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[eng] INTRODUCTION: Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) appear as neurodegenerative disorders with a wide range of symptomatology that differs among patients, among which are different levels of cognitive impairment. For instance, mild cognitive impairment (MCI) in PD contributes to a specific clinical profile with a higher risk of developing dementia. Looking at PD and DLB together provides evidence of the existence of different subtypes within both diseases. In recent years, complex imaging techniques, such as magnetic resonance imaging (MRI), have been used to study pathologies of the brain. MRI measures can be used to better characterize the brain basis of PD and DLB symptomatology, such as MCI. The reconstruction of the whole-brain connectome is a complex approach that can help to describe the heterogeneous symptomatology in neurodegenerative disorders. In addition, MRI in combination with new data-driven methods, such as cluster analysis, has been used to group patients according to their similarities, which allows subtypes to be identified. Until now, most studies in PD have described subtypes based on clinical and neuropsychological data, and just a few have used MRI measures to identify subtypes with different brain patterns. As DLB research is still in a relatively early stage, no cluster analyses have been yet performed based on MRI data. OBJECTIVES AND HYPOTHESES: Given this context, the current Doctoral Thesis focuses on the heterogeneity that characterizes PD and DLB. The main objectives were to identify subtypes based on structural MRI measures in PD and DLB, as well as to characterize the structural brain connectivity of PD associated with MCI. We hypothesized that there would be PD subtypes with different patterns of grey and white matter alterations that would be associated with particular clinical and cognitive profiles. We also hypothesized that there would be DLB subtypes characterized by different grey matter (GM) patterns, and that these patterns would explain specific symptomatology of the disease and would be differentially associated to concomitant brain changes seen in cerebrovascular and Alzheimer’s diseases. Finally, we expected that PD-MCI would present a characteristic pattern of impaired structural connectivity. In order to examine and clarify these issues, the current Doctoral Thesis is presented as a compendium of three studies. METHODS: In Study 1, to identify subtypes in PD, we performed a hierarchical cluster analysis based on multimodal imaging using the Ward’s linkage method. We performed the analysis in a sample of 62 PD patients. GM volumes of cortical and subcortical brain regions as well as fractional anisotropy (FA) white matter (WM) measures were combined. Once the subtypes were identified, voxel-based morphometry (VBM) and tract-based spatial statistics (TBSS) analyses were carried out in order to compare the pattern of GM and WM of the PD subtypes to the 33 healthy control group. Demographical, clinical and neuropsychological data were used to characterize the subtypes. In Study 2, the sample consisted of 27 PD-MCI and 35 PD without MCI, as well as 51 healthy controls. In this study we applied threshold-free network-based statistics (TFNBS), a novel technique based on whole-brain probabilistic tractography data useful to study structural connectivity. We complemented the analysis with TBSS and graph theory analyses (global and local measures). Study 3 included 165 DLB subjects from the Mayo Clinic and 3 centres from the European DLB consortium (E-DLB). We performed a cluster analysis based on GM volumes using a random forest method, and characterized the subtypes based on GM volumes, clinical, demographical data as well as tau, β-amyloid and cerebrovascular biomarkers. Additionally, we characterized cognitive trajectories of the subtypes in a 3-year follow- up. RESULTS: In Study 1, we identified 3 PD subtypes which mainly differed in GM patterns, while WM involvement appeared to be more limited. PD1 (24%) was characterized by temporo-parieto-occipital GM atrophy and subcortical atrophy, as well as FA reductions mainly affecting fronto-occipital WM tracts. This subtype was the oldest and had the worse neuropsychological profile. The second subtype, PD2 (34%), was characterized by GM atrophy limited to frontal and temporal cortical regions, and a third subtype, PD3 (42%), with non-detectable GM atrophy or WM impairment, and preserved cognitive profile. In Study 2, we found that PD patients had fewer streamlines (NOS) compared with healthy controls. Structural connectivity impairments were present in PD with and without MCI. However, the pattern and degree of connectivity impairment were different. PD-MCI showed a higher number of abnormal connections, primarily involving those between deep GM structures and cortical regions and posterior cortico-cortical connections, mainly in the temporal and occipital regions. PD without MCI showed fewer altered connections, and unlike PD-MCI, they were mainly located in the bilateral prefrontal cortex. What is more, the logistic regression and ROC curve analysis showed that the decreased NOS in the impaired connections characteristic of PD-MCI, were able to discriminate between both PD groups with high accuracy. The TBSS analysis revealed that only PD-MCI had reduced FA values compared to controls. The graph theory analysis showed PD groups differed in local graph measures. In Study 3, three DLB subtypes with the same disease evolution were identified based on their GM volumes. The cortical predominant subtype (30%) was characterized by widespread reduced cortical GM, older age, worse cognition at baseline and faster cognitive decline over 3 years. The second subtype, the fronto-occipital subtype (46%), had lower GM volumes in frontal and occipital regions. Finally, the subcortical predominant subtype (24%) was characterized by the greatest GM volumes, and relatively low GM volumes in the basal ganglia, as they were the only brain regions where the 3 subtypes had equivalent GM volumes. The subcortical predominant subtype was also characterized by the highest frequency of cognitive fluctuations. CONCLUSIONS: Our overall findings support the existence of different PD and DLB subtypes that can be identified by means of cluster analyses based on MRI data, which are in turn associated with specific cognitive profiles, and that cognitive impairment in PD is also associated to a specific pattern of structural connectivity impairment. These results contribute to clarifying the basis of heterogeneity in DLB and PD and give further information about which characteristics could be considered biomarkers of worse prognosis, with the final aim of approaching a more personalized medicine., [cat] INTRODUCCIÓ: La malaltia de Parkinson i la Demència amb cossos de Lewy són malalties neurodegeneratives que es presenten amb una àmplia varietat de símptomes, els quals difereixen entre pacients. El deteriorament cognitiu lleu, per exemple, és un possible símptoma de la malaltia de Parkinson que contribueix en un perfil clínic concret amb un elevat risc de desenvolupar demència. Tot plegat aporta evidència de l’existència de diferents subtipus (grups) dins d’ambdues malalties. En els últims anys, s’han utilitzat tècniques d’imatge complexes, com la imatge per ressonància magnètica (IRM), per estudiar les malalties que afecten al cervell. Les diferents mesures obtingudes de la IRM, es poden utilitzar per caracteritzar les bases de la simptomatologia de la malaltia de Parkinson i de la Demència amb cossos de Lewy, com per exemple el deteriorament cognitiu lleu. La reconstrucció del connectoma de tot el cervell és una aproximació complexa que pot ajudar a descriure la simptomatologia heterogènia de les malalties neurodegeneratives, a més, la IRM en combinació amb noves tècniques guiades per les dades, com els anàlisis de clúster, s’han utilitzat per agrupar els pacient d’acord a les seves similituds, el que permet trobar subtipus de pacients. Fins ara, la majoria dels estudis en la malaltia de Parkinson han trobat subtipus basant les anàlisis en dades clíniques i neuropsicològiques, i només escassos treballs s’han basat en dades derivades d’IRM. En la Demència amb cossos de Lewy, degut a que la recerca es troba en fases més primerenques que no pas la recerca en la malaltia Parkinson, encara no s’ha realitzat cap anàlisis de clúster basat en dades d’IRM. OBJECTIUS I HIPÒTESIS: Donat aquest context, la Tesi Doctoral que aquí es presenta es centra en l’heterogeneïtat que caracteritza la malaltia de Parkinson i a la Demència amb cossos de Lewy. Els objectius principals han estat identificar subtipus basats en dades d’IRM en Parkinson i Demència amb cossos de Lewy, així com caracteritzar la connectivitat cerebral a nivell estructural del deteriorament cognitiu lleu en la malaltia de Parkinson. Hem hipotetitzat que hi haurien subtipus en la malaltia de Parkinson que presentarien diferents patrons d’alteració de la substància grisa i la substància blanca que s’associarien amb un perfil clínic i cognitiu específic. En aquesta línia també hem hipotetitzat que hi haurien subtipus dins de la Demència amb cossos de Lewy que es caracteritzarien per diferents patrons d’alteració de la substància grisa, i que aquests patrons, podran explicar diferències en la simptomatologia de la malaltia i es trobaran diferencialment associats a marcadors biològics d’altres processos degeneratius associats a l’envelliment, com els canvis que es troben en processos cerebrovasculars i en la malaltia d’Alzheimer. Finalment, esperaríem que el deteriorament cognitiu lleu en la malaltia de Parkinson presentés un patró característic de connectivitat estructural alterada. Amb la intenció de donar resposta a aquestes qüestions, l’actual Tesi Doctoral es presenta com un compendi de 3 estudis. MÈTODES: En l’estudi 1, per tal d’identificar subtipus en la malaltia de Parkinson, es va dur a terme un anàlisis de clúster jeràrquic aglomeratiu, utilitzant dades multimodals d’IRM. L’anàlisi es va dur a terme en una mostra de 62 pacients i es van combinar volums de substància grisa de regions corticals i subcorticals amb mesures d’anisotropia fraccional de la substància blanca. Un cop identificats els subtipus, es va dur a terme una morfometria basada en vòxels (voxel-based morphometry (VBM), en anglès) i una estadística espacial basada en tractes (Tract-based spatial statistics (TBSS), en anglès) per tal de comparar els patrons de substància grisa i blanca amb un grup de 33 controls sans. A més, vam utilitzar dades demogràfiques, clíniques i neuropsicològiques per caracteritzar els subtipus. A l’estudi 2, la mostra consistia en 27 pacients amb malaltia de Parkinson i deteriorament cognitiu lleu i 35 pacients sense deteriorament cognitiu, així com de 51 controls sans. En aquest estudi vam aplicar una tècnica nova que en anglès rep el nom de threshold-free network-based statistics (TFNBS), aquesta tècnica resulta útil per estudiar la connectivitat estructural i la vam utilitzar amb dades de tractografia probabilística de tot el cervell. A més, vam complementar l’anàlisi amb TBSS i mesures globals i locals de graf. A l’estudi 3, vam incloure 165 pacients amb Demència amb cossos de Lewy que procedien de la Clínica Mayo i de 3 centres que formen part del consorci europeu de Demència amb cossos de Lewy, abreviat com a E-DLB. Vam realitzar un anàlisi de clúster basat en random forest en el que hi vam introduir volums de substància grisa de regions corticals i subcorticals de tot el cervell. En conseqüència, els subtipus els vam caracteritzar en base als volums de substància grisa, però també en base a dades clíniques, demogràfiques i de biomarcadors de tau, β-amiloide i malaltia cerebrovascular. A més, vam caracteritzar les trajectòries cognitives dels subtipus amb un seguiment de 3 anys. RESULTATS: A l’estudi 1, vam definir 3 subtipus en la malaltia de Parkinson que principalment diferien en substància grisa, mentre que la implicació de la substància blanca era molt baixa. El primer subtipus (PD1, 24%) es caracteritzava per presentar atròfia de la substància grisa a nivell de còrtex temporal, parietal i occipital així com en regions subcorticals. A més, presentava reduccions de l’anisotropia fraccional principalment en tractes fronto-occipitals de substància blanca. Aquest subtipus, el PD1, incloïa als pacients de més edat amb el pitjor perfil neuropsicològic. El segon subtipus (PD2, 34%) es caracteritzava per atròfia de la substància grisa en regions frontals i temporals. El tercer subtipus (PD3, 42%), no presentava alteracions detectables ni a nivell de substància grisa ni de blanca, i presentava un perfil cognitiu preservat. En l’estudi 2, vam trobar que els pacients amb malaltia de Parkinson presentaven menor número de fibres en nombroses connexions cerebrals en comparació amb controls sans. Es van trobar alteracions en la connectivitat estructural tant en pacients amb deteriorament cognitiu lleu com sense deteriorament cognitiu, tot i així, el patró i grau de les alteracions en connectivitat estructural eren diferents entre grups de pacients. Els pacients amb deteriorament cognitiu lleu presentaven major número de connexions alterades, les quals principalment involucraven connexions corticals en regions temporals i occipitals així com connexions entre l’escorça cerebral i els nuclis grisos de la base. Els pacients amb malaltia de Parkinson però sense deteriorament cognitiu, presentaven menys connexions alterades que, a diferència dels pacients amb deteriorament cognitiu lleu, es localitzaven principalment en regions bilaterals del còrtex prefrontal. A més, la regressió logística i la corba ROC van mostrar que el número reduït de fibres en les connexions alterades que caracteritzaven el grup de pacients amb deteriorament cognitiu lleu, permetien la discriminació entre els dos grups de pacients amb una precisió elevada. L’anàlisi de TBSS va mostrar que només el pacients amb deteriorament cognitiu lleu presentaven reduccions en l’anisotropia fraccional en comparació al grup de controls sans, i l’anàlisi de graf va mostrar que els 2 grups de pacients diferien en mesures locals. En l’estudi 3, es van identificar 3 subtipus dins de la Demència amb cossos de Lewy en base als volums de substància grisa. El subtipus amb predominança cortical (30%) es caracteritzava per una reducció generalitzada dels volums corticals de substància grisa, incloïa els pacients de més edat, pitjor cognició global i deteriorament cognitiu més ràpid en el transcurs de 3 anys. El segon subtipus, el fronto-occipital (46%), presentava menors volums de substància grisa en regions frontals i occipitals. Finalment, el subtipus amb predominança subcortical (24%), es caracteritzava per majors volums de substància grisa en regions corticals i volums relativament baixos en estructures subcorticals, donat que eren les úniques estructures en les que no hi havia diferències entre els 3 subtipus. Aquest últim subtipus amb predominança subcortical, també es caracteritzava per una major freqüència de pacients amb fluctuacions cognitives. CONCLUSIONS: En conjunt, les troballes presentades en aquesta Tesi Doctoral donen suport a l’existència de diferents subtipus, tant en la malaltia de Parkinson com en la Demència amb cossos de Lewy, que poden ser identificats mitjançant anàlisis de clúster basats en dades d’IMR. A més, aquests subtipus reflecteixen perfils cognitius específics. El deteriorament cognitiu en la malaltia de Parkinson també es troba associat a un patró concret d’alteracions en connectivitat estructural. Així, aquests resultats contribueixen a aclarir les bases de l’heterogeneïtat descrita en la malaltia de Parkinson i en la Demència amb cossos de Lewy, i aporten informació rellevant sobre quines característiques podrien ser considerades com biomarcadors de pitjor pronòstic, amb l’objectiu final d’apropar-nos a una medicina més personalitzada.

