Neurite Branch Points Research Articles

Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10−8 – 3 ×10−3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10−7 - 1 ×10−8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14–28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10−3 M and 5.8 × 10−4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10−7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by βIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Human Induced Pluripotent Stem Cell-Derived Neural Cells from Alzheimer's Disease Patients Exhibited Different Susceptibility to Oxidative Stress.

The cell-type-specific response of neural cells to oxidative stress, a crucial mechanism for accelerating aging and cognitive dysfunction in Alzheimer's disease (AD), is still far from understood. Here, we employed human-induced pluripotent stem cells (hiPSCs)-derived neural stem cells (hiPSC-NSCs), neurons (hiPSC-Neurons), and microglia-like cells (hiPSC-MGLs) from sporadic AD (sAD) patients, age-matched cognitive normal controls (CNCs), and young subjects to observe human neural cell-type response to H2O2 stimulation. Without H2O2 exposure, reactive oxygen species (ROS) cannot be detected in hiPSC-NSCs from all three groups, but the viability of hiPSC-NSCs from AD patients was significantly lower than those of CNCs and young subjects. There were no significant differences in ROS, viabilities, neurite length, and neurite branch points in hiPSC-Neurons among three groups. No significant differences in viabilities, phagocytosis, and secretion of cytokines were observed in hiPSC-MGLs among three groups, but higher ROS levels in sAD hiPSC-MGLs. Under H2O2 exposure, the viability, neurite length, and neurite branch points of hiPSC-Neurons from AD patients reduced more significantly accompanied by more ROS release. H2O2 exposure caused hiPSC-MGLs from AD patients to release more ROS, cytokines, and stronger phagocytosis. Nevertheless, H2O2 exposure had no effect on viability of hiPSC-NSCs. Our results showed hiPSC-Neurons and hiPSC-MGLs were more sensitive to H2O2 than hiPSC-NSCs, which indicated the different response styles of hiPSC-NSCs, hiPSC-Neurons, and hiPSC-MGLs to oxidative stress. HiPSC-derived neural cells from AD patients suffered more severe injury from H2O2 than those of CNCs and young subjects, indicating that the vulnerability to oxidative stress of AD patients can be recapitulated in hiPSCs.

Functions of Small Organic Compounds that Mimic the HNK-1 Glycan.

Because of the importance of the HNK-1 carbohydrate for preferential motor reinnervation after injury of the femoral nerve in mammals, we screened NIH Clinical Collection 1 and 2 Libraries and a Natural Product library comprising small organic compounds for identification of pharmacologically useful reagents. The reason for this attempt was to obviate the difficult chemical synthesis of the HNK-1 carbohydrate and its isolation from natural sources, with the hope to render such compounds clinically useful. We identified six compounds that enhanced neurite outgrowth from cultured spinal motor neurons at nM concentrations and increased their neurite diameter, but not their neurite branch points. Axons of dorsal root ganglion neurons did not respond to these compounds, a feature that is in agreement with their biological role after injury. We refer to the positive functions of some of these compounds in animal models of injury and delineate the intracellular signaling responses elicited by application of compounds to cultured murine central nervous system neurons. Altogether, these results point to the potential of the HNK-1 carbohydrate mimetics in clinically-oriented settings.

N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity

BackgroundDrug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment.MethodsVincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance.ResultsCEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing.ConclusionsThe N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.

A heterogeneous tRNA granule structure exhibiting rapid, bi-directional neuritic transport

mRNA translation is regulated by diverse mechanisms that converge at the initiation and elongation steps to determine the rate, profile, and localization of proteins synthesized. A consistently relevant feature of these mechanisms is the spatial re-distribution of translation machinery, a process of particular importance in neural cells. This process has, however, been largely overlooked with respect to its potential role in regulating the local concentration of cytoplasmic tRNAs, even as a multitude of data suggest that spatial regulation of the tRNA pool may help explain the remarkably high rates of peptide elongation. Here, we report that Cy3/Cy5-labeled bulk tRNAs transfected into neural cells distribute into granule-like structures – “tRNA granules” – that exhibit dynamic mixing of tRNAs between granules and rapid, bi-directional vectorial movement within neurites. Imaging of endogenous tRNAgly and tRNAlys by fluorescent in situ hybridization revealed a similar granular distribution of tRNAs in somata and neurites; this distribution was highly overlapping with granules imaged by introduction of exogenous Cy5-tRNAthr and Cy3-tRNAval. A subset of tRNA granules located in the cell body, neurite branch points and growth cones displayed fluorescence resonance energy transfer (FRET) between Cy3 and Cy5-labeled tRNAs indicative of translation, and co-localization with elongation machinery. A population of smaller, rapidly trafficked granules in neurites lacked FRET and showed poor colocalization with translation initiation and elongation factors, suggesting that they are a translationally inactive tRNA transport particle. Our data suggest that tRNAs are packaged into granules that are rapidly transported to loci where translation is needed, where they may greatly increase the local concentration of tRNAs in support of efficient elongation. The potential implications of this newly described structure for channeling of elongation, local translation, and diseases associated with altered tRNA levels or function are discussed.

