Survival Bias and Crosstalk between Chronological and Behavioral Age: Age- and Genotype-Sensitivity Tests Define Behavioral Signatures in Middle-Aged, Old, and Long-Lived Mice with Normal and AD-Associated Aging

Malaltia d'Alzheimer; Envelliment; SupervivènciaAlzheimer’s disease; Aging; SurvivalEnfermedad de Alzheimer; Envejecimiento; SupervivenciaNew evidence refers to a high degree of heterogeneity in normal but also Alzheimer’s disease (AD) clinical and temporal patterns, increased mortality, and the need to find specific end-of-life prognosticators. This heterogeneity is scarcely explored in very old male AD mice models due to their reduced survival. In the present work, using 915 (432 APP23 and 483 C57BL/6 littermates) mice, we confirmed the better survival curves in male than female APP23 mice and respective wildtypes, providing the chance to characterize behavioral signatures in middle-aged, old, and long-lived male animals. The sensitivity of a battery of seven paradigms for comprehensive screening of motor (activity and gait analysis), neuropsychiatric and cognitive symptoms was analyzed using a cohort of 56 animals, composed of 12-, 18- and 24-month-old male APP23 mice and wildtype littermates. Most variables analyzed detected age-related differences. However, variables related to coping with stress, thigmotaxis, frailty, gait, and poor cognition better discriminated the behavioral phenotype of male APP23 mice through the three old ages compared with controls. Most importantly, non-linear age- and genotype-dependent behavioral signatures were found in long-lived animals, suggesting crosstalk between chronological and biological/behavioral ages useful to study underlying mechanisms and distinct compensations through physiological and AD-associated aging.This work was funded by the BrightFocus Foundation, US (A2017243S). The Neurovascular Research Laboratory is part of the INVICTUS+network, Instituto de Salud Carlos III (ISCIII), Spain [RD16/0019/0021], co-financed by the European Regional Development Fund FEDER. P.M. held a predoctoral fellowship from the Vall d’Hebron Research Institute

Impact of Cerebral Amyloid Angiopathy in Two Transgenic Mouse Models of Cerebral β-Amyloidosis: A Neuropathological Study

Microhemorragias cerebrales; Beta-amiloidosis cerebral; Resonancia magnética preclínicaCerebral microbleeds; Cerebral beta-amyloidosis; Preclinical MRIMicrohemorràgies cerebrals; Beta-amiloidosi cerebral; Ressonància magnètica preclínicaThe pathological accumulation of parenchymal and vascular amyloid-beta (Aβ) are the main hallmarks of Alzheimer’s disease (AD) and Cerebral Amyloid Angiopathy (CAA), respectively. Emerging evidence raises an important contribution of vascular dysfunction in AD pathology that could partially explain the failure of anti-Aβ therapies in this field. Transgenic mice models of cerebral β-amyloidosis are essential to a better understanding of the mechanisms underlying amyloid accumulation in the cerebrovasculature and its interactions with neuritic plaque deposition. Here, our main objective was to evaluate the progression of both parenchymal and vascular deposition in APP23 and 5xFAD transgenic mice in relation to age and sex. We first showed a significant age-dependent accumulation of extracellular Aβ deposits in both transgenic models, with a greater increase in APP23 females. We confirmed that CAA pathology was more prominent in the APP23 mice, demonstrating a higher progression of Aβ-positive vessels with age, but not linked to sex, and detecting a pronounced burden of cerebral microbleeds (cMBs) by magnetic resonance imaging (MRI). In contrast, 5xFAD mice did not present CAA, as shown by the negligible Aβ presence in cerebral vessels and the occurrence of occasional cMBs comparable to WT mice. In conclusion, the APP23 mouse model is an interesting tool to study the overlap between vascular and parenchymal Aβ deposition and to evaluate future disease-modifying therapy before its translation to the clinic.This work was funded by the Instituto de Salud Carlos III (ISCIII), (PI20/00465) and co-financed by the European Regional Development Fund (FEDER). The Neurovascular Research Laboratory is part of the RICORS-ICTUS-Enfermedades Vasculares Cerebrales network, ISCIII, Spain (RD21/0006/0007)

Association of CD2AP neuronal deposits with Braak neurofibrillary stage in Alzheimer’s disease