The mechanism of nigral dopaminergic neuronal degeneration in Parkinson’s disease (PD) is unknown. One of the pathological characteristics of the disease is the deposition of α-synuclein (α-syn) that occurs in the brain from both familial and sporadic PD patients. This paper constitutes a narrative review that takes advantage of information related to genes (SNCA, LRRK2, GBA, UCHL1, VPS35, PRKN, PINK1, ATP13A2, PLA2G6, DNAJC6, SYNJ1, DJ-1/PARK7 and FBXO7) involved in familial cases of Parkinson’s disease (PD) to explore their usefulness in deciphering the origin of dopaminergic denervation in many types of PD. Direct or functional interactions between genes or gene products are evaluated using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The rationale is to propose a map of the interactions between SNCA, the gene encoding for α-syn that aggregates in PD, and other genes, the mutations of which lead to early-onset PD. The map contrasts with the findings obtained using animal models that are the knockout of one of those genes or that express the mutated human gene. From combining in silico data from STRING-based assays with in vitro and in vivo data in transgenic animals, two likely mechanisms appeared: (i) the processing of native α-syn is altered due to the mutation of genes involved in vesicular trafficking and protein processing, or (ii) α-syn mutants alter the mechanisms necessary for the correct vesicular trafficking and protein processing. Mitochondria are a common denominator since both mechanisms require extra energy production, and the energy for the survival of neurons is obtained mainly from the complete oxidation of glucose. Dopamine itself can result in an additional burden to the mitochondria of dopaminergic neurons because its handling produces free radicals. Drugs acting on G protein-coupled receptors (GPCRs) in the mitochondria of neurons may hopefully end up targeting those receptors to reduce oxidative burden and increase mitochondrial performance. In summary, the analysis of the data of genes related to familial PD provides relevant information on the etiology of sporadic cases and might suggest new therapeutic approaches.

Paciente de 61 años con demencia con cuerpos de Lewy, que acude derivado por el servicio de neurología por posible deterioro cognitivo. Se trata de un paciente ficticio basado en mi experiencia durante la realización de las prácticas del máster. La demencia con cuerpos de Lewy es una enfermedad neurodegenerativa de inicio subagudo y curso evolutivo fluctuante que se desarrolla en la edad adulta. Es la segunda causa de demencia degenerativa más frecuente después de la enfermedad de Alzheimer. Los resultados de la evaluación revelan alteraciones en las praxias visuoconstructivas, en el de funcionamiento ejecutivo, en las funciones visuoperceptivas y visuoespaciales, y síntomas leves de ansiedad, depresión, apatía e irritabilidad. Así mismo se observan alteraciones en la atención sostenida, selectiva y alternante, la memoria de trabajo y la memoria visual, aunque estos resultados deben ser tomados con prudencia. El objetivo de la intervención es mejorar o mantener el funcionamiento cognitivo, la independencia y calidad de vida el mayor tiempo posible. Se realizarán 2 sesiones semanales de 60 durante 3 meses, donde se trabajarán tanto las funciones cognitivas alteradas como preservadas, así como los problemas emocionales y conductuales observados. Además, se trabajará también con la familia. 61-year-old patient with dementia with Lewy bodies, referred by the neurology department for possible cognitive impairment. This is a fictitious patient based on my experience during my master's internship. Dementia with Lewy bodies is a neurodegenerative disease with subacute onset and fluctuating course that develops in adulthood. It is the second most common cause of degenerative dementia after Alzheimer's disease. The results of the evaluation reveal alterations in visuoconstructive praxias, executive functioning, visuoperceptive and visuospatial functions, and mild symptoms of anxiety, depression, apathy and irritability. Alterations in sustained, selective and alternating attention, working memory and visual memory are also observed, although these results should be taken with caution. The aim of the intervention is to improve or maintain cognitive functioning, independence and quality of life as long as possible. There will be 2 weekly sessions of 60' during 3 months, where both altered and preserved cognitive functions will be worked on, as well as the emotional and behavioral problems observed. In addition, we will also work with the family. Pacient de 61 anys amb demència amb cossos de Lewy, que acudeix derivat pel servei de neurologia per possible deteriorament cognitiu. Es tracta d'un pacient fictici basat en la meva experiència durant la realització de les pràctiques de l'màster. La demència amb cossos de Lewy és una malaltia neurodegenerativa d'inici subagut i curs evolutiu fluctuant que es desenvolupa en l'edat adulta. És la segona causa de demència degenerativa més freqüent després de la malaltia d'Alzheimer. Els resultats de l'avaluació revelen alteracions en les pràxies visuoconstructives, al de funcionament executiu, en les funcions visuoperceptives i visuoespacials, i símptomes lleus d'ansietat, depressió, apatia i irritabilitat. Així mateix s'observen alteracions en l'atenció sostinguda, selectiva i alternant, la memòria de treball i la memòria visual, encara que aquests resultats s'han de prendre amb prudència. L'objectiu de la intervenció és millorar o mantenir el funcionament cognitiu, la independència i qualitat de vida el major temps possible. Es realitzaran 2 sessions setmanals de 60 durant 3 mesos, on es treballaran tant les funcions cognitives alterades com preservades, així com els problemes emocionals i conductuals observats. A més, es treballarà també amb la família.

