Gliovascular transcriptional perturbations in Alzheimer's disease reveal molecular mechanisms of blood brain barrier dysfunction.

To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer's disease, we performed single nucleus RNA sequencing in 24 Alzheimer's disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands, SMAD3, upregulated in Alzheimer's disease pericytes, has the highest number of ligands including VEGFA, downregulated in Alzheimer's disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer's disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here, we detect vast transcriptome changes in Alzheimer's disease at the gliovascular-unit, prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions, and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer's disease.

Evaluation of Tear Fluid and Aqueous Humor Concentration of Clusterin as Biomarkers for Early Diagnosis of Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma.

PURPOSE: Pseudoexfoliation syndrome (PEX) is an age-related disorder of the extracellular matrix characterized by the accumulation of fibrillary deposits in the anterior chamber of the eye, which leads to the development of pseudoexfoliative glaucoma (PEG). Early identification of subjects with higher susceptibility to PEX and PEG development is very important so that these conditions are managed at earlier stages, which requires that an objective biomarker is defined. Therefore, in the present study, we aimed to determine if aqueous humor and tear fluid concentrations of clusterin, an extracellular chaperone, are objective biomarkers for PEX and PEG risk. METHODS: Tear fluid was obtained from 80 patients with PEG, 80 patients with PEX, and 80 controls, using Schirmer strips. Aqueous humor was also collected during cataract surgery from 12 patients with PEG, 17 patients with PEX, and 22 controls, who also gave tear samples. Clusterin concentration was determined by ELISA. RESULTS: Clusterin concentration in aqueous humor was significantly higher in patients with PEG than in PEX cases (P = .002) and controls (P = .004). Receiver operating characteristics analysis revealed that this parameter is a robust classifier to distinguish PEG and PEX cases. Tear fluid clusterin concentrations did not differ significantly between groups. Aqueous humor and tear fluid levels of clusterin were not significantly correlated. CONCLUSIONS: In conclusion, tear fluid clusterin level in patients with PEG and PEX was determined for the first time, which showed no difference between study groups. Aqueous humor clusterin level was markedly higher in patients with PEG.

Evaluation of tear and aqueous humor level, and genetic variants of connective tissue growth factor as biomarkers for early detection of pseudoexfoliation syndrome/glaucoma.

Pseudoexfoliation syndrome (PEX) may lead to the development of pseudoexfoliative glaucoma (PEG), a potential cause of irreversible blindness, if left untreated. This type of glaucoma often presents with much higher intraocular pressure (IOP) values than observed in primary open angle glaucoma, and patients are often unaware of their condition. Therefore, early diagnosis is of utmost importance in PEX and PEG. Unfortunately, no valid objective biomarkers are available that can be used for this purpose. The excessive synthesis and deposition of elastic microfibrillar pseudoexfoliation material is observed in the pathophysiology of PEX, therefore, growth factors may play roles in this pathology. Thus, in this study, we sought to determine the roles of phenotypes and genotypes of connective tissue growth factor (CTGF) as objective biomarkers for early diagnosis of PEX and PEG. Thus, we investigated possible associations involving tear and aqueous humor CTGF concentrations and four single nucleotide polymorphisms (SNPs) of the CTGF gene in PEX and PEG. The study was designed as a 2-year case-control study in the Turkish population. Study population was composed of 214 patients with PEG, 214 patients with PEX, and 214 age-matched controls for CTGF SNP analysis. Tear fluid study group consisted of 78 patients with PEG, 77 patients with PEX, and 78 controls. Aqueous humor analysis included 8 patients with PEG, 17 patients with PEX, and 23 controls. Tear fluid was collected using Schirmer strips, and aqueous humor samples were taken during cataract surgery. CTGF concentration was determined by ELISA, and total protein concentration was determined by Bradford assay in tear and aqueous humor samples. PCR followed by restriction fragment length polymorphism analysis was used for genotyping of rs6918698 G/C and rs9399005 C/T, while real-time PCR was used for rs9402373 C/G and rs12526196 T/C. Intraocular pressure, visual field score, mean deviation, and pattern standard deviation parameters were also evaluated. CTGF concentration in tear fluid was significantly higher in PEG patients compared with controls (P = 0.001), while it was lower in PEX patients. Similarly, total protein concentration in tear fluid was significantly increased in PEG patients relative to PEX patients (P = 0.026) and controls (P = 0.004). CTGF concentration in aqueous humor did not differ markedly between the groups, whereas total protein was significantly higher in the PEG group compared with the PEX group (P = 0.012) and controls (P = 0.003). Receiver operating characteristic analysis revealed that total protein in aqueous humor was a robust classifier for evaluating the presence of PEG against controls (Area under the curve = 0.897, P = 0.001). The genotypes of the studied SNPs were not significantly correlated with CTGF concentration in aqueous humor or tear fluid, and did not exhibit significant association with PEG or PEX. In conclusion, this was the first study to investigate tear fluid CTGF concentration in PEX and PEG, which came out not to be a good classifier for PEG or PEX. Total protein level in tear fluid and CTGF SNPs also did not predict PEG or PEX status successfully.

