Integrated staging of morphofunctional connectivity in neuronal cultures.

Neurological disorders are defined by synaptic dysfunction. We present a workflow to quantify morphological and functional aspects of synaptic connectivity in neuronal cultures and obtain an integrated readout. We describe steps for measuring synchronous calcium bursting in GCaMP6f-transduced neurons and labeling mature synapses using a proximity ligation assay. The integration of functional and morphological information from the same cultures provides a rich fingerprint of synaptic connectivity, deployable in different experimental conditions. For complete details on the use and execution of this protocol, please refer to Verstraelen et al. and Verschuuren et al.1,2.

Serum Amyloid A3 Fuels a Feed-Forward Inflammatory Response to the Bacterial Amyloid Curli in the Enteric Nervous System.

BACKGROUND & AIMS: Mounting evidence suggests the gastrointestinal microbiome is a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastrointestinal tract and its enteric nervous system. METHODS: To examine the early response, we challenged primary murine myenteric networks with curli, the prototypical bacterial amyloid, and performed shotgun RNA sequencing and multiplex enzyme-linked immunosorbent assay. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. RESULTS: Curli induced a proinflammatory response, with strong up-regulation of Saa3 and the secretion of several cytokines. This proinflammatory state was induced primarily in enteric glia, was accompanied by increased levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant Serum Amyloid A3 (SAA3) was sufficient to recapitulate this specific proinflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Similar to curli, recombinant SAA3 caused a strong up-regulation of Saa3 transcripts, illustrating its self-amplifying potential . Since colonization of curli-producing Salmonella and dextran sulfate sodium-induced colitis triggered a significant increase in Saa3 transcripts as well, we assume SAA3plays a central role in enteric dysfunction. Inhibition of dual leucine zipper kinase, an upstream regulator of the c-Jun N-terminal kinase pathway responsible for SAA3 production, attenuated curli- and recombinant SAA3-induced Saa3 up-regulation, DNA damage, and replication in enteric glia. CONCLUSIONS: Our results position SAA3 as an important mediator of gastrointestinal vulnerability to bacterial-derived amyloids and demonstrate the potential of dual leucine zipper kinase inhibition to dampen enteric pathology.

S100A8-enriched microglia populate the brain of tau-seeded and accelerated aging mice.

Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.

High-throughput Analysis of Synaptic Activity in Electrically Stimulated Neuronal Cultures.

Synaptic dysfunction is a hallmark of various neurodegenerative and neurodevelopmental disorders. To interrogate synapse function in a systematic manner, we have established an automated high-throughput imaging pipeline based on fluorescence microscopy acquisition and image analysis of electrically stimulated synaptic transmission in neuronal cultures. Identification and measurement of synaptic signal fluctuations is achieved by means of an image analysis algorithm based on singular value decomposition. By exploiting the synchronicity of the evoked responses, the algorithm allows disentangling distinct temporally correlated patterns of firing synapse populations or cell types that are present in the same recording. We demonstrate the performance of the analysis with a pilot compound screen and show that the multiparametric readout allows classifying treatments by their spatiotemporal fingerprint. The image analysis and visualization software has been made publicly available on Github ( https://www.github.com/S3Toolbox ). The streamlined automation of multi-well image acquisition, electrical stimulation, analysis, and meta-data warehousing facilitates large-scale synapse-oriented screens and, in doing so, it will accelerate the drug discovery process.

Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction.

Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.

Systematic Quantification of Synapses in Primary Neuronal Culture.

Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

Improving extracellular vesicles visualization: From static to motion.

In the last decade, extracellular vesicles (EVs) have become a hot topic. The findings on EVs content and effects have made them a major field of interest in cancer research. EVs, are able to be internalized through integrins expressed in parental cells, in a tissue specific manner, as a key step of cancer progression and pre-metastatic niche formation. However, this specificity might lead to new opportunities in cancer treatment by using EVs as devices for drug delivery. For future applications of EVs in cancer, improved protocols and methods for EVs isolation and visualization are required. Our group has put efforts on developing a protocol able to track the EVs for in vivo internalization analysis. We showed, for the first time, the videos of labeled EVs uptake by living lung cancer cells.

Single-Cell and Neuronal Network Alterations in an In Vitro Model of Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) is involved in many cellular processes and it regulates synaptic and network development in neurons. Its absence is known to lead to intellectual disability, with a wide range of comorbidities including autism. Over the past decades, FMRP research focused on abnormalities both in glutamatergic and GABAergic signaling, and an altered balance between excitation and inhibition has been hypothesized to underlie the clinical consequences of absence of the protein. Using Fmrp knockout mice, we studied an in vitro model of cortical microcircuitry and observed that the loss of FMRP largely affected the electrophysiological correlates of network development and maturation but caused less alterations in single-cell phenotypes. The loss of FMRP also caused a structural increase in the number of excitatory synaptic terminals. Using a mathematical model, we demonstrated that the combination of an increased excitation and reduced inhibition describes best our experimental observations during the ex vivo formation of the network connections.

