Neuritic Processes Research Articles

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.

Intraganglionic interactions between satellite cells and adult sensory neurons.

Perineuronal satellite cells have an intimate anatomical relationship with sensory neurons that suggests close functional collaboration and mutual support. We examined several facets of this relationship in adult sensory dorsal root ganglia (DRG). Collaboration included the support of process outgrowth by clustering of satellite cells, induction of distal branching behavior by soma signaling, the capacity of satellite cells to respond to distal axon injury of its neighboring neurons, and evidence of direct neuron-satellite cell exchange. In vitro, closely adherent coharvested satellite cells routinely clustered around new outgrowing processes and groups of satellite cells attracted neurite processes. Similar clustering was encountered in the pseudounipolar processes of intact sensory neurons within intact DRG in vivo. While short term exposure of distal growth cones of unselected adult sensory neurons to transient gradients of a PTEN inhibitor had negligible impacts on their behavior, exposure of the soma induced early and substantial growth of their distant neurites and branches, an example of local soma signaling. In turn, satellite cells sensed when distal neuronal axons were injured by enlarging and proliferating. We also observed that satellite cells were capable of internalizing and expressing a neuron fluorochrome label, diamidino yellow, applied remotely to distal injured axons of the neuron and retrogradely transported to dorsal root ganglia sensory neurons. The findings illustrate a robust interaction between intranganglionic neurons and glial cells that involve two way signals, features that may be critical for both regenerative responses and ongoing maintenance.

Vulnerability of synapses in the frontal cortex of mice developmentally exposed to an insecticide: Potential contribution to neuropsychiatric disease.

Increasingly, exposure to various chemicals found in our environment has been found to be a significant contributor to the risk of developing neurological disease, such as Parkinson disease, autism spectrum disorder, as well as other deficits in thought and function. Exposure to these compounds during critical periods of neurodevelopment, encompassing exposures that occur in utero, during infancy, childhood, and adolescence, represents a time period of nervous system growth that is uniquely vulnerable to disruption by environmental chemicals. Indeed, a contemporary hypothesis suggests that the pathological cascade associated with many common neurological disorders has its origin in disturbances of normal neurodevelopment. Moreover, alterations to the ontogeny of the synapse and neurotransmitter signaling during neurodevelopment may be a premier pathological event that underlies neuropsychiatric and neurodegenerative disease. To interrogate the impact of exposure to a ubiquitous environmental chemical, the pesticide, endosulfan, on development of neurotransmitter circuits, we coupled in vitro and in vivo platforms to evaluate its effect on the formation of GABAergic, glutamatergic, and dopaminergic pathways in the frontal cortex. With this approach we found exposure of cortical neurons, in vitro, exhibited a marked reduction in the length of their neurite process as well as the number of synaptic connections. Further investigation using an in vivo model of developmental exposure identified significant alterations to pre and postsynaptic proteins involved in neurotransmitter handling and signaling in each of the neurotransmitter systems investigated. These findings suggest that exposure to endosulfan during vulnerable periods of neurodevelopment can alter the normal development and potential function of neurotransmission in the frontal cortex. Interestingly, the alterations identified in our study closely mimic the pathological markers associated with schizophrenia, which shows disturbances in synaptic proteins important for GABAergic, glutamatergic, and dopaminergic signaling in the frontal cortex. These findings provide important support for the impact of exposure to environmental chemicals during neurodevelopment and risk for neurological disease.

RNA and the synapse.

RNA and the synapse.

Nanoscale neuroelectrode modification via sub-20nm silicon nanowires through self-assembly of block copolymers.

