Neurite Outgrowth In Primary Neurons Research Articles

Loss of the psychiatric risk factor SLC6A15 is associated with increased metabolic functions in primary hippocampal neurons.

Major depressive disorder (MDD) is one of the most severe global health problems with millions of people affected, however, the mechanisms underlying this disorder is still poorly understood. Genome-wide association studies have highlighted a link between the neutral amino acid transporter SLC6A15 and MDD. Additionally, a number of preclinical studies support the function of this transporter in modulating levels of brain neurotransmitters, stress system regulation and behavioural phenotypes related to MDD. However, the molecular and functional mechanisms involved in this interaction are still unresolved. Therefore, to investigate the effects of the SLC6A15 transporter, we used hippocampal tissue from Slc6a15-KO and wild-type mice, together with several in-vitro assays in primary hippocampal neurons. Utilizing a proteomics approach we identified differentially regulated proteins that formed a regulatory network and pathway analysis indicated significantly affected cellular domains, including metabolic, mitochondrial and structural functions. Furthermore, we observed reduced release probability at glutamatergic synapses, increased mitochondrial function, higher GSH/GSSG redox ratio and an improved neurite outgrowth in primary neurons lacking SLC6A15. In summary, we hypothesize that by controlling the intracellular concentrations of neutral amino acids, SLC6A15 affects mitochondrial activity, which could lead to alterations in neuronal structure and activity. These data provide further indication that a pharmacological or genetic reduction of SLC6A15 activity may indeed be a promising approach for antidepressant therapy.

Hippocampal neural stem cells are more susceptible to the neurotoxin BMAA than primary neurons: effects on apoptosis, cellular differentiation, neurite outgrowth, and DNA methylation

Developmental exposure to the environmental neurotoxin β-N-methylamino-l-alanine (BMAA), a proposed risk factor for neurodegenerative disease, can induce long-term cognitive impairments and neurodegeneration in rats. While rodent studies have demonstrated a low transfer of BMAA to the adult brain, this toxin is capable to cross the placental barrier and accumulate in the fetal brain. Here, we investigated the differential susceptibility of primary neuronal cells and neural stem cells from fetal rat hippocampus to BMAA toxicity. Exposure to 250 µM BMAA induced cell death in neural stem cells through caspase-independent apoptosis, while the proliferation of primary neurons was reduced only at 3 mM BMAA. At the lowest concentrations tested (50 and 100 µM), BMAA disrupted neural stem cell differentiation and impaired neurite development in neural stem cell-derived neurons (e.g., reduced neurite length, the number of processes and branches per cell). BMAA induced no alterations of the neurite outgrowth in primary neurons. This demonstrates that neural stem cells are more susceptible to BMAA exposure than primary neurons. Importantly, the changes induced by BMAA in neural stem cells were mitotically inherited to daughter cells. The persistent nature of the BMAA-induced effects may be related to epigenetic alterations that interfere with the neural stem cell programming, as BMAA exposure reduced the global DNA methylation in the cells. These findings provide mechanistic understanding of how early-life exposure to BMAA may lead to adverse long-term consequences, and potentially predispose for neurodevelopmental disorders or neurodegenerative disease later in life.

Discovery of novel compound promotes neurogenesis by activation of mTOR signaling

Neurodegenerative disorders are continually increasing in incidence due to higher life expectancy. Currently, limited therapeutic strategies are available for affected patients, most targeting symptomatic treatment. mTOR pathway is identified as important player in neurogenesis that modulates healthy neuron growth and differentiation. Moreover, many neurodegenerative disorders are associated with downregulated mTOR signaling. However, mTOR activation can also lead to uncontrolled cell proliferation, promoting cancer. This careful balance limits the development of mTOR as a target for modulation of neuronal growth. Recently, selected classes of antidepressants were reported to activate mTOR pathways, causing dendritic outgrowth and increased synaptic protein levels. Although results were promising, they require treatment with up to 100 μM of tested compounds.To identify potential utilization of penfluridol, an anti‐psychotic agent, as a neuron growth modulator, we have screened this compound and its analogs using reported methodology. Although no activity was observed for PFL, one of its analogs, PF331, promoted neurogenesis in primary neurons. In particular, treatment with PF331 (100 nM,1 μM) for 5 days during reoxygenation (post‐stroke conditions) resulted in improved neurite outgrowth. At the same time, no effect on the viability of the primary neurons was observed under normoxic conditions and during the reperfusion stage. Further, we have established that observed activity was not related to the overstimulation of mTOR pathway, but rather to the compound’s ability to restore mTOR activation to the pre‐stress levels. We hypothesized that PF331 will have minimal impact on cancer proliferation and confirmed it in vivo, where our compound had no effect on cancer growth. Stroke is one of the pathological conditions characterized by hypoxia‐induced damage to neurons. Hence, we have proposed to evaluate our compound for the ability to lessen hypoxia‐induced damage using in‐vivo stroke model with prior confirmation of the ability of PF331 to cross the BBB in vivo and to achieve desired levels in the brain.In conclusion, PF331 has shown the ability to promote neurite outgrowth in primary neurons and restore mTOR activation in the hypoxic in vitro model. Because of its biological activity and favorable in vivo PK profile, we envision that this compound is a potential candidate for the treatment of neurodegenerative diseases, such as stroke.Evaluation of the effect of PF331 on primary neuronsEvaluation of the effect of PF331 on of primary neurons. Neurons were subjected to 3 hrs of OGD followed by 4 (A and B)/ 5 (C) days of oxygenation to mimic ischemia reperfusion injury. (A–B) OGD reperfusion (OGDR) down‐regulates mTOR and ERK compared to normoxic (Nor) condition whereas treatment with Compound PF331 activates mTOR and ERK. (C) PF331 facilitates dendritic outgrowth and increased number of synapses.Figure 1