Lewy body diseases (LBD) including dementia with Lewy bodies (DLB) and Parkinson disease (PD) are characterized by alpha-synuclein pathology. DLB is difficult to diagnose and peripheral biomarkers are urgently needed. Therefore, we analyzed the expression of five alpha-synuclein gene (SNCA) transcripts, SNCAtv1, SNCAtv2, SNCAtv3, SNCA126, and SNCA112, in 45 LBD and control temporal cortex samples and in the blood of 72 DLB, 59 PD, and 54 control subjects. The results revealed overexpression of SNCAtv1 and SNCA112 in DLB, and SNCAtv2 in PD temporal cortices. In DLB blood, diminution of all SNCA transcripts was observed. SNCAtv1 and SNCAtv2 were diminished in PD with disease onset before 70 years. SNCAtv3, driven by its own promoter, showed opposite expression in early DLB and PD, suggesting that its amount may be an early, DLB specific biomarker. Correlation between blood transcript levels and disease duration was positive in DLB and negative in PD, possibly reflecting differences in brain alpha-synuclein aggregation rates associated with differences in disease courses. In conclusion, SNCA transcripts showed a disease-specific increase in the brain and were diminished in blood of LBD patients. SNCAtv3 expression was decreased in early DLB and increased in early PD and could be a biomarker for early DLB diagnosis.

Dementia with Lewy bodies (DLB) is a clinically heterogeneous disorder with a substantial burden on healthcare. Despite this, the genetic basis of the disorder is not well defined and its boundaries with other neurodegenerative diseases are unclear. Here, we performed whole exome sequencing of a cohort of 1118 Caucasian DLB patients, and focused on genes causative of monogenic neurodegenerative diseases. We analyzed variants in 60 genes implicated in DLB, Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, and atypical parkinsonian or dementia disorders, in order to determine their frequency in DLB. We focused on variants that have previously been reported as pathogenic, and also describe variants reported as pathogenic which remain of unknown clinical significance, as well as variants associated with strong risk. Rare missense variants of unknown significance were found in APP, CHCHD2, DCTN1, GRN, MAPT, NOTCH3, SQSTM1, TBK1 and TIA1. Additionally, we identified a pathogenic GRN p.Arg493* mutation, potentially adding to the diversity of phenotypes associated with this mutation. The rarity of previously reported pathogenic mutations in this cohort suggests that the genetic overlap of other neurodegenerative diseases with DLB is not substantial. Since it is now clear that genetics plays a role in DLB, these data suggest that other genetic loci play a role in this disease.

