Functionalized Nanocellulose Drives Neural Stem Cells toward Neuronal Differentiation.

Transplantation of differentiated and fully functional neurons may be a better therapeutic option for the cure of neurodegenerative disorders and brain injuries than direct grafting of neural stem cells (NSCs) that are potentially tumorigenic. However, the differentiation of NSCs into a large population of neurons has been a challenge. Nanomaterials have been widely used as substrates to manipulate cell behavior due to their nano-size, excellent physicochemical properties, ease of synthesis, and versatility in surface functionalization. Nanomaterial-based scaffolds and synthetic polymers have been fabricated with topology resembling the micro-environment of the extracellular matrix. Nanocellulose materials are gaining attention because of their availability, biocompatibility, biodegradability and bioactivity, and affordable cost. We evaluated the role of nanocellulose with different linkage and surface features in promoting neuronal differentiation. Nanocellulose coupled with lysine molecules (CNC-Lys) provided positive charges that helped the cells to attach. Embryonic rat NSCs were differentiated on the CNC-Lys surface for up to three weeks. By the end of the three weeks of in vitro culture, 87% of the cells had attached to the CNC-Lys surface and more than half of the NSCs had differentiated into functional neurons, expressing endogenous glutamate, generating electrical activity and action potentials recorded by the multi-electrode array.

Predominant differentiation of rat fetal neural stem cells into functional oligodendrocytes in vitro.

Oligodendrocytes form myelin in the CNS. A fast method to produce large quantity of oligodendrocytes that have the capacity of myelinating CNS neurons would be very useful for treating CNS injuries or demyelinating diseases, or for research purposes. We developed a simple standard protocol for predominant differentiation of rat fetal neural stem cells (NSCs) into oligodendrocytes. We adopted a new method to purify the oligodendrocytes and co-cultured the newly differentiated oligodendrocytes with hippocampal neurons to confirm their myelination capability. NSCs from embryonic day 14 (E14) were propagated at the presence of basic fibroblast growth factor and platelet derived growth factor alpha, and then differentiated in the medium containing triiodothyronine. Four extracellular matrix (ECM), poly-d-lysine (PDL), PDL-laminin, fibronectin, and matrigel, were examined for NSC differentiation. About 90 % of NSCs differentiated into oligodendrocytes on matrigel compared to 32 % on PDL or PDL-laminin, and 26 % on fibronectin after 3 weeks of differentiation, demonstrating the significant influence of ECM. Further, newly differentiated oligodendrocytes were co-cultured with hippocampal neurons from E18 rat embryos resulting in robust myelination of neurites at three weeks. In summary, we present a simplified and efficient method to predominantly generate oligodendrocytes from NSCs that is potentially very useful for CNS demyelination diseases.

Glioma-derived exosomes drive the differentiation of neural stem cells to astrocytes.

Exosomes appear to be effective inter-cellular communicators delivering several types of molecules, such as proteins and RNAs, suggesting that they could influence neural stem cell (NSC) differentiation. Our RNA sequencing studies demonstrated that the RNAs related to cell proliferation and astrocyte differentiation were upregulated in human mesenchymal stem cells (hMSC) when co-cultured with exosomes obtained from the culture medium of human glioma cells (U87). Metallothionein 3 and elastin genes, which are related to cell proliferation, increased 10 and 7.2 fold, respectively. Expression of genes for astrocyte differentiation, such as tumor growth factor alpha, induced protein 3 of the NOTCH1 family, colony stimulating factor and interleukin 6 of the STAT3 family and Hes family bHLH transcription factor 1 also increased by 2.3, 10, 4.7 and 2.9 fold, respectively. We further examined the effects of these exosomes on rat fetal neural stem cell (rNSC) differentiation using the secreted exosomes from U87 glioma cells or exosomes from U87 cells that were stimulated with interleukin 1ß (IL-1ß). The rNSCs, extracted from rat brains at embryonic day 14 (E14), underwent a culture protocol that normally leads to predominant (~90%) differentiation to ODCs. However, in the presence of the exosomes from untreated or IL-1ß-treated U87 cells, significantly more cells differentiated into astrocytes, especially in the presence of exosomes obtained from the IL-1ß-challenged glioma cells. Moreover, glioma-derived exosomes appeared to inhibit rNSC differentiation into ODCs or astrocytes as indicated by a significantly increased population of unlabeled cells. A portion of the resulting astrocytes co-expressed both CD133 and glial fibrillary acidic protein (GFAP) suggesting that exosomes from U87 cells could promote astrocytic differentiation of NSCs with features expected from a transformed cell. Our data clearly demonstrated that exosomes secreted by human glioma cells provide a strong driving force for rat neural stem cells to differentiate into astrocytes, uncovering potential pathways and therapeutic targets that might control this aggressive tumor type.

Plasmonic nano surface for neuronal differentiation and manipulation.

