Vascular Amyloid Deposits Research Articles

β-amyloid protein induces mitophagy-dependent ferroptosis through the CD36/PINK/PARKIN pathway leading to blood–brain barrier destruction in Alzheimer’s disease

IntroductionBlood–brain barrier (BBB) dysfunction may occur at the onset of Alzheimer’s disease (AD). Pericytes are a vital part of the neurovascular unit and the BBB, acting as gatekeepers of the BBB. Amyloid β (Aβ) deposition and neurofibrillary tangles in the brain are the central pathological features of AD. CD36 promotes vascular amyloid deposition and leads to vascular brain damage, neurovascular dysfunction, and cognitive deficits. However, the molecular mechanism by which pericytes of the BBB are disrupted remains unclear.ObjectivesTo investigate the effect of low-dose Aβ1-40 administration on pericyte outcome and the molecular mechanism of BBB injury.MethodsWe selected 6-month-old and 9-month-old APP/PS1 mice and wild-type (WT) mice of the same strain, age, and sex as controls. We assessed the BBB using PET/CT. Brain pericytes were extracted and cocultured with endothelial cells (bEnd.3) to generate an in vitro BBB model to observe the effect of Aβ1-40 on the BBB. Furthermore, we explored the intracellular degradation and related molecular mechanisms of Aβ1-40 in cells.ResultsBBB permeability and the number of pericytes decreased in APP/PS1 mice. Aβ1-40 increased BBB permeability in an in vivo model and downregulated the expression of CD36, which reversed the Aβ-induced changes in BBB permeability. Aβ1-40 was uptaked in pericytes with high CD36 expression. We observed that this molecule inhibited pericyte proliferation, caused mitochondrial damage, and increased mitophagy. Finally, we confirmed that Aβ1-40 induced pericyte mitophagy-dependent ferroptosis through the CD36/PINK1/Parkin pathway.ConclusionPDGFRβ (a marker of pericytes), CD36, and Aβ colocalized in vitro and in vivo, and Aβ1-40 caused BBB disruption by upregulating CD36 expression in pericytes. The mechanism by which Aβ1-40 destroys the BBB involves the induction of pericyte mitophagy-dependent ferroptosis through the CD36/PINK1/Parkin pathway.

The Extracellular Chaperone Clusterin in Aβ and Non‐Aβ Cerebral Amyloidoses

Clusterin, a multifunctional glycoprotein also known as apolipoprotein J, has been implicated in several physiological processes and is an important contributor to many pathological conditions, including Alzheimer’s disease (AD) in which genetic variants and plasma levels of the apolipoprotein were identified as risk factors for the disorder. To investigate whether clusterin exerted a more general role in the pathophysiology of cerebral amyloidoses, we turned to two unrelated hereditary conditions, familial British and Danish dementias (FBD and FDD), which share striking neuropathological similarities with AD, including neurofibrillary degeneration in limbic areas as well as parenchymal and vascular amyloid deposition. Two different 4 kDa molecules – ABri in FBD and ADan in FDD – totally unrelated to the 4 kDa Alzheimer’s Aβ, are the main constituents of the amyloid deposits in these forms of cerebral amyloidoses. Mirroring findings in AD, a panel of immunohistochemical studies demonstrated clusterin co‐localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri‐ and ADan‐binding plasma protein. Solid‐phase enzyme‐linked immunosorbent assays highlighted a specific saturable binding of clusterin to ABri and ADan. Non‐linear regression analysis of the data revealed Kd values within the same low nanomolar range as those demonstrated for the clusterin‐Aβ interaction. Consistent with its reported chaperone activity, clusterin showed a modulatory effect on ABri, ADan, and Aβ aggregation/fibrillization properties evaluated using thioflavin T, a dye that displays enhanced fluorescence upon binding fibrillar and protofibrillar amyloid conformations. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of protein folding disorders, including AD, FBD, and FDD – suggests the likelihood of a more complex role and a translational potential of clusterin in the amelioration/prevention of these pathogenic mechanisms.

