BackgroundThe cellular mechanisms that promote the maintenance of cognitive abilities in very old people designated as successful agers remain under-investigated. Here, we report an episodic memory performance-based criteria that differentiates superior cognitive function from normative cognitive function in adults aged 80 and older.ObjectiveUsing this new criteria, we demonstrate how neuropathological and neurobiological underpinnings of superior cognitive performance can be investigated.MethodsThe most recent verbal episodic memory WMS-R Logical Memory Delayed Recall (LM-DR) score was derived from 144 participants with no cognitive impairment (NCI) 80 years or older participants from the Rush Religious Orders Study classified with Superior Cognitive Performance (SCP, LM-DR ≥ 14) or Normal Cognitive Performance (NCP, LM-DR 13 ≥ 7). Both groups were compared on neuropathological measures for neuritic plaque (NP), diffuse plaque (DP), and neurofibrillary tangle (NFT) load.ResultsNP (p = 0.44), DP (p = 0.27), and NFT (p = 0.28) burden did not differ between SCP and NCP cases. LM-DR scores did not correlate with NP (r = -0.08, p = 0.32), DP (r = -0.14, p = 0.07), or NFT (r = -0.12, p = 0.13) load. Biochemical analysis revealed significantly higher levels of heat-shock protein HSPB6 in SCP compared to NCP (p < 0.001).ConclusionsHeat shock protein differences were observed between NCP and SCP groups. This suggests that our proposed criteria for SCP can help identify neurobiological mechanisms of successful cognitive aging. Our SCP criteria are also concordant with the SuperAger criteria which supports the generalizability of the SCP criteria to other datasets.

A major pathological hallmark of Alzheimer's disease (AD) is plaque deposition of amyloid-β (Aβ) peptide which is cleaved from a larger Aβ precursor protein (APP). One of the most common causes of autosomal dominant AD (ADAD) are mutations in the PSEN1 gene, encoding a component of γ-secretase, which alter APP processing and increase the production and aggregation potential of Aβ. However, the impact of PSEN1 mutations on blood biomarker levels of neurodegeneration-related proteins is largely unexplored. We applied a novel Nucleic Acid Linked Immuno-Sandwich Assay (NULISA), the NULISAseq CNS Disease Panel, to quantify 127 key neurodegenerative proteins in plasma samples from a cohort consisting of ADAD cases with PSEN1 mutations ( n  = 6), neuropathologically diagnosed sporadic AD (sAD; n  = 8), and cognitively unimpaired controls ( n  = 7). Normalized protein quantification (NPQ) was used as a proxy for protein levels. Statistical comparisons were performed using the Kriskal-Wallis and Wilcoxon rank-sum tests. Spearman correlation was employed to evaluate the association between NULISA and Single Molecule Array (SIMOA)-based measurements. The NULISA had high detectability (95.5% ± 13.6%) and reproducibility (median intra-plate coefficient of variation [CV]=6.3%). NULISA and SIMOA correlated robustly for p -tau181, p -tau217, GFAP, NfL, and Aβ42 (Spearman coefficients ρ range 0.721 to 0.947), but less so for Aβ40 (ρ=0.302). Total-tau (MAPT), p -tau181, p -tau217, and p -tau231 were among the proteins most significantly different across the sample groups ( p  values range 0.001 to 0.003), followed by NfL, NfH, CCL2, GFAP, and PDGFRB ( p  values range 0.005 to 0.046). Several targets, including NfH ( p  = 0.001), CCL11 ( p  = 0.013), CHIT1 ( p  = 0.021), PDGFRB ( p  = 0.029), PTN ( p  = 0.029), and CNTN2 ( p  = 0.043), showed statistically significant differences between ADAD and sAD. Proteomic profiling of key neurodegenerative proteins in plasma revealed significant differences among the sample groups and include identified targets that can distinguish ADAD from sAD. Further validation with larger cohorts is needed to confirm these findings.

