Assessing Midbrain Abnormalities In Parkinson’s Disease Using Magnetic Resonance Imaging

Diagnosing early-stage Parkinson’s disease (PD) and its manifestations is still a clinical challenge. Previous imaging studies using iron, neuromelanin (NM) and the Nigrosome-1 (N1) measures in the substantia nigra (SN) by themselves have been unable to provide sufficiently high diagnostic performance for these methods to be adopted clinically. In this dissertation, we start by studying idiopathic PD patients at their intermediate stages of the disease to evaluate the role of global and regional iron in the major deep gray matter nuclei. Then, we only focus on the NM complex in the midbrain and how neuronal loss interact with iron overload as well as their relationship with clinical scores strictly on early PD patients. Finally, by taking one more step back in the disease progression process, we investigate the impact of iron deposition and N1 appearance in idiopathic rapid eye movement sleep behavior disorder (RBD) as the prodromal stage of PD. The results of this work are summarized as the following: 1) the increase in iron in the SN in some PD patients is higher than the normal range in healthy controls (HC) as found in both regional and global analyses and that regional high iron content may provide a means to separate two populations of PD patients; one with and one without iron increases in the SN; 2) we have introduced a rapid five-minute 3D approach to depict NM degeneration and iron deposition simultaneously which provides a practical MR imaging method to differentiate early stage subjects with PD from HCs with an approximately 98% accuracy; and finally 3) iRBD patients were found to have a higher incidence of N1 loss, reduced volume and elevated iron levels in a few brain structures as well as cognitive and motor impairment scores being correlated with iron deposition of several cerebral nuclei. All these in vivo biomarkers put together can significantly contribute to a better understanding of the underlying pathophysiology in PD onset and progression with the ultimate goal being a more confident clinical diagnosis prior to symptomatic dysfunction

Development and optimisation of tools for preclinical studies on Parkinson's disease