P 25 CSF biomarkers in CAA compared to AD

Background: Both, cerebral amyloid angiopathy (CAA) and Alzheimer’s disease (AD) pathologies coexist on a spectrum, where one end represents parenchymal amyloid deposition causing AD-dementia, and the other end represents vascular amyloid deposition leading to intracerebral hemorrhage and vascular cognitive impairment. CAA is present, to varying degrees, in several AD patients, resulting in a large overlap between the two pathologies in the same brain. Cerebrospinal fluid (CSF) biomarkers could aid in the better discrimination between CAA and AD pathology. Objective: To assess core biomarkers of AD pathology and axonal degeneration in CAA compared to AD and healthy controls. Methods: In a hospital-based setting we recruited 234 patients. N=96 (41%) had probable CAA according to the modified Boston criteria, but did not meet the NIA-AA criteria for AD. N=99 (42%) had AD, and of those n=21 (9%) had AD with CAA, while n=78 (33%) had AD without CAA. N=39 (17%) were considered controls without CAA or AD. CSF measures included β-amyloid (Aβ) 1-40, Aβ 1-42, phosphorylated tau (ptau), total tau (ttau) and neurofilament light chain (NfL). NfL data were missing in n=139 (59%) subjects. Group comparisons were conducted applying ANOVA, adjusted for age and sex, respectively. Bonferroni-adjusted p-values ≤ 0.01 were considered significant. Results: Group differences became obvious for Aβ 1-42, ptau and ttau (Figure). Aβ 1-42 was lower in AD with and without CAA compared to CAA and controls. Ptau was higher in AD with and without CAA compared to CAA and controls. Compared to controls, ttau was higher in AD with and without CAA, and in CAA. There were no group differences for Aβ 1-40 and NfL, albeit there was a clear trend towards higher NfL levels in AD with CAA and CAA compared to AD without CAA and controls (Figure). None of the CSF markers differed between AD with CAA compared to AD without CAA. Conclusion: Lower CSF Aβ 1-42 and higher ptau seem to be specific markers for AD pathology independent of the concurrent occurrence of CAA. They could aid in the discrimination between pure CAA and CAA with AD pathology. Outlook: Upcoming analysis will focus on the correlation between CSF biomarkers and CSVD MRI features according to the STRIVE criteria.

Activated endothelial cells induce a distinct type of astrocytic reactivity

Reactive astrogliosis is a universal response of astrocytes to abnormal events and injuries. Studies have shown that proinflammatory microglia can polarize astrocytes (designated A1 astrocytes) toward a neurotoxic phenotype characterized by increased Complement Component 3 (C3) expression. It is still unclear if inflammatory stimuli from other cell types may also be capable of inducing a subset of C3+ neurotoxic astrocytes. Here, we show that a subtype of C3+ neurotoxic astrocytes is induced by activated endothelial cells that is distinct from astrocytes activated by microglia. Furthermore, we show that endothelial-induced astrocytes have upregulated expression of A1 astrocytic genes and exhibit a distinctive extracellular matrix remodeling profile. Finally, we demonstrate that endothelial-induced astrocytes are Decorin-positive and are associated with vascular amyloid deposits but not parenchymal amyloid plaques in mouse models and AD/CAA patients. These findings demonstrate the existence of potentially extensive and subtle functional diversity of C3+-reactive astrocytes.

Prognostic value of histopathological scoring and grading in patients with renal AA amyloidosis.

The amount and distribution pattern of amyloid deposits may contribute to renal function and outcome, given the great diversity of renal involvement in amyloidosis. The aim of this study was to analyze the impact of histological characteristics of patients with biopsy-proven renal AA amyloidosis (AAA)on renal outcome. Renal biopsies of 37 patients with AAA were re-evaluated. The distribution pattern of glomerular amyloid (GA) deposits was classified, the extent of amyloid deposits in glomeruli, vessel, and interstitium and other histopathologic lesions were scored, and renal amyloid prognostic score (RAPS) was determined by summing all scores. Their potential prognostic relevance on renal outcome was investigated. GA and vascular amyloid (VA) deposits were noted in all cases, interstitial amyloid (IA) was detected in 70.2%. GA deposits were predominantly seen in diffuse mesengiocapillary fashions (class IV) (51.4%). GA class, the extent of GA, VA, IA deposit, and RAPS, as well as interstitial fibrosis (IF) and interstitial inflammation were correlated to renal function at diagnosis. During the median follow-up of 52months, 13 patients developed doubling of serum creatinine or end stage renal disease and they had a higher degree of GA and VA load (p = 0.03 and p = 0.042, respectively) as compared to the remaining patients. VA load, but not GA and RAPS grade, was associated with poor renal outcome (HR 3.016, 95% CI 1.45-6.25, p = 0.003). Baseline renal function is closely linked to the extent of AA amyloid deposit in renal parenchyma but only VA load was a predictor of renal outcome in AAA patients.

Amyloid cast nephropathy: A rare presentation of multiple myeloma–associated light chain amyloidosis

Introduction: Monoclonal immunoglobulins can cause a variety of histologic patterns of kidney injury, depending on the physicochemical properties. Multiple myeloma manifests more often as light-chain nephropathy. On the other hand, light-chain amyloidosis leads to glomerular and vascular amyloid deposits, but a less common presentation with amyloid casts has also been described. Rarely, more than one histologic pattern can be present in the same patient. Case report: We report a case of a 73-year-old man, diagnosed with multiple myeloma that 8 months after achieving partial response to chemotherapy develops acute kidney injury and nephrotic syndrome. Kidney biopsy revealed features of light-chain nephropathy, amyloid cast nephropathy, and glomerular and vascular amyloid deposits. Immunofluorescence was positive for IgA (++) and lambda chains (+++) and negative for kappa chains. After the diagnosis of multiple myeloma-associated light-chain amyloidosis, chemotherapy was initiated; unfortunately, the patient died 1 month after the diagnosis. Conclusion: Amyloid casts, isolated or accompanied by other renal or extra-renal amyloid deposits, are another form of tubular toxicity caused by dysproteinemias and should be systematically screened in kidney biopsies.

Recent update on the heterogeneity of the Alzheimer's disease spectrum.

Alzheimer's disease (AD), the most common form of dementia worldwide, is a mixed proteinopathy (β-amyloid, tau and other proteins). Classically defined as a clinicopathological entity, AD is a heterogeneous, multifactorial disorder with various pathobiological subtypes showing different forms of cognitive presentation, currently referred to as the Alzheimer spectrum or continuum. Its morphological hallmarks are extracellular β-amyloid (amyloid plaques) and intraneuronal tau aggregates forming neurofibrillary tangles and neurites, vascular amyloid deposits (cerebral amyloid angiopathy), synapse and neuronal loss as well as neuroinflammation and reactive astrogliosis, leading to cerebral atrophy and progressive mental/cognitive impairment (dementia). In addition to "classical" AD, several subtypes with characteristic regional patterns of tau pathology have been segregated that are characterized by distinct clinical features, differences in age, sex distribution, disease duration, cognitive status, APOE genotype, and biomarker levels. In addition to four major subtypes based on the distribution of tau pathology and brain atrophy (typical, limbic predominant, hippocampal sparing, and minimal atrophy), several other clinical variants (non-amnestic, corticobasal, behavioral/dysexecutive, posterior cortical variants, etc.) have been identified. These heterogeneous AD variants are characterized by different patterns of key neuronal network destructions, in particular the default-mode network that is responsible for cognitive decline. Other frequent age-related co-pathologies, e.g., cerebrovascular lesions, Lewy and TDP-43 pathologies, hippocampal sclerosis, or argyrophilic grain disease, essentially influence the clinical picture and course of AD, and can challenge our understanding of this disorder including the threshold and causal relevance of each individual pathology. Unravelling the clinico-morphological heterogeneity among the AD spectrum entities is important for better elucidation of the pathogenic mechanisms affecting the aging brain that may enable a broader diagnostic coverage of AD as a basis for implementing precision medicine approaches and for developing preventive and ultimately disease-modifying therapies for this devastating disorder.

Fibrillar Aβ causes profound microglial metabolic perturbations in a novel APP knock-in mouse model.

