Longitudinal changes in subcortical morphology in Huntington Disease and the relationship with clinical, motor and neurocognitive outcomes

Huntington disease (HD) is a devastating inherited neurodegenerative disease which causes progressive motor, psychiatric and cognitive disturbances as well as neurodegeneration. Mapping the spatiotemporal progression of neuroanatomical change in HD is fundamental to developing biomeasures suitable for prognostication and to aid in development and testing of potential treatments. The neostriatum is central to HD and is known to start to degenerate more than a decade before observable motor onset. It is central to a number of frontostriatal re-entrant circuits which regulate motor control and other forms of behaviour. Changes in striatal morphology can consequently be correlated with observable clinical, motor and cognitive outcomes. However, the neostriatum is merely one part of the "hubs and spokes" of neural circuitry and neurodegeneration in HD also occurs in other areas of the brain. The hippocampus has been less fully studied in HD and has implications for neural plasticity, particularly given neurogenesis continues into adulthood in this region. Furthermore, thickness of the corpus callosum may be used as a proxy for cortical changes that are known to occur later in HD. This thesis uses data from the IMAGE-HD study to characterise neuroanatomical changes in HD, with the aim to improve knowledge of HD-associated neurodegenerative pathways and to provide further insight to relate quantitative measures of morphology to function. A number of analytical techniques are used to investigate changes in size and shape of neuroanatomical structures and to correlate these with clinical, motor and neurocognitive outcomes. This thesis demonstrates that shape changes in the neostriatum in HD and pre-symptomatic HD correlate with functional measures subserved by corticostriatal circuits, and identifies significant longitudinal differences in putaminal and caudate shape. Only the putamen has a significant group by time interaction, suggesting that it is a better marker for longitudinal change in pre-symptomatic HD and HD. While HD has its most marked effects on the neostriatum, it also has more subtle effects on other subcortical areas. This thesis shows surface contraction occurring in HD in the hippocampus compared to controls, although without correlations to functional measures or significant longitudinal change. Unlike these "hubs", this thesis finds that the large "spoke" of the corpus callosum is not impacted early in the HD process but becomes affected after symptom onset, highlighting the spread of neurodegeneration in other structures. This is the first time that such robust statistical analysis of longitudinal shape change in HD has been able to be performed and shows the neostriatum, particularly the putamen, as a potentially useful structural basis for the characterisation of an endophenotype of HD. This thesis provides a more comprehensive picture of neuroanatomical change in HD by using a "hubs and spokes" approach to analyse key areas, increasing knowledge about neurodegenerative pathways and functional outcomes

Reward circuitry is perturbed in the absence of the serotonin transporter

The serotonin transporter (SERT) modulates the entire serotonergic system in the brain and influences both the dopaminergic and norepinephrinergic systems. These three systems are intimately involved in normal physiological functioning of the brain and implicated in numerous pathological conditions. Here we use high-resolution magnetic resonance imaging (MRI) and spectroscopy to elucidate the effects of disruption of the serotonin transporter in an animal model system: the SERT knock-out mouse. Employing manganese-enhanced MRI, we injected Mn^(2+) into the prefrontal cortex and obtained 3D MR images at specific time points in cohorts of SERT and normal mice. Statistical analysis of co-registered datasets demonstrated that active circuitry originating in the prefrontal cortex in the SERT knock-out is dramatically altered, with a bias towards more posterior areas (substantia nigra, ventral tegmental area, and Raphé nuclei) directly involved in the reward circuit. Injection site and tracing were confirmed with traditional track tracers by optical microscopy. In contrast, metabolite levels were essentially normal in the SERT knock-out by in vivo magnetic resonance spectroscopy and little or no anatomical differences between SERT knock-out and normal mice were detected by MRI. These findings point to modulation of the limbic cortical–ventral striatopallidal by disruption of SERT function. Thus, molecular disruptions of SERT that produce behavioral changes also alter the functional anatomy of the reward circuitry in which all the monoamine systems are involved

Characterising the structural brain changes in Huntington’s disease using translational neuroimaging

This thesis examined the macro-structural and micro-structural changes in Huntington’s disease (HD) in order to improve understanding of the temporal and spatial patterns of neurodegeneration, and the functional relevance of these changes. Translational techniques were employed using genetic mouse models of HD in combination with a patient cohort to examine grey and white matter changes with a particular focus on white matter microstructure. In the patient cohort, the cognitive profile was examined using a cognitive battery not before applied in HD. Specific deficits were found in set-shifting and flexibility, verbal reasoning, working memory and paired associate learning, along with subtle differences in response inhibition that were sensitive to disease burden. A composite cognitive score was produced to examine the relationship between cognitive function and brain structure. A multi-modal examination of white matter tract-specific microstructural measurements revealed abnormalities in the corpus callosum and cingulum bundle that were sensitive to disease burden (chapter 4). In chapter 5, multiple analysis techniques converged to reveal tissue macrostructure abnormalities that were also sensitive to disease burden in HD. Cortical changes were less consistent, and unlike the microstructure findings, white matter macrostructural abnormalities were not related to disease burden. In chapters 6 and 7, genetic mouse models of HD were used to examine changes across the disease course, and to pilot an interventional design. In vivo diffusion MRI and T2-weighted MRI sequences were acquired at 2 different time points in the HdhQ150 knock-in model of HD and imaging data is presented alongside behavioural results and immunohistochemistry. In chapter 7, an environmental modification regime was tested in the YAC128 mouse model using in vivo MRI. Environmental intervention reduced the degree of disease-related atrophy, altered tissue microstructure and improve motor but not cognitive performance in YAC128 mice

Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the HdhQ150/Q150 mouse model of huntington's disease

A variety of mouse models have been developed that express mutant huntingtin (mHTT) leading to aggregates and inclusions that model the molecular pathology observed in Huntington's disease. Here we show that although homozygous HdhQ150 knock-in mice developed motor impairments (rotarod, locomotor activity, grip strength) by 36 weeks of age, cognitive dysfunction (swimming T maze, fear conditioning, odor discrimination, social interaction) was not evident by 94 weeks. Concomitant to behavioral assessments, T2-weighted MRI volume measurements indicated a slower striatal growth with a significant difference between wild type (WT) and HdhQ150 mice being present even at 15 weeks. Indeed, MRI indicated significant volumetric changes prior to the emergence of the "clinical horizon" of motor impairments at 36 weeks of age. A striatal decrease of 27% was observed over 94 weeks with cortex (12%) and hippocampus (21%) also indicating significant atrophy. A hypothesis-free analysis using tensor-based morphometry highlighted further regions undergoing atrophy by contrasting brain growth and regional neurodegeneration. Histology revealed the widespread presence of mHTT aggregates and cellular inclusions. However, there was little evidence of correlations between these outcome measures, potentially indicating that other factors are important in the causal cascade linking the molecular pathology to the emergence of behavioral impairments. In conclusion, the HdhQ150 mouse model replicates many aspects of the human condition, including an extended pre-manifest period prior to the emergence of motor impairments

Topological Biomarker of Alzheimer’s Disease

For years, it has been assumed that the cerebral accumulation of pathologic protein forms is the main trigger of Alzheimer’s disease (AD) pathology; however, recent studies revealed strong evidences that the alternations in synaptic activity precede and affect the homeostasis of amyloid-beta and tau, both of which aggregate during AD. Given that the neuropathological changes, characteristic for AD, start decades before the onset of the first symptoms, when alternations become irreversible, it is crucial to find a biomarker that can detect the preclinical signs of disease, presumably synaptic dysfunction of specific cerebral areas. Here is presented a novel, a high potential neuroimaging biomarker that can detect the postsynaptic dysfunction of specific neural substrate located in medial prefrontal cortex (mPFC) during sensory gating processing of a simple auditory stimulus. The magnetoencephalography-based localization of mPFC gating activation has the potential not only to detect symptomatic AD but also to become a predictor of cognitive decline related to the pathophysiological processes of AD, both at the individual level. The strengths of proposed biomarker lie in the simplicity of using a binary value, i.e., activated or not activated a neural generator along with its potential to follow the evolution of the pathophysiological process of disease from preclinical phase. The novel biomarker does not require estimation of uniform cutoff levels and standardization processes, the main problems of so far proposed biomarkers. Ability to individually detect AD pathology during putative preclinical and clinical stages, absolute noninvasiveness, and large effect size give this biomarker a high translation capacity and clinical potential

Axon pathology in mouse models of Huntington's disease

Axon or synapse dysfunction parallels or precedes symptom onset in many neurodegenerative disorders. In some of these conditions, not only do axon and synapse loss determine the course of pathology, but protection of those neuronal compartments is mandatory to alleviate the disease. Whether this is also the case in Huntington’s disease (HD), a devastating neurodegenerative disorder characterised by progressive deterioration of both physical and mental abilities and inevitable early death, remains unclear. Present therapeutic strategies do not address protection of axons and synapses, which may help explain why there is no effective treatment currently in use. Moreover, an accurate characterisation of the development of axon pathology relative to neuronal loss and to the deposition of mutant-Huntingtin (mHTT) aggregates, neuropathological hallmark of HD, is lacking. In the present thesis I have carried out a detailed study aiming to investigate axon degeneration in the R6/2 transgenic (Tg) and the HdhQ140 knock-in (KI) mice, two HD models, and to assess whether this occurs early in and contributes to the course of pathology. I tested the hypothesis that axon degeneration precedes or at least parallels degeneration of other neuronal compartments in these mice. To characterise axon pathology and its spatio-temporal relationship to aggregate formation, neuronal loss and symptom onset, I crossed R6/2 and HdhQ140 mice with YFP-H transgenic mice that express the yellow fluorescent protein (YFP) in a subset of neurons. Neuronal pathways labelled by YFP in this model include some reported to be affected in HD. In these mice individual fluorescent neurons can be tracked over long distance and axons can be traced back to their cell bodies. Using a powerful axon imaging method that was developed and successfully applied to study Alzheimer’s disease, I was able to place axon degeneration accurately in the sequence of pathological events and develop methods to quantify it as readout for future therapeutic studies from our group and elsewhere. I found that the morphology of axons was strikingly abnormal in some brain areas in HdhQ140 homozygous mice (HdhQ140/Q140) where large axonal swellings were detected at 6 months and at 12 months of age in stria terminalis and striatum. In these mice, the number of axonal swellings increased age-dependently and was significantly higher than that found in wild-type littermates. However, I did not detect degeneration in cell bodies, dendrites or synapses suggesting that axon pathology is the main feature of the disease in this model. To better characterise the KI model, I also performed a battery of behavioural tests to assess motor and cognitive impairment during disease progression. I used tests of locomotor activity, motor coordination and balance and sensorimotor gating to measure motor function and tests of spatial working memory and anxiety-like behaviour to assess cognitive and behavioural symptoms, respectively. A longitudinal study from 1 to 12 months was carried out to detect pathological changes from early stage and relate them to swelling formation. In all tests, I found a strong reduction in locomotor activity in HdhQ140 mice compared to the controls although balance and coordination seemed not to be impaired as rotarod performance was unaltered. Alterations were also detected in prepulse inhibition, suggesting sensorimotor defects occur in these mice, while no abnormal cognitive or psychiatric behaviour was detected in the time-frame of the study. Behavioural symptoms, as well as abnormal morphological changes found in axons, worsened with time and major impairments were found at the latest time-point, 12 months of age. Finally, I asked whether alterations in the NAD biosynthetic pathway could underline the signs of axon pathology detected in HdhQ140 homozygous mice, as it has recently emerged that this pathway regulates axon survival and axon and synapse degeneration in many neurodegenerative disorders. To this purpose, I looked at possible alterations in the level of nucleotides (NMN, NAD) and in the activity of key enzymes in this pathway (NMNAT, NAMPT). I also tested the hypothesis that mHTT interacts with NMNAT enzymes and with the Wallerian degeneration slow protein (WLDs), an NMNAT fusion protein, and interferes/impairs their normal function. As WLDs delays axon degeneration in acute and neurodegeneration models, future works may address beneficial role of WLDs in HD/WldS crossed mice. Despite no detected alterations in nucleotide levels or enzymatic activity in the KI mice compared to the controls, colocalisation was found between mHTT and WLDs and between mHTT and NMNAT2, an important axon survival factor, suggesting a possible interaction between these proteins which could play a role in HD neurodegeneration

