Quantitative estimate of cognitive resilience and its medical and genetic associations.

BACKGROUND: We have proposed that cognitive resilience (CR) counteracts brain damage from Alzheimer's disease (AD) or AD-related dementias such that older individuals who harbor neurodegenerative disease burden sufficient to cause dementia remain cognitively normal. However, CR traditionally is considered a binary trait, capturing only the most extreme examples, and is often inconsistently defined. METHODS: This study addressed existing discrepancies and shortcomings of the current CR definition by proposing a framework for defining CR as a continuous variable for each neuropsychological test. The linear equations clarified CR's relationship to closely related terms, including cognitive function, reserve, compensation, and damage. Primarily, resilience is defined as a function of cognitive performance and damage from neuropathologic damage. As such, the study utilized data from 844 individuals (age = 79 ± 12, 44% female) in the National Alzheimer's Coordinating Center cohort that met our inclusion criteria of comprehensive lesion rankings for 17 neuropathologic features and complete neuropsychological test results. Machine learning models and GWAS then were used to identify medical and genetic factors that are associated with CR. RESULTS: CR varied across five cognitive assessments and was greater in female participants, associated with longer survival, and weakly associated with educational attainment or APOE ε4 allele. In contrast, damage was strongly associated with APOE ε4 allele (P value < 0.0001). Major predictors of CR were cardiovascular health and social interactions, as well as the absence of behavioral symptoms. CONCLUSIONS: Our framework explicitly decoupled the effects of CR from neuropathologic damage. Characterizations and genetic association study of these two components suggest that the underlying CR mechanism has minimal overlap with the disease mechanism. Moreover, the identified medical features associated with CR suggest modifiable features to counteract clinical expression of damage and maintain cognitive function in older individuals.

Unveiling Resilience to Alzheimer's Disease: Insights From Brain Regional Proteomic Markers.

Studying proteomics data of the human brain could offer numerous insights into unraveling the signature of resilience to Alzheimer's disease. In our previous study with rigorous cohort selection criteria that excluded 4 common comorbidities, we harnessed multiple brain regions from 43 research participants with 12 of them displaying cognitive resilience to Alzheimer's disease. Based on the previous findings, this work focuses on 6 proteins out of the 33 differentially expressed proteins associated with resilience to Alzheimer's disease. These proteins are used to construct a decision tree classifier, enabling the differentiation of 3 groups: (i) healthy control, (ii) resilience to Alzheimer's disease, and (iii) Alzheimer's disease with dementia. Our analysis unveiled 2 important regional proteomic markers: Aß peptides in the hippocampus and PA1B3 in the inferior parietal lobule. These findings underscore the potential of using distinct regional proteomic markers as signatures in characterizing the resilience to Alzheimer's disease.

Paradigm Shift: Multiple Potential Pathways to Neurodegenerative Dementia.

Neurodegenerative dementia can result from multiple underlying abnormalities, including neurotransmitter imbalances, protein aggregation, and other neurotoxic events. A major complication in identifying effective treatment targets is the frequent co-occurrence of multiple neurodegenerative processes, occurring either in parallel or sequentially. The path towards developing effective treatments for Alzheimer's disease (AD) and other dementias has been relatively slow and until recently has focused on disease symptoms. Aducanumab and lecanemab, recently approved by the FDA, are meant to target disease structures but have only modest benefit on symptom progression and remain unproven in reversing or preventing dementia. A third, donanemab, appears more promising but awaits FDA approval. Ongoing trials include potential cognition enhancers, new combinations of known drugs for synergistic effects, prodrugs with less toxicity, and increasing interest in drugs targeting neuroinflammation or microbiome. Scientific and technological advances offer the opportunity to move in new therapy directions, such as modifying microglia to prevent or suppress underlying disease. A major challenge, however, is that underlying comorbidities likely influence the effectiveness of therapies. Indeed, the full range of comorbidity, today only definitively identified postmortem, likely contributes to failed clinical trials and overmedication of older adults, since it is difficult to exclude (during life) people unlikely to respond. Our current knowledge thus signals that a paradigm shift towards individualized and multimodal treatments is necessary to effectively advance the field of dementia therapeutics.

Whole genome deconvolution unveils Alzheimer's resilient epigenetic signature.

Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) accurately depicts the chromatin regulatory state and altered mechanisms guiding gene expression in disease. However, bulk sequencing entangles information from different cell types and obscures cellular heterogeneity. To address this, we developed Cellformer, a deep learning method that deconvolutes bulk ATAC-seq into cell type-specific expression across the whole genome. Cellformer enables cost-effective cell type-specific open chromatin profiling in large cohorts. Applied to 191 bulk samples from 3 brain regions, Cellformer identifies cell type-specific gene regulatory mechanisms involved in resilience to Alzheimer's disease, an uncommon group of cognitively healthy individuals that harbor a high pathological load of Alzheimer's disease. Cell type-resolved chromatin profiling unveils cell type-specific pathways and nominates potential epigenetic mediators underlying resilience that may illuminate therapeutic opportunities to limit the cognitive impact of the disease. Cellformer is freely available to facilitate future investigations using high-throughput bulk ATAC-seq data.

Cross-species comparative analysis of single presynapses.

Comparing brain structure across species and regions enables key functional insights. Leveraging publicly available data from a novel mass cytometry-based method, synaptometry by time of flight (SynTOF), we applied an unsupervised machine learning approach to conduct a comparative study of presynapse molecular abundance across three species and three brain regions. We used neural networks and their attractive properties to model complex relationships among high dimensional data to develop a unified, unsupervised framework for comparing the profile of more than 4.5 million single presynapses among normal human, macaque, and mouse samples. An extensive validation showed the feasibility of performing cross-species comparison using SynTOF profiling. Integrative analysis of the abundance of 20 presynaptic proteins revealed near-complete separation between primates and mice involving synaptic pruning, cellular energy, lipid metabolism, and neurotransmission. In addition, our analysis revealed a strong overlap between the presynaptic composition of human and macaque in the cerebral cortex and neostriatum. Our unique approach illuminates species- and region-specific variation in presynapse molecular composition.

Brain proteomic analysis implicates actin filament processes and injury response in resilience to Alzheimer's disease.

Resilience to Alzheimer's disease is an uncommon combination of high disease burden without dementia that offers valuable insights into limiting clinical impact. Here we assessed 43 research participants meeting stringent criteria, 11 healthy controls, 12 resilience to Alzheimer's disease and 20 Alzheimer's disease with dementia and analyzed matched isocortical regions, hippocampus, and caudate nucleus by mass spectrometry-based proteomics. Of 7115 differentially expressed soluble proteins, lower isocortical and hippocampal soluble Aß levels is a significant feature of resilience when compared to healthy control and Alzheimer's disease dementia groups. Protein co-expression analysis reveals 181 densely-interacting proteins significantly associated with resilience that were enriched for actin filament-based processes, cellular detoxification, and wound healing in isocortex and hippocampus, further supported by four validation cohorts. Our results suggest that lowering soluble Aß concentration may suppress severe cognitive impairment along the Alzheimer's disease continuum. The molecular basis of resilience likely holds important therapeutic insights.

Prediction of neuropathologic lesions from clinical data.

INTRODUCTION: Post-mortem analysis provides definitive diagnoses of neurodegenerative diseases; however, only a few can be diagnosed during life. METHODS: This study employed statistical tools and machine learning to predict 17 neuropathologic lesions from a cohort of 6518 individuals using 381 clinical features (Table S1). The multisite data allowed validation of the model's robustness by splitting train/test sets by clinical sites. A similar study was performed for predicting Alzheimer's disease (AD) neuropathologic change without specific comorbidities. RESULTS: Prediction results show high performance for certain lesions that match or exceed that of research annotation. Neurodegenerative comorbidities in addition to AD neuropathologic change resulted in compounded, but disproportionate, effects across cognitive domains as the comorbidity number increased. DISCUSSION: Certain clinical features could be strongly associated with multiple neurodegenerative diseases, others were lesion-specific, and some were divergent between lesions. Our approach could benefit clinical research, and genetic and biomarker research by enriching cohorts for desired lesions.

Human cerebrospinal fluid single exosomes in Parkinson's and Alzheimer's diseases.

Exosomes are proposed to be important in the pathogenesis of prevalent neurodegenerative diseases. We report the first application of solid-state technology to perform multiplex analysis of single exosomes in human cerebrospinal fluid (CSF) obtained from the lumbar sac of people diagnosed with Alzheimer's disease dementia (ADD, n=30) or Parkinson's disease dementia (PDD, n=30), as well as age-matched health controls (HCN, n=30). Single events were captured with mouse monoclonal antibodies to one of three different tetraspanins (CD9, CD63, or CD81) or with mouse (M) IgG control, and then probed with fluorescently labeled antibodies to prion protein (PrP) or CD47 to mark neuronal or presynaptic origin, as well as ADD- and PDD-related proteins: amyloid beta (Aß), tau, α-synuclein, and Apolipoprotein (Apo) E. Data were collected only from captured events that were within the size range of 50 to 200 nm. Exosomes were present at approximately 100 billion per mL human CSF and were similarly abundant for CD9+ and CD81+ events, but CD63+ were only 22% to 25% of CD9+ (P<0.0001) or CD81+ (P<0.0001) events. Approximately 24% of CSF exosomes were PrP+, while only 2% were CD47+. The vast majority of exosomes were surface ApoE+, and the number of PrP-ApoE+ (P<0.001) and PrP+ApoE+ (P<0.01) exosomes were significantly reduced in ADD vs. HCN for CD9+ events only. Aß, tau, and α-synuclein were not detected on the exosome surface or in permeabilized cargo. These data provide new insights into single exosome molecular features and highlight reduction in the CSF concentration of ApoE+ exosomes in patients with ADD.

Understanding the molecular basis of resilience to Alzheimer's disease.

The cellular and molecular distinction between brain aging and neurodegenerative disease begins to blur in the oldest old. Approximately 15-25% of observations in humans do not fit predicted clinical manifestations, likely the result of suppressed damage despite usually adequate stressors and of resilience, the suppression of neurological dysfunction despite usually adequate degeneration. Factors during life may predict the clinico-pathologic state of resilience: cardiovascular health and mental health, more so than educational attainment, are predictive of a continuous measure of resilience to Alzheimer's disease (AD) and AD-related dementias (ADRDs). In resilience to AD alone (RAD), core features include synaptic and axonal processes, especially in the hippocampus. Future focus on larger and more diverse cohorts and additional regions offer emerging opportunities to understand this counterforce to neurodegeneration. The focus of this review is the molecular basis of resilience to AD.

Single-cell spatial proteomic imaging for human neuropathology.

Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimer's disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves broadly as a methodology for spatial proteomic analysis of archival human neuropathology. TEASER: Multiplex Ion beam Imaging enables deep spatial phenotyping of human neuropathology-associated cellular and disease features.

Parkinson's Progression Markers Initiative brain autopsy program.

We report on the initial 17 (11 male:6 female) brain autopsies from across Europe and the United States in the Parkinson's Progression Markers Initiative (PPMI). Clinical diagnoses were Parkinson's disease (n = 15), multiple system atrophy (n = 1), and Dementia with Lewy bodies (n = 1); average age of death = 72 ± 8 yr. Cognitive assessment at last evaluation was 5 with normal cognition, 7 with mild cognitive impairment, and 5 with dementia. Genetic assessment showed 4 participants were heterozygous or homozygous for GBA N370S and 3 were heterozygous carriers for LRRK2 R1441G or G2019S; 1 was an APOE ε2 carrier and 5 were APOE ε4 carriers. Longitudinal DAT neuroimaging as well as CSF and plasma biomarker data are summarized. Neuropathologic examination confirmed all clinical diagnoses and showed the expected frequencies of common comorbidities; no evidence of chronic traumatic encephalopathy was observed. Thus, brain autopsy data can provide confirmation, clarification, and new insights into the PD progression observed during life. As it grows, the PPMI brain autopsy program will provide a deeply-annotated research resource to the community of investigators focused on developing biomarkers for PD progression.

Mass Synaptometry: Applying Mass Cytometry to Single Synapse Analysis.

Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. Synaptic changes are also known to be important in neurocognitive disorders such as schizophrenia and autism spectrum disorders. Several labs, including ours, have demonstrated that conventional (fluorescence-based) flow cytometry of individual synaptosomes is a robust and reproducible method. However, the repertoire of probes needed to assess comprehensively the type of synapse, pathologic proteins (including protein products of risk genes discovered in GWAS), and markers of stress and injury far exceeds what is achievable with conventional flow cytometry. We recently developed a method that applies CyTOF (Cytometry by Time-Of-Flight mass spectrometry) to high-dimensional analysis of individual human synaptosomes, overcoming many of the multiplexing limitations of conventional flow cytometry. We call this new method Mass Synaptometry. Here we describe the preparation of synaptosomes from human and mouse brain, the generation and quality control of the "SynTOF" (Synapse by Time-Of-Flight mass spectrometry) antibody panel, the staining protocol, and CyTOF parameter setup for acquisition, post-acquisition processing, and analysis.

Single-synapse analyses of Alzheimer's disease implicate pathologic tau, DJ1, CD47, and ApoE.

Synaptic molecular characterization is limited for Alzheimer's disease (AD). Our newly invented mass cytometry­based method, synaptometry by time of flight (SynTOF), was used to measure 38 antibody probes in approximately 17 million single-synapse events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), nonhuman primates (NHPs), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar. Although not detected in human synapses, Aß was in PS/APP mice single-synapse events. Clustering and pattern identification of human synapses showed expected disease-specific differences, like increased hippocampal pathologic tau in AD and reduced caudate dopamine transporter in LBD, and revealed previously unidentified findings including increased hippocampal CD47 and lowered DJ1 in AD and higher ApoE in AD with dementia. Our results were independently supported by multiplex ion beam imaging of intact tissue. This highlights the higher depth and breadth of insight on neurodegenerative diseases obtainable through SynTOF.

Enantiomers of 4-aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior.

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain. Mouse cerebral synaptosome uptake (nmol/mg protein over 30 min) of (R)-4APA or (S)-4APA was time and temperature dependent; however, the R enantiomer had greater uptake, reduction of endogenous GABA concentration, and release following membrane depolarization than did the S enantiomer. (S)-4APA exhibited some weak agonist (GABAA α4ß3δ, GABAA α5ß2γ2, and GABAB B1/B2) and antagonist (GABAA α6ß2γ2) activity while (R)-4APA showed weak agonist activity only with GABAA α5ß2γ2. Both 4APA enantiomers (100 mg/kg IP) were detected in mouse brain 10 min after injection, and by 1 hr had reached concentrations that were stable over 6 hr; both enantiomers were cleared rapidly from mouse serum over 6 hr. Two-month-old mice had no mortality following 100-900 mg/kg IP of each 4APA enantiomer but did have similar dose-dependent reduction in distance moved in a novel cage. Neither enantiomer at 30 or 100 mg/kg impacted outcomes in 23 measures of well-being, activity chamber, or withdrawal from hot plate. Our results suggest that enantiomers of 4APA are active in mouse brain, and that (R)-4APA may act as a novel false neurotransmitter of GABA. Future work will focus on disease models and on possible applications as neuroimaging agents.

Mass-tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events.

Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.

Single-cell epigenomic analyses implicate candidate causal variants at inherited risk loci for Alzheimer's and Parkinson's diseases.

Genome-wide association studies of neurological diseases have identified thousands of variants associated with disease phenotypes. However, most of these variants do not alter coding sequences, making it difficult to assign their function. Here, we present a multi-omic epigenetic atlas of the adult human brain through profiling of single-cell chromatin accessibility landscapes and three-dimensional chromatin interactions of diverse adult brain regions across a cohort of cognitively healthy individuals. We developed a machine-learning classifier to integrate this multi-omic framework and predict dozens of functional SNPs for Alzheimer's and Parkinson's diseases, nominating target genes and cell types for previously orphaned loci from genome-wide association studies. Moreover, we dissected the complex inverted haplotype of the MAPT (encoding tau) Parkinson's disease risk locus, identifying putative ectopic regulatory interactions in neurons that may mediate this disease association. This work expands understanding of inherited variation and provides a roadmap for the epigenomic dissection of causal regulatory variation in disease.

The basis of cellular and regional vulnerability in Alzheimer's disease.

Alzheimer's disease (AD) differentially and specifically affects brain regions and neuronal cell types in a predictable pattern. Damage to the brain appears to spread and worsens with time, taking over more regions and activating multiple stressors that can converge to promote vulnerability of certain cell types. At the same time, other cell types and brain regions remain intact in the face of this onslaught of neuropathology. Although neuropathologic descriptions of AD have been extensively expanded and mapped over the last several decades, our understanding of the mechanisms underlying how certain regions and cell populations are specifically vulnerable or resistant has lagged behind. In this review, we detail what is known about the selectivity of local initiation of AD pathology in the hippocampus, its proposed spread via synaptic connections, and the diversity of clinical phenotypes and brain atrophy patterns that may arise from different fibrillar strains of pathologic proteins or genetic predispositions. We summarize accumulated and emerging knowledge of the cellular and molecular basis for neuroanatomic selectivity, consider potential disease-relevant differences between vulnerable and resistant neuronal cell types and isolate molecular markers to identify them.

Mass synaptometry: High-dimensional multi parametric assay for single synapses.

BACKGROUND: Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline. NEW METHOD: We report "Mass Synaptometry" for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells. RESULT: Here we overcome challenges for single synapse analysis by optimizing synaptosome preparations, generating a 'SynTOF panel,' recalibrating acquisition settings, and applying computational analyses. Through the analysis of 390,000 individual synaptosomes, we also provide proof-of principle validation by characterizing changes in synaptic diversity in Lewy Body Disease (LBD), Alzheimer's disease and normal brain. COMPARISON WITH EXISTING METHOD(S): Current imaging methods to study synapses in humans are capable of analyzing a limited number of synapses, and conventional flow cytometric techniques are typically restricted to fewer than 6 parameters. Our method allows for the simultaneous detection of 34 parameters from tens of thousands of individual synapses. CONCLUSION: We applied Mass Synaptometry to analyze 34 parameters simultaneously on more than 390,000 synaptosomes from 13 human brain samples. This new approach revealed regional and disease-specific changes in synaptic phenotypes, including validation of this method with the expected changes in the molecular composition of striatal dopaminergic synapses in Lewy body disease and Alzheimer's disease. Mass synaptometry enables highly parallel molecular profiling of individual synaptic terminals.

TDP-43 Neuropathologic Associations in the Nun Study and the Honolulu-Asia Aging Study.

Transactive response binding protein-43 (TDP-43) cytoplasmic neuronal and glial aggregates (pathologic TDP-43) have been described in multiple brain diseases. We describe the associations between neuropathologically confirmed TDP-43 and cognition in two population-based cohorts: the Nun Study (NS) and the Honolulu-Asia Aging Study (HAAS). In the HAAS, there was a significant association between hippocampal sclerosis (HS) and TDP-43 (OR = 11.04, p < 0.0001, 95% CI 3.57-34.13). In the NS, there were significant associations between TDP-43 and HS (OR = 16.44, p > 0.001 95%, CI 7.10-38.00) and Alzheimer's disease (AD) severity (OR = 1.74, p = 0.009, 95% CI 1.15-2.64). When cognitive scores were added to the model, HS remained significant but the other variables were not. When HS was removed from the model, the overall model remained significant and the associations between cognitive performance and TDP-43 (OR = 2.11, p = 0.022, 95% CI 1.11-4.02) were significant. In the NS, there was a significant association between cognitive performance and TDP-43 (OR 1.94 p = 0.005, 95% CI 1.22-3.09) (HS remained significant, but AD did not). When HS was removed from the model, only CERAD was significant (OR = 2.43 p < 0.001, 95% CI 1.58-3.74). These results support a consistent association between pathologic TDP-43, HS, and the development of cognitive impairment in two large studies of brain aging, while the relationship between AD pathology and TDP-43 may vary according to cohort-specific features.

Flow cytometric evaluation of crude synaptosome preparation as a way to study synaptic alteration in neurodegenerative diseases.

Neurodegenerative diseases, the most common among them Alzheimer's disease (AD) and Lewy body disease (LBD), are a group of progressive incurable illnesses. In both AD and LBD, abundant evidence points to the synapse as the critical and early focus of pathological changes. Here we present a method for the isolation and flow cytometric analysis of synaptosomes prepared from postmortem human brain tissue, which we also applied to animal models, including mice and nonhuman primates. The use of flow cytometry for analysis allows for relatively fast and efficient examination of thousands of synaptosome particles in a matter of minutes, and also makes it possible to use crude, rather than purified, synaptosomal preparation, thus conserving tissue resources. We have applied this method to study synaptic alteration in several brain regions in human research participants and animal models.