Role of canonical and non-canonical cAMP sources in CRHR2α-dependent signaling.

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.

Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells.

Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.

Inositol trisphosphate and ryanodine receptor signaling distinctly regulate neurite pathfinding in response to engineered micropatterned surfaces.

Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants.

TRPM4 inhibition slows neuritogenesis progression of cortical neurons.

TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.

Regenerating enteric neurites navigate the adult intestine using a glial positioning system?

While the enteric nervous system (ENS) is highly dynamic during development, the extent to which it is capable of repair remains unclear. In this issue of Neuron, Stavely et al.1 show that enteric neurons can reinnervate damaged regions to regain functionality using a glial positioning system (GPS) as their guide.

Nuclear Magnetic Resonance Treatment Induces ßNGF Release from Schwann Cells and Enhances the Neurite Growth of Dorsal Root Ganglion Neurons In Vitro.

Peripheral nerve regeneration depends on close interaction between neurons and Schwann cells (SCs). After nerve injury, SCs produce growth factors and cytokines that are crucial for axon re-growth. Previous studies revealed the supernatant of SCs exposed to nuclear magnetic resonance therapy (NMRT) treatment to increase survival and neurite formation of rat dorsal root ganglion (DRG) neurons in vitro. The aim of this study was to identify factors involved in transferring the observed NMRT-induced effects to SCs and consequently to DRG neurons. Conditioned media of NMRT-treated (CM NMRT) and untreated SCs (CM CTRL) were tested by beta-nerve growth factor (ßNGF) ELISA and multiplex cytokine panels to profile secreted factors. The expression of nociceptive transient receptor potential vanilloid 1 (TRPV1) channels was assessed and the intracellular calcium response in DRG neurons to high-potassium solution, capsaicin or adenosine triphosphate was measured mimicking noxious stimuli. NMRT induced the secretion of ßNGF and pro-regenerative-signaling factors. Blocking antibody experiments confirmed ßNGF as the main factor responsible for neurotrophic/neuritogenic effects of CM NMRT. The TRPV1 expression or sensitivity to specific stimuli was not altered, whereas the viability of cultured DRG neurons was increased. Positive effects of CM NMRT supernatant on DRG neurons are primarily mediated by increased ßNGF levels.

Evaluation of a Synthetic Retinoid, Ellorarxine, in the NSC-34 Cell Model of Motor Neuron Disease.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease worldwide and is characterized by progressive muscle atrophy. There are currently two approved treatments, but they only relieve symptoms briefly and do not cure the disease. The main hindrance to research is the complex cause of ALS, with its pathogenesis not yet fully elucidated. Retinoids (vitamin A derivatives) appear to be essential in neuronal cells and have been implicated in ALS pathogenesis. This study explores 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)ethylnyl]benzoic acid (Ellorarxine, or DC645 or NVG0645), a leading synthetic retinoic acid, discussing its pharmacological mechanisms, neuroprotective properties, and relevance to ALS. The potential therapeutic effect of Ellorarxine was analyzed in vitro using the WT and SOD1G93A NSC-34 cell model of ALS at an administered concentration of 0.3-30 nM. Histological, functional, and biochemical analyses were performed. Elorarxine significantly increased MAP2 expression and neurite length, increased AMPA receptor GluA2 expression and raised intracellular Ca2+ baseline, increased level of excitability, and reduced Ca2+ spike during depolarization in neurites. Ellorarxine also displayed both antioxidant and anti-inflammatory effects. Overall, these results suggest Ellorarxine shows relevance and promise as a novel therapeutic strategy for treatment of ALS.

Transcriptome Profiling of Phenylalanine-Treated Human Neuronal Model: Spotlight on Neurite Impairment and Synaptic Connectivity.

Phenylketonuria (PKU) is the most common inherited disorder of amino acid metabolism, characterized by high levels of phenylalanine (Phe) in the blood and brain, leading to cognitive impairment without treatment. Nevertheless, Phe-mediated brain dysfunction is not fully understood. The objective of this study was to address gene expression alterations due to excessive Phe exposure in the human neuronal model and provide molecular advances in PKU pathophysiology. Hence, we performed NT2/D1 differentiation in culture, and, for the first time, we used Phe-treated NT2-derived neurons (NT2/N) as a novel model for Phe-mediated neuronal impairment. NT2/N were treated with 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 30 mM Phe and subjected to whole-mRNA short-read sequencing. Differentially expressed genes (DEGs) were analyzed and enrichment analysis was performed. Under three different Phe concentrations (2.5 mM, 5 mM, and 10 mM), DEGs pointed to the PREX1, LRP4, CDC42BPG, GPR50, PRMT8, RASGRF2, and CDH6 genes, placing them in the context of PKU for the first time. Enriched processes included dendrite and axon impairment, synaptic transmission, and membrane assembly. In contrast to these groups, the 30 mM Phe treatment group clearly represented the neurotoxicity of Phe, exhibiting enrichment in apoptotic pathways. In conclusion, we established NT2/N as a novel model for Phe-mediated neuronal dysfunction and outlined the Phe-induced gene expression changes resulting in neurite impairment and altered synaptic connectivity.

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes.

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Cortical spheroids show strain-dependent cell viability loss and neurite disruption following sustained compression injury.

Sustained compressive injury (SCI) in the brain is observed in numerous injury and pathological scenarios, including tumors, ischemic stroke, and traumatic brain injury-related tissue swelling. Sustained compressive injury is characterized by tissue loading over time, and currently, there are few in vitro models suitable to study neural cell responses to strain-dependent sustained compressive injury. Here, we present an in vitro model of sustained compressive neural injury via centrifugation. Spheroids were made from neonatal rat cortical cells seeded at 4000 cells/spheroid and cultured for 14 days in vitro. A subset of spheroids was centrifuged at 104, 209, 313 or 419 rads/s for 2 minutes. Modeling the physical deformation of the spheroids via finite element analyses, we found that spheroids centrifuged at the aforementioned angular velocities experienced pressures of 10, 38, 84 and 149 kPa, respectively, and compressive (resp. tensile) strains of 10% (5%), 18% (9%), 27% (14%) and 35% (18%), respectively. Quantification of LIVE-DEAD assay and Hoechst 33342 nuclear staining showed that centrifuged spheroids subjected to pressures above 10 kPa exhibited significantly higher DNA damage than control spheroids at 2, 8, and 24 hours post-injury. Immunohistochemistry of ß3-tubulin networks at 2, 8, and 24 hours post-centrifugation injury showed increasing degradation of microtubules over time with increasing strain. Our findings show that cellular injuries occur as a result of specific levels and timings of sustained tissue strains. This experimental SCI model provides a high throughput in vitro platform to examine cellular injury, to gain insights into brain injury that could be targeted with therapeutic strategies.

Enhancing neurite growth and neural functions on polymeric nerve conduit with BMSC-derived ECM coating.

The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andßIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.

NeuroQuantify - An image analysis software for detection and quantification of neuron cells and neurite lengths using deep learning.

BACKGROUND: The segmentation of cells and neurites in microscopy images of neuronal networks provides valuable quantitative information about neuron growth and neuronal differentiation, including the number of cells, neurites, neurite length and neurite orientation. This information is essential for assessing the development of neuronal networks in response to extracellular stimuli, which is useful for studying neuronal structures, for example, the study of neurodegenerative diseases and pharmaceuticals. NEW METHOD: We have developed NeuroQuantify, an open-source software that uses deep learning to efficiently and quickly segment cells and neurites in phase contrast microscopy images. RESULTS: NeuroQuantify offers several key features: (i) automatic detection of cells and neurites; (ii) post-processing of the images for the quantitative neurite length measurement based on segmentation of phase contrast microscopy images, and (iii) identification of neurite orientations. COMPARISON WITH EXISTING METHODS: NeuroQuantify overcomes some of the limitations of existing methods in the automatic and accurate analysis of neuronal structures. It has been developed for phase contrast images rather than fluorescence images. In addition to typical functionality of cell counting, NeuroQuantify also detects and counts neurites, measures the neurite lengths, and produces the neurite orientation distribution. CONCLUSIONS: We offer a valuable tool to assess network development rapidly and effectively. The user-friendly NeuroQuantify software can be installed and freely downloaded from GitHub at https://github.com/StanleyZ0528/neural-image-segmentation.

Dipeptidyl Peptidase (DPP)-4 Inhibitors and Pituitary Adenylate Cyclase-Activating Polypeptide, a DPP-4 Substrate, Extend Neurite Outgrowth of Mouse Dorsal Root Ganglia Neurons: A Promising Approach in Diabetic Polyneuropathy Treatment.

Individuals suffering from diabetic polyneuropathy (DPN) experience debilitating symptoms such as pain, paranesthesia, and sensory disturbances, prompting a quest for effective treatments. Dipeptidyl-peptidase (DPP)-4 inhibitors, recognized for their potential in ameliorating DPN, have sparked interest, yet the precise mechanism underlying their neurotrophic impact on the peripheral nerve system (PNS) remains elusive. Our study delves into the neurotrophic effects of DPP-4 inhibitors, including Diprotin A, linagliptin, and sitagliptin, alongside pituitary adenylate cyclase-activating polypeptide (PACAP), Neuropeptide Y (NPY), and Stromal cell-derived factor (SDF)-1a-known DPP-4 substrates with neurotrophic properties. Utilizing primary culture dorsal root ganglia (DRG) neurons, we meticulously evaluated neurite outgrowth in response to these agents. Remarkably, all DPP-4 inhibitors and PACAP demonstrated a significant elongation of neurite length in DRG neurons (PACAP 0.1 µM: 2221 ± 466 µm, control: 1379 ± 420, p < 0.0001), underscoring their potential in nerve regeneration. Conversely, NPY and SDF-1a failed to induce neurite elongation, accentuating the unique neurotrophic properties of DPP-4 inhibition and PACAP. Our findings suggest that the upregulation of PACAP, facilitated by DPP-4 inhibition, plays a pivotal role in promoting neurite elongation within the PNS, presenting a promising avenue for the development of novel DPN therapies with enhanced neurodegenerative capabilities.

AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

FMRP cooperates with miRISC components to repress translation and regulate neurite morphogenesis in Drosophila.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is caused by mutations in the gene encoding the Fragile X messenger ribonucleoprotein (FMRP). FMRP is an evolutionarily conserved and neuronally enriched RNA-binding protein (RBP) with functions in RNA editing, RNA transport, and protein translation. Specific target RNAs play critical roles in neurodevelopment, including the regulation of neurite morphogenesis, synaptic plasticity, and cognitive function. The different biological functions of FMRP are modulated by its cooperative interaction with distinct sets of neuronal RNA and protein-binding partners. Here, we focus on interactions between FMRP and components of the microRNA (miRNA) pathway. Using the Drosophila S2 cell model system, we show that the Drosophila ortholog of FMRP (dFMRP) can repress translation when directly tethered to a reporter mRNA. This repression requires the activity of AGO1, GW182, and MOV10/Armitage, conserved proteins associated with the miRNA-containing RNA-induced silencing complex (miRISC). Additionally, we find that untagged dFMRP can interact with a short stem-loop sequence in the translational reporter, a prerequisite for repression by exogenous miR-958. Finally, we demonstrate that dFmr1 interacts genetically with GW182 to control neurite morphogenesis. These data suggest that dFMRP may recruit the miRISC to nearby miRNA binding sites and repress translation via its cooperative interactions with evolutionarily conserved components of the miRNA pathway.

