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A multimodal MRI approach to Huntington Disease to disclose a marker of onset and evolution in presymptomatic mutation carriers

Final Report Summary - MULTIMODAL MRI IN HD (A multimodal MRI approach to Huntington Disease to disclose a marker of onset and evolution in presymptomatic mutation carriers)

Huntington’s disease (HD) is a neurodegenerative disease without any cure to date. However, is possible to know which subjects are going to develop the disease by means of a genetic test. This offers the prospective of looking for preventive treatments that may delay the disease onset and evolution. Preventive cures would let patients independent life last longer, enhancing the quality of life of patients and families, and reduce the costs for healthcare and social service systems. Therefore, in vivo biomarkers are needed to monitor the evolution of the disease and the efficacy of preventive treatments. Fundamental questions in HD are: Can we find markers of progression? Can we determine the disease onset? Do presymptomatic patients already present measurable signs of pathology? Which is the roles of the brain cortex on the disease? The present project aimed to answer these questions, providing metrics to evaluate neural change over time especially before symptoms arise, and being an useful outcome for neuroprotective trials and to track disease-induced changes. We studied HD by a multisequence MRI protocol. The acquisition and analysis of gray and white matter volume, DTI and relaxometry data (iron content) as a whole is going to be crucial to understand a) the nature of the subcortical impairment; b) the involvement of cortical structures and cerebral white matter; and d) the influence of the CAG repeat length on cerebral impairment.
The training objectives revolved around obtaining expertise on Huntington’s disease, extending the knowledge to novel MRI techniques and acquiring proficiency in advanced imaging data analysis.

MAIN SCIENTIFIC RESULTS
Regarding basal ganglia, we analyzed the volume, microstructure and iron abnormalities of subcortical nuclei in premanifest gene carriers (PreHD), early-stage HD patients and healthy controls (Sanchez-Castaneda, Hum Brain Mapp 2013). We found two different MRI patterns in presymptomatic subjects: a volume loss in the striatum (caudate and putamen) associated with microstructural impairment; and volume loss in the globus pallidus, associated with iron accumulation and microstructural impairment. HD patients suffered more extensive damage to the striatum and globus pallidus, which further extended to the thalamus and accumbens (Figure 1). We also studied the distribution of iron in the brain of PreHD and HD subjects and how it related to brain atrophy (Sanchez-Castañeda et al., under review). We found that: a) iron accumulation starts in the caudate in presymptomatic stages, probably preceding neurodegeneration; and b) with disease progression, the iron concentration in the caudate maintains relatively stable; but increases in the putamen and globus pallidus. c) Volume instead reduces as the disease progresses in all the three nuclei. These results suggest that there is one primary impairment affecting the caudate nucleus, characterized by an initial iron accumulation that stabilizes later on while gray matter loss progresses; and a secondary neurodegenerative process affecting the putamen and globus pallidus, in which iron accumulates progressively along the disease.
Regarding cortical structures, our data (Sanchez-Castañeda et al., under review) showed that: a) iron levels in the cortex progressively decrease (while they are normal in PreHD subjects, they are reduced in parieto-occipital areas in PreHD-SS subjects, and more extensively reduced in the premotor cortex and parieto-temporo-occipital regions in HD patients); and b) there is also a progressive cortical volume loss, which starts in sensoriomotor and medial temporal areas in PreHD subjects and extends throughout the cortex, with preservation of anterior frontal regions, in HD patients. These results suggest a regional selective process affecting the cortex, starting from parieto-occipital areas.
Regarding white matter, we also analized the largest white matter tract, the corpus callosum (CC) (Di Paola, Luders, Cherubini, Sanchez-Castaneda, et al., Cereb Cortex. 2012). PreHD subjects had impairment within the isthmus subregion, most likely caused by specific damage to myelin sheaths; while HD patients had impairment in all CC subregions, driven by both myelin and axonal damage. Concerning the iron content (Di Paola, Phillips, Sanchez-Castaneda et al., Hum Brain Mapp 2013), at PreHD stage myelin breakdown starts with no iron disturbances. The iron content reduction manifests later, in early HD stage, characterized by lower iron content in the isthmus. Besides, we further examined the CC by parcellating it into seven major tracts (Phillips, Sanchez-Castaneda et al., PlosOne 2013). We found reduced volume and microstructural impairment across all white matter tracts in PreHD subjects; and even more severe deficits in HD patients. CC pathways are compromised prior to disease onset and CAG repeat length is a contributing factor to these deficits. Finally, we studied corticospinal tract (CST), the main white matter pathway that conducts motor impulses from the primary motor cortex to the spinal cord (Phillips, Squitieri, Sanchez-Castaneda et al., Cerebral Cortex 2014). In the presymptomatic phase, there was iron increase in the inferior portion of the CST (between the brainstem and the thalamus), suggesting that axonal damage advances in a retrograde fashion.
Moreover, we evaluated how CAG repeats length influences gray matter volume decrease and iron accumulation in basal ganglia (Sanchez-Castañeda et al., under review). We divided mutation carriers into three groups according to their CAG repeats length: 1) low-CAG repeats (≤ 42); 2) medium-CAG repeats (43-44); and 3) high-CAG repeats, (≥ 45). We found that the higher the CAG length, the less the volume. Regarding iron content, patients with higher number of CAG repeats (>45) had more iron in the globus pallidus bilaterally than the other two groups. So, in the globus pallidus, the higher the number of CAG repeats, the more the iron.
Finally, we investigated two resting-state networks, the Motor (MN) and Default Mode (DMN) networks, and related them to the gray matter loss (Sanchez-Castañeda et al., in preparation). The MN is directly affected by the disease through the striatal involvement; while the latter is involved in a wide range of internally focused tasks and plays a fundamental role of integration across different functional domains. HD patients had structural impairment (gray matter loss) in all the cortical and subcortical areas involved in the Motor network, with preservation of the supplementary motor area (SMA); but structural preservation of all the cortical areas involved in the DMN. Accordingly, the functional connectivity was impaired in the Motor network (there were no connections of SMA with the primary and secondary sensory-motor areas and the putamen); but it was relatively preserved in the DMN.

PROGRESS OF THE RESEARCH TRAINING ACTIVITIES
The development of the present project has: 1) provided the opportunity to a female young researcher to strengthen her competences in the discipline she’s working in, enhancing her knowledge of pathology, statistics and MRI acquisition and analysis; 2) conferred the applicant on flexibility, adaptability, and potentiate her initiative and responsibility working in a different country and under a diverse research setting; 3) implied a translational approach and is expected to have a translation in terms of better understanding and treatment of the patients, enhancing their quality of life; 4) allowed a young researcher to collaborate with the European HD network. To reinforce this intra-European networks is paramount to guarantee the high level of European research, as well as to ensure that younger researchers decide to develop their professional carriers within Europe, instead of moving to other continents; 5) been the starting point of a collaboration between the European research centers.

CONCLUSIONS
The results of this project show that in Huntington’s disease there is a progressive iron increase and volume loss in basal ganglia since presymptomatic stages. The degeneration of the caudate represents the primary early impairment; whereas the impairment in the putamen and globus pallidus might be the consequence of the progressive deafferentiation of the striato-pallidal pathways. This iron increase since presymptomatic stages suggests that iron accumulation is not merely a secondary phenomenon of neurodegeneration, but that early in the HD process may underlie the pattern of regional toxicity observed in HD. In cortical areas and the corpus callosum instead, iron content is normal in PreHD, but decreases in HD patients parallel to the decrease in volume. Nowadays, emerging treatments for neurodegenerative diseases (such as iron chelators and antioxidants) make in vivo regional assessment of iron levels and tissue integrity essential for planning such interventions. The results of this project are a prove that relaxometry may be a useful measure, combined to gray matter and white matter measures, to study the effect of neuroprotective drugs in the HD brain since presymptomatic stages of the disease. Regarding the cerebral connectivity, the motor network is structurally and functionally impaired in HD. However, albeit all the cortical areas of the Motor network have reduced volume, the SMA remains structurally preserved, suggesting a pattern of damage affecting first local and less important nodes, then more central nodes. These results, especially the ones arising from resting state and functional connectivity, might benefit rehabilitation programs.

SOCIO-ECONOMIC IMPACT OF THE PROJECT
Huntington's disease is a neurodegenerative disease that can be diagnosed before symptoms appear. For this reason, finding biomarkers of pathology progression would be helpful for in vivo evaluating the efficacy of treatments that aim at slowing the degeneration in the absence of clinical markers. Preventive cures will let patients independent life last longer, enhancing the quality of life of patients and families, and reducing the costs for healthcare and social service systems. The close contact with all the regional patients’ associations, which are collaborating with the study, contributed to the diffusion of the results to patients and society, enhancing disease knowledge and prevention. Thus, the results obtained from this project will benefit at long-term public health in Europe.
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