Neurodegenerative diseases and traumatic brain injuries can destroy neurons, resulting in sensory and motor function loss. Transplantation of differentiated neurons from stem cells could help restore such lost functions. Plasmonic gold nanorods (AuNR) were integrated in growth surfaces to stimulate and modulate neural cells in order to tune cell physiology. An AuNR nanocomposite system was fabricated, characterized, and then utilized to study the differentiation of embryonic rat neural stem cells (NSCs). Results demonstrated that this plasmonic surface 1) accelerated differentiation, yielding almost twice as many differentiated neural cells as a traditional NSC culture surface coated with poly-D-lysine and laminin for the same time period; and 2) promoted differentiation of NSCs into neurons and astrocytes in a 2:1 ratio, as evidenced by the expression of relevant marker proteins. These results indicate that the design and properties of this AuNR plasmonic surface would be advantageous for tissue engineering to address neural degeneration.

Nicotine is neuroprotective to neonatal neurons of sympathetic ganglion in rat.

Sympathetic neurons of SCG are dependent on availability of nerve growth factor (NGF) for their survival. SCG neurons express nicotinic receptors (nAChR) whose expression levels are modulated by nicotine. Nicotine exerts multiple effects on neurons, including neuroprotection, through nAChR binding. Although sympathetic neurons express robust levels of nAChR, a possible neuroprotective role for nicotine in these neurons is not well-understood. Therefore we determined the effect of nicotine exposure on survival of SCG neurons during NGF withdrawal in a well-established cell culture system. NGF was withdrawn in rat neonatal SCG neuron cultures which were then treated with either 10 µM nicotine alone or with nAChR antagonists 0.1 µM α-bungarotoxin (antagonist for α7 subunit bearing nAChR) and 10 µM mecamylamine (non-specific antagonist for ganglionic nAChR) for 48 h. Apoptotic death was determined by TUNEL staining. Cell survival was also determined by MTS assay. Western blot analysis of ERK1/2 was also performed. Our results showed that exposure to 10 µM nicotine significantly reduced apoptotic cell death in SCG neurons resulting from NGF withdrawal as shown by fewer TUNEL positive cells. The MTS assay results also revealed that 10 µM nicotine concentration significantly increased cell survival thus indicating neuroprotective effect of nicotine against cell death resulting from NGF withdrawal. Nicotinic receptor antagonists (bungarotoxin & mecamylamine) attenuated the effect of nicotine's action of neuroprotection. Western blot analysis showed an increased expression of ERK1/2 in nicotine treated cultures suggesting nicotine provided neuroprotection in SCG neurons by increasing the expression of ERK1/2 through nicotinic receptor dependent mechanisms.

Neuroprotective effects of physical activity on the brain: a closer look at trophic factor signaling.

While the relationship between increased physical activity and cognitive ability has been conjectured for centuries, only recently have the mechanisms underlying this relationship began to emerge. Convergent evidence suggests that physical activity offers an affordable and effective method to improve cognitive function in all ages, particularly the elderly who are most vulnerable to neurodegenerative disorders. In addition to improving cardiac and immune function, physical activity alters trophic factor signaling and, in turn, neuronal function and structure in areas critical for cognition. Sustained exercise plays a role in modulating anti-inflammatory effects and may play a role in preserving cognitive function in aging and neuropathological conditions. Moreover, recent evidence suggests that myokines released by exercising muscles affect the expression of brain-derived neurotrophic factor synthesis in the dentate gyrus of the hippocampus, a finding that could lead to the identification of new and therapeutically important mediating factors. Given the growing number of individuals with cognitive impairments worldwide, a better understanding of how these factors contribute to cognition is imperative, and constitutes an important first step toward developing non-pharmacological therapeutic strategies to improve cognition in vulnerable populations.

Volunteerism is Key to Offering Successful Neuroscience Outreach with Limited Resources.

As sponsors of a university Society for Neuroscience (SfN) organization, we and our student members are committed to neuroscience outreach but with limited resources, it is not feasible for us to host a week-long program during National Brain Awareness Week (BAW). Hence, we decided on a half-day program wherein attendees are provided with information about the workings of the nervous system and current research in the field in a fun and interactive environment. Our volunteers - mostly undergraduate students - select hands-on activities, gather required materials, and actively engage participants of all ages. We coined the event Brain Awareness Day (BAD) and organize the annual program on a budget between $100-$300.

Copper nanoparticles exert size and concentration dependent toxicity on somatosensory neurons of rat.

