Multi-analyte proteomic analysis identifies blood-based neuroinflammation, cerebrovascular and synaptic biomarkers in preclinical Alzheimer's disease.

Background: Blood-based biomarkers are gaining grounds for Alzheimer's disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte assessments and the need for markers of neuroinflammation, vascular, and synaptic dysfunction. Here, we evaluated a novel multi-analyte biomarker platform, NULISAseq CNS disease panel, a multiplex NUcleic acid-linked Immuno-Sandwich Assay (NULISA) targeting ∼120 analytes, including classical AD biomarkers and key proteins defining various disease hallmarks. Methods: The NULISAseq panel was applied to 176 plasma samples from the MYHAT-NI cohort of cognitively normal participants from an economically underserved region in Western Pennsylvania. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aß40, and Aß42, were also measured using Single Molecule Array (Simoa). Amyloid pathology, tau pathology, and neurodegeneration were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and MRI, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA biomarkers and AD pathologies. Spearman correlations were used to compare NULISA and Simoa. Results: NULISA concurrently measured 116 plasma biomarkers with good technical performance, and good correlation with Simoa measures. Cross-sectionally, p-tau217 was the top hit to identify Aß pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aß-PET+ participants, including TIMP3, which regulates brain Aß production, the neurotrophic factor BDNF, the energy metabolism marker MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aß PET-dependent yearly increases in Aß-PET+ participants. Markers with tau PET-dependent longitudinal changes included the microglial activation marker CHIT1, the reactive astrogliosis marker CHI3L1, the synaptic protein NPTX1, and the cerebrovascular markers PGF, PDGFRB, and VEFGA; all previously linked to AD but only reliably measured in cerebrospinal fluid. SQSTM1, the autophagosome cargo protein, exhibited a significant association with neurodegeneration status after adjusting age, sex, and APOE ε4 genotype. Conclusions: Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.

A Neuropathology Case Report of a Woman with Down Syndrome who Remained Cognitively Stable.

In this neuropathology case report, we present findings from an individual with Down syndrome (DS) who remained cognitively stable despite Alzheimer's disease (AD) neuropathology. Clinical assessments, fluid biomarkers, neuroimaging, and neuropathological examinations were conducted to characterize her condition. Notably, her ApoE genotype was E2/3, which is associated with a decreased risk of dementia. Neuroimaging revealed stable yet elevated amyloid profiles and moderately elevated tau levels, while neuropathology indicated intermediate AD neuropathologic change with Lewy body pathology and cerebrovascular pathology. Despite the presence of AD pathology, the participant demonstrated intact cognitive functioning, potentially attributed to factors such as genetic variations, cognitive resilience, and environmental enrichment. The findings suggest a dissociation between clinical symptoms and neuropathological changes, emphasizing the complexity of AD progression in DS. Further investigation into factors influencing cognitive resilience in individuals with DS, including comorbidities and social functioning, is warranted. Understanding the mechanisms underlying cognitive stability in DS could offer insights into resilience to AD neuropathology in people with DS and in the general population and inform future interventions.

Alzheimer blood biomarkers: practical guidelines for study design, sample collection, processing, biobanking, measurement and result reporting.

Alzheimer's disease (AD), the most common form of dementia, remains challenging to understand and treat despite decades of research and clinical investigation. This might be partly due to a lack of widely available and cost-effective modalities for diagnosis and prognosis. Recently, the blood-based AD biomarker field has seen significant progress driven by technological advances, mainly improved analytical sensitivity and precision of the assays and measurement platforms. Several blood-based biomarkers have shown high potential for accurately detecting AD pathophysiology. As a result, there has been considerable interest in applying these biomarkers for diagnosis and prognosis, as surrogate metrics to investigate the impact of various covariates on AD pathophysiology and to accelerate AD therapeutic trials and monitor treatment effects. However, the lack of standardization of how blood samples and collected, processed, stored analyzed and reported can affect the reproducibility of these biomarker measurements, potentially hindering progress toward their widespread use in clinical and research settings. To help address these issues, we provide fundamental guidelines developed according to recent research findings on the impact of sample handling on blood biomarker measurements. These guidelines cover important considerations including study design, blood collection, blood processing, biobanking, biomarker measurement, and result reporting. Furthermore, the proposed guidelines include best practices for appropriate blood handling procedures for genetic and ribonucleic acid analyses. While we focus on the key blood-based AD biomarkers for the AT(N) criteria (e.g., amyloid-beta [Aß]40, Aß42, Aß42/40 ratio, total-tau, phosphorylated-tau, neurofilament light chain, brain-derived tau and glial fibrillary acidic protein), we anticipate that these guidelines will generally be applicable to other types of blood biomarkers. We also anticipate that these guidelines will assist investigators in planning and executing biomarker research, enabling harmonization of sample handling to improve comparability across studies.

