M H Ellisman

University of California, San Diego, San Diego, California, United States

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Publications (193)1130.41 Total impact

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    ABSTRACT: Evidence indicates that nitrosative stress and mitochondrial dysfunction participate in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Aβ) and peroxynitrite induce mitochondrial fragmentation and neuronal cell death by abnormal activation of dynamin-related protein 1 (DRP1), a large GTPase that regulates mitochondrial fission. The exact mechanisms of mitochondrial fragmentation and DRP1 overactivation in AD remain unknown; however, DRP1 serine 616 (S616) phosphorylation is likely involved. Although it is clear that nitrosative stress caused by peroxynitrite has a role in AD, effective antioxidant therapies are lacking. Cerium oxide nanoparticles, or nanoceria, switch between their Ce(3+) and Ce(4+) states and are able to scavenge superoxide anions, hydrogen peroxide and peroxynitrite. Therefore, nanoceria might protect against neurodegeneration. Here we report that nanoceria are internalized by neurons and accumulate at the mitochondrial outer membrane and plasma membrane. Furthermore, nanoceria reduce levels of reactive nitrogen species and protein tyrosine nitration in neurons exposed to peroxynitrite. Importantly, nanoceria reduce endogenous peroxynitrite and Aβ-induced mitochondrial fragmentation, DRP1 S616 hyperphosphorylation and neuronal cell death.Cell Death and Differentiation advance online publication, 6 June 2014; (2014) 0, 000-000.doi:10.1038/cdd.2014.72.
    Cell death and differentiation. 06/2014;
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    ABSTRACT: We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (Vitrification Of Sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.
    Journal of Structural Biology 01/2014; · 3.36 Impact Factor
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    ABSTRACT: The cell-autonomous role of synaptic transmission in the regulation of neuronal structural and electrical properties is unclear. We have now employed a genetic approach to eliminate glutamatergic synaptic transmission onto individual CA1 pyramidal neurons in a mosaic fashion in vivo. Surprisingly, while electrical properties are profoundly affected in these neurons, as well as inhibitory synaptic transmission, we found little perturbation of neuronal morphology, demonstrating a functional segregation of excitatory synaptic transmission from neuronal morphological development.
    Neuron 05/2013; 78(3):433-9. · 15.77 Impact Factor
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    ABSTRACT: 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.
    The Journal of General Physiology 04/2013; · 4.73 Impact Factor
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    ABSTRACT: Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H2O2)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ10 can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ10 not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ10 prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ10 preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ10 may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.
    Cell Death & Disease 01/2013; 4:e820. · 6.04 Impact Factor
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    ABSTRACT: The cell: an image library-CCDB (CIL-CCDB) (http://www.cellimagelibrary.org) is a searchable database and archive of cellular images. As a repository for microscopy data, it accepts all forms of cell imaging from light and electron microscopy, including multi-dimensional images, Z- and time stacks in a broad variety of raw-data formats, as well as movies and animations. The software design of CIL-CCDB was intentionally designed to allow easy incorporation of new technologies and image formats as they are developed. Currently, CIL-CCDB contains over 9250 images from 358 different species. Images are evaluated for quality and annotated with terms from 14 different ontologies in 16 different fields as well as a basic description and technical details. Since its public launch on 9 August 2010, it has been designed to serve as not only an archive but also an active site for researchers and educators.
    Nucleic Acids Research 11/2012; · 8.81 Impact Factor
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    ABSTRACT: 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.
    Science 11/2012; 338(6110):1080-1084. · 31.20 Impact Factor
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    ABSTRACT: Optic atrophy 1 (OPA1) mutations cause dominant optic atrophy (DOA) with retinal ganglion cell (RGC) and optic nerve degeneration. The mechanism for the selective degeneration of RGCs in DOA remains elusive. To address the mechanism, we reduced OPA1 protein expression in cell lines and RGCs by RNA interference. OPA1 loss results in mitochondrial fragmentation, deficiency in oxidative phosphorylation, decreased ATP levels, decreased mitochondrial Ca(2+) retention capacity, reduced mtDNA copy numbers, and sensitization to apoptotic insults. We demonstrate profound cristae depletion and loss of crista junctions in OPA1 knockdown cells, whereas the remaining crista junctions preserve their normal size. OPA1-depleted cells exhibit decreased agonist-evoked mitochondrial Ca(2+) transients and corresponding reduction of NAD(+) to NADH, but the impairment in NADH oxidation leads to an overall more reduced mitochondrial NADH pool. Although in our model OPA1 loss in RGCs has no apparent impact on mitochondrial morphology, it decreases buffering of cytosolic Ca(2+) and sensitizes RGCs to excitotoxic injury. Exposure to glutamate triggers delayed calcium deregulation (DCD), often in a reversible manner, indicating partial resistance of RGCs to this injury. However, when OPA1 is depleted, DCD becomes irreversible. Thus, our data show that whereas OPA1 is required for mitochondrial fusion, maintenance of crista morphology and oxidative phosphorylation, loss of OPA1 also results in defective Ca(2+) homeostasis.Cell Death and Differentiation advance online publication, 9 November 2012; doi:10.1038/cdd.2012.128.
    Cell death and differentiation 11/2012; · 8.24 Impact Factor
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    ABSTRACT: Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments or require light and can be difficult to use. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.
    Nature Biotechnology 10/2012; · 32.44 Impact Factor
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    ABSTRACT: Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central β core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors. PAPERFLICK:
    Cell 10/2012; 151(2):304-19. · 31.96 Impact Factor
  • Microscopy and Microanalysis 07/2012; 18(S2):834-835. · 2.50 Impact Factor
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    ABSTRACT: Electron tomography (ET) has been proven an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. Large-field high-resolution views of biological specimens span more than four orders of magnitude in spatial scale, and, as a consequence, are rather difficult to generate directly. Various techniques have been developed towards generating those views, from increasing the sensor array size to implementing serial sectioning and montaging. Datasets and reconstructions obtained by the latter techniques generate multiple three-dimensional (3D) reconstructions, that need to be combined together to provide all the multiscale information. In this work, we show how to implement montages within TxBR, a tomographic reconstruction software package. This work involves some new application of mathematical concepts related to volume preserving transformations and issues of gauge ambiguity, which are essential problems arising from the nature of the observation in an electron microscope. The purpose of TxBR is to handle those issues as generally as possible in order to correct for most distortions in the 3D reconstructions and allow for a seamless recombination of ET montages.
