Irem L. Atasoy

The interleukin 1 alpha, interleukin 1 beta, interleukin 6 and alpha-2-macroglobulin serum levels in patients with early or late onset Alzheimer's disease, mild cognitive impairment or Parkinson's disease

Alzheimers disease (EOAD, LOAD), mild cognitive impairment (MCI), Parkinsons disease (PD) and healthy controls were included to determine the serum interleukin-1s (IL-1α, IL-1β), IL-6 and alpha-2-macroglobulin (α2M) levels using ELISA. IL-6 might be a significant contributor to the inflammatory response in LOAD. The MCI data indicate that IL-1s, α2M and BDNF are somehow related, and this relationship might allow MCI patients to be more similar to the healthy controls. A correlation analysis of multiple biomarkers in different neurodegenerative disorders might be more useful than determining the levels of a single cytokine in a single disorder.

BDNF, TNFα, HSP90, CFH, and IL-10 Serum Levels in Patients with Early or Late Onset Alzheimer's Disease or Mild Cognitive Impairment

Identifying early-detection biomarkers have become an increasingly important approach in the treatment and prevention of Alzheimers disease (AD). In this study, we investigated the potential of brain-derived neurotrophic factor (BDNF), complement factor H (CFH), tumor necrosis factor-α (TNFα), interleukin 10 (IL-10), and heat shock protein 90 (Hsp90) as serum biomarkers for AD in a cohort of the Turkish population because they have been suggested to be associated with AD. Serum BDNF, CFH, TNFα, IL-10, and Hsp90 levels in three groups of patients, early-onset AD (EOAD; age of onset < 65; n = 22), late-onset AD (LOAD; age of onset > 65; n = 54), and mild cognitive impairment (MCI) (n = 30), were compared with age-matched healthy controls (age < 65, n = 18 and age > 65; n = 32) using ELISA. The serum BDNF levels significantly decreased and TNFα levels significantly increased in the EOAD and LOAD groups compared to the age-matched healthy controls. There was a correlation between serum TNFα and IL-10 levels in the LOAD and healthy control groups. Serum CFH levels in the LOAD and MCI patients were significantly decreased compared with controls. Serum Hsp90 levels in the EOAD, LOAD, and MCI patients were significantly decreased compared with controls. The protein misfolding, the inflammatory response, and decreased neurotrophic factor synthesis are all suggested to be related to AD type brain pathology, and our results indicate these alterations might be traced from serum samples. For accurate early diagnosis of AD, it is important to determine a profile of alterations in multiple biomarkers in large-scale population studies.

Vitamin D deficiency might pose a greater risk for ApoEɛ4 non-carrier Alzheimer’s disease patients

Vitamin D is a secosteroid hormone that shares a synthetic pathway with cholesterol. ApoE, which is involved in the transport of cholesterol, is the most significant genetic risk factor for sporadic Alzheimer’s disease (AD). Surprisingly, recent studies have indicated the presence of an evolutionary juncture between these two molecules. To demonstrate this possible relationship, we investigated serum levels of 25-hydroxyvitamin-D3 (25OHD) in patients with early onset-AD (EOAD; n:22), late onset-AD (LOAD; n:72), mild cognitive impairment (MCI; n:32) and in healthy subjects (n:70). We then analyzed the correlation between 25OHD and cytokines, BDNF and Hsp90 with respect to ApoE alleles, as these molecules were investigated in our previous studies. The LOAD patients had low levels of 25OHD, but these low levels originated only from ApoEɛ4 non-carrier patients. Negative correlations were observed between serum 25OHD and TNFα, IL-1β or IL-6 levels in healthy subjects or MCI patients, but these same correlations were positive in LOAD patients. ApoE alleles indicated that these positive correlations exist only in ɛ4 carrier LOAD patients. Consequently, our results indicate that vitamin D deficiency presents a greater risk for ApoEɛ4 non-carrier AD patients than for ɛ4 carriers. Therefore, it might be beneficial to monitor the vitamin D status of ApoEɛ4 allele non-carrier AD patients.

