Mitochondria modulate programmed neuritic retraction.

Neuritic retraction in the absence of overt neuronal death is a shared feature of normal aging and neurodegenerative disorders, but the intracellular mechanisms modulating this process are not understood. We propose that cumulative distal mitochondrial protein damage results in impaired protein import, leading to mitochondrial dysfunction and focal activation of the canonical apoptosis pathway in neurites. This is a controlled process that may not lead to neuronal death and, thus, we term this phenomenon "neuritosis." Consistent with our hypothesis, we show that in primary cerebrocortical neurons, mitochondrial distance from the soma correlates with increased mitochondrial protein damage, PINK1 accumulation, reactive oxygen species production, and decreased mitochondrial membrane potential and depolarization threshold. Furthermore, we demonstrate that the distance-dependent mitochondrial membrane potential gradient exists in vivo in mice. We demonstrate that impaired distal mitochondria have a lower threshold for focal/nonlethal neuritic caspase-3 activation in normal neurons that is exacerbated in aging, stress, and neurodegenerative conditions, thus delineating a fundamental mechanistic underpinning for synaptic vulnerability.

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Myocyte Enhancer Factor 2A (MEF2A) Defines Oxytocin-Induced Morphological Effects and Regulates Mitochondrial Function in Neurons.

The neuropeptide oxytocin (OT) is a well-described modulator of socio-emotional traits, such as anxiety, stress, social behavior, and pair bonding. However, when dysregulated, it is associated with adverse psychiatric traits, such as various aspects of autism spectrum disorder (ASD). In this study, we identify the transcription factor myocyte enhancer factor 2A (MEF2A) as the common link between OT and cellular changes symptomatic for ASD, encompassing neuronal morphology, connectivity, and mitochondrial function. We provide evidence for MEF2A as the decisive factor defining the cellular response to OT: while OT induces neurite retraction in MEF2A expressing neurons, OT causes neurite outgrowth in absence of MEF2A. A CRISPR-Cas-mediated knockout of MEF2A and retransfection of an active version or permanently inactive mutant, respectively, validated our findings. We also identified the phosphatase calcineurin as the main upstream regulator of OT-induced MEF2A signaling. Further, MEF2A signaling dampens mitochondrial functioning in neurons, as MEF2A knockout cells show increased maximal cellular respiration, spare respiratory capacity, and total cellular ATP. In summary, we reveal a central role for OT-induced MEF2A activity as major regulator of cellular morphology as well as neuronal connectivity and mitochondrial functioning, with broad implications for a potential treatment of disorders based on morphological alterations or mitochondrial dysfunction.

Nuclear receptors and Drosophila neuronal remodeling.

During the development of both vertebrates and invertebrates, neurons undergo a crucial remodeling process that is necessary for their new function. Neuronal remodeling is composed of two stages: first, axons and dendrites are pruned without the loss of the cell body; later, this process is most commonly followed by a regrowth step. Holometabolous insects like the fruitfly Drosophila exhibit striking differences between their larval and adult stages. These neuronal remodeling processes occur during metamorphosis, the period of transformation from a larva to an adult. All axon and dendrite pruning events ultimately depend on the EcR nuclear receptor. Its ligand, the steroid molting hormone ecdysone, binds to heteromeric receptors comprising the nuclear receptor ECR and USP, and this complex regulates target genes involved in neuronal remodeling. Here we review the nuclear receptor-mediated genetic control of the main neuronal remodeling events described so far in Drosophila. These events consist of neurite degeneration in the mushroom bodies (MBs: the brain memory center) and in the dendritic arborizing sensory neurons, of neurite retraction or small scale elimination in the thoracic ventral neurosecretory cells, in the olfactory circuits and in the neuromuscular junction. MB axon regrowth after pruning and the role of MB neuron remodeling in memory formation are also reviewed. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Prostaglandin D2 elicits the reversible neurite retraction in hypothalamic cell line.

Prostaglandins (PGs) play important roles in diverse physiological processes in the central nervous system. PGD2 is the most abundant PG in the brain and acts through specific receptors, DP1 and CRTH2. We investigated the effects of PGD2 on the morphology of the hypothalamic cell line mHypoE-N37 (N37). In N37 cells, serum starvation induced neurite outgrowth and PGD2 elicited neurite retraction, although we failed to detect transcripts for DP1 and CRTH2. Such an effect of PGD2 was efficiently mimicked by its metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2. N-acetyl cysteine completely abolished the effect of PGD2, and reactive oxygen species (ROS) were considered to be important. Notably, neurite outgrowth was restored by PGD2 removal. These results suggest that PGD2 induces reversible neurite retraction in a ROS-mediated mechanism that does not involve any known receptor.

