IgG targeting distinct seasonal coronavirus- conserved SARS-CoV-2 spike subdomains correlates with differential COVID-19 disease outcomes.

Despite SARS-CoV-2 being a "novel" virus, early detection of anti-spike IgG in severe COVID-19 patients may be caused by the amplification of humoral memory responses against seasonal coronaviruses. Here, we examine this phenomenon by characterizing anti-spike IgG responses in non-hospitalized convalescent individuals across a spectrum of COVID-19 severity. We observe that disease severity positively correlates with anti-spike IgG levels, IgG cross-reactivity against other betacoronaviruses (ß-CoVs), and FcγR activation. Analysis of IgG targeting ß-CoV-conserved and non-conserved immunodominant epitopes within the SARS-CoV-2 spike protein revealed epitope-specific relationships: IgG targeting the conserved heptad repeat (HR) 2 region significantly correlates with milder disease, while targeting the conserved S2'FP region correlates with more severe disease. Furthermore, a lower HR2-to-S2'FP IgG-binding ratio correlates with greater disease severity, with ICU-hospitalized COVID-19 patients showing the lowest HR2/S2'FP ratios. These findings suggest that HR2/S2'FP IgG profiles may predict disease severity and offer insight into protective versus deleterious humoral recall responses.

Sustained Antibody-Dependent NK Cell Functions in Mild COVID-19 Outpatients During Convalescence.

The coronavirus disease 2019 (COVID19) pandemic has left researchers scrambling to identify the humoral immune correlates of protection from COVID-19. To date, the antibody mediated correlates of virus neutralization have been extensively studied. However, the extent that non-neutralizing functions contribute to anti-viral responses are ill defined. In this study, we profiled the anti-spike antibody subtype/subclass responses, along with neutralization and antibody-dependent natural killer cell functions in 83 blood samples collected between 4 and 201 days post-symptoms onset from a cohort of COVID-19 outpatients. We observed heterogeneous humoral responses against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Overall, anti-spike profiles were characterized by a rapid rise of IgA and sustained IgG titers. In addition, strong antibody-mediated natural killer effector responses correlated with milder disease and being female. While higher neutralization profiles were observed in males along with increased severity. These results give an insight into the underlying function of antibodies beyond neutralization and suggest that antibody-mediated natural killer cell activity is a key function of the humoral response against the SARS-CoV-2 spike protein.

Wnt/ß-catenin signaling stimulates the expression and synaptic clustering of the autism-associated Neuroligin 3 gene.

Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/ß-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/ß-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous ß-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/ß-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.

Early Transcriptional Changes Induced by Wnt/ß-Catenin Signaling in Hippocampal Neurons.

Wnt/ß-catenin signaling modulates brain development and function and its deregulation underlies pathological changes occurring in neurodegenerative and neurodevelopmental disorders. Since one of the main effects of Wnt/ß-catenin signaling is the modulation of target genes, in the present work we examined global transcriptional changes induced by short-term Wnt3a treatment (4 h) in primary cultures of rat hippocampal neurons. RNAseq experiments allowed the identification of 170 differentially expressed genes, including known Wnt/ß-catenin target genes such as Notum, Axin2, and Lef1, as well as novel potential candidates Fam84a, Stk32a, and Itga9. Main biological processes enriched with differentially expressed genes included neural precursor (GO:0061364, p-adjusted = 2.5 × 10-7), forebrain development (GO:0030900, p-adjusted = 7.3 × 10-7), and stem cell differentiation (GO:0048863 p-adjusted = 7.3 × 10-7). Likewise, following activation of the signaling cascade, the expression of a significant number of genes with transcription factor activity (GO:0043565, p-adjusted = 4.1 × 10-6) was induced. We also studied molecular networks enriched upon Wnt3a activation and detected three highly significant expression modules involved in glycerolipid metabolic process (GO:0046486, p-adjusted = 4.5 × 10-19), learning or memory (GO:0007611, p-adjusted = 4.0 × 10-5), and neurotransmitter secretion (GO:0007269, p-adjusted = 5.3 × 10-12). Our results indicate that Wnt/ß-catenin mediated transcription controls multiple biological processes related to neuronal structure and activity that are affected in synaptic dysfunction disorders.

Alternative RUNX1 Promoter Regulation by Wnt/ß-Catenin Signaling in Leukemia Cells and Human Hematopoietic Progenitors.

Two distantly located promoter regions regulate the dynamic expression of RUNX genes during development: distal P1 and proximal P2 promoters. We have recently described that ß-catenin increases total Runx1 mRNA levels in human CD34(+) hematopoietic progenitors and enhances spatial proximity with its translocation partner ETO. Here, we report that induction of Wnt/ß-catenin signaling in HL60 and Jurkat leukemia-derived cell lines and CD34(+) progenitors selectively activate the production of the longer distal P1-Runx1 mRNA isoform. Gain- and loss-of-function experiments revealed that the differential increase in P1-Runx1 expression is accomplished through a minimal ß-catenin responsive region that includes a highly conserved TCF/LEF-binding element, located -20/-16 bp upstream of the canonical distal P1-Runx1 transcription start site. We conclude that the distal P1-Runx1 promoter is a direct transcriptional target of Wnt/ß-catenin signaling that may be important in normal hematopoiesis or its transition into malignant stem cells during the onset or progression of leukemia.

Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells.

Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/ß-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that ß-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/ß-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia.

Wnt/ß-catenin signaling in Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder that causes a progressive decline of mental and cognitive processes such as memory, judgment and reasoning. We proposed earlier that a sustained loss of function of Wnt/ß- catenin signaling components underlies the onset and progression of the disease. Here, we discuss recent data on the involvement of Wnt/b-catenin signaling on amyloid precursor protein (APP) processing, Aß peptide neurotoxicity, τ phosphorylation, and modulation of Apolipoprotein E function in the brain. We conclude that several components of the cascade are actively engaged in the events leading to AD neuropathology and propose that compounds that mimic activation of this signaling cascade, such as lithium, should be considered for therapeutic intervention in Alzheimer's patients. In summary, data accumulated during the past decade confirm some important predictions of our hypothesis where components of this signaling cascade are actively engaged in the events leading to AD neuropathology and that compounds that mimic activation of this signaling cascade, such as lithium, should be considered for therapeutic intervention in Alzheimer's patients.

A novel functional low-density lipoprotein receptor-related protein 6 gene alternative splice variant is associated with Alzheimer's disease.

We previously found that single nucleotide polymorphisms in the low-density lipoprotein receptor-related protein 6 (LRP6) gene are associated with Alzheimer's disease (AD). Here, we studied the posttranscriptional metabolism of the LRP6 message scanning sequentially the 23 LRP6 exons in human tissues and found a novel LRP6 isoform that completely skips exon 3 (LRP6Δ3) in all tissues examined and was also conserved in mice. Expression levels of the LRP6 isoforms were determined in 47 cortical brain messenger (m)RNA samples including 22 AD cases, 11 control subjects, and 14 individuals with other neurological disorders. LRP6Δ3 mRNA levels were significantly augmented in AD brains compared with controls (1.6-fold; p = 0.037) or other pathological samples (2-fold; p = 0.007). Functional analysis in Wnt/ß-catenin signaling assays revealed that skipping of exon 3 reduced significantly the signaling activity of the LRP6 coreceptor. We conclude that the LRP6Δ3 isoform is a novel splice variant, which shows diminished Wnt/ß-catenin signaling activity and might have a functional role in individuals with AD.