Microglial dysfunction is believed to play a pathogenic role in Alzheimer's disease (AD). Microglia respond to various pathogenic drivers of AD, including amyloid-ß (Aß) and tau, but the mechanisms by which microglia may become dysfunctional and contribute to disease remain unclear. Here, we aim to characterize the amyloid-ß related pathology and microglial responses in an engineered APP knock-in mouse model of familial AD (AppSAA ). To circumvent the numerous limitations inherent to transgenesis, we used a knock-in strategy to humanize the Aß sequence of the murine App gene and introduced three FAD mutations - Swedish (KM670/671NL), Arctic (E693G) and Austrian (T712I) - using homologous recombination. We then conducted an extensive biochemical and histological characterization of various tissues from the three resulting genotypes at various ages. Finally, we conducted a deep-phenotyping analysis of brain-sorted microglia using multi-omics approaches. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered glial responses and increased levels of markers of neurodegeneration such as CSF Tau and neurofilament light chain. We found lipid accumulation and an exacerbated disease-associated transcriptomic response in methoxy-X04-positive, phagocytic microglia. Altogether, our in-depth analysis of the AppSAA knock-in mouse model confirms emergence of disease-relevant biology and progressive accumulation of pathological hallmarks of AD. Our data lends further support to the notion that phagocytic microglia undergo profound cellular alterations, including lysosomal dysfunction, lipid dyshomeostasis, and other metabolic changes. Since this new mouse model can be used to investigate multiple relevant aspects of AD biology, we have made it broadly available to the scientific community.

Amyloid‐βeta peptides in CSF and plasma discriminate cerebral amyloid angiopathy from controls

AbstractBackgroundCerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid‐beta (Abeta) peptides in the cerebral vasculature and has been associated with vascular dementia and intracerebral haemorrhages (ICH). Currently, the diagnosis of CAA is based on neuroimaging and includes the identification of lobar (micro‐)bleeds and cortical superficial siderosis. However, these MRI markers reflect only late‐stage manifestations and are therefore not suitable for an early diagnosis of CAA. We aimed to evaluate the diagnostic potential of the Abeta peptides AB1‐38, AB1‐40, AB1‐42, and AB1‐43 in cerebrospinal fluid (CSF) to discriminate (early) CAA from controls.MethodWe used immunoassays to quantify AB1‐38, AB1‐40 and AB1‐42 in CSF and plasma and AB1‐43 only in CSF. CSF from sporadic CAA patients (n=28) and controls (n=40), and plasma from hereditary Dutch CAA (D‐CAA) patients (asymptomatic and symptomatic), sporadic CAA patients (n=129) and controls (n=99) were analyzed.ResultIn CSF we found decreased levels of AB1‐38 (p=0.03; AUC 0.64), AB1‐40 (p=0.04; AUC 0.63), AB1‐42 (p<0.0001, AUC 0.87) and AB1‐43 (p<0.0001, AUC 0.92) in CAA patients compared to controls. In plasma we found significantly decreased levels of AB1‐38, AB1‐40 and AB1‐42 both in asymptomatic and symptomatic D‐CAA mutation carriers compared to controls. The concentrations of all Abeta peptides strongly correlated with each other (Spearman r>0.6, p<0.0001 for each comparison).ConclusionAbeta peptides have strong biomarker potential for the diagnosis of (early)_CAA. Interestingly, our results indicate that AB1‐43, which has shown to be relatively low‐abundant in vascular amyloid deposits, has the highest diagnostic accuracy to discriminate CAA from controls. In addition, we show that CSF AB1‐38 levels are decreased in CAA compared to controls.

Cerebral amyloid angiopathy and visuospatial functioning: Analysis of the clock drawing test

