βII-spectrin promotes mouse brain connectivity through stabilizing axonal plasma membranes and enabling axonal organelle transport

βII-spectrin is the generally expressed member of the β-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of βII-spectrin in neurons by knockout of βII-spectrin in mouse neural progenitors. βII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration. βII-spectrin–null neurons exhibited reduced axon growth, loss of actin–spectrin-based periodic membrane skeleton, and impaired bidirectional axonal transport of synaptic cargo. We found that βII-spectrin associates with KIF3A, KIF5B, KIF1A, and dynactin, implicating spectrin in the coupling of motors and synaptic cargo. βII-spectrin required phosphoinositide lipid binding to promote axonal transport and restore axon growth. Knockout of ankyrin-B (AnkB), a βII-spectrin partner, primarily impaired retrograde organelle transport, while double knockout of βII-spectrin and AnkB nearly eliminated transport. Thus, βII-spectrin promotes both axon growth and axon stability through establishing the actin–spectrin-based membrane-associated periodic skeleton as well as enabling axonal transport of synaptic cargo.

Download Full-text

Enhanced splenomegaly and severe liver inflammation in haptoglobin/hemopexin double-null mice after acute hemolysis

Blood ◽

10.1182/blood-2002-04-1270 ◽

2002 ◽

Vol 100 (12) ◽

pp. 4201-4208 ◽

Cited By ~ 90

Author(s):

Emanuela Tolosano ◽

Sharmila Fagoonee ◽

Emilio Hirsch ◽

Franklin G. Berger ◽

Heinz Baumann ◽

...

Keyword(s):

Bacterial Infections ◽

Plasma Membranes ◽

Antioxidant Action ◽

Liver Inflammation ◽

Intravascular Hemolysis ◽

Double Knockout ◽

Radical Production ◽

Physiological Relevance ◽

Protective Proteins

Intravascular hemolysis is associated with several pathologic conditions that include hemoglobinopathies, trauma, malaria, and bacterial infections. Among plasma-protective proteins against oxidative damage caused by red blood cell rupture, haptoglobin and hemopexin are thought to play a crucial role. Haptoglobin and hemopexin, by binding with high-affinity hemoglobin and heme, respectively, exert an antioxidant action by preventing heme-catalyzed free radical production. Moreover, these proteins prevent iron loss by inhibiting glomerular filtration of hemoglobin and heme diffusion through plasma membranes. Analysis of single-null mice demonstrated the antioxidant action of haptoglobin and hemopexin in vivo and suggests that the 2 proteins cooperate in the resolution of hemolytic stress. To evaluate the physiological relevance of the haptoglobin-hemopexin system and the principal targets of its action, we generated haptoglobin-hemopexin double-knockout mice and analyzed them under basal conditions and after acute hemolysis. Whereas haptoglobin-hemopexin double-null mice displayed no obvious alteration in phenotype under basal conditions, nonlethal hemolytic stress in these animals led to pronounced splenomegaly as well as liver inflammation and fibrosis. These data demonstrate that haptoglobin and hemopexin together are essential for protection from splenomegaly and liver fibrosis resulting from intravascular hemolysis.

Download Full-text

Sphingosine kills bacteria by binding to cardiolipin

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012325 ◽

2020 ◽

Vol 295 (22) ◽

pp. 7686-7696 ◽

Cited By ~ 3

Author(s):

Rabea Verhaegh ◽

Katrin Anne Becker ◽

Michael J. Edwards ◽

Erich Gulbins

Keyword(s):

Plasma Membrane ◽

Bactericidal Activity ◽

Cell Biology ◽

Plasma Membranes ◽

Central Element ◽

Lipid Binding ◽

Bacterial Cells ◽

Bacterial Killing

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively–charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo. We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

Download Full-text

A PIK3C3–Ankyrin-B–Dynactin pathway promotes axonal growth and multiorganelle transport

The Journal of Cell Biology ◽

10.1083/jcb.201407063 ◽

2014 ◽

Vol 207 (6) ◽

pp. 735-752 ◽

Cited By ~ 48

Author(s):

Damaris Nadia Lorenzo ◽

Alexandra Badea ◽

Jonathan Davis ◽

Janell Hostettler ◽

Jiang He ◽

...