Vitamin D Receptor Regulates Amyloid Beta 1–42 Production with Protein Disulfide Isomerase A3

The challenge of understanding the biology of neuronal amyloid processing could provide a basis for understanding the amyloid pathology in Alzheimers disease (AD). Based on our previous studies, we have suggested that AD might be the consequence of a hormonal imbalance in which the critical hormone is vitamin D. The present study primarily focused on the creation of a condition that prevents the genomic or nongenomic action of vitamin D by disrupting vitamin D receptors (VDR or PDIA3/1,25MARRS); the effects of these disruptions on the series of proteins involved in secretases that play a crucial role in amyloid pathology and on amyloid beta (Aβ) production in primary cortical neurons were observed. VDR and PDIA3/1,25MARRS genes were silenced separately or simultaneously in E16 primary rat cortical neurons. The expression of target genes involved in APP processing, including Presenilin1, Presenilin2, Nicastrin, BACE1, ADAM10, and APP, was investigated with qRT-PCR and Western blot in this model. 1,25-Dihydroxyvitamin D3 treatments were used to verify any transcriptional regulation data gathered from siRNA treatments by determining the mRNA expression of the target genes. Immunofluorescence labeling was used for the verification of silencing experiments and intracellular Aβ1-42 production. Extracellular Aβ1-42 level was assessed with ELISA. mRNA and protein expression results showed that 1,25-dihydroxyvitamin D3 might affect the transcriptional regulation of the genes involved in APP processing. The intracellular and extracellular Aβ1-42 measurements in our study support this suggestion. Consequently, we suggest that 1,25-dihydroxyvitamin D3 and its receptors are important parts of the amyloid processing pathway in neurons.

Okadaic acid–induced tau hyperphosphorylation and the downregulation of Pin1 expression in primary cortical neurons

Hyperphosphorylation of tau leading to neurofibrillary tangles (NFT) is one of the key pathological hallmarks in neurodegenerative disorders such as Alzheimer disease (AD). Peptidyl-prolyl cis-trans isomerase (Pin1) regulates the phosphorylation of Ser/Thr sites of tau protein, and promotes microtubule assembly. In this study, we aimed to determine the effect of tau hyperphosphorylation on Pin1 expression in primary cortical neurons in order to investigate the results of the pathological process on Pin1, an important enzyme involved in various cellular mechanisms. Primary cortical neurons were prepared from embryonic day 16 -Sprague Dawley rat embryos. The cultures were treated with 25 nM okadaic acid (OKA) on day 7 in order to promote tau hyperphosphorylation. The cytotoxicity was determined with LDH release and measured by ELISA. Tau phosphorylation was confirmed by western blot using anti-tau antibodies Thr231 and Tau-1. Pin1 mRNA expression level was determined by qRT-PCR at 8 and 24 h. Pin1 protein expression was analyzed with immunofluorescent labeling at 8 and 24 h. Tau phosphorylation on Thr231 was increased and non-phosphorylated Tau-1 was decreased in OKA treated group compared with the untreated control at 8 h of treatment. While Pin1 mRNA expression levels at 8 h post-OKA treatment were lower than that of control groups, there were no differences between OKA-treated group and control groups in Pin1 protein expression. Whereas no significant differences for Pin1 mRNA expression, protein expression levels were decreased OKA-treated group compared to control groups at 24 h of treatment. The LDH release of OKA-treated group was significantly increased at 24 h. Our study indicates that although OKA treatment suppressed Pin1 mRNA expression and induced tau phosphorylation at 8 h of treatment, its influence on Pin1 protein expression has 16 h phase delay. Given the important role of Pin1 in many cellular mechanisms these results might indicate that tau hyperphosphorylation involved in many neurodegenerative disorders may cause some alterations in brain microenvironment via Pin1.This is the first demonstration of the alteration of the Pin1 mRNA and protein expression in OKA induced model in primary cortical neurons.

Both secreted and the cellular levels of BDNF attenuated due to tau hyperphosphorylation in primary cultures of cortical neurons

Intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of taupathies such as Alzheimers disease. Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is selectively downregulated in AD. In this study, we investigated the effects of OKA induced tau hyperphosphorylation on secreted and cellular levels of BDNF in primary cortical neurons that were treated with 25nM OKA. Tau phosphorylation at threonine 231 (Thr231) sites was assessed by Western blot using antibodies against phospho-Thr231. Non-phosphorylated tau protein was detected with the Tau-1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 8th and 24th hours of treatment. Cellular localization and protein expression of BDNF and tau were assessed by immunofluorescent labeling and fluorescent intensity measurements at 24h of treatment. Tau hyperphosphorylation was confirmed with increase in Thr231 and the decrease in Tau-1 signals after 8h of OKA treatment, compared with the control groups, secreted BDNF levels in the OKA-treated group were significantly lower after 24h of treatment but were not significantly different at 8h of treatment. BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control group. BDNF immunoreactivity significantly decreased in the OKA treated group and this attenuation was significant especially at neurites. Our results suggest that the decrease in BDNF secretion and the BDNF expression might depend on the disruption of microtubule structure caused by tau hyperphosphorylation.

The Transcriptional Regulatory Properties of Amyloid Beta 1–42 may Include Regulation of Genes Related to Neurodegeneration

Our previous study demonstrated the translocation of Aβ1–42 to the nucleus in response to antibiotic treatment, and interpreted it as a possible transcriptional response of Aβ1–42 to antibiotics. The present study aims to investigate how amyloid acts on the key elements of neurodegeneration and the molecules involved in the induction of Aβ1–42 production. For this purpose, we investigated the acute effect of Aβ1–42 on the transcriptional levels of genes that have roles in the mechanisms that produce Aβ itself: alpha secretase (ADAM10), beta secretase (BACE1), the gamma secretase complex (PS-1, PS-2, Nicastrin), the substrate APP, APOE (the significant risk factor for sporadic form of the AD), TREM2 (recently indicated as a contributor to AD risk), NMDAR subunits and PKCzeta (contributors of memory and learning), and key elements of tau pathology such as tau, GSK3α, GSK3β, and Cdk5. Additionally, we examined cholecalciferol metabolism-related enzyme 1α-hydroxylase (1αOHase) in primary cortical neurons with qRT-PCR. Our results indicate that Aβ1–42 has an effect on most of the target genes. This effect involves regulation of the amyloidogenic pathway in a complex manner, specifically, a general downregulation in NMDARs, ApoE, Trem2, and 1αOHase genes, and general up-regulation of tau pathway-related genes. We speculate that the presence of Aβ impacts the neurons not only with toxic events but also at the transcriptional level. The nuclear localization of Aβ1–42 and its regulatory effects on the target genes that we investigated in present study indicates Aβ1–42 as a transcriptional regulator of genes related to neurodegeneration.

The role of astaxanthin on transcriptional regulation of NMDA receptors, voltage sensitive calcium channels and calcium binding proteins in primary cortical neurons

Introduction Calcium (Ca) is the phenomenon intracellular molecule that regulate many cellular process in neurons physiologically. Calcium dysregulation may occur in neurons due to excessive synaptic release of glutamate or other reasons related with neurodegeneration. Astaxanthin is a carotenoid that has antioxidant effect in cell. The purpose of this study was to investigate whether astaxanthin affects NMDA subunits, calcium binding proteins and L Type voltage sensitive Ca-channels (LVSCC) in primary cortical neuron cultures in order to see its role in calcium metabolism. Methods Primary cortical neurons were prepared from embryonic day 16-Sprague Dawley rat embryos. The cultures were treated with 10 nM and 20 nM astaxanthin on day 7. NMDA subunits, LVSCC-A1C and LVSCC-A1D, calbindinD28k and parvalbumin mRNA expression levels was determined by qRT-PCR at 4, 24 and 48 hours. Results Our findings indicate that astaxanthin could have direct or indirect outcome on calcium homeostasis by regulating mRNA expression levels of NMDA subunits, LVSCC-A1C and LVSCC-A1D, calbindinD28k and parvalbumin by a dose and time dependent manner. Conclusion Neuroprotective effects of astaxanthin as a Ca homeostasis regulator should be noted throughout neurodegenerative disorders, and neurosurgery applications.