AbstractBackgroundCerebral amyloid angiopathy (CAA) contributes to cognitive impairment as well as dementia in the elderly. In clinical cohorts, symptomatic CAA is associated with impairments in executive function and processing speed. Because CAA severity tends to be highest in the occipital and posterior temporal and parietal lobes, it may also affect visuospatial function. We hypothesized that participants with a clinical diagnosis of CAA will have impaired performance on tests of visuospatial function when compared to control, mild cognitive impairment (MCI) and Alzheimer’s disease (AD) participants.MethodParticipants with CAA presented with lobar intracerebral hemorrhage, transient focal neurological episodes, or mild cognitive impairment; met Boston criteria for probable CAA; and did not have dementia. Clocks were scored based on a published 20‐point system (Mendez, JAGS 1992). Clock items were subcategorized by general contours, number items, and hand items (Mendez, JAGS 1992); and by a subset of 9 items correlated in prior literature with visuospatial function and temporal‐parietal hypoperfusion (visuospatial items).ResultData from 53 CAA without dementia, 43 MCI, 30 AD with mild dementia (MMSE ≥20), and 40 control participants were analyzed (mean age 71.6 [SD 7.5]; 47% women). Compared to controls, CAA participants performed significantly worse on total score, hand items, and visuospatial items (Table 1). AD participants performed worse than controls on all components except general contour, and MCI participants did not differ from controls. Among CAA participants, total and visuospatial scores did not correlate with history of stroke, number of microbleeds, or white matter hyperintensity volume (p>0.10).ConclusionMean performance on the clock drawing test is lower in CAA patients without dementia than controls, suggesting visuospatial impairment. This impairment may be related to vascular amyloid deposition in posterior brain regions; however, it was not correlated with typical markers of CAA severity suggesting it may be an early but not necessarily progressive feature. Future research should investigate the mechanisms of visuospatial impairment in CAA—including whether it is related to posterior white matter damage or parieto‐occipital atrophy—and its association with daily function and risk for cognitive decline.

Current findings on the coincidence of cerebral amyloid angiopathy and Alzheimer's disease

Die zerebrale Amyloidangiopathie (CAA) tritt trotz verschiedener Pathomechanismen häufig koinzident zur Alzheimer-Demenz auf. Sie moduliert kognitive Defizite im Rahmen der Alzheimer-Erkrankung (AD) annehmbar durch additive Effekte, auch wenn die diesbezüglichen Zusammenhänge komplex sind. Die pathophysiologische Gemeinsamkeit beider Erkrankungen besteht in einem gestörten Amyloidmetabolismus, distinkt ist jedoch die pathologische Prozessierung von Amyloidvorläuferproteinen. Die CAA mit ihren verschiedenen Subtypen ist eine pathomechanistisch heterogene Gefäßerkrankung des Gehirns. Vaskuläre und parenchymatöse Amyloidablagerungen kommen gemeinsam, aber auch isoliert und unabhängig voneinander vor. Um den spezifischen Beitrag der CAA zu kognitiven Defiziten im Rahmen der AD zu untersuchen, bedarf es daher geeigneter diagnostischer Methoden, die der Komplexität der histopathologischen bzw. bildmorphologischen Charakteristika der CAA gerecht werden, sowie differenzierender testpsychometrischer Verfahren, anhand derer der Beitrag der CAA zu kognitiven Defiziten deskriptiv erfasst und damit ätiologisch besser zuordenbar wird.

Cerebral amyloid angiopathy related inflammation: steroid responsive or spontaneously remissive brain dysfunction?

Definite diagnosis of cerebral amyloid angiopathy related inflammation (CAA-ri) requires histopathological features of vascular inflammation and amyloid deposition. However clinico-radiological criteria for CAA-ri have been recently introduced1 and validated2. Clinical syndrome shows dramatic improvement after immunotherapy3. However, spontaneous remission has been described in some patients4.

Cerebrospinal fluid levels of the neurotrophic factor neuroleukin are increased in early Alzheimer\u2019s disease, but not in cerebral amyloid angiopathy

BackgroundNeuroleukin (NLK) is a protein with neurotrophic properties and is present in a proportion of senile plaques and amyloid laden vessels. It has been suggested that NLK is part of a neuroprotective response to amyloid β-induced cell death. The aim of our study was to investigate the value of cerebrospinal fluid (CSF) NLK levels as a biomarker of vascular amyloid deposition in patients with cerebral amyloid angiopathy (CAA) and in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD).MethodsCSF NLK levels were quantified by ELISA in CAA patients (n = 25) and controls (n = 27) and in two independent samples of aMCI patients, AD patients, and controls: (1) From the Radboud University Medical Center (Nijmegen), we included n = 19 aMCI patients, n = 40 AD patients, and n = 32 controls. (2) From the Hospital of Sant Pau (Barcelona), we included n = 33 aMCI patients, n = 17 AD patients, and n = 50 controls.ResultsCSF NLK levels were similar in CAA patients and controls (p = 0.95). However, we found an elevated CSF concentration of NLK in aMCI (p < 0.0001) and AD patients (p < 0.0001) compared to controls in both samples sets. In addition, we found a correlation of CSF NLK with CSF YKL-40 (age-adjusted-spearman-rank-coefficient = 0.82, p < 0.0001) in aMCI/AD patients, a well-known glial marker of neuro-inflammation.ConclusionsWe found that CSF NLK levels are elevated in aMCI and AD patients compared to controls, but are not increased in CAA patients. CSF NLK levels may be related to an increased neuroinflammatory state in early stages of AD, given its association with YKL-40.