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity.

Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity.

Image-Based Profiling of Synaptic Connectivity in Primary Neuronal Cell Culture.

Neurological disorders display a broad spectrum of clinical manifestations. Yet, at the cellular level, virtually all these diseases converge into a common phenotype of dysregulated synaptic connectivity. In dementia, synapse dysfunction precedes neurodegeneration and cognitive impairment by several years, making the synapse a crucial entry point for the development of diagnostic and therapeutic strategies. Whereas high-resolution imaging and biochemical fractionations yield detailed insight into the molecular composition of the synapse, standardized assays are required to quickly gauge synaptic connectivity across large populations of cells under a variety of experimental conditions. Such screening capabilities have now become widely accessible with the advent of high-throughput, high-content microscopy. In this review, we discuss how microscopy-based approaches can be used to extract quantitative information about synaptic connectivity in primary neurons with deep coverage. We elaborate on microscopic readouts that may serve as a proxy for morphofunctional connectivity and we critically analyze their merits and limitations. Finally, we allude to the potential of alternative culture paradigms and integrative approaches to enable comprehensive profiling of synaptic connectivity.

Selective Labeling of Individual Neurons in Dense Cultured Networks With Nanoparticle-Enhanced Photoporation.

Neurodevelopmental and neurodegenerative disorders are characterized by subtle alterations in synaptic connections and perturbed neuronal network functionality. A hallmark of neuronal connectivity is the presence of dendritic spines, micron-sized protrusions of the dendritic shaft that compartmentalize single synapses to fine-tune synaptic strength. However, accurate quantification of spine density and morphology in mature neuronal networks is hampered by the lack of targeted labeling strategies. To resolve this, we have optimized a method to deliver cell-impermeable compounds into selected cells based on Spatially resolved NAnoparticle-enhanced Photoporation (SNAP). We show that SNAP enables efficient labeling of selected individual neurons and their spines in dense cultured networks without affecting short-term viability. We compare SNAP with widely used spine labeling techniques such as the application of lipophilic dyes and genetically encoded fluorescent markers. Using SNAP, we demonstrate a time-dependent increase in spine density in healthy cultures as well as a reduction in spine density after chemical mimicry of hypoxia. Since the sparse labeling procedure can be automated using an intelligent acquisition scheme, SNAP holds promise for high-content screening campaigns of neuronal connectivity in the context of neurodevelopmental and neurodegenerative disorders.

Fast spatial-selective delivery into live cells.

Intracellular delivery of functional compounds into living cells is of great importance for cell biology as well as therapeutic applications. Often it is sufficient that the compound of interest (being a molecule or nanoparticle) is delivered to the cell population as a whole. However, there are applications that would benefit considerably from the possibility of delivering a compound to a certain subpopulation of cells, or even in selected single cells. Here we report on an integrated platform for high-throughput spatially resolved nanoparticle-enhanced photoporation (SNAP) of adherent cells. SNAP enables safe, intracellular delivery of exogenously administered nanomaterials in selected subpopulations of cells, even down to the single cell level. We demonstrate the power of SNAP by selectively delivering a safe contrast agent into a subpopulation of polynucleated keratinocytes, enabling their downstream purification for unraveling their role in neoplasm formation. The flexibility and speed with which individual cells can be labeled make SNAP an ideal tool for high-throughput applications, not only for selective labeling but also for targeted drug delivery.

Dysregulation of Microtubule Stability Impairs Morphofunctional Connectivity in Primary Neuronal Networks.

Functionally related neurons assemble into connected networks that process and transmit electrochemical information. To do this in a coordinated manner, the number and strength of synaptic connections is tightly regulated. Synapse function relies on the microtubule (MT) cytoskeleton, the dynamics of which are in turn controlled by a plethora of MT-associated proteins, including the MT-stabilizing protein Tau. Although mutations in the Tau-encoding MAPT gene underlie a set of neurodegenerative disorders, termed tauopathies, the exact contribution of MT dynamics and the perturbation thereof to neuronal network connectivity has not yet been scrutinized. Therefore, we investigated the impact of targeted perturbations of MT stability on morphological (e.g., neurite- and synapse density) and functional (e.g., synchronous calcium bursting) correlates of connectivity in networks of primary hippocampal neurons. We found that treatment with MT-stabilizing or -destabilizing compounds impaired morphofunctional connectivity in a reversible manner. We also discovered that overexpression of MAPT induced significant connectivity defects, which were accompanied by alterations in MT dynamics and increased resistance to pharmacological MT depolymerization. Overexpression of a MAPT variant harboring the P301L point mutation in the MT-binding domain did far less, directly linking neuronal connectivity with Tau's MT binding affinity. Our results show that MT stability is a vulnerable node in tauopathies and that its precise pharmacological tuning may positively affect neuronal network connectivity. However, a critical balance in MT turnover causes it to be a difficult therapeutic target with a narrow operating window.

Sustained synchronized neuronal network activity in a human astrocyte co-culture system.

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

Image Informatics Strategies for Deciphering Neuronal Network Connectivity.

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Amongst the neuronal structures that show morphological plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular communication and the associated calcium bursting behaviour. In vitro cultured neuronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardization of both image acquisition and image analysis, it has become possible to extract statistically relevant readouts from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies.

Pharmacological characterization of cultivated neuronal networks: relevance to synaptogenesis and synaptic connectivity.

Mental disorders, such as schizophrenia or Alzheimer's disease, are associated with impaired synaptogenesis and/or synaptic communication. During development, neurons assemble into neuronal networks, the primary supracellular mediators of information processing. In addition to the orchestrated activation of genetic programs, spontaneous electrical activity and associated calcium signaling have been shown to be critically involved in the maturation of such neuronal networks. We established an in vitro model that recapitulates the maturation of neuronal networks, including spontaneous electrical activity. Upon plating, mouse primary hippocampal neurons grow neurites and interconnect via synapses to form a dish-wide neuronal network. Via live cell calcium imaging, we identified a limited period of time in which the spontaneous activity synchronizes across neurons, indicative of the formation of a functional network. After establishment of network activity, the neurons grow dendritic spines, the density of which was used as a morphological readout for neuronal maturity and connectivity. Hence, quantification of neurite outgrowth, synapse density, spontaneous neuronal activity, and dendritic spine density allowed to study neuronal network maturation from the day of plating until the presence of mature neuronal networks. Via acute pharmacological intervention, we show that synchronized network activity is mediated by the NMDA-R. The balance between kynurenic and quinolinic acid, both neuro-active intermediates in the tryptophan/kynurenine pathway, was shown to be decisive for the maintenance of network activity. Chronic modulation of the neurotrophic support influenced the network formation and revealed the extreme sensitivity of calcium imaging to detect subtle alterations in neuronal physiology. Given the reproducible cultivation in a 96-well setup in combination with fully automated analysis of the calcium recordings, this approach can be used to build a high-content screening assay usable for neurotoxicity screening, target identification/validation, or phenotypic drug screening.

Quantitation of chronic and acute treatment effects on neuronal network activity using image and signal analysis: toward a high-content assay.

Upon maturation, primary neuronal cultures form an interconnected network based on neurite outgrowth and synaptogenesis in which spontaneous electrical activity arises. Measurement of network activity allows quantification of neuronal health and maturation. A fluorescent indicator was used to monitor secondary calcium influxes after the occurrence of action potentials, allowing us to examine activity of hippocampal cultures via confocal live cell imaging. Subsequently, nuclear staining with DAPI allows accurate cell segmentation. To analyze the calcium recording in a robust, observer-independent manner, we implemented an automated image- and signal-processing algorithm and validated it against a visual, interactive procedure. Both methods yielded similar results on the emergence of synchronized activity and allowed robust quantitative measurement of acute and chronic modulation of drugs on network activity. Both the number of days in vitro (DIV) and neutralization of nerve growth factor (NGF) have a significant effect on synchronous burst frequency and correlation. Acute effects are demonstrated using 5-HT (serotonin) and ethylene glycol tetra-acetic acid. Automated analysis allowed measuring additional features, such as peak decay times and bursting frequency of individual neurons. Based on neuronal cell cultures in 96-well plates and accurate calcium recordings, the analysis method allows development of an integrated high-content screening assay. Because molecular biological techniques can be applied to assess the influence of genes on network activity, it is applicable for neurotoxicity or neurotrophics screening as well as development of in vitro disease models via, for example, pharmacologic manipulation or RNAi.

High-content imaging in cervical cancer screening.

A shift from conventional cytology to a molecular approach could improve cervical cancer screening. This proof-of-concept study aims to develop a high-content imaging platform for the simultaneous detection of multiple biomarkers for cervical disease. Liquid-based cytology (LBC) samples were used to optimize a dual ProExC/Ki-67 immunofluorescence staining protocol for SurePath-fixed cells. The simultaneous and automated detection of these biomarkers was performed using the BD Pathway 435 system. The ability of high-content imaging to detect dysplastic cervical cells was assessed using keratinocytes spiked with immunopositive SiHa cells and a high-grade squamous intraepithelial lesion (HSIL) LBC sample. The percentages of Ki-67- and ProExC-immunopositive objects correlated significantly with the percentages of spiked SiHa cells. The dysplastic cells of the HSIL sample could be detected using high-content cell analysis. In conclusion, high-content imaging allows the simultaneous and automated detection of Ki-67- and ProExC-immunopositive dysplastic cells in LBC specimens.