Total Neurite Outgrowth Research Articles

Static Magnetic Stimulation and Magnetic Microwires Synergistically Enhance and Guide Neurite Outgrowth.

Axonal growth is heavily influenced by topography and biophysical stimuli including magnetic and electrical fields. Despite extensive investigation, the degree of influence and the underlying genetic mechanisms remain poorly understood. Here, a novel approach to guide neurite growth is undertaken using an innovative ferromagnetic composite material - glass-coated magnetic microwire - to furnish a synergistic combination of magnetic and topographical cues. Whole rat dorsal root ganglia (DRG) are cultured under five different conditions: control, static magnetic field, magnetic microwire, static magnetic field + glass fiber, and static magnetic field + magnetic microwire. DRG outgrowth responses under each condition, including total neurite outgrowth and directionality, are compared. The combination of both magnetic stimulation and topography significantly increases total neurite outgrowth compared to the controls. The combination of magnetic stimulation and magnetic microwire lead to a strong directional bias of growth along the microwire, double what is observed with the glass fiber. Next generation RNA sequencing of DRG exposed to static magnetic field + magnetic microwire reveals the downregulation of genes relating to the immune response, interleukin signaling, and signal transduction. These results set the stage for contemplating future biophysical stimulation for axonal guidance and improved understanding of material-tissue interactions.

An efficient cellular image-based platform for high-content screening of neuroprotective agents against chemotherapy-induced neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting side effect, with no approved therapy for prevention or treatment. Here, we aimed to establish a high-content image platform based on the neurite outgrowth of dorsal root ganglia (DRG)-derived neuron cells for the discovery of neuroprotective agents against paclitaxel-induced CIPN. ND7/23 cells, an immortalized hybrid DRG cell line, were maturely differentiated by induction with nerve growth factor and upregulation of intracellular cAMP levels. High-content image analyses of the neurofilament-stained neurite network showed that paclitaxel disrupted the neurite outgrowth of well-differentiated ND7/23 DRG neuron cells, recapitulating characteristic effects of paclitaxel on primary cultured DRG neurons. This process coincided with the upregulated activity of store-operated Ca2+ entry, similar to those found in rodent models of paclitaxel-induced CIPN. The previously identified neuroprotective agents, minoxidil and 8-Br-cyclic adenosine monophosphate ribose (8-Br-cADPR), attenuated the reduction in total neurite outgrowth in paclitaxel-damaged ND7/23 cells. Additionally, the total neurite outgrowth of well-differentiated ND7/23 cells was concentration-dependently reduced by the neurotoxic chemotherapeutic agents, oxaliplatin and bortezomib, but not the less neurotoxic 5-fluorouracil. We demonstrated that high-content analyses of neurite morphology in well-differentiated DRG neuron-derived cells provide an effective, reproducible, and high-throughput strategy for developing therapeutics against CIPN.

Synaptosome microRNAs: emerging synapse players in aging and Alzheimer's disease.

Synaptosome microRNAs: emerging synapse players in aging and Alzheimer's disease.

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis. There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation. Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy. We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress. This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis, inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models.

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer's disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.

Growth and regeneration of sympathetic neuron axons induced by pulsed low intensity ultrasound

Over a quarter million Americans are currently living with spinal cord injuries (SCIs), costing $3 billion a year to manage [1]. SCI results in myelin loss, axonal degradation, and death of sympathetic neurons, leading to lesion formation and disruption of the primary communication pathway in the spinal cord. Low intensity ultrasound has been shown to improve axonal regeneration in the peripheral nervous system, but its effect on cells in the central nervous systems is not yet studied. The objective of this work is to investigate the growth-stimulating effects of low to mid-intensity ultrasound on axons of sympathetic neurons. To find optimal ultrasound intensities, neuroendocrine-type PC-12 cells were cultured and treated with pulsed ultrasound at varying intensities. Cells were cultured in deprived states to model a post-SCI environment, while being treated with neurotrophic factor (NGF) to induce their neuron differentiation. Images of cells were analyzed for number of cells and neurite outgrowth. Experiments with PC-12 cells found that an ultrasound intensity of 1.02 W/cm2 led to optimal neurite outgrowth when analyzing the rate of neurite outgrowth and total neurite outgrowth at the final day of experimentation. Our ultimate goal is to develop an effective, noninvasive SCI treatment based on the ultrasound technology.

Long non-coding RNA ANRIL knockdown suppresses apoptosis and pro-inflammatory cytokines while enhancing neurite outgrowth via binding microRNA-125a in a cellular model of Alzheimer's disease.

The present study aimed to investigate the effect of the long non-coding RNA antisense non-coding RNA in the INK4 locus (lnc-ANRIL) knockdown on apoptosis, neurite outgrowth and inflammation based on a PC12 cellular Alzheimer's disease (AD) model. A cellular AD model was constructed by treating nerve growth factor stimulated PC12 cells with amyloid β (Aβ) 1–42 and then control knockdown plasmid and lnc-ANRIL knockdown plasmid were transfected in the PC12 cellular AD model as the KD- negative control (NC) group or the AD-ANRIL group respectively. Apoptosis, neurite outgrowth, pro-inflammatory cytokines and microRNA (miR)-125a were assessed. Rescue experiments were conducted by transfecting lnc-ANRIL knockdown plasmid and lnc-ANRIL knockdown plasmid and miR-125a inhibitor in the PC12 cellular AD model as the KD-ANRIL group or KD-ANRIL + KD-miR-125a group respectively. Following transfection, cell apoptosis deccreased while neurite outgrowth increased in the KD-ANRIL group compared with the KD-NC group (all P<0.01). Concerning inflammation, tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, IL-6 and IL-17 were decreased in the KD-ANRIL group compared with the KD-NC group (all P<0.01). miR-125a was negatively regulated by lnc-ANRIL and therefore rescue experiments were subsequently conducted. In the rescue experiments, cell apoptosis was increased while total neurite outgrowth was inhibited in the KD-ANRIL + KD-miR-125a group compared with the KD-ANRIL group (all P<0.01), and TNF-α, IL-1β, IL-6 and IL-17 were increased in the KD-ANRIL + KD-miR-125a group compared with the KD-ANRIL group (all P<0.01). A luciferase reporter assay demonstrated that lnc-ANRIL directly bound miR-125a. lnc-ANRIL knockdown suppressed cell apoptosis and inflammation while promoting neurite outgrowth via binding of miR-125a in AD.

Directed and enhanced neurite outgrowth following exogenous electrical stimulation on carbon nanotube-hydrogel composites

Objective. The objective of this work was to test the synergistic effects of substrate stiffness, electro-conductivity, composition and electrical stimulation on the morphology, alignment and directional neurite outgrowth of neuron-like PC12 cells. The use of exogenous electrical stimulation has emerged as a promising new intervention to promote neural regeneration following injury. For critical gap size nerve injuries, a permissive biomaterial coupled to electrical stimulation may be needed to provide guidance and support for neurite outgrowth. Thus, the combinatorial effects of biomaterial composition and properties and exogenous electrical stimulation need interrogation to develop successful therapeutic interventions. Carefully designed in vitro models are ideally suited to perform such multidimensional detailed studies. Approach. We assembled a simple electrical stimulation device to deliver uniform electrical current with minimum voltage field variation through a hydrogel. We used polyacrylamide (PA), polyethylene glycol (PEG), and multi-walled carbon nanotubes (MWCNT)-PEG nanocomposite hydrogels of varying stiffness, resistivity and MWCNT concentration. Cells were seeded on the substrates for 24 h, stimulated for 1 h at 30 V m−1 DC, and then cultured for additional 24 h. Non-stimulated cells were used as controls. To induce neurite outgrowth, cells were primed with nerve growth factor (100 ng ml−1). Main results. For all substrates tested, electrical stimulation induced neurite alignment at 60–90° angle to the applied current. It also increased total neurite outgrowth by 18%–49% and mean neurite length by 20%–46% (increase dependent on the underlying substrate) compared to non-stimulated cells. The nanocomposite composed of 20% w/v PEG and 0.1% w/v MWCNTs resulted in the highest total neurite outgrowth and mean neurite length, which were further significantly enhanced by electrical stimulation by 2-fold and 1.8-fold, respectively. Significance. Our results indicate that nanocomposites, where carbon nanotubes have been added to hydrogel substrates, in combination with electrical stimulation provided improved conditions for neural growth and regeneration.

Enhanced total neurite outgrowth and secondary branching in dorsal root ganglion neurons elicited by low intensity pulsed ultrasound

Objective. Despite the prevalence of peripheral nerve injuries (PNI), challenges remain in restoring full functionality to those afflicted. For recovery to occur, axons must extend across the injury site to connect with distal targets, where injury gap size is a critical factor in the probability of restoration of function. Current clinical therapies often achieve limited neural regeneration, motivating the development of new therapeutic interventions such as biophysical stimulation. Approach. To investigate the potential for low intensity, pulsed ultrasonic simulation (LIPUS) to impact peripheral nerve regeneration, primary neonatal rat dorsal root ganglion neurons were examined in vitro in response to ultrasound (US). Dissociated neurons were stimulated with varied acoustic power (low, medium, high) and their morphometrics, including total outgrowth, branching, and length, were analyzed acutely after 18 h of growth. Main results. Results show US increases total neurite outgrowth by 2.83-fold compared to unstimulated controls at the highest power. Neurite branching at medium and high-power US increased approximately 2-fold compared to controls, while low stimulation exhibited more muted trends. Neurite branching is also impacted by US, with medium and high power eliciting the highest branching, of approximately 2-fold compared to low power and unstimulated controls. Significance. These results demonstrate that US stimulation of DRG neurons in vitro impacts neurite morphology and enhances total extension, indicating the potential for advancing and understanding driving mechanisms of ultrasonic therapies for peripheral nerve regeneration.

MiR-103 Promotes Neurite Outgrowth and Suppresses Cells Apoptosis by Targeting Prostaglandin-Endoperoxide Synthase 2 in Cellular Models of Alzheimer's Disease.

miR-103 has been reported to be decreased in brain of transgenic mouse model of Alzheimer’s disease (AD) and in cerebrospinal fluid (CSF) of AD patients, while the detailed mechanism of its effect on AD is obscure, thus this study aimed to investigate the effect of miR-103 expression on neurite outgrowth and cells apoptosis as well as its targets in cellular models of AD. Blank mimic (NC1-mimic), miR-103 mimic, blank inhibitor (NC2-mimic) and miR-103 inhibitor plasmids were transferred into PC12 cellular AD model and Cellular AD model of cerebral cortex neurons which were established by Aβ1–42 insult. Rescue experiment was subsequently performed by transferring Prostaglandin-endoperoxide synthase 2 (PTGS2) and miR-103 mimic plasmid. mRNA and protein expressions were detected by qPCR and Western Blot assays. Total neurite outgrowth was detected by microscope, cells apoptosis was determined by Hoechst/PI assay, and apoptotic markers Caspase 3 and p38 expressions were determined by Western Blot assay. In both PC12 and cerebral cortex neurons cellular AD models, miR-103 mimic increases the total neurite outgrowth compared with NC1-mimic, while miR-103 inhibitor decreases the total neurite outgrowth than NC2-inhibitor. The apoptosis rate was decreased in miR-103 mimic group than NC1-mimic group while increased in miR-103 inhibitor group than NC2-inhibitor group. PTGS2, Adisintegrin and metalloproteinase 10 (ADAM10) and neprilysin (NEP) were selected as target genes of miR-103 by bioinformatics analysis. And PTGS2 was found to be conversely regulated by miR-103 expression while ADAM10 and NEP were not affected. After transfection by PTGS2 and miR-103 mimic plasmid in PC12 cellular AD model, the total neurite growth was shortened compared with miR-103 mimic group, and cells apoptosis was enhanced which indicated PTGS2 mimic attenuated the influence of miR-103 mimic on progression of AD. In conclusion, miR-103 promotes total neurite outgrowth and inhibits cells apoptosis by targeting PTGS2 in cellular models of AD.

Bioactive Organic Rosette Nanotubes Support Sensory Neurite Outgrowth.

Regardless of the intervention for peripheral nerve repair, slow rates of axonal regeneration often result in poor clinical outcomes. Thus, using new materials such as biologically inspired, biocompatible, organic rosette nanotubes (RNTs) could provide a tailorable scaffold to modulate neurite extension and attachment for improved nerve repair. RNTs are obtained through the spontaneous self-assembly of a synthetic DNA base analogue featuring the hydrogen bond triads of both guanine and cytosine, the G∧C base. Here, we investigated the potential of RNTs functionalized with lysine and Arg-Gly-Asp-Ser-Lys (RGDSK) peptide to support neural growth. We hypothesized that (a) due to their dimensions, the RNTs would support neuron attachment, and (b) their conjugation to the integrin-binding peptide RGDSK would further enhance neurite outgrowth compared to unfunctionalized RNT. Neurite extension was examined on a variety of RNT structures, including RNT with a lysine side chain (K1), a mixture of the K1 and a free RGDS peptide, RNT alone, an RGDSK-functionalized RNT, in addition to poly-d-lysine and laminin controls. Both whole dorsal root ganglion (DRG) and single dissociated DRG neurons were seeded onto RNT-coated substrates containing various ratios of peptides. Analysis of neuron morphometrics showed that RNT blends support DRG neuron attachment and neurite extension, with RGDS presentation increasing neurite outgrowth from whole DRG by up to 47% over a 7-day period compared to K1 alone (p < 0.013). In addition, while RNTs increased the sprouting of primary neurites extending from dissociated DRG neurons, the total neurite outgrowth per neuron remained the same. These results show that functionalized biomimetic RNTs provide a support for neurite growth and extension and have the ability to modulate neuronal morphology. These results also pave the way for the design of injectable RNT-based nanomaterials that support guided neural regeneration following traumatic injury.

Cross-Talk between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson's Disease.

Parkinson's disease (PD) is a progressive movement disorder characterized by neuroinflammation and dopaminergic neurodegeneration in the brain. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces the release of inflammatory mediators from glial cells and neurons. Glia maturation factor (GMF), a brain proinflammatory protein, MPP+, and mast cell-derived inflammatory mediators induce neurodegeneration which eventually leads to PD. However, the precise mechanisms underlying interaction between glial cells, neurons and mast cells in PD still remain elusive. In the present study, mouse bone marrow-derived mast cells (BMMCs) and mouse fetal brain-derived mixed glia/neurons, astrocytes and neurons were incubated with MPP+, GMF and mast cell-derived inflammatory mediators mouse mast cell protease-6 (MMCP-6), MMCP-7 or tryptase/brain-specific serine protease-4 (tryptase/BSSP-4). Inflammatory mediators released from these cells in the culture medium were quantitated by enzyme-linked immunosorbent assay. Neurodegeneration was quantified by measuring total neurite outgrowth following microtubule-associated protein-2 immunocytochemistry. MPP+-induced significant neurodegeneration with reduced total neurite outgrowth. MPP+induced the release of tryptase/BSSP-4 from the mouse mast cells, and tryptase/BSSP-4 induced chemokine (C-C motif) ligand 2 (CCL2) release from astrocytes and glia/neurons. Overall our results suggest that MPP+, GMF, MMCP-6 or MMCP-7 stimulate glia/neurons, astrocytes or neurons to release CCL2 and matrix metalloproteinase-3. Additionally, CD40L expression is increased in BMMCs after incubation with MPP+ in a co-culture system consisting of BMMCs and glia/neurons. We propose that mast cell interaction with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD.

The proinflammatory cytokine, interleukin-17A, augments mitochondrial function and neurite outgrowth of cultured adult sensory neurons derived from normal and diabetic rats

BackgroundDiabetic neuropathy comprises dying back of nerve endings that reflects impairment in axonal plasticity and regenerative nerve growth. Metabolic changes in diabetes can lead to a dysregulation of hormonal mediators, such as cytokines, that may constrain distal nerve fiber growth. Interleukin-17 (IL-17A), a proinflammatory and neurotropic cytokine produced by T-cells, was significantly reduced in sciatic nerve of streptozotocin (STZ)-diabetic rats. Thus we studied the effect of IL-17A on the phenotype of sensory neurons derived from age matched control or type 1 diabetic rats. The aims were to determine the ability of IL-17A to enhance neurite outgrowth in cultured sensory neurons, investigate the signaling pathways activated by IL-17A, study the role of mitochondria and mechanistically link to neurite outgrowth. ResultsIL-17A (10ng/ml; p<0.05) significantly and dose-dependently increased total neurite outgrowth in cultures of adult dorsal root ganglia (DRG) sensory neurons derived from both control and streptozotocin (STZ)-diabetic rats. This enhancement was mediated by IL-17A-dependent activation of extracellular-regulated protein kinase (ERK) and phosphoinositide-3 kinase (PI-3K) signal transduction pathways. Pharmacological blockade of one of these activated pathways triggered complete inhibition of neurite outgrowth. IL-17A augmented mitochondrial bioenergetic function of sensory neurons derived from control or diabetic rats and this was also mediated via ERK or PI-3K. IL-17A-dependent elevation of bioenergetic function was associated with augmented expression of proteins of the mitochondrial electron transport system complexes. ConclusionsIL-17A enhanced axonal plasticity through activation of ERK and PI-3K pathways and was associated with augmented mitochondrial bioenergetic function in sensory neurons.