Neuroprosthetic technologies for therapeutic neuromodulation have seen major advances in recent years but these advances have been impeded due to electrode failure or a temporal deterioration in the device recording or electrical stimulation potential. This deterioration is attributed to an intrinsic host tissue response, namely glial scarring or gliosis, which prevents the injured neurons from sprouting, drives neurite processes away from the neuroelectrode and increases signal impedance by increasing the distance between the electrode and its target neurons. To address this problem, there is a clinical need to reduce tissue encapsulation of the electrodes in situ and improve long-term neuroelectrode function. Nanotopographical modification has emerged as a potent methodology for the disruption of protein adsorption and cellular adhesion in vitro. This study investigates the use of block copolymer self-assembly technique for the generation of sub-20nm nanowire features on silicon substrates. Critically, these nanostructures were observed to significantly reduce electrical impedance and increase conductivity. Human neuroblastoma SH-SY5Y cells cultured on nanowire substrates for up to 14days were associated with enhanced focal adhesion reinforcement and a reduction in proliferation. We conclude that nanowire surface modulation may offer significant potential as an electrode functionalization strategy.

A targeted secretome profiling by multiplexed immunoassay revealed that secreted chemokine ligand 2 (MCP-1/CCL2) affects neural differentiation in mesencephalic neural progenitor cells.

Chemokines and cytokines, primarily known for their roles in the immune and inflammatory response, have also been identified as key components of the neurogenic niche where they are involved in the modulation of neural stem cell proliferation and differentiation. However, a complete understanding of the functional role played in neural differentiation and a comprehensive profiling of these secreted molecules are lacking. By exploiting the multiplexing capability of magnetic bead-based immunoassays, we have investigated the changes of the expression levels of a set of chemokines and cytokines released from the pluripotent neural cell line mes-c-myc A1 following its differentiation from a proliferating phenotype (A1P) toward a neural (A1D) phenotype. We found a subset of molecules exclusively released from A1P, whereas others were differentially detected in A1P and A1D conditioned media. Among them, we identified monocyte chemoattractant protein-1/chemokine ligand 2 (MCP-1/CCL2) as a proneurogenic factor able to affect neuronal differentiation of A1 cells as well as of neuroblasts from primary cultures and to induce the elongation and/or formation of neuritic processes. Altogether, data are suggestive of a main role played by the CCL2/CCR2 signaling pathway and in general of the network of secreted cytokines/chemokines in the differentiation of neural progenitor cells toward a neural fate.

Silk–tropoelastin protein films for nerve guidance

Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk–tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly-d-lysine. When patterned by drying on grooved polydimethylsiloxane (3.5μm groove width, 0.5μm groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk–tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs.

A knockin mouse model of spinocerebellar ataxia type 3 exhibits prominent aggregate pathology and aberrant splicing of the disease gene transcript.

Polyglutamine diseases, including spinocerebellar ataxia type 3 (SCA3), are caused by CAG repeat expansions that encode abnormally long glutamine repeats in the respective disease proteins. While the mechanisms underlying neurodegeneration remain uncertain, evidence supports a proteotoxic role for the mutant protein dictated in part by the specific genetic and protein context. To further define pathogenic mechanisms in SCA3, we generated a mouse model in which a CAG expansion of 82 repeats was inserted into the murine locus by homologous recombination. SCA3 knockin mice exhibit region-specific aggregate pathology marked by intranuclear accumulation of the mutant Atxn3 protein, abundant nuclear inclusions and, in select brain regions, extranuclear aggregates localized to neuritic processes. Knockin mice also display altered splicing of the disease gene, promoting expression of an alternative isoform in which the intron immediately downstream of the CAG repeat is retained. In an independent mouse model expressing the full human ATXN3 disease gene, expression of this alternatively spliced transcript is also enhanced. These results, together with recent findings in other polyglutamine diseases, suggest that CAG repeat expansions can promote aberrant splicing to produce potentially more aggregate-prone isoforms of the disease proteins. This report of a SCA3 knockin mouse expands the repertoire of existing models of SCA3, and underscores the potential contribution of alternative splicing to disease pathogenesis in SCA3 and other polyglutamine disorders.

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

Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.

Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.

