Astrocyte tau deposition in progressive supranuclear palsy is associated with dysregulation of MAPT transcription.

Progressive supranuclear palsy (PSP) is a neurodegenerative disease characterized by 4R tau deposition in neurons as well as in astrocytes and oligodendrocytes. While astrocytic tau deposits are rarely observed in normal aging (so-called aging-related tau astrogliopathy, ARTAG) and Alzheimer's disease (AD), astrocytic tau in the form of tufted astrocytes is a pathognomonic hallmark of PSP. Classical biochemical experiments emphasized tau synthesis in neurons in the central nervous system, suggesting that astrocytic tau inclusions might be derived from uptake of extracellular neuronal-derived tau. However, recent single-nucleus RNAseq experiments highlight the fact that MAPT, the gene encoding tau, is also expressed by astrocytes, albeit in lower amounts. We, therefore, revisited the question of whether astrocyte-driven expression of tau might contribute to astrocytic tau aggregates in PSP by performing fluorescent in situ hybridization/immunohistochemical co-localization in human postmortem brain specimens from individuals with PSP and AD with ARTAG as well as normal controls. We find that, in PSP but not in AD, tau-immunoreactive astrocytes have higher levels of MAPT mRNA compared to astrocytes that do not have tau aggregates. These results suggest that astrocytic responses in PSP are unique to this tauopathy and support the possibility that fundamental changes in PSP astrocyte-endogenous mRNA biology contribute to increased synthesis of tau protein and underlies the formation of the astrocytic tau deposits characteristic of PSP.

Astrocyte-Neuron Interactions in Alzheimer's Disease.

Besides its two defining misfolded proteinopathies-Aß plaques and tau neurofibrillary tangles-Alzheimer's disease (AD) is an exemplar of a neurodegenerative disease with prominent reactive astrogliosis, defined as the set of morphological, molecular, and functional changes that astrocytes suffer as the result of a toxic exposure. Reactive astrocytes can be observed in the vicinity of plaques and tangles, and the relationship between astrocytes and these AD neuropathological lesions is bidirectional so that each AD neuropathological hallmark causes specific changes in astrocytes, and astrocytes modulate the severity of each neuropathological feature in a specific manner. Here, we will review both how astrocytes change as a result of their chronic exposure to AD neuropathology and how those astrocytic changes impact each AD neuropathological feature. We will emphasize the repercussions that AD-associated reactive astrogliosis has for the astrocyte-neuron interaction and highlight areas of uncertainty and priorities for future research.

Interneuron diversity in the human dorsal striatum.

Deciphering the striatal interneuron diversity is key to understanding the basal ganglia circuit and to untangling the complex neurological and psychiatric diseases affecting this brain structure. We performed snRNA-seq and spatial transcriptomics of postmortem human caudate nucleus and putamen samples to elucidate the diversity and abundance of interneuron populations and their inherent transcriptional structure in the human dorsal striatum. We propose a comprehensive taxonomy of striatal interneurons with eight main classes and fourteen subclasses, providing their full transcriptomic identity and spatial expression profile as well as additional quantitative FISH validation for specific populations. We have also delineated the correspondence of our taxonomy with previous standardized classifications and shown the main transcriptomic and class abundance differences between caudate nucleus and putamen. Notably, based on key functional genes such as ion channels and synaptic receptors, we found matching known mouse interneuron populations for the most abundant populations, the recently described PTHLH and TAC3 interneurons. Finally, we were able to integrate other published datasets with ours, supporting the generalizability of this harmonized taxonomy.

Multifaceted roles of APOE in Alzheimer disease.

For the past three decades, apolipoprotein E (APOE) has been known as the single greatest genetic modulator of sporadic Alzheimer disease (AD) risk, influencing both the average age of onset and the lifetime risk of developing AD. The APOEε4 allele significantly increases AD risk, whereas the ε2 allele is protective relative to the most common ε3 allele. However, large differences in effect size exist across ethnoracial groups that are likely to depend on both global genetic ancestry and local genetic ancestry, as well as gene-environment interactions. Although early studies linked APOE to amyloid-ß - one of the two culprit aggregation-prone proteins that define AD - in the past decade, mounting work has associated APOE with other neurodegenerative proteinopathies and broader ageing-related brain changes, such as neuroinflammation, energy metabolism failure, loss of myelin integrity and increased blood-brain barrier permeability, with potential implications for longevity and resilience to pathological protein aggregates. Novel mouse models and other technological advances have also enabled a number of therapeutic approaches aimed at either attenuating the APOEε4-linked increased AD risk or enhancing the APOEε2-linked AD protection. This Review summarizes this progress and highlights areas for future research towards the development of APOE-directed therapeutics.

Characterization of monoamine oxidase-B (MAO-B) as a biomarker of reactive astrogliosis in Alzheimer's disease and related dementias.

Reactive astrogliosis accompanies the two neuropathological hallmarks of Alzheimer's disease (AD)-Aß plaques and neurofibrillary tangles-and parallels neurodegeneration in AD and AD-related dementias (ADRD). Thus, there is growing interest in developing imaging and fluid biomarkers of reactive astrogliosis for AD/ADRD diagnosis and prognostication. Monoamine oxidase-B (MAO-B) is emerging as a target for PET imaging radiotracers of reactive astrogliosis. However, a thorough characterization of MAO-B expression in postmortem control and AD/ADRD brains is lacking. We sought to: (1) identify the primary cell type(s) expressing MAO-B in control and AD brains; (2) quantify MAO-B immunoreactivity in multiple brain regions of control and AD donors as a proxy for PET radiotracer uptake; (3) correlate MAO-B level with local AD neuropathological changes, reactive glia, and cortical atrophy; (4) determine whether the MAOB rs1799836 SNP genotype impacts MAO-B expression level; (5) compare MAO-B immunoreactivity across AD/ADRD, including Lewy body diseases (LBD) and frontotemporal lobar degenerations with tau (FTLD-Tau) and TDP-43 (FTLD-TDP). We found that MAO-B is mainly expressed by subpial and perivascular cortical astrocytes as well as by fibrous white matter astrocytes in control brains, whereas in AD brains, MAO-B is significantly upregulated by both cortical reactive astrocytes and white matter astrocytes across temporal, frontal, and occipital lobes. By contrast, MAO-B expression level was unchanged and lowest in cerebellum. Cortical MAO-B expression was independently associated with cortical atrophy and local measures of reactive astrocytes and microglia, and significantly increased in reactive astrocytes surrounding Thioflavin-S+ dense-core Aß plaques. MAO-B expression was not affected by the MAOB rs1799836 SNP genotype. MAO-B expression was also significantly increased in the frontal cortex and white matter of donors with corticobasal degeneration, Pick's disease, and FTLD-TDP, but not in LBD or progressive supranuclear palsy. These findings support ongoing efforts to develop MAO-B-based PET radiotracers to image reactive astrogliosis in AD/ADRD.

Landscape of brain myeloid cell transcriptome along the spatiotemporal progression of Alzheimer's disease reveals distinct sequential responses to Aß and tau.

Human microglia are critically involved in Alzheimer's disease (AD) progression, as shown by genetic and molecular studies. However, their role in tau pathology progression in human brain has not been well described. Here, we characterized 32 human donors along progression of AD pathology, both in time-from early to late pathology-and in space-from entorhinal cortex (EC), inferior temporal gyrus (ITG), prefrontal cortex (PFC) to visual cortex (V2 and V1)-with biochemistry, immunohistochemistry, and single nuclei-RNA-sequencing, profiling a total of 337,512 brain myeloid cells, including microglia. While the majority of microglia are similar across brain regions, we identified a specific subset unique to EC which may contribute to the early tau pathology present in this region. We calculated conversion of microglia subtypes to diseased states and compared conversion patterns to those from AD animal models. Targeting genes implicated in this conversion, or their upstream/downstream pathways, could halt gene programs initiated by early tau progression. We used expression patterns of early tau progression to identify genes whose expression is reversed along spreading of spatial tau pathology (EC > ITG > PFC > V2 > V1) and identified their potential involvement in microglia subtype conversion to a diseased state. This study provides a data resource that builds on our knowledge of myeloid cell contribution to AD by defining the heterogeneity of microglia and brain macrophages during both temporal and regional pathology aspects of AD progression at an unprecedented resolution.

Alzheimer DataLENS: An Open Data Analytics Portal for Alzheimer's Disease Research.

