Citations

AbstractBackgroundA complex, multicellular disease with genetic and immunological elements, Alzheimer’s disease (AD) affects millions worldwide. There has been previous research linking AD to the missense variants ABI3‐rs616338‐T and PLCG2‐rs72824905‐G, and the altered expression of these genes has been shown to disrupt microglial function. In our understanding of AD risk and resilience, limited research has been conducted on how these variants affect microglial subtypes and states in AD.MethodsWe previously identified DOCK8 protein as a target for fluorescent activated nuclei sorting (FANS) to enrich microglia nuclei from frozen human brains. Using this enrichment strategy, we generated microglia enriched snRNAseq data from temporal cortex tissue of 30 donors harboring ABI3 or PLCG2 missense mutations, or neither mutation. We introduced PLCG2 variant in either homozygote, and heterozygote forms into an AD patient derived iPSC through CRISPR/Cas9 approach, differentiated these cells into microglia cells (IMGLs) following established protocols, and generated scRNAseq data after treatment with Amyloidβ. We applied standard alignment and quality control pipelines to the snRNAseq and scRNAseq datasets in parallel. Established markers of microglia states and subtypes were utilized to annotate the clusters; analyzed for their hub gene expression using comparisons between cluster differential profiling to determine signature pathways. Differential gene expression was assessed using pseudo bulk and MAST approaches.ResultsWe obtained single nuclei transcriptome profiles of 54,000 frozen human brain nuclei, of which 35,000 are microglia, through snRNAseq, and single cell profiles of 63,000 iMGLs through scRNAseq, using our standard alignment and quality control procedures. Our differential gene expression analysis identified novel genes and pathways which were identified as part of resilience or AD risk networks in microglia subtypes and states. These alterations were validated using orthogonal experimental methods and further explored for their conservation using in vivo external datasets. Pseudotime inference will be completed to probe microglia marker gene expression dynamics, treatment conditions, along with continuous cell‐state changes.ConclusionOur study uncovers microglia subtype specific resilience and risk factors provided by AD related genetic variants using snRNAseq from frozen human brain and scRNAseq from IPSC derived microglia cells. These findings nominate novel targets and pathways with therapeutic potential.

AbstractBackgroundGenetic variations have emerged as crucial players in the etiology of Alzheimer’s disease (AD), and they serve for a better understanding of the disease mechanisms; yet the specific roles of these genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Therefore, we generated zebrafish models for AD variants to analyze the in depth molecular and biological functions of these variants.MethodUsing CRISPR/Cas9, we generated a knockout model for abca7, orthologous to human ABCA7. We performed single cell transcriptomics and analyzed the altered genes and molecular pathways in zebrafish. We leveraged data from multiethnic AD cohorts at Mayo Clinic and Columbia University, to perform genetic association studies, co‐expression analyses, in silico interaction mapping, family based variant segregation analyses and epigenetic association studies, and the functional and histological studies in zebrafish.ResultThe abca7± zebrafish reduced astroglial proliferation, synaptic integrity, and microglial response after Aβ42 toxicity. We found that the abca7 loss‐of‐function (LOF) reduced neuropeptide Y (npy) expression as well as Brain‐derived neurotrophic factor (bdnf) and Nerve growth factor receptor (ngfr). Human brain analysis showed reduced NPY in AD, regulatory interaction between NPY and BDNF, genetic variants in NPY associated with AD, and segregation of variants in ABCA7, BDNF and NGFR in families. ABCA7 variants altered the epigenetic codes in NPY, BDNF, and NGFR promoter regions. Human results paralleled with zebrafish findings to indicate an evolutionarily conserved disease mechanism through ABCA7‐NPY signalling axis. NPY administration to zebrafish rescued the phenotypes in abca7 knockout, suggesting a true biological relevance.ConclusionOur results demonstrate a previously unknown link between ABCA7 and NPY in regulation of synaptic integrity and neurogenesis in AD. We propose that ABCA7‐dependent NPY is a resilience factor in vertebrate brains, and this reserve mechanism is impaired in AD.

