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

Oxidative stress and mitochondrial dysfunction in Kindler syndrome

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease. [Methods]: Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.[Results]: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state. [Conclusions]: This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.FL was supported by grants from Instituto de Salud Carlos III (PI11/01225) and Comunidad de Madrid (S2010/BMD- 2359; SKINMODEL). MDR was supported by grants from the Science and Innovation Ministry of Spain SAF2010-16976), Comunidad de Madrid (S2010/BMD-2420; CELLCAM), GENEGRAFT - contract N° HEALTH-F2-2011-261392 and CIBERER ACCI 13-714/172.04. MG is supported in part by ERA-NET grant: E-Rare-2 (SpliceEB). EZ was in part supported by a fellowship from CIBERER and SAF2010-16976.Peer reviewe

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

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

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

Adaptability and reproducibility of a memory disruption rTMS protocol in the PharmaCog IMI European project

Transcranial magnetic stimulation (TMS) can interfere with cognitive processes, such as transiently impairing memory. As part of a multi-center European project, we investigated the adaptability and reproducibility of a previously published TMS memory interfering protocol in two centers using EEG or fMRI scenarios. Participants were invited to attend three experimental sessions on different days, with sham repetitive TMS (rTMS) applied on day 1 and real rTMS on days 2 and 3. Sixty-eight healthy young men were included. On each experimental day, volunteers were instructed to remember visual pictures while receiving neuronavigated rTMS trains (20 Hz, 900 ms) during picture encoding at the left dorsolateral prefrontal cortex (L-DLPFC) and the vertex. Mixed ANOVA model analyses were performed. rTMS to the L-DLPFC significantly disrupted recognition memory on experimental day 2. No differences were found between centers or between fMRI and EEG recordings. Subjects with lower baseline memory performances were more susceptible to TMS disruption. No stability of TMS-induced memory interference could be demonstrated on day 3. Our data suggests that adapted cognitive rTMS protocols can be implemented in multi-center studies incorporating standardized experimental procedures. However, our center and modality effects analyses lacked sufficient statistical power, hence highlighting the need to conduct further studies with larger samples. In addition, inter and intra-subject variability in response to TMS might limit its application in crossover or longitudinal studies

Neolit dövründə Cənubi Qafqazda keramika istehsalının yaranması və inkişafı

Aging is accompanied by a decline in memory and other brain functions. Physical exercise may mitigate this decline through the modulation of factors participating in the crosstalk between skeletal muscle and the brain, such as neurotrophins and oxidative stress parameters. We aimed to determine whether long term exercise training (35 ± 15 years) promotes memory maintenance in middle-aged men, and to characterize the changes in neurotrophic factors and lipid oxidation markers in peripheral blood samples in both middle-aged and young men. The neuropsychological analysis showed significant improvements in memory through the Free and Cued Immediate Recall tests, in the middle-aged trained individuals when compared to the sedentary ones. We found a significant decrease in the resting serum BDNF and plasma Cathepsin B (CTSB) levels in the trained groups at both middle and young ages. BDNF and CTSB levels were inversely correlated with weekly hours of exercise. We also found a significant decrease in plasma malondialdehyde, an index of lipid peroxidation, in middle-aged and young trained subjects. The positive impact of long-term exercise training by delaying the onset of physiological memory loss and the associated neurotrophic and redox peripheral modulation, suggests the effectiveness of exercise as preventive strategy against age-related memory loss and neurodegeneration.This work was supported by the following grants: Ajut Mario Sàlvia i Ferret 2014 de l’lnstitut d’Estudis Catalans “per incentivar la recerca en biomedicine i estil de vida”; PIE15/00013 from Instituto de Salut Carlos III and FEDER; SAF2016-75508 from the Spanish MINECO and FEDER; CB16/10/00435, CIBERFES; PROMETEOII2014/056 from “Conselleria de Sanitat de la Generalitat Valenciana” and EU Funded CM1001 and FRAILOMIC-HEALTH.2012.2.1.1-2 and ADVANTAGE-724099 Join Action (HP-JA) 3rd EU Health Programme