Desvendando os mecanismos fisiopatológicos das lesões da substância branca: um estudo sobre marcadores no líquido cefalorraquidiano e no sangue em pacientes com doença de pequenos vasos cerebrais

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e TecnologiaA doença de pequenos vasos cerebrais (DPV) é um grupo heterogéneo de distúrbios resultante de danos na estrutura ou função da microcirculação cerebral (pequenas artérias, arteríolas, capilares, pequenas veias e vênulas), afetando o fornecimento de sangue ao tecido da substância branca e cinzenta profundas. Como consequência da DPV formam-se lesões localizadas em estruturas subcorticais (como as hiperintensidades da substância branca) que se manifestam habitualmente sob a forma de eventos vasculares cerebrais recorrentes e um compromisso progressivo da cognição, do humor e da marcha. Além de um percurso clínico variável (alguns indivíduos apresentam sintomas logo nas fases iniciais da doença, enquanto outros permanecem assintomáticos mesmo na presença de uma grande carga lesional), a DPV compreende formas esporádicas e familiares associadas a múltiplos defeitos genéticos, podendo também estar associada a vários fatores de risco (idade, hipertensão, tabagismo e enxaquecas). Devido à escassez de estudos patológicos, os mecanismos subjacentes à DPV encontram-se ainda pouco esclarecido.O objetivo deste projeto foi contribuir para a compreensão dos mecanismos da DPV e identificar marcadores bioquímicos associados a diferentes fenótipos clínicos. O estudo incluiu doentes esporádicos não idosos com DPV (n = 63), com e sem hipertensão, e um pequeno grupo com formas hereditárias (n = 12), submetidos a uma avaliação clínica completa, que incluiu estudo neuropsicológico e de neuroimagem. Nestes doentes avaliámos um amplo painel de biomarcadores no líquido cefalorraquidiano (LCR) e soro que refletem: disfunção da barreira hematoencefálica (BHE) (metaloproteínase da matriz 9 – MMP-9; inibidor tecidual de metaloproteínase – TIMP-1; folato – 5-MTHF), disfunção endotelial (molecula de adesão intercelular – ICAM-1; adenosina deaminase 2 – ADA2), neuroinflamação (YKL-40), deposição amilóide (peptídeo beta amilóide com 42 e 40 resíduos – Aβ42 e Aβ40), neurodegeneração cortical (proteína Tau total – t-Tau; e fosforilada – p-Tau) e subcortical (neurofilamento de cadeia ligeira – NFL), tendo os resultados sido comparados com um grupo de controlos neurológicos (n = 12). Os nossos resultados não mostraram sinais de neurodegeneração cortical nos doentes com DPV esporádica, como evidenciado pelos níveis normais de t-Tau e p-Tau. Foi observado contudo um aumento dos níveis de MMP-9 e Aβ42 no LCR, refletindo provavelmente um aumento da permeabilidade da BHE e disfunção endotelial. Encontrámos também uma forte associação entre os níveis de NFL no LCR e os vários marcadores de disfunção da BHE. Os doentes do género masculinos e os doentes com hipertensão revelaram níveis mais elevados de NFL, ICAM-1, TIMP-1 e YKL-40 no LCR relativamente a doentes femininos e não hipertensos, respetivamente, o que poderá ser explicado pela rigidez arterial e stresse endotelial associado a estas condições. Foram encontradas associações entre o volume das lesões da substância branca e níveis elevados de NFL, YKL-40, TIMP-1, t-Tau e ICAM-1 no LCR de doentes com DPV esporádica. Além disso, observou-se uma correlação negativa entre o funcionamento cognitivo e o aumento de NFL, YKL-40 e t-Tau no LCR, e também entre a velocidade de processamento e os níveis de t-Tau. Contudo, não encontrámos associação entre os marcadores no LCR e a função executiva. Os resultados obtidos no pequeno grupo de casos de formas familiares de DPV mostraram uma grande heterogeneidade, mas parecem apontar para diferentes vias fisiopatológicas entre diferentes causas genéticas.Os nossos resultados apoiam a hipótese de que o dano da BHE e a disfunção endotelial estarão na génese da neuroinflamação