Alzheimer; CD2AP; Enfermedad de PickAlzheimer's disease; CD2AP; Pick's diseaseAlzheimer; CD2AP; Malaltia de PickGenome-wide association studies have described several genes as genetic susceptibility loci for Alzheimer's disease (AD). Among them, CD2AP encodes CD2-associated protein, a scaffold protein implicated in dynamic actin remodeling and membrane trafficking during endocytosis and cytokinesis. Although a clear link between CD2AP defects and glomerular pathology has been described, little is known about the function of CD2AP in the brain. The aim of this study was to analyze the distribution of CD2AP in the AD brain and its potential associations with tau aggregation and β-amyloid (Aβ) deposition. First, we performed immunohistochemical analysis of CD2AP expression in brain tissue from AD patients and controls (N = 60). Our results showed granular CD2AP immunoreactivity in the human brain endothelium in all samples. In AD cases, no CD2AP was found to be associated with Aβ deposits in vessels or parenchymal plaques. CD2AP neuronal inclusions similar to neurofibrillary tangles (NFT) and neuropil thread-like deposits were found only in AD samples. Moreover, immunofluorescence analysis revealed that CD2AP colocalized with pTau. Regarding CD2AP neuronal distribution, a hierarchical progression from the entorhinal to the temporal and occipital cortex was detected. We found that CD2AP immunodetection in neurons was strongly and positively associated with Braak neurofibrillary stage, independent of age and other pathological hallmarks. To further investigate the association between pTau and CD2AP, we included samples from cases of primary tauopathies (corticobasal degeneration [CBD], progressive supranuclear palsy [PSP], and Pick's disease [PiD]) in our study. Among these cases, CD2AP positivity was only found in PiD samples as neurofibrillary tangle-like and Pick body-like deposits, whereas no neuronal CD2AP deposits were detected in PSP or CBD samples, which suggested an association of CD2AP neuronal expression with 3R-Tau-diseases. In conclusion, our findings open a new road to investigate the complex cellular mechanism underlying the tangle conformation and tau pathology in the brain.This work was funded by Instituto de Salud Carlos III (ISCIII) (PI17/00275, PI20/00465), cofinanced by the European Regional Development Fund (FEDER). The Neurovascular Research Laboratory is part of the INVICTUS+ network, ISCIII, Spain (RD16/0019/0021). M.H.-G. is supported by the Miguel Servet Programme, ISCIII, Spain (CPII17/00010

MFG-E8 (LACTADHERIN): a novel marker associated with cerebral amyloid angiopathy

Malaltia d'Alzheimer; Biomarcadors; Microdissecció de captura làserAlzheimer's disease; Biomarkers; Laser capture microdissectionEnfermedad de Alzheimer; Biomarcadores; Microdisección por captura láserBrain accumulation of amyloid-beta (Aβ) is a crucial feature in Alzheimer´s disease (AD) and cerebral amyloid angiopathy (CAA), although the pathophysiological relationship between these diseases remains unclear. Numerous proteins are associated with Aβ deposited in parenchymal plaques and/or cerebral vessels. We hypothesized that the study of these proteins would increase our understanding of the overlap and biological differences between these two pathologies and may yield new diagnostic tools and specific therapeutic targets. We used a laser capture microdissection approach combined with mass spectrometry in the APP23 transgenic mouse model of cerebral-β-amyloidosis to specifically identify vascular Aβ-associated proteins. We focused on one of the main proteins detected in the Aβ-affected cerebrovasculature: MFG-E8 (milk fat globule-EGF factor 8), also known as lactadherin. We first validated the presence of MFG-E8 in mouse and human brains. Immunofluorescence and immunoblotting studies revealed that MFG-E8 brain levels were higher in APP23 mice than in WT mice. Furthermore, MFG-E8 was strongly detected in Aβ-positive vessels in human postmortem CAA brains, whereas MFG-E8 was not present in parenchymal Aβ deposits. Levels of MFG-E8 were additionally analysed in serum and cerebrospinal fluid (CSF) from patients diagnosed with CAA, patients with AD and control subjects. Whereas no differences were found in MFG-E8 serum levels between groups, MFG-E8 concentration was significantly lower in the CSF of CAA patients compared to controls and AD patients. Finally, in human vascular smooth muscle cells MFG-E8 was protective against the toxic effects of the treatment with the Aβ40 peptide containing the Dutch mutation. In summary, our study shows that MFG-E8 is highly associated with CAA pathology and highlights MFG-E8 as a new CSF biomarker that could potentially be used to differentiate cerebrovascular Aβ pathology from parenchymal Aβ deposition.This work was funded by the Instituto de Salud Carlos III (ISCIII), (PI20/00465), co-financed by the European Regional Development Fund (FEDER). The Neurovascular Research Laboratory is part of the INVICTUS + network, ISCIII, Spain (RD16/0019/0021). P.M. held a predoctoral fellowship from the Vall d’Hebron Research Institute. MMV is supported by the BIONIC project (no. 733050822, which has been made possible by ZonMW as part of ‘Memorabel’, the research and innovation program for dementia, as part of the Dutch national ‘Deltaplan for Dementia’:the CAFÉ project (the National Institutes of Health, USA, grant number 5R01NS104147-02), and a grant from the Selfridges Group Foundation (NR170024). The BIONIC project is a consortium of Radboudumc, LUMC, ADX Neurosciences, and Rhode Island University