Potential role of calcifying nanoparticles in the etiology of multiple sclerosis.

Nanobacteria or calcifying nanoparticles are 80-500 nm sized nano-organisms that are physically associated with carbonate apatite mineral formations. They have been indicated in various diseases, including kidney stone formation, Alzheimer's disease, and atherosclerosis. Nanoparticles contain calcium and apatite-binding protein fetuin-A, a calcification inhibitor. However, recent evidence indicates that fetuin-A can form nucleation seeds or nidi that grow in size through ion sedimentation to become larger amorphous nanoparticles in the presence of excess calcium and apatite ions. Fetuin-A also functions as an inhibitor of meprin, a metalloproteinase implicated in inflammation and neurodegenerative diseases. During inflammation, meprin functions to regulate chemokine activity of monocyte chemotactic protein 1, which is associated with chronic inflammatory diseases, including atherosclerosis, renal inflammatory diseases, and multiple sclerosis (MS). In addition, calcium phosphate nanocrystals that contain fetuin-A are pro-inflammatory to macrophages and promote vascular smooth muscle cell mineralization, potentiating a vicious cycle of inflammation and calcification. Thus, mineral stress and inflammation appear to be associated with each other. Furthermore, fetuin-A deficient mice exhibited reduced experimental autoimmune encephalomyelitis severity. Thus, fetuin-A plays a direct role in the neuroinflammatory response. Indeed, the level of fetuin-A in cerebrospinal fluid has been defined as a biomarker of disease activity in MS. MS is a chronic, inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) with an unknown etiology. The "inside-out" model of MS, supported by recent data, states that the initial axonal degeneration in the CNS occurs before demyelination, which then stimulates an auto-immune attack. It was shown very recently that influx of calcium from the extracellular space through nanoscale ruptures of the axonal plasma membrane predict axon degeneration in neuroinflammation. Calcium is an activator of calpains, proteases that function to break down the cytoskeleton, leading to neurodegeneration. Nanoruptures of the plasma membrane were suggested to occur at the early stages of axon damage, especially at nodes of Ranvier, which are devoid of myelin. Here, I propose that calcifying nanoparticles may have a role in the etiology and/or pathophysiology of MS. The initial event causing neurodegeneration may be due to the nanoparticles that have been suggested to easily cross the blood-brain barrier. Following this, the nanoparticles may create nanoruptures in the axonal membrane and also increase the calcium concentration around and within the neurons by forming nidi for calcification, eventually causing neurodegeneration. Nanoparticles can self-replicate; hence, they may represent an infectious causative agent for the development of MS.

Genetic polymorphisms of vitamin D3 metabolizing CYP24A1 and CYP2R1 enzymes in Turkish patients with ischemic stroke.

Objective Vitamin D deficiency is known as an important risk factor in pathogenesis of atherosclerosis, which contributes to stroke development. Genetic variations including single nucleotide polymorphisms (SNPs) in enzymes involved in vitamin D metabolism can affect susceptibility to the development of stroke. Therefore, the objective of this study was to investigate the association between polymorphisms of vitamin D metabolizing enzymes (rs927650 SNP in CYP24A1, and rs10741657 SNP in CYP2R1 genes,) and ischemic stroke risk in Turkish population. Materials and methods To test this hypothesis, we designed a case-control study which consisted of 256 ischemic stroke patients and 132 controls. Genotypes were determined by PCR-RFLP technique. Results No significant differences were found between patients and controls in terms of CYP24A1 rs927650 and CYP2R1 rs10741657 genotype frequencies. Polymorphic allele frequencies of CYP24A1 rs927650 and CYP2R1 rs10741657 were 0.414 and 0.660 in stroke patients, respectively. Conclusion This is the first study conducted regarding the association of CYP24A1 rs927650 and CYP2R1 rs10741657 genetic polymorphisms and ischemic stroke risk. The polymorphic genotypes of these polymorphisms, together with hypertension, diabetes, smoking, and obesity, were found as significant risk factors for ischemic stroke.