Background: Recent Alzheimer's disease (AD) discoveries are increasingly based on studies from a variety of omics technologies on large cohorts. Currently, there is no easily accessible resource for neuroscientists to browse, query, and visualize these complex datasets in a harmonized manner. Objective: Create an online portal of public omics datasets for AD research. Methods: We developed Alzheimer DataLENS, a web-based portal, using the R Shiny platform to query and visualize publicly available transcriptomics and genetics studies of AD on human cohorts. To ensure consistent representation of AD findings, all datasets were processed through a uniform bioinformatics pipeline. Results: Alzheimer DataLENS currently houses 2 single-nucleus RNA sequencing datasets, over 30 bulk RNA sequencing datasets from 19 brain regions and 3 cohorts, and 2 genome-wide association studies (GWAS). Available visualizations for single-nucleus data include bubble plots, heatmaps, and UMAP plots; for bulk expression data include box plots and heatmaps; for pathways include protein-protein interaction network plots; and for GWAS results include Manhattan plots. Alzheimer DataLENS also links to two other knowledge resources: the AD Progression Atlas and the Astrocyte Atlas. Conclusions: Alzheimer DataLENS is a valuable resource for investigators to quickly and systematically explore omics datasets and is freely accessible at https://alzdatalens.partners.org.

Update on modifiable risk factors for Alzheimer's disease and related dementias.

PURPOSE OF REVIEW: All human beings undergo a lifelong cumulative exposure to potentially preventable adverse factors such as toxins, infections, traumatisms, and cardiovascular risk factors, collectively termed exposome. The interplay between the individual's genetics and exposome is thought to have a large impact in health outcomes such as cancer and cardiovascular disease. Likewise, a growing body of evidence is supporting the idea that preventable factors explain a sizable proportion of Alzheimer's disease and related dementia (ADRD) cases. RECENT FINDINGS: Here, we will review the most recent epidemiological, experimental preclinical, and interventional clinical studies examining some of these potentially modifiable risk factors for ADRD. We will focus on new evidence regarding cardiovascular risk factors, air pollution, viral and other infectious agents, traumatic brain injury, and hearing loss. SUMMARY: While greater and higher quality epidemiological and experimental evidence is needed to unequivocally confirm their causal link with ADRD and/or unravel the underlying mechanisms, these modifiable risk factors may represent a window of opportunity to reduce ADRD incidence and prevalence at the population level via health screenings, and education and health policies.

Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Distinct transcriptomic responses to Aß plaques, neurofibrillary tangles, and APOE in Alzheimer's disease.

INTRODUCTION: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE-linked differences remain unclear. METHODS: We performed laser capture microdissection of amyloid beta (Aß) plaques, the 50 µm halo around them, tangles with the 50 µm halo around them, and areas distant (> 50 µm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing. RESULTS: Aß plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aß plaques had more differentially expressed genes than tangles. We identified a gradient Aß plaque > peri-plaque > tangle > distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aß plaques. DISCUSSION: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aß plaques, and are exacerbated by the APOE ε4 allele.

Interneuron diversity in the human dorsal striatum.

Deciphering the striatal interneuron diversity is key to understanding the basal ganglia circuit and to untangle the complex neurological and psychiatric diseases affecting this brain structure. We performed snRNA-seq of postmortem human caudate nucleus and putamen samples to elucidate the diversity and abundance of interneuron populations and their transcriptional structure in the human dorsal striatum. We propose a new taxonomy of striatal interneurons with eight main classes and fourteen subclasses and provide their specific markers and some quantitative FISH validation, particularly for a novel PTHLH-expressing population. For the most abundant populations, PTHLH and TAC3, we found matching known mouse interneuron populations based on key functional genes such as ion channels and synaptic receptors. Remarkably, human TAC3 and mouse Th populations share important similarities including the expression of the neuropeptide tachykinin 3. Finally, we were able to integrate other published datasets supporting the generalizability of this new harmonized taxonomy.

Endothelial Cells Are Heterogeneous in Different Brain Regions and Are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 human AD and non-AD donors (19 female, 13 male) each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex, and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid ß plaques and cerebral amyloid angiopathy. This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain.SIGNIFICANCE STATEMENT In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Distinct Transcriptomic Responses to Aß plaques, Neurofibrillary Tangles, and APOE in Alzheimer's Disease.

INTRODUCTION: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE -linked differences remain unclear. METHODS: We performed laser capture microdissection of Aß plaques, the 50µm halo around them, tangles with the 50µm halo around them, and areas distant (>50µm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing. RESULTS: Aß plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aß plaques had more differentially expressed genes than tangles. We identified a gradient Aß plaque>peri-plaque>tangle>distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aß plaques. DISCUSSION: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aß plaques, and are exacerbated by the APOE ε4 allele.

