role of reactive oxygen in peroxisomal disorder, sites of their production and molecular targets remain to be determined. Three key facts put us in a unique position to accomplish this. Firstly, we have developed unique reactive oxygen sensitive probes which are not only directly applicable to intact cells but can also be targete4d to different sub cellular localisations such as the peroxisome. This is accomplished by coupling the probe to a short targeting sequence specific for peroxisomes. Secondly, we have a large collection of fibroblasts, tissue specimens and plasma samples from many patients at our disposal. Thirdly, excellent mouse models for various peroxisomal disorders are available to us. Overall aim and key objectives: Resolution of the role of reactive oxygen in the pathogenesis of peroxisomal disorders. To achieve this objective we aim: 1) To identify the organs and cell types that are most prominently damaged as a consequence of enhanced reactive oxygen production in knock-out mice with impaired peroxisomal a/ß-oxidation (SCPx, Prof. U. Seedorf, Munster, Germany). 2) To analyse the reactive oxygen activities with sub-cellular resolution in fibroblasts of patients suffering from impaired perosisomal a- (Refsum) or ß-oxidation (XALD, deficiencies in bifunctional enzyme, thiolase or acyl-CoA oxidase). 3) To identify in the above systems the major proteins which are oxidatively modified as a consequence of the peroxisomal dysfunction. 4) To establish whether these proteins are also oxidatively modified in patients' tissues. Flow diagram summarising the experimental phases of the project. PhD-1: a map will be produced of the most prominent reactive oxygen producing tissues in the SCPx mice (as compared to the control). The fluorescence will be quantified on paraffin embedded sections of the mouse using advanced confocal microscopy (1). Oxidative damage to proteins will be assessed by high resolution PAGE, microsequencing and mass spectroscopy (2). PhD-2: The latest ROS assays will be applied in combination with more conventional approaches to quantify ROS production in living cells of patients and mice (3). In addition oxidation products in tissues (blood, brain liver) of patients and mice will be analysed (4). Reactive oxygen production in mice and cultured cells will be investigated by the use of oxidation-sensitive fluorophores that are specific for lipids, proteins and DNA. These probes have recently been developed in our laboratory and prove to be very suitable for ratio-imaging so that corrections can be made for heterogeneous probe distribution in tissues (Pap & Wirtz et al, 1999). The probes will be administered to mice as food additive or intravenously and to cultured cells in the growth medium. -Lipid oxidation will be evaluated by measuring the oxidation of the fluorescent fatty acid Bodipy. Upon oxidation, the fluorescence excitation and emission of this fluorophore is shifted from red to green. The oxidative sensitivity of the fluorophore is similar to that of natural unsaturated fatty acids. Its metabolic incorporation into lipids has the advantage that its diffusion in cells and tissues is low (high spatial resolution) and that its reactive oxygen-induced signal can be accumulated for days (high sensitivity). Protein oxidation will be evaluated using a membrane-permeable fluorescein-tyrosine conjugate that upon conversion into a tyrosyl radical cross-links to cellular proteins. Prolonged exposure of mice and cultured cells to this conjugate leads to accumulation of the fluorescent conjugate in reactive oxygen producing sites. DNA oxidation will be detected with a commercial fluorescent antibody against the oxidised DNA base: 8- hydroxydeoxyguanosine, and by HPLC analysis of tissue extracts. Reactive oxygen activities in the nucleus will also be determined by deoxy-nucleotides that are labelled with oxidative-sensitive fluorophores and incorporate into the DNA of living cells (technology available). Peroxisome specific reactive oxygen activities will be analysed in cultured cells using oxidation-sensitive probes that are specifically targeted to these organelles by a membrane-permeable peptide carrying the peroxisomal targeting signal. Model systems: A mouse model (SCPx) deficient in the peroxisomal oxidation of branched chain fatty acids has recently been developed by targeted gene disruption. May biochemical and clinical features of this mouse resemble those found in human patients. Therefore, the SCP mouse is an excellent model for establishing the role of reactive oxygen in tissue degeneration. The SCP mouse (Seedorf & Assmann, et al. 1998) has a deficiency in the peroxisomal branched chain ß-ketothiolase. This deficiency causes (1) an impaired a and ß-oxidation of branched chain fatty acids, (2) marked alterations in gene expressions, (3) peroxisome proliferation, (4) hypolipidemia and (5) neuropathy. The reactive oxygen activities will be analysed in cultured fibroblasts of patients suffering from distinct effects in the peroxisomal a or ß-oxidation. The cell culture conditions in these experiments will be adapted such that they resemble the biochemical abnormalities in these patients as much as possible. Two photon confocal microscopy and advanced protein analysis. After incorporation of the reactive oxygen sensitive probes in mice or in cultured cells, their fluorescence will be visualised at the macroscopic and microscopic level using two-photon excitation and confocal laser scanning microscopy (in collaboration with Dr. H. Gerritsen, Debye Institue, Utrecht University and Mr. W. Hage, Hubrecht Laboratories, Utrecht). By using two-photon excitation, light scattering and background fluorescence in dense tissues are reduced to minimum levels. Further background suppression can be achieved by time-gated detection of the fluorescence signals. Form cells and tissues with enhanced reactive oxygen activities, major protein targets (labelled with the fluorescein-tyrosine conjugate) will be isolated by affinity chromatography (using immobilised antibodies). The proteins will be identified using high resolution PAGE coupled to automated micro sequencing and mass spectroscopy (in collaboration with Prof. Dr. A. Heck, Utrecht University). Elements of innovation: Although the role of reactive oxygen in the progression of disease is appreciated by an increasing number of scientists, hardly any experimental data are currently available. This lack of data can be explained by the wide range of expertise needed which can not be covered by any single research group. In addition, only recently proper mouse models, novel methods for the detection of reactive oxygen and ultra sensitive biophysical technologies have become available. With the participating laboratories, it is now within reach to relate reactive oxygen activities at the macroscopic, microscopic and even molecular level with tissue degeneration. Relevance for health care: We aim to advance our understanding of the pathogenesis of peroxisomal diseases. Organs, cell types, cellular organelles and proteins that are most prominently damaged by reactive oxygen will be identified. The study may provide a basis for the development of rational strategies for treatment of patients with peroxisomal deficiencies.

Onderwerpen

Kenmerken

Projectnummer:
90103097
Looptijd: 100%
Looptijd: 100 %
2001
2007
Onderdeel van programma:
Projectleider en penvoerder:
Prof. dr. K.W.A. Wirtz