Correspondence amongst the PENO Test Battery Cognitive Results and Hippocampal Lesions in Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by a decline of cognitive functions. Distinctive histopathological hallmarks are neuritic plaques, neurofibrillary tangles, and synaptic alterations. Abnormally enlarged synaptic structures called “Meganeurite clusters” have been linked to plasticity changes. The aims of this study were to determine if cognitive impairment was related to specific neuritic and synaptic degeneration processes in patients with AD, and if the results of a cognitive test could be correlated with the histopathological damage. The neuropsychological evaluation obtained by the Protocole d’evaluation neuropsychologique optimal (PENO) test battery was used in live AD and control individuals. The histopathological evaluation of their brain after their death was carried out with specific polyclonal and monoclonal antibodies to Aβ, pTau protein, synaptophysin, and GAP-43. Images were obtained by confocal microscopy. The results showed a significant difference between healthy controls and Alzheimer’s patients in neuropsychological evaluation and histopathological hallmarks expression. The most significant positive correlation in AD patients was between memory and language results with the PENO test and the presence of Aβ +pTau+ plaques in the hippocampus. An interesting negative correlation was between cognitive impairment and the presence of Meganeuritic clusters, considered as “plasticity” markers. These results strongly supported the use of the PENO battery test to evaluate the progression of cognitive impairment in AD prone individuals and patients due to the strong correlation of the test results with histopathological brain lesions characteristic of Alzheimer’s disease.

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.

Spatial Wavelet Analysis of Calcium Oscillations in Developing Neurons

Calcium signals play a major role in the control of all key stages of neuronal development, and in particular in the growth and orientation of neuritic processes. These signals are characterized by high spatial compartmentalization, a property which has a strong relevance in the different roles of specific neuronal regions in information coding. In this context it is therefore important to understand the structural and functional basis of this spatial compartmentalization, and in particular whether the behavior at each compartment is merely a consequence of its specific geometry or the result of the spatial segregation of specific calcium influx/efflux mechanisms. Here we have developed a novel approach to separate geometrical from functional differences, regardless on the assumptions on the actual mechanisms involved in the generation of calcium signals. First, spatial indices are derived with a wavelet-theoretic approach which define a measure of the oscillations of cytosolic calcium concentration in specific regions of interests (ROIs) along a cell, in our case developing chick ciliary ganglion neurons. The resulting spatial profile demonstrates clearly that different ROIs along the neuron are characterized by specific patterns of calcium oscillations. Next we have investigated whether this inhomogeneity is due just to geometrical factors, namely the surface to volume ratio in the different subcompartments (e.g. soma vs. growth cone) or it depends on their specific biophysical properties. To this aim correlation functions are computed between the activity indices and the surface/volume ratio along the cell: the data thus obtained are validated by a statistical analysis on a dataset of different cells. This analysis shows that whereas in the soma calcium dynamics is highly correlated to the surface/volume ratio, correlations drop in the growth cone-neurite region, suggesting that in this latter case the key factor is the expression of specific mechanisms controlling calcium influx/efflux.

Tau Oligomers and Fibrils Induce Activation of Microglial Cells

Neuroinflammation is a process related to the onset of several neurodegenerative disorders, including Alzheimer's disease (AD). Increasing sets of evidence support the major role of deregulation of the interaction patterns between glial cells and neurons in the pathway toward neuronal degeneration, a process we are calling neuroimmunomodulation in AD. On the basis of the hypothesis that pathological tau aggregates induce microglial activation with the subsequent events of the neuroinflammatory cascade, we have studied the effects of tau oligomeric species and filamentous structures over microglial cells in vitro. Tau oligomers and fibrils were induced by arachidonic acid and then their actions assayed upon addition to microglial cells. We showed activation of the microglia, with significant morphological alterations as analyzed by immunofluorescence. The augmentation of nitrites and the proinflammatory cytokine IL-6 was evaluated in ELISA assays. Furthermore, conditioned media of stimulated microglia cells were exposed to hippocampal neurons generating altered patterns in these cells, including shortening of neuritic processes and cytoskeleton reorganization.