Abstract BACKGROUND Lynch syndrome is caused by pathogenic germline variants in the DNA mismatch repair (MMR) genes, predisposing individuals to malignancies, including glioblastoma (GBM). MMR-deficient tumors are resistant to monofunctional alkylators such as temozolomide but not to bifunctional alkylators such as the nitrosourea lomustine. Anti-programmed cell death protein 1 (PD1) immune checkpoint inhibitor (ICI) pembrolizumab represents another treatment option, which is approved by the United States Food and Drug Administration (FDA) for the treatment of mismatch repair deficient cancers. METHODS We present a patient with multifocal GBM and Lynch syndrome who experienced rapid progression following standard-of-care chemoradiation, followed by durable responses to lomustine and then to pembrolizumab. RESULTS A 58-year-old man with Lynch syndrome (MSH2 mutation) and prior history of colon cancer presented with seizures. Magnetic resonance imaging (MRI) brain identified right frontal and left parietal masses. Right frontal tumor was resected; pathology was consistent with GBM, IDH wildtype, MGMT-unmethylated. Both lesions were treated with radiation therapy (60 Gy, 30 fractions) with concurrent and adjuvant temozolomide. After two cycles of adjuvant temozolomide, his left parietal tumor progressed and was subtotally resected, followed by four cycles of lomustine (stopped due to myelotoxicity). He remained progression-free for 15 months. Surveillance MRI identified new enhancement involving the left frontal lobe, left periventricular white matter, and right internal auditory canal, concerning for leptomeningeal dissemination. MRI spine identified no additional lesions. Cerebrospinal fluid cytology was negative for tumor cells on two occasions. Patient was treated with pembrolizumab. He remains on treatment with stable disease four months after ICI initiation and 31 months after GBM diagnosis. CONCLUSIONS Patients with MMR-deficient GBM resist standard chemoradiation. Alternatives such as nitrosourea or ICI therapy may achieve durable responses. While data is insufficient to administer these agents as upfront therapy in this specific setting, both should be considered as options for recurrent disease.

Abstract INTRODUCTION Multi‐omics studies in Alzheimer's disease (AD) revealed many potential disease pathways and therapeutic targets. Despite their promise of precision medicine, these studies lacked Black Americans (BA) and Latin Americans (LA), who are disproportionately affected by AD. METHODS To bridge this gap, Accelerating Medicines Partnership in Alzheimer's Disease (AMP‐AD) expanded brain multi‐omics profiling to multi‐ethnic donors. RESULTS We generated multi‐omics data and curated and harmonized phenotypic data from BA ( n = 306), LA ( n = 326), or BA and LA ( n = 4) brain donors plus non‐Hispanic White ( n = 252) and other ( n = 20) ethnic groups, to establish a foundational dataset enriched for BA and LA participants. This study describes the data available to the research community, including transcriptome from three brain regions, whole genome sequence, and proteome measures. DISCUSSION The inclusion of traditionally underrepresented groups in multi‐omics studies is essential to discovering the full spectrum of precision medicine targets that will be pertinent to all populations affected with AD. Highlights Accelerating Medicines Partnership in Alzheimer's Disease Diversity Initiative led brain tissue profiling in multi‐ethnic populations. Brain multi‐omics data is generated from Black American, Latin American, and non‐Hispanic White donors. RNA, whole genome sequencing and tandem mass tag proteomicsis completed and shared. Multiple brain regions including caudate, temporal and dorsolateral prefrontal cortex were profiled.

Deep learning offers significant advancements in the accuracy of prostate identification and classification, underscoring its potential for clinical integration. However, the opacity of deep learning models presents interpretability challenges, critical for their acceptance and utility in medical diagnosis and detection. While explanation methods have been proposed to demystify these models, enhancing their clinical viability, the efficacy and acceptance of these methods in medical tasks are not well documented. This pilot study investigates the effectiveness of deep learning explanation methods in clinical settings and identifies the attributes that radiologists consider crucial for explainability, aiming to direct future enhancements. This study reveals that while explanation methods can improve clinical task performance by up to 20%, their perceived usefulness varies, with some methods being rated poorly. Radiologists prefer explanation methods that are robust against noise, precise, and consistent. These preferences underscore the need for refining explanation methods to align with clinical expectations, emphasizing clarity, accuracy, and reliability. The findings highlight the importance of developing explanation methods that not only improve performance but also are tailored to meet the stringent requirements of clinical practice, thereby facilitating deeper trust and a broader acceptance of deep learning in medical diagnostics.