Neurovascular dysfunction and vascular amyloid accumulation as early events in Alzheimer's disease.

Alzheimer's disease (AD) is clinically characterized by a progressive loss of cognitive functions and short-term memory. AD patients present two distinctive neuropathological lesions: neuritic plaques and neurofibrillary tangles (NFTs), constituted of beta-amyloid peptide (Aβ) and phosphorylated and truncated tau proteins. Aβ deposits around cerebral blood vessels (cerebral amyloid angiopathy, CAA) is a major contributor to vascular dysfunction in AD. Vascular amyloid deposits could be early events in AD due to dysfunction in the neurovascular unit (NVU) and the blood-brain barrier (BBB), deterioration of the gliovascular unit, and/or decrease of cerebral blood flow (CBF). These pathological events can lead to decreased Aβ clearance, facilitate a neuroinflammatory environment as well as synaptic dysfunction and, finally, lead to neurodegeneration. Here, we review the histopathological AD hallmarks and discuss the two-hit vascular hypothesis of AD, emphasizing the role of neurovascular dysfunction as an early factor that favors vascular Aβ aggregation and neurodegeneration. Addtionally, we emphasize that pericyte degeneration is a key and early element in AD that can trigger amyloid vascular accumulation and NVU/BBB dysfunction. Further research is required to better understand the early pathophysiological mechanisms associated with NVU alteration and CAA to generate early biomarkers and timely treatments for AD.

Postmortem Neocortical 3H-PiB Binding and Levels of Unmodified and Pyroglutamate Aβ in Down Syndrome and Sporadic Alzheimer's Disease.

Individuals with Down syndrome (DS) have a genetic predisposition for amyloid-β (Aβ) overproduction and earlier onset of Aβ deposits compared to patients with sporadic late-onset Alzheimer’s disease (AD). Positron emission tomography (PET) with Pittsburgh Compound-B (PiB) detects fibrillar Aβ pathology in living people with DS and AD, but its relationship with heterogeneous Aβ forms aggregated within amyloid deposits is not well understood. We performed quantitative in vitro 3H-PiB binding assays and enzyme-linked immunosorbent assays of fibrillar (insoluble) unmodified Aβ40 and Aβ42 forms and N-terminus truncated and pyroglutamate-modified AβNpE3-40 and AβNpE3-42 forms in postmortem frontal cortex and precuneus samples from 18 DS cases aged 43–63 years and 17 late-onset AD cases aged 62–99 years. Both diagnostic groups had frequent neocortical neuritic plaques, while the DS group had more severe vascular amyloid pathology (cerebral amyloid angiopathy, CAA). Compared to the AD group, the DS group had higher levels of Aβ40 and AβNpE3-40, while the two groups did not differ by Aβ42 and AβNpE3-42 levels. This resulted in lower ratios of Aβ42/Aβ40 and AβNpE3-42/AβNpE3-40 in the DS group compared to the AD group. Correlations of Aβ42/Aβ40 and AβNpE3-42/AβNpE3-40 ratios with CAA severity were strong in DS cases and weak in AD cases. Pyroglutamate-modified Aβ levels were lower than unmodified Aβ levels in both diagnostic groups, but within group proportions of both pyroglutamate-modified Aβ forms relative to both unmodified Aβ forms were lower in the DS group but not in the AD group. The two diagnostic groups did not differ by 3H-PiB binding levels. These results demonstrate that compared to late-onset AD cases, adult DS individuals with similar severity of neocortical neuritic plaques and greater CAA pathology have a preponderance of both pyroglutamate-modified AβNpE3-40 and unmodified Aβ40 forms. Despite the distinct molecular profile of Aβ forms and greater vascular amyloidosis in DS cases, cortical 3H-PiB binding does not distinguish between diagnostic groups that are at an advanced level of amyloid plaque pathology. This underscores the need for the development of CAA-selective PET radiopharmaceuticals to detect and track the progression of cerebral vascular amyloid deposits in relation to Aβ plaques in individuals with DS.

Association of clusterin with the BRI2-derived amyloid molecules ABri and ADan

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aβ in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aβ in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aβ interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of these protein folding disorders – suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms.

Pro-inflammatory interleukin-6 signaling links cognitive impairments and peripheral metabolic alterations in Alzheimer\u2019s disease

Alzheimer’s disease (AD) is associated with memory impairment and altered peripheral metabolism. Mounting evidence indicates that abnormal signaling in a brain-periphery metabolic axis plays a role in AD pathophysiology. The activation of pro-inflammatory pathways in the brain, including the interleukin-6 (IL-6) pathway, comprises a potential point of convergence between memory dysfunction and metabolic alterations in AD that remains to be better explored. Using T2-weighted magnetic resonance imaging (MRI), we observed signs of probable inflammation in the hypothalamus and in the hippocampus of AD patients when compared to cognitively healthy control subjects. Pathological examination of post-mortem AD hypothalamus revealed the presence of hyperphosphorylated tau and tangle-like structures, as well as parenchymal and vascular amyloid deposits surrounded by astrocytes. T2 hyperintensities on MRI positively correlated with plasma IL-6, and both correlated inversely with cognitive performance and hypothalamic/hippocampal volumes in AD patients. Increased IL-6 and suppressor of cytokine signaling 3 (SOCS3) were observed in post-mortem AD brains. Moreover, activation of the IL-6 pathway was observed in the hypothalamus and hippocampus of AD mice. Neutralization of IL-6 and inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling in the brains of AD mouse models alleviated memory impairment and peripheral glucose intolerance, and normalized plasma IL-6 levels. Collectively, these results point to IL-6 as a link between cognitive impairment and peripheral metabolic alterations in AD. Targeting pro-inflammatory IL-6 signaling may be a strategy to alleviate memory impairment and metabolic alterations in the disease.

Apolipoprotein E genotype predicts subarachnoid extension in spontaneous intracerebral haemorrhage

Spontaneous intracerebral haemorrhage (ICH) with subarachnoid extension (SAHE) predicts poor outcomes and haematoma expansion in spontaneous ICH and is also a potential predictor of the severity of vascular amyloid deposition. The biological underpinnings of SAHE remain elusive. A study was conducted to identify risk factors associated with SAHE. A retrospective analysis was performed of an ongoing prospective cohort of primary spontaneous supratentorial ICH patients admitted to Tongji Hospital. SAHE was rated on baseline noncontrast computed tomography images by investigators blinded to the clinical data. A total of 189 patients were enrolled. Apolipoprotein E (APOE) ε2 copies (p=0.020), but not APOE ε4 copies (p>0.2), were more common in patients with SAHE in univariate analysis. After controlling for confounding factors in multiple logistic regression, lobar haematoma (odds ratio [OR] 14.21, 95% confidence interval [CI] 5.89-34.33; p<0.001), large haematoma volume (OR 1.04, 95% CI 1.02-1.06; p<0.001) and APOE ε2 copies (OR 3.07, 95% CI 1.05-8.97; p=0.041) were three independent predictors of SAHE. For subgroup analysis stratified by location, APOE ε2 showed a possible association with SAHE in lobar ICH (p=0.026) but not in deep ICH (p>0.2). No significant association was found between APOE ε4 copies and either lobar (p>0.2) or deep ICH (p>0.2). The APOE ε2 allele predicts SAHE in spontaneous supratentorial ICH. The association may predominantly apply to lobar ICH. Given the established relationship between the APOE ε2 allele and pathological cerebrovascular changes, our findings suggest that SAHE involves genetically driven vessel pathology.

