Longitudinal alterations in brain perfusion and vascular reactivity in the zQ175DN mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is marked by a CAG-repeat expansion in the huntingtin gene that causes neuronal dysfunction and loss, affecting mainly the striatum and the cortex. Alterations in the neurovascular coupling system have been shown to lead to dysregulated energy supply to brain regions in several neurological diseases, including HD, which could potentially trigger the process of neurodegeneration. In particular, it has been observed in cross-sectional human HD studies that vascular alterations are associated to impaired cerebral blood flow (CBF). To assess whether whole-brain changes in CBF are present and follow a pattern of progression, we investigated both resting-state brain perfusion and vascular reactivity longitudinally in the zQ175DN mouse model of HD. METHODS: Using pseudo-continuous arterial spin labelling (pCASL) MRI in the zQ175DN model of HD and age-matched wild-type (WT) mice, we assessed whole-brain, resting-state perfusion at 3, 6 and 9 and 13 months of age, and assessed hypercapnia-induced cerebrovascular reactivity (CVR), at 4.5, 6, 9 and 15 months of age. RESULTS: We found increased perfusion in cortical regions of zQ175DN HET mice at 3 months of age, and a reduction of this anomaly at 6 and 9 months, ages at which behavioural deficits have been reported. On the other hand, under hypercapnia, CBF was reduced in zQ175DN HET mice as compared to the WT: for multiple brain regions at 6 months of age, for only somatosensory and retrosplenial cortices at 9 months of age, and brain-wide by 15 months. CVR impairments in cortical regions, the thalamus and globus pallidus were observed in zQ175DN HET mice at 9 months, with whole brain reactivity diminished at 15 months of age. Interestingly, blood vessel density was increased in the motor cortex at 3 months, while average vessel length was reduced in the lateral portion of the caudate putamen at 6 months of age. CONCLUSION: Our findings reveal early cortical resting-state hyperperfusion and impaired CVR at ages that present motor anomalies in this HD model, suggesting that further characterization of brain perfusion alterations in animal models is warranted as a potential therapeutic target in HD.

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.

Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity.

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers.

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

Altered basal forebrain function during whole-brain network activity at pre- and early-plaque stages of Alzheimer's disease in TgF344-AD rats.

BACKGROUND: Imbalanced synaptic transmission appears to be an early driver in Alzheimer's disease (AD) leading to brain network alterations. Early detection of altered synaptic transmission and insight into mechanisms causing early synaptic alterations would be valuable treatment strategies. This study aimed to investigate how whole-brain networks are influenced at pre- and early-plague stages of AD and if these manifestations are associated with concomitant cellular and synaptic deficits.  METHODS: To this end, we used an established AD rat model (TgF344-AD) and employed resting state functional MRI and quasi-periodic pattern (QPP) analysis, a method to detect recurrent spatiotemporal motifs of brain activity, in parallel with state-of-the-art immunohistochemistry in selected brain regions. RESULTS: At the pre-plaque stage, QPPs in TgF344-AD rats showed decreased activity of the basal forebrain (BFB) and the default mode-like network. Histological analyses revealed increased astrocyte abundance restricted to the BFB, in the absence of amyloid plaques, tauopathy, and alterations in a number of cholinergic, gaba-ergic, and glutamatergic synapses. During the early-plaque stage, when mild amyloid-beta (Aß) accumulation was observed in the cortex and hippocampus, QPPs in the TgF344-AD rats normalized suggesting the activation of compensatory mechanisms during this early disease progression period. Interestingly, astrogliosis observed in the BFB at the pre-plaque stage was absent at the early-plaque stage. Moreover, altered excitatory/inhibitory balance was observed in cortical regions belonging to the default mode-like network. In wild-type rats, at both time points, peak activity in the BFB preceded peak activity in other brain regions-indicating its modulatory role during QPPs. However, this pattern was eliminated in TgF344-AD suggesting that alterations in BFB-directed neuromodulation have a pronounced impact in network function in AD. CONCLUSIONS: This study demonstrates the value of rsfMRI and advanced network analysis methods to detect early alterations in BFB function in AD, which could aid early diagnosis and intervention in AD. Restoring the global synaptic transmission, possibly by modulating astrogliosis in the BFB, might be a promising therapeutic strategy to restore brain network function and delay the onset of symptoms in AD.

Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics.

Human papillomaviruses (HPV) are small, non-enveloped DNA viruses, which upon chronic infection can provoke cervical and head-and-neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer-causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high-content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time-resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image-based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.

MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells.

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as ß-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation.

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.

Effects of metformin on tumor hypoxia and radiotherapy efficacy: a [18F]HX4 PET imaging study in colorectal cancer xenografts.

BACKGROUND: In a colorectal cancer xenograft model, we investigated the therapeutic effect of metformin on tumor hypoxia with [18F]flortanidazole ([18F]HX4) small-animal positron emission tomography (µPET). We also assessed the additive effect of metformin on long-term radiotherapy outcome and we studied the potential of [18F]HX4 as a predictive and/or prognostic biomarker within this setup. METHODS: Colo205-bearing mice (n = 40) underwent a baseline [18F]HX4 hypoxia µPET/computed tomography (CT) scan. The next day, mice received 100 mg/kg metformin or saline intravenously (n = 20/group) and [18F]HX4 was administered intravenously 30 min later, whereupon a second µPET/CT scan was performed to assess changes in tumor hypoxia. Two days later, mice were further divided into four therapy groups (n = 10/group): control (1), metformin (2), radiotherapy (3), and metformin + radiotherapy, i.e., combination (4). Then, they received a second dose of metformin (groups 2 and 4) or saline (groups 1 and 3), followed by a single radiotherapy dose of 15 Gy (groups 3 and 4) or sham irradiation (groups 1 and 2) 30 min later. Tumor growth was followed three times a week by caliper measurements to assess the therapeutic outcome. RESULTS: [18F]HX4 uptake decreased in metformin-treated tumors with a mean intratumoral reduction in [18F]HX4 tumor-to-background ratio (TBR) from 2.53 ± 0.30 to 2.28 ± 0.26 (p = 0.04), as opposed to saline treatment (2.56 ± 0.39 to 3.08 ± 0.39; p = 0.2). The median tumor doubling time (TDT) was 6, 8, 41, and 43 days in the control, metformin, radiotherapy and combination group, respectively (log-rank p < 0.0001), but no metformin-specific therapy effects could be detected. Baseline [18F]HX4 TBR was a negative prognostic biomarker for TDT (hazard ratio, 2.39; p = 0.02). CONCLUSIONS: Metformin decreased [18F]HX4 uptake of Colo205-tumors, but had no additive effect on radiotherapy efficacy. Nevertheless, [18F]HX4 holds promise as a prognostic imaging biomarker.

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.

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.

Accurate Detection of Dysmorphic Nuclei Using Dynamic Programming and Supervised Classification.

A vast array of pathologies is typified by the presence of nuclei with an abnormal morphology. Dysmorphic nuclear phenotypes feature dramatic size changes or foldings, but also entail much subtler deviations such as nuclear protrusions called blebs. Due to their unpredictable size, shape and intensity, dysmorphic nuclei are often not accurately detected in standard image analysis routines. To enable accurate detection of dysmorphic nuclei in confocal and widefield fluorescence microscopy images, we have developed an automated segmentation algorithm, called Blebbed Nuclei Detector (BleND), which relies on two-pass thresholding for initial nuclear contour detection, and an optimal path finding algorithm, based on dynamic programming, for refining these contours. Using a robust error metric, we show that our method matches manual segmentation in terms of precision and outperforms state-of-the-art nuclear segmentation methods. Its high performance allowed for building and integrating a robust classifier that recognizes dysmorphic nuclei with an accuracy above 95%. The combined segmentation-classification routine is bound to facilitate nucleus-based diagnostics and enable real-time recognition of dysmorphic nuclei in intelligent microscopy workflows.

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.

BiDiFuse: a FIJI plugin for fusing bi-directionally recorded microscopic image volumes.

Deep tissue imaging is increasingly used for non-destructive interrogation of intact organs and small model organisms. An intuitive approach to increase the imaging depth by almost a factor of 2 is to record a sample from two sides and fuse both image stacks. However, imperfect three-dimensional alignment of both stacks presents a computational challenge. We have developed a FIJI plugin, called BiDiFuse, which merges bi-directionally recorded image stacks via 3D rigid transformations. The method is broadly applicable, considering it is compatible with all optical sectioning microscopes and it does not rely on fiducial markers for image registration. AVAILABILITY AND IMPLEMENTATION: The method is freely available as a plugin for FIJI from https://github.com/JanDetrez/BiDiFuse/ CONTACT: winnok.devos@uantwerpen.be.

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.