Isocitrate dehydrogenase (IDH) mutant gliomas are a primary malignancy of the central nervous system (CNS) malignancies, most commonly affecting adults under the age of 55. Standard of care therapy for IDH-mutant gliomas involves maximal safe resection, radiotherapy, and chemotherapy. However, despite good initial responses to multimodality treatment, recurrence is virtually universal. IDH-mutant gliomas represent a life-limiting prognosis. For this reason, there is a great need for novel treatments that can prolong survival. Uniquely for IDH-mutant gliomas, the IDH mutation is the direct driver of oncogenesis through its oncometabolite 2-hydroxygluterate. Inhibition of this mutated IDH with a corresponding reduction in 2-hydroxygluterate offers an attractive treatment target. Researchers have tested several IDH inhibitors in glioma through preclinical and early clinical trials. A phase III clinical trial of an IDH1 and IDH2 inhibitor vorasidenib yielded promising results among patients with low-grade IDH-mutant gliomas who had undergone initial surgery and no radiation or chemotherapy. However, many questions remain regarding optimal use of IDH inhibitors in clinical practice. In this review, we discuss the importance of IDH mutations in oncogenesis of adult-type diffuse gliomas and current evidence supporting the use of IDH inhibitors as therapeutic agents for glioma treatment. We also examine unresolved questions and propose potential directions for future research.

Mechanical cardiac alternans is usually associated with left ventricular dysfunction. Atrial alternans has been described on invasive assessment of atrial action potentials, however cannot be detected clinically. Herein we report a patient who had transthyretin type cardiac amyloidosis with prior history of recurrent atrial fibrillation requiring ablations as well as ablation for atrial flutter and a recent history of cardioversion for atrial flutter. The patient presented with diastolic heart failure and recurrent atrial flutter and was referred for a TEE guided cardioversion. Mechanical left and right atrial alternans was seen on Doppler interrogation of both atrial appendages with velocities corresponding to the flutter P wave on the ultrasound ECG monitor. The patient was successfully cardioverted with the TEE probe in situ. Post cardioversion TEE showed resumption of normal albeit reduced mechanical atrial function in the left atrium and preserved right atrial function in tissue Doppler imaging. Our case demonstrates the mechanical phenomenon of atrial alternans in both atria on TEE with disappearance of atrial alternans during sinus rhythm suggesting rate related atrial alternans likely related to atrial stiffness from amyloid infiltration and as well as possibly scar formation from prior ablations.

AbstractBackgroundDementia with Lewy bodies (DLB), the second most prevalent form of degenerative dementia, is characterized by pathological α‐synuclein (αSyn) protein aggregation in neurons. Pathological αSyn shows a protein‐seeding phenomenon that may result in aggregation years before clinical symptoms. Studies using Real‐time quaking‐induced conversion (RT‐QuIC) assay show αSyn seeding from peripheral tissue including skin and cerebrospinal Fluid (CSF). Identifying early biomarkers for DLB is essential for accurate diagnosis, and developing effective treatments to slow progression. Herein, we investigate αSyn seeding activities in CSF from patients with mild cognitive impairment with Lewy bodies (MCI‐LB) phenoconverting to DLB.MethodCSF samples were obtained from 3 MCI‐LB and 2 DLB patients at baseline and two‐year follow‐up appointments. CSF samples were tested for their potential to seed α‐synuclein aggregation using an RT‐QuIC assay. CSF samples were tested by seeding 2 μl of CSF in 98 ul of RT‐QuIC reaction mixture with human recombinant αSyn protein, analyzed on a FLUOstar Omega plate reader. Results were recorded every 30 minutes over a period of 45 hours analyzed based on the reaction kinetics to determine the seeding ability of the α‐synuclein in the CSF samples.ResultWe observed seed amplification at first visit for both DLB patients and one MCI‐LB patient. The absolute quantification of aggregation increased at second visit (2 years apart) for one DLB patient and one MCI‐LB. The other two MCI‐LB patients showed no amplification at baseline visit but showed signal at two‐year visit, by which time all three had phenoconverted to DLB. Interestingly the second DLB patient showed a decline at two‐year follow‐up. The medical and neuroimaging records of that patient indicate that the patient’s cognitive decline began as early‐onset disease (first symptoms at age 50 years).ConclusionWe demonstrate that measures of seed aggregation in CSF with RT‐QuIC are informative in MCI‐LB and DLB, and may also reflect changes in disease progression and dynamics.