INTRODUCTION:Neuroimaging studies report associations of amyloid beta (Aβ) positron emission tomography (PET) with [18F](S)-(2-methylpyrid-5-yl)-6-[(3-fluoro-2-hydroxy)propoxy]quinoline ([18F]SMBT-1), a novel reactive astrogliosis surrogate radiotracer with high affinity for monoamine oxidase B (MAO-B) in Alzheimer's disease (AD). Postmortem association of [18F]SMBT-1 binding with pathology of AD and non-AD tauopathies is undefined. METHODS:[18F]SMBT-1 binding, [3H]Pittsburgh compound B ([3H]PiB) binding, and MAO-B activity assays in brain homogenates, with [18F]SMBT-1 autoradiography and MAO-B immunoreactivity in relation to glial fibrillary acidic protein (GFAP), major histocompatibility complex II (MHC-II), Aβ, phosphorylated tau, cyano-Pittsburgh compound B (cyano-PiB), and X-34 on brain sections from AD, non-AD tauopathies, and nondemented controls. RESULTS:[18F]SMBT-1 binding correlated with MAO-B activity and [3H]PiB binding, with highest levels in AD. [18F]SMBT-1 autoradiography corresponded to MAO-B/GFAP-immunoreactive astrocytes associated with Aβ deposits in plaques and vasculature, more closely than to tau pathology. [18F]SMBT-1 binding and MAO-B activity in non-AD tauopathies partially overlapped controls and AD, with MAO-B/GFAP-immunoreactive astrocytes in areas with tau pathology. DISCUSSION:[18F]SMBT-1 is a promising biomarker of MAO-B reactive astrocytes in AD and non-AD tauopathies.

INTRODUCTION:Most available phosphorylated tau (p-tau)217 immunoassays have similar performance. It is unclear if this is due to the use of the same antibody (the "ALZpath antibody"). We established and evaluated a novel p-tau217 assay that uses an alternative antibody and benchmarked the results against ALZpath-p-tau217. METHODS:After development and analytical validation of the University of Pittsburgh ("Pitt-p-tau217") method, clinical verification was performed in three independent cohorts (n = 363). RESULTS:Pitt-p-tau217 demonstrated high between-run stability, linearity, and specificity. Clinically, Pitt-p-tau217 differentiated neuropathologically confirmed PSEN1 mutation carriers from controls with area under the curve (AUC) = 0.94, and amyloid beta (Aβ) positron emission tomography (PET)-positive from Aβ PET-negative cognitively normal older adults with AUC up to 0.84, equivalent to ALZpath-p-tau217 results. Both Pitt-p-tau217 and ALZpath-p-tau217 were slightly elevated in tau PET-positive versus tau PET-negative participants. Between-assay correlations were up to 0.93. DISCUSSION:The new Pitt-p-tau217 assay exhibits high and reproducible classification accuracies for identifying individuals with biological evidence of Alzheimer's disease, equivalent to the widely used ALZpath-p-tau217. HIGHLIGHTS:We designed and developed an alternative assay to quantify plasma phosphorylated tau (p-tau)217, aiming to enhance accuracy and enable early detection of Alzheimer's disease (AD). Comprehensive analytical and clinical validation demonstrated that the new p-tau217 assay is a valuable and affordable resource for investigating AD pathophysiology. The new p-tau217 assay showed similar performance to the established ALZpath assay in staging and monitoring early AD.

Neurofibrillary tangles (NFT), consisting of hyperphosphorylated tau aggregates, are one of the major pathological hallmarks of Alzheimer’s disease (AD). The burden of NFTs correlates with cognitive decline, and in vivo detection of NFT may help predict the clinical progression of AD. Mass spectrometry-based proteomic analysis of brain regions affected by NFTs holds the potential to unveil the molecular mechanisms underlying tau pathogenesis and uncover novel diagnostic/prognostic biomarkers and therapeutic targets. Post-mortem frozen samples of inferior temporal and middle frontal cortex were collected from 9 cases with different severity of tau pathology assessed by Braak NFT stage. Proteins were fractionated into three fractions using sequential extraction with lysis buffers with different solubility strengths (TBS, Na2CO3, and Urea), trypsin-digested, and analyzed using label-free nano-flow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Linear mixed models were employed to assess the significance of proteins showing a differential abundance in tissues with Braak NFT stages 0-III and IV-VI or displaying Braak stage-dependent solubility changes. A total of 64908 peptides originating from 5475 protein groups were identified through the analysis of 18 brain tissue samples. Differential proteomic analysis revealed several proteins with significantly varied abundance in tissues with different severity of tau pathology. The most significant proteins among these include COL25A1, SNRNP70, MDK, ASPRV1, CLMN, DOCK5, HTRA1, LNPEP, SQSTM1 and Aβ peptides. Additionally, a number of proteins exhibited Braak-stage dependent solubility change; a significant subset of these proteins, including SNRNP70, SNRPGP15, PRPF6, SNRPE, SNRPD1, SNRPB, and SNRPD3, is involved in the spliceosome-mediated mRNA splicing. Our study reveals numerous proteins exhibiting a significant association with tau pathology. These results align well with published findings that suggest the involvement of alternative splicing in reprogramming gene expression in the pathogenesis of AD. Further validation will be needed to assess their potential as therapeutic targets and/or diagnostic/prognostic biomarkers for AD pathologies.

