Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition.

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy.

The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here, we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

Morphological principles of neuronal mitochondria.

In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like adenosine triphosphate and heat often represent mitochondria as idealized geometries, and therefore, can miscalculate the metabolic fluxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial transmission electron microscopy (TEM) tomography images and converted to watertight meshes with minimal distortion of the original microscopy volumes with a granularity of 1.64 nanometer isotropic voxels. The resulting in-silico representations were subsequently quantified by differential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs present across this population of mitochondria, which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons.

Mitochondrial morphology provides a mechanism for energy buffering at synapses.

Mitochondria as the main energy suppliers of eukaryotic cells are highly dynamic organelles that fuse, divide and are transported along the cytoskeleton to ensure cellular energy homeostasis. While these processes are well established, substantial evidence indicates that the internal structure is also highly variable in dependence on metabolic conditions. However, a quantitative mechanistic understanding of how mitochondrial morphology affects energetic states is still elusive. To address this question, we here present an agent-based multiscale model that integrates three-dimensional morphologies from electron microscopy tomography with the molecular dynamics of the main ATP producing components. We apply our modeling approach to mitochondria at the synapse which is the largest energy consumer within the brain. Interestingly, comparing the spatiotemporal simulations with a corresponding space-independent approach, we find minor spatial effects when the system relaxes toward equilibrium but a qualitative difference in fluctuating environments. These results suggest that internal mitochondrial morphology is not only optimized for ATP production but also provides a mechanism for energy buffering and may represent a mechanism for cellular robustness.

Two Pathways for the Activity-Dependent Growth and Differentiation of Synaptic Boutons in Drosophila.

Synapse formation can be promoted by intense activity. At the Drosophila larval neuromuscular junction (NMJ), new synaptic boutons can grow acutely in response to patterned stimulation. We combined confocal imaging with electron microscopy and tomography to investigate the initial stages of growth and differentiation of new presynaptic boutons at the Drosophila NMJ. We found that the new boutons can form rapidly in intact larva in response to intense crawling activity, and we observed two different patterns of bouton formation and maturation. The first pathway involves the growth of filopodia followed by a formation of boutons that are initially devoid of synaptic vesicles (SVs) but filled with filamentous matrix. The second pathway involves rapid budding of synaptic boutons packed with SVs, and these more mature boutons are sometimes capable of exocytosis/endocytosis. We demonstrated that intense activity predominantly promotes the second pathway, i.e., budding of more mature boutons filled with SVs. We also showed that this pathway depends on synapsin (Syn), a neuronal protein which reversibly associates with SVs and mediates their clustering via a protein kinase A (PKA)-dependent mechanism. Finally, we took advantage of the temperature-sensitive mutant sei to demonstrate that seizure activity can promote very rapid budding of new boutons filled with SVs, and this process occurs at scale of minutes. Altogether, these results demonstrate that intense activity acutely and selectively promotes rapid budding of new relatively mature presynaptic boutons filled with SVs, and that this process is regulated via a PKA/Syn-dependent pathway.

Nano-structural analysis of engrafted human induced pluripotent stem cell-derived cardiomyocytes in mouse hearts using a genetic-probe APEX2.

Many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments. However, nano-structural confirmation of the successful incorporation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of graft cells in EM has proven to be difficult. Using APEX2, an engineered ascorbate peroxidase imaging tag, we successfully localized and analyzed the fine structure of sarcomeres and the excitation contraction machinery of iPSC-CMs 6 months after their engraftment in infarcted mouse hearts. APEX2 made iPSC-CMs visible in multiple imaging modalities including light microscopy, X-ray microscopic tomography, transmission EM, and scanning EM. EM tomography allowed assessment of the differentiation state of APEX2-positive iPSC-CMs and analysis of the fine structure of the sarcomeres including T-tubules and dyads.

Iron-specific Signal Separation from within Heavy Metal Stained Biological Samples Using X-Ray Microtomography with Polychromatic Source and Energy-Integrating Detectors.

Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- "High" and "Low", we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals. With proper selection of scan configuration, achieving strong overlap of source characteristic emission lines and iron K-edge absorption, iron absorption was enhanced enabling K-edge imaging. Specifically, iron images were obtained by scatter plot material analysis, after selecting specific regions within scatter plots generated from the "High" and "Low" scans. Using this method, we identified iron rich regions associated with an iron staining reaction that marks the nodes of Ranvier along nerve axons within mouse spinal roots, also stained with osmium metal commonly used for electron microscopy.

Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain.

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIß or the RIIß subunit results in distinct neuronal phenotypes. Although RIß contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIß is enriched in dendrites and co-localizes with MAP2, whereas RIIß is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIß in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIß, but not of RIIß, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

Cellular and subcellular localization of the neuron-specific plasma membrane calcium ATPase PMCA1a in the rat brain.

Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca(2+) extrusion from the cell, the plasma membrane calcium-dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a "housekeeping" function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca(2+) transients.