Neurite Network Research Articles

Geometrically Mediated Topographic Steering of Neurite Behaviors and Network Formation

AbstractThe relationship between different mechanoregulatory factors and mechanisms by which neuronal development is regulated by diverse biophysical cues in the immediate vicinity of neural cells remains sparsely understood. Considering the extreme physical complexity of neuronal niches, it is imperative to look beyond the chemical cues for a holistic understanding in these lines. Here the role of geometrically diverse physical microcues is studied in morphologically regulating neurite branching, direction, growth, and network formation in nascent primary cortical neurons on a simple cytocompatible interface. The results indicate distinct differences in neurite branching, microgap sensing, and path‐finding tendencies in response to the underlying angled/curved cues on the micropatterned interface. While the microtraps along curved cues promote higher branching, the highly angled cues promote relatively lower branching with higher neurite lengths. In this study specific morphological changes induced by these cues on the collective conformity, branching, and mean length of neurites in both isolated neurons and neurons in networks are investigated. Finally, a model is proposed to study mechanoregulation of isolated neurons and neuronal networks in response to diverse physical cues. Overall, this platform aims to propel efficient morphological modulation for the study of neurodevelopmental anomalies or physical guidance inspired repair strategies.

The plasma membrane calcium pumps-The old and the new.

The plasma membrane Ca2+-ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca2+) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca2+ [Ca2+]i. Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca2+]i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration.

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry.

Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure culture of CGN from postnatal rats/mice and an easy procedure for their intoxication with CNT. We also illustrate handy methods to evaluate CNT activity and their inhibition.

NeuronRead, an open source semi-automated tool for morphometric analysis of phase contrast and fluorescence neuronal images.

Neurons are specialized cells of the Central Nervous System whose function is intricately related to the neuritic network they develop to transmit information. Morphological evaluation of this network and other neuronal structures is required to establish relationships between neuronal morphology and function, and may allow monitoring physiological and pathophysiologic alterations. Fluorescence-based microphotographs are the most widely used in cellular bioimaging, but phase contrast (PhC) microphotographs are easier to obtain, more affordable, and do not require invasive, complicated and disruptive techniques. Despite the various freeware tools available for fluorescence-based images analysis, few exist that can tackle the more elusive and harder-to-analyze PhC images. To surpass this, an interactive semi-automated image processing workflow was developed to easily extract relevant information (e.g. total neuritic length, average cell body area) from both PhC and fluorescence neuronal images. This workflow, named 'NeuronRead', was developed in the form of an ImageJ macro. Its robustness and adaptability were tested and validated on rat cortical primary neurons under control and differentiation inhibitory conditions. Validation included a comparison to manual determinations and to a golden standard freeware tool for fluorescence image analysis. NeuronRead was subsequently applied to PhC images of neurons at distinct differentiation days and exposed or not to DAPT, a pharmacological inhibitor of the γ-secretase enzyme, which cleaves the well-known Alzheimer's amyloid precursor protein (APP) and the Notch receptor. Data obtained confirms a neuritogenic regulatory role for γ-secretase products and validates NeuronRead as a time- and cost-effective useful monitoring tool.

EFFECTS OF CIRCULATING AMYLOID PEPTIDE ON BBB INTEGRITY, CONSEQUENCES ON THE NEURODEGENERATION PROCESS

Recent papers have shown that Alzheimer disease [AD] is associated with alterations of circulating amyloid peptide 1–42 levels [Aβ1–42], possibly due to alteration of blood brain barrier permeability [BBB]. However, no study deals with the effect of circulating Aβ1–42 on BBB permeability, especially on normal one. The purpose of the present work is to study, in an in vitro model of BBB, fluxes of Aβ1–42 between cerebral and blood compartments, under normal and pathological conditions. The model used here is a tridimensional one, consisting of two different compartments mimicking cerebral (neurons) and blood compartments, separated by a complex cell layer made of vascular endothelial cells and astrocytes, grown on two opposed faces of the artificial membrane of an insert. In the models presented here, we analyzed the effect of cerebral or blood Aβ1–42 on BBB permeability (fluorescein passage), and the protective effects of β-estradiol and BDNF, under the two conditions (integrity of neurons evaluated by maintenance of neurite network integrity and survival). Additionally, we studied the modification of BBB resistance and physical integrity (detection of occludine and ZO1) of endothelial layer tight junctions. We showed that circulating Aβ1–42 induced BBB damages (junctions and permeability) with severe consequences on the neuronal integrity. These effect could be partially protected in presence of β-estradiol and BDNF. Our findings link BBB dysfunction with amyloid toxicity. These findings prompt for future studies investigating molecular mechanisms of BBB damages in AD and impact of therapeutic interventions that target these mechanisms.

