DEVELOPMENT OF IMAGING MARKERS TO TRACK ALZHEIMER¿S DISEASE PROGRESSION IN HUMANS AND MOUSE MODELS

La Malattia di Alzheimer (AD) \ue8 la forma pi\uf9 comune di demenza nella popolazione anziana e affligge pi\uf9 35 milioni di persone nel mondo. Ad oggi, le uniche terapie approvate per la sua cura sono dirette a ridurre i sintomi. Lo sviluppo di nuovi farmaci \ue8 lungo e costoso. Il processo di scoperta \ue8 arduo in quanto i trial clinici coinvolgono un ampio campione di pazienti e implicano dei follow-up troppo lunghi. Inoltre il valore predittivo dei modelli sperimentali \ue8 limitato a causa della mancanza di marcatori omologhi nell\u2019uomo e nei modelli animali. Questo lavoro si inserisce in Pharmacog, un progetto europeo che vede la collaborazione di universit\ue0 ed industrie allo scopo di identificare biomarcatori affidabili e sensibili alla progressione di malattia in pazienti affetti da decadimento cognitivo lieve (MCI) e modelli animali di AD allo scopo di colmare il vuoto tra risultati clinici e preclinici. Nell\u2019uomo, i marcatori di neuroimmagine sono tra i pi\uf9 promettenti candidati nel tracciare la progressione di malattia. Innovazioni nelle tecniche di risonanza magnetica (MRI) rendono possibile l\u2019identificazione di marcatori omologhi nell\u2019uomo e nel topo. Prima dello studio di neuroimmagine nei pazienti MCI, \ue8 necessario verificare che eventuali cambiamenti individuati siano dovuti all\u2019effettiva progressione di malattia e non causati dalla variabilit\ue0 intra e tra i diversi scanner utilizzati nel progetto. Il primo scopo di questo lavoro \ue8 lo studio dei cambiamenti morfometrici e di diffusione in tre diversi modelli murini di Malattia Alzheimer (TASTPM, TauPS2APP e PDAPP da 3 a 22 mesi) tramite l\u2019utilizzo di tecniche MRI. A nove mesi abbiamo trovato una significativa riduzione rispetto ai controlli del volume del caudato-putamen e della corteccia frontale nei TASTPM e nei TauPS2APP (p< 0.001). L\u2019assottigliamento della corteccia entorinale era significativo alla stessa et\ue0 in tutte e tre i modelli (p< 0.001). Abbiamo inoltre individuato delle anormalit\ue0 dipendenti dall\u2019et\ue0 anche in diverse regione di sostanza bianca. Quelle pi\uf9 precoci erano nella commissura anteriore e nel corpo calloso dei TASTPM di 13 mesi (p< 0.001). I danni dei TASTPM sono associabili al pesante carico di amiloide ed alla marcata attivazione della glia e degli astrociti. Il secondo scopo dello studio \ue8 la valutazione e la comparazione della riproducibilit\ue0 di misure volumetriche e di spessore tra test e retest ottenute utilizzando due diversi metodi di processazione esistenti (Freesurfer sulla singola acquisizione o Freesurfer longitudinale). Inoltre abbiamo saggiato la riproducibilit\ue0 di un\u2019analisi per le immagini di diffusione messa a punto nel nostro laboratorio. A questo scopo ognuno degli otto centri europei coinvolti nel progetto e con diversi scanner a 3T ha arruolato un gruppo di 5 volontari sani e anziani sottoponendoli a 2 acquisizioni di risonanza ad almeno una settimana di distanza l\u2019una dall\u2019altra. Abbiamo trovato che la variabilit\ue0 intra e tra i diversi centri nei volumi estratti da queste acquisizioni era inferiore al 3% per le strutture pi\uf9 grandi (come il talamo) e minore del 6% per quelle pi\uf9 piccole (es. amigdala). La variabilit\ue0 degli spessori era meno del 6% e le variazioni dei parametri di diffusione erano prevalentemente nell\u2019intervallo del 2-3%. In conclusione, abbiamo identificato nei modelli analizzati dei marcatori di immagine sensibili alla progressione dell\u2019AD simili a quelli visti nell\u2019uomo e questo apre la strada al possibile utilizzo di una \u201cdistintiva collezione\u201d di marcatori murini di immagine nei trial clinici. I dati collezionati nella parte umana mostrano un pi\uf9 altra riproducibilit\ue0 dei risultati morfometrici ottenuti con l\u2019analisi longitudinale rispetto a quella sulla singola acquisizione (p< 0.01). Infine, abbiamo dimostrato che l\u2019analisi delle immagini di diffusione messa a punto nel nostro laboratorio d\ue0 risultati ugualmente riproducibili a quelli riportati in letteratura.Alzheimer\u2019s disease (AD) is the most common form of dementia in elderly population, affecting more than 35 million people worldwide. To date, the only approved therapies for AD focus on symptomatic relief. The development of new therapeutic agents is time consuming and costly. Drug discovery process is arduous because clinical trials are currently involving too wide sample of patients and long follow-up. Moreover, the predicting value of experimental models used nowadays is limited due to the lack of homologous markers in humans and animals. This work is a branch of Pharmacog, an industry-academic European project aimed at identifying reliable biomarkers that are sensitive to disease progression in patients with Mild Cognitive Impairment (MCI) and in AD animal models in order to bridge the gap between preclinical and clinical outcomes. Human neuroimaging markers are among the most promising candidates to track disease progression. In addition, advanced magnetic resonance imaging (MRI) allow the identification of homologous biomarkers in humans and mice. Prior to investigate neuroimaging biomarkers on MCI patients, we have to test that there is no significant effect of within and across MRI sites variability on brain AD-related longitudinal changes. The first aim of this work is the study of the morphometric and diffusion changes in three different AD mouse model (TASTPM, TauPS2APP and PDAPP from 3 to 22 months of age) through MRI. We found significant volume reduction starting at 9 months in the caudate-putamen and frontal cortex of TASTPM and TauPS2APP (p< 0.001) compared to non transgenic mice. The decrease in the enthorinal cortex thickness was significantly lower in all the strains (p< 0.001). We also found age-related diffusion abnormalities in different white matter regions of TASTPM. The earlier changes were found in the corpus callosum and anterior commissure of 13 months old mice (p< 0.001). In TASTPM, deficits detected with MRI are related to heavy amyloid pathology, marked gliosis and astrocitosys. The second aim of this study is the evaluation and comparison of test-retest reproducibility of brain volumes and thicknesses by two existing Freesurfer pipelines (longitudinal and cross-sectional). Moreover, we assessed the reliability of a diffusion pipeline developed in our lab. Eight different 3T MRI sites in Europe enrolled a group of 5 healthy elderly subjects scanned twice at least a week apart. We found that the within and across sites variability of volumes was less than 3% for larger brain structures (such as thalamus) and less than 6% for smaller regions (i.e., hippocampus). The thickness variability was less than 6% and diffusion indices variations were mostly within the range 2-3%. In conclusion, the present data identify imaging biomarkers of disease progression in mice similar to that seen in humans and pave the way of a murine \u201cimaging signature\u201d usefulness in clinical trials. Human data show significantly higher reproducibility of brain morphometry using the longitudinal pipeline than using the cross-sectional one (p< 0.01). Finally, we demonstrated that the reliability of the analysis of brain diffusion we implemented in our lab is comparable to data reported in the literature