Endothelial Cells are Heterogeneous in Different Brain Regions and are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 AD and non-AD donors each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid beta plaques and cerebral amyloid angiopathy (CAA). This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain. Significance Statement: In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Neuropathology-Independent Association Between APOE Genotype and Cognitive Decline Rate in the Normal Aging-Early Alzheimer Continuum.

Background and Objectives: We previously found that the APOE genotype affects the rate of cognitive decline in mild-to-moderate Alzheimer disease (AD) dementia independently of its effects on AD neuropathologic changes (ADNC) and copathologies. In this study, we tested the hypothesis that the APOE alleles differentially affect the rate of cognitive decline at the normal aging-early AD continuum and that this association is independent of their effects on classical ADNC and copathologies. Methods: We analyzed APOE associations with the cognitive trajectories (Clinical Dementia Rating scale Sum of Boxes [CDR-SOB] and Mini-Mental State Examination [MMSE]) of more than 1,000 individuals from a national clinicopathologic sample who had either no, mild (sparse neuritic plaques and the Braak neurofibrillary tangle [NFT] stage I/II), or intermediate (moderate neuritic plaques and the Braak NFT stage III/IV) ADNC levels at autopsy via 2 latent classes reverse-time longitudinal modeling. Results: Carrying the APOEε4 allele was associated with a faster rate of cognitive decline by both CDR-SOB and MMSE relative to APOEε3 homozygotes. This association remained statistically significant after adjusting for ADNC severity, comorbid pathologies, and the effects of ADNC on the slope of cognitive decline. Our modeling strategy identified 2 latent classes in which APOEε4 carriers declined faster than APOEε3 homozygotes, with latent class 1 members representing slow decliners (CDR-SOB: 76.7% of individuals, 0.195 vs 0.146 points/y in APOEε4 vs APOEε3/ε3; MMSE: 88.6% of individuals, -0.303 vs -0.153 points/y in APOEε4 vs APOEε3/ε3), whereas latent class 2 members were fast decliners (CDR-SOB: 23.3% of participants, 1.536 vs 1.487 points/y in APOEε4 vs APOEε3/ε3; MMSE: 11.4% of participants, -2.538 vs -2.387 points/y in APOEε4 vs APOEε3/ε3). Compared with slow decliners, fast decliners were more likely to carry the APOEε4 allele, younger at initial visit and death, more impaired at initial and last visits, and more likely to have intermediate (vs none or mild) ADNC levels, as well as concurrent Lewy bodies and hippocampal sclerosis at autopsy. Discussion: In a large national sample selected to represent the normal aging-early AD continuum, the APOEε4 allele is associated with a modest but statistically significant acceleration of the cognitive decline rate even after controlling for its effects on ADNC and comorbid pathologies.

Cyclic Multiplex Fluorescent Immunohistochemistry Protocol to Phenotype Glial Cells in Formalin-Fixed Paraffin-Embedded Human Brain Sections.

There is a growing interest in expanding the multiplexing capability of immunohistochemistry to achieve a deeper phenotyping of various cell types in health and disease. Here, we describe a protocol of cyclic multiplex fluorescent immunohistochemistry that enables the labeling of up to 16 antigens on the same formalin-fixed paraffin-embedded section using "off-the-shelf," commercially available, primary antibodies as well as fluorescently conjugated secondary antibodies. Key steps include the denaturing/stripping of the antibodies by microwaving and the quenching of any remaining fluorescent signal between the cycles of otherwise traditional multiplexed fluorescent immunohistochemistry. We have successfully applied this protocol to characterize astrocytic and microglial responses to Aß plaques and neurofibrillary tangles in Alzheimer's disease brains, but it could be easily adapted to other user's needs regarding cell types, disease, and organ.

Development and Evaluation of a Natural Language Processing Annotation Tool to Facilitate Phenotyping of Cognitive Status in Electronic Health Records: Diagnostic Study.