ANGIOTENSIN II-INDUCED ER STRESS RESPONSE CONTRIBUTES TO PROFIBROTIC VSMC PHENOTYPE AND SUBSEQUENT HYPERTENSIVE ARTERIAL STIFFNESS

Objective: The renin-angiotensin-aldosterone system is known to play a pivotal role in human hypertension, and specifically angiotensin II (Ang II) is believed to mediate arterial stiffness through maladaptive responses from vascular smooth muscle cells (VSMCs). The objective of this study was to elucidate Ang II activation of the endoplasmic reticulum unfolded protein response (ER UPR) in VSMCs, how this contributes to hypertrophic and fibrotic responses, and how inhibition of ER UPR may mitigate increased aortic pulse propagation velocity (PPV, a surrogate measurement for aortic stiffness). Design and method: ApoE-/- mice were infused with 1 ug/kg/min angiotensin II for 14 days with or without 1 mg/mL 4-PBA For in vitro studies, rat VSMCs were explanted and used from P4-P8. 4-PBA 30 min pretreatment at 1 mM before Ang II 48 hr stimulation or adeno GRP78 infection used to assess chaperoning effect on sirius red collagen assay, THP-1 adhesion, and protein aggregate accumulation. Results: Chronic 14 day infusion of Ang II in apoE-/- mice leads to increased PPV from 0.06 m/s to 0.12 m/s, whereas supplementing drinking water with 1 mg/mL chemical ER chaperone 4-PBA mitigates this induction (PPV = 0.055 m/s), P = 0.039. Fibrosis of the heart, kidney and aorta were reduced with 4-PBA treatment as well. ER stress markers pIRE1a and XBP1S were activated in the thoracic aorta by Ang II infusion and inhibited by 4-PBA. Preamyloid oligomer accumulation induced by Ang II in the aorta was also reduced by 4-PBA. Rat VSMCs stimulated with Ang II were stained with aggregate dye Proteostat, which was increased with 48 hour Ang II treatment, and inhibited with 4-PBA pretreatment (P &lt; 0.05). To evaluate VSMC stiffness promoting phenotype, collagen production and immune cell adhesion were assessed by Sirius Red assay and THP-1 adhesion, respectively. Both inductions by Ang II were decreased by 4-PBA. Overexpression of GRP78 significantly mitigated aggregate formation (P &lt; 0.05) and immune cell infiltration (P &lt; 0.05). Conclusions: Ang II induced pulse pressure and vascular amyloid deposition is decreased by chaperoning nascent protein folding. Targeting GRP78 to reduce ER UPR activation and subsequent disturbances in proteostasis may be a therapeutic strategy for aortic stiffness.

Abstract P367: Brain-Wide Quantification of Amyloid in Transgenic Mice Using Volumetric Imaging and Automated Image Analysis

Localizing and quantifying amyloid in three dimensions furthers understanding of spatial patterns of amyloid deposition and disease progression in experimental models of cerebral amyloid angiopathy (CAA). Serial two-photon tomography (STPT) is ideal for this purpose because it produces 3D datasets of the whole mouse brain in a matter of hours. We present an optimized pipeline for fluorescent whole-brain labeling of amyloid and vasculature, STPT imaging of labeled brains, machine learning-aided classification of vascular and parenchymal amyloid, registration of images to a standard reference atlas, and automated quantification of these signals across brain regions. We used traditional histological sectioning and slide mounted imaging to compare and optimize published methods of fluorescent amyloid and vascular labeling. An intraperitoneal injection of methoxy-X04 delivered the day before euthanasia was chosen to label amyloid. Cerebral vessels are visualized by transcardial perfusion of rhodamine-B isothiocyanate in gelatin. Labeled brains are imaged by STPT and rendered in 3D to appreciate regional amyloid distribution. Volumetric image datasets are quantified using a modification of our custom image analysis pipeline. Pixels are classified as vascular or parenchymal amyloid with supervised machine learning methods. Classified signals are registered into the Allen Institute for Brain Science Common Coordinate Framework version 3.0 for quantification. This strategy produces high resolution volumetric images amenable to automatic quantification of parenchymal and vascular amyloid deposits throughout the brain, appropriate for studies involving large numbers of experimental brains in rodent models of CAA. Figure 1: A 3D rendering of prediction probability maps of a whole labelled mouse brain depicting fluorescent signals that were automatically classified as parenchymal amyloid (cyan), vascular amyloid (yellow) and brain parenchyma.