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Metastasis suppressor 1 regulates neurite outgrowth in primary neuron cultures

Metastasis suppressor 1 (MTSS1) or missing in metastasis (MIM) is an actin- and membrane-binding protein with tumor suppressor functions. MTSS1 is important for cell morphology, motility, metastasis. The role of MTSS1 in cell morphology has been widely investigated in non-neuronal tissues; however the role of MTSS1 in neurite outgrowth remains unclear. Here we investigated the effect of MTSS1 on neurite outgrowth in primary cerebellar granule and hippocampal neurons of mouse. We found that overexpression of MTSS1 in cerebellar granule neurons significantly enhanced dendrite elaboration but inhibited axon elongation. This phenotype was significantly reduced by deletion of the Wiskott–Aldrich homology 2 (WH2) motif and point mutation in the insulin receptor substrate p53 (IRSp53) and MIM/MTSS1 homology (IMD) domain. Furthermore, inhibition of Rac1 activity or blocking of phosphatidyl inositol phosphates (PIPs) signaling decreased the effect of MTSS1 markedly. In accordance with the over-expression data, knockdown of MTSS1 in cerebellar granule neurons could increase the axon length but decrease the dendrite length and the number of dendrites. In addition, MTSS1 knock down in embryonic hippocampal neurons suppressed neurite branching and reduced dendrite length. Our findings have demonstrated that MTSS1 modulates neuronal morphology, possibly through a Rac1–PIPs signaling pathway.

Casein kinase signaling in axon regeneration.

Casein kinase signaling in axon regeneration.

2,4-dinitrophenol induces neural differentiation of murine embryonic stem cells

2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20μM DNP (or vehicle) for 4days. Significant increases in the proportion of nestin- and β-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells.

Exome sequencing to identify de novo mutations in sporadic ALS trios.

ALS is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, here we assess the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n=141 exomes). We found that amino acid-altering de novo mutations are enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex component SS18L1/CREST. CREST mutations inhibit activity-dependent neurite outgrowth in primary neurons, and CREST associates with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.

AT2-receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating BDNF expression

It is widely accepted that the angiotensin AT2-receptor (AT2R) has neuroprotective features. In the present study we tested pharmacological AT2R-stimulation as a therapeutic approach in a model of spinal cord compression injury (SCI) in mice using the novel non-peptide AT2R-agonist, Compound 21 (C21). Complementary experiments in primary neurons and organotypic cultures served to identify underlying mechanisms. Functional recovery and plasticity of corticospinal tract (CST) fibers following SCI were monitored after application of C21 (0.3mg/kg/dayi.p.) or vehicle for 4weeks. Organotypic co-culture of GFP-positive entorhinal cortices with hippocampal target tissue served to evaluate the impact of C21 on reinnervation. Neuronal differentiation, apoptosis and expression of neurotrophins were investigated in primary murine astrocytes and neuronal cells.C21 significantly improved functional recovery after SCI compared to controls, and this significantly correlated with the increased number of CST fibers caudal to the lesion site. In vitro, C21 significantly promoted reinnervation in organotypic brain slice co-cultures (+50%) and neurite outgrowth of primary neurons (+25%). C21-induced neurite outgrowth was absent in neurons derived from AT2R-KO mice. In primary neurons, treatment with C21 further induced RNA expression of anti-apoptotic Bcl-2 (+75.7%), brain-derived neurotrophic factor (BDNF) (+53.7%), the neurotrophin receptors TrkA (+57.4%) and TrkB (+67.9%) and a marker for neurite growth, GAP43 (+103%), but not TrkC.Our data suggest that selective AT2R-stimulation improves functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms. Thus, AT2R-stimulation may be considered for the development of a novel therapeutic approach for the treatment of spinal cord injury.