e da perda axonal subcortical na DPV. O esclarecimento adicional do potencial prognóstico destes marcadores no LCR e sangue na avaliação da progressão da doença será importante tanto para o seguimento dos doentes como para a identificação de novos alvos para intervenção.Cerebral small vessel disease (SVD) refers to a group of conditions that results from damage to the structure or function of cerebral microcirculation (small arteries, arterioles, capillaries, small veins and venules), affecting blood supply and tissue of cerebral deep white and grey matter areas. Consequences of SVD are mainly lesions located in the subcortical structures (such as white matter hyperintensities) that represent an important cause of cognitive impairment, stroke, and also of other disabling symptoms such as gait disturbance and late-life depression. Apart from a highly variable clinical course (with some individuals being symptomatic at early stages while others have large radiological disease burdens and no apparent symptoms), SVD also encounters sporadic and familial forms with diverse underlying genetic defects and can be associated with several risk factors (age, hypertension, smoking and migraine). Owing to limited pathology studies, the underlying mechanism of SVD remains imprecise. The aim of this project was to gain a better understanding of the disease mechanisms in SVD and to identify biochemical markers associated with different clinical phenotypes and outcomes. We studied non-elderly sporadic SVD patients (n = 63), both with and without hypertension, as well as a small group with familial forms (n = 12), for whom a complete clinical evaluation, including imaging and neuropsychological/behavioral assessment was performed. A wide panel of cerebrospinal fluid (CSF)/blood markers assessing BBB disruption (matrix metalloproteinase 9 - MMP-9; tissue inhibitor of metalloproteinase 1 - TIMP-1; folate – 5-MTHF), endothelial dysfunction (intercellular adhesion molecule 1 - ICAM-1; adenosine deaminase 2 – ADA2), neuroinflammation (YKL-40), amyloid deposition (amyloid beta with 42 and 40 residues - Aβ42 and Aβ40), cortical (total and phosphorylated Tau protein - t-Tau and p-Tau) and subcortical (neurofilament light chain - NFL) neurodegeneration was assessed and compared to a small group of neurological controls (n = 12).Our results showed no noticeable signs of cortical neurodegeneration in sporadic SVD patients, as depicted by normal CSF t-Tau and p-Tau levels. An increased permeability of the BBB and endothelial dysfunction, reflected through an increase in both CSF MMP-9 and Aβ levels was found. Additionally, we found strong correlations between NFL and the several CSF biomarkers of BBB disruption. Both male and hypertensive patients showed increased NFL, ICAM-1, TIMP-1, YKL-40 in CSF compared to female and non-hypertensive patients, respectively, possibly explained by the related arterial stiffness and endothelial stress. An association between white matter lesions volume and increased levels of CSF NFL, YKL-40, TIMP-1, t-Tau and ICAM-1 in sporadic SVD patients was found. Moreover, a negative correlation between cognitive status and CSF NFL, YKL-40 and t-Tau was shown, as well as between speed of processing and t-Tau. However, no associations were found between CSF biomarkers and executive function. The results obtained in the small group of familial SVD cases showed a large heterogeneity between cases and seem to point towards different pathophysiological pathways between different genetic causes.These results support the notion that damage of the BBB and endothelial dysfunction are the damaging effects driving neuroinflammation and subcortical axonal loss in SVD. Further elucidation of the potential prognostic value of CSF/blood markers in assessing disease progression will be important both for patient management and for the identification of novel targets for intervention