Keyword(s):

Axonal Transport ◽

Synaptic Vesicles ◽

Hippocampal Neurons ◽

Axon Growth ◽

Retrograde Transport ◽

Organelle Transport ◽

Class Iii ◽

Functional Relationships ◽

Range Transport ◽

Ankyrin B

Axon growth requires long-range transport of organelles, but how these cargoes recruit their motors and how their traffic is regulated are not fully resolved. In this paper, we identify a new pathway based on the class III PI3-kinase (PIK3C3), ankyrin-B (AnkB), and dynactin, which promotes fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes. We show that dynactin associates with cargo through AnkB interactions with both the dynactin subunit p62 and phosphatidylinositol 3-phosphate (PtdIns(3)P) lipids generated by PIK3C3. AnkB knockout resulted in shortened axon tracts and marked reduction in membrane association of dynactin and dynein, whereas it did not affect the organization of spectrin–actin axonal rings imaged by 3D-STORM. Loss of AnkB or of its linkages to either p62 or PtdIns(3)P or loss of PIK3C3 all impaired organelle transport and particularly retrograde transport in hippocampal neurons. Our results establish new functional relationships between PIK3C3, dynactin, and AnkB that together promote axonal transport of organelles and are required for normal axon length.

Download Full-text

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2845 ◽

2007 ◽

Vol 30 (4) ◽

pp. 77

Author(s):

Y. Y. Chen ◽

C. L. Hehr ◽

K. Atkinson-Leadbeater ◽

J. C. Hocking ◽

S. McFarlane

Keyword(s):

Ganglion Cell ◽

Axon Guidance ◽

Retinal Ganglion Cell ◽

Growth Cone ◽

Expression Patterns ◽

Optic Tract ◽

Axon Growth ◽

Proteolytic Processing ◽

Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

Download Full-text

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

Science Advances ◽

10.1126/sciadv.abg3013 ◽

2021 ◽

Vol 7 (15) ◽

pp. eabg3013

Author(s):

Laura Fumagalli ◽

Florence L. Young ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Arpan R. Mehta ◽

...

Keyword(s):

Axonal Transport ◽

Motor Neurons ◽

Single Molecule ◽

Repeat Expansion ◽

Physical Interaction ◽

Hexanucleotide Repeat ◽

Hexanucleotide Repeat Expansion ◽

Axonal Trafficking ◽

Inhibitory Interactions

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell–derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

Download Full-text

Tractography Alterations in the Arcuate and Uncinate Fasciculi in Post-Stroke Aphasia

Brain Sciences ◽

10.3390/brainsci11010053 ◽

2021 ◽

Vol 11 (1) ◽

pp. 53

Author(s):

Sara Kierońska ◽

Milena Świtońska ◽

Grzegorz Meder ◽

Magdalena Piotrowska ◽

Paweł Sokal

Keyword(s):

White Matter ◽

Brain Connectivity ◽

Three Dimensional ◽

Cerebral White Matter ◽

Neural Pathways ◽

Uncinate Fasciculus ◽

Arcuate Fasciculus ◽

Fiber Tractography ◽

Post Stroke

Fiber tractography based on diffuse tensor imaging (DTI) can reveal three-dimensional white matter connectivity of the human brain. Tractography is a non-invasive method of visualizing cerebral white matter structures in vivo, including neural pathways surrounding the ischemic area. DTI may be useful for elucidating alterations in brain connectivity resulting from neuroplasticity after stroke. We present a case of a male patient who developed significant mixed aphasia following ischemic stroke. The patient had been treated by mechanical thrombectomy followed by an early rehabilitation, in conjunction with transcranial direct current stimulation (tDCS). DTI was used to examine the arcuate fasciculus and uncinate fasciculus upon admission and again at three months post-stroke. Results showed an improvement in the patient’s symptoms of aphasia, which was associated with changes in the volume and numbers of tracts in the uncinate fasciculus and the arcuate fasciculus.

