Microtubule-associated protein 1B dysregulates microtubule dynamics and neuronal mitochondrial transport in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating childhood disease primarily affecting lower motoneurons in the spinal cord. SMA is caused by the loss of functional survival of motoneuron (SMN) protein, leading to structural and functional alterations of the cytoskeleton in motoneurons and other cells. Loss of SMN results in impairments of microtubule architecture, but the underlying mechanisms are not completely understood. In this study, we mechanistically analyzed the effects of SMN deficiency on microtubules, demonstrating a reduced stability together with a reduction in alpha tubulin detyrosination. This was caused by increased levels of microtubule-associated protein 1B and tubulin tyrosine ligase, resulting in mitochondrial mislocalization in SMA. Our findings suggest that altered tubulin post-translational modifications and microtubule-associated proteins are involved in the pathomechanisms of SMA, such as an impaired axonal transport of mitochondria.

SMN loss dysregulates microtubule-associated proteins in spinal muscular atrophy model.

Spinal muscular atrophy (SMA) is a rare neurodegenerative disease caused by the absence of survival motor neuron (SMN) protein. SMN loss results in impairments of the cytoskeleton, including microtubules and regulatory proteins. However, the contribution of microtubule-associated proteins (MAPs) to microtubule dysregulations in SMA is not fully understood. In this study, we investigated neuronal MAPs responsible for the microtubule stability and growth, including MAP1A, MAP2, MAP6, MAP7, EB1, and EB3 using an in vitro model of SMA. Decreased MAP2 and EB3 levels were found in SMN-deficient motor neuron-like cells, and EB3 protein level was also relevant to MAP1B. SMN loss leads to an increase in EB3 comet numbers at proximal neurites, indicating increased microtubule growth. Our findings suggest that SMN deficiency simultaneously causes dysregulations of several MAPs, contributing to the perturbations of microtubule dynamics in SMA.

Rutin increases alpha-tubulin acetylation via histone deacetylase 6 inhibition.

Microtubules are dynamic cytoskeletal filaments composed of alpha- (α) and beta (ß)-tubulin proteins. α-tubulin proteins are posttranslationally acetylated, and loss of acetylation is associated with axonal transport defects, a common alteration contributing to the pathomechanisms of several neurodegenerative diseases. Restoring α-tubulin acetylation by pharmacological inhibition of HDAC6, a primary α-tubulin deacetylase, can rescue impaired transport. Therefore, HDAC6 is considered a promising therapeutic target for neurodegenerative diseases, but currently, there is no clinically approved inhibitor for this purpose. In this study, using drug repurposing strategy, we aimed to identify compounds possessing HDAC6 inhibition activity and inducing α-tubulin acetylation. We systematically analyzed the FDA-approved library by utilizing virtual screening and consensus scoring approaches. Inhibition activities of promising compounds were tested using in vitro assays. Motor neuron-like NSC34 cells were treated with the candidate compounds, and α-tubulin acetylation levels were determined by Western blot. Our results demonstrated that rutin, a natural flavonoid, inhibits cellular HDAC6 activity without inducing any toxicity, and it significantly increases α-tubulin acetylation level in motor neuron-like cells.

Effects of flavonoid quercetin on survival of motor neuron gene expression.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that results in muscle weakness and atrophy. To attenuate disease severity, drug development studies have been applied mainly to target the Survival of Motor Neuron 2 (SMN2) gene, which is an important modifier of SMA. Although several compounds have been tested, there is still no cure for SMA. In this study, SMN2-inducing effects of quercetin, an abundant flavonoid polyphenol in human diet, was investigated in the fibroblast cell lines of two SMA type I patients. Gene expression studies showed that quercetin upregulates SMN2 mRNA up to fourfold, but not the SMN protein level.

The relationship between vitamin D receptor gene polymorphisms and bone density, osteocalcin level and growth in adolescents.

BACKGROUND/OBJECTIVE: The relationship between vitamin D receptor gene polymorphisms and bone density, osteocalcin and growth was investigated. SUBJECTS: Eighty eight adolescents aged between 8-15, with no history of illness influencing the level of bone parameters, were examined in our study. METHODS: Areal BMD for lumbar spine (L1-4) was assessed by dual energy X-ray absorptiometry (DEXA). Height and weight were measured on the day of the DEXA scans. Serum osteocalcin level was determined by using ELISA method. DNA was extracted from white blood cells, amplified by the polymerase chain reaction (PCR) and the polymorphic sites were analyzed by using ApaI, TaqI and FokI restriction enzymes. RESULTS: The most frequent genotypes were FF (% 54.6), Aa (% 53.4) and Tt (% 48.8). No significant relationship was found between VDR genotypes and areal BMD, osteocalcin level or growth in either sex. But there was a strong tendency for a higher BMD at the lumbar spine of TT and AA genotypes compared to tt and Aa genotypes. The children with TT genotype were taller and heavier than the children with tt genotype CONCLUSION: Our results suggest that VDR gene TaqI polymorphism may be associated with body weight and bone mass, but more studies with larger groups should be conducted.

