Identificación de nuevos mecanismos fisiopatológicos en el Síndrome de Kindler

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología MolecularEl Síndrome de Kindler (SK), es una genodermatosis caracterizada clínicamente por la presencia de ampollas acrales, fotosensibilidad y envejecimiento prematuro, atrofia extensiva de la piel y una elevada predisposición a desarrollar carcinomas de la piel y mucosas. La proteína afectada es la kindlina-1 (codificada por el gen FERMT1), un componente de las adhesiones focales de los queratinocitos basales. Las mutaciones en este gen pueden explicar las características clínicas provocadas por la falta de adhesión entre la dermis y la epidermis, pero no aquellas no relacionadas con la fragilidad cutánea (envejecimiento prematuro, fotosensibilidad y predisposición a padecer tumores de piel). El objetivo principal de esta tesis doctoral es el estudio de las bases patológicas de estas manifestaciones, que están firmemente asociadas con alteraciones en el estado redox celular, generado principalmente por las especies reactivas de oxígeno (ROS). Al caracterizar el papel del estrés oxidativo en la patogénesis de la enfermedad, se observó que los queratinocitos de pacientes con SK presentaban un desequilibrio importante en su estado redox. Por un lado, exhibían un estado pro-oxidativo observable mediante el ratio GSSG/GSH (glutatión oxidado/glutatión reducido), así como una menor capacidad de síntesis de novo de este glutatión, que es una de las principales defensas celulares frente a las ROS. Por otra parte, las células SK mostraban un daño oxidativo acentuado en los lípidos de membrana, lo que sugiere la cronicidad del fenómeno. Este estado pro-oxidativo también lo presentaba la línea celular de queratinocitos HaCaT shFERMT1, en la que se ha silenciado parcialmente el gen FERMT1, comportándose de forma análoga a los queratinocitos de pacientes SK respecto a la gestión de las ROS. Además, los análisis de la ultraestructura y función de los principales orgánulos productores de ROS, las mitocondrias, revelaron en los pacientes SK pronunciadas anormalidades morfológicas y funcionales. Al evaluar la fotosensibilidad a la luz ultravioleta, se observó que tanto los queratinocitos de pacientes SK como la línea celular HaCaT shFERMT1 presentaban una mayor fotosensibilidad que sus respectivos controles, la cual se evidenció a través de una menor viabilidad celular. Los análisis transcriptómicos realizados, apoyaron estos datos al revelar la expresión aberrante de algunos genes implicados en la replicación y la reparación del daño al ADN en células SK, eventos que alterados y en paralelo potencian la disminución de la tasa de viabilidad celular. Además de estas alteraciones, los análisis transcriptómicos mostraron múltiples genes desregulados en rutas oncogénicas que podrían favorecer el desarrollo de cáncer de piel en los pacientes SK. Estos datos, en conjunto, señalan bases patológicas novedosas implicadas en las manifestaciones clínicas no relacionadas, a priori, con adhesión presentes en esta genodermatosis. Por último, se generó un modelo de ratón humanizado de SK, que reproducía las principales características histopatológicas de la enfermedad. Además, en este modelo se llevó a cabo la corrección de la enfermedad mediante terapia génica, demostrando que al restaurar la expresión de la proteína kindlina-1 se restauraba el fenotipo fisiológico de la piel.Kindler Syndrome (KS) is a rare heritable skin disorder clinically characterized by acral skin blisters, atrophy of skin, photosensitivity, premature ageing and a high risk of mucocutaneous malignancies. KS results from recessive loss-of-function mutations in the FERMT1 gene that encodes the protein Kindlin-1, a component of the focal adhesions in epithelial cells. However the complex phenotype of KS cannot be exclusively explained based on the adhesive function of Kindlin-1. Therefore, the mechanisms responsible for its clinical features such as photosensitivity, premature ageing and cancer are still unknown. The main goal of this study was to analyze the pathological basis of these clinical features of KS that are not related to cutaneous fragility but to the altered cellular redox state, produced mainly by reactive oxygen species (ROS). Our results, obtained with patient derived keratinocytes and HaCaT shFERMT1 (a keratinocyte cell line with FERMT1 partially silenced), followed a clear trend regarding their oxidative status and mitochondrial alterations. On one hand, they exhibited a pro-oxidative status pointed out by the GSSG/GSH ratio (oxidized glutathione/reduced glutathione), a lower capability to synthesize this glutathione (one of the main cellular defenses against ROS) and also, a high oxidative damage of membrane lipids in KS cells, which suggests a chronic phenomenon. On the other hand, mitochondria were studied, since they are the main source of endogenous ROS. KS patients’ cells showed morphological and functional alterations which are consistent with dysfunctional mitochondria. Our results suggest that alterations of the redox balance and mitochondria in KS may be a potential explanation of the premature skin ageing, photosensitivity and cancer prone phenotype of these patients. Furthermore, our viability assays showed a decrease after ultraviolet light exposure in KS keratinocytes and HaCaT shFERMT1 viability. Using transcriptomic analysis we observed that KS keratinocytes also have a number of altered genes involved in DNA replication and in damage repair. Taking together these data, with the fact that UVB is the main exogenous ROS source and that KS keratinocytes have dysfunctional mitochondria and alterations in the redox balance, we have a possible explanation for the higher risk to develop skin cancer in KS patients. Besides, in the transcriptomic analysis we found that there are several oncogenic genes altered in KS keratinocytes. Finally, we were able to generate a humanized mouse model for KS, which reproduces the main histopathological features of the disease. Also, we could restore collagen IV expression and the physiological phenotype of skin by gene therapy when FERMT1 was reestablished in KS keratinocytes

Decreased Default Mode Network connectivity correlates with age-associated structural and cognitive changes

Ageing entails cognitive and motor decline as well as brain changes such as loss of gray (GM) and white matter (WM) integrity, neurovascular and functional connectivity alterations. Regarding connectivity, reduced resting-state fMRI connectivity between anterior and posterior nodes of the Default Mode Network (DMN) relates to cognitive function and has been postulated to be a hallmark of ageing. However, the relationship between age-related connectivity changes and other neuroimaging-based measures in ageing is fragmentarily investigated. In a sample of 116 healthy elders we aimed to study the relationship between antero-posterior DMN connectivity and measures of WM integrity, GM integrity and cerebral blood flow (CBF), assessed with an arterial spin labeling sequence. First, we replicated previous findings demonstrating DMN connectivity decreases in ageing and an association between antero-posterior DMN connectivity and memory scores. The results showed that the functional connectivity between posterior midline structures and the medial prefrontal cortex was related to measures of WM and GM integrity but not to CBF. Gray and WM correlates of anterio-posterior DMN connectivity included, but were not limited to, DMN areas and cingulum bundle. These results resembled patterns of age-related vulnerability which was studied by comparing the correlates of antero-posterior DMN with age-effect maps. These age-effect maps were obtained after performing an independent analysis with a second sample including both young and old subjects. We argue that antero-posterior connectivity might be a sensitive measure of brain ageing over the brain. By using a comprehensive approach, the results provide valuable knowledge that may shed further light on DMN connectivity dysfunctions in ageing

Cognitive and motor improvement after retesting in normal-pressure hydrocephalus: a real change or merely a learning effect?

Object The test-retest method is commonly used in the management of patients with normal-pressure hydrocephalus (NPH). One of the most widely used techniques in the diagnosis of this condition is evaluation of the patient's response to CSF evacuation by lumbar puncture (a so-called tap test or spinal tap). However, interpretation of improved results in subsequent evaluations is controversial because higher scores could reflect a real change in specific abilities or could be simply the result of a learning effect. Methods To determine the effect of testing-retesting in patients with NPH, the authors analyzed changes documented on 5 neuropsychological tests (the Toulouse-Pieron, Trail Making Test A, Grooved Pegboard, Word Fluency, and Bingley Memory tests) and several motor ability scales (motor performance test, length of step, and walking speed tests) in a series of 32 patients with NPH who underwent the same battery on 4 consecutive days. The same tests were also applied in 30 healthy volunteers. In both groups, the authors used the generalized least-squares regression method with random effects to test for learning effects. To evaluate possible differences in response depending on the degree of cognitive impairment at baseline, the results were adjusted by using the Mini-Mental State Examination scores of patients and controls when these scores were significant in the model. Results In patients with NPH there were no statistically significant differences in any of the neuropsychological or motor tests performed over the 4 consecutive days, except in the results of the Toulouse-Pieron test, which were significantly improved on Day 3. In contrast, healthy volunteers had statistically significant improvement in the results of the Toulouse-Pieron test, Trail Making Test A, and Grooved Pegboard test but not in the remaining neuropsychological tests. Patients in the healthy volunteer group also exhibited statistically significant improvement in the motor performance test but not in step length or walking speed. Conclusions No learning effect was found in patients with NPH on any of the neuropsychological or motor tests. Clinical improvement after retesting in these patients reflects real changes, and this strategy can therefore be used in both the diagnosis and evaluation of surgical outcomes

Neuroanatomical correlates of olfactory loss in normal aged subjects

In non-demented older persons, smell dysfunction, measured premortem, has been associated with postmortem brain degeneration similar to that of Alzheimer's disease. We hypothesized that distinct measures of gray and white matter integrity evaluated through magnetic resonance imaging (MRI) techniques could detect degenerative changes associated with age-related olfactory dysfunction. High-resolution T1-weighted images and diffusion-tensor images (DTI) of 30 clinically healthy subjects aged 51 to 77 were acquired with a 3-Tesla MRI scanner. Odor identification performance was assessed by means of the University of Pennsylvania Smell Identification Test (UPSIT). UPSIT scores correlated with right amygdalar volume and bilateral perirhinal and entorhinal cortices gray matter volume. Olfactory performance also correlated with postcentral gyrus cortical thickness and with fractional anisotropy and mean diffusivity levels in the splenium of the corpus callosum and the superior longitudinal fasciculi. Our results suggest that age-related olfactory loss is accompanied by diffuse degenerative changes that might correspond to the preclinical stages of neurodegenerative processes

Fully automated delineation of the optic radiation for surgical planning using clinically feasible sequences

[EN] Quadrantanopia caused by inadvertent severing of Meyer's Loop of the optic radiation is a well-recognised complication of temporal lobectomy for conditions such as epilepsy. Dissection studies indicate that the anterior extent of Meyer's Loop varies considerably between individuals. Quantifying this for individual patients is thus an important step to improve the safety profile of temporal lobectomies. Previous attempts to delineate Meyer's Loop using diffusion MRI tractography have had difficulty estimating its full anterior extent, required manual ROI placement, and/or relied on advanced diffusion sequences that cannot be acquired routinely in most clinics. Here we present CONSULT: a pipeline that can delineate the optic radiation from raw DICOM data in a completely automated way via a combination of robust pre-processing, segmentation, and alignment stages, plus simple improvements that bolster the efficiency and reliability of standard tractography. We tested CONSULT on 696 scans of predominantly healthy participants (539 unique brains), including both advanced acquisitions and simpler acquisitions that could be acquired in clinically acceptable timeframes. Delineations completed without error in 99.4% of the scans. The distance between Meyer's Loop and the temporal pole closely matched both averages and ranges reported in dissection studies for all tested sequences. Median scan-rescan error of this distance was 1¿mm. When tested on two participants with considerable pathology, delineations were successful and realistic. Through this, we demonstrate not only how to identify Meyer's Loop with clinically feasible sequences, but also that this can be achieved without fundamental changes to tractography algorithms or complex post-processing methods.Advance Queensland, Grant/Award Number: R-09964-01; Fundacion Merck Salud; Proyecto Societat Catalana Neurologia; Ministerio de Economia, Industria y Competitividad of Spain, Grant/Award Number: DPI2017-87743-R; Red Espanola de Esclerosis Multiple, Grant/Award Numbers: RD12/0032/0002, RD12/0060/01-02, RD16/0015/0002, RD16/0015/0003; Spanish Government; Instituto de Salud Carlos III, Grant/Award Numbers: FIS 2015 PI15/00061, FIS 2015 - PI15/00587, FIS 2018 - PI18/01030Reid, LB.; Martínez-Heras, E.; Manjón Herrera, JV.; Jeffree, RL.; Alexander, H.; Trinder, J.; Solana, E.... (2021). Fully automated delineation of the optic radiation for surgical planning using clinically feasible sequences. Human Brain Mapping. 42(18):5911-5926. https://doi.org/10.1002/hbm.25658S59115926421

White matter changes measured by multi-component MR Fingerprinting in multiple sclerosis

T2-hyperintense lesions are the key imaging marker of multiple sclerosis (MS). Previous studies have shown that the white matter surrounding such lesions is often also affected by MS. Our aim was to develop a new method to visualize and quantify the extent of white matter tissue changes in MS based on relaxometry properties. We applied a fast, multi-parametric quantitative MRI approach and used a multi-component MR Fingerprinting (MC-MRF) analysis. We assessed the differences in the MRF component representing prolongedrelaxation time between patients with MS and controls and studied the relation between this component's volume and structural white matter damage identified on FLAIR MRI scans in patients with MS. A total of 48 MS patients at two different sites and 12 healthy controls were scanned with FLAIR and MRF-EPI MRI scans. MRF scans were analyzed with a joint-sparsity multi-component analysis to obtain magnetization fraction maps of different components, representing tissues such as myelin water, white matter, gray matter and cerebrospinal fluid. In the MS patients, an additional component was identified with increased transverse relaxation times compared to the white matter, likely representing changes in free water content. Patients with MS had a higher volume of the long- component in the white matter of the brain compared to healthy controls (B (95%-CI) = 0.004 (0.0006–0.008), p = 0.02). Furthermore, this MRF component had a moderate correlation (correlation coefficient R 0.47) with visible structural white matter changes on the FLAIR scans. Also, the component was found to be more extensive compared to structural white matter changes in 73% of MS patients. In conclusion, our MRF acquisition and analysis captured white matter tissue changes in MS patients compared to controls. In patients these tissue changes were more extensive compared to visually detectable white matter changes on FLAIR scans. Our method provides a novel way to quantify the extent of white matter changes in MS patients, which is underestimated using only conventional clinical MRI scans.</p

Reduced hippocampal subfield volumes and memory performance in preterm children with and without germinal matrix intraventricular hemorrhage.

Preterm newborns with germinal matrix-intraventricular hemorrhage (GM-IVH) are at a higher risk of evidencing neurodevelopmental alterations. Present study aimed to explore the long-term efects that GM-IVH have on hippocampal subfelds, and their correlates with memory. The sample consisted of 58 participants, including 36 preterm-born (16 with GM-IVH and 20 without neonatal brain injury), and 22 full-term children aged between 6 and 15 years old. All participants underwent a cognitive assessment and magnetic resonance imaging study. GM-IVH children evidenced lower scores in Full Intelligence Quotient and memory measures compared to their low-risk preterm and full-term peers. High-risk preterm children with GM-IVH evidenced signifcantly lower total hippocampal volumes bilaterally and hippocampal subfeld volumes compared to both low-risk preterm and full-term groups. Finally, signifcant positive correlations between memory and hippocampal subfeld volumes were only found in preterm participants together; memory and the right CA-feld correlation remained signifcant after Bonferroni correction was applied (p= .002). In conclusion, memory alterations and both global and regional volumetric reductions in the hippocampus were found to be specifcally related to a preterm sample with GM-IVH. Nevertheless, results also suggest that prematurity per se has a long-lasting impact on the association between the right CA-feld volume and memory during childhood

Removing the effects of the site in brain imaging machine-learning Measurement and extendable benchmark

Multisite machine-learning neuroimaging studies, such as those conducted by the ENIGMA Consortium, need to remove the differences between sites to avoid effects of the site (EoS) that may prevent or fraudulently help the creation of prediction models, leading to impoverished or inflated prediction accuracy. Unfortunately, we have shown earlier that current Methods Aiming to Remove the EoS (MAREoS, e.g., ComBat) cannot remove complex EoS (e.g., including interactions between regions). And complex EoS may bias the accuracy. To overcome this hurdle, groups worldwide are developing novel MAREoS. However, we cannot assess their effectiveness because EoS may either inflate or shrink the accuracy, and MAREoS may both remove the EoS and degrade the data. In this work, we propose a strategy to measure the effectiveness of a MAREoS in removing different types of EoS. FOR MAREOS DEVELOPERS, we provide two multisite MRI datasets with only simple true effects (i.e., detectable by most machine-learning algorithms) and two with only simple EoS (i.e., removable by most MAREoS). First, they should use these datasets to fit machine-learning algorithms after applying the MAREoS. Second, they should use the formulas we provide to calculate the relative accuracy change associated with the MAREoS in each dataset and derive an EoS-removal effectiveness statistic. We also offer similar datasets and formulas for complex true effects and EoS that include first-order interactions. FOR MACHINE-LEARNING RESEARCHERS, we provide an extendable benchmark website to show: a) the types of EoS they should remove for each given machine-learning algorithm and b) the effectiveness of each MAREoS for removing each type of EoS. Relevantly, a MAREoS only able to remove the simple EoS may suffice for simple machine-learning algorithms, whereas more complex algorithms need a MAREoS that can remove more complex EoS. For instance, ComBat removes all simple EoS as needed for predictions based on simple lasso algorithms, but it leaves residual complex EoS that may bias the predictions based on standard support vector machine algorithms

Applying multilayer analysis to morphological, structural, and functional brain networks to identify relevant dysfunction patterns

In recent years, research on network analysis applied to MRI data has advanced significantly. However, the majority of the studies are limited to single networks obtained from resting-state fMRI, diffusion MRI, or gray matter probability maps derived from T1 images. Although a limited number of previous studies have combined two of these networks, none have introduced a framework to combine morphological, structural, and functional brain connectivity networks. The aim of this study was to combine the morphological, structural, and functional information, thus defining a new multilayer network perspective. This has proved advantageous when jointly analyzing multiple types of relational data from the same objects simultaneously using graph- mining techniques. The main contribution of this research is the design, development, and validation of a framework that merges these three layers of information into one multilayer network that links and relates the integrity of white matter connections with gray matter probability maps and resting-state fMRI. To validate our framework, several metrics from graph theory are expanded and adapted to our specific domain characteristics. This proof of concept was applied to a cohort of people with multiple sclerosis, and results show that several brain regions with a synchronized connectivity deterioration could be identified