    Journal of Structural Biology 06/2012; 180(1):154-64. · 3.36 Impact Factor
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    ABSTRACT: While diet-induced obesity has been exclusively attributed to increased caloric intake from fat, animals fed a high-fat diet (HFD) ad libitum (ad lib) eat frequently throughout day and night, disrupting the normal feeding cycle. To test whether obesity and metabolic diseases result from HFD or disruption of metabolic cycles, we subjected mice to either ad lib or time-restricted feeding (tRF) of a HFD for 8 hr per day. Mice under tRF consume equivalent calories from HFD as those with ad lib access yet are protected against obesity, hyperinsulinemia, hepatic steatosis, and inflammation and have improved motor coordination. The tRF regimen improved CREB, mTOR, and AMPK pathway function and oscillations of the circadian clock and their target genes' expression. These changes in catabolic and anabolic pathways altered liver metabolome and improved nutrient utilization and energy expenditure. We demonstrate in mice that tRF regimen is a nonpharmacological strategy against obesity and associated diseases.
    Cell metabolism 05/2012; 15(6):848-60. · 17.35 Impact Factor
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    ABSTRACT: Chronic hypoxia (CH) occurs under certain physiological or pathological conditions, including in people who reside at high altitude or suffer chronic cardiovascular or pulmonary diseases. As mitochondria are the predominant oxygen-consuming organelles to generate ATP through oxidative phosphorylation in cells, their responses, through structural or molecular modifications, to limited oxygen supply play an important role in the overall functional adaptation to hypoxia. Here, we report the adaptive mitochondrial ultrastructural modifications and the functional impacts in a recently generated hypoxia-adapted Drosophila melanogaster strain that survives severe, otherwise lethal, hypoxic conditions. Using electron tomography, we discovered increased mitochondrial volume density and cristae abundance, yet also cristae fragmentation and a unique honeycomb-like structure in the mitochondria of hypoxia-adapted flies. The homeostatic levels of adenylate and energy charge were similar between hypoxia-adapted and naïve control flies and the hypoxia-adapted flies remained active under severe hypoxia as quantified by negative geotaxis behavior. The equilibrium ATP level was lower in hypoxia-adapted flies than those of the naïve controls tested under severe hypoxia that inhibited the motion of control flies. Our results suggest that the structural rearrangement in the mitochondria of hypoxia-adapted flies may be an important adaptive mechanism that plays a critical role in preserving adenylate homeostasis and metabolism as well as muscle function under chronic hypoxic conditions.
    PLoS ONE 01/2012; 7(9):e45344. · 3.53 Impact Factor
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    ABSTRACT: Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.
    Nature Communications 01/2012; 3:1227. · 10.02 Impact Factor
  • Biophysical Journal 01/2012; 102(3):408-. · 3.67 Impact Factor
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    ABSTRACT: Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.
    Microscopy and Microanalysis 06/2011; 17:276 - 277. · 2.50 Impact Factor
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    ABSTRACT: The dopamine transporter knockout (DAT KO) mouse is a model of chronic hyperdopaminergia used to study a wide range of neuropsychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), drug abuse, depression, and Parkinson's disease (PD). Early studies characterizing this mouse model revealed a subtle, but significant, decrease in the anterior striatal volume of DAT KO mice accompanied by a decrease in neuronal cell body numbers (Cyr et al., 2005). The present studies were conducted to examine medium spiny neuron (MSN) morphology by extending these earlier reports to include multiscale imaging studies using correlated light microscopy (LM) and electron microscopy (EM) techniques. Specifically, we set out to determine if chronic hyperdopaminergia results in quantifiable or qualitative changes in DAT KO mouse MSNs relative to wild-type (WT) littermates. Using Neurolucida Explorer's morphometric analysis, we measured spine density, dendritic length and synapse number at ages that correspond with the previously reported changes in striatal volume and progressive cell loss. Light microscopic analysis using Neurolucida tracings of photoconverted striatal MSNs revealed a highly localized loss of dendritic spines on the proximal portion of the dendrite (30 μm from the soma) in the DAT KO group. Next, thick sections containing MSN dendritic segments located at a distance of 20-60 μm from the cell soma, a region of the dendrite where spine density is reported to be the highest, were analyzed using electron microscope tomography (EMT). Because of the resolution limits of LM, the EM analysis was an extra measure taken to assure that our analysis included nearly all spines. Spine density measurements collected from the EMT data revealed only a modest decrease in the DAT KO group (n=3 mice) compared to age-matched WT controls (n=3 mice), a trend that supports the LM findings. Finally, a synaptic quantification using unbiased stereology did not detect a difference between DAT KO mice (n=6 mice) and WT controls (n=7 mice) at the EM level, supporting the focal nature of the early synaptic loss. These findings suggest that DAT KO mice have MSNs with highly localized spine loss and not an overall morphologically distinct cell shape. The characterization of morphological changes in DAT KO mice may provide information about the neural substrates underlying altered behaviors in these mice, with relevance for human neurological disorders thought to involve altered dopaminergic homeostasis. Results from this study also indicate the difficulty in correlating structural changes across scales, as the results on fine structure revealed thus far are subtle and non-uniform across striatal MSNs. The complexities associated with multiscale studies are driving the development of shared online informatics resources by gaining access to data where it is being analyzed.
    Brain research 03/2011; 1390:41-9. · 2.46 Impact Factor
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    ABSTRACT: To circumvent the limited spatial resolution of fluorescent protein imaging, we are developing genetically encoded tags for electron microscopy (EM).
    Faraday Discussions 01/2011; 149:9; discussion 63-77. · 3.82 Impact Factor
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    ABSTRACT: Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1(enu/+) mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1(enu/+) mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.
    Cell Death & Disease 01/2011; 2:e240. · 6.04 Impact Factor