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA.

BackgroundNeurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.Methodology/Principal FindingsHere we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.ConclusionsproBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.

Novel Soft Alginate Hydrogel Strongly Supports Neurite Growth and Protects Neurons Against Oxidative Stress

Neural tissue engineering focuses on development of biomaterials that could support regeneration of neurons after trauma as well as injury caused by degenerative diseases. In this work we describe novel soft alginate hydrogels, which provide an adhesive matrix for rat and human neurons and facilitate neurite outgrowth. Only soft hydrogels, prepared with sub-stoichiometric concentrations of Ca²⁺, Ba²⁺, and Sr²⁺ cations by cross-linking with no >10% of all potentially available gelation sites in alginate, facilitated rapid and abundant neurite outgrowth in primary neuronal monolayer cultures, neural spheroids, and neurons derived from rat and human neural stem cells. To support neurite growth, hydrogels did not require modification by any extracellular matrix components and were prepared from high as well as low viscous alginates of different origin. In addition, neurons cultured on soft hydrogels were resistant to oxidative stress injury induced by hydrogen peroxide. These findings, which apply both to rat and human neurons, go beyond the well-described role of alginates as inert materials for cell encapsulation. Such soft alginate hydrogels may be useful for the preparation of pharmaceutical compositions for prophylaxis and treatment of neurodegenerative disorders, for promoting neuronal regeneration in the peripheral and central nervous system and for neural tissue engineering applications.

Foxp2 regulates gene networks implicated in neurite outgrowth in the developing brain.

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.

P53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Neuronal Differentiation Is Regulated by Leucine-rich Acidic Nuclear Protein (LANP), a Member of the Inhibitor of Histone Acetyltransferase Complex

Neuronal differentiation is a tightly regulated process characterized by temporal and spatial alterations in gene expression. A number of studies indicate a significant role for histone acetylation in the regulation of genes involved in development. Histone acetylation is regulated by histone deacetylases and histone acetyltransferases. Recent findings suggest that these catalytic activities, in turn, are modulated by yet another set of regulators. Of considerable interest in this context is the possible role of the INHAT (inhibitor of histone acetyltransferase) complex, comprised of a group of acidic proteins that suppress histone acetylation by a novel "histone-masking" mechanism. In this study, we specifically examined the role of the leucine-rich acidic nuclear protein (LANP), a defining member of the INHAT complex whose expression is tightly regulated in neuronal development. We report that depleting LANP in neuronal cell lines promotes neurite outgrowth by inducing changes in gene expression. In addition, we show that LANP directly regulates expression of the neurofilament light chain, an important neuron-specific cytoskeletal gene, by binding to the promoter of this gene and modulating histone acetylation levels. Finally, we corroborated our findings in vivo by demonstrating increased neurite outgrowth in primary neurons obtained from LANP null mice, which is also accompanied by increased histone acetylation at the NF-L promoter. Taken together, these results implicate INHATs as a distinct class of developmental regulators involved in the epigenetic modulation of neuronal differentiation.

S4-04-05: Dimebon: A phase 3 investigational agent for Alzheimer's disease with a novel mitochondrial mechanism of action

Dimebon is an investigational oral small molecule currently in Phase 3 development for the treatment of Alzheimer's disease (AD). Dimebon is hypothesized to stabilize and enhance mitochondrial and neuronal function. Patients with mild-to-moderate AD treated with Dimebon for one year were stabilized on measures of cognition and memory, activities of daily living, behavior and global function, while placebo-treated patients declined significantly. The magnitude and durability of clinical benefits and excellent safety and tolerability profile observed for Dimebon in Alzheimer's patients suggests a mechanism of action (MOA) distinct from currently marketed AD drugs. Preclinical experiments confirm Dimebon's lack of significant activity against cholinesterases and NMDA receptors, consistent with its unique MOA. Similarly, Dimebon does not appear to interact with other targets implicated in cognition and memory, including the muscarinic receptors, nicotinic receptors and phosphodiesterases. Neurodegenerative diseases such as AD are characterized by synapse loss and neuron death. A body of literature suggests that abnormal depolarization of mitochondrial membranes plays a significant role in the loss of neuron function in several neurodegenerative diseases, including AD. Dimebon is a highly potent (pM to low nM) stabilizer of mitochondria as measured by JC-1 staining of primary neurons in culture. Dimebon is also a potent (pM to low nM) stimulator of neurite outgrowth in primary neurons derived from the cortex, hippocampus and spinal cord of rodents. Dimebon's neurotrophic effects are believed to be related to its ability to stabilize, protect and enhance mitochondrial and neuronal function. Dimebon's efficacy in animal models of memory impairment at picomolar plasma concentrations is consistent with a potent MOA. Neuroprotection measured by mitochondrial vital stains occurs within minutes to hours of Dimebon exposure, while the trophic effects on neurite outgrowth occur over longer exposures. These activities may be related to the short term and longer term effects of Dimebon seen in the completed pivotal Dimebon AD trial. Dimebon is a promising potential therapeutic agent for AD, with a unique and novel target, the mitochondria. The most potent activity of Dimebon identified to date is the stabilization and enhancement of mitochondrial and neuronal function.