Búsqueda de nuevos biomarcadores en la Angiopatía Amiloide Cerebral

La β-amiloidosis cerebral es caracteritza per l'acumulació anòmala del pèptid beta amiloide (Aß) en el parènquima i en la paret dels vasos cerebrals, constituint una de les principals característiques neuropatològiques de la Malaltia d'Alzheimer (MA) i de l'angiopatia amiloide cerebral (AAC). Tot i que en les dues patologies s'observen dipòsits de Aß, la localització d'aquestes difereix entre elles, implicant diferents presentacions clíniques. Fins ara, no existeixen marcadors biològics que identifiquin pacients amb AAC o prediguin la seva evolució en la pràctica clínica. Per això, l'objectiu principal d'aquesta tesi es centra en la recerca de nous biomarcadors involucrats en el procés d'acumulació vascular de Aß que ens ajudin a millorar el diagnòstic i pronòstic de l'AAC, i comprendre millor la fisiopatologia que engloba la β-amiloidosis cerebral. La primera part de la tesi es centra en la caracterització de dos models de ratolins transgènics de β-amiloïdosi cerebral, el APP23 i el 5xFAD, en termes neuropatològics de l'AAC. En aquest context, confirmem que la patologia de l'AAC era més abundant en el model APP23, observant una major progressió de vasos Aß positius amb l'edat i detectant microhemorràgies cerebrals per ressonància magnètica, motiu pel qual va ser el model utilitzat per abordar el següent objectiu. A continuació, vam utilitzar un enfocament de microdisecció làser combinat amb espectrometria de masses per a la identificació de proteïnes vasculars associades a Aß en cervells de ratolins APP23. Ens centrem en l'estudi d'una de les principals proteïnes detectades, MFG-E8 (de l'anglès, Milk Fat Globule-EGF factor-8), i validem la seva localització en cervells de ratolins APP23 i en teixit cerebral post mortem de pacients amb AAC, on confirmem que MFG-E8 estava absent en plaques neurítiques i la seva presència era elevada en vasos cerebrals positius per Aß. A més, analitzem els nivells circulants d'aquesta proteïna en el sèrum i en el líquid cefaloraquidi (LCR) d'una cohort de pacients amb AAC, MA i controls. No vam trobar diferències en els nivells sèrics de MFG-E8 entre els grups. No obstant, els nivells de MFG-E8 en el LCR de pacients amb AAC van ser significativament més baixos que a la resta de grups, i els nivells de Aß en LCR es correlacionen positivament amb els nivells de MFG-E8 en tota la cohort. A l'avaluació de la implicació de la modulació de MFG-E8 en cèl·lules vasculars humanes de múscul llis in vitro, trobem que nivells elevats de MFG-E8 eren protectors enfront de la toxicitat induïda pel pèptid Aβ40 contenint la mutació familiar E693Q (Aβ40-Dutch), demostrant l'associació de MFG-E8 amb la patologia AAC. Finalment, en un tercer estudi, avaluem els nivells circulants de AQP4 (aquoporina-4) en el sèrum d'una cohort de pacients que havia patit una hemorràgia intracerebral (HIC) amb un diagnòstic clínic de l'AAC i analitzem la seva possible associació amb les principals característiques radiològiques de l'AAC. La AQP4 estava associat a la MA i a l'AAC per ser una de les principals proteïnes involucrades en l'eliminació de soluts intersticials de el cervell a través del sistema glinfàtic, però no s'havia estudiat en el sèrum d'aquests pacients. Tot i que no trobem diferències en els nivells circulants de AQP4 entre els pacients amb AAC-HIC i els controls, observem una associació negativa entre la concentració de AQP4 i la càrrega hemorràgica en la cohort de AAC-HIC. Els nostres resultats van suggerir també que la determinació de AQP4 sèrica podria predir el resultat funcional a llarg termini en pacients amb AAC després d'una HIC lobar. En resum, considerem que aquesta tesi doctoral ha permès caracteritzar un model experimental vàlid de l'AAC i proposar nous marcadors que podrien ajudar a millorar el diagnòstic de l'AAC.La β-amiloidosis cerebral se caracteriza por la acumulación anómala del péptido beta amiloide (Aβ) en el parénquima y en la pared de los vasos cerebrales, constituyendo una de las principales características neuropatológicas de la Enfermedad de Alzheimer (EA) y de la Angiopatía Amiloide Cerebral (AAC), respectivamente. Aunque en ambas patologías se observan depósitos de Aβ, la localización de los mismos difiere entre ellas, implicando también diferentes presentaciones clínicas. No existen hasta la fecha marcadores biológicos que nos permitan identificar pacientes con AAC o predecir su evolución en la práctica clínica. Por ello, el objetivo principal de esta tesis se centra en la búsqueda de nuevos biomarcadores involucrados en el proceso de acumulación vascular de Aβ que nos puedan ayudar a mejorar el diagnóstico y pronóstico de la AAC, así como a comprender mejor la fisiopatología que engloba la β-amiloidosis cerebral. La primera parte de la tesis se centró en la caracterización de dos modelos de ratones transgénicos de β-amiloidosis cerebral, el APP23 y el 5xFAD, en términos neuropatológicos de AAC. En este contexto, confirmamos que la patología de AAC era más abundante en el modelo APP23, observando una mayor progresión de vasos Aβ positivos con la edad y detectando microhemorragias cerebrales por resonancia magnética, por lo que fue el modelo utilizado para abordar el siguiente objetivo. A continuación, utilizamos un enfoque de microdisección laser combinado con espectrometría de masas para la identificación de proteínas vasculares asociadas a Aβ. Nos centramos en el estudio de una de las principales proteínas detectadas, MFG-E8 (Lactadherin), y validamos su presencia en cerebros de ratones APP23 y en tejido cerebral post mortem de pacientes con AAC, donde confirmamos que MFG-E8 estaba