Patients with Alzheimer's disease (AD) with little or no quantifiable insoluble brain tau neurofibrillary tangle (NFT) pathology demonstrate stronger clinical benefits of therapies than those with advanced NFTs. The formation of NFTs can be prevented by targeting the intermediate soluble tau assemblies (STAs). However, biochemical understanding and biomarkers of STAs are lacking. We show that Tris-buffered saline-soluble tau aggregates from autopsy-verified AD brain tissues include the core sequence ~tau258-368. In neuropathological assessments, antibodies against the phosphorylation sites serine-262 and serine-356 within the STA core almost exclusively stained granular (that is, prefibrillar) tau aggregates in pre-NFTs while antibodies against phosphorylation at serine-202 and threonine-205 and threonine-231, outside the STA core, stained the entire spectrum of tau aggregates in pre-NFTs and mature NFTs, dystrophic neurites and neuropil threads in the hippocampus. Functionally, a recombinantly produced STA core peptide robustly altered neuronal excitability and synaptic transmission in mouse hippocampal brain slices. Furthermore, we developed a cerebrospinal fluid assay that differentiated STAs in AD from non-AD tauopathies, correlated with the severity of NFT burden and cognitive decline independently of amyloid beta deposition, and with tau positron emission tomography uptake across Braak NFT stages. Together, our findings inform about the status of early-stage tau aggregation, reveal aggregation-relevant phosphorylation epitopes in tau and offer a diagnostic biomarker and targeted therapeutic opportunities for AD.

Specific PSEN1 mutations cause early-onset AD but their effects on blood biomarker levels are unknown. We evaluated autopsy-confirmed individuals affected by six different PSEN1 mutations; two of known (L381V, C410Y) and three (A426P/E318G, M233L, and V261I) of unknown pathogenic status. The sixth patient had Autosomal Dominant AD (ADAD) not yet genotyped. Neuropathologically diagnosed sporadic AD (sAD; n=8) and unaffected controls (n=7) were included for comparison. The participants, except the controls, had brain autopsy performed under informed consent at the University of Pittsburgh Alzheimer’s Disease Research Center. Genetic testing was performed to confirm the presence of PSEN1 mutation(s). Blood was collected, processed, and stored during life. Plasma p-tau217, p-tau181, neurofilament light (NfL), glial fibrillary acidic protein (GFAP), amyloid beta (Ab) 40 and 42 were measured using commercial assays on Simoa HDX at the Biofluid Biomarker Laboratory, Department of Psychiatry, University of Pittsburgh. Plasma p-tau217 and p-tau181 were increased 5-8-fold and 3-4-fold in the PSEN1 and sAD groups respectively versus controls. GFAP had 4-fold increase in both sAD and PSEN1 groups while NfL was highest in sAD. Ab42, Ab40 and Ab42/40 showed no between-group differences. Individuals with the two pathogenic mutations recorded the highest levels of p-tau217, p-tau181, GFAP and NfL, with the A426P/E318G carrier also having a comparably high p-tau181. Marked elevation of plasma p-tau and GFAP suggest increased tau phosphorylation and astrogliosis in ADAD while neurodegeneration intensity (by NfL) may be comparable to sAD.