Novel ultrasensitive immunoassay for the selective quantification of tau oligomers and related soluble aggregates.

INTRODUCTION: Tau aggregation into paired helical filaments and neurofibrillary tangles is characteristic of Alzheimer's disease (AD) and related disorders. However, biochemical assays for the quantification of soluble, earlier-stage tau aggregates are lacking. We describe an immunoassay that is selective for tau oligomers and related soluble aggregates over monomers. METHODS: A homogeneous (single-antibody) immunoassay was developed using a novel anti-tau monoclonal antibody and validated with recombinant and brain tissue-derived tau. RESULTS: The assay signals were concentration dependent for recombinant tau aggregates in solution but not monomers, and recognized peptides within, but not outside, the aggregation-prone microtubule binding region. The signals in inferior and middle frontal cortical tissue homogenates increased with neuropathologically determined Braak staging, and were higher in insoluble than soluble homogenized brain fractions. Autopsy-verified AD gave stronger signals than other neurodegenerative diseases. DISCUSSION: The quantitative oligomer/soluble aggregate-specific assay can identify soluble tau aggregates, including oligomers, from monomers in human and in vitro biospecimens. HIGHLIGHTS: The aggregation of tau to form fibrils and neurofibrillary tangles is a key feature of Alzheimer's disease. However, biochemical assays for the quantification of oligomers/soluble aggregated forms of tau are lacking. We developed a new assay that preferentially binds to soluble tau aggregates, including oligomers and fibrils, versus monomers. The assay signal increased corresponding to the total protein content, Braak staging, and insolubility of the sequentially homogenized brain tissue fractions in an autopsy-verified cohort. The assay recognized tau peptides containing the microtubule binding region but not those covering the N- or C-terminal regions only.

Association Between ß-Amyloid Accumulation and Incident Dementia in Individuals 80 Years or Older Without Dementia.

BACKGROUND AND OBJECTIVES: While the highest prevalence of dementia occurs in individuals older than 80 years, most imaging studies focused on younger populations. The rates of ß-amyloid (Aß) accumulation and the effect of Alzheimer disease (AD) pathology on progression to dementia in this age group remain unexplored. In this study, we examined the relationship between changes in Aß deposition over time and incident dementia in nondemented individuals followed during a period of 11 years. METHODS: We examined 94 participants (age 85.9 + 2.8 years) who had up to 5 measurements of Pittsburgh compound-B (PiB)-PET and clinical evaluations from 2009 to 2020. All 94 participants had 2 PiB-PET scans, 76 participants had 3 PiB-PET scans, 18 participants had 4 PiB-PET scans, and 10 participants had 5 PiB-PET scans. The rates of Aß deposition were compared with 120 nondemented individuals younger than 80 years (69.3 ± 5.4 years) from the Australian Imaging, Biomarker, and Lifestyle (AIBL) study who had 3 or more annual PiB-PET assessments. RESULTS: By 2020, 49% of the participants developed dementia and 63% were deceased. There was a gradual increase in Aß deposition in all participants whether they were considered Aß positive or negative at baseline. In a Cox model controlled for age, sex, education level, APOE-4 allele, baseline Mini-Mental State Examination, and mortality, short-term change in Aß deposition was not significantly associated with incident dementia (HR 2.19 (0.41-11.73). However, baseline Aß burden, cortical thickness, and white matter lesions volume were the predictors of incident dementia. Aß accumulation was faster (p = 0.01) in the older cohort (5.6%/year) when compared with AIBL (4.1%/year). In addition, baseline Aß deposition was a predictor of short-term change (mean time 1.88 years). DISCUSSION: There was an accelerated Aß accumulation in cognitively normal individuals older than 80 years. Baseline Aß deposition was a determinant of incident dementia and short-term change in Aß deposition suggesting that an active Aß pathologic process was present when these participants were cognitively normal. Consequently, age may not be a limiting factor for the use of the emergent anti-Aß therapies.

Biostatistical Estimation of Tau Threshold Hallmarks (BETTH) Algorithm for Human Tau PET Imaging Studies.

A methodology for determining tau PET thresholds is needed to confidently detect early tau deposition. We compared multiple threshold-determining methods in participants who underwent either 18F-flortaucipir or 18F-MK-6240 PET scans. Methods: 18F-flortaucipir (n = 798) and 18F-MK-6240 (n = 216) scans were processed and sampled to obtain regional SUV ratios. Subsamples of the cohorts were based on participant diagnosis, age, amyloid-ß status (positive or negative), and neurodegeneration status (positive or negative), creating older-adult (age ≥ 55 y) cognitively unimpaired (amyloid-ß-negative, neurodegeneration-negative) and cognitively impaired (mild cognitive impairment/Alzheimer disease, amyloid-ß-positive, neurodegeneration-positive) groups, and then were further subsampled via matching to reduce significant differences in diagnostic prevalence, age, and Mini-Mental State Examination score. We used the biostatistical estimation of tau threshold hallmarks (BETTH) algorithm to determine sensitivity and specificity in 6 composite regions. Results: Parametric double receiver operating characteristic analysis yielded the greatest joint sensitivity in 5 of the 6 regions, whereas hierarchic clustering, gaussian mixture modeling, and k-means clustering all yielded perfect joint specificity (2.00) in all regions. Conclusion: When 18F-flortaucipir and 18F-MK-6240 are used, Alzheimer disease-related tau status is best assessed using 2 thresholds, a sensitivity one based on parametric double receiver operating characteristic analysis and a specificity one based on gaussian mixture modeling, delimiting an uncertainty zone indicating participants who may require further evaluation.

Vesicular Glutamate Transporter Changes in the Cortical Default Mode Network During the Clinical and Pathological Progression of Alzheimer's Disease.

BACKGROUND: Altered glutamatergic neurotransmission may contribute to impaired default mode network (DMN) function in Alzheimer's disease (AD). Among the DMN hub regions, frontal cortex (FC) was suggested to undergo a glutamatergic plasticity response in prodromal AD, while the status of glutamatergic synapses in the precuneus (PreC) during clinical-neuropathological AD progression is not known. OBJECTIVE: To quantify vesicular glutamate transporter VGluT1- and VGluT2-containing synaptic terminals in PreC and FC across clinical stages of AD. METHODS: Unbiased sampling and quantitative confocal immunofluorescence of cortical VGluT1- and VGluT2-immunoreactive profiles and spinophilin-labeled dendritic spines were performed in cases with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild-moderate AD (mAD), or moderate-severe AD (sAD). RESULTS: In both regions, loss of VGluT1-positive profile density was seen in sAD compared to NCI, MCI, and mAD. VGluT1-positive profile intensity in PreC did not differ across groups, while in FC it was greater in MCI, mAD, and sAD compared to NCI. VGluT2 measures were stable in PreC while FC had greater VGluT2-positive profile density in MCI compared to sAD, but not NCI or mAD. Spinophilin measures in PreC were lower in mAD and sAD compared to NCI, while in FC they were stable across groups. Lower VGluT1 and spinophilin measures in PreC, but not FC, correlated with greater neuropathology. CONCLUSION: Frank loss of VGluT1 in advanced AD relative to NCI occurs in both DMN regions. In FC, an upregulation of VGluT1 protein content in remaining glutamatergic terminals may contribute to this region's plasticity response in AD.

Astrocyte reactivity influences amyloid-ß effects on tau pathology in preclinical Alzheimer's disease.

An unresolved question for the understanding of Alzheimer's disease (AD) pathophysiology is why a significant percentage of amyloid-ß (Aß)-positive cognitively unimpaired (CU) individuals do not develop detectable downstream tau pathology and, consequently, clinical deterioration. In vitro evidence suggests that reactive astrocytes unleash Aß effects in pathological tau phosphorylation. Here, in a biomarker study across three cohorts (n = 1,016), we tested whether astrocyte reactivity modulates the association of Aß with tau phosphorylation in CU individuals. We found that Aß was associated with increased plasma phosphorylated tau only in individuals positive for astrocyte reactivity (Ast+). Cross-sectional and longitudinal tau-positron emission tomography analyses revealed an AD-like pattern of tau tangle accumulation as a function of Aß only in CU Ast+ individuals. Our findings suggest astrocyte reactivity as an important upstream event linking Aß with initial tau pathology, which may have implications for the biological definition of preclinical AD and for selecting CU individuals for clinical trials.

Plasma Glial Fibrillary Acidic Protein Is Associated with 18F-SMBT-1 PET: Two Putative Astrocyte Reactivity Biomarkers for Alzheimer's Disease.