Background: α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of α- synucleinopathies, such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB). Main body: Understanding of the molecular basis as well as the factors or conditions promoting α-synuclein misfolding and aggregation is an important step towards the comprehension of pathological mechanism of α- synucleinopathies and for the development of efficient therapeutic strategies. Based on the conversion and aggregation mechanism of α-synuclein, novel diagnostic tests, such as protein misfolding seeded conversion assays, e.g. the real-time quaking-induced conversion (RT-QuIC), had been developed. In diagnostics, α-synuclein RT-QuIC exhibits a specificity between 82 and 100% while the sensitivity varies between 70 and 100% among different laboratories. In addition, the α-synuclein RT-QuIC can be used to study the α-synuclein-seeding-characteristics of different α-synucleinopathies and to differentiate between DLB and PD. Conclusion: The variable diagnostic accuracy of current α-synuclein RT-QuIC occurs due to different protocols, cohorts and material etc.. An impact of micro-environmental factors on the α-synuclein aggregation and conversion process and the occurrence and detection of differential misfolded α-synuclein types or strains might underpin the clinical heterogeneity of α-synucleinopathies.

Altres ajuts: This work was supported by the Marató TV3 grant 1405/10. Altres ajuts: MSCBS/PI12-1702 Altres ajuts: MSCBS/PI15-216 Parkinson disease (PD) and dementia with Lewy bodies (DLB) are Lewy body diseases characterized by abnormal alpha-synuclein deposits and overlapping pathological features in the brain. Several studies have shown that glucocerebrosidase (GBA) deficiency is involved in the development of LB diseases. Here, we aimed to find out if this deficiency starts at the transcriptional level, also involves alternative splicing, and if GBA expression changes in brain are also detectable in blood of patients with LB diseases. The expression of three GBA transcript variants (GBAtv1, GBAtv2 and GBAtv5) was analyzed in samples from 20 DLB, 25 PD and 17 control brains and in blood of 20 DLB, 26 PD patients and 17 unaffected individuals. Relative mRNA expression was determined by real-time PCR. Expression changes were evaluated by the ΔΔCt method. In brain, specific expression profiles were identified in the temporal cortex of DLB and in the caudate nucleus of PD. In blood, significant GBA mRNA diminution was found in both DLB and PD patients. Early PD and early-onset DLB patients showed lowest GBA levels which were normal in PD patients with advanced disease and DLB patients who developed disease after 70 years of age. In conclusion, disease group specific GBA expression profiles were found in mostly affected areas of LBD. In blood, GBA expression was diminished in LB diseases, especially in patients with early onset DLB and in patients with early PD. Age of disease onset exerts an opposite effect on GBA expression in DLB and PD.

Objectives: The goal of this study was to assess mitochondrial function, energy and purine metabolism, protein synthesis machinery from the nucleolus to the ribosome, inflammation, and expression of newly identified ectopic olfactory and taste receptors in the frontal cortex of typical cases of Dementia with Lewy Bodies (DLB) and cases with rapid clinical course (rpDLB: 2 years or less) compared with middle-aged non-affected individuals, in order to learn about the biochemical abnormalities underlying Lewy body pathology. Methods: RT-qPCR, mitochondrial enzymatic assays, and analysis of β-amyloid, tau and synuclein species were used. Results: The main alterations in DLB and rpDLB, which are more marked in the rapidly progressive forms, include: i) deregulated expression of several mRNAs and proteins of mitochondrial subunits, and reduced activity of complexes I, II, III and IV of the mitochondrial respiratory chain; ii) reduced expression of selected molecules involved in energy metabolism and increased expression of enzymes involved in purine metabolism; iii) abnormal expression of nucleolar proteins, rRNA18S, genes encoding ribosomal proteins and initiation factors of the transcription at the ribosome; iv) discrete inflammation; and v) marked deregulation of brain olfactory and taste receptors (ORs and TASRs, respectively). Severe mitochondrial dysfunction involving activity of four complexes, minimal inflammatory responses, and dramatic altered expression of ORs and TASRs discriminate DLB from Alzheimer’s disease. Altered solubility and aggregation of α-synuclein, increased β-amyloid bound to membranes and absence of soluble tau oligomers are common in DLB and rpDLB. Low levels of soluble β-amyloid are found in DLB. However, increased soluble β-amyloid 1-40 and β-amyloid 1-42, and increased TNFα mRNA and protein expression, distinguish rpDLB. Conclusions: Molecular alterations in frontal cortex in DLB involve key biochemical pathways such as mitochondria and energy metabolism, protein synthesis, purine metabolism, among others and are accompanied by discrete innate inflammatory response.Rapid DLB can be distinguished from DLB by increased levels of β-amyloid species and TNF-α mRNA brain expression, being both factors harmful to nerve cells

Graphical abstract, Highlights • Sensitive detection of total iron content from cells, rodent and human brain extracts. • There are regional differences in iron concentrations in rodent and human brain. • Iron dyshomeostasis in the substantia nigra correlates with Lewy body disease. • Iron imaging studies may aid in clinical diagnosis and/or serve as biomarker for disease progression., Iron levels in the healthy human brain are known to be high in certain areas such as the substantia nigra (SN), and increase further with age. In addition, there is some evidence for a further increase in iron load in the SN of Parkinsońs disease (PD) patients as compared to controls, which correlates with motor disability. Here, we have analyzed total iron levels in cells as well as mouse and human brain samples by atomic absorption spectroscopy (AAS). Our data indicate that iron load is more pronounced in cells with dopaminergic features. Moreover, region-specific differences in iron load reflecting those in the human brain were detected in rodent brains as well. Whilst altered iron load was not observed in other regions also affected in PD patients, we report a significant increase in iron load in the SN of Lewy body disease patients as compared to Alzheimeŕs disease (AD) patients or controls, which correlates with neurodegeneration in this brain area.

