Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease.

Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-µm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.

Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver.

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.

A cellular hierarchy in melanoma uncouples growth and metastasis.

Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.

Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization.

Identifying protein-protein interactions (PPIs) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.

An in situ sequencing approach maps PLASTOCHRON1 at the boundary between indeterminate and determinate cells.

The plant shoot apex houses the shoot apical meristem, a highly organized and active stem-cell tissue where molecular signaling in discrete cells determines when and where leaves are initiated. We optimized a spatial transcriptomics approach, in situ sequencing (ISS), to colocalize the transcripts of 90 genes simultaneously on the same section of tissue from the maize (Zea mays) shoot apex. The RNA ISS technology reported expression profiles that were highly comparable with those obtained by in situ hybridizations (ISHs) and allowed the discrimination between tissue domains. Furthermore, the application of spatial transcriptomics to the shoot apex, which inherently comprised phytomers that are in gradual developmental stages, provided a spatiotemporal sequence of transcriptional events. We illustrate the power of the technology through PLASTOCHRON1 (PLA1), which was specifically expressed at the boundary between indeterminate and determinate cells and partially overlapped with ROUGH SHEATH1 and OUTER CELL LAYER4 transcripts. Also, in the inflorescence, PLA1 transcripts localized in cells subtending the lateral primordia or bordering the newly established meristematic region, suggesting a more general role of PLA1 in signaling between indeterminate and determinate cells during the formation of lateral organs. Spatial transcriptomics builds on RNA ISH, which assays relatively few transcripts at a time and provides a powerful complement to single-cell transcriptomics that inherently removes cells from their native spatial context. Further improvements in resolution and sensitivity will greatly advance research in plant developmental biology.

The STE20 kinase TAOK3 controls the development of house dust mite-induced asthma in mice.

BACKGROUND: The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. OBJECTIVE: We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model. METHODS: Wild-type Taok3+/+ and gene-deficient Taok3-/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4+ T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3-/- and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. RESULTS: Taok3-/- mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. CONCLUSIONS: Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma.

Excess LINC complexes impair brain morphogenesis in a mouse model of recessive TOR1A disease.

Heterozygosity for the TOR1A-Δgag mutation causes semi-penetrant childhood-onset dystonia (OMIM #128100). More recently, homozygous TOR1A mutations were shown to cause severe neurological dysfunction in infants. However, there is little known about the recessive cases, including whether existing reports define the full spectrum of recessive TOR1A disease. Here we describe abnormal brain morphogenesis in ∼30% of Tor1a-/- mouse embryos while, in contrast, this is not found in Tor1aΔgag/Δgag mice. The abnormal Tor1a-/- brains contain excess neural tissue, as well as proliferative zone cytoarchitectural defects related to radial glial cell polarity and cytoskeletal organization. In cultured cells torsinA effects the linker of nucleoskeleton and cytoskeleton (LINC) complex that couples the nucleus and cytoskeleton. Here we identify that torsinA loss elevates LINC complex levels in the proliferative zone, and that genetic reduction of LINC complexes prevents abnormal brain morphogenesis in Tor1a-/- embryos. These data show that Tor1a affects radial glial cells via a LINC complex mediated mechanism. They also predict human TOR1A disease will include incompletely penetrant defects in embryonic brain morphogenesis in cases where mutations ablate TOR1A function.

A Label-free Multicolor Optical Surface Tomography (ALMOST) imaging method for nontransparent 3D samples.

BACKGROUND: Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples-for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples-cannot be assessed. We have developed "ALMOST," a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters. RESULTS: As well as demonstrating image formation, we provide background information and explain the reconstruction-and consequent rendering-using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole. CONCLUSION: Thus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed.

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice.

BACKGROUND: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk. OBJECTIVE: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma. METHODS: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3-/- mice, and overexpressing Ormdl3Tg/wt mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, TH2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured. RESULTS: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3Tg/wt mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3-/- mice, whereas they were decreased in Ormdl3Tg/wt mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wild-type, Ormdl3Tg/wt, and Ormdl3-/- mice across several models of experimental asthma. CONCLUSION: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.

Three-dimensional reconstruction of the intercalated disc including the intercellular junctions by applying volume scanning electron microscopy.

The intercalated disc (ID) contains different kinds of intercellular junctions: gap junctions (GJs), desmosomes and areae compositae, essential for adhesion and communication between adjacent cardiomyocytes. The junctions can be identified based on their morphology when imaged using transmission electron microscopy (TEM), however, only with very limited information in the z-dimension. The application of volume EM techniques can give insight into the three-dimensional (3-D) organization of complex biological structures. In this study, we generated 3-D datasets using serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam SEM (FIB-SEM), the latter resulting in datasets with 5 nm isotropic voxels. We visualized cardiomyocytes in murine ventricular heart tissue and, for the first time, we could three-dimensionally reconstruct the ID including desmosomes and GJs with 5 nm precision in a large volume. Results show in three dimensions a highly folded structure of the ID, with the presence of GJs and desmosomes in both plicae and interplicae regions. We observed close contact of GJs with mitochondria and a variable spatial distribution of the junctions. Based on measurements of the shape of the intercellular junctions in 3-D, it is seen that GJs and desmosomes vary in size, depending on the region within the ID. This demonstrates that volume EM is essential to visualize morphological changes and its potential to quantitatively determine structural changes between normal and pathological conditions, e.g., cardiomyopathies.

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane.

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided.

Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

Accumulation of APP C-terminal fragments causes endolysosomal dysfunction through the dysregulation of late endosome to lysosome-ER contact sites.

Neuronal endosomal and lysosomal abnormalities are among the early changes observed in Alzheimer's disease (AD) before plaques appear. However, it is unclear whether distinct endolysosomal defects are temporally organized and how altered γ-secretase function or amyloid precursor protein (APP) metabolism contribute to these changes. Inhibiting γ-secretase chronically, in mouse embryonic fibroblast and hippocampal neurons, led to a gradual endolysosomal collapse initiated by decreased lysosomal calcium and increased cholesterol, causing downstream defects in endosomal recycling and maturation. This endolysosomal demise is γ-secretase dependent, requires membrane-tethered APP cytoplasmic domains, and is rescued by APP depletion. APP C-terminal fragments (CTFs) localized to late endosome/lysosome-endoplasmic reticulum contacts; an excess of APP-CTFs herein reduced lysosomal Ca2+ refilling from the endoplasmic reticulum, promoting cholesterol accretion. Tonic regulation by APP-CTFs provides a mechanistic explanation for their cellular toxicity: failure to timely degrade APP-CTFs sustains downstream signaling, instigating lysosomal dyshomeostasis, as observed in prodromal AD. This is the opposite of substrates such as Notch, which require intramembrane proteolysis to initiate signaling.

Inflammasome signaling is dispensable for ß-amyloid-induced neuropathology in preclinical models of Alzheimer's disease.

Background: Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting memory and cognition. The disease is accompanied by an abnormal deposition of ß-amyloid plaques in the brain that contributes to neurodegeneration and is known to induce glial inflammation. Studies in the APP/PS1 mouse model of ß-amyloid-induced neuropathology have suggested a role for inflammasome activation in ß-amyloid-induced neuroinflammation and neuropathology. Methods: Here, we evaluated the in vivo role of microglia-selective and full body inflammasome signalling in several mouse models of ß-amyloid-induced AD neuropathology. Results: Microglia-specific deletion of the inflammasome regulator A20 and inflammasome effector protease caspase-1 in the AppNL-G-F and APP/PS1 models failed to identify a prominent role for microglial inflammasome signalling in ß-amyloid-induced neuropathology. Moreover, global inflammasome inactivation through respectively full body deletion of caspases 1 and 11 in AppNL-G-F mice and Nlrp3 deletion in APP/PS1 mice also failed to modulate amyloid pathology and disease progression. In agreement, single-cell RNA sequencing did not reveal an important role for Nlrp3 signalling in driving microglial activation and the transition into disease-associated states, both during homeostasis and upon amyloid pathology. Conclusion: Collectively, these results question a generalizable role for inflammasome activation in preclinical amyloid-only models of neuroinflammation.

Parkinsonism mutations in DNAJC6 cause lipid defects and neurodegeneration that are rescued by Synj1.

Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. Here, we generated a new Drosophila knock-in model of DNAJC6/Auxilin and find that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants, membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function, are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and metabolize specific phosphoinositides, rescues the DNAJC6/Auxilin lipid alterations, the neuronal function defects and neurodegeneration. Our work reveals a functional relation between two proteins mutated in Parkinsonism and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival.

PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.

The integrity of ER-mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within the ER-mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER-mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER-plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker α-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.