BACKGROUND: Astrocyte reactivity is an early event along the Alzheimer's disease (AD) continuum. Plasma glial fibrillary acidic protein (GFAP), posited to reflect astrocyte reactivity, is elevated across the AD continuum from preclinical to dementia stages. Monoamine oxidase-B (MAO-B) is also elevated in reactive astrocytes observed using 18F-SMBT-1 PET in AD. OBJECTIVE: The objective of this study was to evaluate the association between the abovementioned astrocyte reactivity biomarkers. METHODS: Plasma GFAP and Aß were measured using the Simoa® platform in participants who underwent brain 18F-SMBT-1 and Aß-PET imaging, comprising 54 healthy control (13 Aß-PET+ and 41 Aß-PET-), 11 mild cognitively impaired (3 Aß-PET+ and 8 Aß-PET-) and 6 probable AD (5 Aß-PET+ and 1 Aß-PET-) individuals. Linear regressions were used to assess associations of interest. RESULTS: Plasma GFAP was associated with 18F-SMBT-1 signal in brain regions prone to early Aß deposition in AD, such as the supramarginal gyrus (SG), posterior cingulate (PC), lateral temporal (LT) and lateral occipital cortex (LO). After adjusting for age, sex, APOE ɛ4 genotype, and soluble Aß (plasma Aß42/40 ratio), plasma GFAP was associated with 18F-SMBT-1 signal in the SG, PC, LT, LO, and superior parietal cortex (SP). On adjusting for age, sex, APOE ɛ4 genotype and insoluble Aß (Aß-PET), plasma GFAP was associated with 18F-SMBT-1 signal in the SG. CONCLUSION: There is an association between plasma GFAP and regional 18F-SMBT-1 PET, and this association appears to be dependent on brain Aß load.

The flutemetamol analogue cyano-flutemetamol detects myocardial AL and ATTR amyloid deposits: a post-mortem histofluorescence analysis.

BACKGROUND: [18F]flutemetamol is a PET radioligand used to image brain amyloid, but its detection of myocardial amyloid is not well-characterized. This histological study characterized binding of fluorescently labeled flutemetamol (cyano-flutemetamol) to amyloid deposits in myocardium. METHODS: Myocardial tissue was obtained post-mortem from 29 subjects with cardiac amyloidosis including transthyretin wild-type (ATTRwt), hereditary/variant transthyretin (ATTRv) and immunoglobulin light-chain (AL) types, and from 10 cardiac amyloid-free controls. Most subjects had antemortem electrocardiography, echocardiography, SPECT and cardiac MRI. Cyano-flutemetamol labeling patterns and integrated density values were evaluated relative to fluorescent derivatives of Congo red (X-34) and Pittsburgh compound-B (cyano-PiB). RESULTS: Cyano-flutemetamol labeling was not detectable in control subjects. In subjects with cardiac amyloidosis, cyano-flutemetamol labeling matched X-34- and cyano-PiB-labeled, and transthyretin- or lambda light chain-immunoreactive, amyloid deposits and was prevented by formic acid pre-treatment of myocardial sections. Cyano-flutemetamol mean fluorescence intensity, when adjusted for X-34 signal, was higher in the ATTRwt than the AL group. Cyano-flutemetamol integrated density correlated strongly with echocardiography measures of ventricular septal thickness and posterior wall thickness, and with heart mass. CONCLUSION: The high selectivity of cyano-flutemetamol binding to myocardial amyloid supports the diagnostic utility of [18F]flutemetamol PET imaging in patients with ATTR and AL types of cardiac amyloidosis.

Application of robust regression in translational neuroscience studies with non-Gaussian outcome data.

Linear regression is one of the most used statistical techniques in neuroscience, including the study of the neuropathology of Alzheimer's disease (AD) dementia. However, the practical utility of this approach is often limited because dependent variables are often highly skewed and fail to meet the assumption of normality. Applying linear regression analyses to highly skewed datasets can generate imprecise results, which lead to erroneous estimates derived from statistical models. Furthermore, the presence of outliers can introduce unwanted bias, which affect estimates derived from linear regression models. Although a variety of data transformations can be utilized to mitigate these problems, these approaches are also associated with various caveats. By contrast, a robust regression approach does not impose distributional assumptions on data allowing for results to be interpreted in a similar manner to that derived using a linear regression analysis. Here, we demonstrate the utility of applying robust regression to the analysis of data derived from studies of human brain neurodegeneration where the error distribution of a dependent variable does not meet the assumption of normality. We show that the application of a robust regression approach to two independent published human clinical neuropathologic data sets provides reliable estimates of associations. We also demonstrate that results from a linear regression analysis can be biased if the dependent variable is significantly skewed, further indicating robust regression as a suitable alternate approach.

Chronic effects of blast injury on the microvasculature in a transgenic mouse model of Alzheimer's disease related Aß amyloidosis.

BACKGROUND: Altered cerebrovascular function and accumulation of amyloid-ß (Aß) after traumatic brain injury (TBI) can contribute to chronic neuropathology and increase the risk for Alzheimer's disease (AD). TBI due to a blast-induced shock wave (bTBI) adversely affects the neurovascular unit (NVU) during the acute period after injury. However, the chronic effects of bTBI and Aß on cellular components of the NVU and capillary network are not well understood. METHODS: We exposed young adult (age range: 76-106 days) female transgenic (Tg) APP/PS1 mice, a model of AD-like Aß amyloidosis, and wild type (Wt) mice to a single bTBI (~ 138 kPa or ~ 20 psi) or to a Sham procedure. At 3-months or 12-months survival after exposure, we quantified neocortical Aß load in Tg mice, and percent contact area between aquaporin-4 (AQP4)-immunoreactive astrocytic end-feet and brain capillaries, numbers of PDGFRß-immunoreactive pericytes, and capillary densities in both genotypes. RESULTS: The astroglia AQP4-capillary contact area in the Tg-bTBI group was significantly lower than in the Tg-Sham group at 3-months survival. No significant changes in the AQP4-capillary contact area were observed in the Tg-bTBI group at 12-months survival or in the Wt groups. Capillary density in the Tg-bTBI group at 12-months survival was significantly higher compared to the Tg-Sham control and to the Tg-bTBI 3-months survival group. The Wt-bTBI group had significantly lower capillary density and pericyte numbers at 12-months survival compared to 3-months survival. When pericytes were quantified relative to capillary density, no significant differences were detected among the experimental groups, for both genotypes. CONCLUSION: In conditions of high brain concentrations of human Aß, bTBI exposure results in reduced AQP4 expression at the astroglia-microvascular interface, and in chronic capillary proliferation like what has been reported in AD. Long term microvascular changes after bTBI may contribute to the risk for developing chronic neurodegenerative disease later in life.

Assessing Reactive Astrogliosis with 18F-SMBT-1 Across the Alzheimer Disease Spectrum.

A neuroinflammatory reaction in Alzheimer disease (AD) brains involves reactive astrocytes that overexpress monoamine oxidase-B (MAO-B). 18F-(S)-(2-methylpyrid-5-yl)-6-[(3-fluoro-2-hydroxy)propoxy]quinoline (18F-SMBT-1) is a novel 18F PET tracer highly selective for MAO-B. We characterized the clinical performance of 18F-SMBT-1 PET across the AD continuum as a potential surrogate marker of reactive astrogliosis. Methods: We assessed 18F-SMBT-1 PET regional binding in 77 volunteers (76 ± 5.5 y old; 41 women, 36 men) across the AD continuum: 57 who were cognitively normal (CN) (44 amyloid-ß [Aß]-negative [Aß-] and 13 Aß-positive [Aß+]), 12 who had mild cognitive impairment (9 Aß- and 3 Aß+), and 8 who had AD dementia (6 Aß+ and 2 Aß-). All participants also underwent Aß and tau PET imaging, 3-T MRI, and neuropsychologic evaluation. Tau imaging results were expressed in SUV ratios using the cerebellar cortex as a reference region, whereas Aß burden was expressed in centiloids. 18F-SMBT-1 outcomes were expressed as SUV ratio using the subcortical white matter as a reference region. Results: 18F-SMBT-1 yielded high-contrast images at steady state (60-80 min after injection). When compared with the Aß- CN group, there were no significant differences in 18F-SMBT-1 binding in the group with Aß- mild cognitive impairment. Conversely, 18F-SMBT-1 binding was significantly higher in several cortical regions in the Aß+ AD group but also was significantly lower in the mesial temporal lobe and basal ganglia. Most importantly, 18F-SMBT-1 binding was significantly higher in the same regions in the Aß+ CN group as in the Aß- CN group. When all clinical groups were considered together, 18F-SMBT-1 correlated strongly with Aß burden and much less with tau burden. Although in most cortical regions 18F-SMBT-1 did not correlate with brain volumetrics, regions known for high MAO-B concentrations presented a direct association with hippocampal and gray matter volumes, whereas the occipital lobe was directly associated with white matter hyperintensity. 18F-SMBT-1 binding was inversely correlated with Mini Mental State Examination and the Australian Imaging Biomarkers and Lifestyle's Preclinical Alzheimer Cognitive Composite in some neocortical regions such as the frontal cortex, lateral temporal lobe, and supramarginal gyrus. Conclusion: Cross-sectional human PET studies with 18F-SMBT-1 showed that Aß+ AD patients, but most importantly, Aß+ CN individuals, had significantly higher regional 18F-SMBT-1 binding than Aß- CN individuals. Moreover, in several regions in the brain, 18F-SMBT-1 retention was highly associated with Aß load. These findings suggest that increased 18F-SMBT-1 binding is detectable at the preclinical stages of Aß accumulation, providing strong support for its use as a surrogate marker of astrogliosis in the AD continuum.