Impact of normal ageing and cerebral hypoperfusion on myelinated axons and its relation to the development of Alzheimer’s disease

Cerebral hypoperfusion can occur in normal ageing and is proposed to underlie white matter disturbances observed in the ageing brain. Moreover, cerebral hypoperfusion and white matter attenuation are early events in the progression of Alzheimer’s disease (AD). White matter mostly consists of myelinated axons which have distinct protein architecture, segregated into defined regions; the axon initial segment (AIS), the node of Ranvier, paranode, juxtaparanode, and internode. These sites are essential for action potential initiation and/or propagation and subsequently effective brain function. At the outset of the studies in the thesis there was evidence that the different regions within the myelinated axons are vulnerable to injury and disease. Thus it is hypothesised that in response to normal ageing and/or cerebral hypoperfusion these structures are altered and associated with cognitive impairment and that these effects are exacerbated in a transgenic mouse model (APPSw,Ind, J9 line) which develops age-dependent amyloid-β (Αβ) pathology. The first study aims to investigate the effect of normal ageing and Aβ deposition on myelinated axons and on learning and memory. To address this, the effects of normal ageing on the integrity of the AIS, nodes of Ranvier, myelin, axons, synapses and spatial working memory are examined in young and aged wild-type and TgAPPSw,Ind mice. A significant reduction in the length of nodes of Ranvier is demonstrated in aged wild-type and TgAPPSw,Ind mice. In addition, the length of AIS, is significantly reduced in the aged wild-type animals while the young TgAPPSw,Ind have significantly shorter AIS than the young wild-type mice. These effects are not influenced by the presence of Aβ. Myelin integrity is affected by age but this is more prominent in the wild-type animals whilst axonal integrity is intact. Moreover, there is an age-related decrease of presynaptic boutons only in the TgAPPSw,Ind mice. Contrary to the original hypothesis, working memory performance is not altered with age or influenced by increasing Aβ levels. The second study aims to examine the effects of cerebral hypoperfusion in combination with Αβ pathology and/or ageing on cognitive performance and the structure of myelinated axons. To address this, the effects of surgically induced cerebral hypoperfusion on the integrity of the nodes of Ranvier, paranodes, myelin, axons and spatial working memory performance are investigated in young and aged wild-type and TgAPPSw,Ind mice. A decrease in nodal length is observed in response to hypoperfusion in young and aged animals. This effect is shown to be exacerbated in the young TgAPPSw,Ind animals. Moreover, the disruption of the nodal domain is shown to occur without any gross alterations in myelin and axonal integrity. It is also demonstrated that in response to hypoperfusion, spatial working memory performance is defected in young and aged animals of both genotypes. This deficit is exacerbated in the young TgAPPSw,Ind. The observed changes in the nodal structure are associated with poor working memory performance indicating functional implication for the nodal changes. These data highlight that structures within myelinated axons are vulnerable to ageing and cerebral hypoperfusion. Therefore, the development of strategies that minimize injury or drive repair to these regions is necessary together with therapeutic approaches against the vascular insults that induce hypoperfusion and lead to white matter attenuation and cognitive decline. In the future, it would be interesting to investigate how alterations at the AIS/nodes of Ranvier affect neuronal excitability

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