CSF complement proteins are elevated in prodromal to moderate Alzheimer's disease patients and are not altered by the anti-tau antibody semorinemab.

INTRODUCTION: Growing evidence suggests a role for neuroinflammation in Alzheimer's disease (AD). We investigated complement pathway activity in AD patient cerebrospinal fluid (CSF) and evaluated its modulation by the anti-tau antibody semorinemab. METHODS: Immunoassays were applied to measure CSF complement proteins C4, factor B (FB), C3 and their cleavage fragments C4a, C3a, and factor Bb (Bb) in AD patients and a separate cognitively unimpaired (CU) cohort. RESULTS: All measured CSF complement proteins were increased in AD versus CU subjects, with C4a displaying the most robust increase. Finally, semorinemab did not have a significant pharmacodynamic effect on CSF complement proteins. DISCUSSION: Elevated levels of CSF C4a, C4, C3a, C3, Bb, and FB are consistent with complement activation in AD brains. Despite showing a reduction in CSF soluble tau species, semorinemab did not impact complement protein levels or activity. Further studies are needed to determine the value of complement proteins as neuroinflammation biomarkers in AD. HIGHLIGHTS: Cerebrospinal fluid (CSF) complement proteins C4a, C3a, Bb, C4, C3, and factor B levels were increased in Alzheimer's disease (AD) patients compared to a separate cognitively unimpaired (CU) cohort. Baseline CSF complement protein levels were correlated with neuro-axonal degeneration and glial activation biomarkers in AD patients. The investigational anti-tau antibody semorinemab did not impact CSF complement protein levels or activity relative to the placebo arm.

Toward a human brain extracellular vesicle atlas: Characteristics of extracellular vesicles from different brain regions, including small RNA and protein profiles.

Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a "bdEV Atlas" of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 in all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are warranted to provide more insight into the links between EV heterogeneity and function in the CNS.

NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications.

Towards a human brain EV atlas: Characteristics of EVs from different brain regions, including small RNA and protein profiles.

Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a "bdEV atlas" of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 for all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are merited to lend more insight into the links between EV heterogeneity and function in the CNS.

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

Relationships of APOE Genotypes With Small RNA and Protein Cargo of Brain Tissue Extracellular Vesicles From Patients With Late-Stage AD.

Background and Objectives: Variants of the apolipoprotein E (APOE) gene are the greatest known risk factors for sporadic Alzheimer disease (AD). Three major APOE isoform alleles, ε2, ε3, and ε4, encode and produce proteins that differ by only 1-2 amino acids but have different binding partner interactions. Whereas APOE ε2 is protective against AD relative to ε3, ε4 is associated with an increased risk for AD development. However, the role of APOE in gene regulation in AD pathogenesis has remained largely undetermined. Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by cells to dispose of unwanted materials and mediate intercellular communication, and they are implicated in AD pathophysiology. Brain-derived EVs (bdEVs) could act locally in the tissue and reflect cellular changes. To reveal whether APOE genotype affects EV components in AD brains, bdEVs were separated from patients with AD with different APOE genotypes for parallel small RNA and protein profile. Methods: bdEVs from late-stage AD brains (BRAAK stages 5-6) from patients with APOE genotypes ε2/3 (n = 5), ε3/3 (n = 5), ε3/4 (n = 6), and ε4/4 (n = 6) were separated using our published protocol into a 10,000g pelleted extracellular fraction (10K) and a further purified EV fraction. Counting, sizing, and multiomic characterization by small RNA sequencing and proteomic analysis were performed for 10K, EVs, and source tissue. Results: Comparing APOE genotypes, no significant differences in bdEV total particle concentration or morphology were observed. Overall small RNA and protein profiles of 10K, EVs, and source tissue also did not differ substantially between different APOE genotypes. However, several differences in individual RNAs (including miRNAs and tRNAs) and proteins in 10K and EVs were observed when comparing the highest and lowest risk groups (ε4/4 and ε2/3). Bioinformatic analysis and previous publications indicate a potential regulatory role of these molecules in AD. Discussion: For patients with late-stage AD in this study, only a few moderate differences were observed for small RNA and protein profiles between APOE genotypes. Among these, several newly identified 10K and EV-associated molecules may play roles in AD progression. Possibly, larger genotype-related differences exist and are more apparent in or before earlier disease stages.

Brain Tissue-Derived Extracellular Vesicles in Alzheimer's Disease Display Altered Key Protein Levels Including Cell Type-Specific Markers.

BACKGROUND: Brain tissue-derived extracellular vesicles (bdEVs) play neurodegenerative and protective roles, including in Alzheimer's disease (AD). Extracellular vesicles (EVs) may also leave the brain to betray the state of the CNS in the periphery. Only a few studies have profiled the proteome of bdEVs and source brain tissue. Additionally, studies focusing on bdEV cell type-specific surface markers are rare. OBJECTIVE: We aimed to reveal the pathological mechanisms inside the brain by profiling the tissue and bdEV proteomes in AD patients. In addition, to indicate targets for capturing and molecular profiling of bdEVs in the periphery, CNS cell-specific markers were profiled on the intact bdEV surface. METHODS: bdEVs were separated and followed by EV counting and sizing. Brain tissue and bdEVs from age-matched AD patients and controls were then proteomically profiled. Total tau (t-tau), phosphorylated tau (p-tau), and antioxidant peroxiredoxins (PRDX) 1 and 6 were measured by immunoassay in an independent bdEV separation. Neuron, microglia, astrocyte, and endothelia markers were detected on intact EVs by multiplexed ELISA. RESULTS: Overall, concentration of recovered bdEVs was not affected by AD. Proteome differences between AD and control were more pronounced for bdEVs than for brain tissue. Levels of t-tau, p-tau, PRDX1, and PRDX6 were significantly elevated in AD bdEVs compared with controls. Release of certain cell-specific bdEV markers was increased in AD. CONCLUSION: Several bdEV proteins are involved in AD mechanisms and may be used for disease monitoring. The identified CNS cell markers may be useful tools for peripheral bdEV capture.

Elevation of neural injury markers in patients with neurologic sequelae after hospitalization for SARS-CoV-2 infection.

Patients with SARS-CoV-2 infection (COVID-19) risk developing long-term neurologic symptoms after infection. Here, we identify biomarkers associated with neurologic sequelae one year after hospitalization for SARS-CoV-2 infection. SARS-CoV-2-positive patients were followed using post-SARS-CoV-2 online questionnaires and virtual visits. Hospitalized adults from the pre-SARS-CoV-2 era served as historical controls. 40% of hospitalized patients develop neurological sequelae in the year after recovery from acute COVID-19 infection. Age, disease severity, and COVID-19 infection itself was associated with additional risk for neurological sequelae in our cohorts. Glial fibrillary astrocytic protein (GFAP) and neurofilament light chain (NF-L) were significantly elevated in SARS-CoV-2 infection. After adjusting for age, sex, and disease severity, GFAP and NF-L remained significantly associated with longer term neurological symptoms in patients with SARS-CoV-2 infection. GFAP and NF-L warrant exploration as biomarkers for long-term neurologic complications after SARS-CoV-2 infection.

High Density Display of an Anti-Angiogenic Peptide on Micelle Surfaces Enhances Their Inhibition of αvß3 Integrin-Mediated Neovascularization In Vitro.

Diabetic retinopathy (DR), Retinopathy of Pre-maturity (ROP), and Age-related Macular Degeneration (AMD) are multifactorial manifestations associated with abnormal growth of blood vessels in the retina. These three diseases account for 5% of the total blindness and vision impairment in the US alone. The current treatment options involve heavily invasive techniques such as frequent intravitreal administration of anti-VEGF (vascular endothelial growth factor) antibodies, which pose serious risks of endophthalmitis, retinal detachment and a multitude of adverse effects stemming from the diverse physiological processes that involve VEGF. To overcome these limitations, this current study utilizes a micellar delivery vehicle (MC) decorated with an anti-angiogenic peptide (aANGP) that inhibits αvß3 mediated neovascularization using primary endothelial cells (HUVEC). Stable incorporation of the peptide into the micelles (aANGP-MCs) for high valency surface display was achieved with a lipidated peptide construct. After 24 h of treatment, aANGP-MCs showed significantly higher inhibition of proliferation and migration compared to free from aANGP peptide. A tube formation assay clearly demonstrated a dose-dependent angiogenic inhibitory effect of aANGP-MCs with a maximum inhibition at 4 µg/mL, a 1000-fold lower concentration than that required for free from aANGP to display a biological effect. These results demonstrate valency-dependent enhancement in the therapeutic efficacy of a bioactive peptide following conjugation to nanoparticle surfaces and present a possible treatment alternative to anti-VEGF antibody therapy with decreased side effects and more versatile options for controlled delivery.