Direct Comparison of the Tau PET Tracers 18F-Flortaucipir and 18F-MK-6240 in Human Subjects.

Tau PET tracers exhibit varying levels of specific signal and distinct off-target binding patterns that are more diverse than amyloid PET tracers. This study compared 2 frequently used tau PET tracers, 18F-flortaucipir and 18F-MK-6240, in the same subjects. Methods:18F-flortaucipir and 18F-MK-6240 scans were collected within 2 mo in 15 elderly subjects varying in clinical diagnosis and cognition. FreeSurfer, version 5.3, was applied to 3-T MR images to segment Braak pathologic regions (I-VI) for PET analyses. Off-target binding was assessed in the choroid plexus, meninges, and striatum. SUV ratio (SUVR) outcomes were determined over 80-100 min (18F-flortaucipir) or 70-90 min (18F-MK-6240) normalized to cerebellar gray matter. Masked visual interpretation of images was performed by 5 raters for both the medial temporal lobe and the neocortex, and an overall (majority) rating was determined. Results: Overall visual ratings showed complete concordance between radiotracers for both the medial temporal lobe and the neocortex. SUVR outcomes were highly correlated (r2 > 0.92; P ≪ 0.001) for all Braak regions except Braak II. The dynamic range of SUVRs in target regions was approximately 2-fold higher for 18F-MK-6240 than for 18F-flortaucipir. Cerebellar SUVs were similar for 18F-MK-6240 and 18F-flortaucipir, suggesting that differences in SUVRs are driven by specific signals. Apparent off-target binding was observed often in the striatum and choroid plexus with 18F-flortaucipir and most often in the meninges with 18F-MK-6240. Conclusion: Both 18F-MK-6240 and 18F-flortaucipir are capable of quantifying signal in a common set of brain regions that develop tau pathology in Alzheimer disease; these tracers perform equally well in visual interpretations. Each also shows distinct patterns of apparent off-target binding. 18F-MK-6240 showed a greater dynamic range in SUVR estimates, which may be an advantage in detecting early tau pathology or in performing longitudinal studies to detect small interval changes.

11C-PiB PET can underestimate brain amyloid-ß burden when cotton wool plaques are numerous.

