Correction: Hoshi et al. High Correlation among Brain-Derived Major Protein Levels in Cerebrospinal Fluid: Implication for Amyloid-Beta and Tau Protein Changes in Alzheimer's Disease. Metabolites 2022, 12, 355.

In the original publication [...].

Brain-Derived Major Glycoproteins Are Possible Biomarkers for Altered Metabolism of Cerebrospinal Fluid in Neurological Diseases.

Cerebrospinal fluid (CSF) plays an important role in the homeostasis of the brain. We previously reported that CSF major glycoproteins are biosynthesized in the brain, i.e., lipocalin-type prostaglandin D2 synthase (L-PGDS) and transferrin isoforms carrying unique glycans. Although these glycoproteins are secreted from distinct cell types, their CSF levels have been found to be highly correlated with each other in cases of neurodegenerative disorders. The aim of this study was to examine these marker levels and their correlations in other neurological diseases, such as depression and schizophrenia, and disorders featuring abnormal CSF metabolism, including spontaneous intracranial hypotension (SIH) and idiopathic normal pressure hydrocephalus (iNPH). Brain-derived marker levels were found to be highly correlated with each other in the CSF of depression and schizophrenia patients. SIH is caused by CSF leakage, which is suspected to induce hypovolemia and a compensatory increase in CSF production. In SIH, the brain-derived markers were 2-3-fold higher than in other diseases, and, regardless of their diverse levels, they were found to be correlated with each other. Another abnormality of the CSF metabolism, iNPH, is possibly caused by the reduced absorption of CSF, which secondarily induces CSF accumulation in the ventricle; the excess CSF compresses the brain's parenchyma to induce dementia. One potential treatment is a "shunt operation" to bypass excess CSF from the ventricles to the peritoneal cavity, leading to the attenuation of dementia. After the shunt operation, marker levels began to increase within a week and then further increased by 2-2.5-fold at three, six, and twelve months post-operation, at which point symptoms had gradually attenuated. Notably, the marker levels were found to be correlated with each other in the post-operative period. In conclusion, the brain-derived major glycoprotein markers were highly correlated in the CSF of patients with different neurological diseases, and their correlations were maintained even after surgical intervention. These results suggest that brain-derived proteins could be biomarkers of CSF production.

High Correlation among Brain-Derived Major Protein Levels in Cerebrospinal Fluid: Implication for Amyloid-Beta and Tau Protein Changes in Alzheimer's Disease.

The cerebrospinal fluid (CSF) plays an important role in homeostasis of the brain. We previously demonstrated that major CSF proteins such as lipocalin-type prostaglandin D2 synthase (L-PGDS) and transferrin (Tf) that are biosynthesized in the brain could be biomarkers of altered CSF production. Here we report that the levels of these brain-derived CSF proteins correlated well with each other across various neurodegenerative diseases, including Alzheimer's disease (AD). In addition, protein levels tended to be increased in the CSF samples of AD patients compared with the other diseases. Patients at memory clinics were classified into three categories, consisting of AD (n = 61), mild cognitive impairment (MCI) (n = 42), and cognitively normal (CN) (n = 23), with MMSE scores of 20.4 ± 4.2, 26.9 ± 1.7, and 29.0 ± 1.6, respectively. In each category, CSF protein levels were highly correlated with each other. In CN subjects, increased CSF protein levels correlated well with those of AD markers, including amyloid-ß and tau protein, whereas in MCI and AD subjects, correlations declined with AD markers except p-tau. Future follow-up on each clinical subject may provide a clue that the CSF proteins would be AD-related biomarkers.

Transferrin Biosynthesized in the Brain Is a Novel Biomarker for Alzheimer's Disease.

Glycosylation is a cell type-specific post-translational modification that can be used for biomarker identification in various diseases. Aim of this study is to explore glycan-biomarkers on transferrin (Tf) for Alzheimer's disease (AD) in cerebrospinal fluid (CSF). Glycan structures of CSF Tf were analyzed by ultra-performance liquid chromatography followed by mass spectrometry. We found that a unique mannosylated-glycan is carried by a Tf isoform in CSF (Man-Tf). The cerebral cortex contained Man-Tf as a major isofom, suggesting that CSF Man-Tf is, at least partly, derived from the cortex. Man-Tf levels were analyzed in CSF of patients with neurological diseases. Concentrations of Man-Tf were significantly increased in AD and mild cognitive impairment (MCI) comparing with other neurological diseases, and the levels correlated well with those of phosphorylated-tau (p-tau), a representative AD marker. Consistent with the observation, p-tau and Tf were co-expressed in hippocampal neurons of AD, leading to the notion that a combined p-tau and Man-Tf measure could be a biomarker for AD. Indeed, levels of p-tau x Man-Tf showed high diagnostic accuracy for MCI and AD; 84% sensitivities and 90% specificities for MCI and 94% sensitivities and 89% specificities for AD. Thus Man-Tf could be a new biomarker for AD.

Total transferrin in cerebrospinal fluid is a novel biomarker for spontaneous intracranial hypotension.

Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Patients with SIH experience postural headaches, nausea, etc., due to CSF hypovolemia. Imaging studies and clinical examinations, such as radioisotope (RI) scintigraphy, are useful for diagnosing SIH. However, 20-30% of patients do not show typical morphology and clinical test results. We previously reported that CSF contains transferrin (Tf) isoforms:"brain-type" Tf derived from the choroid plexus and "serum-type" Tf derived from blood. We showed that both isoforms increased in the CSF of patients with SIH by Western blotting. In the present study, we demonstrate that conventional ELISA for quantifying total Tf is useful for diagnosing SIH more accurately than Western blotting. In addition, SIH with chronic subdural hematoma (CSDH) was also accurately diagnosed.　Total Tf in the CSF can serve as a useful biomarker for diagnosing SIH with or without CSDH.

Spontaneous intracranial hypotension is diagnosed by a combination of lipocalin-type prostaglandin D synthase and brain-type transferrin in cerebrospinal fluid.

BACKGROUND: Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Definitive diagnosis can be difficult by clinical examinations and imaging studies. METHODS: SIH was diagnosed with the following criteria: (i) evidence of CSF leakage by cranial magnetic resonance imaging (MRI) findings of intracranial hypotension and/or low CSF opening pressure; (ii) no recent history of dural puncture. We quantified CSF proteins by ELISA or Western blotting. RESULTS: Comparing with non-SIH patients, SIH patients showed significant increase of brain-derived CSF glycoproteins such as lipocalin-type prostaglandin D synthase (L-PGDS), soluble protein fragments generated from amyloid precursor protein (sAPP) and "brain-type" transferrin (Tf). Serum-derived proteins such as albumin, immunoglobulin G, and serum Tf were also increased. A combination of L-PGDS and brain-type Tf differentiated SIH from non-SIH with sensitivity 94.7% and specificity 72.6%. CONCLUSION: L-PGDS and brain-type Tf can be biomarkers for diagnosing SIH. GENERAL SIGNIFICANCE: L-PGDS and brain-type Tf biosynthesized in the brain appears to be markers for abnormal metabolism of CSF.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr134/Thr138/Thr143/Thr147/Thr152) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr134/Thr138 or Thr143/Thr147/Thr152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvß3 and ß1 integrins, as well as αvß3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.

Osteopontin O-glycosylation contributes to its phosphorylation and cell-adhesion properties.

OPN (osteopontin) is a multiphosphorylated extracellular glycoprotein, which has important roles in bone remodelling, inflammation and cancer metastasis. OPN regulates cell spreading and adhesion primarily through its association with several integrins such as αvß3, and its phosphorylation affects these processes. However, the mechanism by which OPN O-glycosylation affects these processes is not completely understood. In the present study, we demonstrated that OPN O-glycosylation self-regulates its biological activities and also affects its phosphorylation status. We prepared two recombinant OPNs, WT (wild-type)-OPN and mutant OPN (ΔO-OPN), which lacks five O-glycosylation sites at a threonine/proline-rich region. O-glycan defects in OPN increased its phosphorylation level, as observed by dephosphorylation assays. Moreover, compared with WT-OPN, ΔO-OPN exhibited enhanced cell spreading and adhesion activities and decreased associations with ß1 integrins. This suggested that defects in O-glycans in OPN altered these activities, and that ß1 integrins have a less important role in adhesion to ΔO-OPN. The cell-adhesion activity of dephosphorylated ΔO-OPN was higher than the cell-adhesion activities of ΔO-OPN and dephosphorylated WT-OPN. This suggested that some of the phosphorylation in ΔO-OPN caused by O-glycan defects and O-glycans of OPN suppressed the OPN cell-adhesion activity. Thus functional activities of OPN can be determined by the combined glycosylation and phosphorylation statuses and not by either status alone.

Siglec-7 mediates nonapoptotic cell death independently of its immunoreceptor tyrosine-based inhibitory motifs in monocytic cell line U937.

Siglec-7, a sialic acid binding immunoglobulin-like lectin, predominantly transduces inhibitory signals through cytosolic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Here, we report that clustering of Siglec-7 with a specific F(ab')(2) elicited cell death. Interestingly, a truncated Siglec-7 lacking the cytosolic ITIM domain still induced the cell death, suggesting that the ITIMs are not essential for the death signaling. Further analyses of the death signaling revealed that an oxygen radical scavenger, N-acetyl cysteine, completely inhibited the cell death, whereas a pancaspase inhibitor did not. In addition, caspase-3 activation, DNA ladder formation, and nuclear condensation were not detected during the death process, suggesting that the cell death is nonapoptotic. To identify the critical region for the death signaling, we prepared a series of shuffling chimeras between Siglec-7 and Siglec-9, the latter of which did not transduce a death signal. The critical region was mapped to the middle of the membrane-proximal C2-set domain, which contained only six amino acid differences between Siglec-7 and Siglec-9. Point mutation analyses of each of these six amino acids revealed that four of the six amino acids were critical for the death signal. A computer-assisted 3D modeling revealed that these four amino acids were proximally located on the surface of the C2-set domain. In conclusion, Siglec-7 induces nonapoptotic cell death, the signal for which is transduced by an extracellular C2-set domain.