Publication Stats

8k Citations
1,130.41 Total Impact Points

Institutions

  • 1979–2014
    • University of California, San Diego
      • • Department of Neurosciences
      • • Department of Pharmacology
      • • Center for Research in Biological Systems (CRBS)
      • • Department of Medicine
      San Diego, California, United States
  • 2007–2012
    • Sanford-Burnham Medical Research Institute
      • Apoptosis and Cell Death Research Program
      La Jolla, California, United States
  • 2009
    • University of Oxford
      • Oxford e-Research Centre
      Oxford, ENG, United Kingdom
  • 1999
    • The Scripps Research Institute
      • Department of Cell and Molecular Biology
      La Jolla, California, United States
  • 1988–1999
    • Stanford University
      • • Department of Molecular and Cellular Physiology
      • • Department of Neurobiology
      Stanford, CA, United States
  • 1996–1998
    • Yokohama City University
      • Department of Medicine
      Yokohama, Kanagawa, Japan
  • 1994
    • University of Ottawa
      Ottawa, Ontario, Canada
  • 1993
    • National University (California)
      San Diego, California, United States
    • University of Nevada School of Medicine
      • Department of Pharmacology
      Reno, Nevada, United States
  • 1990–1991
    • University of Nevada, Reno
      • Department of Pharmacology
      Reno, NV, United States
  • 1989–1990
    • Hebrew University of Jerusalem
      Yerushalayim, Jerusalem District, Israel