The Road to Personalized Medicine in Alzheimer's Disease: The Use of Artificial Intelligence

Dementia remains an extremely prevalent syndrome among older people and represents a major cause of disability and dependency. Alzheimer's disease (AD) accounts for the majority of dementia cases and stands as the most common neurodegenerative disease. Since age is the major risk factor for AD, the increase in lifespan not only represents a rise in the prevalence but also adds complexity to the diagnosis. Moreover, the lack of disease-modifying therapies highlights another constraint. A shift from a curative to a preventive approach is imminent and we are moving towards the application of personalized medicine where we can shape the best clinical intervention for an individual patient at a given point. This new step in medicine requires the most recent tools and analysis of enormous amounts of data where the application of artificial intelligence (AI) plays a critical role on the depiction of disease-patient dynamics, crucial in reaching early/optimal diagnosis, monitoring and intervention. Predictive models and algorithms are the key elements in this innovative field. In this review, we present an overview of relevant topics regarding the application of AI in AD, detailing the algorithms and their applications in the fields of drug discovery, and biomarkers

Exploring the potential of fully automated LUMIPULSE G plasma assays for detecting Alzheimer’s disease pathology

Abstract Background LUMIPULSE G-automated immunoassays represent a widely used method for the quantification of Alzheimer’s disease (AD) biomarkers in the cerebrospinal fluid (CSF). Less invasive blood-based markers confer a promising tool for AD diagnosis at prodromal stages (mild cognitive impairment (MCI)). Highly sensitive assays for the quantification of amyloid-beta (Aβ) and phosphorylated Tau-181 (p-Tau181) in the blood are showing promising results. In this study, we evaluated the clinical performance of the recently available fully automated LUMIPULSE plasma marker assays for detecting brain AD pathology and for predicting progression from MCI to AD dementia stage. Methods A retrospective exploratory cohort of 138 individuals (22 neurological controls [NC], 72 MCI, and 44 AD dementia patients) was included. Data regarding baseline CSF concentrations of Aβ42, Aβ40, t-Tau, and p-Tau181 was available and used to establish the presence of AD brain pathology. Baseline Aβ42, Aβ40, and p-Tau181 concentrations were determined in stored plasma samples using high-throughput fully automated LUMIPULSE assays. Progression from MCI to AD dementia was evaluated during follow-up (mean 6.4 ± 2.5 years). Moreover, a prospective validation cohort of 72 individuals with memory complaints underwent AD biomarker quantification, closely mirroring typical clinical practice. This cohort aimed to confirm the study’s main findings. Results In the exploratory cohort, correlations between CSF and plasma were moderate for p-Tau181 (ρ = 0.61, p < 0.001) and weak for Aβ42/Aβ40 ratio (ρ = 0.39, p < 0.001). Plasma p-Tau181 and p-Tau181/Aβ42 concentrations were significantly increased while Aβ42/Aβ40 was significantly decreased (p < 0.001) in patients with AD dementia and prodromal AD, as well as in individuals with CSF abnormal amyloid concentrations (A +). Plasma p-Tau181 showed a robust performance in differentiating patients clinically diagnosed as AD (AUC = 0.89; 95% CI 0.83–0.94); A + vs. A − (AUC = 0.84, 95% CI 0.77–0.91) and also in predicting conversion to AD dementia in MCI patients (AUC = 0.89, 95% CI 0.81–0.96). When tested in the validation cohort, plasma p-Tau181 displayed 83.3% of the overall percentage of agreement according to amyloid status. Conclusions Our results show that the measurement of p-Tau181 in plasma has great potential as a non-invasive prognostic screening tool for implementation in a clinical setting

Clinical validation of the Lumipulse G cerebrospinal fluid assays for routine diagnosis of Alzheimer's disease

Background: Ongoing efforts within the Alzheimer’s disease (AD) field have focused on improving the intra- and inter-laboratory variability for cerebrospinal fluid (CSF) biomarkers. Fully automated assays offer the possibility to eliminate sample manipulation steps and are expected to contribute to this improvement. Recently, fully automated chemiluminescence enzyme immunoassays for the quantification of all four AD biomarkers in CSF became available. The aims of this study were to (i) evaluate the analytical performance of the Lumipulse G β-Amyloid 1-42 (restandardized to Certified Reference Materials), β-Amyloid 1-40, total Tau, and pTau 181 assays on the fully automated LUMIPULSE G600II; (ii) compare CSF biomarker results of the Lumipulse G assays with the established manual ELISA assays (INNOTEST®) from the same company (Fujirebio); and (iii) establish cut-off values and the clinical performance of the Lumipulse G assays for AD diagnosis. Methods: Intra- and inter-assay variation was assessed in CSF samples with low, medium, and high concentrations of each parameter. Method comparison and clinical evaluation were performed on 40 neurological controls (NC) and 80 patients with a diagnosis of probable AD supported by a follow-up ≥ 3 years and/or positive amyloid PET imaging. A small validation cohort of 10 NC and 20 AD patients was also included to validate the cut-off values obtained on the training cohort. Results: The maximal observed intra-assay and inter-assay coefficients of variation (CVs) were 3.25% and 5.50%, respectively. Method comparisons revealed correlation coefficients ranging from 0.89 (for Aβ40) to 0.98 (for t-Tau), with those for Aβ42 (0.93) and p-Tau (0.94) in-between. ROC curve analysis showed area under the curve values consistently above 0.85 for individual biomarkers other than Aβ40, and with the Aβ42/40, Aβ42/t-Tau, and Aβ42/ p-Tau ratios outperforming Aβ42. Validation of the cut-off values in the independent cohort showed a sensitivity ranging from 75 to 95% and a specificity of 100%. The overall percentage of agreement between Lumipulse and INNOTEST was very high (> 87.5%). Conclusions: The Lumipulse G assays show a very good analytical performance that makes them well-suited for CSF clinical routine measurements. The good clinical concordance between the Lumipulse G and INNOTEST assays facilitates the implementation of the new method in routine practice