Preparation of Hydrogels Based on Modified Pectins by Tuning Their Properties for Anti-Glioma Therapy.

The extracellular matrix (ECM) of the central nervous system (CNS), characterized by low stiffness and predominance of carbohydrates on protein components, mediates limited cell proliferation and migration. Pectins are polysaccharides derived from plants and could be very promising for a tunable hydrogel design that mimics the neural ECM. Aiming to regulate gel structure and viscoelastic properties, we elaborated 10 variants of pectin-based hydrogels via tuning the concentration of the polymer and the number of free carboxyl groups expressed in the degree of esterification (DE). Viscoelastic properties of hydrogels varied in the range of 3 to 900 Pa for G' and were chosen as the first criteria for the selection of variants suitable for CNS remodeling. For extended reciprocal characterization, two pairs of hydrogels were taken to test pectins with opposite DEs close to 0% and 50%, respectively, but with a similar rheology exceeding 100 Pa (G'), which was achieved by adjusting the concentration of pectin. Hydrogel swelling properties and in vitro stability, together with structure characterization using SEM and FTIR spectroscopy, displayed some differences that may sense for biomedical application. Bioassays on C6 and U87MG glioblastoma cultures testified the potential prospects of the anti-glioma activity of hydrogels developed by decreasing cell proliferation and modulating migration but supporting the high viability of neural cells.

Gap junctions and hemichannels: communicating cell death in neurodevelopment and disease.

Gap junctions are unique membrane channels that play a significant role in intercellular communication in the developing and mature central nervous system (CNS). These channels are composed of connexin proteins that oligomerize into hexamers to form connexons or hemichannels. Many different connexins are expressed in the CNS, with some specificity with regard to the cell types in which distinct connexins are found, as well as the timepoints when they are expressed in the developing and mature CNS. Both the main neuronal Cx36 and glial Cx43 play critical roles in neurodevelopment. These connexins also mediate distinct aspects of the CNS response to pathological conditions. An imbalance in the expression, translation, trafficking and turnover of connexins, as well as mutations of connexins, can impact their function in the context of cell death in neurodevelopment and disease. With the ever-increasing understanding of connexins in the brain, therapeutic strategies could be developed to target these membrane channels in various neurological disorders.

Neuronal gap junction coupling as the primary determinant of the extent of glutamate-mediated excitotoxicity.

In the mammalian central nervous system (CNS), coupling of neurons by gap junctions (electrical synapses) increases during early postnatal development, then decreases, but increases in the mature CNS following neuronal injury, such as ischemia, traumatic brain injury and epilepsy. Glutamate-dependent neuronal death also occurs in the CNS during development and neuronal injury, i.e., at the time when neuronal gap junction coupling is increased. Here, we review our recent studies on regulation of neuronal gap junction coupling by glutamate in developing and injured neurons and on the role of gap junctions in neuronal cell death. A modified model of the mechanisms of glutamate-dependent neuronal death is discussed, which includes neuronal gap junction coupling as a critical part of these mechanisms.

Rational Design of Pectin-Chitosan Polyelectrolyte Nanoparticles for Enhanced Temozolomide Delivery in Brain Tumor Therapy.

Conventional chemotherapeutic approaches currently used for brain tumor treatment have low efficiency in targeted drug delivery and often have non-target toxicity. Development of stable and effective drug delivery vehicles for the most incurable diseases is one of the urgent biomedical challenges. We have developed polymer nanoparticles (NPs) with improved temozolomide (TMZ) delivery for promising brain tumor therapy, performing a rational design of polyelectrolyte complexes of oppositely charged polysaccharides of cationic chitosan and anionic pectin. The NPs' diameter (30 to 330 nm) and zeta-potential (-29 to 73 mV) varied according to the initial mass ratios of the biopolymers. The evaluation of nanomechanical parameters of native NPs demonstrated changes in Young's modulus from 58 to 234 kPa and adhesion from -0.3 to -3.57 pN. Possible mechanisms of NPs' formation preliminary based on ionic interactions between ionogenic functional groups were proposed by IR spectroscopy and dynamic rheology. The study of the parameters and kinetics of TMZ sorption made it possible to identify compounds that most effectively immobilize and release the active substance in model liquids that simulate the internal environment of the body. A polyelectrolyte carrier based on an equal ratio of pectin-chitosan (0.1% by weight) was selected as the most effective for the delivery of TMZ among a series of obtained NPs, which indicates a promising approach to the treatment of brain tumors.

Neuronal gap junction coupling is regulated by glutamate and plays critical role in cell death during neuronal injury.