BACKGROUND: Electronic health records (EHRs) with large sample sizes and rich information offer great potential for dementia research, but current methods of phenotyping cognitive status are not scalable. OBJECTIVE: The aim of this study was to evaluate whether natural language processing (NLP)-powered semiautomated annotation can improve the speed and interrater reliability of chart reviews for phenotyping cognitive status. METHODS: In this diagnostic study, we developed and evaluated a semiautomated NLP-powered annotation tool (NAT) to facilitate phenotyping of cognitive status. Clinical experts adjudicated the cognitive status of 627 patients at Mass General Brigham (MGB) health care, using NAT or traditional chart reviews. Patient charts contained EHR data from two data sets: (1) records from January 1, 2017, to December 31, 2018, for 100 Medicare beneficiaries from the MGB Accountable Care Organization and (2) records from 2 years prior to COVID-19 diagnosis to the date of COVID-19 diagnosis for 527 MGB patients. All EHR data from the relevant period were extracted; diagnosis codes, medications, and laboratory test values were processed and summarized; clinical notes were processed through an NLP pipeline; and a web tool was developed to present an integrated view of all data. Cognitive status was rated as cognitively normal, cognitively impaired, or undetermined. Assessment time and interrater agreement of NAT compared to manual chart reviews for cognitive status phenotyping was evaluated. RESULTS: NAT adjudication provided higher interrater agreement (Cohen κ=0.89 vs κ=0.80) and significant speed up (time difference mean 1.4, SD 1.3 minutes; P<.001; ratio median 2.2, min-max 0.4-20) over manual chart reviews. There was moderate agreement with manual chart reviews (Cohen κ=0.67). In the cases that exhibited disagreement with manual chart reviews, NAT adjudication was able to produce assessments that had broader clinical consensus due to its integrated view of highlighted relevant information and semiautomated NLP features. CONCLUSIONS: NAT adjudication improves the speed and interrater reliability for phenotyping cognitive status compared to manual chart reviews. This study underscores the potential of an NLP-based clinically adjudicated method to build large-scale dementia research cohorts from EHRs.

Plasma biomarkers for prognosis of cognitive decline in patients with mild cognitive impairment.

Plasma-based biomarkers present a promising approach in the research and clinical practice of Alzheimer's disease as they are inexpensive, accessible and minimally invasive. In particular, prognostic biomarkers of cognitive decline may aid in planning and management of clinical care. Although recent studies have demonstrated the prognostic utility of plasma biomarkers of Alzheimer pathology or neurodegeneration, such as pTau-181 and NF-L, whether other plasma biomarkers can further improve prediction of cognitive decline is undetermined. We conducted an observational cohort study to determine the prognostic utility of plasma biomarkers in predicting progression to dementia for individuals presenting with mild cognitive impairment due to probable Alzheimer's disease. We used the Olink™ Proximity Extension Assay technology to measure the level of 460 circulating proteins in banked plasma samples of all participants. We used a discovery data set comprised 60 individuals with mild cognitive impairment (30 progressors and 30 stable) and a validation data set consisting of 21 stable and 21 progressors. We developed a machine learning model to distinguish progressors from stable and used 44 proteins with significantly different plasma levels in progressors versus stable along with age, sex, education and baseline cognition as candidate features. A model with age, education, APOE genotype, baseline cognition, plasma pTau-181 and 12 plasma Olink protein biomarker levels was able to distinguish progressors from stable with 86.7% accuracy (mean area under the curve = 0.88). In the validation data set, the model accuracy was 78.6%. The Olink proteins selected by the model included those associated with vascular injury and neuroinflammation (e.g. IL-8, IL-17A, TIMP-4, MMP7). In addition, to compare these prognostic biomarkers to those that are altered in Alzheimer's disease or other types of dementia relative to controls, we analyzed samples from 20 individuals with Alzheimer, 30 with non-Alzheimer dementias and 34 with normal cognition. The proteins NF-L and PTP-1B were significantly higher in both Alzheimer and non-Alzheimer dementias compared with cognitively normal individuals. Interestingly, the prognostic markers of decline at the mild cognitive impairment stage did not overlap with those that differed between dementia and control cases. In summary, our findings suggest that plasma biomarkers of inflammation and vascular injury are associated with cognitive decline. Developing a plasma biomarker profile could aid in prognostic deliberations and identify individuals at higher risk of dementia in clinical practice.

Cyclic multiplex fluorescent immunohistochemistry and machine learning reveal distinct states of astrocytes and microglia in normal aging and Alzheimer's disease.