Modest Static Pressure Can Cause Enteric Nerve Degeneration Through Ectodomain Shedding of Cell Adhesion Molecule 1.

Internal pressure is often involved in neurodegeneration; intraocular and intraventricular pressure elevations over 20-30cmH2O cause glaucoma and hydrocephalus, respectively. Here, we investigated enteric nerve degeneration in colon segments having tumor-induced stenosis and dilation and examined the mechanism of intraluminal pressure involvement. Histological examination revealed that the enteric ganglion neurons and neurites decreased in density in the dilated colons proportionate to the degree of dilation. Western blot analysis for cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member expressed in enteric neurons, revealed that ectodomain shedding of CADM1 increased proportionate to colon dilation, with increased production of its C-terminal fragment αCTF, a proapoptotic intracellular molecule. To link these neurodegenerative events to increased intraluminal pressure, we devised a two-chamber culture system wherein cells cultured on a semipermeable membrane were subjected to increased medium height (water pressure up to 50cmH2O). Mouse dorsal root ganglion (DRG) neurons were examined for expansion of their neurite networks in this system. As the pressure increased to 15, 30, and 45cmH2O, the neurites decreased in density and became thinner. In addition, CADM1 shedding increased with more αCTF production. CADM1 immunofluorescence and Mitotracker mitochondrial labeling revealed that as the pressure increased, neuritic CADM1 distribution changed from uniform to punctate staining patterns, and neuritic mitochondria decreased in number and appeared as course particles. These pressure-induced phenotypes were reproduced by exogenous expression of αCTF in standard DRG neuron cultures. Therefore, increases in colonic intraluminal pressure might cause enteric nerve degeneration by inducing CADM1 shedding and αCTF production.

Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy.

Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell‐signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small‐molecule inhibitors of key signaling pathways in an 8‐day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin‐sensitive action potentials in patch‐clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact‐dependent cues to commit bone marrow‐derived Schwann cell‐like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow‐derived Schwann cells become available for autologous transplantation and remyelination therapy. Stem Cells Translational Medicine 2017;6:369–381

Simultaneous recording of the action potential and its whole-cell associated ion current on NG108-15 cells cultured over a MWCNT electrode

It is well known that, in excitable cells, the dynamics of the ion currents (Ii) is extremely important to determine both the magnitude and time course of an action potential (Ap). To observe these two processes simultaneously, we cultured NG108-15 cells over a multi-walled carbon nanotubes electrode (MWCNTe) surface and arranged a two independent Patch Clamp system configuration (Bi-Patch Clamp). The first system was used in the voltage or current clamp mode, using a glass micropipette as an electrode. The second system was modified to connect the MWCNTe to virtual ground. While the Ap was recorded through the micropipette electrode, the MWCNTe was used to measure the underlying whole-cell current. This configuration allowed us to record both the membrane voltage (Vm) and the current changes simultaneously. Images acquired by atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicate that cultured cells developed a complex network of neurites, which served to establish the necessary close contact and strong adhesion to the MWCNTe surface. These features were a key factor to obtain the recording of the whole-cell Ii with a high signal to noise ratio (SNR). The experimental results were satisfactorily reproduced by a theoretical model developed to simulate the proposed system. Besides the contribution to a better understanding of the fundamental mechanisms involved in cell communication, the developed method could be useful in cell physiology studies, pharmacology and diseases diagnosis.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone‐induced schwannopathy

Amiodarone hydrochloride (AMD), an anti-arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor-primed PC12 cells. Treatment with AMD (1, 5, and 10μm) induced time- and dose-dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2-related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3-II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD-induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time- and dose-dependent manner. These findings suggest that AMD-induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

Assessment of neurotoxic effects of tri-cresyl phosphates (TCPs) and cresyl saligenin phosphate (CBDP) using a combination of in vitro techniques