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI), and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. We report here that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluRs). Specifically, using electrotonic coupling, Western blots, and siRNA in the mouse somatosensory cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36) (neuronal gap junction protein), and inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. We also show that the regulation is via cAMP/PKA (cAMP-dependent protein kinase)-dependent signaling and posttranscriptional control of Cx36 expression and that other glutamate receptors are not involved in these regulatory mechanisms. Furthermore, using the analysis of neuronal death, we show that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemia-mediated neuronal death in vitro and in vivo. Similar results are obtained using in vitro models of TBI and epilepsy. Our results indicate that neuronal gap junction coupling is a critical component of glutamate-dependent neuronal death. They also suggest that causal link among group II mGluR function, neuronal gap junction coupling, and neuronal death has a universal character and operates in different types of neuronal injuries.

How to Develop Drug Delivery System Based on Carbohydrate Nanoparticles Targeted to Brain Tumors.

Brain tumors are the most difficult to treat, not only because of the variety of their forms and the small number of effective chemotherapeutic agents capable of suppressing tumor cells, but also limited by poor drug transport across the blood-brain barrier (BBB). Nanoparticles are promising drug delivery solutions promoted by the expansion of nanotechnology, emerging in the creation and practical use of materials in the range from 1 to 500 nm. Carbohydrate-based nanoparticles is a unique platform for active molecular transport and targeted drug delivery, providing biocompatibility, biodegradability, and a reduction in toxic side effects. However, the design and fabrication of biopolymer colloidal nanomaterials have been and remain highly challenging to date. Our review is devoted to the description of carbohydrate nanoparticle synthesis and modification, with a brief overview of the biological and promising clinical outcomes. We also expect this manuscript to highlight the great potential of carbohydrate nanocarriers for drug delivery and targeted treatment of gliomas of various grades and glioblastomas, as the most aggressive of brain tumors.

Preparation and Characterization of Hydrogel Films and Nanoparticles Based on Low-Esterified Pectin for Anticancer Applications.

Prospective adjuvant anticancer therapy development includes the establishing of drug delivery systems based on biocompatible and biodegradable carriers. We have designed films and nanoparticles (NPs) based on low-esterified pectin hydrogel using the ionic gelation method. We investigated morphology, nanomechanical properties, biocompatibility and anticancer activity. Hydrogel films are characterized by tunable viscoelastic properties and surface nanoarchitectonics through pectin concentration and esterification degree (DE), expressed in variable pore frequency and diameter. An in vitro study showed a significant reduction in metabolic activity and the proliferation of the U87MG human glioblastoma cell line, probably affected via the adhesion mechanism. Glioma cells formed neurosphere-like conglomerates with a small number of neurites when cultured on fully de-esterified pectin films and they did not produce neurites on the films prepared on 50% esterified pectin. Pectin NPs were examined in terms of size distribution and nanomechanical properties. The NPs' shapes were proved spherical with a mean diameter varying in the range of 90-115 nm, and a negative zeta potential from -8.30 to -7.86 mV, which indicated their stability. The NPs did not demonstrate toxic effect on cells or metabolism inhibition, indicating good biocompatibility. Nanostructured biomaterials prepared on low-esterified pectins could be of interest for biomedical applications in adjuvant anticancer therapy and for designing drug delivery systems.

Interplay of chemical neurotransmitters regulates developmental increase in electrical synapses.

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development. We report here that the developmental increase in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) are regulated by an interplay between the activity of group II metabotropic glutamate receptors (mGluRs) and GABA(A) receptors. Specifically, using dye coupling, electrotonic coupling, Western blots and small interfering RNA in the rat and mouse hypothalamus and cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs augments, and inactivation prevents, the developmental increase in neuronal gap junction coupling and Cx36 expression. However, changes in GABA(A) receptor activity have the opposite effects. The regulation by group II mGluRs is via cAMP/PKA-dependent signaling, and regulation by GABA(A) receptors is via Ca(2+)/PKC-dependent signaling. Furthermore, the receptor-mediated upregulation of Cx36 requires a neuron-restrictive silencer element in the Cx36 gene promoter, and the downregulation involves the 3'-untranslated region of the Cx36 mRNA, as shown using reverse-transcription quantitative real-time PCR and luciferase reporter activity analysis. In addition, the methyl thiazolyl tetrazolium analysis indicates that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons. Together, the results suggest a multitiered strategy for chemical synapses in developmental regulation of electrical synapses.

Novel assay to measure chromosome instability identifies Punica granatum extract that elevates CIN and has a potential for tumor- suppressing therapies.

Human artificial chromosomes (HACs) have provided a useful tool to study kinetochore structure and function, gene delivery, and gene expression. The HAC propagates and segregates properly in the cells. Recently, we have developed an experimental high-throughput imaging (HTI) HAC-based assay that allows the identification of genes whose depletion leads to chromosome instability (CIN). The HAC carries a GFP transgene that facilitates quantitative measurement of CIN. The loss of HAC/GFP may be measured by flow cytometry or fluorescence scanning microscope. Therefore, CIN rate can be measured by counting the proportion of fluorescent cells. Here, the HAC/GFP-based assay has been adapted to screen anticancer compounds for possible induction or elevation of CIN. We analyzed 24 cytotoxic plant extracts. Punica granatum leaf extract (PLE) indeed sharply increases CIN rate in HT1080 fibrosarcoma cells. PLE treatment leads to cell cycle arrest, reduction of mitotic index, and the increased numbers of micronuclei (MNi) and nucleoplasmic bridges (NPBs). PLE-mediated increased CIN correlates with the induction of double-stranded breaks (DSBs). We infer that the PLE extract contains a component(s) that elevate CIN, making it a candidate for further study as a potential cancer treatment. The data also provide a proof of principle for the utility of the HAC/GFP-based system in screening for natural products and other compounds that elevate CIN in cancer cells.

Cell and Tissue Nanomechanics: From Early Development to Carcinogenesis.

Cell and tissue nanomechanics, being inspired by progress in high-resolution physical mapping, has recently burst into biomedical research, discovering not only new characteristics of normal and diseased tissues, but also unveiling previously unknown mechanisms of pathological processes. Some parallels can be drawn between early development and carcinogenesis. Early embryogenesis, up to the blastocyst stage, requires a soft microenvironment and internal mechanical signals induced by the contractility of the cortical actomyosin cytoskeleton, stimulating quick cell divisions. During further development from the blastocyst implantation to placenta formation, decidua stiffness is increased ten-fold when compared to non-pregnant endometrium. Organogenesis is mediated by mechanosignaling inspired by intercellular junction formation with the involvement of mechanotransduction from the extracellular matrix (ECM). Carcinogenesis dramatically changes the mechanical properties of cells and their microenvironment, generally reproducing the structural properties and molecular organization of embryonic tissues, but with a higher stiffness of the ECM and higher cellular softness and fluidity. These changes are associated with the complete rearrangement of the entire tissue skeleton involving the ECM, cytoskeleton, and the nuclear scaffold, all integrated with each other in a joint network. The important changes occur in the cancer stem-cell niche responsible for tumor promotion and metastatic growth. We expect that the promising concept based on the natural selection of cancer cells fixing the most invasive phenotypes and genotypes by reciprocal regulation through ECM-mediated nanomechanical feedback loop can be exploited to create new therapeutic strategies for cancer treatment.

Transgenic expression of Glud1 (glutamate dehydrogenase 1) in neurons: in vivo model of enhanced glutamate release, altered synaptic plasticity, and selective neuronal vulnerability.

The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDAR-mediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

In vitro antitumor and immunotropic activity of carrageenans from red algae Chondrus armatus and their low-molecular weight degradation products.

Antitumor and immunotropic effects of κ-, λ-carrageenan from red marine algae Chondrus armatus and their low-molecular weight (LMW) degradation products were explored. Effects on human esophageal cancer cell lines KYSE30 and FLO1 viability and ability to induce production of pro- and anti-inflammatory cytokines by human monocytes was assessed. All polysaccharides demonstrated antimetabolic and cytostatic activity towards cancer lines, with high-molecular weight carrageenans possessing higher antimetabolic and lower cytostatic activity than their LMW degradation products. All carrageenans induced monocytes to produce pro-inflammatory cytokines IL1ß, IL6, IL18, and TNFα. However, secretion of anti-inflammatory cytokine IL10 was induced only by LMW λ-carrageenan, which exhibited the highest cytokine production inducing efficacy overall. We demonstrate that LMW carrageenan degradation products not only retain biological activity of their precursors, but also increase their efficacy in type-dependent manner, allowing for their future development for pharmacological practice.

Hydrogels based on modified pectins capable of modulating neural cell behavior as prospective biomaterials in glioblastoma treatment.

Glioblastoma is the most common malignant tumor of the brain, but its treatment outcomes can be improved by new therapeutic techniques using biocompatible materials. Utilizing controllable alkaline de-esterification we obtained pectin preparation with 27.4% esterification degree and used it for bio-artificial matrix production. We discovered optimal gelation conditions in the presence of Ca2+ by the analysis of visco-elastic properties of the gels and produced a series of biomaterials in hydrogel forms. Hydrogels based on low-esterified pectin significantly slow down the metabolism of C6 glioma cells and neural stem cells (NSCs) and slightly decrease the viability of the C6 glioma, but not of NSCs. This happens due to a decrease in cell proliferation rate, while apoptosis degrees remain stable or negligibly decrease. We created a set of pectin hydrogels supplemented with different ratios of two ECM proteins-collagens I and IV. We have shown that the formation of cell processes in glioma C6 can be regulated by varying the ratio of two ECM proteins in gels used for 3D cell cultivation. Thus, composite matrix materials obtained can be used for modeling brain tumor invasion. The results presented suggest that modified pectins supplemented with two collagen types may serve as prospective biomaterials for glioblastoma treatment due to their ability to regulate glioma cell dynamics.

NMDA receptors regulate developmental gap junction uncoupling via CREB signaling.

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.

The Extracellular Matrix and Biocompatible Materials in Glioblastoma Treatment.

During cancer genesis, the extracellular matrix (ECM) in the human brain undergoes important transformations, starting to resemble embryonic brain cell milieu with a much denser structure. However, the stiffness of the tumor ECM does not preclude cancer cells from migration. The importance of the ECM role in normal brain tissue as well as in tumor homeostasis has engaged much effort in trials to implement ECM as a target and an instrument in the treatment of brain cancers. This review provides a detailed analysis of both experimental and applied approaches in combined therapy for gliomas in adults. In general, matrix materials for glioma treatment should have properties facilitating the simplest delivery into the body. Hence, to deliver an artificial implant directly into the operation cavity it should be packed into a gel form, while for bloodstream injections matrix needs to be in the form of polymer micelles, nanoparticles, etc. Furthermore, the delivered material should mimic biomechanical properties of the native tissue, support vital functions, and slow down or stop the proliferation of surrounding cells for a prolonged period. The authors propose a two-step approach aimed, on the one hand, at elimination of remaining cancer cells and on the other hand, at restoring normal brain tissue. Thereby, the first bioartificial matrix to be applied should have relatively low elastic modulus should be loaded with anticancer drugs, while the second material with a higher elastic modulus for neurite outgrowth support should contain specific factors stimulating neuroregeneration.

Use of calcium imaging for analysis of neuronal gap junction coupling.

We recently used Western blots for connexin 36 and neuronal dye coupling with neurobiotin to measure developmental decrease in neuronal gap junction coupling in cell cultures. To ask whether Ca2+ imaging also can be used to measure changes in the amount of neuronal gap junction coupling, we defined a Ca2+ coupling coefficient as the percentage of neurons with bicuculline-induced increases in intracellular Ca2+ that are suppressed by blocking gap junctions. We demonstrate in rat and mouse hypothalamic neuronal cultures that the Ca2+ coupling coefficient decreases during culture development, this decrease is prevented by manipulations that also prevent developmental decrease in neuronal gap junction coupling, and the coefficient is low in cultures lacking connexin 36. The results indicate that Ca2+ imaging is a useful tool to quantify the amount of neuronal gap junction coupling in cultures.

A potential role for neuronal connexin 36 in the pathogenesis of amyotrophic lateral sclerosis.

Neuronal gap junctional protein connexin 36 (Cx36) contributes to neuronal death following a range of acute brain insults such as ischemia, traumatic brain injury and epilepsy. Whether Cx36 contributes to neuronal death and pathological outcomes in chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), is not known. We show here that the expression of Cx36 is significantly decreased in lumbar segments of the spinal cord of both human ALS subjects and SOD1G93A mice as compared to healthy human and wild-type mouse controls, respectively. In purified neuronal cultures prepared from the spinal cord of wild-type mice, knockdown of Cx36 reduces neuronal death caused by overexpression of the mutant human SOD1-G93A protein. Taken together, these data suggest a possible contribution of Cx36 to ALS pathogenesis. A perspective for the use of blockers of Cx36 gap junction channels for ALS therapy is discussed.

Homeostatic plasticity: comparing and contrasting cortical and hippocampal studies. A review.

Homeostatic plasticity is an important physiological process in the mammalian nervous system. In this review, we discuss methodological and mechanistic similarities and differences in cortical and hippocampal studies of homeostatic plasticity. Although there are many similarities, there are also region-specific differences in the effects and/or mechanisms that regulate homeostatic plasticity in these two regions. In this review, we propose a new experimental paradigm to study homeostatic plasticity that may address some unanswered questions in the field.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.