BACKGROUND: Astrocytes and microglia react to Aß plaques, neurofibrillary tangles, and neurodegeneration in the Alzheimer's disease (AD) brain. Single-nuclei and single-cell RNA-seq have revealed multiple states or subpopulations of these glial cells but lack spatial information. We have developed a methodology of cyclic multiplex fluorescent immunohistochemistry on human postmortem brains and image analysis that enables a comprehensive morphological quantitative characterization of astrocytes and microglia in the context of their spatial relationships with plaques and tangles. METHODS: Single FFPE sections from the temporal association cortex of control and AD subjects were subjected to 8 cycles of multiplex fluorescent immunohistochemistry, including 7 astroglial, 6 microglial, 1 neuronal, Aß, and phospho-tau markers. Our analysis pipeline consisted of: (1) image alignment across cycles; (2) background subtraction; (3) manual annotation of 5172 ALDH1L1+ astrocytic and 6226 IBA1+ microglial profiles; (4) local thresholding and segmentation of profiles; (5) machine learning on marker intensity data; and (6) deep learning on image features. RESULTS: Spectral clustering identified three phenotypes of astrocytes and microglia, which we termed "homeostatic," "intermediate," and "reactive." Reactive and, to a lesser extent, intermediate astrocytes and microglia were closely associated with AD pathology (≤ 50 µm). Compared to homeostatic, reactive astrocytes contained substantially higher GFAP and YKL-40, modestly elevated vimentin and TSPO as well as EAAT1, and reduced GS. Intermediate astrocytes had markedly increased EAAT2, moderately increased GS, and intermediate GFAP and YKL-40 levels. Relative to homeostatic, reactive microglia showed increased expression of all markers (CD68, ferritin, MHC2, TMEM119, TSPO), whereas intermediate microglia exhibited increased ferritin and TMEM119 as well as intermediate CD68 levels. Machine learning models applied on either high-plex signal intensity data (gradient boosting machines) or directly on image features (convolutional neural networks) accurately discriminated control vs. AD diagnoses at the single-cell level. CONCLUSIONS: Cyclic multiplex fluorescent immunohistochemistry combined with machine learning models holds promise to advance our understanding of the complexity and heterogeneity of glial responses as well as inform transcriptomics studies. Three distinct phenotypes emerged with our combination of markers, thus expanding the classic binary "homeostatic vs. reactive" classification to a third state, which could represent "transitional" or "resilient" glia.

Systematic review of human post-mortem immunohistochemical studies and bioinformatics analyses unveil the complexity of astrocyte reaction in Alzheimer's disease.

AIMS: Reactive astrocytes in Alzheimer's disease (AD) have traditionally been demonstrated by increased glial fibrillary acidic protein (GFAP) immunoreactivity; however, astrocyte reaction is a complex and heterogeneous phenomenon involving multiple astrocyte functions beyond cytoskeletal remodelling. To better understand astrocyte reaction in AD, we conducted a systematic review of astrocyte immunohistochemical studies in post-mortem AD brains followed by bioinformatics analyses on the extracted reactive astrocyte markers. METHODS: NCBI PubMed, APA PsycInfo and WoS-SCIE databases were interrogated for original English research articles with the search terms 'Alzheimer's disease' AND 'astrocytes.' Bioinformatics analyses included protein-protein interaction network analysis, pathway enrichment, and transcription factor enrichment, as well as comparison with public human -omics datasets. RESULTS: A total of 306 articles meeting eligibility criteria rendered 196 proteins, most of which were reported to be upregulated in AD vs control brains. Besides cytoskeletal remodelling (e.g., GFAP), bioinformatics analyses revealed a wide range of functional alterations including neuroinflammation (e.g., IL6, MAPK1/3/8 and TNF), oxidative stress and antioxidant defence (e.g., MT1A/2A, NFE2L2, NOS1/2/3, PRDX6 and SOD1/2), lipid metabolism (e.g., APOE, CLU and LRP1), proteostasis (e.g., cathepsins, CRYAB and HSPB1/2/6/8), extracellular matrix organisation (e.g., CD44, MMP1/3 and SERPINA3), and neurotransmission (e.g., CHRNA7, GABA, GLUL, GRM5, MAOB and SLC1A2), among others. CTCF and ESR1 emerged as potential transcription factors driving these changes. Comparison with published -omics datasets validated our results, demonstrating a significant overlap with reported transcriptomic and proteomic changes in AD brains and/or CSF. CONCLUSIONS: Our systematic review of the neuropathological literature reveals the complexity of AD reactive astrogliosis. We have shared these findings as an online resource available at www.astrocyteatlas.org.