Molecular modifications on carboxylic acid derivatives as potent histone deacetylase inhibitors: Activity and docking studies.

In the light of known HDAC inhibitors, 33 carboxylic acid derivatives were tested to understand the structural requirements for HDAC inhibition activity. Several modifications were applied to develop the structure-activity relationships of carboxylic acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor-enzyme complexes. Of these compounds, caffeic acid derivatives such as chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, which is a well-known HDAC inhibitor.

A preliminary report on spinal muscular atrophy lymphoblastoid cell lines: are they an appropriate tool for drug screening?

INTRODUCTION: Spinal muscular atrophy (SMA) is a neurodegenerative disease of the motor neurons that results in progressive muscle weakness. It is also the leading hereditary cause of infant mortality. Homozygous loss of the survival motor neuron (SMN1) gene causes SMA, and the number of copies of the SMN2 gene modulates the severity of the disease. Increasing the expression of the SMN2 gene by pharmacological agents is one of the therapeutic approaches currently being implemented. METHODS: In this preliminary study, we investigated the effect of phenylbutyrate, a histone deacetylase (HDAC) inhibitor, on SMN2 expression in two SMA type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines to understand the suitability of lymphoblastoid cell lines in drug screening. These cell lines are regarded as a good source as they can easily be established from the peripheral leucocytes of patients. Quantitative analysis of SMN2 mRNA was performed on established cell lines treated with various concentrations of phenylbutyrate and for a range of incubation periods using real-time polymerase chain reaction. Western blot analysis was used to determine SMN protein levels. RESULTS: Real-time polymerase chain reaction and Western blot analysis demonstrated that the levels of SMN2 full-length (fl-SMN2) transcripts and protein were not increased in phenylbutyrate-treated cell lines compared to non-treated controls. CONCLUSION: These results suggest that EBV-transformed lymphoblastoid cell lines are not suitable for studying the effect of certain HDAC inhibitors on SMN2 gene expression.

Vitamin D receptor gene polymorphisms in Turkish children with vitamin D deficient rickets.

Vitamin D deficient rickets is prevalent in Turkey and a considerable number of children are at risk of growth retardation, impaired bone formation and fracture. In order to check whether vitamin D receptor (VDR) gene polymorphism relates to the vitamin D deficient rickets, we analyzed VDR gene FokI, TaqI and ApaI polymorphisms in 24 Turkish vitamin D deficient rickets patients and 100 healthy controls. We found that "A" (ApaI) allele is more abundant in patients than controls (83 vs 57%, p = 0.002) but there were no significant differences for FokI (p = 0.693) and TaqI (p = 0.804) allele frequencies between patients and controls. We also showed that the frequency of Tt and Aa genotypes was significantly decreased in patients. Our results indicated that VDR gene polymorphisms might be an important factor for genetic susceptibility to vitamin D deficient rickets in the Turkish population.

Effects of Arm Cycling Exercise in Spinal Muscular Atrophy Type II Patients: A Pilot Study.

Exercise studies in neuromuscular diseases like spinal muscular atrophy (SMA), a devastating disease caused by survival of motor neuron 1 ( SMN1) gene mutations, are drawing attention due to its beneficial effects. In this study, we presented a constructed arm cycling exercise protocol and evaluated the benefits on SMA patients. Five SMA type II patients performed 12 weeks of supervised arm cycling exercise. The physical functions were evaluated together with the SMN2 copy numbers, SMN protein levels, insulin-like growth factor 1(IGF1) and binding protein 3 (IGFBP3) levels. The active cycling distance and duration of patients significantly improved. Significant changes could not have detected either SMN or IGF1 and IGFBP3 levels in response to exercise. The findings demonstrated that the patients tolerated the exercise protocol and gained a benefit from arm cycling but benefits could not be associated with SMN2 copy number, SMN protein level, IGF1, or IGFBP3 levels.

Polymorphisms of vitamin D receptor gene in Turkish familial psoriasis patients.

