Apoptosis of Cortical Neurons in Ischemic Insult

Acute brain ischemia is accompanied by the intense apoptotic and/or necrotic death of cortical neurons. This review deals with the molecular mechanisms underlying apoptosis, in particular those activated in progressive cerebral ischemic insult. We analyze the data of experimental studies and clinical findings that confirm the principal role of caspase-dependent cell death resulting from acute disorder of the brain circulation. The prospects for the use of apoptosis inhibitors in neurological practice for prevention or minimization of cerebral ischemic injury and reduction of neuronal degeneration within a penumbral zone are discussed.     

Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression

Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.     

A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

Background

Tissue inhibitor of metalloproteinases-1 (TIMP-1) displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons.

Principal Findings

We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, βIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1.

Conclusions

Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.     

T-588 Enhances Neurite Outgrowth and Choline Acetyltransferase in Cultured Rat Spinal Ventral Horn Neurons

T-588(R(-)-1-(benzo(b)thiophen-5yl)-2-[2(N,N-diethylamino)ethoxy]ethanol hydrochloride) is a novel compound which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro. This compound can slow the motor deterioration of wobbler mouse motor neuron disease. However, it is not known whether this compound has a trophic effect on spinal motor neurons. We have studied the effect of T-588 on neurite outgrowth and choline acetyltransferase(ChAT) activity in primary explant cultures of ventral spinal cord of fetal rats(VSCC). Cultures were treated with T-588 from day 1 to 1 week. T-588 treated VSCC, compared with control VSCC, had a significant neurite promoting effect at ranged between 10^–8 molar(M) and 10^–5 M, with 2.3 to 5.3 fold increased over that of control VSCC. In T-588 treated VSCC, ChAT activity was increased 1.5 times over that of control at 10^–6, and 10^–5 M respectively. Our data showing T-588 has neurotrophic action on VSCC and suggests a potential use of T-588 in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and motor neuron disease.     

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions¹.At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods^1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology⁴, cellular and molecular biology^5-8, biochemistry⁵, imaging and microscopy^4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses¹¹, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously^4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x10⁶ cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.Download video file.^{(75M, mov)}     

Effect of Apolipoprotein E on Neurite Outgrowth and β-Amyloid-Induced Toxicity in Developing Rat Primary Hippocampal Cultures

Abstract: The correlation between the ε4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established. However, the role of apoE in normal as well as pathological brain processes remains unclear. We evaluated the effect of apoE treatment on development and β-amyloid (Aβ)-induced toxicity using primary cultures of developing rat hippocampal neurons. The source of apoE was conditioned media from HEK cells stably transfected with human apoE3 or apoE4 cDNA, a preparation where apoE is lipid-associated. Morphological and biochemical changes in the cultures were assessed at 1 and 3 days following low- and high-density plating with either apoE3 or E4 with or without Aβ. Both apoE isoforms were neurotrophic, as measured by increased neurite length. Aged Aβ(1–42), a peptide preparation exhibiting extensive fibril and aggregate formation, is toxic to these cultures. Addition of apoE3 and E4 significantly and comparably attenuated the Aβ-induced reduction in both neurite length and cell viability. The level of protection against this toxicity was proportional to the neurotrophic actions of the two apoE isoforms. Thus, apoE acts as a potent growth factor in both the absence and the presence of Aβ, supporting a potentially important role for apoE in neurobiology.     

Hepatoma-derived Growth Factor-related Protein-3 Interacts with Microtubules and Promotes Neurite Outgrowth in Mouse Cortical Neurons

Hepatoma-derived growth factor-related proteins (HRP) comprise a family of 6 members, which the biological functions are still largely unclear. Here we show that during embryogenesis HRP-3 is strongly expressed in the developing nervous system. At early stages of development HRP-3 is located in the cytoplasm and neurites of cortical neurons. Upon maturation HRP-3 relocalizes continuously to the nuclei and in the majority of neurons of adult mice it is located exclusively in the nucleus. This redistribution from neurites to nuclei is also found in embryonic cortical neurons maturing in cell culture. We show that HRP-3 is necessary for proper neurite outgrowth in primary cortical neurons. To identify possible mechanisms of how HRP-3 modulate neuritogenesis we isolated HRP-3 interaction partners and demonstrate that it binds tubulin through the N-terminal so called HATH region, which is strongly conserved among members of the HRP family. It promotes tubulin polymerization, stabilizes and bundles microtubules. This activity depends on the extranuclear localization of HRP-3. HRP-3 thus could play an important role during neuronal development by its modulation of the neuronal cytoskeleton.Neuritogenesis is a key step in nervous system development in which neurons extend dendrites and axons and connect to different targets in and outside the nervous system. The proper regulation of this process is controlled by a number of extra- and intracellular molecules expressed by neurons themselves or non-neuronal cells in their surroundings. Multiple studies indicate that rearrangement of the neuronal cytoskeleton in response to extracellular signals is an important mechanism during neurite extension and pathfinding (1-3). Manipulation of the polymerization and depolymerization of microtubules has shown that regulation of microtubule assembly and maintenance is important for neuritogenesis (4). Microtubule dynamics are regulated by a huge number of regulatory proteins like tau or other microtubule-associated proteins (MAPs)⁴ (5). In addition, proteins like CRMP-2 that interact with tubulin dimers and accelerate the assembly of tubulin into microtubles have been shown to be involved in the regulation of neuronal polarity and neuritogenesis (6-10). Despite all advances, however, made in the understanding of the role of the cytoskeleton and its regulatory proteins during neuritic growth there are still many open questions regarding the regulation of these processes. Therefore identifying new molecules binding to and modulating the turnover of microtubules is of high interest for the understanding of how neurite outgrowth is regulated.Hepatoma-derived growth factor (HDGF) is a protein that was purified from secretions of hepatoma cells by virtue of its growth factor activity. Subsequently 5 additional proteins were identified in which the 97 N-terminal amino acid residues show strong similarity to HDGF. Accordingly this family of proteins has been termed HDGF-related proteins (HRP) (11-13). HDGF has neurotrophic activity for hippocampal, spinal, and facial motor neurons (14, 15). So far, however, no receptor or signal transduction pathway involved has been identified for any of the HRPs.Most HRPs are expressed in a variety of tissues. HRP-3, however, is the only family member in whose expression is restricted. It is only expressed in neurons and to a low extent in glial cells (16, 17). Like HDGF after transfection into human embryonic kidney cells HRP-3 exhibits proliferative activity (12). The strong and almost exclusive expression of HRP-3 in postmitotic neurons, however, suggests biological functions other than its growth factor activity (16).In the present study we examine the expression and function of HRP-3 protein during mouse embryonic neuronal development. We demonstrate that the protein locates to the cytoplasm and neurites during early nervous system development, whereas most of HRP-3 can be found in the nucleus in adult neurons. We show that HRP-3 promotes neurite growth and suggest that this is due to the interaction of HRP-3 with the tubulin component of the neuronal cytoskeleton.     

Effects of GnRH on Neurite Outgrowth,Neurofilament and Spinophilin Proteins Expression in Cultured Spinal Cord Neurons of Rat Embryos

It has been previously described the presence of GnRH receptor in spinal cord neurons of rat embryos and adult rats. However, the functional role of these receptors has not been studied. In this work, the effect of GnRH on neurite outgrowth and cytoskeletal protein expression in cultured spinal cord neurons of rat embryos was analyzed. Specifically, neurofilaments of 68 and 200 kDa by immunoblot assays and spinophilin mRNA expression by RT-PCR. Results show that GnRH stimulates neurite outgrowth in addition to an increase in neurofilaments and spinophilin expression. These findings suggest that GnRH may play a role as neuromodulator in neuronal plasticity and that could be considered as a potential factor for neuronal regeneration in spinal cord injuries.     

Proteomic Analysis of Primary Cultured Rat Cortical Neurons in Chemical Ischemia

To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.     

Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.     

Characterization of Chick Gizzard Extract That Promotes Neurite Outgrowth in Cultured Ciliary Neurons

Abstract: Chicken gizzard extract contains a macromolecule(s) that promotes the neurite outgrowth of dissociated neurons from the ciliary ganglia (CG) of chick embryos. The factor in gizzard extract was partially purified and estimated to be about 12S (M.W. 200,000-300,000) on sucrose density gradient centrifugation. The neurite outgrowth of CG neurons by the factor strictly depends on the embryonal age. The maximal neurite outgrowth was observed when CG neurons were dissociated from the embryos younger than 10 days. After that time the response of CG neurons to the factor rapidly declined and was almost lost at day 14. The amount of factor in the gizzard began to increase rapidly from 12-day-old embryo and reached the maximal level at day 16, and thereafter a fairly steady level was maintained. When CG neurons were co-cultured with rat myotubes, the ratio of muscle cells with synaptic responses (miniature end-plate potentials) was significantly higher in the presence of gizzard factor than its absence. The results suggest that this factor acts as an external signal on CG neurons to form synaptic connections in vivo.     

Radicicol Potentiates Neurotrophin-Mediated Neurite Outgrowth and Survival of Cultured Sensory Neurons from Chick Embryo

Radicicol, an antifungal antibiotic with markedly low toxicity, is a potent inhibitor of the Src family of protein tyrosine kinases and causes morphological reversion of v-src-transformed fibroblasts. Recently, this antibiotic was also found to inhibit Raf kinase. In the present study, we found that nanomolar concentrations of radicicol (10 ng/ml) enhanced the survival and neurite outgrowth of neurons from embryonic chick dorsal root ganglia (DRGs) and sympathetic ganglia. It potentiated the trophic effects of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 on the cultured DRG neurons. This concentration of radicicol did not alter the tyrosine phosphorylation of Trk receptors or the activity of mitogen-activated protein (MAP) kinases. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not inhibit radicicol, excluding the involvement of PI3-kinase in the radicicol-dependent trophic actions. These results suggest that radicicol mediates neuronal growth presumably via a mechanism not involving the activation of Trk receptors, MAP kinase, or PI3-kinase.     

Effect of AMPA on Cerebral Cortical Oxygen Balance of Ischemic Rat Brain

We tested the hypothesis that the excitatory neurotransmitter receptor agonist, alpha amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), would worsen cerebral cortical oxygen supply/consumption balance during focal ischemia. In this study, we compared regional cerebral blood flow, arterial and venous O₂ saturation, O₂ extraction and oxygen consumption of ischemic and AMPA treated ischemic and control regions of rat brain. Ischemia was induced by middle cerebral artery (MCA) occlusion in isoflurane (1.4%) anesthetized Wistar rats. Twenty minutes after MCA occlusion, 10^–5 M AMPA was applied to the ischemic cortex (IC) for a period of 40 min; the fluid was changed every 10 min. After 1 hr of ischemia, animals were sacrificed and regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined microspectrophotometrically. In control, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 46 ± 20 vs. 81 ± 39 ml/min/100g, O₂ consumption: 2.8 ± 1.4 vs. 3.6 ± 1.4 ml O₂/min/100g). 10^–5 M AMPA did not significantly alter regional cerebral blood flow and oxygen consumption of the IC, but did decrease the average venous O₂ saturation of the IC from 50.2 ± 3.9% to 46.7 ± 1.6%. AMPA also significantly increased the frequency of small veins with less than 45% O₂ saturation in the IC (8 out of 56 veins in IC vs. 18 out of 56 veins in AMPA treated IC). Thus, topical application of 10^–5 M AMPA to the ischemic area worsens cerebral O₂ balance and suggests that excitatory amino acids contribute to the degree of cerebral ischemia.     

