Uptake of surface-functionalized thermo-responsive polymeric nanocarriers in corticospinal tract motor neurons.

Nanocarrier drug delivery systems are attractive options for targeted delivery of survival- and regeneration-enhancing therapeutics to neurons damaged by degenerative or traumatic central nervous system (CNS) lesions. Functional groups on nanocarrier surfaces allow derivatization with molecules to target specific cells but may affect cellular interactions and nanocarrier uptake. We synthesized differently sized -COOH and -NH2 surface functionalized polymeric nanocarriers (SFNCs) by emulsion copolymerization and assessed uptake by different cell types in mixed cortical cultures. Following 60-min incubation with SFNCs, mean intensity measurements of fluorescently labeled SFNCs indicated that corticospinal tract motor neurons (CSMNs) took up more COOH- or NH2- functionalized SFNCs with similar sizes (150 nm), compared to glia. However, larger diameter (750 nm) SFNCs were taken up at higher concentrations compared to smaller COOH-derivatized SFNCs (150 nm). These data suggest that larger SFNCs may provide an advantage for enhanced uptake by targeted neurons.

Cytomegalovirus infection induces Alzheimer's disease-associated alterations in tau.

Alzheimer's disease (AD) manifests with loss of neurons correlated with intercellular deposition of amyloid (amyloid plaques) and intracellular neurofibrillary tangles of hyperphosphorylated tau. However, targeting AD hallmarks has not as yet led to development of an effective treatment despite numerous clinical trials. A better understanding of the early stages of neurodegeneration may lead to development of more effective treatments. One underexplored area is the clinical correlation between infection with herpesviruses and increased risk of AD. We hypothesized that similar to work performed with herpes simplex virus 1 (HSV1), infection with the cytomegalovirus (CMV) herpesvirus increases levels and phosphorylation of tau, similar to AD tauopathy. We used murine CMV (MCMV) to infect mouse fibroblasts and rat neuronal cells to test our hypothesis. MCMV infection increased steady-state levels of primarily high molecular weight forms of tau and altered the patterns of tau phosphorylation. Both changes required viral late gene products. Glycogen synthase kinase 3 beta (GSK3ß) was upregulated in the HSVI model, but inhibition with lithium chloride suggested that this enzyme is unlikely to be involved in MCMV infection mediated tau phosphorylation. Thus, we confirm that MCMV, a beta herpes virus, like alpha herpes viruses (e.g., HSV1), can promote tau pathology. This suggests that CMV infection can be useful as another model system to study mechanisms leading to neurodegeneration. Since MCMV infects both mice and rats as permissive hosts, our findings from tissue culture can likely be applied to a variety of AD models to study development of abnormal tau pathology.

G1P3/IFI6, an interferon stimulated protein, promotes the association of RAB5+ endosomes with mitochondria in breast cancer cells.

G1P3/IFI6 is an interferon stimulated gene with antiapoptotic, prometastatic, and antiviral functions. Despite its pleiotropic functions, subcellular localization of G1P3 remains unclear. Using biochemical- and confocal microscopic approaches, this study identified the localization of G1P3 in organelles of the endomembrane system and in the mitochondria of breast cancer cells. In cell fractionation studies, both interferon-induced endogenous- and stably expressed G1P3 cofractionated with affinity-isolated mitochondria. Results of the protease protection assay have suggested that ~24% of mitochondrial G1P3 resides within the mitochondria. Conforming to this, confocal microscopy studies of cells stably expressing epitope-tagged G1P3 (MCF-7/G1P3-FLAG), identified its localization in mitochondria (~38%) as well as in ER, trans-Golgi network (TGN), lysosomes, and in RAB5 positive (RAB5+ ) endosomes. These results suggested the trafficking of G1P3 from TGN into endolysosomes. Both G1P3 and RAB5 were known to confer apoptosis resistance through mitochondrial stabilization. Therefore, the effects of G1P3 on the localization of RAB5 in mitochondria were tested. Compared to vector control, the co-occurrence of RAB5 with the mitochondria was increased by 1.5-fold in MCF-7/G1P3-FLAG expressing cells (p ≤ .005). Taken together, our results demonstrate a role for G1P3 to promote the association of RAB5+ endosomes with mitochondria and provide insight into yet another mechanism of G1P3-induced cancer cell survival.

Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet ß-cells: reversal of such metabolic defects by metformin.

Emerging evidence suggests that long-term exposure of insulin-secreting pancreatic ß-cells to hyperglycemic (HG; glucotoxic) conditions promotes oxidative stress, which, in turn, leads to stress kinase activation, mitochondrial dysfunction, loss of nuclear structure and integrity and cell apoptosis. Original observations from our laboratory have proposed that Rac1 plays a key regulatory role in the generation of oxidative stress and downstream signaling events culminating in the onset of dysfunction of pancreatic ß-cells under the duress of metabolic stress. However, precise molecular and cellular mechanisms underlying the metabolic roles of hyperactive Rac1 remain less understood. Using pharmacological and molecular biological approaches, we now report mistargetting of biologically-active Rac1 [GTP-bound conformation] to the nuclear compartment in clonal INS-1 cells, normal rat islets and human islets under HG conditions. Our findings also suggest that such a signaling step is independent of post-translational prenylation of Rac1. Evidence is also presented to highlight novel roles for sustained activation of Rac1 in HG-induced expression of Cluster of Differentiation 36 [CD36], a fatty acid transporter protein, which is implicated in cell apoptosis. Finally, our findings suggest that metformin, a biguanide anti-diabetic drug, at a clinically relevant concentration, prevents ß-cell defects [Rac1 activation, nuclear association, CD36 expression, stress kinase and caspase-3 activation, and loss in metabolic viability] under the duress of glucotoxicity. Potential implications of these findings in the context of novel and direct regulation of islet ß-cell function by metformin are discussed.

Regulation of Small GTPase Prenylation in the Nervous System.

Mevalonate pathway inhibitors have been extensively studied for their roles in cholesterol depletion and for inhibiting the prenylation and activation of various proteins. Inhibition of protein prenylation has potential therapeutic uses against neurological disorders, like neural cancers, neurodegeneration, and neurotramatic lesions. Protection against neurodegeneration and promotion of neuronal regeneration is regulated in large part by Ras superfamily small guanosine triphosphatases (GTPases), particularly the Ras, Rho, and Rab subfamilies. These proteins are prenylated to target them to cellular membranes. Prenylation can be specifically inhibited through altering the function of enzymes of the mevalonate pathway necessary for isoprenoid production and attachment to target proteins to elicit a variety of effects on neural cells. However, this approach does not address how prenylation affects a specific protein. This review focuses on the regulation of small GTPase prenylation, the different techniques to inhibit prenylation, and how this inhibition has affected neural cell processes.

Differential activation of Rac1 and RhoA in neuroblastoma cell fractions.

The Rho guanine nucleotide triphosphatases (GTPases) Rac1 and RhoA are important regulators of axon growth. However, the specific roles each plays are complicated by implications that each is involved in promoting and inhibiting neurite outgrowth. Differential regulation of Rac1 and RhoA activation in cell bodies and growth cones may be important in directing axon growth. To test this, we separated neuroblastoma cells into growth cone and cell body fractions and assessed Rac1 and RhoA activation in response to outgrowth promoters, serum withdrawal and 8-bromoadeosine-5',3'-cyclic monophosphate (8-Br-cAMP), and outgrowth inhibitors, chondroitin sulfate proteoglycans (CSPGs) or semaphorin 3A (Sema 3A). In whole cell lysates, serum withdrawal decreased and CSPGs or Sema 3A increased RhoA activity, but no treatments affected Rac1 activity. In growth cones, serum withdrawal or 8-Br-cAMP increased Rac1 activation and serum withdrawal decreased RhoA activation. Conversely, outgrowth inhibitors decreased Rac1 activity. Additionally, 8-Br-cAMP reversed increases in RhoA activity induced by Sema 3A in whole cell lysates and CSPGs in growth cones. These data suggest that activation of RhoA and Rac1 is differentially regulated in specific cellular regions, perhaps contributing to the complexity of Rho GTPase-mediated axon growth.

Non-prenylatable, cytosolic Rac1 alters neurite outgrowth while retaining the ability to be activated.

Rac1 is an important regulator of axon extension, cell migration and actin reorganization. Like all Rho guanine triphosphatases (GTPases), Rac1 is targeted to the membrane by the addition of a geranylgeranyl moiety, an action thought to result in Rac1 guanosine triphosphate (GTP) binding. However, the role that Rac1 localization plays in its activation (GTP loading) and subsequent activation of effectors is not completely clear. To address this, we developed a non-prenylatable emerald green fluorescent protein (EmGFP)-Rac1 fusion protein (EmGFP-Rac1(C189A)) and assessed how expressing this construct affected neurite outgrowth, Rac1 localization and activation in neuroblastoma cells. Expression of EmGFP-Rac1(C189A) increased localization to the cytosol and induced cell clustering while increasing neurite initiation. EmGFP-Rac1(C189A) expression also increased Rac1 activation in the cytosol, compared to cells expressing wild-type Rac1 (EmGFP-Rac1). These results suggest that activation of Rac1 may not require plasma membrane localization, potentially leading to differential activation of cytosolic signaling pathways that alter cell morphology. Understanding the consequences of differential localization and activation of Rho GTPases, including Rac1, could lead to new therapeutic targets for treating neurological disorders.

A semi-automated image analysis method to quantify neurite preference/axon guidance on a patterned substratum.

Axon outgrowth and guidance are differentially promoted or inhibited by specific extracellular matrix (ECM) molecules. The effects of these molecules can be examined by culturing neuronal explants on patterned substrata consisting of alternating stripes adsorbed with the molecules of interest. While outgrowth on substrata adsorbed with homogenous molecules can be reliably quantified, current methods of quantifying neurite preference on patterned substrata are subjective, labor intensive, and overall less reliable. Here, we present a quick, semi-automated, lowly subjective macro-based method to quantify the effects of a change in substratum on axon extension and guidance. We plated chick dorsal root ganglion explants on a substratum consisting of alternating stripes of laminin-1 (outgrowth supportive) and chondroitin sulfate proteoglycans (CSPGs, outgrowth inhibitory). We evaluated neurite preference for laminin or CSPG-coated regions by measuring total neurite area, and produced an inhibition index. The quantitative data confirmed previous qualitative data showing that increasing concentrations of CSPGs induced increases in inhibition. The methods presented here: (1) require less stringent image capture criteria; (2) are quicker; (3) are less subjective compared to previously described methods; and (4) are versatile in that they can be used to assay neurite preference for any substratum-bound molecules in living or fixed cultures.

Inhibiting geranylgeranylation increases neurite branching and differentially activates cofilin in cell bodies and growth cones.

Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 µM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 µM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization.

Role of engineered nanocarriers for axon regeneration and guidance: current status and future trends.

There are approximately 1.5 million people who experience traumatic injuries to the brain and 265,000 who experience traumatic injuries to the spinal cord each year in the United States. Currently, there are few effective treatments for central nervous system (CNS) injuries because the CNS is refractory to axonal regeneration and relatively inaccessible to many pharmacological treatments. Smart, remotely tunable, multifunctional micro- and nanocarriers hold promise for delivering treatments to the CNS and targeting specific neurons to enhance axon regeneration and synaptogenesis. Furthermore, assessing the efficacy of treatments could be enhanced by biocompatible nanovectors designed for imaging in vivo. Recent developments in nanoengineering offer promising alternatives for designing biocompatible micro- and nanovectors, including magnetic nanostructures, carbon nanotubes, and quantum dot-based systems for controlled release of therapeutic and diagnostic agents to targeted CNS cells. This review highlights recent achievements in the development of smart nanostructures to overcome the existing challenges for treating CNS injuries.

Mevalonate depletion mediates the suppressive impact of geranylgeraniol on murine B16 melanoma cells.