Fibroblast growth factor 8 promotes in vitro neurite outgrowth of placode-derived petrosal and nodose ganglia to varying degrees.

Fibroblast growth factors (FGFs) are required for the specification and formation of the epibranchial placodes, which give rise to the distal part of the cranial sensory ganglia. However, it remains unclear whether FGFs play a role in regulating the neurite outgrowth of the epibranchial placode-derived ganglia during further development. Previous studies have shown that Fibroblast growth factor 8 (FGF8) promotes neurite outgrowth from the statoacoustic ganglion in vitro. However, these studies did not distinguish between the neural crest- and placode-derived components of the sensory ganglia. In this study, we focused on the petrosal and nodose ganglia as representatives of the epibranchial ganglia and investigated their axonal outgrowth under the influence of FGF8 signaling protein in vitro. To precisely isolate the placode-derived ganglion part, we labeled the placode and its derivatives with enhanced green fluorescent protein (EGFP) through electroporation. The isolated ganglia were then collected for qRT-PCR assay and cultured in a collagen gel with and without FGF8 protein. Our findings revealed that both placode-derived petrosal and nodose ganglia expressed FGFR1 and FGFR2. In culture, FGF8 exerted a neural trophic effect on the axon outgrowth of both ganglia. While the expression levels of FGFR1/2 were similar between the two ganglia, the petrosal ganglion exhibited greater sensitivity to FGF8 compared to the nodose ganglion. This indicates that the placode-derived ganglia have differential responsiveness to FGF8 signaling during axonal extension. Thus, FGF8 is not only required for the early development of the epibranchial placode, as shown in previous studies, but also promotes neurite outgrowth of placode-derived ganglia.

Analysis of Neurite and Spine Formation in Neurons In Vitro.

Primary neuronal cultures are commonly used to study genetic and exogenous factors influencing neuronal development and maturation. During development, neurons undergo robust morphological changes involving expansion of dendritic arbor, formation of dendritic spines, and expression of synaptic proteins. In this chapter, we will cover methodological approaches allowing quantitative assessment of in vitro cultured neurons. Various quantitative characteristics of dendritic arbor can be derived based on immunostaining against anti-microtubule-associated protein 2 followed by dendrite tracing with the SNT plug-in of the FIJI software package. The number and subtypes of dendritic spines can be assessed by double labeling with DiI and Phalloidin iFluor448 followed by laser scanning confocal microscopy analysis. Finally, expression of presynaptic and postsynaptic proteins can be determined by immunohistochemistry and quantification using several available software packages including FIJI and Imaris, which also allows for 3D rendering and statistical displaying of the expression level of synaptic proteins.

Analyses of Neurite and Spine Formation by In Utero Electroporation in Mice.

Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons.

Proteomic Identification and Label-Free Quantification of Proteins Implicated in Neurite and Spine Formation.

The molecular mechanisms underlying neurite formation include multiple crosstalk between pathways such as membrane trafficking, intracellular signaling, and actin cytoskeletal rearrangement. To study the proteins involved in such complex pathways, we present a detailed workflow of the sample preparation for mass spectrometry-based proteomics and data analysis. We have also included steps to perform label-free quantification of proteins that will help researchers quantify changes in the expression levels of key regulators of neuronal morphogenesis on a global scale.

Ex Vivo Live Imaging in Brain Slices for Analysis of Neurite Formation in Combination with In Utero Electroporation.

The use of time-lapse live imaging enables us to track the dynamic changes in neurites during their formation. Ex vivo live imaging with acute brain slices provides a more physiological environment than cultured cells. To accomplish this, a certain method of labeling is necessary to visualize and identify neurite morphology. To understand the dynamics of neurite structure at early stages of neurite formation, we describe in this chapter ex vivo live imaging using a confocal microscope at P0 in combination with in utero electroporation (IUE).