Environmental exposures to tri-cresyl phosphates (TCPs) and the possible formation of toxic metabolites (e.g. cresyl saligenin phosphate; CBDP) may cause a variety of neurotoxic effects in humans. As reported for other organophosphorus compounds (OPs), the inhibition of acetylcholine esterase (AChE) has also been proposed as the underlying mechanism for TCP neurotoxicity. The ortho-isomer, ToCP and its metabolite CBDP are also known to affect neuropathy target esterase (NTE) leading to organophosphate-induced delayed neuropathy (OPIDN). Recently, in vitro testing has led to the identification of other molecular targets and alternative mechanisms of ToCP toxicity. The metabolite CBDP and other isomers, as well as commercial mixtures have not been tested for such additional modes of actions. Accordingly, the present study investigates alterations of neurobiological correlates of central nervous processes using different in vitro techniques. The three symmetric TCP isomers – ToCP, TpCP, and TmCP – that contain a methyl group at the ortho-, para-, or meta-position of the aromatic ring system, respectively, together with a commercial TCP mixture, and CBDP were all tested using concentrations not exceeding their cytotoxic concentrations. Isolated cortical neurons were kept in culture for 6days followed by 24h incubation with different concentrations of the test compounds. Thus, all endpoints were assessed after 7days in vitro (DIV 7), at which time cell viability, neurite microstructure, and the function of glutamate receptors and voltage-gated calcium cannels (VGCC) were measured. While the cytotoxic potential of the TCP isomers and their mixture were comparable (IC50≥80μM), CBDP was more cytotoxic (IC50: 15μM) to primary cortical neurons. In contrast, CBDP (up to 10μM) did not compromise the microstructure of neurites. Ten μM of ToCP significantly reduced the size and complexity of neurite networks, but neither TmCP and TpCP nor the mixture affected this second endpoint of neurotoxicity assessment. TCPs and their mixture significantly reduced the Ca2+ influx in response to glutamate and KCl stimulation in concentrations of 10μM. Only ToCP showed a specific effect on glutamate receptors with 100nM reducing the evoked Ca2+ influx. The effects of CBDP on the provoked Ca2+ influx were much weaker than those observed for TCPs. These results confirmed that ToCP has a unique mode of action on glutamate receptors that are not observed with the metabolite CBDP and the other symmetric TCP isomers. In addition, the TmCP isomer seems to have the lowest potency with respect to inducing neurotoxic effects. CBDP did not affect the neurospecific endpoints investigated in this study. Therefore, the specific affinity of CBDP for NTE and the reported general cytotoxicity might be the most relevant modes of action of this toxic metabolite in the context of ToCP-induced neurotoxicity, including OPIDN.

Natural lecithin promotes neural network complexity and activity.

Phospholipids in the brain cell membranes contain different polyunsaturated fatty acids (PUFAs), which are critical to nervous system function and structure. In particular, brain function critically depends on the uptake of the so-called “essential” fatty acids such as omega-3 (n-3) and omega-6 (n-6) PUFAs that cannot be readily synthesized by the human body. We extracted natural lecithin rich in various PUFAs from a marine source and transformed it into nanoliposomes. These nanoliposomes increased neurite outgrowth, network complexity and neural activity of cortical rat neurons in vitro. We also observed an upregulation of synapsin I (SYN1), which supports the positive role of lecithin in synaptogenesis, synaptic development and maturation. These findings suggest that lecithin nanoliposomes enhance neuronal development, which may have an impact on devising new lecithin delivery strategies for therapeutic applications.

Repositioning again of zonisamide for nerve regeneration.

Repositioning again of zonisamide for nerve regeneration.

Simultaneous Scanning Ion Conductance Microscopy and Atomic Force Microscopy with Microchanneled Cantilevers.

We combined scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) into a single tool using AFM cantilevers with an embedded microchannel flowing into the nanosized aperture at the apex of the hollow pyramid. An electrode was positioned in the AFM fluidic circuit connected to a second electrode in the bath. We could thus simultaneously measure the ionic current and the cantilever bending (in optical beam deflection mode). First, we quantitatively compared the SICM and AFM contact points on the approach curves. Second, we estimated where the probe in SICM mode touches the sample during scanning on a calibration grid and applied the finding to image a network of neurites on a Petri dish. Finally, we assessed the feasibility of a double controller using both the ionic current and the deflection as input signals of the piezofeedback. The experimental data were rationalized in the framework of finite elements simulations.

Calcium-responsive transactivator (CREST) protein shares a set of structural and functional traits with other proteins associated with amyotrophic lateral sclerosis.

