Preclinical Experimental Therapeutics

This chapter begins by reviewing the mammalian models of Huntington’s disease (HD) that have been developed using mice, rats, and a number of large animals, including sheep, pigs, and nonhuman primates. Analysis of these models, together with genetically engineered mice created through specific manipulations of the mouse genome, has provided considerable insights into the molecular pathogenesis of HD. The number of potential therapeutic targets that have been proposed for HD is considerable, and their preclinical evaluation in HD mouse models is being used to select targets that should be pursued in drug development programs. Hence, mouse models have been used extensively to validate therapeutic targets and in the preclinical testing of therapeutic strategies. The limitations of these studies are discussed, and best-practice approaches are highlighted. The chapter concludes with a summary of the gene therapy approaches that are being developed, including strategies to lower the levels of huntingtin

The role of ASK1 in selective striatal lesion formation induced by neuronal injury

Dept. of Medical Science/박사Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. The systemic administration of 3-nitropropionic acid (3-NP) facilitates the development of selected striatal lesions and it remains unclear whether specific neurons are selectively targeted in 3-NP infused striatal degeneration. Although not entirely elucidated, the mechanisms of neurotoxicity induced by 3-NP have been shown to include the exhaustion of adenosine triphosphate, mitochondrial membrane depolarization, dysregulation of intracellular calcium homeostasis, calpain activation, and the release of pro-apoptotic proteins from mitochondria. The present study is to characterize the regulation of BDNF in each cortical and striatal subregion. This study investigates that mild and chronic exposure of mitochondrial toxin can modulate the C1q level both in cortex and striatum via regulation of TGF-beta from astrocyte. Consequently we investigate how the BDNF is dominantly depleted in striatum, and eventually whether striatal lesion is established in involving in ASK1 pathway.The results of the present work show an alteration of ASK1 pathway molecules, TGF-beta, C1q level, and BDNF level as a final standard to striatal degeneration. By ASK1 down-regulation, improvement in each molecules containing behavioral impairment was evaluated in 3NP- infused mice and 3-NP treated primary neuronal cells. We propose the hypothesis that (1) ASK1 overexpression by systemic infusion of 3-NP promotes the formation of selective striatal lesions, and this occurs apart from just ROS generation. (2) ASK1 may differentially regulate C1q secretion level via active TGF-beta in each brain subregion of cortex and striatum, consequently involved in axon degeneration of corticostriatal projection neuron. When brain is mildly and chronically exposed to mitochondrial toxin, presynaptic neuron (in cortical neuron) degrades first, and then postsynaptic neuron of striatal MSN neuron withers as a consequence of it.Consolidating these results, we suggest that the increased ASK1 is linked to regulation of TGF-beta secreted in astrocytes, and differential C1q expression in neurons triggered by TGF-beta leads degradation of cortical projection and depletion of BDNF in striatal neuron in mice brains systemically infused with 3-NP.ope

CHARACTERIZATION OF BRAIN TISSUE MICROSTRUCTURES WITH DIFFUSION MRI

Diffusion MRI is a useful medical imaging tool for noninvasive mapping of the neuroanatomy and brain connectivity. In this dissertation, we worked on developing diffusion MRI techniques to probe brain tissue microstructures from various perspectives. Spatial resolution of the diffusion MRI is the key to obtain accurate microstructural information. In Chapter 2 and 3, we focused on developing high-resolution in vivo diffusion MRI techniques, such as 3D fast imaging sequence and a localized imaging approach using selective excitation RF pulses. We demonstrated the power of the superior resolution in delineating complex microstructures in the live mouse brain. With the high resolution diffusion MRI data, we were able to map the intra-hippocampal connectivity in the mouse brain, which showed remarkable similarity with tracer studies (Chapter 4). Using the localized fast imaging technique, we were the first to achieve in utero diffusion MRI of embryonic mouse brain, which revealed the microstructures in the developing brains and the changes after inflammatory injury (Chapter 5). The second half of the dissertation explores the restricted water diffusion at varying diffusion times and microstructure scales, using the oscillating gradient spin-echo (OGSE) diffusion MRI. We showed in the live normal mouse brains that unique tissue contrasts can be obtained at different oscillating frequency. We demonstrated in a neonatal mouse model of hypoxia-ischemia, that in the edema brain tissues, diffusion MRI signal changed much faster with oscillating frequency compared to the normal tissue, indicating significant changes in cell size associated with cytotoxic edema (Chapter 6). In the mild injury mice, OGSE showed exquisite sensitivity in detecting subtle injury in the hippocampus, which may relate to microstructural changes in smaller scales, such as the subcellular organelles (Chapter 7). Finally, we addressed the technical issues of OGSE diffusion MRI, and proposed a new hybrid OGSE sequence with orthogonally placed pulsed and oscillating gradients to suppress the perfusion related pseudo-diffusion (Chapter 8). In conclusion, we developed in vivo high-resolution diffusion techniques, and time-dependent diffusion measurements to characterize brain tissue microstructures in the normal and diseased mouse brains. The knowledge gained from this dissertation study may advance our understanding on microstructural basis of diffusion MRI

Imaging mouse models of neurodegeneration using multi-parametric MRI

Alzheimer’s disease (AD) is a devastating condition characterised by significant cognitive impairment and memory loss. Transgenic mouse models are increasingly being used to further our knowledge of the cause and progression of AD, and identify new targets for therapeutic intervention. These mice permit the study of specific pathological hallmarks of the disease, including intracellular deposits of hyperphosphorylated tau protein and extracellular amyloid plaques. In order to characterise these transgenic mice, robust biomarkers are required to evaluate neurodegenerative changes and facilitate preclinical evaluation of emerging therapeutics. In this work, a platform for in vivo structural imaging of the rTg4510 mouse model of tauopathy was developed and optimised. This was combined with a range of other clinically relevant magnetic resonance imaging (MRI) biomarkers including: arterial spin labelling, diffusion tensor imaging and chemical exchange saturation transfer. These techniques were applied in a single time-point study of aged rTg4510 mice, as well as a longitudinal study to serially assess neurodegeneration in the same cohort of animals. Doxycycline was administered to a subset of rTg4510 mice to suppress the tau transgene; this novel intervention strategy permitted the evaluation of the sensitivity of MRI biomarkers to the accumulation and suppression of tau. Follow-up ex vivo scans were acquired in order to assess the sensitivity of in vivo structural MRI to the current preclinical gold standard. High resolution structural MRI, when used in conjunction with advanced computational analysis, yielded high sensitivity to pathological changes occurring in the rTg4510 mouse. Atrophy was reduced in animals treated with doxycycline. All other MRI biomarkers were able to discriminate between doxycycline-treated and untreated rTg4510 mice as well as wildtype controls, and provided insight into complimentary pathological mechanisms occurring within the disease process. In addition, this imaging protocol was applied to the J20 mouse model of familial AD. This mouse exhibits widespread plaque formation, enabling the study of amyloid-specific pathological changes. Atrophy and deficits in cerebral blood flow were observed; however, the changes occurring in this model were markedly less than those observed in the rTg4510 mouse. This study was expanded to investigate the early-onset AD observed in individuals with Down’s syndrome (DS) by breeding the J20 mouse with the Tc1 mouse model of DS, permitting the relationship between genetics and neurodegeneration to be dissected. This thesis demonstrates the application of in vivo multi-parametric MRI to mouse models of neurodegeneration. All techniques were sensitive to pathological changes occurring in the models, and may serve as important biomarkers in clinical studies of AD. In addition, in vivo multi-parametric MRI permits longitudinal studies of the same animal cohort. This experimental design produces more powerful results, whilst contributing to worldwide efforts to reduce animal usage with respect to the 3Rs principles

Development of magnetic resonance imaging techniques for mouse models of Alzheimer's Disease

Due to increasing life expectancy in western societies, a rise in the prevalence of Alzheimer’s Disease (AD) is expected to have adverse social and economic consequences. The success of emerging treatments for AD relies heavily on the ability to test their efficacy. Sensitive biomarkers are required that provide information specific to the therapeutic targets. Through manipulation of the genome, transgenic mice have been bred to exhibit particular pathological features of AD in isolation. Magnetic Resonance Imaging (MRI) of these mouse models can be used to observe phenotypic abnormalities in-vivo in a controlled environment. As summarised in the introductory chapter, the aim of this work was to develop MRI techniques for inclusion in multi-parametric protocols to characterise AD models in-vivo. Structural MRI has become an increasingly popular tool in the measurement of atrophy of brain tissue over time and requires both accuracy and stability of the imaging system. In chapter 3, a protocol for the calibration of system gradients for high resolution, pre-clinical MRI is described. A structural phantom has been designed and 3D printed for use in a 9.4T small bore MRI and micro CT system. Post processing software is used to monitor gradient stability and provide corrections for scaling errors and non-linearity. Diffusion Tensor Imaging (DTI) and Quantitative Susceptibility Mapping (QSM) are MRI techniques that have shown sensitivity to changes in white matter regions of the brain. QSM may also provide a non invasive method for measurement of increased iron concentration in grey matter tissue observed in AD. Chapters 4 and 5 evaluate the utility of these measurements as imaging biomarkers in a mouse model that exhibits tau pathology associated with AD. Discrepancies between transgenic and wild-type groups were identified for both MRI techniques indicating the potential benefit of their inclusion in a multi-parametric in-vivo protocol

High-throughput transgenic mouse phenotyping using microscopic-MRI

With the completion of the human genome sequence in 2003, efforts have shifted towards elucidating gene function. Such phenotypic investigations are aided by advances in techniques for genetic modification of mice, with whom we share ~99% of genes. Mice are key models for both examination of basic gene function and translational study of human conditions. Furthering these efforts, ambitious programmes are underway to produce knockout mice for the ~25,000 mouse genes. In the coming years, methods to rapidly phenotype mouse morphology will be in great demand. This thesis demonstrates the development of non-invasive microscopic magnetic resonance imaging (\muMRI) methods for high-resolution ex-vivo phenotyping of mouse embryo and mouse brain morphology. It then goes on to show the application of computational atlasing techniques to these datasets, enabling automated analysis of phenotype. First, the issue of image quality in high-throughput embryo MRI was addressed. After investigating preparation and imaging parameters, substantial gains in signal- and contrast-to-noise were achieved. This protocol was applied to a study of Chd7+/- mice (a model of CHARGE syndrome), identifying cardiac defects. Combining this protocol with automated segmentation-propagation techniques, phenotypic differences were shown between three groups of mice in a volumetric analysis involving a number of organ systems. Focussing on the mouse brain, the optimal preparation and imaging parameters to maximise image quality and structural contrast were investigated, producing a high-resolution in-skull imaging protocol. Enhanced delineation of hippocampal and cerebellar structures was observed, correlating well to detailed histological comparisons. Subsequently this protocol was applied to a phenotypic investigation of the Tc1 model of Down syndrome. Using both visual inspection and automated, tensor based morphometry, novel phenotypic findings were identified in brain and inner ear structures. It is hoped that a combination of \muMRI with computational analysis techniques, as presented in this work, may help ease the burden of current phenotyping efforts