The clinical utility of multidisciplinary rehabilitation in individuals with Huntington’s Disease

Background Huntington’s disease (HD) is a chronic neurodegenerative disorder characterised by a progressive loss of cognitive function, motor control and psychiatric features. Individuals also display a variety of systemic features. Progressive neuronal dysfunction and neuronal cell death are thought to underlie the onset and progression of many clinical features of HD. Despite scientific progress, there is still no cure or disease modifying therapy for HD, and available pharmaceutical agents only provide partial relief of motor and psychiatric features. An emerging body of evidence indicates that lifestyle enrichment may delay the onset and progression of clinical features, and exert favourable effects on neuropathological aspects of HD. Few studies have evaluated the effects of lifestyle enrichment strategies like multidisciplinary rehabilitation on the clinical features of HD. Moreover, no study has evaluated the effects of multidisciplinary rehabilitation on neuropathological aspects of HD. Aims The initial aim of this thesis was to determine factors that contribute to features of the disease that negatively impact on activities of daily living such as mobility and balance (Chapter 2), and to identify, using a literature review, a rehabilitation strategy that could positively impact on these features of HD (Chapter 3). These studies informed our ultimate aim which was to investigate the clinical utility of multidisciplinary rehabilitation on clinical and neuropathological features of HD (Chapters 4, 5 and 6) Methods In study 1 (Chapter 2), 22 participants were assessed using a battery of balance, mobility, cognitive tests, assessments of muscle strength and body composition measures. Data was . then statistically examined using stepwise linear regression to identify factors that contribute to balance and mobility impairments in individuals with manifest HD. In study 2 (Chapter 3), a systematic search of journal databases was made from inception to July 2014 for studies reporting on resistance exercise in patients with neurodegenerative disorders. Selected studies were abstracted and critically appraised using a quality control checklist. For the intervention studies, (3 and 4 Chapters 4 and 5), 20 participants with manifest HD were randomly assigned to either a control or training group. Individuals randomised to the intervention group were provided with a nine month multidisciplinary intervention comprising once weekly supervised clinical exercise, thrice weekly home based exercise and fortnightly occupational therapy, while those randomised to the control group were asked to continue with their standard care and daily activities. Participants were assessed using motor, cognitive, psychological, body composition and quality of life measures at baseline and at the completion of the intervention. In study 5 (Chapter 6), 15 participants with manifest HD were assessed using magnetic resonance imaging and a battery of cognitive assessments after nine months of multidisciplinary rehabilitation to see whether such a therapy is capable of inducing favourable changes in brain structure and cognitive function. Results The main factors that contribute to mobility and balance impairments in patients with manifest HD were found to be lower limb muscle weakness and a loss of cognitive function (Study 1). Systematic evaluation of the effects of resistance exercise for neurodegenerative disorders showed that it is beneficial for multiple sclerosis and Parkinson’s disease. In particular, improvements in muscle strength, mobility, balance, clinical disease progression, fatigue, functional capacity, quality of life, disease biology, electromyography activity, mood, skeletal muscle volume and architecture were reported in individuals with multiple sclerosis or Parkinson’s disease (PD) after resistance exercise. The most robust effects of resistance exercise were found for muscle strength outcomes, and were more pronounced in individuals with PD (Study 2). The multidisciplinary rehabilitation intervention studies conducted as part of this thesis significantly improved isometric and isokinetic muscle strength, self-perceived balance, body mass, lean tissue mass and fat mass in patients with HD (Studies 3 and 4). Moreover, multidisciplinary rehabilitation also increased grey matter (GM) volume in the caudate nucleus and dorsolateral prefrontal cortex of patients. The significant increases in GM volume were accompanied by, and correlated to, a significant improvement in performance in verbal learning and memory. Conclusions The work presented here shows that lower extremity muscle weakness and a loss of cognitive function significantly contribute to impairments in mobility and balance. This work also shows that strength training has favourable effects on motor function, including strength, mobility and balance, as well as other clinical features in similar neurodegenerative disorders, and thus should be integrated into multidisciplinary rehabilitation interventions for HD. In addition, this study provides evidence that multidisciplinary rehabilitation can significantly improve aspects of motor control, cognitive function and body composition. Finally we show, for the first time, that multidisciplinary rehabilitation can increase GM volume in structures known to degenerate in HD, and that such increases are functionally related to changes in verbal learning and memory. Future work is urgently required to confirm and expand on these exciting findings, particularly with respect to the neurorestorative properties of multidisciplinary rehabilitation