Effect of Testosterone on Neuronal Morphology and Neuritic Growth of Fetal Lamb Hypothalamus-Preoptic Area and Cerebral Cortex in Primary Culture.

Testosterone plays an essential role in sexual differentiation of the male sheep brain. The ovine sexually dimorphic nucleus (oSDN), is 2 to 3 times larger in males than in females, and this sex difference is under the control of testosterone. The effect of testosterone on oSDN volume may result from enhanced expansion of soma areas and/or dendritic fields. To test this hypothesis, cells derived from the hypothalamus-preoptic area (HPOA) and cerebral cortex (CTX) of lamb fetuses were grown in primary culture to examine the direct morphological effects of testosterone on these cellular components. We found that within two days of plating, neurons derived from both the HPOA and CTX extend neuritic processes and express androgen receptors and aromatase immunoreactivity. Both treated and control neurites continue to grow and branch with increasing time in culture. Treatment with testosterone (10 nM) for 3 days significantly (P < 0.05) increased both total neurite outgrowth (35%) and soma size (8%) in the HPOA and outgrowth (21%) and number of branch points (33%) in the CTX. These findings indicate that testosterone-induced somal enlargement and neurite outgrowth in fetal lamb neurons may contribute to the development of a fully masculine sheep brain.

Olfactory ensheathing cell–neurite alignment enhances neurite outgrowth in scar-like cultures

The regenerative capacity of adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth-inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC–neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core.

Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN), the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs) as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05). The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011). The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05), demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism.

Regenerative endodontic procedures have become a valuable alternative for the treatment of immature teeth with pulp necrosis. In addition to resolution of periradicular pathosis and promotion of continued root development, positive vitality testing has been observed in some regenerative clinical cases. Importantly, the positive response to electric stimulation of the regenerated tissue requires targeting of periradicular axons into the previously empty root canal space. However, the mechanism by which this process occurs is largely unknown. Since stem cells of the apical papilla (SCAP) have been proposed to populate the root canal following regenerative endodontic procedures, we hypothesized that SCAP regulate neurite outgrowth and axonal targeting. To test this hypothesis, we established primary co-cultures of human SCAP and rat trigeminal neurons, and performed neurite outgrowth assays using ELISA and confocal microscopy to determine the effect of increasing concentration of SCAP on the total neurite outgrowth and axonal targeting. In addition, we evaluated whether SCAP evoked axonal targeting in vivo using a matrigel subcutaneous implant assay. Data were analyzed by ANOVA with Bonferroni's post hoc test, and significance was set at p<0.05. The results demonstrated that SCAP release a soluble factor that regulates neurite outgrowth from cultured trigeminal neurons. Next, we demonstrated that this effect is completely abolished by pretreatment with a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by antibodies to other neurotrophins. Further, SCAP release BDNF in a concentration-dependent manner as detected by ELISA, and trigger directed axonal targeting both in vitro and in vivo as demonstrated by microfluidic and matrigel implant experiments, respectively. Collectively, these results suggest that SCAP may be responsible for the chemical signal driving axons to target regenerated tissue via a BDNF-dependent mechanism.

Citrate-stabilized gold nanoparticles as negative controls for measurements of neurite outgrowth

Gold nanoparticles (AuNPs) are promising candidates for medical diagnostics and therapeutics, due to their chemical stability, optical properties, and ease of functionalization. Citrate-stabilized reference materials also have potential as negative controls in toxicology studies of other nanoparticles. Here we examine the impact of 30nm particles on the in vitro development of rat-cortex neural progenitor cells (NPCs), which mimic aspects of the developing neurological environment. AuNPs dispersed in a low-serum culture medium initially agglomerated, but then remained stable during a three day incubation period, and agglomerated only slightly during a ten day incubation period, as determined by dynamic light scattering. Transmission electron microscopy indicated the presence of individual nanoparticles at all timepoints examined. Fixed cells were cross-sectioned by ion milling and imaged by scanning electron microscopy and helium-ion microscopy to evaluate particle incorporation. Individual nanoparticles could be resolved inside cross-sectioned cells. AuNPs were incubated with developing NPCs for ten days at concentrations of 0.5μg/mL Au, 0.1μg/mL Au, or 0.05μg/mL Au. Adenosine triphosphate levels, as determined by bioluminescence measurements sensitive to low cell numbers, were not affected by AuNPs and the particles did not interfere with the assay. Multiple endpoints of neurite outgrowth were not altered by AuNPs, in particular, total neurite outgrowth per cell, a sensitive measure of neuronal development. Slide-level comparisons demonstrated the consistent response of NPCs to gold nanoparticles and a positive control chemical, neuroactive lithium. These results indicate that 30nm citrate-stabilized AuNPs could serve as negative-control reference materials for in vitro measurements of neurite outgrowth.