Download Full-text

A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors

Cancers ◽

10.3390/cancers13020230 ◽

2021 ◽

Vol 13 (2) ◽

pp. 230

Author(s):

Barbara Costa ◽

Michael N.C. Fletcher ◽

Pavle Boskovic ◽

Ekaterina L. Ivanova ◽

Tanja Eisemann ◽

...

Keyword(s):

Cell Lines ◽

Immune Cell ◽

Treatment Options ◽

Morphological Characteristics ◽

Molecular Heterogeneity ◽

Double Knockout ◽

In Vivo Models ◽

Human Glioblastoma ◽

Human Gbm

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.

Download Full-text

DIPG-43. CAN WE REPROGRAM DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)? EXPLORING THE ROLE OF DISTALLESS/DLX HOMEOBOX GENE REGULATION OF OLIGODENDROGLIAL PROGENITOR CELLS (OPC) IN THE DEVELOPING VERTEBRATE NERVOUS SYSTEM

Neuro-Oncology ◽

10.1093/neuonc/noaa222.090 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii295-iii295

Author(s):

Mikaela Nevin ◽

Janine Gallego ◽

Xiaohua Song ◽

Qiang Jiang ◽

Alan Underhill ◽

...

Keyword(s):

Progenitor Cells ◽

Homeobox Gene ◽

Diffuse Intrinsic Pontine Glioma ◽

Cell Of Origin ◽

Double Knockout ◽

Promoter Dna ◽

And Migration ◽

Developmental Reprogramming

Abstract BACKGROUND The identification of H3.3/H3.1K27M in most DIPG has changed our understanding of this disease. H3K27M mutations usually demonstrate global loss of H3K27 trimethylation (me3) with gain of H3K27 acetylation (ac). Single cell RNAseq has identified the putative cell of origin as oligodendroglial progenitor cells (OPC). The distalless gene family is necessary for the differentiation and tangential migration of committed neural progenitors to become GABAergic interneurons. Dlx1/Dlx2 double knockout (DKO) cells from the ganglionic eminences (GE) transplanted into a wild-type environment become oligodendrocytes. RESULTS We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo and confirmed direct DLX2 protein-promoter DNA binding in vitro. Co-expression of Dlx2 with target sequences reduced reporter gene expression in vitro. There was increased expression of OLIG2, NKX2.2 and PLP-1 expression in vivo, consistent with de-repression in the absence of Dlx1/Dlx2 function. Transient over-expression of a Dlx2-GFP construct into murine DIPG cells from a GEMM that develops DIPG resulted in significant increases in expression of Gad isoforms with concomitant decreases in Olig2 and Nkx2.2. Dlx2-transfected mDIPG cells also demonstrated reduced migration, invasion and colony formation in vitro. Of significance, there was global restoration of H3K27me3 with corresponding loss of H3K27ac expression in transfected cells compared to controls. CONCLUSIONS DLX2 promotes GABAergic differentiation and migration while concomitantly repressing OPC differentiation in vivo. Developmental reprogramming of mDIPG cells by DLX2 demonstrates the potential role for directed differentiation strategies towards improving patient outcomes for this devastating pediatric cancer.

Download Full-text

Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes

Biochemical Journal ◽

10.1042/bj1780217 ◽

1979 ◽

Vol 178 (1) ◽

pp. 217-221 ◽

Cited By ~ 27

Author(s):

M D Houslay ◽

R W Palmer

Keyword(s):

Adenylate Cyclase ◽

Rat Liver ◽

Plasma Membranes ◽

Hormone Receptors ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Liver Plasma Membranes ◽

Rat Liver Plasma Membranes ◽

Increased Sensitivity

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.

Download Full-text

Continuation of fast axonal transport in regenerating axons in vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y79-189 ◽

1979 ◽

Vol 57 (11) ◽

pp. 1251-1255

Author(s):

M. A. Bisby ◽

C. E. Hilton

Keyword(s):

Sciatic Nerve ◽

Vagus Nerve ◽

Axonal Transport ◽

Nerve Injury ◽

Axon Regeneration ◽

Free Medium ◽

Fast Axonal Transport ◽

Sensory Axons

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.

Download Full-text