Mutant Huntingtin toxicity modifiers revealed by a spatiotemporal proteomic profiling

Huntington's disease (HD) is a genetic hereditary disorder characterized by aggregation of polyQ-expanded mutant Huntingtin (mHTT) protein and progressive neurodegeneration within different brain regions, but specially in cortex and striatum. The pathology is associated with motor, cognitive and psychiatric symptoms. A hallmark of HD is the aggregation of polyglutamine-expanded (polyQ) huntingtin from soluble oligomers to inclusion bodies. Still nowadays, the character of these aggregates and the transition to the neuronal functional disorder, is poorly understood. In this thesis, the progression of the disease was assessed in a spatiotemporal manner in the R6/2 mice, a HD model, in order to find molecular signatures that could lead first, to a more detailed description of the disorder and second, to the elucidation of possible protein candidates that eventually have the ability of modify HD-related toxicity. Initially, it was approached by mass spectrometry-based quantitative proteomics to break down the spatiotemporal mechanisms of degeneration in HD. The formation of insoluble inclusion bodies throughout the disease progression correlated with the profound remodeling of the soluble proteome. The complexity in protein numbers of the aggregates was detailed through a quantitative characterization. This deep analysis unraveled the dependency of the aggregates' protein sequestration on specific biophysical features and sequence domains. Based on the proteomic data and applying different criteria, a follow-up study of some proteins was carried out. Overexpression of a selected group of the sequestered proteins improved the cellular viability in a cell line model of HD and reduced in most cases the inclusion body size. The effect of most of those proteins was specific to a mHTT-toxicity induced context. These results suggest that widespread loss of function contributes to aggregate-mediated toxicity. The strong effect of one of the protein candidates in the viability assays, Hepatome-derived grow factor (HDGF), lead to a closer examination. The effect of the protein was confirmed in primary neurons, in both transient transfection and in long-term viral transduction. Overexpression of HDGF in the striatum of R6/2 mice significantly rescued their exploratory behavior and ameliorated their clasping phenotype. In summary, the thesis represents a multi-disciplinary study in the R6/2 mouse model, spanning from proteomics to in vivo overexpression of a validated sequestered protein, which appears to be a potential therapeutic mHTT-toxicity modifier. Collectively, the study provides an integrative approach to solve HD molecular mechanisms and contributes to fill in the gap between identification of disease-associated pathways and their corresponding phenotypes

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