Identification of pharmacogenetic target genes associated with chemotherapy-induced peripheral neuropathy.

e13541 Background: Chemotherapy-induced Peripheral Neuropathy (CIPN) is a dose-limiting toxicity of many anticancer drugs, including taxanes, vinca alkaloids and platinating agents. There are no effective means to predict which patients are at risk for CIPN nor are there effective treatments. Therefore, we have used a whole genome approach in lymphoblastoid cell lines (LCLs) alone or combined with clinical study data to identify target genes associated with CIPN that we tested further in Neuroscreen-1 (NS-1: rat pheochromocytoma) cells and human neurons derived from induced pluripotent stem cells (iCell neurons). Methods: For selecting candidate target genes, we compared published genome-wide association studies (GWAS) results of sensory peripheral neuropathy in cancer patients receiving paclitaxel with GWAS results of pharmacologic phenotypes in LCLs after paclitaxel treatment. We evaluated neurotoxic drug-induced cell growth inhibition using Cell Titer Glo and neurite parameters of outgrowth, processes and branching through imaging following transient knockdown of target genes in NS-1 cells and iCell neurons. Results: After treatment of paclitaxel, vincristine and cisplatin, iCell neurons showed decrease of neurite outgrowth, processes and branching. Whereas, these parameters did not decrease for hydroxyurea, which is not reported to induce neuropathy. Through transient knockdown in NS-1 cells of RFX2, a gene identified through clinical and preclinical studies to be associated with paclitaxel-induced neuropathy, sensitivity to paclitaxel increased as measured by a decrease of neurite outgrowth. Conclusions: These findings imply our model system could identify pharmacogenetic target genes associated with CIPN. These genes have potential for biomarkers or therapeutic targets and enable future personalized treatments for cancer patients at risk for CIPN.

Abstract 2210: Integration of preclinical and clinical studies to identify pharmacogenetic markers associated with paclitaxel-induced neuropathy.

Abstract Paclitaxel is a tubulin-targeting agent used in the treatment of many cancers, including breast, lung, head and neck, and ovarian. Peripheral neuropathy is a common side effect of many chemotherapeutic agents, including paclitaxel, and limits their efficacy in patients. The overall goal of our work is to identify patients at risk for peripheral neuropathy using whole genome preclinical and clinical studies. Epstein-Barr virus transformed lymphoblastoid cell lines (LCLs) have emerged as a promising preclinical model system in the study of the genetics of drug toxicity because the cells provide a cost-effective testing system in which environmental factors such as concomitant medications can be controlled. Most importantly, publicly available genotype and sequencing data is available, to allow for genome-wide association studies between HapMap/1000 Genomes variants and pharmacologic phenotypes measured in the LCLs after drug treatment such as paclitaxel induced growth inhibition and apoptosis. The overall genetic architecture of paclitaxel-induced neuropathy is well captured by the cell-based models as evidenced by significant enrichment of paclitaxel-induced toxicity SNPs from the preclinical model in clinical genome-wide association studies of peripheral neuropathy. We utilized iCell Neurons, which are commercially available cells derived from induced pluripotent stem cells, to study the effects of gene modulation on paclitaxel-induced cell growth inhibition using Cell Titer Glo and on neurite parameters of outgrowth, processes and branching using the ImageXpress Micro system. Through transient knockdown in iCell Neurons of RFX2, a gene identified through clinical and preclinical studies to be associated with paclitaxel neuropathy, sensitivity to paclitaxel increased as measured by a decrease of neurite outgrowth, processes and branching. The ability to identify cancer patients at risk of paclitaxel-induced peripheral neuropathy could influence treatment choice, outcome and quality of life for breast, lung, head and neck, and ovarian cancer patients. This work was supported in part by NIH/NIGMS UO1GM61393. Citation Format: Heather E. Wheeler, Cristina Rodriguez-Antona, Claudia Wing, Masaaki Komatsu, M. Eileen Dolan. Integration of preclinical and clinical studies to identify pharmacogenetic markers associated with paclitaxel-induced neuropathy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2210. doi:10.1158/1538-7445.AM2013-2210

Abstract 5040: CASZ1 haploinsufficiency in murine embryonic stem cells causes increased MYC expression, aberrant lineage commitment, and prevents neurogenesis.