ausente en depósitos parenquimatosos de Aβ y su presencia era elevada en vasos cerebrales positivos para Aβ. Además analizamos los niveles circulantes de esta proteína en el suero y en el líquido cefalorraquídeo (LCR) de una cohorte de pacientes con AAC, EA y en controles. No encontramos diferencias en los niveles séricos de MFG-E8 entre los grupos. Sin embargo, los niveles de MFG-E8 en el LCR de pacientes con AAC fueron significativamente más bajos que en controles y en los pacientes con EA, y los niveles de Aβ en LCR se correlacionaron positivamente con los niveles de MFG-E8 en toda la cohorte. Además, evaluamos la implicación de la modulación de MFG-E8 en células vasculares del músculo liso humanas y encontramos que niveles aumentados de MFG-E8 era protectores frente al tratamiento con la forma tóxica del péptido Aβ40-Dutch, demostrando así que MFG-E8 estaba altamente asociada con la patología de AAC. Por último, en un tercer estudio, evaluamos los niveles circulantes de aquoporina-4 (AQP4) en el suero de una cohorte de pacientes que había sufrido una hemorragia intracerebral (HIC) con un diagnóstico clínico de AAC y analizamos su posible asociación con las principales características radiológicas de la AAC. La AQP4 se había asociado anteriormente a la EA y a la AAC por ser una de las principales proteínas involucradas en la eliminación de solutos intersticiales del cerebro a través del sistema glinfático, pero nunca antes se había estudiado en el suero de estos pacientes. Aunque no encontramos diferencias entre AAC-HIC y controles, observamos una asociación entre los niveles circulantes de AQP4 y la carga hemorrágica en la cohorte de AAC-HIC. Además nuestros resultados sugirieron que AQP4 podría predecir el resultado funcional a largo plazo en pacientes con AAC tras una HIC lobar. En resumen, consideramos que esta tesis doctoral ha permitido caracterizar un modelo experimental válido de AAC y proponer nuevos marcadores que podrían ayudar a mejorar el diagnóstico de la AAC.Cerebral β-amyloidosis is characterized by parenchymal and vascular deposition of amyloid beta-peptide (Aβ), which are major features of Alzheimer's disease (AD) and Cerebral Amyloid Angiopathy (CAA), respectively. Although Aβ is accumulated in both pathologies, the localization differs between them, involving different clinical presentations. To date, there are no biological markers that identify CAA patients or predict their evolution in clinical practice. Thus, the main objective of this thesis is to find out new biomarkers related to vascular Aβ accumulation that might improve the diagnosis and prognosis of CAA and help to understand the pathophysiology of cerebral β-amyloidosis. The first part of the thesis focused on the characterization of two transgenic mice models of cerebral β-amyloidosis, APP23 and 5xFAD transgenic mice, in terms of neuropathological features of CAA. In this context, we confirmed that CAA pathology was more prominent in the APP23 model, observing a higher progression of Aβ-positive vessels with age and detecting cerebral microbleeds by magnetic resonance imaging. Thus, this was the model used to address the following objective of this thesis. Next, we used a laser microdissection approach combined with mass spectrometry to identify Aβ-associated vascular proteins in the brains of APP23 mice. We focused on the study of one of the main proteins detected in old APP23 mouse brains, MFG-E8 (Milk Fat Globule-EGF factor 8), and we validated its localization in APP23 mouse brains and in post mortem brain tissue of CAA patients, where we confirmed that MFG-E8 was absent in neuritic plaques and its presence was strongly detected in Aβ-positive cerebral vessels. We also analyzed the circulating levels of this protein in the serum and cerebrospinal fluid (CSF) of a cohort of patients with CAA, AD and controls. No differences in MFG-E8 serum levels were found between the diagnostic groups. However, MFG-E8 levels were significantly lower in the CSF of CAA patients than all the groups studied and the levels of Aβ in the CSF were positively correlated with the levels of MFG-E8 in the total cohort. When evaluating the involvement of MFG-E8 modulation in human vascular smooth muscle cells in vitro, we found that increased levels of MFG-E8 were protective against the toxicity induced by the Aβ40 peptide containing the familial E693Q (Aβ40-Dutch) mutation, demonstrating the association of MFG-E8 with CAA pathology. Finally, in a third study, we evaluated the circulating levels of AQP4 (aquaporin-4) in the serum of a cohort of patients who had suffered an intracerebral hemorrhage (ICH) with a clinical diagnosis of CAA and we analyzed its potential association with the main neuroimaging hallmarks of CAA. AQP4 had previously been associated with AD and CAA as one of the main proteins involved in the clearance of interstitial solutes from the brain through the glymphatic system, however, no studies had reported the modulation of AQP4 in the circulation of CAA patients. Although no differences were found in AQP4 serum levels between CAA-ICH and controls, we observed a negative association between circulating AQP4 concentration and specific hemorrhagic neuroimaging features in the CAA-ICH cohort. Furthermore, our results suggested that AQP4 could potentially predict a long-term functional outcome in patients presenting with lobar ICH. In summary, we consider that this doctoral thesis allowed the characterization of a valid experimental model for CAA and highlighted new markers that could help improve the diagnosis of CAA and provide valuable insights for the understanding of cerebral β-amyloidosis pathophysiology

