Propensity of <Emphasis Type="Italic">Withania somnifera</Emphasis> to Attenuate Behavioural,Biochemical, and Histological Alterations in Experimental Model of Stroke

The present study was designed to evaluate the beneficial effects of Withania somnifera (WS) pre-supplementation on middle cerebral artery occlusion (MCAO) model of ischemic stroke. Ischemic stroke was induced in the rats by inserting intraluminal suture for 90 min, followed by reperfusion injury for 24 h. The animals were assessed for locomotor functions (by neurological deficit scores, narrow beam walk and rotarod test), cognitive and anxiety-like behavioural functions (by morris water maze and elevated plus maze test). MCAO animals showed significant impairment in locomotor and cognitive functions. Neurobehavioural changes were accompanied by decreased acetylcholinesterase activity, increased oxidative stress in terms of enhanced lipid peroxidation and lowered thiol levels in the MCAO animals. In addition, MCAO animals had cerebral infarcts and the presence of pycnotic nuclei. Single-photon emission computerized tomography (SPECT) of MCAO animals revealed a cerebral infarct as a hypoactive area. On the other hand, pre-supplementation with WS (300 mg/kg body weight) for 30 days to MCAO animals was effective in restoring the acetylcholinesterase activity, lipid peroxidation, thiols and attenuated MCAO induced behavioural deficits. WS significantly reduced the cerebral infarct volume and ameliorated histopathological alterations. Improved blood flow was observed in the SPECT images from the brain regions of ischemic rats pre-treated with WS. The results of the study showed a protective effect of WS supplementation in ischemic stroke and are suggestive of its potential application in stroke management.     

Deletion of <Emphasis Type="Italic">mTOR</Emphasis> in Reactive Astrocytes Suppresses Chronic Seizures in a Mouse Model of Temporal Lobe Epilepsy

Germline and somatic mutations in key genes of the mammalian target of rapamycin (mTOR) pathway have been identified in seizure-associated disorders. mTOR mutations lead to aberrant activation of mTOR signaling, and, although affected neurons are critical for epileptogenesis, the role of mTOR activation in glial cells remains poorly understood. We previously reported a consistent activation of the mTOR pathway in astrocytes in the epileptic foci of temporal lobe epilepsy. In this study, it was demonstrated that mTOR deletion from reactive astrocytes prevents increases in seizure frequency over the disease course. By using a tamoxifen-inducible mTOR conditional knockout system and kainic acid, a model was developed that allowed astrocyte-specific mTOR gene deletion in mice with chronic epilepsy. Animals in which mTOR was deleted from 44 % of the astrocyte population exhibited a lower seizure frequency compared with controls. Down-regulation of mTOR significantly ameliorated astrogliosis in the sclerotic hippocampus but did not rescue mossy fiber sprouting. In cultured astrocytes, the mTOR pathway modulated the stability of the astroglial glutamate transporter 1 (Glt1) and influenced the ability of astrocytes to remove extracellular glutamate. Taken together, these data indicate that astrocytes with activated mTOR signaling may provide conditions that are favorable for spontaneous recurrent seizures.     

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM ^SA/SA ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM ^SA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM ^SA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM ^SA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.     

Steroid compounds from two pacific starfish of the genus <Emphasis Type="Italic">Evasterias</Emphasis>

Three new steroid glycosides (evasteriosides C, D, and E) along with six known compounds were isolated from two Pacific starfish of the genus Evasterias. Evasterioside C from E. retiferacollected from the Sea of Japan was identified as (20R, 22E)-3-O-(β-D-xylopyranosyl)-24-nor-5α-cholest-22-ene-3β,6β,15α,26-pentaol 26-sulfate sodium salt. The structures of evasteriosides D and E from E. echinosoma (collected from the Gulf of Shelichov, the Sea of Okhotsk) were established as (20R, 24S)-24-O-(β-D-glucopyranosyl)-5α-cholestane-3β,6α,8,15β,24-pentaol and (20R,24S)-3,24-di-O-(β-D-xylopyranosyl)-cholest-4-ene-3β,6β,8,15α,24-pentaol, respectively. In addition, the known compounds pycnopodiosides A and C, luridoside A, 5α-cholestane-3β,6α,8,15β,16β,26-hexaol. 5α-Cholestane-3β,6α,8,15β,24-pentaol 24-sulfate sodium saltand marthasterone sulfate sodium salt were identified in E. echinosoma. The structures of the isolated compounds were established on the basis of spectroscopic analyses, using 1D and 2D NMR techniques, mass spectrometry, and some chemical transformations.     

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM’s effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM’s effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM’s effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.     

Amino acid substitutions [K16A] and [K28A] distinctly affect amyloid <Emphasis Type="Italic">β</Emphasis>-protein oligomerization

Amyloid β-protein (A β) assembles into oligomers that play a seminal role in Alzheimer’s disease (AD), a leading cause of dementia among the elderly. Despite undisputed importance of A β oligomers, their structure and the basis of their toxicity remain elusive. Previous experimental studies revealed that the [K16A] substitution strongly inhibits toxicity of the two predominant A β alloforms in the brain, A β ₄₀ and A β ₄₂, whereas the [K28A] substitution exerts only a moderate effect. Here, folding and oligomerization of [A16]A β ₄₀, [A28]A β ₄₀, [A16]A β ₄₂, and [A28]A β ₄₂ are examined by discrete molecular dynamics (DMD) combined with a four-bead implicit solvent force field, DMD4B-HYDRA, and compared to A β ₄₀ and A β ₄₂ oligomer formation. Our results show that both substitutions promote A β ₄₀ and A β ₄₂ oligomerization and that structural changes to oligomers are substitution- and alloform-specific. The [K28A] substitution increases solvent-accessible surface area of hydrophobic residues and the intrapeptide N-to-C terminal distance within oligomers more than the [K16A] substitution. The [K16A] substitution decreases the overall β-strand content, whereas the [K28A] substitution exerts only a modest change. Substitution-specific tertiary and quaternary structure changes indicate that the [K16A] substitution induces formation of more compact oligomers than the [K28A] substitution. If the structure-function paradigm applies to A β oligomers, then the observed substitution-specific structural changes in A β ₄₀ and A β ₄₂ oligomers are critical for understanding the structural basis of A β oligomer toxicity and correct identification of therapeutic targets against AD.     

Elastic band resistance training influences transforming growth factor-ß receptor I mRNA expression in peripheral mononuclear cells of institutionalised older adults: the Vienna Active Ageing Study (VAAS)

Background

Ageing, inactivity and obesity are associated with chronic low-grade inflammation contributing to a variety of lifestyle-related diseases. Transforming growth factor-β (TGF-β) is a multimodal protein with various cellular functions ranging from tissue remodelling to the regulation of inflammation and immune functions. While it is generally accepted that aerobic exercise exerts beneficial effects on several aspects of immune functions, even in older adults, the effect of resistance training remains unclear. The aim of this study was to investigate whether progressive resistance training (6 months) with or without nutritional supplementation (protein and vitamins) would influence circulating C-reactive protein and TGF-β levels as well as TGF-β signalling in peripheral mononuclear cells (PBMCs) of institutionalised adults with a median age of 84.5 (65.0–97.4) years.

Results

Elastic band resistance training significantly improved performance as shown by the arm-lifting test (p?=?0.007), chair stand test (p?=?0.001) and 6-min walking test (p?=?0.026). These results were paralleled by a reduction in TGF-β receptor I (TGF-βRI) mRNA expression in PBMCs (p?=?0.006), while circulating inflammatory markers were unaffected. Protein and vitamin supplementation did not provoke any additional effects. Interestingly, muscular endurance of upper and lower body and aerobic performance at baseline were negatively associated with changes in circulating TGF-β at the early phase of the study. Furthermore, drop-outs of the study were characterised not only by lower physical performance but also higher TGF-β and TGF-βRI mRNA expression, and lower miRNA-21 expression.

Conclusions

Progressive resistance training with elastic bands did not influence chronic low-grade inflammation but potentially affected TGF-β signalling in PBMCs through altered TGF-βRI mRNA expression. There appears to be an association between physical performance and TGF-β expression in PBMCs of older adults, in which the exact mechanisms need to be clarified.

     

Role of RAGE in Alzheimer’s Disease

Receptor for advanced glycation end products (RAGE) is a receptor of the immunoglobulin super family that plays various important roles under physiological and pathological conditions. Compelling evidence suggests that RAGE acts as both an inflammatory intermediary and a critical inducer of oxidative stress, underlying RAGE-induced Alzheimer-like pathophysiological changes that drive the process of Alzheimer’s disease (AD). A critical role of RAGE in AD includes beta-amyloid (Aβ) production and accumulation, the formation of neurofibrillary tangles, failure of synaptic transmission, and neuronal degeneration. The steady-state level of Aβ depends on the balance between production and clearance. RAGE plays an important role in the Aβ clearance. RAGE acts as an important transporter via regulating influx of circulating Aβ into brain, whereas the efflux of brain-derived Aβ into the circulation via BBB is implemented by LRP1. RAGE could be an important contributor to Aβ generation via enhancing the activity of β- and/or γ-secretases and activating inflammatory response and oxidative stress. However, sRAGE–Aβ interactions could inhibit Aβ neurotoxicity and promote Aβ clearance from brain. Meanwhile, RAGE could be a promoting factor for the synaptic dysfunction and neuronal circuit dysfunction which are both the material structure of cognition, and the physiological and pathological basis of cognition. In addition, RAGE could be a trigger for the pathogenesis of Aβ and tau hyper-phosphorylation which both participate in the process of cognitive impairment. Preclinical and clinical studies have supported that RAGE inhibitors could be useful in the treatment of AD. Thus, an effective measure to inhibit RAGE may be a novel drug target in AD.     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Sex differences in the inflammatory response of primary astrocytes to lipopolysaccharide

Background

Numerous neurological and psychiatric disorders show sex differences in incidence, age of onset, symptomatology or outcome. Astrocytes, one of the glial cell types of the brain, show sex differences in number, differentiation and function. Since astrocytes are involved in the response of neural tissue to injury and inflammation, these cells may participate in the generation of sex differences in the response of the brain to pathological insults. To explore this hypothesis, we have examined whether male and female astrocytes show a different response to an inflammatory challenge and whether perinatal testosterone influences this response.

Methods

Cortical astrocyte cultures were prepared from postnatal day 1 (one day after birth) male or female CD1 mice pups. In addition, cortical astrocyte cultures were also prepared from female pups that were injected at birth with 100 μg of testosterone propionate or vehicle. Cultures were treated for 5 hours with medium containing lipopolysaccharide (LPS) or with control medium. The mRNA levels of IL6, interferon-inducible protein 10 (IP10), TNFα, IL1β, Toll-like receptor 4 (TLR4), steroidogenic acute regulatory protein and translocator protein were assessed by quantitative real-time polymerase chain reaction. Statistical significance was assessed by unpaired t-test or by one-way analysis of variance followed by the Tukey post hoc test.

Results

The mRNA levels of IL6, TNFα and IL1β after LPS treatment were significantly higher in astrocytes derived from male or androgenized females compared to astrocytes derived from control or vehicle-injected females. In contrast, IP10 mRNA levels after LPS treatment were higher in astrocytes derived from control or vehicle-injected females than in those obtained from males or androgenized females. The different response of male and female astrocytes to LPS was due neither to differences in the basal expression of the inflammatory molecules nor to differences in the expression of the LPS receptor TLR4. In contrast, the different inflammatory response was associated with increased mRNA levels of translocator protein, a key steroidogenic regulator, in female astrocytes that were treated with LPS.

Conclusions

Male and female cortical astrocytes respond differentially to an inflammatory challenge and this may be predetermined by perinatal testosterone exposure.

     

Sudden death due to paralysis and synaptic and behavioral deficits when <Emphasis Type="Italic">Hip14/Zdhhc17</Emphasis> is deleted in adult mice

Background

Palmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is an important regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have been implicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis, Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylates many synaptic proteins. Hip14 hypomorphic mice have behavioral and synaptic deficits. However, the phenotype is developmental; thus, a model of post-developmental loss of Hip14 was generated to examine the role of HIP14 in synaptic function in the adult.

Results

Ten weeks after Hip14 deletion (iHip14 ^Δ/Δ ), mice die suddenly from rapidly progressive paralysis. Prior to death the mice exhibit motor deficits, increased escape response during tests of anxiety, anhedonia, a symptom indicative of depressive-like behavior, and striatal synaptic deficits, including reduced probability of transmitter release and increased amplitude but decreased frequency of spontaneous post-synaptic currents. The mice also have increased brain weight due to microgliosis and astrogliosis in the cortex.

Conclusions

Behavioral changes and electrophysiological measures suggest striatal dysfunction in iHip14 ^Δ/Δ mice, and increased cortical volume due to astrogliosis and microgliosis suggests a novel role for HIP14 in glia. These data suggest that HIP14 is essential for maintenance of life and neuronal integrity in the adult mouse.

     

Serum levels of transforming growth factor β1 and C-reactive protein as possible markers of intra uterine insemination outcome

Objective

Maternal immunity is important for the implantation phase, and exaggerated inflammatory responses may reduce the chance of implantation and pregnancy. Transforming growth factor β1 (TGF-β1) plays a role in the modulation of cellular growth, maturation and differentiation, extracellular matrix formation, immunoregulation, and apoptosis. In this study, we aimed to evaluate the changes in serum TGF-β1 and C-reactive protein (CRP) levels in infertile women following intrauterine insemination (IUI) according to the presence of pregnancy.

Methods

Sixty-three infertile patients were selected for the study in a nine-month period. Clomiphene citrate or recombinant gonadotropins were used for ovulation induction, and all patients underwent IUI following human chorionic gonadotropin (hCG) trigger. The pregnant and non-pregnant groups’ TGF-β1 and CRP levels were measured.

Results

The CRP levels increased significantly from the day of the hCG trigger to the 8th day after hCG trigger in the non-pregnant group (P = 0.003) whereas TGF-β1 levels decreased in the pregnant group (P = 0.001).

Conclusion

Maternal inflammatory responses play an important role in the occurrence of pregnancy. Changes in the levels of TGF-β1 and CRP may have a role in the outcome of IUI. Serial measurements of TGF-β1 and C-reactive protein, if confirmed by larger studies, may become valuable in predicting the outcome of IUI.

     

Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane

Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors’ and receptors’ in the glycerol cryopreserved HAM.     

Downregulation of expression of mater genes <Emphasis Type="Italic">SOX9, FOXA2</Emphasis>, and <Emphasis Type="Italic">GATA4</Emphasis> in pancreatic cancer cells stimulated with TGFβ1 epithelial–mesenchymal transition

We show characteristic morphological changes corresponding to epithelial–mesenchymal transition (EMT) program fulfillment in PANC1 cell line stimulated with TGFβ1. Our results support downregulation of E-cadherin protein. We show 5- and 28-fold increase in SNAI1 and SNAI2 expression levels and 25- and 15-fold decrease in CDH1 and KRT8 expression levels, respectively, which confirms the EMT-program fulfillment. We demonstrate downregulation of expression of pancreatic master genes SOX9, FOXA2, and GATA4 (2-, 5-, and 4-fold, respectively) and absence of significant changes in HES1, NR5A2, and GATA6 expression levels in the cells stimulated with TGFβ1. Our results indicate the absence of induction of expression of PTF1A, PDX1, HNF1b, NEUROG3, RPBJL, NKX6.1, and ONECUT1 genes, which are inactive in PANC1 cell line after the EMT stimulated by TGFβ1.     

Involvement of Nuclear Receptor REV-ERBβ in Formation of Neurites and Proliferation of Cultured Adult Neural Stem Cells

Neural stem cells (NSCs) serve as the source of both neurons and support cells, and neurogenesis is reportedly linked to the circadian clock. This study aimed to clarify the functional role of the circadian rhythm-related nuclear receptor, REV-ERBβ, in neurogenesis of NSCs from adult brain. Accordingly, Rev-erbβ expression and the effect of Rev-erbβ gene-specific knockdown on neurogenesis in vitro was examined in adult rodent NSCs. Initial experiments confirmed REV-ERBβ expression in cultured adult NSCs, while subsequent gene expression and gene ontogeny analyses identified functional genes upregulated or downregulated by REV-ERBβ. In particular, expression levels of factors associated with proliferation, stemness, and neural differentiation were affected. Knockdown of Rev-erbβ showed involvement of REV-ERBβ in regulation of cellular proliferation and self-renewal of cultured adult NSCs. Moreover, Rev-erbβ-knockdown cells formed neurons with a slightly shrunken morphology, fewer new primary neurites, and reduced length and branch formation of neurites. Altogether, this suggests that REV-ERBβ is involved in neurite formation during neuronal differentiation of cultured adult NSCs. In summary, REV-ERBβ is a known circadian regulatory protein that appears to be involved in neurogenesis via regulation of networks for cell proliferation and neural differentiation/maturation in adult NSCs.