The impact of developmental stress on the functioning and vulnerability of CNS neurons

[摘要] The overall objective of this thesis is to provide additional data to assist clinicians andexperimental neurologists alike in the quest for better understanding, more accuratelydiagnosing and more successfully treating patients suffering from Parkinson's disease (PD).The general theme of the thesis is the interaction between certain environmental stimuli,including the exposure to adverse events during early central nervous system (CNS)development and the manifestation of elements of neurodegeneration, whether by means ofneurochemical changes or expressed as a dysfunctional voluntary motor system.The first chapter provides a general introduction to the research theme of the thesis. Thisincludes, in particular, a discussion on current understanding concerning the etiology andclinical profile of PD, the relative contribution made by genetic factors compared toenvironmental ones, and current treatment strategies for treating the disease. Mention is alsomade of the failure of these therapeutic applications for reversing or protecting against thedisease, due to the side-effects associated with them. The material covered in chapter 1provides the basis for the more complete discussion concerning these various aspects,contained in the chapters to follow.The overall aim was also to characterise the effects of commonly used toxin-induced animalmodels of PD, and the extent of vulnerability that the CNS displays towards them. Thedestruction of dopaminergic neurons following the administration of 6-OHDA at targeted pointsalong the nigrostriatal tract is used extensively to model PD pathology in rats and is anestablished animal model of the disease. However, mature or even aged animals are mainlyused in these studies, while the effects that the toxin might have on the developing CNS remainunclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA's actions on theyoung adolescent (35 days-old) CNS by comparing the motor and biochemical effects of aunilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medialforebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either adirect delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum.Although both lesion types were used, the MFB model is considered a more accurate portrayalof end-stage PD, while the striatum-model better reflects the long-term progressive pathology ofthe disease. The different lesions' effects on motor function were determined by observinganimal's asymmetrical forelimb use to correct for weigh shifting during the vertical exploration ofa cylindrical enclosure. Following the final behavioral assessment, the concentration ofdopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using4HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. TheHPLC-EC results revealed a compensatory effect for DA production and DA turnover on thelesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment,there appears to be extensive adaptive mechanisms in place within the remaining dopaminergicterminals that may be sufficient for maintaining relatively high extracellular and synapticconcentrations of DA. However, since substantial changes in motor-function were observed, it issuggested that the capacity of the remaining dopaminergic neurons to respond to increasedfunctional demands may be limited. In addition, the behavioral results indicate that the distinctindices relating to different functional deficits depend on the lesioning of anatomically distinctstructures along the nigrostrial tract.It has long been known that far fewer women are diagnosed with PD than men are. Thisseeming protection offered to females against degenerative disease of the CNS may relate toestrogen, although the hormone's mechanism of action on the dopaminergic system is poorlydefined. With an estimated 10-15 million women using oral contraceptives (OCs) in the UnitedStates alone, the aim of chapter 2 was to examine the evidence for a possible relationshipbetween PD and the female reproductive hormone estrogen. A review of the current literatureavailable on the topic was performed by consulting Medline, and by performing a search of thecase-reports contained within the World Health Organization's (WHO) International DrugMonitoring database, for possible PD-related symptoms that may arise from estrogenreplacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogenprotects women from obtaining the disease, or at least some features of it. Intensive researchefforts are called for, with sufficient power to establish the relationship between ERT and theonset and development of parkinsonism. Chapter 3 reports on the results obtained from anexperiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higherdose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6-OHDA the female rats were less impaired than males in making adjustment steps in responseto a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosinehydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early genderdifferences in cell survival factors and/or other promoters of neuroplasticity may havecontributed to the beneficial outcome seen in the females. For example, nerve growth factor(NGF) was found to be higher in the female rats following administration of the DA neurotoxin. Itis unclear whether gonadal steroids are involved, and, if so, whether female hormones areprotective or whether male hormones are prodegenerative. Determining the mechanisms for theimproved outcome seen in the young female rats may lead to potential treatment strategiesagainst PD.5Many studies have shown that early life stress may lead to impaired brain development, andmay be a risk factor for developing psychiatric diseases, including clinical depression. However,few studies have investigated the impact that early stress may have on the onset anddevelopment of neurodegenerative disorders such as PD. The study reported on in chapter 5conjointly subjected rat pups to a maternal separation (MS) paradigm that is a wellcharacterised model of adverse early life events, and a unilateral, intrastriatal injection of 6-OHDA. The combined effects of these models on motor deficits and brain protein levels wereinvestigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesionwith a battery of tests that are sensitive to the degree of DA loss sustained. The resultsshow that animals that had been subjected to MS display poorer performance in the vibrissaeand single-limb akinesia test compared to non-MS control animals (that had also beensubjected to the toxin exposure). In addition, there was a significant increase in the loss of THstaining in MS rats compared to non-MS ones. The results from this study therefore suggestthat exposure to adverse experiences during the early stages of life may contribute towardsmaking dopaminergic neurons more susceptible to subsequent insults to the CNS occurringduring mature stages of life. Therefore, taken together, early exposure to stress may predisposean individual towards the onset and development of neurodegenerative disease, whichespecially becomes a threat during the later stages of adult life.Moreover, within the framework of these characteristics, the capacity of a widely-usedpharmacological agent (statins) was tested for possible future therapeutic application in PD(chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggeststhat it may associate with defective activity of complex I of the mitochondrial electron transportchain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increasedoxygen free radical production, depressed antioxidant enzyme activities, and greatersusceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used forlowering cholesterol levels in patients who display elevated concentrations of low-densitylipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have onmotor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex Iinhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either withsimvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motorfunction were used: The apomorphine-rotation test, and the vibrissae-elicited forelimbplacement test. Following the drug administration protocol, the nigrostriatal tract was unilaterallylesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing thevehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purifiedconcentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2-6Dimensional electrophoresis (2-DE) with subsequent identification of the spots usingelectronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) wasperformed and the results BLAST-searched using bio-informatics tools for naming the identifiedpeptides. The motor test results indicate that while unilateral rotenone causes behavioralasymmetries, treatment with simvastatin improved motor function relative to the rotenoneinducedones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly inprotein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadlyclassified according to their cellular function into 6 categories, with the majority involved inenergy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticatedtechniques and non-biased ability to quantify proteins, provides a methodology with which tostudy the changes in neurons associated with neurodegeneration. As an emerging tool forestablishing disease-associated protein profiles, it also generates a greater understanding as tohow these proteins interact and undergo post-translational modifications. Furthermore, due tothe advances made in bioInformatics, insight is created concerning their functionalcharacteristics. Chapter 4 summarises the most prominent proteomics techniques and discussmajor advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hopedthat the application of this technology will lead towards a presymptomatic diagnosis of PD, andthe identification of risk factors and new therapeutic targets at which pharmacologicalintervention can be aimed.The final chapter (chapter 8) provides a retrospective look at the academic work that hadbeen performed for the purpose of this thesis, recaps on the main findings, and also highlightscertain aspects of the project and provides relevant suggestions for future research. Lastly, theappendix provides a detailed overview of the methods followed for the experiments described inthis thesis. It provides not only a comprehensive description of the techniques that had beenfollowed, but provides information concerning the care taken with the animals (i.e. post-surgery)in order to control for the potential influence of experimental variables on the results.

[发布机构] Stellenbosch University