AbstractBackgroundCerebral amyloid angiopathy (CAA), characterized by the accumulation of amyloid‐beta in the cerebrovasculature, effects blood vessel integrity leading to brain hemorrhages and an accelerated cognitive decline in Alzheimer’s disease (AD) patients. In a previous genome‐wide association study (GWAS) of 853 neuropathologically confirmed AD cases, we identified a LINC‐PINT splice variant that associated with lower CAA levels amongst individuals that did not have a copy of the APOEe4 AD risk allele (Reddy. J et al 2021). In this study, we perform GWAS to identify further genetic risk factors for CAA and AD neuropathology (Braak stage, Thal phase) in additional AD cases, and participants lacking significant AD pathology (non‐AD).MethodWe expanded the original AD cohort (N = 853) with 400 additional AD cases genotyped using the Infinium Omni2.5+Exome array. We also identified 110 AD and 502 non‐AD donors from the Mayo Clinic Brain Bank, with existing CAA scores and genome‐wide genotypes. We performed QC and imputation of these genotypes to TOPMed. We will conduct GWAS in AD only (N = 1,363), non‐AD only, as well as the combined cohort (N = 1,865). We will test imputed variant dosages for association with square root transformed CAA, Braak and Thal, using linear regression in PLINK, adjusting for relevant covariates. We will perform interaction analysis of CAA‐associated variants with APOEe4 and sex; and pursue stratified analyses. Summary statistics will be utilized to perform SNP‐based gene set enrichment analysis to identify biological pathways implicated in CAA.ResultWe expect that the increased power of this expanded dataset will lead to the identification of additional novel genetic risk factors for CAA and AD neuropathology. Genetic interaction analysis with sex and APOEe4 may further elucidate the role of these risk factors. The addition of 1,102 participants will enable assessment of the LINC‐PINT locus for association with CAA in an independent dataset.ConclusionWe expect this study will provide further insights into the genetic architecture underlying risk for CAA, both in the context of significant AD pathology, and without. Characterization of genetic variants in the context of neuropathology may lead to new avenues of research aimed at identifying biomarkers and therapies to treat CAA, and AD.

AbstractBackgroundAlzheimer’s disease (AD) is a complex, multicellular disease with immunological and genetic components. Previous studies demonstrated associations between AD and the missense variants ABI3‐rs616338‐T and PLCG2‐rs72824905‐G. The perturbed expression of these genes has been demonstrated to disrupt microglial function. To date, the transcriptomic profiles of variant carriers in the context of microglial subtype and state have not been explored in human brain. This represents a knowledge gap in our understanding of AD risk and resilience. We have developed and optimized a single nucleus RNAseq (snRNAseq) approach to study the impact of ABI3 and PLCG2 variants on microglial enriched nuclei from frozen human brain.MethodDOCK8 was identified as a nuclear microglial marker based on previous brain snRNAseq data. DOCK8 based microglial enrichment using fluorescent activated nuclei sorting (FANS) was validated using RT‐qPCR and snRNAseq in human cerebellum and temporal cortex (TCX), respectively. We are applying this enrichment and snRNAseq approach to TCX tissue from a cohort of 30 donors harboring ABI3 or PLCG2 missense mutations, or neither. Standard snRNAseq alignment and quality control pipelines will be applied, and nuclei annotated based on putative microglial markers. Differential gene expression will be assessed using pseudo bulk and MAST approaches, for association with ABI3 and PLCG2 genotype, diagnosis groups, risk factors, and pathology scores.ResultDOCK8 has several commercially available antibodies, making it an ideal microglia‐selection marker. Using RT‐qPCR in microglia enriched nuclei, we demonstrated an enrichment of microglia marker genes and depletion of other brain cell type markers. This is further supported by snRNA‐seq data from DOCK8‐sorted TCX tissue that yielded a 5x increase in microglia proportion (56% vs. 11% in unselected nuclei), enabling analyses of microglia sub‐populations. Leveraging this approach, we will investigate affected genes and pathways and uncover the resilience and risk mechanisms within microglia sub‐populations in ABI3 and PLCG2 variant‐carriers.ConclusionOur study presents a novel method for enriching microglial nuclei from frozen human brains and enables transcriptomic profiling of microglial subpopulations in ABI3 and PLCG2 missense variant‐carriers. We expect these insights will reveal novel genes and pathways that may play a key role in microglial function in AD.