Neurotrophic factor-α1 modulates NGF-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and cortical neurons.

Wnt-3a and Wnt-5a signaling activities inhibit and promote neurite outgrowth, respectively, to regulate dendritic and axonal genesis during neurodevelopment. NF-α1, a neurotrophic factor, has been shown to modulate dendritic remodeling and negatively regulate the canonical Wnt-3a pathway. Here, we investigated whether NF-α1 could modify nerve growth factor (NGF)-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and mouse primary cortical neurons. We showed that NGF-induced neurite outgrowth was inhibited by Wnt-3a, and this inhibition was prevented by NF-α1. Western blot analysis revealed that NF-α1 reduced the expression of both ß-catenin in the canonical Wnt-3a pathway and Rho, a downstream effector of Wnt-3a's non-canonical signaling pathway. Treatment of PC12 cells with a ROCK inhibitor prevented the inhibition of NGF-induced neurite outgrowth by Wnt-3a, suggesting that NF-α1 promotes neurite outgrowth in the presence of Wnt-3a by down-regulating its canonical and non-canonical activities. Interestingly, treatment of PC12 cells with Wnt-5a, which formed a complex with NF-α1, induced neurite outgrowth that was enhanced by treatment with the combination of Wnt-5a, NGF, and NF-α1. These effects of NF-α1 on Wnt 3a's and Wnt 5a's regulation of neurite outgrowth in PC12 cells were also demonstrated in primary cultures of mouse cortical neurons. In addition, we showed in PC12 cells that NF-α1 acts by upregulating adenomatous polyposis coli (APC) accumulation at neurite tips, thereby providing positive and negative Wnt-3a/Wnt-5a mediated cues to modulate neurite outgrowth, a process important during neurodevelopment.

Fat3 acts through independent cytoskeletal effectors to coordinate asymmetric cell behaviors during polarized circuit assembly.

The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with the directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense the position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during the assembly of polarized circuits in the murine retina. We find that the Fat3 intracellular domain (ICD) binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites form but do not make ectopic synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development.

Pathological Aspects of Neuronal Hyperploidization in Alzheimer's Disease Evidenced by Computer Simulation.

When subjected to stress, terminally differentiated neurons are susceptible to reactivate the cell cycle and become hyperploid. This process is well documented in Alzheimer's disease (AD), where it may participate in the etiology of the disease. However, despite its potential importance, the effects of neuronal hyperploidy (NH) on brain function and its relationship with AD remains obscure. An important step forward in our understanding of the pathological effect of NH has been the development of transgenic mice with neuronal expression of oncogenes as model systems of AD. The analysis of these mice has demonstrated that forced cell cycle reentry in neurons results in most hallmarks of AD, including neurofibrillary tangles, Aß peptide deposits, gliosis, cognitive loss, and neuronal death. Nevertheless, in contrast to the pathological situation, where a relatively small proportion of neurons become hyperploid, neuronal cell cycle reentry in these mice is generalized. We have recently developed an in vitro system in which cell cycle is induced in a reduced proportion of differentiated neurons, mimicking the in vivo situation. This manipulation reveals that NH correlates with synaptic dysfunction and morphological changes in the affected neurons, and that membrane depolarization facilitates the survival of hyperploid neurons. This suggests that the integration of synaptically silent, hyperploid neurons in electrically active neural networks allows their survival while perturbing the normal functioning of the network itself, a hypothesis that we have tested in silico. In this perspective, we will discuss on these aspects trying to convince the reader that NH represents a relevant process in AD.

Nitrite Protects Neurons Against Hypoxic Damage Through S-nitrosylation of Caspase-6.

Aims: The coordination of neurons to execute brain functions requires plenty of oxygen. Thus, it is not surprising that the chronic hypoxia resulting from chronic obstructive pulmonary diseases (COPD) can cause neuronal damage. Injury in the cortex can give rise to anxiety and cognitive dysfunction. This study investigated what causes hypoxia-induced neuronal injury and what strategies might be used to protect neurons against such damage. Results: This study found that hypoxia in primary cortical neurons caused neurite retraction, a caspase-6-dependent process. The hypoxic stress activated caspase-6 within the neurite, leading to microtubule disassembly and neurite retraction. The effect of hypoxia on caspase-6 activation, microtubule disassembly, and neurite retraction was alleviated by nitrite treatment. The protective role of nitrite was further supported by the observation that the active-site Cys146 of caspase-6 was S-nitrosylated in hypoxic neuro-2a cells treated with nitrite. We further validated the beneficial effect of nitrite on neuronal function against hypoxic stress in vivo. Using the wild-type or Apo E-/- mice exposed to chronic hypoxia as a model, we demonstrated that supplementing drinking water with nitrite suppressed active caspase-6 in the cortex of the brain, concomitant with the prevention of hypoxia-induced anxiety in the animals. Innovation: These results are the first evidence of a new pathway for the activation of caspase-6 and the first to indicate that nitrite can protect neurons against chronic hypoxic insult. Conclusion: Our findings suggest that nitrite holds great potential for the treatment of diseases such as COPD associated with hypoxia-induced neuronal injury.

Oxytocin alters the morphology of hypothalamic neurons via the transcription factor myocyte enhancer factor 2A (MEF-2A).

Oxytocin (OT) has gained attention not only as anxiolytic drug and as potential treatment option for autistic children; it also acts as a growth and differentiation factor in neuronal cells. While behavioral effects of OT have been studied in detail, knowledge about the cellular effects of OT is relatively sparse. In this study, we present evidence for three hypotheses: 1) OT leads to neurite retraction in hypothalamic neurons via the OT receptor (OTR) 2) The transcription factor MEF-2A is a central regulator of OT-induced neurite retraction, and 3) The MAPK pathway is critical for OT-induced MEF-2A activation. Incubation of rat hypothalamic H32 cells with 10 nM to 1 µM OT, vasopressin, and the specific OTR agonist TGOT, over the course of 12 h resulted in a time-dependent, significant retraction of neurites. In addition, the size of the nuclear compartment increased, whereas the overall cell size remained unchanged. OT treatment for 10 h increased the cellular viability significantly, and this effect could be blocked by a specific OTR antagonist, providing evidence for a specific and pro-active effect of OT on neurite retraction, and not as an unspecific side effect of apoptosis. The molecular mechanism that controls OT-induced neurite retraction includes a reduced phosphorylation of the transcription factor MEF-2A at Serine 408 (S408). This dephosphorylation is under the control of the OTR-coupled MAPK pathway, as blocking MEK1/2 by U0126 inhibited MEF-2A activation and subsequent neurite retraction. The siRNA-mediated knockdown of MEF-2A prevented the OT-induced neurite retraction, providing direct evidence for a role of MEF-2A in morphological alterations induced by OT treatment. In summary, the present study reveals a previously unknown OTR-coupled MAPK-MEF-2A pathway, which is responsible for OT-induced neurite retraction of hypothalamic neurons.

Digital quantification of neurite outgrowth and retraction by phase-contrast microscopy: A tau perspective.

The proper organization and function of the mammalian nervous system relies on neuronal processes or "neurites," extended morphological projections that include axons and dendrites. Tau is a structural microtubule-associated protein that is widely expressed in the nervous system that mediates the establishment of cell polarity, neurite outgrowth, and axonal transport. A useful model for studying the establishment and maintenance of these neuronal structures are rat neuronal PC12 cells, which can be induced to express tau and project neurites by treating the cells with nerve growth factor. Here, we present a simple method for continuously measuring the rate of neurite outgrowth and retraction over time by neurite length and neurite area analyses. This method uses freely available ImageJ software and widely available phase-contrast imaging.

The effect and mechanism of astragaloside Ⅳ on lysophosphatidic acid-induced neurite retraction of N1E-115 cells / 国际中医中药杂志

Objective:To observe the effect of astragaloside Ⅳ on lysophosphatidic acid(LPA)- induced neurite retraction of N1E-115 cells and its potential mechanism.Methods:N1E-115 cells were divided into blank group, model group, the low, medium and high dose groups of astragaloside Ⅳ. The blank group and model group was not intervened by astragaloside; while the low, medium and high dose groups were treated with 20,40 and 80 μg/ml astragaloside Ⅳ for 24 h. Each group was cultured with serum-free medium for 12 h. The model group and astragaloside Ⅳ groups were intervened by 40 μmol/L LPA for 10 min. Each group was observed and photographed with the inverted microscope, and the number of neurites in N1E-115 cells was counted by Image J software. The fluorescence expression of recombinant ras homolog gene family member A (RhoA), rho associated coiledcoil protein kinase 2 (ROCK2), phospho-rho associated coiledcoil protein kinase 2 (p-ROCK2) and phospho-myosin light chain 2 (p-MLC2) proteins was detected by immunohistochemistry. Real-time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expression levels of RhoA and ROCK2 ; the protein expression levels of RhoA, ROCK2, p-MLC2 and myosin light chain 2 (MLC2) were detected by Western blotting.Results:Compared with 20 μg/ml astragaloside Ⅳ group, the inhibition rate of neurite retraction in 40 and 80 μg/ml astragalosideⅣ groups increased ( P<0.05). Compared with model group, the average fluorescence intensity of RhoA, p-ROCK2, p-MLC2 in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the ROCK2 average fluorescence intensity in 40 μg/ml astragaloside Ⅳ group were decreased ( P<0.05, P<0.01); the expression of RhoA mRNA (0.89±0.09, 0.41±0.01, 0.09±0.03 vs. 1.50±0.01) and ROCK2 mRNA (0.89±0.09, 0.14±0.01, 0.20±0.01 vs. 1.62±0.17) decreased in 20, 40, 80 μg/ml astragaloside Ⅳ groups ( P<0.05, P<0.01); the ROCK2 protein (0.75±0.06, 0.57±0.02, 0.66±0.01 vs. 1.08±0.02), p-MLC2 protein (1.72±0.03, 1.40±0.04, 1.29±0.03 vs. 2.19±0.11), MLC2 protein (1.13±0.02, 0.68±0.03, 0.75±0.03 vs. 1.60±0.03) in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the RhoA protein (0.35±0.01, 0.40±0.03 vs. 0.57±0.08) in 20, 40 μg/ml astragaloside Ⅳ groups were decreased ( P<0.05, P<0.01). Conclusion:Astragaloside Ⅳ can prevent LPA-induced neurite retraction and promote damaged nerve regeneration. The mechanism may down-regulae the protein expression levels of RhoA, ROCK2, p-ROCK2, p-MLC2 and MLC2 in RhoA-ROCK2 signaling pathway, and inhibite nerve growth cone collapse.

Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation.

The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons.

Serum-induced neurite retraction in CAD cells--involvement of an ATP-actin retractile system and the lack of microtubule-associated proteins.

Cultured catecholamine-differentiated cells [which lack the microtubule-associated proteins (MAPs): MAP1B, MAP2, Tau, STOP, and Doublecortin] proliferate in the presence of fetal bovine serum, and, in its absence, cease dividing and generate processes similar to the neurites of normal neurons. The reintroduction of serum induces neurite retraction, and proliferation resumes. The neurite retraction process in catecholamine-differentiated cells was partially characterized in this study. Microtubules in the cells were found to be in a highly dynamic state, and tubulin in the microtubules consisted primarily of the tyrosinated and deacetylated isotypes. Increased levels of acetylated or Δ2-tubulin (which are normally absent) did not prevent serum-induced neurite retraction. Treatment of differentiated cells with lysophosphatidic acid or adenosine deaminase induced neurite retraction. Inhibition of Rho-associated protein kinase, ATP depletion and microfilament disruption each (individually) blocked serum-induced neurite retraction, suggesting that an ATP-dependent actomyosin system underlies the mechanism of neurite retraction. Nocodazole treatment induced neurite retraction, but this effect was blocked by pretreatment with the microtubule-stabilizing drug paclitaxel (Taxol). Paclitaxel did not prevent serum-induced or lysophosphatidic acid-induced retraction, suggesting that integrity of microtubules (despite their dynamic state) is necessary to maintain neurite elongation, and that paclitaxel-induced stabilization alone is not sufficient to resist the retraction force induced by serum. Transfection with green fluorescent protein-Tau conferred resistance to retraction caused by serum. We hypothesize that, in normal neurons (cultured or in vivo), MAPs are necessary not only to stabilize microtubules, but also to establish interactions with other cytoskeletal or membrane components to form a stable structure capable of resisting the retraction force.