Abstract 3489: A novel investigation into chemotherapy-induced peripheral neuropathy using iPSC-derived human neurons

Abstract Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting side effect of many anti-cancer drugs including taxanes, vinca alkaloids and platinating agents. These drugs represent standard of care for common cancers, however there are no effective treatments and no means to identify patients at risk for CIPN. The mechanisms underlying CIPN have not been precisely determined and few human neuronal models to study CIPN exist. Recent technology has allowed for the differentiation of induced pluripotent stem cells (iPSC) to human neurons. We have utilized genetically diverse human neurons as a model to study the effects on chemotherapy on neuronal health and on neurite morphology including neurite outgrowth, processes and branching. Upon treatment of human neurons with paclitaxel, vincristine or cisplatin (1 nM to 100 µM) for 72 hours, we identified reproducible decreases in cell median neurite process length per magnitude of drug concentration of 12-14%, 6-18% and 2-4%, respectively. No decrease in neurite length was observed with hydroxyurea, an agent that does not cause neuropathy. The morphological changes differed across drugs, with vincristine treatment showing the most dramatic effect on neurite outgrowth, paclitaxel showing an intermediate effect, and cisplatin showing no effect up to 1 µM of drug. To determine whether the model could be used for evaluation of genes important in CIPN, we used siRNA to decrease expression of TUBB2A, a gene known to be associated with paclitaxel-induced neuropathy. Transient knockdown of TUBB2A caused decreases of mean neurite process length (interaction P = 2.2 x 10-8) and total neurite outgrowth (interaction P = 1.8 x 10-10) of neurons 48 hours post-paclitaxel treatment. We are using this novel approach to screen agents that may be used to prevent or treat CIPN. Citation Format: Masaaki Komatsu, Heather E. Wheeler, Claudia Wing, Shannon Delaney, M Eileen Dolan. A novel investigation into chemotherapy-induced peripheral neuropathy using iPSC-derived human neurons. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3489. doi:10.1158/1538-7445.AM2014-3489

Abstract P3-15-10: Human neuronal model to study chemotherapeutic-induced peripheral neuropathy

Abstract Chemotherapy-induced peripheral neuropathy is the major dose-limiting toxicity for several anti-cancer drugs including taxanes, platinating agents and microtubule inhibitors. In general, animal models have been used to study neuropathy. Our goals are twofold: 1) to create a human model for study of chemotherapeutic induced neuropathy using iCell Neurons derived from human induced pluripotent stem cells (iPSCs) and; 2) to elucidate the molecular genetic mechanisms underlying chemotherapeutic induced peripheral neuropathy by identifying genetic variants and genes that increase the likelihood of this devastating adverse event. We have used this model system to perform functional studies on candidate targets of interest from clinical studies of chemotherapeutic induced neuropathy. Upon treatment of iCell Neurons with increasing concentrations of paclitaxel (0.001-100 μM) for 72 hours, we identified a reproducible 3-5 μm (12-14%) decrease in cell median neurite process length and a 13-19 μm (10-13%) decrease in cell total neurite outgrowth per order of magnitude increase in drug. The same concentrations of vincristine and cisplatin for 72 hours result in a decrease in cell median neurite process length of 6-18% and 2-4% per order of magnitude increase in drug, respectively. No decrease in neurite length is observed with hydroxyurea, a drug that does not result in neuropathy. Paclitaxel binds to beta-tubulin to exert its cytotoxic effect and genetic variants within the promoter of TUBB2A were shown to be associated with paclitaxel induced neuropathy (Clin Cancer Res, 18(16):4441-8, 2012). As a proof of concept, we have shown that decreased expression of the beta-tubulin isotype TUBB2A by siRNA transfection causes decreased median neurite process length (interaction P = 2.0 × 10-4) and decreased total neurite outgrowth (interaction P = 6.2 × 10-10) of iCell Neurons 48 hours post-paclitaxel treatment. To determine clinical relevance, we have developed a protocol to collect blood of breast and ovarian cancer patients experiencing severe paclitaxel-induced peripheral neuropathy and matched patients without neuropathy following similar paclitaxel regimens to create iPSCs and eventually neurons. We hope to create a resource of these cells for the scientific community. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-15-10.