Individuals with familial Alzheimer's disease due to PSEN1 mutations develop high cortical fibrillar amyloid-ß load but often have lower cortical 11C-Pittsburgh compound B (PiB) retention than Individuals with sporadic Alzheimer's disease. We hypothesized this is influenced by limited interactions of Pittsburgh compound B with cotton wool plaques, an amyloid-ß plaque type common in familial Alzheimer's disease but rare in sporadic Alzheimer's disease. Histological sections of frontal and temporal cortex, caudate nucleus and cerebellum were obtained from 14 cases with sporadic Alzheimer's disease, 12 cases with familial Alzheimer's disease due to PSEN1 mutations, two relatives of a PSEN1 mutation carrier but without genotype information and three non-Alzheimer's disease cases. Sections were processed immunohistochemically using amyloid-ß-targeting antibodies and the fluorescent amyloid stains cyano-PiB and X-34. Plaque load was quantified by percentage area analysis. Frozen homogenates from the same brain regions from five sporadic Alzheimer's disease and three familial Alzheimer's disease cases were analysed for 3H-PiB in vitro binding and concentrations of amyloid-ß1-40 and amyloid-ß1-42. Nine sporadic Alzheimer's disease, three familial Alzheimer's disease and three non-Alzheimer's disease participants had 11C-PiB PET with standardized uptake value ratios calculated using the cerebellum as the reference region. Cotton wool plaques were present in the neocortex of all familial Alzheimer's disease cases and one sporadic Alzheimer's disease case, in the caudate nucleus from four familial Alzheimer's disease cases, but not in the cerebellum. Cotton wool plaques immunolabelled robustly with 4G8 and amyloid-ß42 antibodies but weakly with amyloid-ß40 and amyloid-ßN3pE antibodies and had only background cyano-PiB fluorescence despite labelling with X-34. Relative to amyloid-ß plaque load, cyano-Pittsburgh compound B plaque load was similar in sporadic Alzheimer's disease while in familial Alzheimer's disease it was lower in the neocortex and the caudate nucleus. In both regions, insoluble amyloid-ß1-42 and amyloid-ß1-40 concentrations were similar in familial Alzheimer's disease and sporadic Alzheimer's disease groups, while 3H-PiB binding was lower in the familial Alzheimer's disease than the sporadic Alzheimer's disease group. Higher amyloid-ß1-42 concentration associated with higher 3H-PiB binding in sporadic Alzheimer's disease but not familial Alzheimer's disease. 11C-PiB retention correlated with region-matched post-mortem amyloid-ß plaque load; however, familial Alzheimer's disease cases with abundant cotton wool plaques had lower 11C-PiB retention than sporadic Alzheimer's disease cases with similar amyloid-ß plaque loads. PiB has limited ability to detect amyloid-ß aggregates in cotton wool plaques and may underestimate total amyloid-ß plaque burden in brain regions with abundant cotton wool plaques.

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

Individuals with Down syndrome (DS) have a genetic predisposition for amyloid-ß (Aß) overproduction and earlier onset of Aß deposits compared to patients with sporadic late-onset Alzheimer's disease (AD). Positron emission tomography (PET) with Pittsburgh Compound-B (PiB) detects fibrillar Aß pathology in living people with DS and AD, but its relationship with heterogeneous Aß 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 Aß40 and Aß42 forms and N-terminus truncated and pyroglutamate-modified AßNpE3-40 and AßNpE3-42 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 Aß40 and AßNpE3-40, while the two groups did not differ by Aß42 and AßNpE3-42 levels. This resulted in lower ratios of Aß42/Aß40 and AßNpE3-42/AßNpE3-40 in the DS group compared to the AD group. Correlations of Aß42/Aß40 and AßNpE3-42/AßNpE3-40 ratios with CAA severity were strong in DS cases and weak in AD cases. Pyroglutamate-modified Aß levels were lower than unmodified Aß levels in both diagnostic groups, but within group proportions of both pyroglutamate-modified Aß forms relative to both unmodified Aß 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 AßNpE3-40 and unmodified Aß40 forms. Despite the distinct molecular profile of Aß 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 Aß plaques in individuals with DS.

Visual assessment of [18F]flutemetamol PET images can detect early amyloid pathology and grade its extent.

PURPOSE: To investigate the sensitivity of visual read (VR) to detect early amyloid pathology and the overall utility of regional VR. METHODS: [18F]Flutemetamol PET images of 497 subjects (ALFA+ N = 352; ADC N = 145) were included. Scans were visually assessed according to product guidelines, recording the number of positive regions (0-5) and a final negative/positive classification. Scans were quantified using the standard and regional Centiloid (CL) method. The agreement between VR-based classification and published CL-based cut-offs for early (CL = 12) and established (CL = 30) pathology was determined. An optimal CL cut-off maximizing Youden's index was derived. Global and regional CL quantification was compared to VR. Finally, 28 post-mortem cases from the [18F]flutemetamol phase III trial were included to assess the percentage agreement between VR and neuropathological classification of neuritic plaque density. RESULTS: VR showed excellent agreement against CL = 12 (κ = .89, 95.2%) and CL = 30 (κ = .88, 95.4%) cut-offs. ROC analysis resulted in an optimal CL = 17 cut-off against VR (sensitivity = 97.9%, specificity = 97.8%). Each additional positive VR region corresponded to a clear increase in global CL. Regional VR was also associated with regional CL quantification. Compared to mCERADSOT-based classification (i.e., any region mCERADSOT > 1.5), VR was in agreement in 89.3% of cases, with 13 true negatives, 12 true positives, and 3 false positives (FP). Regional sparse-to-moderate neuritic and substantial diffuse Aß plaque was observed in all FP cases. Regional VR was also associated with regional plaque density. CONCLUSION: VR is an appropriate method for assessing early amyloid pathology and that grading the extent of visual amyloid positivity could present clinical value.