Repositioning again of zonisamide for nerve regeneration.

Repositioning again of zonisamide for nerve regeneration.

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

PurposeRGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.Methods661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.ResultsTreatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.Conclusions661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.

Transcriptional response of rat frontal cortex following acute In Vivo exposure to the pyrethroid insecticides permethrin and deltamethrin

BackgroundPyrethroids are neurotoxic pesticides that interact with membrane bound ion channels in neurons and disrupt nerve function. The purpose of this study was to characterize and explore changes in gene expression that occur in the rat frontal cortex, an area of CNS affected by pyrethroids, following an acute low-dose exposure.ResultsRats were acutely exposed to either deltamethrin (0.3 – 3 mg/kg) or permethrin (1 – 100 mg/kg) followed by collection of cortical tissue at 6 hours. The doses used range from those that cause minimal signs of intoxication at the behavioral level to doses well below apparent no effect levels in the whole animal. A statistical framework based on parallel linear (SAM) and isotonic regression (PIR) methods identified 95 and 53 probe sets as dose-responsive. The PIR analysis was most sensitive for detecting transcripts with changes in expression at the NOAEL dose. A sub-set of genes (Camk1g, Ddc, Gpd3, c-fos and Egr1) was then confirmed by qRT-PCR and examined in a time course study. Changes in mRNA levels were typically less than 3-fold in magnitude across all components of the study. The responses observed are consistent with pyrethroids producing increased neuronal excitation in the cortex following a low-dose in vivo exposure. In addition, Significance Analysis of Function and Expression (SAFE) identified significantly enriched gene categories common for both pyrethroids, including some relating to branching morphogenesis. Exposure of primary cortical cell cultures to both compounds resulted in an increase (~25%) in the number of neurite branch points, supporting the results of the SAFE analysis.ConclusionIn the present study, pyrethroids induced changes in gene expression in the frontal cortex near the threshold for decreases in ambulatory motor activity in vivo. The penalized regression methods performed similarly in detecting dose-dependent changes in gene transcription. Finally, SAFE analysis of gene expression data identified branching morphogenesis as a biological process sensitive to pyrethroids and subsequent in vitro experiments confirmed this predicted effect. The novel findings regarding pyrethroid effects on branching morphogenesis indicate these compounds may act as developmental neurotoxicants that affect normal neuronal morphology.

The phosphatidylinositol-3 kinase (PI3K)-Akt pathway suppresses neurite branch formation in NGF-treated PC12 cells.

Previous studies have shown that phosphatidylinositol-3 kinase (PI3K) plays an important role in NGF (nerve growth factor)-induced neurite elongation. However, the roles of the PI3K pathway in neurite branch formation were not fully understood. Also, it was not clear where the PI3K pathway is activated during branch formation. We found that the treatment of PC12 cells with the PI3K inhibitor LY294002 resulted in a marked increase in the number of neurite branch points, suggesting a suppressive role of PI3K in neurite branch formation. Expression of a constitutively active form of Akt, a downstream effector of PI3K, decreased the number of branch points, whereas that of a dominant-negative form of Akt increased it. In contrast, inhibition of neither Rac, mTOR nor GSK3, other effectors of PI3K, promoted branch formation. Importantly, the phosphorylated form of endogenous Akt was localized at the tips of growth cones, but devoid of small branches in NGF-treated PC12 cells. A GFP-fusion protein of the plekstrin-homology (PH) domain of Akt was also localized at the tips of growth cones. The PI3K-Akt pathway thus plays a key role in suppression of neurite branch formation in NGF-treated PC12 cells.

Morphological Deficits in Noradrenergic Neurons in GEPR-9s Stem from Abnormalities in both the Locus Coeruleus and Its Target Tissues

The epileptic condition of the genetically epilepsy-prone rat (GEPR) appears to be caused partially by deficiencies in the locus coeruleus (LC) innervation of the superior colliculus (SC). Previous studies provide quantitative documentation of noradrenergic morphological deficits in the moderately epileptic GEPR-3. The present findings extend these studies by applying cell culture methodology to assessments of the severely epileptic GEPR-9. Our data show that total neurite length, the number of neurite branch points per cell, the cross-sectional area of cell bodies, and the cell perimeter are deficient in noradrenergic neurons in LC + SC cocultures derived exclusively from GEPR-9s compared to analogous cocultures obtained solely from nonepileptic control rats. Partial restoration of LC neuron morphology toward normal occurs when the GEPR-9 SC component of the coculture is replaced with nonepileptic control SC. Finally, when the GEPR-9 SC is cocultured with the control LC, a partial morphological deficit occurs in the otherwise normal noradrenergic neurons. However, the magnitude of this deficit is less than that observed in noradrenergic neurons of the GEPR-9 LC cocultured with the control SC. These data support the hypothesis that the developmental deficiencies of noradrenergic neurons of the GEPR-9 are derived from two sources, the LC and its target tissue, in this case, the SC. Also, intrinsic abnormalities of the LC appear to make a more pronounced contribution to the noradrenergic deficits than do those which reside in the SC.

NMDA sensitivity is neurite enhanced.

Glutamate toxicity in retinal ganglion cells has been well documented both in vitro and in vitro, and may play a role in both normal neuronal development and a variety of pathological states. Glutamate receptors are found on cell bodies and neuronal processes, both axons and dendrites. Other work has suggested that one or more of these locales may play a more pronounced role in glutamate-mediated toxicity. We now report that N-methyl-D-aspartate (NMDA) is more toxic to retinal ganglion cells with neurites. Cells without neurites were relatively unaffected by glutamate or NMDA. Cells with longer neurites or more neurite branch points were more likely to sustain NMDA-mediated neurotoxicity. These observations suggest that glutamate-mediated loss may be mediated through NMDA receptors found on neurites, rather than through a direct effect on the cell body.

The NeurotroDhins BDNF, NT‐3 and NT‐4/5 Promote Survival and Morphological and Biochemical Differentiation of Striatal Neurons In Vitro

The neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5) and nerve growth factor (NGF), were compared for their effects on the survival and differentiation of embryonic rat striatal neurons grown in low-density cultures. Treatment with BDNF for 8 days resulted in a 40% increase in overall neuronal survival, a 3- to 5-fold increase in the number of calbindin-immunoreactive neurons, and an 80% increase in GABA-positive neurons. Treatment with NT-3 or NT-4/5 produced a 2- to 3-fold increase in the number of calbindin-positive neurons and an increase in GABA-positive cell number similar to that induced by BDNF, BDNF treatment produced a striking morphological differentiation of striatal GABAergic neurons, which was characterized by a doubling of the number of neurite branch points, the total area of aborization and the perikaryal area compared to control cultures. All three of these factors increased high-affinity GABA uptake 2-fold. NGF had no effect on any of the parameters examined. Our results show that BDNF, NT-3 and NT-4/5 promote the survival and/or differentiation of calbindin-immunopositive and GABAergic striatal neurons.

Neurite Branch Development of an Identified Serotonergic Neuron from Embryonic Helisoma: Evidence for Autoregulation by Serotonin

Previous studies have shown that in select neurons, neurite outgrowth can be regulated by the same neurotransmitter that is synthesized and released by those neurons. However, it is not known whether such an autoregulatory mechanism is utilized during the normal course of nervous system development in either invertebrates or vertebrates. In the present study, we tested this hypothesis on the first pair of identified serotonergic neurons to be expressed in embryos of the pulmonate gastropod, Helisoma trivolvis. Embryonic neurons C1 (ENC1) elaborate a stereotyped pattern of neurite outgrowth prior to the differentiation of subsequent serotonergic neurons. Embryos were treated with either p-chlorophenylalanine (pCPA) or 5-hydroxytryptophan (5-HTP) to lower or raise embryonic serotonin content, respectively. High-performance liquid chromatography with electrochemical detection was used to measure the effects of these treatments on serotonin content, and serotonin immunohistochemistry was carried out to quantify the extent of neurite outgrowth of ENC1. Embryonic serotonin content was significantly reduced at both 24 and 48 hr after treatment with 0.02% pCPA, whereas dopamine levels were unchanged. Although the proximal neurite outgrowth of ENC1 appeared unaffected by the pCPA treatment at both of these time points, the distal outgrowth in the target cell region appeared more profuse. This effect on outgrowth was quantified by counting the number of neurite branch points, which was significantly increased both 24 and 48 hr after pCPA treatment. In contrast, 5-HTP treatment resulted in an increase in embryonic serotonin content and a significant decrease in the number of ENC1 branch points. Treatment with dopamine had no effect on the pattern of ENC1 neurite outgrowth. Together, these results support the hypothesis that a neuron my utilize its own transmitter in an autoregulatory fashion to regulate neurite formation during embryonic development.

Analysis of microtubule number and length in cytoskeletons of cultured chick sensory neurons

In order to examine the continuity of individual microtubules in growing neurites, microtubules were observed and counted in whole mounted cytoskeletons of chick embryo sensory neurons. At neurite branch points, the microtubules of the proximal segment are divided between the distal segments without the addition of microtubules to the distal branches. Few microtubules ends were seen in the cytoskeletons, and on occasion, the same number of microtubules was counted at widely separated points along a neurite segment. I conclude that microtubules are long in embryonal neurites, and some may extend the entire length from perikaryon to neurite tip. This may limit where microtubule growth occurs to the perikaryon and the neurite tip, where many microtubule ends are. In addition, the great length of most neuritic microtubules may explain why collateral sprouting is rare along embryonal neurites.