Psoriasis is characterized by hyperproliferation and abnormal differentiation of keratinocytes, and inflammation. 1,25-Dihydroxyvitamin D3, which is used for the treatment of psoriasis, binds to vitamin D receptor (VDR) and modulates gene transcription. We analyzed VDR gene FokI, ApaI and TaqI polymorphisms in 51 Turkish familial psoriasis patients (psoriasis vulgaris and psoriatic arthritis) and 100 healthy subjects, and evaluated the correlation between VDR genotypes and calcipotriol response. We found that the TT genotype was significantly more frequent in the patients than in the controls (51 vs. 35%: P < or = 0.05). The frequency of the T allele in patients was also significantly higher than that in the controls (73.5 vs. 59.5%: P < or = 0.025). In psoriatic arthritis patients, T allele frequency was even higher (91.7%: P < or = 0.05). With regard to response to calcipotriol treatment, in nonresponsive patients TT genotype and T allele frequencies were higher than they were in the controls (63.6 vs. 35%: P < or = 0.025, 81.8 vs. 59.5%: P < or = 0.01, respectively). In conclusion, we show that VDR gene TaqI polymorphism is associated with familial psoriasis in the Turkish population. We also demonstrate that VDR gene polymorphisms may play a role in partial resistance to calcipotriol therapy.

Effects of Exercise on Low Density Lipoprotein Receptor Related Protein 5 Gene Expression in Patients With Postmenopausal Osteoporosis.

OBJECTIVES: This study aims to investigate the effects of aerobic exercise on low density lipoprotein receptor related protein 5 (LRP5) gene messenger ribonucleic acid expression and evaluate the relationship between the clinical parameters and gene expression in patients with postmenopausal osteoporosis (OP). PATIENTS AND METHODS: Seven patients with postmenopausal OP (mean age 60.0±5.3 years; range 51 to 66 years) were included in the study. An exercise protocol/program consisting of treadmill exercising for 30 minutes three days a week for six weeks was performed at a moderate intensity. LRP5 gene expression levels were evaluated before the onset of the exercise program and then four hours after the end of the first session and 12th (fourth week) and 18th (sixth week) sessions of exercise. RESULTS: Our results demonstrated variable changes in the LRP5 gene expression after the aerobic exercise sessions. Excluding one patient, the LRP5 gene expression levels showed a slight tendency to increase. In spite of this tendency, gene expression differences during the exercise sessions were not significant. CONCLUSION: Our results suggest that interindividual variations of LRP5 gene expression exist after moderate intensity aerobic exercises in patients with postmenopausal OP. Despite of this variability, LRP5 gene expression levels increased slightly, except in peripheral blood in one patient. Future studies with larger sample sizes and different sampling time/tissues are required to shed more light on the impact of exercise at molecular level in OP.

Spinal muscular atrophy type III: Molecular genetic characterization of Turkish patients.

Spinal Muscular Atrophy (SMA) is a neurodegenerative disease with autosomal recessive inheritance. Homozygous loss of exon 7 of the Survival of motor neuron 1 (SMN1) gene is the main cause of SMA. Although progressive muscle weakness and atrophy are common symptoms, disease severity varies from severe to mild. Type III is one of the milder and less frequent forms of SMA. In this study, we report molecular genetic characteristics of 24 Turkish type III SMA patients. Homozygous loss of SMN1 exon 7 and 8 was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex ligation dependent probe amplification (MLPA). SMN2, homologue of SMN1, and Neuronal apoptosis inhibitory protein (NAIP) genes were also evaluated considering their influence on disease severity. We determined that male patients who were born in consanguineous families were predominant in our cohort and these patients mostly carry the homozygous loss of SMN1 exon 7 and 8 and four copies of SMN2 gene without NAIP deletions.

Analysis of the modifying effects of TAP 1/2 genes on cystic fibrosis phenotype.

Phenotypic variability has been reported in cystic fibrosis (CF) patients. TAP1 and TAP2 genes are encoding "the transporter associated with antigen processing" proteins. The aim of the present study was to analyze the frequency of TAP 1/2 variants in the Turkish population and to investigate a possible modifying role of these variants in CF phenotype. Sixty-three CF patients of known genotypes and 100 healthy control subjects were analyzed. There was a significant difference in the frequencies at positions 333 and 637 of TAP 1 gene and at position 665 of TAP 2 gene between patients and controls. Comparison of TAP gene polymorphisms in 36 CF patients homozygous for AF508 mutation with control subjects revealed a significant difference at position 665 of TAP 2 gene. These findings may be useful to assess the predisposition and to predict severity of the disease. We demonstrated that TAP genes might have modifying effects on the CF phenotype.

Investigations of curcumin and resveratrol on neurite outgrowth: perspectives on spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease with progressive muscle weakness and atrophy. SMA is caused by low levels of the Survival of Motor Neuron (SMN) protein, which also leads to neurite outgrowth defects in neuronal cells. Rescue of the outgrowth defect is thought to be a strategy for SMA treatment. Polyphenolic histone deacetylase (HDAC) inhibitors might be good candidates due to their neuritogenic properties. In the present study, it was investigated whether neurite outgrowth defects could be rescued by curcumin and resveratrol, which are SMN-inducing polyphenols, having HDAC inhibition activity. According to our results, although curcumin and resveratrol failed to restore the neurite outgrowth defects, the SMN protein was found to be necessary for the neurite-promoting activity of curcumin in neuron-like PC12 cells.

