Citations

AbstractBackgroundNeuron‐derived extracellular vesicles (NDEVs) are a valuable resource for understanding brain conditions and discovering neurodegenerative diseases biomarkers, notably Alzheimer’s disease (AD). Recent interest focuses on capturing neuron‐specific EVs from patient‐derived samples, characterizing their contents as a pathological reflection of the central nervous system (CNS). Our recent study identified ATPase Na+/K+ Transporting Subunit Alpha 3 (ATP1A3) as a prevalent neuron‐specific EV marker specifically expressed in brains. This study systematically analyzes neuronal EVs to assess the specificity of ATP1A3 to NDEVs and its potential as a target for NDEV pulldown from accessible biofluids for disease monitoring. Additionally, ATP1A3 is compared with other proposed NDEV markers.MethodWe applied immunoelectron microscopy to detect ATP1A3, L1 cell adhesion molecule (L1CAM) and neural cell adhesion molecule 1 (NCAM1) in EVs isolated from iPSC‐derived excitatory neurons, brain tissue, cerebrospinal fluid (CSF) and plasma. Neuronal EV enrichment was achieved through immunoaffinity isolation, utilizing anti‐ATP1A3, L1CAM and NCAM1 antibodies separately. Evaluation involved quantitative mass‐spectrometry, immunoblotting and ELISA. To compare the enrichment of neuronal markers, EVs isolated from CSF and plasma samples were analyzed using ExoView and Nanoimager. Additionally, we investigated the potential of ATP1A3+ plasma EVs as AD biomarker by comparing the value of amyloid‐beta peptide (Aβ) in ATP1A3+ plasma EVs measured by Nanoimager and other plasma AD biomarkers as determined by SIMOA.ResultATP1A3, identified as a neuron‐specific protein, exhibits substantial enrichment in NDEVs isolated from induced human neurons, brain tissue, CSF, and plasma samples, surpassing NCAM1 or L1CAM. Both single‐ and bulk‐EV analysis consistently demonstrate a higher enrichment of ATP1A3 associated EVs in human samples. Immunoprecipitation of ATP1A3+ EVs from human brain tissues reveals superior neuronal cell‐type specificity over NCAM1+ and L1CAM+ EVs by label‐free mass spectrometry. Increased Aβ levels are observed in ATP1A3+ EVs derived from CSF and plasma of AD cases by SIMOA. Moreover, Aβ+ populations in ATP1A3+ EVs from plasma can distinguish AD from mild cognitive impairment and control cases via Nanoimager compared to the conventional quantification of AD markers.ConclusionOur findings demonstrate that ATP1A3 as a promising target for isolating NDEVs from biofluids, offering potential diagnostic advancements in neurological research.

AbstractBackgroundTau aggregates, a hallmark of Alzheimer’s disease (AD) and other tauopathies, spread throughout the brain, contributing to neurodegeneration. How this propagation occurs remains elusive. Previous research suggests that tau‐seed interactors play a crucial role. Based on this, the study aimed to identify novel tau‐seed interactors in AD brains and validate their impact in vivo.MethodAD and control brain extracts were separated in fractions by Size Exclusion Chromatography. Fractions with the highest tau seeding activity, measured using a tai‐biosensor cell line, were analyzed by mass spectrometry to identify interacting proteins. Bioinformatic tools dissected enriched pathways, identifying interactors that were validated in a Drosophila tauopathy model by genetically interfering with their homologs and assessing tau accumulation and eye degeneration.ResultsTau seeding activity was concentrated in high molecular weight fractions containing only a small portion of total tau in the AD brains. Compared to controls, AD brains revealed a distinct interactome for tau‐seeds, enriched in proteins associated with synaptic and mitochondrial pathways. Notably, Drosophila screening confirmed that several novel interactors significantly reduced tau accumulation and eye degeneration, suggesting their potential therapeutic relevance.ConclusionThis study sheds light on tau propagation mechanisms in AD by identifying novel tau‐seed interactors. These interactors, particularly those involved in synaptic and mitochondrial pathways, offer promising targets for therapeutic interventions aimed at decreasing tau spread and potentially preventing neurodegeneration in tauopathies. The findings add to the growing evidence that targeting tau‐seed interactors, like previously identified BSN, could represent a novel strategy for treating these debilitating conditions.

AbstractBackgroundTauopathies are a group of neurodegenerative disorders which are characterized by the accumulation of abnormal tau protein in the brain. However, the mechanistic understanding of pathogenic tau formation and spread within the brain remains elusive. Astrocytes are major immune reactive cells in the brain and have been implicated in exacerbating tau pathology by releasing extracellular vesicles (AEVs) containing pro‐inflammatory cytokines and chemokines upon activation. Our prior investigation revealed a significant association between AEVs and tau pathology development, as well as cognitive function, by analyzing brain‐derived EV proteins from AD patients. In this study, we explore the potential roles of AEVs in tau pathogenesis using a human induced pluripotent stem cell (iPSC) model.MethodWe obtained two male P301L tau mutant iPSC lines from a Polish family with frontotemporal dementia. By including two male control lines, these iPSCs were differentiated into astrocytes (iAs) and characterized by immunocytochemistry and subjected for bulk RNAseq. Bioinformatics analysis was conducted to compare the transcriptome profile between wild‐type (WT) and P301L iAs. EVs from WT and P301L iAs were isolated by ultracentrifugation combined with size exclusion chromatography. Characterization of WT and P301L iAEVs involved nanoparticle tracking analysis, nano‐flow cytometry and super‐resolution microscopy.ResultWe successfully differentiated WT and P301L mutant iPSC lines into astrocytes with >99% purity. P301L iAs displayed distinctive astrocyte reactivity compared to WT cells, with elevated levels of pan‐reactive astrocyte genes (e.g., GFAP, CD44) and decreased expression of neuroprotective A2 astrocyte‐specific genes (e.g., TM4SF1, PTGS2). Additionally, gene enrichment set analysis of RNAseq data revealed dysregulation in the endo‐lysosomal pathway and extracellular matrix in P301L iAs compared to WT iAs. The count of intraluminal vesicles marked by CD9+ were reduced in P301L iAs compared to WT cells. Moreover, we observed a significant increase in the internalization of Tau by P301L iAs compared to WT iAs following incubation with preformed Tau fibrils, resulting in an augmented release of tau‐containing EVs from P301L iAs.ConclusionOur findings suggest a potential alteration in EV biogenesis in P301L iAs, potentially contributing to astrocyte‐mediated tau pathology. Future investigations will focus on understanding how AEVs contribute to tau propagation and accumulation.

AbstractBackgroundTau aggregation is the major cause of several neurodegenerative tauopathies. Tau interaction with other proteins affects the formation of tau aggregates with seeding activity but less is known about its effects on tau‐seed properties. Our previous study revealed that Bassoon (BSN), a presynaptic protein, interacts with tau‐seed, exacerbating its toxicity in vivo. Bsndownregulation reduced tau spreading and overall pathology. Intriguingly, a parallel study associated missense mutations in BSN with tau aggregation in patients, prompting an investigation into the influence of genetic mutations in BSN on tau pathology for potential therapeutic insights.MethodWe generated a knock‐in mouse model (BSNKI) harboring the disease‐associated p.Pro3866Ala mutation in endogenous Bsn. Cognitive and motor abilities were assessed in aged heterozygous and homozygous BSNKI mice, followed by analyses of BSN and tau patterns, gliosis, and gene expression changes in their brains. Additionally, we validated our findings in a human BSN mutation carrier.ResultAt 10 months, BSNKI mice displayed motor impairments on the rotarod, and grip strength assays compared to WT mice. Their brains displayed somatic BSN and pathological tau accumulation, with the gene expression changes indicating alterations in microglia activation, protein degradation pathways, complement activation, and synapse pruning. We also observed an accumulation of pathological tau at the synapses and synapse engulfment by microglia. Histopathological analyses revealed robust microglia activation and co‐deposition of proteasomal subunits with BSN. The human BSN mutation carrier displayed inclusions of BSN and tau pathology similar to observations in the BSNKI model.ConclusionOur BSNKI mouse model, reflecting a disease‐associated BSN mutation, revealed motor impairments and pathological tau and BSN deposits, mirroring observations in BSN mutation carriers. Notably, BSN seems to play a dual role, promoting tau aggregation and sequestering protein degradation molecules, leading to tau and protein accumulation at the soma and synapse, triggering microgliosis and neuroinflammation. These findings propose BSN as a promising therapeutic target for tauopathies, underscoring the need for further exploration to elucidate underlying mechanisms and therapeutic implications.

Abstract Background and Aims Artificial intelligence (AI) algorithms in 12-lead electrocardiogram (ECG) provides promising age prediction methods. This study investigated whether the discrepancy between ECG-derived AI-predicted age (AI-ECG age) and chronological age, termed electrocardiographic aging (ECG aging), is associated with atrial fibrillation (AF) risk. Methods An AI-ECG age prediction model was developed using a large-scale dataset (1 533 042 ECGs from 689 639 participants) and validated with six independent and multi-national datasets (737 133 ECGs from 330 794 participants). The AI-ECG age gap was calculated across two South Korean cohorts [mean (standard deviation) follow-up: 4.1 (4.3) years for 111 483 participants and 6.1 (3.8) years for 37 517 participants], one UK cohort [3.0 (1.6) years; 40 973 participants], and one US cohort [12.9 (8.6) years; 90 639 participants]. Participants were classified into two groups: normal group (age gap < 7 years) and ECG-aged group (age gap ≥ 7 years). The predictive capability of ECG aging for new- and early-onset AF risk was assessed. Results The mean AI-ECG ages were 51.9 (16.2), 47.4 (12.5), 68.4 (7.8), and 56.7 (14.6) years with age gaps of .0 (6.8), −.1 (6.0), 4.7 (8.7), and −1.4 (8.9) years in the two South Korean, UK, and US cohorts, respectively. In the ECG-aged group, increased risks of new-onset AF were observed with hazard ratios (95% confidence intervals) of 2.50 (2.24–2.78), 1.89 (1.46–2.43), 1.90 (1.55–2.33), and 1.76 (1.67–1.86) in the two South Korean, UK, and US cohorts, respectively. For early-onset AF, odds ratios were 2.89 (2.47–3.37), 1.94 (1.39–2.70), 1.58 (1.06–2.35), and 1.79 (1.62–1.97) in these cohorts compared with the normal group. Conclusions The AI-derived ECG aging was associated with the risk of new- and early-onset AF, suggesting its potential utility to identify individuals for AF prevention across diverse populations.

Abstract Disclosure: R. Kennedy: None. D.A. D'Alessio: None. A.V. Vella: None. Introduction: Selective arterial calcium stimulation testing (SACST) can be utilized in patients to localize solitary insulinomas when imaging is non diagnostic. While a heterogenous stimulation pattern suggests NIPHS, or multiple insulinomas this case uniquely demonstrates such a pattern with a solitary insulinoma. Clinical Case: The patient is a 48 year old female who presented to the emergency department (ED) for bizarre behavior which resolved with a sweetened drink. Serum glucose was 62 mg/dL, she was treated with IV dextrose and was discharged from the ED. She was referred to outpatient endocrinology for further evaluation where continuous glucose monitoring confirmed predominantly fasting hypoglycemia. Adrenal insufficiency was excluded. She was admitted for a 72 hour inpatient fast where the patient achieved hypoglycemia within 24 hours, with glucose 46 mg/dL (associated with neuroglycopenic symptoms), beta hydroxybutyrate < 0.18 mmol/L, insulin 3.9 mcIU/mL, proinsulin 157pmol/L, c-peptide 1.4 ng/mL and a glucose rise greater than 25mg/dL after glucagon administration. Sulfonylurea screen and insulin antibodies were negative; IGF1 within normal limits. These labs were consistent with hyperinsulinemic hypoglycemia. An MRI pancreas however did not reveal an insulinoma. The patient therefore underwent a SACST, demonstrating a 10 fold response in the territory of the gastroduodenal artery and splenic artery, as well as a 3 fold response in the superior mesenteric artery. This heterogenous pattern in addition to the marked response in each arterial territory raised the possibility of multiple insulinomas. However as there were no tumors present on imaging, medical management was initiated with close follow up. She was trialed with months of somatostatin analogs and diazoxide with minimal improvement in the frequency and severity of her hypoglycemia. About a year after her initial diagnosis, repeat abdominal imaging (CT and MRI) revealed an 8 mm arterial hyper-enhancing lesion in the body of the pancreas concerning for neuroendocrine tumor. Repeat SACST was performed, this time showing a greater than 10 fold response in all three arterial territories. Angiography showed a tumor blush that was visible when each of the three major arteries were injected sequentially. She underwent laparoscopic pancreatic enucleation. The lesion was located on the body of the pancreas, with histology confirming an insulinoma. The patient responded promptly post operatively; her symptoms resolved without further need of medical management. Conclusion: Our case shows that the interpretation of an SACST should take into account the arterial anatomy before ascribing a non-focal response to multiple territories of insulin hypersecretion. Presentation: 6/1/2024