Lipid rafts are highly dynamic membrane microdomains intimately associated with cell signaling. Compelling evidence has demonstrated that alterations in lipid rafts are associated with neurodegenerative diseases such Alzheimer's disease, but at present, whether alterations in lipid raft microdomains occur in other types of dementia such dementia with Lewy bodies (DLB) remains unknown. Our analyses reveal that lipid rafts from DLB exhibit aberrant lipid profiles including low levels of n-3 long-chain polyunsaturated fatty acids (mainly docosahexaenoic acid), plasmalogens and cholesterol, and reduced unsaturation and peroxidability indexes. As a consequence, lipid raft resident proteins holding principal factors of the β-amyloidogenic pathway, including β-amyloid precursor protein, presenilin 1, β-secretase, and PrP, are redistributed between lipid rafts and nonraft domains in DLB frontal cortex. Meta-analysis discloses certain similarities in the altered composition of lipid rafts between DLB and Parkinson's disease which are in line with the spectrum of Lewy body diseases. In addition, redistribution of proteins linked to the β-amyloidogenic pathway in DLB can facilitate generation of β-amyloid, thus providing mechanistic clues to the intriguing convergence of Alzheimer's disease pathology, particularly β-amyloid deposition, in DLB.

[eng] Parkinson's disease (PD) is a neurodegenerative disorder characterized by movement impairment, or parkinsonism, for which there is still no cure. The manifest clinical signs result from neuronal loss of more than 60% in the substantia nigra pars compacta. Cognitive disorders and dementia in PD usually occur, thus leading to Parkinson disease with dementia (PDD). Moreover, Dementia with Lewy bodies (DLB) is also considered a neurodegenerative disease and one of the most common causes of dementia, with cognitive impairment symptoms similar to Alzheimer-type dementia, and with parkinsonism. Its onset is insidious and is characterized by a slow progression in comparison with its fast form, also known as Dementia with Lewy bodies rapidly progressive (rpDCL), which appears suddenly and progresses quickly. In these pathologies there occurs a neural degeneration not only related to the accumulation of altered proteins, but more likely as a result of multiple deleterious factors. The hypothesis of this work is that the identification of molecular changes analyzed through the application of "-omics" techniques will be useful to obtain information about a risk transcriptome/proteome in the aforementioned diseases. Thus, the main objective of the present thesis is the identification of molecular alterations underlying functional cerebral changes and anatomical modifications in different brain regions and different Braak stages of PD, as well as DCL and DCLrp, with the use of post-mortem human brain samples compared with controls, combining microarray, mRNA, protein and enzyme assays studies. The obtained results have identified molecular alterations in PD, DLB, and rpDLB of different metabolic pathways including changes in the machinery of protein synthesis, in the mitochondrial energy metabolism, in neuroinflammation, in the purine pathway, and in new signaling pathways comprising olfactory and taste receptors paths., [spa] La enfermedad de Parkinson (EP) es una patología neurodegenerativa perteneciente al grupo de afecciones conocidas como trastornos del movimiento, o parkinsonismo, para la cual actualmente no existe cura. Los signos clínicos que manifiesta son resultado de una pérdida neuronal superior al 60% en el área cerebral más afectada, la sustancia nigra pars compacta. Asimismo, la aparición de demencia y los desórdenes cognitivos en la EP conducen a una EP con demencia. A su vez, la Demencia con cuerpos de Lewy (DCL) es también una enfermedad neurodegenerativa considerada como una de las causas más comunes de demencia, con una sintomatología de deterioro cognitivo similar a la observable en la demencia de tipo Alzheimer y con la aparición de síntomas de parkinsonismo. Su aparición es insidiosa y se caracteriza por presentar una progresión lenta, a diferencia de su forma rápida también conocida como Demencia con cuerpos de Lewy rápidamente progresiva (DCLrp), que aparece de forma súbita y evoluciona vertiginosamente. En todas estas enfermedades se produce una degeneración neural debida no únicamente a la acumulación de proteínas alteradas, sino más probablemente consecuencia de múltiples factores deletéreos convergentes. La hipótesis de este trabajo es considerar que la identificación de cambios moleculares analizados gracias a la aplicación de métodos “-ómicos” servirá para obtener información sobre un trascriptoma/proteoma de riesgo en las anteriormente citadas enfermedades. El principal objetivo abordado en la presente tesis es la identificación de las alteraciones moleculares subyacentes a los cambios cerebrales funcionales y anatómicos presentes en diferentes regiones cerebrales y en distintos estadiajes de Braak de la EP, así como en la DCL y en la DCLrp, por medio del uso de muestras de cerebro humano post-mortem comparando con controles, combinando estudios de microarrays, mRNA, proteínas y ensayos enzimáticos. Los resultados obtenidos por medio de métodos de transcriptómica con su posterior validación y ampliación a proteómica han permitido identificar alteraciones moleculares en la EP, DCL y DCLrp de distintas vías metabólicas incluyendo cambios en la maquinaria de síntesis de proteínas, en el metabolismo mitocondrial y energético, en la neuroinflamación, en la vía de las purinas y en nuevas vías de señalización comprendiendo las vías de receptores olfatorios y gustativos.

[eng] Mitochondria dysfunction is documented in the substantia nigra in Parkinson's disease (PD), a region which plays a central role in the characteristic motor manifestations of the disease. Mitochondria! dysfunction is a cause of neuron energy exhaustion, oxidative stress and dopaminergic cell death. However, little is known about mitochondria! dysfunction in other brain regions in PD, the impairment of which is causative of other symptoms such as cognitive impairment and, in some cases, dementia. The present study was undertaken to analyse mitochondria! function in the frontal cortex area 8 and angular gyrus. On the other hand, protein synthesis has also been shown to play role in neurodegenerative diseases which leads to neuronal atrophy. It is reported that significant percentage of protein synthesis results in the generation of defective ribosomal products, occurring as the result of faulty coding/or transfer within the ribosomal complex. The present study was undertaken to analyse impairment of ribosomal subunits in substantia nigra, frontal cortex area 8, angular gyrus, and precuneus. The frozen human post-mortem brain samples obtained following generous donation after informed consent, and stored at the Institute of Neuropathology Brain Bank (a branch of the HUB-ICO-IDIBELL brain bank). The study contemplates different stages of disease progression, according to the findings of the neuropathological study, and includes expression levels of mRNA and protein of selected subunits of the mitochondria! complexes and ribosomal subunits, and also enzymatic activities of each one of these complexes compared to those seen in age-matched controls processed in parallel. Results show altered activity of selected complexes of the respiratory chain thus supporting the concept that mitochondria! alterations in PD are not restricted to the substantia nigra but they rather represent a more widespread region-dependent alteration in PD. On the other hand the results of ribosomal proteins show disease-dependent differences in ribosomal protein gene expression.

Importance Mutations in leucine-rich repeat kinase 2 ( LRRK2 ) are the most common cause of genetic Parkinson disease (PD) known to date. The clinical features of manifesting LRRK2 mutation carriers are generally indistinguishable from those of patients with sporadic PD. However, some PD cases associated with LRRK2 mutations lack Lewy bodies (LBs), a neuropathological hallmark of PD. We investigated whether the presence or absence of LBs correlates with different clinical features in LRRK2 -related PD. Observations We describe genetic, clinical, and neuropathological findings of 37 cases of LRRK2 -related PD including 33 published and 4 unpublished cases through October 2013. Among the different mutations, the LRRK2 p.G2019S mutation was most frequently associated with LB pathology. Nonmotor features of cognitive impairment/dementia, anxiety, and orthostatic hypotension were correlated with the presence of LBs. In contrast, a primarily motor phenotype was associated with a lack of LBs. Conclusions and Relevance To our knowledge, this is the first report of clinicopathological correlations in a series of LRRK2 -related PD cases. Findings from this selected group of patients with PD demonstrated that parkinsonian motor features can occur in the absence of LBs. However, LB pathology in LRRK2 -related PD may be a marker for a broader parkinsonian symptom complex including cognitive impairment.

ObjectiveTo estimate the risk for developing a defined neurodegenerative syndrome in a large cohort of idiopathic REM sleep behavior disorder (IRBD) patients with long follow-up.MethodsUsing the Kaplan-Meier method, we estimated the disease-free survival rate from defined neurodegenerative syndromes in all the consecutive IRBD patients diagnosed and followed-up in our tertiary referal sleep center between November 1991 and July 2013.ResultsThe cohort comprises 174 patients with a median age at diagnosis of IRBD of 69 years and a median follow-up of four years. The risk of a defined neurodegenerative syndrome from the time of IRBD diagnosis was 33.1% at five years, 75.7% at ten years, and 90.9% at 14 years. The median conversion time was 7.5 years. Emerging diagnoses (37.4%) were dementia with Lewy bodies (DLB) in 29 subjects, Parkinson disease (PD) in 22, multiple system atrophy (MSA) in two, and mild cognitive impairment (MCI) in 12. In six cases, in whom postmortem was performed, neuropathological examination disclosed neuronal loss and widespread Lewy-type pathology in the brain in each case.ConclusionsIn a large IRBD cohort diagnosed in a tertiary referal sleep center, prolonged follow-up indicated that the majority of patients are eventually diagnosed with the synucleinopathies PD, DLB and less frequently MSA. IRBD represented the prodromal period of these conditions. Our findings in IRBD have important implications in clinical practice, in the investigation of the early pathological events occurring in the synucleinopathies, and for the design of interventions with potential disease-modifying agents.

Previous studies have demonstrated the presence of hemoglobin α-chain and β-chain in neurons of the rodent and human brain thus indicating that hemoglobin is a normal component of nerve cells and that hemoglobin may play a role in intraneuronal oxygen homeostasis. Progressing with these studies, hemoglobin expression has been examined in selected cell population in the brains of Alzheimer's disease (AD), argyrophilic grain disease (AGD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Double labeling immunofluorescence and confocal microscopy revealed reduced hemoglobin α-chain and β-chain in practically all neurons with small amounts of granular or punctuate hyperphosphorylated tau deposits and in neurons with tangles in the hippocampus and frontal cortex in AD and in the hippocampus in AGD; in ballooned neurons containing αB-crystallin in the amygdala in AD and AGD; and in about 80% of neurons with punctuate α-synuclein deposits and in neurons with Lewy bodies in the substantia nigra pars compacta and in vulnerable neurons of the medulla oblongata in PD and DLB; and in neurons with Lewy bodies in the frontal cortex in DLB. Hemoglobin immunoreactivity was also observed in the core of neuritic plaques and in diffuse plaques, but not in dystrophic neurites. Loss of hemoglobin was specific as neuroglobin was present equally in neurons with and without abnormal protein inclusions, and erythropoietin receptor was expressed equally in neurons without and in neurons with abnormal protein aggregates in AD, AGD, PD, and DLB.