Abstract Background: Tumor suppressor CASZ1(1p36.22) is often lost in poor prognosis neuroblastomas (NB) with an undifferentiated histopathology. Differentiation of NB cells upon CASZ1 restoration indicates CASZ1 loss may contribute to the undifferentiated phenotype during NB tumorigenesis. How CASZ1 functions during normal mammalian development is unknown. Using a murine embryonic stem cell (mESC) embryogenesis model, we investigated the effects of CASZ1 haploinsufficiency and CASZ1 reconstitution on differentiation programs. Methods: Differentiation in mESCs, wild-type (WT) and Casz1 haploinsufficient (CAS+/- mESCs), was evaluated using mESC embryoid body (EB) neurogenesis and adipogenesis assays. For rescue experiments, CAS+/- mESCs were transfected with a bacterial artificial chromosome containing the full human CASZ1 genomic locus (CASBAC). Gene expression was evaluated by qPCR and western blot. Results: Analysis of CAS+/- mESCs revealed pluripotency gene expression was relatively unaffected by CASZ1 loss, but there is higher Myc mRNA (∼5.5 fold) compared to WT mESCs. During neural differentiation Myc is aberrantly expressed as evidenced by increased protein levels compared to the mESC state. Four days after LIF withdrawal, CAS+/- EBs have ∼2.5 fold higher MYC levels as compared to WT EBs that have half the MYC levels as compared to the mESC state. Coincident with aberrant MYC expression CAS+/- mESCs exhibit a ∼2.8 fold increase in Gata4 mRNA (endoderm lineage) and a ∼3.2 fold increase in Gata2 mRNA (mesoderm lineage) compared to WT mESCs. Functionally, CAS+/- mESCs retain adipogenesis potential. However after neural induction, CAS+/- EBs exhibit decreased neuroectoderm lineage commitment with neural lineage marker Pax6 mRNA levels some ∼8.9 fold less than WT EBs. Neurogenesis potential is lost as evidenced by a failure to extend ßIII tubulin positive neuritic processes. However, CASBAC mESCs restore Myc regulation, lineage commitment, and neurogenesis potential. Four days after LIF withdrawal, CASBAC EBs have ∼0.6 fold MYC levels as compared to CASBAC mESCs, much like WT EBs. Lineage markers Gata4 and Gata2 also normalize to ∼0.7 and ∼0.5 times WT EB levels, respectively. Pax6 levels in CASBAC EBs are also restored to ∼1.8 fold WT EB levels. Most noticeable is the restoration of full neurogenesis potential as evidenced by ßIII tubulin positive neuritic processes. Conclusions: The data shows CAS+/- mESCs are unable to implement a neural differentiation program. We propose that CASZ1 haploinsufficiency causes misalignment of spatial and temporal transcription factor networks necessary for proper neurogenesis. Perturbation of neurogenesis results in latent proliferative states in neural precursors, which may contribute to tumorigenesis and the undifferentiated histopathology of poor prognosis NBs. Citation Format: Stanley He, Ryan Virden, Zhihui LIu, Carol J. Thiele. CASZ1 haploinsufficiency in murine embryonic stem cells causes increased MYC expression, aberrant lineage commitment, and prevents neurogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5040. doi:10.1158/1538-7445.AM2013-5040

Calcium signals and FGF-2 induced neurite growth in cultured parasympathetic neurons: spatial localization and mechanisms of activation

The growth of neuritic processes in developing neurons is tightly controlled by a wide set of extracellular cues that act by initiating downstream signaling cascades, where calcium signals play a major role. Here we analyze the calcium dependence of the neurite growth promoted by basic fibroblast growth factor (bFGF or FGF-2) in chick embryonic ciliary ganglion neurons, taking advantage of dissociated, organotypic, and compartmentalized cultures. We report that signals at both the growth cone and the soma are involved in the promotion of neurite growth by the factor. Blocking calcium influx through L- and N-type voltage-dependent calcium channels and transient receptor potential canonical (TRPC) channels reduces, while release from intracellular stores does not significantly affect, the growth of neuritic processes. Simultaneous recordings of calcium signals elicited by FGF-2 at the soma and at the growth cone show that the factor activates different patterns of responses in the two compartments: steady and sustained responses at the former, oscillations at the latter. At the soma, both voltage-dependent channel and TRPC blockers strongly affect steady-state levels. At the growth cone, the changes in the oscillatory pattern are more complex; therefore, we used a tool based on wavelet analysis to obtain a quantitative evaluation of the effects of the two classes of blockers. We report that the oscillatory behavior at the growth cone is dramatically affected by all the blockers, pointing to a role for calcium influx through the two classes of channels in the generation of signals at the leading edge of the elongating neurites.