The diterpene geranylgeraniol (all trans-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraen-1-ol) suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach and blood tumors with undefined mechanisms. We evaluated the growth-suppressive activity of geranylgeraniol in murine B16 melanoma cells. Geranylgeraniol induced dose-dependent suppression of B16 cell growth (IC(50) = 55 ± 13 µmol/L) following a 48-h incubation in 96-well plates. Cell cycle arrest at the G1 phase, manifested by a geranylgeraniol-induced increase in the G1/S ratio and decreased expression of cyclin D1 and cyclin-dependent kinase 4, apoptosis detected by Guava Nexin™ assay and fluorescence microscopy following acridine orange and ethidium bromide dual staining, and cell differentiation shown by increased alkaline phosphatase activity, contributed to the growth suppression. Murine 3T3-L1 fibroblasts were 10-fold more resistant than B16 cells to geranylgeraniol-mediated growth suppression. Geranylgeraniol at near IC(50) concentration (60 µmol/L) suppressed the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by 50%. The impact of geranylgeraniol on B16 cell growth, cell cycle arrest and apoptosis were attenuated by supplemental mevalonate, the product of HMG-CoA reductase that is essential for cell growth. Geranylgeraniol and d-δ-tocotrienol, a down-regulator of HMG-CoA reductase, additively suppressed the growth of B16 cells. These results support our hypothesis that mevalonate depletion mediates the tumor-specific growth-suppressive impact of geranylgeraniol. Geranylgeraniol may have potential in cancer chemoprevention and/or therapy.

RHO GTPase signaling for axon extension: is prenylation important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.

Alternating Magnetic Field Controlled, Multifunctional Nano-Reservoirs: Intracellular Uptake and Improved Biocompatibility.

Biocompatible magnetic nanoparticles hold great therapeutic potential, but conventional particles can be toxic. Here, we report the synthesis and alternating magnetic field dependent actuation of a remotely controllable, multifunctional nano-scale system and its marked biocompatibility with mammalian cells. Monodisperse, magnetic nanospheres based on thermo-sensitive polymer network poly(ethylene glycol) ethyl ether methacrylate-co-poly(ethylene glycol) methyl ether methacrylate were synthesized using free radical polymerization. Synthesized nanospheres have oscillating magnetic field induced thermo-reversible behavior; exhibiting desirable characteristics comparable to the widely used poly-N-isopropylacrylamide-based systems in shrinkage plus a broader volumetric transition range. Remote heating and model drug release were characterized for different field strengths. Nanospheres containing nanoparticles up to an iron concentration of 6 mM were readily taken up by neuron-like PC12 pheochromocytoma cells and had reduced toxicity compared to other surface modified magnetic nanocarriers. Furthermore, nanosphere exposure did not inhibit the extension of cellular processes (neurite outgrowth) even at high iron concentrations (6 mM), indicating minimal negative effects in cellular systems. Excellent intracellular uptake and enhanced biocompatibility coupled with the lack of deleterious effects on neurite outgrowth and prior Food and Drug Administration (FDA) approval of PEG-based carriers suggest increased therapeutic potential of this system for manipulating axon regeneration following nervous system injury.

Rit promotes MEK-independent neurite branching in human neuroblastoma cells.

Rit, by sequence homology, is a member of the Ras subfamily of small guanine triphosphatases (GTPases). In PC6 cells, Rit signals through pathways both common to and different from those activated by Ras to promote cell survival and neurite outgrowth. However, the specific morphological changes induced by Rit in human cells are not known. Here, we show in a human neuronal model that Rit increases neurite outgrowth and branching through MEK-dependent and MEK-independent signaling mechanisms, respectively. Adenoviral expression of wild-type or constitutively active Rit increased neurite initiation, elongation and branching on endogenous matrix or a purified laminin-1 substratum of SH-SY5Y cells as assessed using image analysis. This outgrowth was morphologically distinct from that promoted by constitutively active Ras or Raf (evidenced by increased branching and elongation). Constitutively active Rit increased phosphorylation of ERK 1/2, but not Akt, and the MEK inhibitor PD 098059 blocked constitutively active Rit-induced neurite initiation but not elongation or branching. These results suggest that Rit plays a key role in human neuronal development and regeneration through activating both known and as yet undefined signaling pathways.

Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures.

Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury.

Human intervertebral disc aggrecan inhibits nerve growth in vitro.

OBJECTIVE: To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques. METHODS: Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared. RESULTS: Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentration-dependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects. CONCLUSION: Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.