DiAnna L. Hynds

Neurite outgrowth inhibition by chondroitin sulfate proteoglycan : Stalling/stopping exceeds turning in human neuroblastoma growth cones

Chondroitin sulfate proteoglycan (CSPG) inhibits outgrowth from embryonic chick and rodent neurons in vivo and in vitro and is upregulated during development and following injury. The role of CSPG in outgrowth from human neurons has been largely untested, but is critical for our understanding of regeneration in humans following nervous system injury. Here we determined the effects of CSPG on platelet-derived growth factor (PDGF)-stimulated neurite outgrowth from SH-SY5Y human neuroblastoma cells, a well-accepted model of neuronal differentiation. Cells were plated on glass coverslips adsorbed with laminin (LN), CSPG, or a patterned substratum consisting of alternating stripes of the two molecules. Similar to other studies using chick or rodent neurons, SH-SY5Y cells extend neurites on LN, displaying a 15.2% increase in the total neurite length/cell as compared to cells plated on glass. Cells plated on CSPG alone exhibited reduced neurite outgrowth compared to cells plated on glass or LN. Interestingly, SH-SY5Y growth cones extending on LN and then encountering a CSPG border display more stopping/stalling (62.3%) than turning (27.9%) behaviors. Soluble CSPG inhibits neurite initiation from SH-SY5Y cells plated on glass, but not on LN. These data demonstrate that several CSPG-elicited responses of human neuron-like cells are similar to those from nonhuman neurons. However, approximately 70% of SH-SY5Y growth cones stop or stall at a CSPG border while over 80% of chick sensory neurons turn at a CSPG border. The experimental difference between these models may well indicate a functional difference between animal and human neuronal regeneration.

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.

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.

Fibronectin and laminin elicit differential behaviors from SH-SY5Y growth cones contacting inhibitory chondroitin sulfate proteoglycans

Neuronal growth cones integrate signals from outgrowth‐promoting molecules, e.g., laminin (LN) or fibronectin (FN), and outgrowth‐inhibiting molecules, e.g., chondroitin sulfate proteoglycans (CSPGs), to navigate through extracellular matrix (ECM). Sensory neurons on LN typically turn to avoid areas rich in inhibitory CSPGs, whereas neuron‐like cells of human origin (SH‐SY5Y) preferentially stop/stall. These different behaviors may reflect differences in neuron type, response to outgrowth‐promoters, or the mechanisms involved in outgrowth vs. inhibition. We used image analysis to determine the effects of different outgrowth promoters on the response of SH‐SY5Y cells to inhibitory CSPGs. LN increased neurite initiation and elongation compared to cells plated either on endogenous matrix or FN. On a patterned substratum consisting of alternating stripes of FN and CSPGs, 59.6 ± 9.3% of SH‐SY5Y growth cones turned upon CSPG contact, whereas only 31.9 ± 8.2% of growth cones turned at a LN/CSPG border. Growth cones on LN spread more upon contact with CSPG than growth cones on FN, whereas growth cones on LN or FN not contacting CSPGs were morphologically similar. Because it is known that integrins are involved in outgrowth on promoters, we analyzed integrin expression in response to inhibitory CSPGs in a choice assay. CSPGs did not induce increases or redistribution of several integrin subunits in SH‐SY5Y cells. Furthermore, an anti‐β1 integrin function‐blocking antibody did not alter growth cone behavior at a CSPG border. These results indicate that significant mechanistic differences may exist between outgrowth on homogenous outgrowth promoters and growth cone turning at inhibitory molecules. J. Neurosci. Res. 66:630–642, 2001.

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.

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.

Excellent biocompatibility of semiconductor quantum dots encased in multifunctional poly(N-isopropylacrylamide) nanoreservoirs and nuclear specific labeling of growing neurons

Quantum dots (QDs) have received attention for labeling biomolecules; however, toxicity of these nanostructures in the intracellular environment has prevented a biomedical breakthrough. Here we report biocompatibility of a QD based multifunctional system on neuronal cells. Moreover, the designed nanostructures bind with high affinity in the cell nucleus. Nucleus specific binding and enhanced biocompatibility, coupled with no deleterious effects on neurite outgrowth, even at high dosages (500 μg/ml sphere conc.) suggest increased therapeutic potential of this system for specific targeting followed by controlled release of drugs in treating neurodegenerative 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.

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 (IC50 = 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 IC50 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.

L- and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) inhibit neurite outgrowth from SH-SY5Y cells

Gangliosides and extracellular matrix molecules influence neurite outgrowth, but the combinatorial effects of these endogenous agents on outgrowth are unclear. Exogenous gangliosides inhibit neurite outgrowth from SH-SY5Y cells stimulated with platelet-derived growth factor-BB, and different isoforms of the ceramide analog threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) stimulate (L-PDMP) or inhibit (D-PDMP) glycosphingolipid biosynthesis. In this study, we determined whether altering the endogenous ganglioside levels with PDMP in SH-SY5Y cells regulates neurite outgrowth on the outgrowth-supporting extracellular matrix molecule, laminin. In cells stimulated with 20 ng/ml platelet-derived growth factor-BB to promote outgrowth, we used image analysis to evaluate neurite outgrowth from SH-SY5Y cells grown on endogenous matrix or laminin and exposed to L- or D-PDMP. Both L- and D-PDMP decreased neurite initiation (the number of neurites/cell, the percent of neurite-bearing cells), elongation (the length of the longest neurite/cell, the total neurite length/cell), and branching (the number of branch points/neurite) from SH-SY5Y cells on endogenous matrix or laminin in a dose-dependent manner in serum-free or serum-containing medium. The inhibitory effects of each PDMP isoform were reversible. Inhibition of neurite outgrowth by L-PDMP could be mimicked by addition of exogenous gangliosides or C2-ceramide. Our analyses of neurite outgrowth in SH-SY5Y cells, a model of developing or regenerating noradrenergic neurons, demonstrate that increasing or decreasing endogenous ganglioside levels decreases neurite outgrowth. These results may indicate that SH-SY5Y cells undergo tight regulation by gangliosides, possibly through modulation of growth/trophic factor- and/or extracellular matrix-activated signaling cascades.