Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques ...and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10-10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.
Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate ...some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we have developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tauP301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, the tauP301S assembloids showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders.
Alzheimer's disease (AD) is a neurodegenerative disease that is clinically characterized by progressive cognitive decline. More than 200 pathogenic mutations have been identified in amyloid-β ...precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2). Additionally, common and rare variants occur within APP, PSEN1, and PSEN2 that may be risk factors, protective factors, or benign, non-pathogenic polymorphisms. Yet, to date, no single study has carefully examined the effect of all of the variants of unknown significance reported in APP, PSEN1 and PSEN2 on Aβ isoform levels in vitro. In this study, we analyzed Aβ isoform levels by ELISA in a cell-based system in which each reported pathogenic and risk variant in APP, PSEN1, and PSEN2 was expressed individually. In order to classify variants for which limited family history data is available, we have implemented an algorithm for determining pathogenicity using available information from multiple domains, including genetic, bioinformatic, and in vitro analyses. We identified 90 variants of unknown significance and classified 19 as likely pathogenic mutations. We also propose that five variants are possibly protective. In defining a subset of these variants as pathogenic, individuals from these families may eligible to enroll in observational studies and clinical trials.
•We sought to classify variants of unknown significance in APP, PSEN1, and PSEN2.•We applied a pathogenicity algorithm using genetic, bioinformatic, and in vitro data.•90 variants of unknown significance were evaluated.•19 variants were classified as likely pathogenic mutations.•5 variants were classified as possibly protective.
Alzheimer's disease (AD) is the most dominant form of dementia characterized by the deposition of extracellular amyloid plaques and intracellular neurofibrillary tau tangles (NFTs). In addition to ...these pathologies, an emerging pathophysiological mechanism that influences AD is neuroinflammation. Astrocytes are a vital type of glial cell that contribute to neuroinflammation, and reactive astrocytes, or astrogliosis, are a well-known pathological feature of AD. However, the mechanisms by which astrocytes contribute to the neurodegenerative process in AD have not been fully elucidated. Here, we showed that astrocytic α2-Na
/K
adenosine triphosphatase (α2-NKA) is elevated in postmortem human brain tissue from AD and progressive nuclear palsy, a primary tauopathy. The increased astrocytic α2-NKA was also recapitulated in a mouse model of tauopathy. Pharmacological inhibition of α2-NKA robustly suppressed neuroinflammation and reduced brain atrophy. In addition, α2-NKA knockdown in tauopathy mice halted the accumulation of tau pathology. We also demonstrated that α2-NKA promoted tauopathy, in part, by regulating the proinflammatory protein lipocalin-2 (Lcn2). Overexpression of Lcn2 in tauopathy mice increased tau pathology, and prolonged Lcn2 exposure to primary neurons promoted tau uptake in vitro. These studies collectively highlight the contribution of reactive astrocytes to tau pathogenesis in mice and define α2-NKA as a major regulator of astrocytic-dependent neuroinflammation.
Background TREM2 is a transmembrane receptor that is predominantly expressed by microglia in the central nervous system. Rare variants in the TREM2 gene increase the risk for late-onset Alzheimer's ...disease (AD). Soluble TREM2 (sTREM2) resulting from shedding of the TREM2 ectodomain can be detected in the cerebrospinal fluid (CSF) and is a surrogate measure of TREM2-mediated microglia function. CSF sTREM2 has been previously reported to increase at different clinical stages of AD, however, alterations in relation to Amyloid beta-peptide (Abeta) deposition or additional pathological processes in the amyloid cascade (such as tau pathology or neurodegeneration) remain unclear. In the current cross-sectional study, we employed the biomarker-based classification framework recently proposed by the NIA-AA consensus guidelines, in combination with clinical staging, in order to examine the CSF sTREM2 alterations at early asymptomatic and symptomatic stages of AD. Methods A cross-sectional study of 1027 participants of the Alzheimer's Disease Imaging Initiative (ADNI) cohort, including 43 subjects carrying TREM2 rare genetic variants, was conducted to measure CSF sTREM2 using a previously validated enzyme-linked immunosorbent assay (ELISA). ADNI participants were classified following the A/T/N framework, which we implemented based on the CSF levels of Abeta.sub.1-42 (A), phosphorylated tau (T) and total tau as a marker of neurodegeneration (N), at different clinical stages defined by the clinical dementia rating (CDR) score. Results CSF sTREM2 differed between TREM2 variants, whereas the p.R47H variant had higher CSF sTREM2, p.L211P had lower CSF sTREM2 than non-carriers. We found that CSF sTREM2 increased in early symptomatic stages of late-onset AD but, unexpectedly, we observed decreased CSF sTREM2 levels at the earliest asymptomatic phase when only abnormal Abeta pathology (A+) but no tau pathology or neurodegeneration (TN-), is present. Conclusions Abeta pathology (A) and tau pathology/neurodegeneration (TN) have differing associations with CSF sTREM2. While tau-related neurodegeneration is associated with an increase in CSF sTREM2, Abeta pathology in the absence of downstream tau-related neurodegeneration is associated with a decrease in CSF sTREM2. Keywords: Alzheimer's disease, Biomarkers, Microglia, Neurodegeneration, Neuroinflammation, Shedding, TREM2
Genetic studies of Alzheimer disease (AD) have prioritized variants in genes related to the amyloid cascade, lipid metabolism, and neuroimmune modulation. However, the cell-specific effect of ...variants in these genes is not fully understood. Here, we perform single-nucleus RNA-sequencing (snRNA-seq) on nearly 300,000 nuclei from the parietal cortex of AD autosomal dominant (APP and PSEN1) and risk-modifying variant (APOE, TREM2 and MS4A) carriers. Within individual cell types, we capture genes commonly dysregulated across variant groups. However, specific transcriptional states are more prevalent within variant carriers. TREM2 oligodendrocytes show a dysregulated autophagy-lysosomal pathway, MS4A microglia have dysregulated complement cascade genes, and APOEε4 inhibitory neurons display signs of ferroptosis. All cell types have enriched states in autosomal dominant carriers. We leverage differential expression and single-nucleus ATAC-seq to map GWAS signals to effector cell types including the NCK2 signal to neurons in addition to the initially proposed microglia. Overall, our results provide insights into the transcriptional diversity resulting from AD genetic architecture and cellular heterogeneity. The data can be explored on the online browser ( http://web.hararilab.org/SNARE/ ).