Biocompatibility of pristine graphene for neuronal interface

Graphene possesses unique electrical, physical, and chemical properties that may offer significant potential as a bioscaffold for neuronal regeneration after spinal cord injury. The purpose of this investigation was to establish the in vitro biocompatibility of pristine graphene for interface with primary rat cortical neurons. Graphene films were prepared by chemical vapor deposition on a copper foil catalytic substrate and subsequent apposition on bare Permanox plastic polymer dishes. Rat neuronal cell culture was grown on graphene-coated surfaces, and cell growth and attachment were compared with those on uncoated and poly-d-lysine (PDL)-coated controls; the latter surface is highly favorable for neuronal attachment and growth. Live/dead cell analysis was conducted with flow cytometry using ethidium homodimer-1 and calcein AM dyes. Lactate dehydrogenase (LDH) levels-indicative of cytotoxicity-were measured as markers of cell death. Phase contrast microscopy of active cell culture was conducted to assess neuronal attachment and morphology. Statistically significant differences in the percentage of live or dead neurons were noted between graphene and PDL surfaces, as well as between the PDL-coated and bare surfaces, but there was little difference in cell viability between graphene-coated and bare surfaces. There were significantly lower LDH levels in the graphene-coated samples compared with the uncoated ones, indicating that graphene was not more cytotoxic than the bare control surface. According to phase contrast microscopy, neurons attached to the graphene-coated surface and were able to elaborate long, neuritic processes suggestive of normal neuronal metabolism and morphology. Further use of graphene as a bioscaffold will require surface modification that enhances hydrophilicity to increase cellular attachment and growth. Graphene is a nanomaterial that is biocompatible with neurons and may have significant biomedical applications.

Cellular and ultra structural evidence for cytoskeletal localization of prolyl endopeptidase-like protein in neurons

The biochemical properties and subcellular localization of prolyl endopeptidase (PREP) in brain are well characterized and its implications in the realization of cognitive processes and in the pathogenesis of neurodegenerative disorders are a matter of intensive investigation. In contrast, very little is known about its homolog, the PREP-like protein (PREPL). In order to obtain initial hints about the involvement of PREPL in physiological processes, a differential proteomic screen was performed with human skin fibroblasts from controls and patients with PREPL deficiency (hypotonia–cystinuria syndrome). The majority of affected proteins represented cytoskeletal proteins, including caldesmon, tropomyosin α3 chain, lamin A, β-actin, γ-actin, vimentin and zyxin. Therefore, the analysis of PREPL subcellular localization by confocal laser scanning and electron microscopy in mouse neurons was focused on the cytoskeleton. The co-localization of PREPL with cytoskeletal marker proteins such as β-actin and microtubulin-associated protein-2 was observed, in addition to the presence of PREPL within Golgi apparatus and growth cones. In the mouse brain, PREPL is neuronally expressed and highly abundant in neocortex, substantia nigra and locus coeruleus. This mirrors to some extent the distribution pattern of PREP and points toward redundant functions of both proteins. In the human neocortex, PREPL immunostaining was found in the cytoplasm and in neuropil, in particular of layer V pyramidal neurons. This staining was reduced in the neocortex of Alzheimer’s disease (AD) patients. Moreover, in AD brains, PREPL immunoreactivity was observed in the nucleus and in varicose neuritic processes. Our data indicate physiological functions of PREPL associated with the cytoskeleton, which may be affected under conditions of cytoskeletal degeneration.