Plasma phospho-tau217 (p-tau217) is a promising blood-based biomarkers for Alzheimer's disease (AD). However, the accessibility of pTau217 tests for both research and clinical applications has been constrained. Previous studies focused on highly-phenotyped cohorts that differ substantially from the wider population. To broaden the access to this highly precise biomarker for AD, we developed a novel blood-based immunoassay for p-tau217 at the University of Pittsburgh (Pitt-p-tau217). Following thorough analytical validation, we evaluated the clinical utility of the assay in four independent real-world cohorts (n=503 total) reflective of the wider population. A two-step ultra-sensitive p-tau217 immunoassay was developed on the Quanterix HDX Simoa platform. Analytical validation focused on dilution linearity, competitive binding, day-to-day stability, and spike recovery. Clinical validation was performed in four cohorts including: two population-based, racially-diverse cohorts in medically underserved communities (the MYHAT-Neuroimaging and Human Connectome cohorts); and a cohort of neuropathologically-confirmed familial AD participants carrying pathogenic PSEN1 mutations. The fourth cohort included older adults >65 years attending a memory clinic. Assay performance was compared with the commercially-available ALZpath-p-tau217 assay. The new plasma Pitt-p-tau217 assay demonstrated high between-run stability, linearity of dilution, and substantial signal reduction when a p-tau217-positive antigen was allowed to competitively bind to the p-tau217 target in plasma samples before assay measurement. Clinically, the assay differentiated amyloid-beta (Aβ) PET-positive and -negative cognitively unimpaired individuals with areas under the curve (AUCs) of 0.84-0.87, equivalent to 0.87-0.90 for ALZPath and correlations of 0.40-0.73. According to centiloids (CL), the Pitt-p-tau217 assay showed stepwise higher values from Aβ-negative (CL<15) to low-burden Aβ (CL15-25) and Aβ-positive (CL>25). By tau-PET status, Pitt-p-tau217 was elevated in tau-PET-positive versus negative (P<0.001) and separated the groups with the same AUC of 0.71 as ALZpath-p-tau217. Furthermore, Pitt-p-tau217 was 5.3-fold and 3.3-fold higher in neuropathologically-confirmed PSEN1 mutation carriers and sporadic AD participants respectively versus controls, with correlation of 0.93 with ALZpath-p-tau217. Our results indicate that the newly-developed Pitt-p-tau217 assay, the first from an academic laboratory in North America, exhibits reliable and reproducible diagnostic accuracy for identifying individuals with biological evidence of AD. Demonstration of assay performance in inclusive cohorts suggests its suitability for use in the general population.

Abstract Background Molecular biomarkers of chronic traumatic encephalopathy (CTE) are lacking. We evaluated 18F-MK-6240 tau PET as a biomarker for CTE. Two studies were done: (1) 3H-MK-6240 autoradiography and an in-vitro brain homogenate binding studies on postmortem CTE tissue, (2) an in-vivo 18F-MK-6240 tau PET study in former American football players. Methods Autoradiography and in-vitro binding studies were done using 3H-MK-6240 on frozen temporal and frontal cortex tissue from six autopsy cases with stage III CTE compared to Alzheimer’s disease. Thirty male former National Football League (NFL) players with cognitive concerns (mean age = 58.9, SD = 7.8) completed tau (18F-MK-6240) and Aβ (18F-Florbetapir) PET. Controls included 39 Aβ-PET negative, cognitively normal males (mean age = 65.7, SD = 6.3). 18F-MK-6240 SUVr images were created using 70–90 min post-injection data with inferior cerebellar gray matter as the reference. We compared SUVr between players and controls using voxelwise and region-of-interest approaches. Correlations between 18F-MK-6240 SUVr and cognitive scores were tested. Results All six CTE stage III cases had Braak NFT stage III but no neuritic plaques. Two had Thal Phase 1 for Aβ; one showed a laminar pattern of 3H-MK-6240 autoradiography binding in the superior temporal cortex and less so in the dorsolateral frontal cortex, corresponding to tau-immunoreactive lesions detected using the AT8 antibody (pSer202/pThr205 tau) in adjacent tissue sections. The other CTE cases had low frequencies of cortical tau-immunoreactive deposits and no well-defined autoradiography binding. In-vitro 3H-MK-6240 binding studies to CTE brain homogenates in the case with autoradiography signal indicated high binding affinity (KD = 2.0 ± 0.9 nM, Bmax = 97 ± 24 nM, n = 3). All NFL players had negative Aβ-PET. There was variable, low-to-intermediate intensity 18F-MK-6240 uptake across participants: 16 had no cortical signal, 7 had medial temporal lobe (MTL) uptake, 2 had frontal uptake, and 4 had MTL and frontal uptake. NFL players had higher SUVr in the entorhinal cortex (d = 0.86, p = 0.001), and the parahippocampal gyrus (d = 0.39, p = 0.08). Voxelwise regressions showed increased uptake in NFL players in two bilateral anterior MTL clusters (p < 0.05 FWE). Higher parahippocampal and frontal–temporal SUVrs correlated with worse memory (r = -0.38, r = -0.40) and semantic fluency (r = -0.38, r = -0.48), respectively. Conclusion We present evidence of 3H-MK-6240 in-vitro binding to post-mortem CTE tissue homogenates and in vivo 18F-MK-6240 PET binding in the MTL among a subset of participants. Additional studies in larger samples and PET-to-autopsy correlations are required to further elucidate the potential of 18F-MK-6240 to detect tau pathology in CTE.

Phosphorylated tau proteins accumulate in pathological aggregates which define neurodegenerative tauopathies, including Alzheimer’s disease (AD). Insight into the early stages of tau polymerization/aggregation, including early hyperphosphorylation events, is critical for identification of biomarkers of incipient disease as well as novel therapy targets. We analyzed postmortem tissue sections of hippocampus from AD cases and middle frontal gyrus from non-AD cases with mainly 4R tau isoforms (progressive supranuclear palsy, PSP; corticobasal degeneration, CBD; aging related tau astrogliopathy, ARTAG) or 3R tau (Pick’s disease, PiD). Immunohistochemical and multiple fluorescence methods were used to assess the labeling patterns of antibodies directed to phospo-epitopes within soluble non-fibrillar tau core (pSer262), both soluble and fibril core (pSer356), or outside of either core region (pSer202/pThr205, pThr231). In sections of hippocampus from AD cases, pSer262 and pSer356 stained punctate/granular tau aggregates in pretangles while pSer202/pThr205 and pThr231 antibodies stained tau in pretangles as well as classic NFT, neuritic component of plaques, and neuropil threads. Both pSer262 and pSer356 signals co-distributed with pSer202/pThr205 signal in pretangles but were rarely detected in pSer202/pThr205 immunoreactive classic NFT. In sections of middle frontal gyrus from PSP and CBD cases, tufted astrocytes and astrocytic plaques, respectively, were robustly labeled with the pSer202/pThr205 antibody but lacked significant immunoreactivity to pSer262 and pSer356 antibodies. Similarly, thorn shaped astrocytes in ARTAG cases were robustly labeled with the pSer202/pThr205 antibody but were weakly labeled with the pSer262 and pSer356 antibodies. Pick bodies were labeled robustly with the pSer202/pThr205 antibody, moderately with the pSer356 antibody, and weak and sparsely with the pSer262 antibody. Antibodies directed against phosphorylated epitopes within core region of soluble and fibrillar tau distinguish hippocampal pretangles in AD and appear to have less affinity for tau pathology forms in non-AD tauopathies. Thus, they may be novel biomarkers for detecting early stages of neurofibrillary pathology development in AD.

[ 18 F]MK-6240 was developed for PET imaging of AD tau pathology, but the exact molecular signature of specific binding remains unclear. This study quantified levels of four phospho-tau forms and total tau in postmortem brain tissues from [ 18 F]MK-6240 imaged cases to investigate associations with antemortem [ 18 F]MK-6240 PET. This study included four participants from the Wisconsin ADRC or WRAP with antemortem [ 18 F]MK-6240 and [ 11 C]PiB PET imaging and postmortem brain tissue obtained on average 32-months after imaging (Table 1). Parametric SUVR were generated using T1-w MRI to delineate regions matched to equivalent ROIs on fresh frozen brain tissue slabs. Tissue ROI dissected from ten brain regions per case were homogenized and ELISA was used to quantify concentrations of guanidine-extracted total tau and tau phosphorylated at epitopes pThr181, pSer199, pThr231, and pSer396. Matched PET-autopsy ROI analysis showed significant correlations of higher [ 18 F]MK-6240 SUVR with higher levels of all measured p-tau forms or their ratios to total tau across all regions and cases. Analyses of two A+T+ cases with clinical AD dementia and postmortem Thal Phase 5/Braak NFT Stage VI showed higher [ 18 F]MK-6240 SUVR correlated significantly with higher levels of pThr181, pSer199, pThr231, and pSer396 tau or their ratios to total tau in a 79-yo APOE-ε4ε4 case, and with higher levels of pThr231 and pSer396 tau in a 78-yo APOE-ε3ε3 case. In two T- cases, we observed weak correlations of [ 18 F]MK-6240 SUVR with pThr181 and pSer396 tau in an A-T- cognitively unimpaired case (Thal Phase 2/Braak NFT Stage II; 70-yo; APOE-ε3ε3), but no correlations in A+T- early-onset AD dementia case with Thal Phase 4/Braak NFT Stage V (63-yo; APOE-ε4ε4). This latter case also had high [ 18 F]MK-6240 uptake outside the brain and a low tangle density outside the MTL. Higher [ 18 F]MK-6240 PET binding reflects high brain concentrations of pThr181, pSer199, pThr231, and pSer396 tau in AD dementia cases with high AD neuropathological change. Lack of associations between PET and p-tau biochemistry in the T-, Braak V early-onset dementia case suggests that the T- PET status is more closely associated with p-tau biochemistry than postmortem neuropathological staging, likely due to low tangle density.