What Is T+? A Gordian Knot of Tracers, Thresholds, and Topographies.

In this review we examine, in the context of the amyloid, tau, and neurodegeneration framework, the available evidence and potential alternatives on how to establish tau positivity (T+) for multiple tau-imaging tracers in order to reach a consensus on normal and abnormal tau imaging values that can be universally implemented in clinical research and therapeutic trials.

Development of a PET radioligand selective for cerebral amyloid angiopathy.

INTRODUCTION: Positron emission tomography (PET) using radiolabeled amyloid-binding compounds has advanced the field of Alzheimer's disease (AD) by enabling detection and longitudinal tracking of fibrillar amyloid-ß (Aß) deposits in living people. However, this technique cannot distinguish between Aß deposits in brain parenchyma (amyloid plaques) from those in blood vessels (cerebral amyloid angiopathy, CAA). Development of a PET radioligand capable of selectively detecting CAA would help clarify its contribution to global brain amyloidosis and clinical symptoms in AD and would help to characterize side-effects of anti-Aß immunotherapies in AD patients, such as CAA. METHODS: A candidate CAA-selective compound (1) from a panel of analogues of the amyloid-binding dye Congo red was synthesized. The binding affinity to Aß fibrils and lipophilicity of compound 1 were determined and selectivity for CAA versus parenchymal plaque deposits was assessed ex-vivo and in-vivo in transgenic APP/PS1 mice and in postmortem human brain affected with AD pathology. RESULTS: Compound 1 displays characteristics of Aß binding dyes, such as thioflavin-S, in that it labels both parenchymal Aß plaques and CAA when applied to histological sections from both a transgenic APP/PS1 mouse model of Aß amyloidosis and AD brain. Thus, compound 1 lacks molecular selectivity to distinguish Aß deposits in CAA from those in plaques. However, when administered to living APP/PS1 mice intravenously, compound 1 preferentially labels CAA when assessed using in-vivo two-photon microscopy and ex-vivo histology and autoradiography. CONCLUSION: We hypothesize that selectivity of compound 1 for CAA is attributable to its limited penetration of the blood-brain barrier due to the highly polar nature of the carboxylate moiety, thereby limiting access to parenchymal plaques and promoting selective in-vivo labeling of Aß deposits in the vascular wall (i.e., "delivery selectivity").

Modeling Aß42 Accumulation in Response to Herpes Simplex Virus 1 Infection: 2D or 3D?

Alzheimer's disease is a progressive neurodegenerative disease characterized neuropathologically by presence of extracellular amyloid plaques composed of fibrillar amyloid beta (Aß) peptides and intracellular neurofibrillary tangles. Post-mortem and in vivo studies implicate HSV-1 infection in the brain as a precipitating factor in disease/pathology initiation. HSV-1 infection of two-dimensional (2D) neuronal cultures causes intracellular accumulation of Aß42 peptide, but these 2D models do not recapitulate the three-dimensional (3D) architecture of brain tissue.We employed human induced pluripotent stem cells (hiPSCs) to compare patterns of Aß42 accumulation in HSV-1 infected 2D (neuronal monolayers) and 3D neuronal cultures (brain organoids). Akin to prior studies, HSV-1-infected 2D cultures showed Aß42 immunoreactivity in cells expressing the HSV-1 antigen ICP4 (ICP4+). Conversely, accumulation of Aß42 in ICP4+ cells in infected organoids was rarely observed. These results highlight the importance of considering 3D cultures to model host-pathogen interaction.IMPORTANCE The "pathogen" hypothesis of Alzheimer's disease (AD) proposes that brain HSV-1 infection could be an initial source of amyloid beta (Aß) peptide-containing amyloid plaque development. Aß accumulation was reported in HSV-1-infected 2D neuronal cultures and neural stem cell cultures, as well as in HSV-1-infected 3D neuronal culture models.The current study extends these findings by showing different patterns of Aß42 accumulation following HSV-1 infection of 2D compared to 3D neuronal cultures (brain organoids). Specifically, 2D neuronal cultures showed Aß42-immunoreactivity mainly in HSV-1-infected cells and only rarely in uninfected cells or infected cells exposed to antivirals. Conversely, 3D brain organoids showed accumulation of Aß42 mainly in non-infected cells surrounding HSV-1-infected cells. We suggest that because brain organoids better recapitulate architectural features of a developing brain than 2D cultures, they may be a more suitable model to investigate the involvement of HSV-1 in the onset of AD pathology.