ASSOCIATION BETWEEN NADSYN1/DHCR7 AND CYP2R1 GENOTYPES AND PARKINSON’S DISEASE AND ITS CLINICAL FEATURES

dysfunction, however, the association between MT genetic variants and AD has not been fully explored in large datasets. Methods:The Alzheimer’s Disease Genetics Consortium (ADGC) recently genotyped 8,706 AD cases and 7,002 healthy controls of European ancestry from five cohorts using the Illumina Exome-Chip 1.0 containing 227MT single nucleotide variants (SNVs). Each cohort was analyzed separately for variants with minor allele count (MAC) 10 and call rate 0.95 (94 variants on average). In gene-based tests, only SNVs with minor allele frequency (MAF) <0.05 were considered, and genes with 2 SNVs and cumulative MAC 10 were tested. Associations with AD status were tested using single variant SCORE tests and gene-based (SKAT-O) tests in seqMeta in R package, controlling for age, sex, and principal components of ancestry. Results were combined across cohorts using meta-analysis. Bonferroni-corrected thresholds were used to determine statistical significance for single variant (a1⁄48.6310) and gene-based (a1⁄44.5310) associations. Finally, SNVs (MAF<0.05) in genes that encode subunits of the respiratory oxidative phosphorylation (OXPHOS) complexes were aggregated for testing association with AD. Results: In single variant analyses, one missense SNV in MT-ND3which encodes NADH dehydrogenase 3 involved in oxidative phosphorylation of ATP (rs2853826, Thr114Ala, MAF1⁄40.22) was significantly associated with AD risk (OR1⁄41.043; P1⁄42.50310). The most significant gene-based association finding was obtained withMT-TP (P1⁄4 7.9310), but this result did not survive correction for multiple testing. However, the hypothesis-driven test of aggregated rare variants in the OXPHOS complex was significant (P1⁄40.02, cumulative MAF1⁄40.015), which is consistent with previous reports that the expression level of OXPHOS genes is significantly lower in AD than in MCI or cognitively healthy subjects. Conclusions:We identified significant association of AD risk with one common MT SNV and the collective group of OXPHOS pathway genes. A replication study of independent cohorts from the International Genomics of Alzheimer’s Project is currently underway. Additional replication and discovery of novel rare AD-associated MT variants in datasets from the Alzheimer’s Disease Sequencing Project data are also in progress.

THE BLOOD PROFILE OF MTDNA ENCODED GENES EXPRESSION IN PARKINSON’S DISEASE PATIENTS AND HEALTHY INDIVIDUALS

0.00001), 24(s)-hydroxycholesterol (1.27, 1.03-1.57, p1⁄40.02), SAP (1.30, 1.01-1.67, p1⁄40.05), SAM (0.90, 0.83-0.98, p1⁄40.01), 5-MTHF (0.91, 0.85-0.97, p1⁄40.008), and homocysteine: (plasma 1.19, 95% CI 1.11-1.28, p< 0.00001; CSF 1.15, 95% CI 1.101.34 p1⁄40.06). Conclusions: The neurovascular hypothesis implicates glial, vascular, and neuronal changes in AD pathology. Glial involvement was suggested by highly significant elevations of glial markers YKL-40 and MCP-1. Vascular (endothelial) dysfunction is not implicated as homocysteine was significantly elevated in blood, but NOT CSF. Vascular involvement via reduced cholesterol clearance is suggested by elevation of CSF 24(s)-hydroxycholesterol levels. Additional data analysis yielded unforeseen results, such as possible dysfunction of methylation reactions in the AD brain (SAM, 5MTHF). Our meta-analysis validates glial involvement in the neurovascular hypothesis, suggest that homocysteine lowering therapies may be ineffective, and compels the investigation of further therapeutic targets.