Carboxylic acid derivatives of histone deacetylase inhibitors induce full length SMN2 transcripts: a promising target for spinal muscular atrophy therapeutics.

INTRODUCTION: Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder. It is caused by homozygous absence of the survival motor neuron 1 (SMN1) gene. SMN2, which modulates the severity of the disease, represents a major target for therapy. The aim of this study was to investigate whether SMN2 expression can be increased by caffeic acid, chlorogenic acid and curcumin, which are designed by modifications of the carboxylic acid class of histone deacetylase (HDAC) inhibitors. MATERIAL AND METHODS: Using quantitative real-time PCR, we analysed the levels of full-length SMN2 and Δ7SMN2 mRNA. We performed LDH cytotoxicity assay to analyse whether SMN2 activating concentrations of caffeic acid, chlorogenic acid and curcumin were cytotoxic to fibroblasts. RESULTS: We found that caffeic acid and curcumin were more efficient than chlorogenic acid and increased full-length SMN2 mRNA levels 1.5 and 1.7-fold, respectively. Δ7SMN2 mRNA levels were measured to investigate alternative splicing of exon 7. We also found that cytotoxicity was not observed at SMN2 activating concentrations. CONCLUSIONS: Our data suggest that carboxylic acid derivatives including phenolic structure and symmetry could be a good candidate for SMA treatment.

Evaluation of the effects of vitamin D receptor and estrogen receptor 1 gene polymorphisms on bone mineral density in postmenopausal women.

The aim of this study is to evaluate the effects of estrogen receptor 1 (ESR1) and vitamin D receptor (VDR) gene polymorphisms on bone mineral density (BMD) in a group of previously untreated osteoporotic women. Effects of demographic, environmental, and hormonal factors were also evaluated in this context. Fifty women who did not have a prior diagnosis or treatment of osteoporosis were compared with 50 nonosteoporotic postmenopausal women. Demographic and morphometric characteristics, medical history, dietary habits, exercise history, and sunlight exposure were recorded. The diagnosis of osteoporosis was made with regard to BMD measurements with DEXA. Blood samples were obtained for serum biochemistry, bone turnover markers, and VDR and ESR1 gene polymorphism analysis. Polymorphic sites of VDR and ESR1 genes were amplified by polymerase chain reaction and examined using restriction fragment length polymorphism. Bb genotype was significantly higher in the osteoporotic group when compared to controls (p=0.022). Each 1 U decrease in the body mass index (BMI) increased the risk of osteoporosis by 8% independent of the genotype. We could not observe a significant effect of ESR1 polymorphism on BMD or osteoporosis risk. The interaction of ApaI and BsmI genotypes were found to be significant (p=0.041) and the AaBb genotype, when corrected for BMI, was shown to increase the risk of osteoporosis five times (p=0.005). However, the results demonstrated insignificant p values when correction for multiple testing was performed with the Bonferroni method in the logistic regression model. A predominance of Bb genotype of the VDR gene was evident in this group of postmenopausal Turkish women. Moreover, the combined genotype AaBb conferred a five times increased risk for osteoporosis when corrected for clinical variables.

Histone deacetylase inhibition activity and molecular docking of (e )-resveratrol: its therapeutic potential in spinal muscular atrophy.

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is caused by mutation of the survival motor neuron gene (SMN1) but all patients retain a nearly identical copy, SMN2. The disease severity correlates inversely with increased SMN2 copy. Currently, the most promising therapeutic strategy for spinal muscular atrophy is induction of SMN2 gene expression by histone deacetylase inhibitors. Polyphenols are known for protection against oxidative stress and degenerative diseases. Among our candidate prodrug library, we found that (E )-resveratrol, which is one of the polyphenolic compounds, inhibited histone deacetylase activity in a concentration-dependent manner and half-maximum inhibition was observed at 650 microM. Molecular docking studies showed that (E )-resveratrol had more favorable free energy of binding (-9.09 kcal/mol) and inhibition constant values (0.219 microM) than known inhibitors. To evaluate the effect of (E )-resveratrol on SMN2 expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E )-resveratrol. The level of full-length SMN2 mRNA and protein showed 1.2- to 1.3-fold increase after treatment with 100 microM (E )-resveratrol in only one cell line. These results indicate that response to (E )-resveratrol treatment is variable among cell lines. This data demonstrate a novel activity of (E )-resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy.