Alzheimer's disease risk associated with changes in Epstein-Barr virus nuclear antigen 1-specific epitope targeting antibody levels.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder influenced by age, sex, genetic factors, immune alterations, and infections. Multiple lines of evidence suggest that changes in antibody response are linked to AD pathology. METHODS: To elucidate the mechanisms underlying AD development, we investigated antibodies that target autoimmune epitopes using high-resolution epitope microarrays. Our study compared two groups: individuals with AD (n = 19) and non-demented (ND) controls (n = 19). To validate the results, we measured antibody levels in plasma samples from AD patients (n = 96), mild cognitive impairment (MCI; n = 91), and ND controls (n = 97). To further explore the invlovement of EBV, we performed epitope masking immunofluorescence microscopy analysis and tests to induce lytic replication using the B95-8 cell line. RESULTS: In this study, we analyzed high-resolution epitope-specific serum antibody levels in AD, revealing significant disparities in antibodies targeting multiple epitopes between the AD and control groups. Particularly noteworthy was the significant down-regulation of antibody (anti-DG#29) targeting an epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1). This down-regulation increased AD risk in female patients (odds ratio up to 6.6), but not in male patients. Our investigation further revealed that the down-regulation of the antibody (anti-DG#29) is associated with EBV reactivation in AD, as indicated by the analysis of EBV VCA IgG or IgM levels. Additionally, our data demonstrated that the epitope region on EBNA1 for the antibody is hidden during the EBV lytic reactivation of B95-8 cells. CONCLUSION: Our findings suggest a potential relationship of EBV in the development of AD in female. Moreover, we propose that antibodies targeting the epitope (DG#29) of EBNA1 could serve as valuable indicators of AD risk in female.

Elevation of phospholipase C-ß1 expression by amyloid-ß facilitates calcium overload in neuronal cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia. Amyloid-ß (Aß) has long been considered a key cause of neurodegeneration in the AD brain. Although the mechanisms underlying Aß-induced neurodegeneration are not fully understood, a number of recent studies have suggested that intracellular calcium overload mediates this process. In this study, we focused on the cellular function of phospholipase C-ß1 (PLCB1), which regulates calcium signaling by mediating hydrolysis of phosphatidylinositol 4,5-bisphosphate through G-protein coupled receptor pathways. First, we confirmed that acetylcholine-induced calcium release from intracellular stores of SH-SY5Y cells was significantly increased with Aß42 oligomer treatment. We further found that PLCB1 expression was upregulated in Aß42-treated cells, and PLCB1 overexpression in SH-SY5Y cells elicited the calcium overload observed in Aß-treated cells. In addition, Aß42 oligomer-induced calcium overload in SH-SY5Y cells was alleviated by knockdown of PLCB1, indicating that PLCB1 plays an essential role in the neurotoxic process initiated by Aß. The elevation of PLCB1 expression was confirmed in the brain tissues from the 5× familial AD (5×FAD) model mice. These findings suggest that PLCB1 may represent a potential therapeutic target for protecting neuronal cells against excitotoxicity in AD progression.

Tonicity-responsive enhancer-binding protein promotes diabetic neuroinflammation and cognitive impairment via upregulation of lipocalin-2.

BACKGROUND: Diabetic individuals have increased circulating inflammatory mediators which are implicated as underlying causes of neuroinflammation and memory deficits. Tonicity-responsive enhancer-binding protein (TonEBP) promotes diabetic neuroinflammation. However, the precise role of TonEBP in the diabetic brain is not fully understood. METHODS: We employed a high-fat diet (HFD)-only fed mice or HFD/streptozotocin (STZ)-treated mice in our diabetic mouse models. Circulating TonEBP and lipocalin-2 (LCN2) levels were measured in type 2 diabetic subjects. TonEBP haploinsufficient mice were used to investigate the role of TonEBP in HFD/STZ-induced diabetic mice. In addition, RAW 264.7 macrophages were given a lipopolysaccharide (LPS)/high glucose (HG) treatment. Using a siRNA, we examined the effects of TonEBP knockdown on RAW264 cell' medium/HG-treated mouse hippocampal HT22 cells. RESULTS: Circulating TonEBP and LCN2 levels were higher in experimental diabetic mice or type 2 diabetic patients with cognitive impairment. TonEBP haploinsufficiency ameliorated the diabetic phenotypes including adipose tissue macrophage infiltrations, neuroinflammation, blood-brain barrier leakage, and memory deficits. Systemic and hippocampal LCN2 proteins were reduced in diabetic mice by TonEBP haploinsufficiency. TonEBP (+ / -) mice had a reduction of hippocampal heme oxygenase-1 (HO-1) expression compared to diabetic wild-type mice. In particular, we found that TonEBP bound to the LCN2 promoter in the diabetic hippocampus, and this binding was abolished by TonEBP haploinsufficiency. Furthermore, TonEBP knockdown attenuated LCN2 expression in lipopolysaccharide/high glucose-treated mouse hippocampal HT22 cells. CONCLUSIONS: These findings indicate that TonEBP may promote neuroinflammation and cognitive impairment via upregulation of LCN2 in diabetic mice.

Enhanced Expression of microRNA-1273g-3p Contributes to Alzheimer's Disease Pathogenesis by Regulating the Expression of Mitochondrial Genes.

Alzheimer's disease (AD) is the most common form of dementia in the elderly population, but its underlying cause has not been fully elucidated. Recent studies have shown that microRNAs (miRNAs) play important roles in regulating the expression levels of genes associated with AD development. In this study, we analyzed miRNAs in plasma and cerebrospinal fluid (CSF) from AD patients and cognitively normal (including amyloid positive) individuals. miR-1273g-3p was identified as an AD-associated miRNA and found to be elevated in the CSF of early-stage AD patients. The overexpression of miR-1273g-3p enhanced amyloid beta (Aß) production by inducing oxidative stress and mitochondrial impairments in AD model cell lines. A biotin-streptavidin pull-down assay demonstrated that miR-1273g-3p primarily interacts with mitochondrial genes, and that their expression is downregulated by miR-1273g-3p. In particular, the miR-1273g-3p-target gene TIMM13 showed reduced expression in brain tissues from human AD patients. These results suggest that miR-1273g-3p expression in an early stage of AD notably contributes to Aß production and mitochondrial impairments. Thus, miR-1273g-3p might be a biomarker for early diagnosis of AD and a potential therapeutic target to prevent AD progression.

High-throughput epitope profiling of antibodies in the plasma of Alzheimer's disease patients using random peptide microarrays.

The symptoms of Alzheimer's disease (AD), a major cause of dementia in older adults, are linked directly with neuronal cell death, which is thought to be due to aberrant neuronal inflammation. Autoantibodies formed during neuronal inflammation show excellent stability in blood; therefore, they may be convenient blood-based diagnostic markers of AD. Here, we performed microarray analysis of 29,240 unbiased random peptides to be used for comprehensive screening of AD-specific IgG and IgM antibodies in the blood. The results showed that (1) sequence-specific and isotype-specific antibodies are regulated differentially in AD, and combinations of these antibodies showing high area under the receiver operating characteristic curve values (0.862-0.961) can be used to classify AD, (2) AD-specific IgG antibodies arise from IgM antibody-secreting cells that existed before disease onset and (3) target protein profiling of the antibodies identified some AD-related proteins, some of which are involved in AD-related signalling pathways. Therefore, we propose that these epitopes may facilitate the development of biomarkers for AD diagnosis and form the basis for a mechanistic study related to AD progression.

Swiprosin-1 stimulates cancer invasion and metastasis by increasing the Rho family of GTPase signaling.

Ectopic expression of Swiprosin-1, an actin-binding protein (also known as EF hand domain containing 2; EFHD2), enhanced motile protrusions associated with actin, such as lamellipodia and membrane ruffles. Swiprosin-1 levels were increased in various human cancer tissues, particularly at highly invasive stages of malignant melanoma. Expression of Swiprosin-1 was correlated with that of epidermal growth factor receptor (EGFR) and induced by EGF. In a mouse metastasis model, Swiprosin-1 overexpression induced pulmonary metastasis whereas its knockdown led to marked inhibition of metastasis of highly invasive melanoma cells. Swiprosin-1 at the lamellipodia and membrane ruffles controlled the direction of cell protrusion and enhanced migration velocity through activating the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Our collective findings support the potential utility of Swiprosin-1 as a therapeutic target to prevent cancer invasion and metastasis.

Expression of the metabotropic glutamate receptor 5 (mGluR5) induces melanoma in transgenic mice.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synaptic transmission upon activation of glutamate-gated ion channels. In addition, glutamate modulates a variety of other synaptic responses and intracellular signaling by activating metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors. The mGluRs are also expressed in nonneuronal tissues and are implicated in a variety of normal biological functions as well as diseases. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in the forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, tyrosinase-related protein 1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3-5 d after birth for most of the mGluR5 transgene-positive mice. There was 100% penetrance in the progeny from the tyrosinase-related protein 1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by RT-PCR, immunoblotting, and immunohistochemistry. We evaluated the expression of several cancer-related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5-mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype make these mouse lines unique and a potentially powerful tool for therapeutic studies.

Regulation of dendritic spine morphology by SPIN90, a novel Shank binding partner.

Dendritic spines are highly specialized actin-rich structures on which the majority of excitatory synapses are formed in the mammalian CNS. SPIN90 is an actin-binding protein known to be highly enriched in postsynaptic densities (PSDs), though little is known about its function there. Here, we show that SPIN90 is a novel binding partner for Shank proteins in the PSD. SPIN90 and Shank co-immunoprecipitate from brain lysates and co-localize in postsynaptic dendrites and act synergistically to mediate spine maturation and spine head enlargement. At the same time, SPIN90 causes accumulation of Shank and PSD-95 within dendritic spines. In addition, we found that the protein composition of PSDs in SPIN90 knockout mice is altered as is the actin cytoskeleton of cultured hippocampal SPIN90 knockout neurons. Taken together, these findings demonstrate that SPIN90 is a Shank1b binding partner and a key contributor to the regulation of dendritic spine morphogenesis and brain function.

Modulation of beta-catenin by cyclin-dependent kinase 6 in Wnt-stimulated cells.

Beta-catenin is implicated in quite different cellular processes, which require a fine-tuned regulation of its function. Here we demonstrate that cyclin-dependent kinase 6 (CDK6), in association with cyclin D1 (CCND1), directly binds to beta-catenin. We showed that CCND1-CDK6 phosphorylates beta-catenin on serine 45 (S45). This phosphorylation creates a priming site for glycogen synthase kinase 3beta (GSK3beta) and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Moreover, co-immunoprecipitation assays using Wnt3a-conditioned medium reveals that while Wnt stimulation leads to the dissociation of beta-catenin from axin and casein kinase Ialpha (CKIalpha), Wnt treatment promotes an increase in CCND1 level and the association of beta-catenin with CCND1-CDK6. Furthermore, Wnt3a-stimulated cytosolic beta-catenin levels were higher in CDK6 knockout mouse embryonic fibroblasts (CDK6-/- MEFs) compared to wild-type MEFs. Thus, the CCND1-CDK6 complex is like to negatively regulate Wnt signaling by mediating beta-catenin phosphorylation and its subsequent degradation in Wnt-stimulated cells.