Probing Relevant Molecules in Modulating the Neurite Outgrowth of Hippocampal Neurons on Substrates of Different Stiffness

Hippocampal neurons play a critical role in learning and memory; however, the effects of environmental mechanical forces on neurite extension and associated underlying mechanisms are largely unexplored, possibly due to difficulties in maintaining central nervous system neurons. Neuron adhesion, neurite length, and mechanotransduction are mainly influenced by the extracellular matrix (ECM), which is often associated with structural scaffolding. In this study, we investigated the relationship between substrate stiffness and hippocampal neurite outgrowth by controlling the ratios of polydimethylsiloxane (PDMS) base to curing agent to create substrates of varying stiffness. Immunostaining results demonstrated that hippocampal neurons have longer neurite elongation in 35∶1 PDMS substrate compared those grown on 15∶1 PDMS, indicating that soft substrates provide a more optimal stiffness for hippocampal neurons. Additionally, we discovered that pPKCα expression was higher in the 15∶1 and 35∶1 PDMS groups than in the poly-l-lysine-coated glass group. However, when we used a fibronectin (FN) coating, we found that pFAKy397 and pFAKy925 expression were higher in glass group than in the 15∶1 or 35∶1 PDMS groups, but pPKCα and pERK1/2 expression were higher in the 35∶1 PDMS group than in the glass group. These results support the hypothesis that environmental stiffness influences hippocampal neurite outgrowth and underlying signaling pathways.     

Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

Abstract: We reported previously that stereoisomers of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the d - threo and l - threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l - or d - threo -PDMP. These isomers exhibited opposite effects on neurite outgrowth: d -PDMP was inhibitory at concentrations ranging from 5 to 20 µ M , whereas l -PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µ M . Rat neocortical explants were doubly labeled with [¹⁴C]serine and [³H]galactose at 15 µ M l - or d -PDMP. l -PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d -PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l -PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N -acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l -PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.     

Inhibitory Effects of Carvone Isomers on the GABAA Receptor in Primary Cultures of Rat Cortical Neurons

Carvone is a natural terpene which can be purified as R‐(?) or S‐(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA_A receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA_A by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA‐induced stimulation of [³H]flunitrazepam binding, suggesting their interaction with the GABA_A receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R‐(?)‐carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA_A receptor at its noncompetitive blocker site. Chirality 26:368–372, 2014. © 2014 Wiley Periodicals, Inc.     

Respiration in Primary Cultured Cerebellar Granule Neurons and Cerebral Cortical Neurons

Respiration was measured polarographically in primary cultures enriched with cerebellar granule neurons or cerebral cortical neurons. The basal respiratory rate, measured on the sixth day after culturing, was 12.00 natom equiv. O/mg protein/min for the cortical neurons and 12.70 natom equiv. O/mg protein/min for the granule neurons. Maximal stimulation by 2,4-dinitrophenol produced a 20-40% increase over the basal rate for both neuronal types. Oligomycin inhibited neuronal basal respiration by 45%. These respiratory rates in neurons from primary culture are markedly lower than those measured in astrocytes grown under similar conditions.     

A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.     

A Peptide Antagonist of ErbB Receptors, Inherbin3, Induces Neurite Outgrowth from Rat Cerebellar Granule Neurons Through ErbB1 Inhibition

ErbB receptors not only function in cancer, but are also key developmental regulators in the nervous system. We previously identified an ErbB1 peptide antagonist, Inherbin3, that is capable of inhibiting tumor growth in vitro and in vivo. In this study, we found that inhibition of ErbB1 kinase activity and activation of ErbB4 by NRG-1β induced neurite extension, suggesting that ErbB1 and ErbB4 act as negative and positive regulators, respectively, of the neuritogenic response. Inherbin3, inhibited activation not only of ErbB1 but also of ErbB4 in primary neurons, strongly induced neurite outgrowth in rat cerebellar granule neurons, indicating that this effect mainly was due to inhibition of ErbB1 activation.