Eric Hwang

NeurphologyJ: An automatic neuronal morphology quantification method and its application in pharmacological discovery

BackgroundAutomatic quantification of neuronal morphology from images of fluorescence microscopy plays an increasingly important role in high-content screenings. However, there exist very few freeware tools and methods which provide automatic neuronal morphology quantification for pharmacological discovery.ResultsThis study proposes an effective quantification method, called NeurphologyJ, capable of automatically quantifying neuronal morphologies such as soma number and size, neurite length, and neurite branching complexity (which is highly related to the numbers of attachment points and ending points). NeurphologyJ is implemented as a plugin to ImageJ, an open-source Java-based image processing and analysis platform. The high performance of NeurphologyJ arises mainly from an elegant image enhancement method. Consequently, some morphology operations of image processing can be efficiently applied. We evaluated NeurphologyJ by comparing it with both the computer-aided manual tracing method NeuronJ and an existing ImageJ-based plugin method NeuriteTracer. Our results reveal that NeurphologyJ is comparable to NeuronJ, that the coefficient correlation between the estimated neurite lengths is as high as 0.992. NeurphologyJ can accurately measure neurite length, soma number, neurite attachment points, and neurite ending points from a single image. Furthermore, the quantification result of nocodazole perturbation is consistent with its known inhibitory effect on neurite outgrowth. We were also able to calculate the IC50 of nocodazole using NeurphologyJ. This reveals that NeurphologyJ is effective enough to be utilized in applications of pharmacological discoveries.ConclusionsThis study proposes an automatic and fast neuronal quantification method NeurphologyJ. The ImageJ plugin with supports of batch processing is easily customized for dealing with high-content screening applications. The source codes of NeurphologyJ (interactive and high-throughput versions) and the images used for testing are freely available (see Availability).

Collagen VI protects against neuronal apoptosis elicited by ultraviolet irradiation via an Akt/Phosphatidylinositol 3-kinase signaling pathway

Collagen VI, one of the extracellular matrix proteins, has been implicated in regulating cell proliferation and reducing apoptosis in several different systems. However, the role of collagen VI in the central nervous system remains unclear. In this manuscript, we demonstrated that upon ultraviolet (UV) irradiation, mouse primary hippocampal neurons specifically up-regulate the expression of Col6a1, Col6a2, and Col6a3 mRNA and secreted collagen VI protein. Augmentation of collagen VI mRNA and protein after UV irradiation may have a neuroprotective role as suggested by the fact that extracellular supplying soluble collagen VI protein, but not other collagen proteins, reduced UV induced DNA damage, mitochondria dysfunction, and neurite shrinkage. We also tried to determine the signaling molecules that mediate the protective effect of collagen VI via Western blot and inhibitor analysis. After collagen VI treatment, UV irradiated neurons increased phosphorylation of Akt and decreased phosphorylation of JNK. Inhibiting Akt/phosphatidylinositol 3-kinases (PI3K) pathway diminished the protective effect of collagen VI. Our study suggested a potential protective mechanism by which neurons up-regulate collagen VI production under stress conditions to activate Akt/PI3K anti-apoptotic signaling pathway.

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

NeuriteQuant: An open source toolkit for high content screens of neuronal Morphogenesis

BackgroundTo date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems.ResultsHere we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology.ConclusionsNeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.

The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis

Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).

Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.

An inverted method for culturing dissociated mouse hippocampal neurons.

Dissociated hippocampal neuron culture has long been the model system of choice for many neuroscientists. The ability to culture dissociated hippocampal neurons from genetically modified mice provides an invaluable tool for studying many neuronal processes. In this study, we established a novel method to culture dissociated hippocampal neurons from embryonic and neonatal mice. Dissociated neurons were cultured in a microchamber between the glass coverslip and the plastic cell container without the use of glial feeder cells. Our method significantly simplifies the preparation while produces healthy and long-lived neuronal cultures that are difficult to achieve without the use of feeder cells.

HCS-Neurons: identifying phenotypic changes in multi-neuron images upon drug treatments of high-content screening

BackgroundHigh-content screening (HCS) has become a powerful tool for drug discovery. However, the discovery of drugs targeting neurons is still hampered by the inability to accurately identify and quantify the phenotypic changes of multiple neurons in a single image (named multi-neuron image) of a high-content screen. Therefore, it is desirable to develop an automated image analysis method for analyzing multi-neuron images.ResultsWe propose an automated analysis method with novel descriptors of neuromorphology features for analyzing HCS-based multi-neuron images, called HCS-neurons. To observe multiple phenotypic changes of neurons, we propose two kinds of descriptors which are neuron feature descriptor (NFD) of 13 neuromorphology features, e.g., neurite length, and generic feature descriptors (GFDs), e.g., Haralick texture. HCS-neurons can 1) automatically extract all quantitative phenotype features in both NFD and GFDs, 2) identify statistically significant phenotypic changes upon drug treatments using ANOVA and regression analysis, and 3) generate an accurate classifier to group neurons treated by different drug concentrations using support vector machine and an intelligent feature selection method. To evaluate HCS-neurons, we treated P19 neurons with nocodazole (a microtubule depolymerizing drug which has been shown to impair neurite development) at six concentrations ranging from 0 to 1000 ng/mL. The experimental results show that all the 13 features of NFD have statistically significant difference with respect to changes in various levels of nocodazole drug concentrations (NDC) and the phenotypic changes of neurites were consistent to the known effect of nocodazole in promoting neurite retraction. Three identified features, total neurite length, average neurite length, and average neurite area were able to achieve an independent test accuracy of 90.28% for the six-dosage classification problem. This NFD module and neuron image datasets are provided as a freely downloadable MatLab project at http://iclab.life.nctu.edu.tw/HCS-Neurons.ConclusionsFew automatic methods focus on analyzing multi-neuron images collected from HCS used in drug discovery. We provided an automatic HCS-based method for generating accurate classifiers to classify neurons based on their phenotypic changes upon drug treatments. The proposed HCS-neurons method is helpful in identifying and classifying chemical or biological molecules that alter the morphology of a group of neurons in HCS.

Ran-dependent TPX2 activation promotes acentrosomal microtubule nucleation in neurons

The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.

Application of aurintricarboxylic acid for the adherence of mouse P19 neurons and primary hippocampal neurons to noncoated surface in serum-free culture

Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix‐coated surface to provide a proper environment for cell anchorage under serum‐free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum‐free medium in a dosage‐dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 μg/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 μg/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly‐L‐lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed.