Development of a Cell Culture Chamber for Investigating the Therapeutic Effects of Electrical Stimulation on Neural Growth.

Natural electric fields exist throughout the body during development and following injury, and, as such, EFs have the potential to be utilized to guide cell growth and regeneration. Electrical stimulation (ES) can also affect gene expression and other cellular behaviors, including cell migration and proliferation. To investigate the effects of electric fields on cells in vitro, a sterile chamber that delivers electrical stimuli is required. Here, we describe the construction of an ES chamber through the modification of an existing lid of a 6-well cell culture plate. Using human SH-SY5Y neuroblastoma cells, we tested the biocompatibility of materials, such as Araldite®, Tefgel™ and superglue, that were used to secure and maintain platinum electrodes to the cell culture plate lid, and we validated the electrical properties of the constructed ES chamber by calculating the comparable electrical conductivities of phosphate-buffered saline (PBS) and cell culture media from voltage and current measurements obtained from the ES chamber. Various electrical signals and durations of stimulation were tested on SH-SY5Y cells. Although none of the signals caused significant cell death, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that shorter stimulation times and lower currents minimized negative effects. This design can be easily replicated and can be used to further investigate the therapeutic effects of electrical stimulation on neural cells.

INPP5D/SHIP1: Expression, Regulation and Roles in Alzheimer's Disease Pathophysiology.

Alzheimer's disease (AD) is the most common form of dementia, accounting for approximately 38.5 million cases of all-cause dementia. Over 60% of these individuals live in low- and middle-income countries and are the worst affected, especially by its deleterious effects on the productivity of both patients and caregivers. Numerous risk factors for the disease have been identified and our understanding of gene-environment interactions have shed light on several gene variants that contribute to the most common, sporadic form of AD. Microglial cells, the innate immune cells of the central nervous system (CNS), have long been established as guardians of the brain by providing neuroprotection and maintaining cellular homeostasis. A protein with a myriad of effects on various important signaling pathways that is expressed in microglia is the Src Homology 2 (SH2) domain-containing Inositol 5' Phosphatase 1 (SHIP1) protein. Encoded by the INPP5D (Inositol Polyphosphate-5-Phosphatase D) gene, SHIP1 has diminutive effects on most microglia signaling processes. Polymorphisms of the INPP5D gene have been found to be associated with a significantly increased risk of AD. Several studies have elucidated mechanistic processes by which SHIP1 exerts its perturbations on signaling processes in peripheral immune cells. However, current knowledge of the controllers of INPP5D/SHIP1 expression and the idiosyncrasies of its influences on signaling processes in microglia and their relevance to AD pathophysiology is limited. In this review, we summarize these discoveries and discuss the potential of leveraging INPP5D/SHIP1 as a therapeutic target for Alzheimer's disease.

Fiber and Electrical Field Alignment Increases BDNF Expression in SH-SY5Y Cells following Electrical Stimulation.

The limited expression of neurotrophic factors that can be included in neural tissue engineering scaffolds is insufficient for sustained neural regeneration. A localized and sustained method of introducing neurotrophic factors is required. We describe our attempt at inducing neuroblastoma cells to express trophic factors following electrical stimulation. Human SH-SY5Y neuroblastoma cells, cultured on polycaprolactone electrospun nanofibers, were electrically stimulated using a 100 mV/mm electric field. Nuclear morphology and brain-derived neurotrophic factor (BDNF) expression were analyzed. Cells were classified based on the type of fiber orientation and the alignment of these fibers in relation to the electric field. Nuclear deformation was mainly influenced by fiber orientation rather than the electrical field. Similarly, fiber orientation also induced BDNF expression. Although electrical field alone had no significant effect on BDNF expression, combining fiber orientation with electrical field resulted in BDNF expression in cells that grew on electrospun fibers that were aligned perpendicular to the electrical field.

Neuroprotective Effects of Carnosic Acid: Insight into Its Mechanisms of Action.

Carnosic acid is a diterpenoid abundantly present in plants belonging to the genus Rosmarinus and Salvia of the family Lamiaceae, accounting for their application in traditional medicine. The diverse biological properties of carnosic acid that include antioxidant, anti-inflammatory, and anticarcinogenic activities have instigated studies on its mechanistic role, providing further insights into its potential as a therapeutic agent. Accumulating evidence has established the relevance of carnosic acid as a neuroprotective agent exhibiting therapeutic efficacy in combatting neuronal-injury-induced disorders. The physiological importance of carnosic acid in the mitigation of neurodegenerative disorders is just beginning to be understood. This review summarizes the current data on the mode of action through which carnosic acid exerts its neuroprotective role that may serve to strategize novel therapeutic approaches for these debilitating neurodegenerative disorders.

Author Correction: Selective, high-contrast detection of syngeneic glioblastoma in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Selective, high-contrast detection of syngeneic glioblastoma in vivo.

Glioblastoma is a highly malignant, largely therapy-resistant brain tumour. Deep infiltration of brain tissue by neoplastic cells represents the key problem of diffuse glioma. Much current research focuses on the molecular makeup of the visible tumour mass rather than the cellular interactions in the surrounding brain tissue infiltrated by the invasive glioma cells that cause the tumour's ultimately lethal outcome. Diagnostic neuroimaging that enables the direct in vivo observation of the tumour infiltration zone and the local host tissue responses at a preclinical stage are important for the development of more effective glioma treatments. Here, we report an animal model that allows high-contrast imaging of wild-type glioma cells by positron emission tomography (PET) using [18 F]PBR111, a selective radioligand for the mitochondrial 18 kDa Translocator Protein (TSPO), in the Tspo-/- mouse strain (C57BL/6-Tspotm1GuMu(GuwiyangWurra)). The high selectivity of [18 F]PBR111 for the TSPO combined with the exclusive expression of TSPO in glioma cells infiltrating into null-background host tissue free of any TSPO expression, makes it possible, for the first time, to unequivocally and with uniquely high biological contrast identify peri-tumoral glioma cell invasion at preclinical stages in vivo. Comparison of the in vivo imaging signal from wild-type glioma cells in a null background with the signal in a wild-type host tissue, where the tumour induces the expected TSPO expression in the host's glial cells, illustrates the substantial extent of the peritumoral host response to the growing tumour. The syngeneic tumour (TSPO+/+) in null background (TSPO-/-) model is thus well suited to study the interaction of the tumour front with the peri-tumoral tissue, and the experimental evaluation of new therapeutic approaches targeting the invasive behaviour of glioblastoma.

Identification of molecular signatures and pathways to identify novel therapeutic targets in Alzheimer's disease: Insights from a systems biomedicine perspective.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. However, there are no peripheral biomarkers available that can detect AD onset. This study aimed to identify the molecular signatures in AD through an integrative analysis of blood gene expression data. We used two microarray datasets (GSE4226 and GSE4229) comparing peripheral blood transcriptomes of AD patients and controls to identify differentially expressed genes (DEGs). Gene set and protein overrepresentation analysis, protein-protein interaction (PPI), DEGs-Transcription Factors (TFs) interactions, DEGs-microRNAs (miRNAs) interactions, protein-drug interactions, and protein subcellular localizations analyses were performed on DEGs common to the datasets. We identified 25 common DEGs between the two datasets. Integration of genome scale transcriptome datasets with biomolecular networks revealed hub genes (NOL6, ATF3, TUBB, UQCRC1, CASP2, SND1, VCAM1, BTF3, VPS37B), common transcription factors (FOXC1, GATA2, NFIC, PPARG, USF2, YY1) and miRNAs (mir-20a-5p, mir-93-5p, mir-16-5p, let-7b-5p, mir-708-5p, mir-24-3p, mir-26b-5p, mir-17-5p, mir-193-3p, mir-186-5p). Evaluation of histone modifications revealed that hub genes possess several histone modification sites associated with AD. Protein-drug interactions revealed 10 compounds that affect the identified AD candidate biomolecules, including anti-neoplastic agents (Vinorelbine, Vincristine, Vinblastine, Epothilone D, Epothilone B, CYT997, and ZEN-012), a dermatological (Podofilox) and an immunosuppressive agent (Colchicine). The subcellular localization of molecular signatures varied, including nuclear, plasma membrane and cytosolic proteins. In the present study, it was identified blood-cell derived molecular signatures that might be useful as candidate peripheral biomarkers in AD. It was also identified potential drugs and epigenetic data associated with these molecules that may be useful in designing therapeutic approaches to ameliorate AD.

Calcium-axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes.

We have used cell culture of astrocytes aligned within microchannels to investigate calcium effects on primary cilia morphology. In the absence of calcium and in the presence of flow of media (10 µL.s-1) the majority (90%) of primary cilia showed reversible bending with an average curvature of 2.1 ± 0.9 × 10-4 nm-1. When 1.0 mM calcium was present, 90% of cilia underwent bending. Forty percent of these cilia demonstrated strong irreversible bending, resulting in a final average curvature of 3.9 ± 1 × 10-4 nm-1, while 50% of cilia underwent bending similar to that observed during calcium-free flow. The average length of cilia was shifted toward shorter values (3.67 ± 0.34 µm) when exposed to excess calcium (1.0 mM), compared to media devoid of calcium (3.96 ± 0.26 µm). The number of primary cilia that became curved after calcium application was reduced when the cell culture was pre-incubated with 15 µM of the microtubule stabilizer, taxol, for 60 min prior to calcium application. Calcium caused single microtubules to curve at a concentration ≈1.0 mM in vitro, but at higher concentration (≈1.5 mM) multiple microtubule curving occurred. Additionally, calcium causes microtubule-associated protein-2 conformational changes and its dislocation from the microtubule wall at the location of microtubule curvature. A very small amount of calcium, that is 1.45 × 1011 times lower than the maximal capacity of TRPPs calcium channels, may cause gross morphological changes (curving) of primary cilia, while global cytosol calcium levels are expected to remain unchanged. These findings reflect the non-linear manner in which primary cilia may respond to calcium signaling, which in turn may influence the course of development of ciliopathies and cancer.

Linagliptin, a Dipeptidyl Peptidase-4 Inhibitor, Mitigates Cognitive Deficits and Pathology in the 3xTg-AD Mouse Model of Alzheimer's Disease.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone shown to be active in the treatment of type-2 diabetes (T2D) and has also been shown as efficacious in Alzheimer's disease (AD). Dipeptidyl peptidase-4 (DPP-4), an enzyme that is expressed in numerous cells, rapidly inactivates endogenous GLP-1. Therefore, DPP-4 inhibition is employed as a therapeutic avenue to increase GLP-1 levels in the management of T2D. The effectiveness of DPP-4 inhibitors in the treatment of AD has been reported in various animal models of AD. With this background, the present study was designed to examine the effectiveness of linagliptin, a DPP-4 inhibitor in the 3xTg-AD mouse model of Alzheimer's disease. Nine-month-old 3xTg-AD mice were administered linagliptin orally (5, 10, and 20 mg/kg) for 8 weeks. At the end of the linagliptin treatment, mice were evaluated for cognitive ability on the Morris Water Maze and Y-maze. Following cognitive evaluation, mice were sacrificed to determine the effect of the linagliptin on brain incretin levels, amyloid burden, tau phosphorylation, and neuroinflammation. We confirm that linagliptin treatment for 8 weeks mitigates the cognitive deficits present in 3xTg-AD mice. Moreover, linagliptin also improves brain incretin levels and attenuates amyloid beta, tau phosphorylation as well as neuroinflammation. In conclusion, linagliptin possesses neuroprotective properties that may be attributed to the improvement of incretin levels in the brain.

Saxagliptin: a dipeptidyl peptidase-4 inhibitor ameliorates streptozotocin induced Alzheimer's disease.

Type 2 diabetes (T2D) is one of the major risk factors associated with Alzheimer's disease (AD). Recent studies have found similarities in molecular mechanisms that underlie the respective degenerative developments in the two diseases. Pharmacological agents, such as dipeptidyl peptidase-4 (DPP-4) inhibitors, which increase the level of glucagon-like peptide-1 (GLP-1) and ameliorate T2D, have become valuable candidates as disease modifying agents in the treatment of AD. In addition, endogenous GLP-1 levels decrease amyloid beta (Aß) peptide and tau phosphorylation in AD. The present study examines the efficacy of Saxagliptin, a DPP-4 inhibitor in a streptozotocin (STZ) induced rat model of AD. Three months following induction of AD by intracerebral administration of streptozotocin, animals were orally administered Saxagliptin (0.25, 0.5 and 1 mg/kg) for 60 days. The effect of the DPP-4 inhibitor on hippocampal GLP-1 levels, Aß burden, tau phosphorylation, inflammatory markers and memory retention were evaluated. The results reveal an attenuation of Aß, tau phosphorylation and inflammatory markers and an improvement in hippocampal GLP-1 and memory retention following treatment. This remarkable therapeutic effect of Saxagliptin mediated through DPP-4 inhibition demonstrates a unique mechanism for Aß and tau clearance by increasing GLP-1 levels and reverses the behavioural deficits and pathology observed in AD.

Degradation of the Alzheimer disease amyloid beta-peptide by metal-dependent up-regulation of metalloprotease activity.

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid beta-peptide (Abeta) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu(2+) or Zn(2+) resulted in an approximately 85-90% reduction of secreted Abeta-(1-40) and Abeta-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Abeta was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu(2+). MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu(2+) also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Abeta-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of Abeta deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients.

Heparin activates beta-secretase (BACE1) of Alzheimer's disease and increases autocatalysis of the enzyme.

BACE1 is an aspartic protease that generates the N-terminus of the beta-amyloid protein (Alphabeta) from the beta-amyloid precursor protein (APP). BACE1 is a key target for Alzheimer drug development. However, little is known about the physiological regulation of the enzyme. Heparin can promote beta-secretase cleavage of APP in neuroblastoma cells. However, heparin has also been reported to directly inhibit BACE1 activity in vitro. To clarify the role of heparin in regulating BACE1, we examined the effect of heparin on the activity of recombinant human BACE1 (rBACE1) in vitro. Low concentrations (1 microg/mL) of heparin were found to stimulate rBACE1, increasing enzyme V(max) and decreasing the K(M). In contrast, higher concentrations of heparin (10 or 100 microg/mL) were inhibitory. Heparin affinity chromatography demonstrated that heparin interacted strongly with the zymogen form of rBACE1 and bound to a peptide homologous to the N-terminal pro sequence of BACE1. Mature (pro sequence cleaved) enzyme lacked the capacity to be stimulated by heparin, indicating that the pro domain was necessary for the stimulation by heparin. Furthermore, in the presence of stimulatory concentrations of heparin, there was an increase in autocatalytic cleavage of the protease domain and a subsequent loss of enzyme activity in vitro. Our results strongly suggest that heparin stimulates the partially active BACE1 zymogen, and we propose that the activation is mediated by high-affinity binding of heparin to the pro domain. Our study provides evidence that heparan sulfate proteoglycans could regulate the rate of Alphabeta production in vivo.

Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease.

Beta-secretase cleavage represents the first step in the generation of Abeta polypeptides and initiates the amyloid cascade that leads to neurodegeneration in Alzheimer's disease. By comparative Western blot analysis, we show a 2.7-fold increase in protein expression of the beta-secretase enzyme BACE in the brain cortex of Alzheimer's disease patients as compared to age-matched controls. Similarly, we found the levels of the amyloid precursor protein C-terminal fragment produced by beta-secretase to be increased by nearly twofold in Alzheimer's disease cortex.