Metal nanoparticles, due to their unique properties and important applications in optical, magnetic, thermal, electrical, sensor devices and cosmetics, are beginning to be widely manufactured and used. This new and rapidly growing field of technology warrants a thorough examination of the material's bio-compatibility and safety. Ultra-small particles may adversely affect living cells and organisms since they can easily penetrate the body through skin contact, inhalation and ingestion. Retrograde transport of copper nanoparticles from nerve endings on the skin can reach the somatosensory neurons in dorsal root ganglion (DRG). Since copper nanoparticles have industrial and healthcare applications, we determined the concentration and size-dependant effects of their exposure on survival of DRG neurons of rat in cell culture. The neurons were exposed to copper nanoparticles of increasing concentrations (10-100 muM) and sizes (40, 60 and 80 nm) for 24 h. Light microscopy, histochemical staining for copper, lactate dehydrogenase (LDH) assay for cell death, and MTS [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay for cell viability were performed to measure the resultant toxicity and cell survival. DRG neurons exposed to copper nanoparticles displayed vacuoles and detachment of some neurons from the substratum. Neurons also exhibited disrupted neurite network. LDH and MTS assays revealed that exposure to copper nanoparticles had significant toxic effect with all the sizes tested when compared to unexposed control cultures. Further analysis of the results showed that copper nanoparticles of smaller size and higher concentration exerted the maximum toxic effects. Rubeanic acid staining showed intracellular deposition of copper. These results demonstrate that copper nanoparticles are toxic in a size- and concentration-dependent manner to DRG neurons.

The effects of functional magnetic nanotubes with incorporated nerve growth factor in neuronal differentiation of PC12 cells.

In this in vitro study the efficiency of magnetic nanotubes to bind with nerve growth factor (NGF) and the ability of NGF-incorporated magnetic nanotubes to release the bound NGF are investigated using rat pheochromocytoma cells (PC12 cells). It is found that functional magnetic nanotubes with NGF incorporation enabled the differentiation of PC12 cells into neurons exhibiting growth cones and neurite outgrowth. Microscope observations show that filopodia extending from neuron growth cones were in close proximity to the NGF-incorporated magnetic nanotubes, at times appearing to extend towards or into them. These results show that magnetic nanotubes can be used as a delivery vehicle for NGF and thus may be exploited in attempts to treat neurodegenerative disorders such as Parkinson's disease with neurotrophins. Further neurite outgrowth can be controlled by manipulating magnetic nanotubes with external magnetic fields, thus helping in directed regeneration.

Nicotine alters nicotinic receptor subunit levels differently in developing Mammalian sympathetic neurons.

The subunit specific expression of nicotinic acetylcholine receptors (nAChRs) undergo changes in the superior cervical sympathetic ganglion (SCG) of rat pups during neonatal growth. Since nAChRs play a significant role in sympathetic transmission, and the effect of nicotine on the expression of nAChR subunits in neurons of neonatal SCG is not known, we determined the effects of nicotine on receptor profiles using primary cultures of SCG neurons of 1-day-old rat pups. Neurons in culture were exposed to 1 and 10 microM of nicotine in the presence and absence of nerve growth factor (NGF). After 24 h, protein from the control and experimental neuron cultures was analyzed for the presence of nAChR containing alpha7 and alpha3 subunits using subunit specific antibodies. Exposure to 1 microM of nicotine marginally increased alpha7 subunits only in the absence of NGF. However it increased the level of alpha3 subunits significantly by 18% and 33.6% in the presence and absence of NGF, respectively. Exposure to 10 microM of nicotine did not alter the levels of either of the subunits. Interestingly, when the neurons were pre-exposed to alpha-bungarotoxin (antagonist of alpha7 nAChR), exposure to 10 microM of nicotine resulted in significant increases not only in alpha7 nAChR (25.5%) but also in alpha3 nAChR (32.2%). These results show that exposure to nicotine alters the nAChR levels differently in the neonatal sympathetic neurons in a subunit specific manner and suggest that the level of alpha7 as well as alpha3 nAChR is linked to the functional status of alpha7 nAChR in these neurons.

An analysis of acetylcholinesterase sequence for predicting mechanisms of its non-catalytic actions.

The enzyme acetylcholinesterase (AChE) which belongs to the family of alpha/beta hydrolases is well known for hydrolyzing the neurotransmitter acetylcholine (ACh). In addition to its catalytic function, AChE appears to play a significant non-catalytic role in development, regeneration and modulation of properties of neurons. However the mechanisms underlying these important actions of AChE are unknown. It prompted the analysis of the sequence of fetal bovine serum AChE to seek clue(s) for the mechanisms of AChE non-catalytic actions. The searches for motifs, finger prints and domains demonstrated the presence of a highly conserved carboxylesterase type B signature in AChE from slime molds to human. Interestingly, the presence of finger prints for a metabotropic glutamate receptor, gap junction protein connexin, a calcium binding motif, several phosphorylation sites, and a motif similar to nicotinic acetylcholine receptor were also found in the sequence of AChE suggesting a role for AChE in cell signalling. The similarity search using gapped BLAST retrieved previously known similar proteins, namely neurotactin, neuroligin and thyroglobulin. When aligned neurotactin, neuroligin and bovine AChE showed the presence of three stretches of consensus, one of which is carboxylesterase B signature. The identity of the other two are not known. Amino acids in these stretches in AChE may be involved in possible growth factor like action and awaits experimental verification. Thus sequence analysis can be very useful for biologists while searching for a possible mechanism of action of a protein. Further, these results underscore the significance of performing both local and global searches to identify possible functional domains as well as consensus sequence.