Gene expression profiling of primary fibrochondrocyte cultures in traumatic and degenerative meniscus lesions.

PURPOSE: This study aimed to investigate how fibroblastic and chondrocytic properties of human meniscal fibrochondrocytes are affected in culture conditions according to the type of meniscal pathology and localization, and to provide basic information for tissue-engineering studies. METHODS: Primary fibrochondrocyte cultures were prepared from meniscus samples of patients who had either traumatic tear or degeneration due to osteoarthritis. Cultures were compared in terms of mRNA expression levels of COL1A1, COL2A1, COMP1, HIF1A, HIF2A, and SOX9 and secreted total collagen and sulfated sGAG levels according to the type of meniscal pathology, anatomical localization, and the number of subcultures. RESULTS: mRNA expression levels of COL1A1, COMP1, HIF1A, HIF2A, and SOX9 were found to be increased in subsequent subcultures in all specimens. COL1A1 mRNA expression levels of both lateral and medial menisci of patients with traumatic tear were significantly higher than in patients with degenerative pathology, indicating a more fibroblastic character. P1 subculture of lateral and P3 or further subculture of medial meniscus showed more fibroblastic characteristics in patients with degenerative pathology. Furthermore, in patients with degenerative pathology, the subcultures of the lateral meniscus (especially on the inner part) presented more chondrocytic characteristics than did those of medial meniscus. CONCLUSIONS: The mRNA expression levels of the cultures showed significant differences according to the anatomical localization and pathology of the meniscus, indicating distinct chondrocytic and fibroblastic features. This fundamental knowledge would help researchers to choose more efficient cell sources for cell-seeding of a meniscus scaffold, and to generate a construct resembling the original meniscus tissue.

Low Levels of LRRK2 Gene Expression are Associated with LRRK2 SNPs and Contribute to Parkinson's Disease Progression.

Parkinson's disease (PD) is a chronic neurodegenerative disease that has relatively slow progression with motor symptoms. Leucine-rich repeat kinase 2 (LRRK2) gene mutations and polymorphisms are suggested to be associated with PD. In this study, we aimed to investigate the association between single-nucleotide polymorphisms (SNPs) of the LRRK2 gene, namely, rs11176013, rs10878371, rs11835105, and PD. Genotypes of 132 PD cases and 133 healthy individuals were determined by qRT-PCR. Haplotype analysis was performed. Additionally, LRRK2 mRNA expression levels were determined in 83 PD cases and 55 healthy subjects. The relationship between LRRK2 mRNA levels, the target SNPs, and clinical data was also investigated. Our results indicated that the "GG" genotype and "G" allele of rs11176013 and the "CC" genotype and "C" allele of rs10878371 were more frequent in cases. The "GCG" haplotype was significantly more frequent in cases. LRRK2 mRNA expression levels in patients were significantly lower than those in healthy individuals. The patients with the "CC" genotype for rs10878371 and the "GG" genotype for rs11176013 had decreased LRRK2 mRNA levels. We found that the rs11176013 "GG" genotype and the rs10878371 "CC" genotype were less frequently seen in cases with akinetic rigid or combined akinetic rigid and tremor-dominant initial symptoms. Consequently, our results demonstrate that the rs11176013 and rs10878371 polymorphisms are associated with PD in a Turkish cohort, and moreover, these results suggest that these polymorphisms may affect the expression of the LRRK2 gene and disease progression and thus play a role in the pathogenesis of PD.

Amyloid Beta 1-42 Alters the Expression of miRNAs in Cortical Neurons.

Recently, Aß1-42 was demonstrated to have the potential to translocate into the nucleus and to be involved in the transcriptional regulation of certain neurodegeneration-related genes. This data raises the question of whether Aß-induced neurodegeneration might include the expression of miRNAs. Thus, our aim in this study was to investigate the effects of Aß1-42 on certain miRNAs which are related with vitamin D metabolism, neuronal differentiation, development, and memory. This question was investigated in primary cortical neurons that were treated with 10 µM Aß and/or 10-8 M 1,25-dihydroxyvitamin D3 at different time points by expression analysis of let-7a-5p, miR-26b-5p, miR-27b-3p, miR-31a-5p, miR-125b-5p, and miR-192-5p with qRT-PCR. Our data indicate that amyloid pathology has effects on the expression of miRNAs. Furthermore, some of these miRNAs simultaneously regulate the proteins or the enzymes involved in neuronal metabolism. The experimental setup that we used and the data we acquired supply valuable information about the miRNAs that play a part in the Aß pathology and suggested Aß as a counterpart of vitamin D at the crossroads of neuronal differentiation, development, and memory.

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

Our previous study demonstrated the translocation of Aß1-42 to the nucleus in response to antibiotic treatment, and interpreted it as a possible transcriptional response of Aß1-42 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 Aß1-42 production. For this purpose, we investigated the acute effect of Aß1-42 on the transcriptional levels of genes that have roles in the mechanisms that produce Aß 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, GSK3ß, and Cdk5. Additionally, we examined cholecalciferol metabolism-related enzyme 1α-hydroxylase (1αOHase) in primary cortical neurons with qRT-PCR. Our results indicate that Aß1-42 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 Aß impacts the neurons not only with toxic events but also at the transcriptional level. The nuclear localization of Aß1-42 and its regulatory effects on the target genes that we investigated in present study indicates Aß1-42 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.

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 Alzheimer's 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 (Aß) 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 Aß1-42 production. Extracellular Aß1-42 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 Aß1-42 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.

GC and VDR SNPs and Vitamin D Levels in Parkinson's Disease: The Relevance to Clinical Features.

Vitamin D deficiency is suggested to be associated with Parkinson's disease (PD). Our aim was to investigate the serum 25-hydroxyvitamin D3 (25OHD) levels of PD patients in Turkish cohort, to investigate any association of vitamin D binding protein (GC) genotypes with PD due to the significant role of GC in vitamin D transport, to determine whether vitamin D receptor (VDR) haplotype that we previously demonstrated to be a risk haplotype for AD is also a common haplotype for PD and to investigate any relevant consequence of serum 25OHD levels, GC or VDR genotypes on clinical features of PD. Three hundred eighty-two PD patients and 242 healthy subjects were included in this study. The serum 25OHD levels were investigated by CLIA, and GC and VDR SNPs were evaluated with LightSnip. Our results indicated a strong relationship between low serum 25OHD levels and PD (p < 0.001). rs7041 of GC and ApaI of VDR were associated with the PD risk (p < 0.05). Minor allele carriers for BsmI of VDR gene in both PD patients and healthy subjects had significantly higher levels of serum 25OHD (p < 0.05). The homozygous major allele carriers for rs2282679, rs3755967 and rs2298850 of GC gene in PD patients with slower progression had significantly higher levels of serum 25OHD (p  < 0.05). Minor allele carriers for FokI of VDR gene were more frequent in patients with advanced-stage PD (p < 0.05). Consequently, this is the first study demonstrating GC gene as a risk factor for PD. The relationship between PD's clinical features and low 25OHD or risk genotypes might have effects on PD independently.

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-1ß 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.