S4-04-06: Presenilin complex modulation of APP and notch molecule at gammma and epsilon sites

and neuronal function. Patients with mild-to-moderate AD treated with Dimebon for one year were stabilized on measures of cognition and memory, activities of daily living, behavior and global function, while placebo-treated patients declined significantly. The magnitude and durability of clinical benefits and excellent safety and tolerability profile observed for Dimebon in Alzheimer’s patients suggests a mechanism of action (MOA) distinct from currently marketed AD drugs. Preclinical experiments confirm Dimebon’s lack of significant activity against cholinesterases and NMDA receptors, consistent with its unique MOA. Similarly, Dimebon does not appear to interact with other targets implicated in cognition and memory, including the muscarinic receptors, nicotinic receptors and phosphodiesterases. Neurodegenerative diseases such as AD are characterized by synapse loss and neuron death. A body of literature suggests that abnormal depolarization of mitochondrial membranes plays a significant role in the loss of neuron function in several neurodegenerative diseases, including AD. Results: Dimebon is a highly potent (pM to low nM) stabilizer of mitochondria as measured by JC-1 staining of primary neurons in culture. Dimebon is also a potent (pM to low nM) stimulator of neurite outgrowth in primary neurons derived from the cortex, hippocampus and spinal cord of rodents. Dimebon’s neurotrophic effects are believed to be related to its ability to stabilize, protect and enhance mitochondrial and neuronal function. Dimebon’s efficacy in animal models of memory impairment at picomolar plasma concentrations is consistent with a potent MOA. Neuroprotection measured by mitochondrial vital stains occurs within minutes to hours of Dimebon exposure, while the trophic effects on neurite outgrowth occur over longer exposures. These activities may be related to the short term and longer term effects of Dimebon seen in the completed pivotal Dimebon AD trial. Conclusions: Dimebon is a promising potential therapeutic agent for AD, with a unique and novel target, the mitochondria. The most potent activity of Dimebon identified to date is the stabilization and enhancement of mitochondrial and neuronal function.

Immobilized Concentration Gradients of Neurotrophic Factors Guide Neurite Outgrowth of Primary Neurons in Macroporous Scaffolds

Immobilized Concentration Gradients of Neurotrophic Factors Guide Neurite Outgrowth of Primary Neurons in Macroporous Scaffolds

Immobilized Concentration Gradients of Neurotrophic Factors Guide Neurite Outgrowth of Primary Neurons in Macroporous Scaffolds

Neurotrophic factors present as concentration gradients are neurotropic cues that direct axonal growth toward their targets. Multiple factors work together in vivo to ensure axons reach the proper targets, likely interacting with one another via intracellular signalling pathways. Nerve growth factor (NGF) and neurotrophin-3 (NT-3) are neurotrophins known to guide axons as well as promote axonal growth following injury to both the spinal cord and peripheral nerve. These molecules interact with neurons through different tyrosine kinase receptors. In this study, the receptors for these growth factors were shown to be co-localized on E10 chick dorsal root ganglion (DRG) cells, providing an opportunity for synergism. Well-defined concentration gradients of NGF and NT-3 were immobilized for the first time in a cell-penetrable, cell-adhesive scaffold of poly(2-hydroxyethylmethacrylate) and poly(L-lysine). An NGF concentration gradient of 310 ng/mL/mm was required to guide chick DRG neurites. A lower concentration gradient of 200 ng/mL/mm of NGF was shown to elicit guidance when an NT-3 concentration gradient of 200 ng/mL/mm was also present, indicating a synergistic response in the DRG neurons. These gradient scaffolds may be useful for guided regeneration following injury to the spinal cord or peripheral nerve and may also elucidate the mechanism for intracellular signaling of neurotrophic factors.