BackgroundMutations in calcium-responsive transactivator (CREST) encoding gene have been recently linked to ALS. Similar to several proteins implicated in ALS, CREST contains a prion-like domain and was reported to be a component of paraspeckles.ResultsWe demonstrate that CREST is prone to aggregation and co-aggregates with FUS but not with other two ALS-linked proteins, TDP-43 and TAF15, in cultured cells. Aggregation of CREST affects paraspeckle integrity, probably by trapping other paraspeckle proteins within aggregates. Like several other ALS-associated proteins, CREST is recruited to induced stress granules. Neither of the CREST mutations described in ALS alters its subcellular localization, stress granule recruitment or detergent solubility; however Q388stop mutation results in elevated steady-state levels and more frequent nuclear aggregation of the protein. Both wild-type protein and its mutants negatively affect neurite network complexity of unstimulated cultured neurons when overexpressed, with Q388stop mutation being the most deleterious. When overexpressed in the fly eye, wild-type CREST or its mutants lead to severe retinal degeneration without obvious differences between the variants.ConclusionsOur data indicate that CREST and certain other ALS-linked proteins share several features implicated in ALS pathogenesis, namely the ability to aggregate, be recruited to stress granules and alter paraspeckle integrity. A change in CREST levels in neurons which might occur under pathological conditions would have a profound negative effect on neuronal homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0014-y) contains supplementary material, which is available to authorized users.

Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway

Trophic factors (TFs) play important role during development and adult tissue maintenance. In neurodegenerative diseases (ND) TF supplementation provides protection. Stromal cells (HUMS) derived from the human umbilical cord matrix provide neuroprotection in the ND models of mice. Though TF mediated protection is known, the exact mechanism of protection is not clear. So, here the essential TFs (secreted by HUMS cells) and the pathway of induction of neurite extension, differentiation and networking is addressed. The HUMS cells from the human umbilical cord matrix were derived and the mouse spinal cord motor neuron cell line, NSC-34 was extensively used. Flow cytometry, immunohistochemistry, RT- PCR, western blot, ELISA and antibody/inhibitor treatment were carried out to figure out the TF pathway. The HUMS cells secrete six neurotrophic factors (sTFs), namely, NT-3, NGF, BDNF, VEGF, IGF-1 and GDNF (TFs). These TFs are sufficient to induce differentiation, neurite extension and neural networking in a motor neuron cell line, NSC34. All the 5 TFs need to be neutralized simultaneously with their antibodies to abrogate neurite extension. These motor neurons express the concomitant receptors, which are either receptor tyrosine kinase (TrK) coupled or to the receptor followed by the TrKs, for the above trophic factors (except for BDNF). The tyrosine kinase inhibitor, K252a, drastically reduces neurite extension. In NSC34, the TFs are coupled to the PI3K-Akt-pathway and the RAS-MAP kinase signaling through phosphorylation of ERK1 and ERK2. PI3K inhibitor, Ly 294002, abolishes neural differentiation and neurite extension. Thus, differentiation, neurite extension and networking could be achieved through the PI3K pathway. Intriguingly, the cAMP second messenger system coupling was not required. H89, PKA-inhibitor caused extensive cell death. But, had no effect in the presence of HUMS-secreted-TFs(HSTFs) suggesting a pathway switch for cell survival itself. HUMS cells and their secreted factors could be of great use in regenerative medicine (RM). The activators of PI3K pathway, the major route of these HUMS-TFs action could be explored in RM and in the neurobiology of neural differentiation and extension.

Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity.

The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HTo is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein-gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HTo sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca(2+) influx, vesicular monoamine transporter 2-mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HTo in the DRN and suggest that 5-HTo may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.

Self-Sufficient Stem Cells: Stem Cell-Derived Serotonergic Neurons Rely on Endogenous BDNF Release to Establish Serotonergic Networks during Terminal Differentiation

The neurotransmitter serotonin plays an essential role in a variety of physiological processes including learning and memory, mood, and neuromodulation. Consequently a dysfunction of serotonergic neurotransmission is associated with psychiatric diseases such as depression, anxiety disorders, and schizophrenia. Because the small number and vast aborization of serotonergic neurons in vivo impedes their use in primary cell culture, stem cell-derived serotonergic neurons are applied as in vitro-models for serotonergic neurotransmission. Among others, 1C11 stem cells are used to generate serotonergic neurons in vitro, for example, to analyze the effect of antidepressant treatment on serotonin re-uptake or the function of microRNAs in serotonergic neurons. Since 1C11 stem cells differentiate uniformly into serotonergic neurons, there is no need to optimize terminal differentiation. Therefore the contribution of neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), to terminal differentiation of 1C11 stem cells is currently unknown. To bridge this gap, we differentiated 1C11 stem cells and determined the effect of endogenous BDNF release as well as of supplemented recombinant BDNF on neurite growth. With regard to the application of recombinant BDNF we also differentiated 1C11 stem cells in microfluidic isolation devices in order to determine whether the location of recombinant BDNF application may exert differential effects on neurite growth. Our data showed that endogenously synthesized and released BDNF is sufficient to form functional neuritic networks during terminal differentiation and that an exogenous BDNF source did not extensively promote neurite growth. However, the location of recombinant BDNF application did affect the microgrooves targeting behaviour of differentiating 1C11 stem cells in microfluidic isolation devices. In summary, our data shows that 1C11 stem cells rely on synthesis and release of endogenous BDNF for serotonergic network formation during terminal differentiation.

Caffeic acid phenethyl ester (CAPE) protects PC12 cells from MPP+ toxicity by inducing the expression of neuron-typical proteins

Neurite loss is an early event in neurodegenerative diseases; therefore, the regeneration of the network of neurites constitutes an interesting strategy of treatment for such disorders. Neurotrophic factors play a critical role in neuronal regeneration, but their clinical use is limited by their inability to cross the blood brain barrier. Oxidative and inflammatory events are implicated in neurodegeneration and antioxidant compounds have been suggested as potential neuroprotectors. The protective potential of CAPE (caffeic acid phenethyl ester) has been shown in different models of neurotoxicity (in vitro and in vivo) and it has been associated with immune-modulatory, antioxidant and anti-inflammatory properties; however, other mechanisms might be involved. The present study demonstrates that CAPE protects PC12 cells from the cellular death induced by the dopaminergic neurotoxin MPP+ by increasing the network of neurites. Results showed that CAPE induced the formation, elongation and ramification of neurites in PC12 cells non-stimulated with NGF (nerve growth factor) and inhibited the shortage of neurites induced by the dopaminergic neurotoxin. These effects were associated with increased expression of neuron-typical proteins responsible for axonal growth (GAP-43) and synaptogenesis (synaptophysin and synapsin I). It is noteworthy that, unlike neurotrophins, CAPE would be able to cross the blood brain barrier and exert its neurotrophic effects in the brain. This study corroborates the therapeutic potential of CAPE in neurodegenerative diseases while proposes the involvement of neuroplasticity in the mechanism of neuroprotection.

MiR-21 is an Ngf-Modulated MicroRNA That Supports Ngf Signaling and Regulates Neuronal Degeneration in PC12 Cells

The neurotrophins Ngf, Bdnf, NT-3, NT4–5 have key roles in development, survival, and plasticity of neuronal cells. Their action involves broad gene expression changes at the level of transcription and translation. MicroRNAs (miRs)—small RNA molecules that control gene expression post-transcriptionally—are increasingly implicated in regulating development and plasticity of neural cells. Using PC12 cells as a model system, we show that Ngf modulates changes in expression of a variety of microRNAs, including miRs known to be modulated by neurotrophins—such as the miR-212/132 cluster—and several others, such as miR-21, miR-29c, miR-30c, miR-93, miR-103, miR-207, miR-691, and miR-709. Pathway analysis indicates that Ngf-modulated miRs may regulate many protein components of signaling pathways involved in neuronal development and disease. In particular, we show that miR-21 enhances neurotrophin signaling and controls neuronal differentiation induced by Ngf. Notably, in a situation mimicking neurodegeneration—differentiated neurons deprived of Ngf—this microRNA is able to preserve the neurite network and to support viability of the neurons. These findings uncover a broad role of microRNAs in regulating neurotrophin signaling and suggest that aberrant expression of one or more Ngf-modulated miRs may be involved in neurodegenerative diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-014-8292-z) contains supplementary material, which is available to authorized users.

Juliprosopine and Juliprosine from Prosopis juliflora Leaves Induce Mitochondrial Damage and Cytoplasmic Vacuolation on Cocultured Glial Cells and Neurons

Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 μg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 μg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 μg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in β-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.