Circulating AQP4 Levels in Patients with Cerebral Amyloid Angiopathy-Associated Intracerebral Hemorrhage

Aquaporina 4; Angiopatia amiloide cerebral; Marcadors d'imatge per ressonància magnèticaAcuaporina 4; Angiopatía amiloide cerebral; Marcadores de imágenes por resonancia magnéticaAquaporin 4; Cerebral amyloid angiopathy; Magnetic resonance imaging markersCerebral amyloid angiopathy (CAA) is a major cause of lobar intracerebral hemorrhage (ICH) in elderly patients. Growing evidence suggests a potential role of aquaporin 4 (AQP4) in amyloid-beta-associated diseases, including CAA pathology. Our aim was to investigate the circulating levels of AQP4 in a cohort of patients who had suffered a lobar ICH with a clinical diagnosis of CAA. AQP4 levels were analyzed in the serum of 60 CAA-related ICH patients and 19 non-stroke subjects by enzyme-linked immunosorbent assay (ELISA). The CAA–ICH cohort was divided according to the time point of the functional outcome evaluation: mid-term (12 ± 18.6 months) and long-term (38.5 ± 32.9 months) after the last ICH. Although no differences were found in AQP4 serum levels between cases and controls, lower levels were found in CAA patients presenting specific hemorrhagic features such as ≥2 lobar ICHs and ≥5 lobar microbleeds detected by magnetic resonance imaging (MRI). In addition, CAA-related ICH patients who presented a long-term good functional outcome had higher circulating AQP4 levels than subjects with a poor outcome or controls. Our data suggest that AQP4 could potentially predict a long-term functional outcome and may play a protective role after a lobar ICH.This research was funded by the Instituto de Salud Carlos III (ISCIII), (PI17/00275 and PI20/00465), co-financed by the European Regional Development Fund FEDER. The Neurovascular Research Laboratory is part of the INVICTUS+ network, ISCIII, Spain (RD16/0019/0021). M.H.-G. was supported by the Miguel Servet programme, ISCIII, Spain (CPII17/00010). P.M. held a predoctoral fellowship from the Vall d’Hebron Research Institute