[cat] L'objectiu general d'aquest treball és el d'aprofundir en l'estudi de les sinucleïnopaties, principalment a nivell bioquímic, en les interaccions de l'alfa-sinucleïna amb altres proteïnes del cervell humà per tal d'estudiar l'efecte de la unió en aquestes patologies. També hem volgut comprovar la similitud de les sinucleïnopaties humanes amb un model animal transgènic per a la forma humana mutada de l'alfa-sinucleïna. D'altra banda i també relacionat amb aquestes neurodegenarions, s'ha estudiat de quina manera resultaven afectats diferents marcadors de l'estrès oxidatiu, que se sap juga un important paper en el desenvolupament d'aquestes patologies. Mitjançant estudis d'immunoprecipitació i de pull-down en cervell humà s'ha trobat interacció de l'alfa-sinucleïna amb les proteïnes sinàptiques rab3a i rabfilina a córtex de la sinucleïnopatia anomenada Demència amb cossos de Lewy (DLBD), i no en cervells control. Donada la interacció anòmala amb aquestes proteïnes sinàptiques podem afirmar que a DLBDs la transmissió sinàptica es troba alterada, i una de les causes és la interacció anòmala de l'alfa-sinucleïna amb rab3a i rabfilina. Aquest tipus d'interacció també s'ha estudiat a l'MSA o Atròfia Sistèmica Múltiple, una sinucleïnopatia que té afectat sobretot el cerebel, i no el córtex, com les DLBDs. Doncs, tot i estar classificada com a sinucleïnopatia a part, hem observat la mateixa interacció anòmala tan a cerebel com escorça de pacients d'MSA, suggerint-se un mecanisme comú de neurodegeneració per a totes les sinucleïnopaties. Paral.lelament també hem estudiat la via de transducció glutamatèrgica a pacients amb DLBDs, i s'ha trobat que en condicions normals exiteix interacció de l'alfa-sinucleïna amb un efector del receptors metabotròpics del glutmat tipus I, com és la PLC-beta-1. Al mateix temps, i a través d'estudis de solubilitat, s'han observat canvis en la solubilitat de PLC-beta-1 en aquestes patologies, tal com succeeix amb l'alfa-sinucleïna. Quan s'han estudiat marcadors d'estrès oxidatiu per una forma primerenca de la malaltia de Parkinson s'han trobat alterats a córtex d'aquest pacients, quan la patología, en els primers estadis només es detecta fins ara a substància negra. Per tant, l'estrès oxidatiu a córtex d'una forma pre-clínica de la malaltia de Parkinson, ja té un paper en el desenvolupament d'estadis posterior de la malaltia, com podrien ser les DLBDs., [eng] The aim of this work is to study alpha-synuclein interactions in human brain to deep on the knowledge of a group of neurodegenerative diseases called synucleinopathies in which alpha-synuclein accumulates in the form of Lewy bodies. In human cortex we found alpha-synuclein interactions with the synaptic proteins rab3a and rabphilin in Dementia with Lewy Bodies (DLBDs) patients, and not in control brains. This interaction is corroborated in transgenic mice expressing human A30P human mutated alpha-synuclein. When a different synucleipathy , like Multiple Systemic Atrophy (MSA), is studied we found the same anomalous interaction even different areas were affected, cerebellum vs cortex in the case of MSA and DLBDs, indicating a common neurodegenerative mechanism for the synucleinopathies group. Also in DLBDs we studied the glutamatergic signal transduction pathway immunoprecipitating alpha-synuclein from brain cortex and we found that PLC-beta-1, an effector of the metabotropic glutamate receptors (mGLuR1) binds to alpha-synuclein only in control conditions and not in DLBDs. At the same time PLC-beta-1 showed changes in solubility like alpha-synuclein in DLBDs. So, the results presented here indicate a kind of relation between alpha-synuclein and PLC-beta-1 in DLBDs which damage can affect the mGLuR1 transduction pathway which is altered in cortex from DLBDs. The other factor studied in this work is the oxidative stress effect in brain cortex from early stages Parkinson's disease (PD) patients. We found an increase of the oxidative stress markers used in this work indicating that oxidative stress in cortex from PD patients starts at early stages in human cortex, even though in early stages only the substantia nigra seems to be affected. This markers of oxidative stress were also studied in Alzheimer's disease (AD) obtaining similar results. Finally another point of this work is the study of the Amyloid Precursor Protein (A-beta-PP) mRNA isoforms in human cortex from DLBDs. We found a relative increase of the isoforms contained the Kunitz Protease Inhibitor, implicating A-beta-PP the isoforms processing in the developing of DLBDs

The present study investigates global transcriptional changes in frontal cortex area 8 in incidental Lewy Body disease (iLBD), Parkinson disease (PD) and Dementia with Lewy bodies (DLB). We identified different co-expressed gene sets associated with disease stages, and gene ontology categories enriched in gene modules and differentially expressed genes including modules or gene clusters correlated to iLBD comprising upregulated dynein genes and taste receptors, and down-regulated innate inflammation. Focusing on DLB, we found modules with genes significantly enriched in functions related to RNA and DNA production, mitochondria and energy metabolism, purine metabolism, chaperone and protein folding system, and synapses and neurotransmission (particularly the GABAergic system). The expression of more than fifty selected genes was assessed with RT-qPCR. Our findings provide, for the first time, evidence of molecular cortical alterations in iLBD and involvement of several key metabolic pathways and gene hubs in DLB which may underlie cognitive impairment and dementia. Key words: Lewy body diseases, dementia with Lewy bodies, cerebral cortex, transcriptome, mitochondria, synapses, neurotransmission, GABA, purine metabolism, chaperones, dynein, axonema, taste receptors.