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.

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.

The binding substrates of [18F]flutemetamol and [11C]PiB PET consist of fibrillar amyloid-ß (Aß) aggregates in morphologically and structurally distinct diffuse plaques (DP) and cored plaques (CP) which are present at different proportions across brain regions in Alzheimer’s disease (AD). To account for this heterogeneity, an unbiased quantitative measure integrating plaque size and density of amyloid-ß (Aß) fibrils in plaques could help avoid false positive results, improve imaging-to-autopsy correlations, and further advance our understanding of pathological substrates for binding of amyloid PET tracers. Sixteen subjects underwent [18F]flutemetamol PET imaging in the pivotal Phase III end-of-life study (Study GE067-007 and GE067-026) and later came to autopsy (mean imaging-to-autopsy interval was 111 ± 87.6 days). [18F]flutemetamol SUVRpons values in four neocortical regions were correlated with region-matched postmortem measures of cyano-flutemetamol labeling of all plaques, as well as CP and DP separately, using percent area coverage, mean grayscale intensity of fluorescence, or a combination of the two measures referred to as integrated density. In the analysis of all cyano-flutemetamol-labeled plaques, [18F]flutemetamol SUVRpons values were more closely associated with integrated density measures than percent area coverage and mean grayscale values. Similarly, when CP and DP were analyzed separately, the integrated density values showed the closest association with neocortical region-matched [18F]flutemetamol SUVRpons values. When area coverage of CP and DP were each expressed as a fraction of total plaque area coverage with or without integrating fluorescence intensity values, [18F]flutemetamol SUVRpons values within each region of interest were associated with the DP, but not the CP, area coverage measure. The association of antemortem [18F]flutemetamol PET and region-matched postmortem integrated density measurements of cyano-flutemetamol labeled plaques further supports the hypothesis that amyloid PET signal is influenced both by plaque size and Aß fibril density within DP and CP. Further elucidating the relative contributions of each plaque type to the retention of amyloid PET radioligands could influence how a positive amyloid PET signal is interpreted in relation to postmortem pathology data.

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.

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.

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.

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-beta (A beta) deposits in living people. However, this technique cannot distinguish between A beta deposits in brain parenchyma (amyloid plaques) from those in blood vessels (cerebral amyloid angiopathy, CM). 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-A beta 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 A beta 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 Ali binding dyes, such as thioflavin-S, in that it labels both parenchymal Ali plaques and CAA when applied to histological sections from both a transgenic APP/PS1 mouse model of Ali amyloidosis and AD brain. Thus, compound 1 lacks molecular selectivity to distinguish A beta deposits in CAA from those in plaques. However, when administered to living APP / PS1 mice intravenously, compound 1 preferentially labels CM 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 A beta deposits in the vascular wall (i.e., "delivery selectivity"). Published by Elsevier Inc.

Alzheimer's disease is a progressive neurodegenerative disease characterized neuropathologically by presence of extracellular amyloid plaques composed of fibrillar amyloid beta (Aβ) 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 Aβ42 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 Aβ42 accumulation in HSV-1 infected 2D (neuronal monolayers) and 3D neuronal cultures (brain organoids). Akin to prior studies, HSV-1-infected 2D cultures showed Aβ42 immunoreactivity in cells expressing the HSV-1 antigen ICP4 (ICP4+). Conversely, accumulation of Aβ42 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 (Aβ) peptide-containing amyloid plaque development. Aβ 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 Aβ42 accumulation following HSV-1 infection of 2D compared to 3D neuronal cultures (brain organoids). Specifically, 2D neuronal cultures showed Aβ42-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 Aβ42 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.