Successful drug development requires numerous tests to deem a drug safe and efficacious. Before clinical trials, preclinical testing is needed to ensure that the drug can be safely tried out in humans. In preclinical testing, efficacy is also assessed to minimise the risk of a drug failing in clinical trials. Parkinson’s disease (PD) is a common age-associated neurodegenerative disease characterised by distinct debilitating motor symptoms caused by the dysfunction of the dopaminergic nigrostriatal pathway. For PD, a plethora of cellular and animal models have been developed to study the pathophysiology of the disease and to test potential new therapeutic interventions for treating the disease. New models are constantly created. However, methods to study outcomes also need to be developed and refined for reliable and reproducible results, which is pivotal to demonstrating the efficacy of drugs. This dissertation work developed new tools and refined current methods to study PD in preclinical models and studied the characteristics of the cytomegalovirus (CMV) promoter and a primary culture of postnatal dopamine neurons used to model PD. First, we used infrared analysis of optical densities to assess the striatal innervation of tyrosine hydroxylase-positive (TH+) fibres in rat brain sections, a useful alternative to colourimetric optical density analyses. We also developed a novel method based on convolutional neural network algorithms to count dopaminergic neurons from rodent brain sections. The number of neurons counted had a high degree of correlation with results obtained using other counting methods, and counting was substantially faster with the algorithm. Additionally, we developed reporter assays, both reporter plasmids, and cell lines, to measure the activity of a PD-associated drug target, Dicer. These assays, using either exogenous or endogenous fluorescent and bioluminescent indicators, were validated and produced comparable results to previously published similar assays in more physiologically relevant conditions. We also found out that a commonly used promoter in gene therapy, the CMV promoter, could be activated by neurotransmission. We showed in vivo that methamphetamine – a potent dopamine-releasing drug – activated the CMV promoter in the rat brain. Moreover, we observed differences in the distribution of the endoplasmic reticulum between different compartments of cultured mouse TH+ neurons. In summary, the methods and refined tools obtained in these studies expand the toolbox of researchers engaged in studying PD preclinically and may be applicable to other disease areas and human clinical studies as well. Furthermore, our findings on the activation of the CMV promoter are important to consider when designing gene expression systems, reporter assays, or gene therapies for preclinical PD studies utilising amphetamines. And finally, we gained novel insight into the ultrastructural characteristics of cultured postnatal dopamine neurons and provided a valuable resource for the research community.Onnistunut lääkekehitys vaatii useita tutkimuksia, jolla lääkeaine voidaan osoittaa turvalliseksi ja tehokkaaksi. Ennen kliiniisiä kokeita, prekliinissisä kokeissa lääkeaineen turvallisuudesta tulee varmistua voidakseen sitä tutkia potilailla. Prekliinisissä kokeissa myös lääkeaineen tehoa tarkastellaan minimoidakseen riski epäonnistua kliinisissä tutkimuksissa. Parkinsonin tauti on yleinen hermostoa rappeuttava sairaus, joka aiheuttaa haitallisia ja tunnusomaisia liikehäiriöitä, jotka johtuvat keskiaivojen dopamiinijärjestelmän toimintahäiriöstä. Parkinsonin tautiin on kehitetty valtava määrä solu- ja eläinmalleja taudin patofysiologian tutkimiseksi ja uusien lääkkeiden tehon osoittamiseksi. Uusia malleja kehitetään jatkuvasti lisää, mutta myös lopputuloksia määrittäviä menetelmiä tulisi kehittää ja parantaa luotettavien ja toistettavien tulosten aikaansaamiseksi. Tämä on erityisen tärkeää, kun aikeena on osoittaa lääkeaine tehokkaaksi. Tässä väitöskirjatyössä kehitettiin uusia työkaluja ja paranneltiin aiempia menetelmiä Parkinsonin taudin tutkimiseksi prekliinisissä malleissa, ja tutkittiin cytomegalovirus (CMV) promoottorin sekä Parkinsonin taudin mallintamiseen soveltuvan dopaamiinihermosoluviljelmän ominaisuuksia. Ensiksi hyödynsimme rotan striatumin tyrosiinihydroksylaasi positiivisten (TH+) hermosoluyhteyksien optisten tiheyksien mittaamisessa infrapuna-analyysiä, tavallisen väriaineanalyysin sijaan, tarjoten vaihtoehtoisen menetelmän optisten tiheyksien mittaamiseksi. Lisäksi kehitimme uuden hermoverkkoalgoritmeihin perustuvan menetelmän solujen laskemiseksi jyrsijöiden aivoleikkeistä. Solulaskelmat korreloivat voimakkaasti muilla laskentamenetelmillä saatujen tulosten kanssa ja algoritmilla saadut laskelmat olivat huomattavasti nopeampia. Kehitimme myös, sekä plasmidi- että solupohjaisia, reportterikokeita Parkinsonin tautiin liitetyn entsyymin, Dicerin, aktivaation mittaamiseksi. Nämä reportterikokeet, jotka hyödynsivät ekso- ja endogeenisiä fluoresenssi- sekä luminesenssi-indikaattoreita, validoitiin ja tulokset olivat verrattavissa aiempiin julkaistuihin reporttereihin, paranneltujen fysiologisesti suotuisien olosuhteiden myötä. Näiden lisäksi huomasimme, että CMV promoottori, jota käytetään geeniterapioissa, voi aktivoitua neurotransmission myötä. Osoitimme in vivo, että metamfetamiinin – voimakas dopamiinia vapautta aine – annostelun myötä CMV promoottori aktivoitui rottien aivoissa. Viimeiseksi huomasimme eroja solulimakalvoston rakenteissa viljeltyjen hiiren TH+ hermosolujen eri osissa. Yhteenvetona, tässä työssä kehitetyt ja parannellut työkalut laajentavat Parkinsonin taudin prekliinisten tutkijoiden työkalurepertuaaria, mutta ne ovat myös mahdollisesti sovellettavissa muiden tautien, sekä kliinisten tutkimusten, tutkimiseen. Lisäksi löydöksemme CMV-promoottorin aktivaatiosta, on tärkeä tieto ottaa huomioon suunnitellessa ekspressiovektoreita, reportterikokeita, ja geeniterapioita käytettäväksi prekliiniisissä Parkinsonin taudin kokeissa, joissa käytetään amfetamiineja. Ja lopuksi, saimme uusia havaintoja viljeltyjen dopamiinihermosolujen ultrarakenteellisista ominaisuuksista ja tuotimme hyödyllisen resurssin tutkijoiden käytettäväksi

Investigation of pathophysiological mechanisms in clinically isolated syndrome using advanced imaging techniques

This thesis concerns an observational study of patients recruited after their first episode of neurological symptoms suggestive of demyelination in the central nervous system and diagnosed either with clinically isolated syndrome or relapsing-remitting multiple sclerosis. In multiple sclerosis, brain tissues can exhibit extensive neuroaxonal microstructural and metabolic abnormalities, but little is known about their presence and significance at the time of the first demyelinating episode. I used a novel multi-parametric quantitative MRI approach, combining neurite orientation dispersion and density imaging (NODDI), which gives information about tissue microstructure, and 23Na MRI, which estimates total sodium concentration, a marker of metabolic dysfunction, in the brains of clinically isolated syndrome patients. I found microstructural and sodium homeostasis alterations in cortical areas of patients that showed clinical relevance. Within the diffuse axonal dispersion found in the normal-appearing white matter, the corpus callosum shared with lesions, signs of axonal damage and metabolic dysfunction, thus emerging as a possible target for early neuroprotective interventions. Structural cortical networks (SCNs) represent patterns of coordinated morphological modifications in cortical areas and they have shown pathophysiological changes in many brain disorders, including multiple sclerosis. I investigated alterations of SCNs at the individual level in this early cohort. Patients showed altered small-world topology, an efficient network organization combining dense local clustering with relatively few long-distance connections. These disruptions were worse for patients with higher lesion load and worse cognitive processing speed indicating that pathophysiological changes in the cortical morphology can influence clinical outcomes. Finally, I hypothesised that the patients in the cohort presenting with optic neuritis may have disturbances in neuropsychological functions related to visual processes. I found that cognitive visuospatial processing is affected after unilateral optic neuritis and improves over time with visual recovery, independently of the structural damage in the visual and central nervous system

Immunohistochemical and electrophysiological investigation of E/I balance alterations in animal models of frontotemporal dementia

Behavioural variant frontotemporal dementia (bvFTD) is a neurodegenerative disease characterised by changes in behaviour. Apathy, behavioural disinhibition and stereotyped behaviours are the first symptoms to appear and all have a basis in reward and pleasure deficits. The ventral striatum and ventral regions of the globus pallidus are involved in reward and pleasure. It is therefore reasonable to suggest alterations in these regions may underpin bvFTD. One postulated contributory factor is alteration in E/I balance in striatal regions. GABAergic interneurons play a role in E/I balance, acting as local inhibitory brakes, they are therefore a rational target for research investigating early biological predictors of bvFTD. To investigate this, we will carry out immunohistochemical staining for GABAergic interneurons (parvalbumin and neuronal nitric oxide synthase) in striatal regions of brains taken from CHMP2B mice, a validated animal model of bvFTD. We hypothesise that there will be fewer GABAergic interneurons in the striatum which may lead to ‘reward-seeking’ behaviour in bvFTD. This will also enable us to investigate any preclinical alterations in interneuron expression within this region. Results will be analysed using a mixed ANOVA and if significant, post hoc t-tests will be used. The second part of our study will involve extracellular recordings from CHMP2B mouse brains using a multi-electrode array (MEA). This will enable us to determine if there are alterations in local field potentials (LFP) in preclinical and symptomatic animals. We will also be able to see if neuromodulators such as serotonin and dopamine effect LFPs after bath application. We will develop slice preparations to preserve pathways between the ventral tegmental area and the ventral pallidum, an output structure of the striatum, and the dorsal raphe nucleus and the VP. Using the MEA we will stimulate an endogenous release of dopamine and serotonin using the slice preparations as described above. This will enable us to see if there are any changes in LFPs after endogenous release of neuromodulators. We hypothesise there will be an increase in LFPs due to loss of GABAergic interneurons

Oxidoreductase

Oxidoreductase enzymes are a group of enzymes that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. Oxidoreductase enzymes utilize NADP+ or NAD+ as cofactors. Oxidoreductase enzymes include the following: oxidase, dehydrogenase, peroxidase, hydroxylase, oxygenase, and reductase. Most oxidoreductase enzymes are dehydrogenases. However, reductases are also common. The accepted nomenclature for dehydrogenases is “donor dehydrogenase”, where the donor is the oxidized substrate. Metabolic abnormalities disorders resulting from a deficiency (quantitative and qualitative) or from over-activity of oxidoreductase, which may contribute to the decreased normal performance of life, are becoming common. This book covers the potential applications of oxidoreductases on the growth of oxidoreductase-based diagnostic tests and better biosensors in the design of inventive systems for crucial co-enzyme generations and in the synthesis of polymers and organic substrates. The book describes the role of oxidoreductase as essential in medical drug formation. It can be employed to produce a huge amount of compounds that act as medical mediators like Cephalosporin (beta lactam antibiotic). Furthermore, the idea of how to use different enzymes as targets for medical treatment in different types of cancers is also described in this book

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Imaging diffusional variance by MRI [public] : The role of tensor-valued diffusion encoding and tissue heterogeneity

Diffusion MRI provides a non-invasive probe of tissue microstructure. We recently proposed a novel method for diffusion-weighted imaging, so-called q-space trajectory encoding, that facilitates tensor-valued diffusion encoding. This method grants access to b-tensors with multiple shapes and enables us to probe previously unexplored aspects of the tissue microstructure. Specifically, we can disentangle diffusional heterogeneity that originates from isotropic and anisotropic tissue structures; we call this diffusional variance decomposition (DIVIDE).In Paper I, we investigated the statistical uncertainty of the total diffusional variance in the healthy brain. We found that the statistical power was heterogeneous between brain regions which needs to be taken into account when interpreting results.In Paper II, we showed how spherical tensor encoding can be used to separate the total diffusional variance into its isotropic and anisotropic components. We also performed initial validation of the parameters in phantoms, and demonstrated that the imaging sequence could be implemented on a high-performance clinical MRI system. In Paper III and V, we explored DIVIDE parameters in healthy brain tissue and tumor tissue. In healthy tissue, we found that diffusion anisotropy can be probed on the microscopic scale, and that metrics of anisotropy on the voxel scale are confounded by the orientation coherence of the microscopic structures. In meningioma and glioma tumors, we found a strong association between anisotropic variance and cell eccentricity, and between isotropic variance and variable cell density. In Paper IV, we developed a method to optimize waveforms for tensor-valued diffusion encoding, and in Paper VI we demonstrated that whole-brain DIVIDE is technically feasible at most MRI systems in clinically feasible scan times