Mutations in the microtubule-associated protein tau (MAPT) gene are known to cause familial frontotemporal dementia (FTD). The R406W tau mutation is a unique missense mutation whose patients have ...been reported to exhibit Alzheimer’s disease (AD)-like phenotypes rather than the more typical FTD phenotypes. In this study, we established patient-derived induced pluripotent stem cell (iPSC) models to investigate the disease pathology induced by the R406W mutation. We generated iPSCs from patients and established isogenic lines using CRISPR/Cas9. The iPSCs were induced into cerebral organoids, which were dissociated into cortical neurons with high purity. In this neuronal culture, the mutant tau protein exhibited reduced phosphorylation levels and was increasingly fragmented by calpain. Furthermore, the mutant tau protein was mislocalized and the axons of the patient-derived neurons displayed morphological and functional abnormalities, which were rescued by microtubule stabilization. The findings of our study provide mechanistic insight into tau pathology and a potential for therapeutic intervention.
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•Phenotype analysis of FTD R406W mutant tau using iPSC-derived neurons•Homogeneous neuronal population obtained via dissociation of cerebral organoids•Altered phosphorylation and fragmentation state of R406W mutant tau•Multiple axonal defects in patient neurons rescued with microtubule stabilization
In this article, Nakamura and colleagues establish an iPSC-derived neuronal model from frontotemporal dementia patients with the tau R406W mutation and gain insight into the disease pathology. Here, the R406W mutant tau exhibited reduced phosphorylation levels and was prone to fragmentation by calpain. Furthermore, the patients’ neurons displayed multiple axonal defects caused by microtubule destabilization.
One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, ...disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.
Unbiased data-driven omic approaches are revealing the molecular heterogeneity of Alzheimer disease. Here, we used machine learning approaches to integrate high-throughput transcriptomic, proteomic, ...metabolomic, and lipidomic profiles with clinical and neuropathological data from multiple human AD cohorts. We discovered 4 unique multimodal molecular profiles, one of them showing signs of poor cognitive function, a faster pace of disease progression, shorter survival with the disease, severe neurodegeneration and astrogliosis, and reduced levels of metabolomic profiles. We found this molecular profile to be present in multiple affected cortical regions associated with higher Braak tau scores and significant dysregulation of synapse-related genes, endocytosis, phagosome, and mTOR signaling pathways altered in AD early and late stages. AD cross-omics data integration with transcriptomic data from an SNCA mouse model revealed an overlapping signature. Furthermore, we leveraged single-nuclei RNA-seq data to identify distinct cell-types that most likely mediate molecular profiles. Lastly, we identified that the multimodal clusters uncovered cerebrospinal fluid biomarkers poised to monitor AD progression and possibly cognition. Our cross-omics analyses provide novel critical molecular insights into AD.
Cognitive resilience is an important modulating factor of cognitive decline in Alzheimer's disease, but the functional brain mechanisms that support cognitive resilience remain elusive. Given ...previous findings in normal ageing, we tested the hypothesis that higher segregation of the brain's connectome into distinct functional networks represents a functional mechanism underlying cognitive resilience in Alzheimer's disease. Using resting-state functional MRI, we assessed both resting-state functional MRI global system segregation, i.e. the balance of between-network to within-network connectivity, and the alternate index of modularity Q as predictors of cognitive resilience. We performed all analyses in two independent samples for validation: (i) 108 individuals with autosomal dominantly inherited Alzheimer's disease and 71 non-carrier controls; and (ii) 156 amyloid-PET-positive subjects across the spectrum of sporadic Alzheimer's disease and 184 amyloid-negative controls. In the autosomal dominant Alzheimer's disease sample, disease severity was assessed by estimated years from symptom onset. In the sporadic Alzheimer's sample, disease stage was assessed by temporal lobe tau-PET (i.e. composite across Braak stage I and III regions). In both samples, we tested whether the effect of disease severity on cognition was attenuated at higher levels of functional network segregation. For autosomal dominant Alzheimer's disease, we found higher functional MRI-assessed system segregation to be associated with an attenuated effect of estimated years from symptom onset on global cognition (P = 0.007). Similarly, for patients with sporadic Alzheimer's disease, higher functional MRI-assessed system segregation was associated with less decrement in global cognition (P = 0.001) and episodic memory (P = 0.004) per unit increase of temporal lobe tau-PET. Confirmatory analyses using the alternate index of modularity Q revealed consistent results. In conclusion, higher segregation of functional connections into distinct large-scale networks supports cognitive resilience in Alzheimer's disease.
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