Membrane Biology and Axonal Repair

Membrane Biology and Axonal Repair

Membrane Biology Laboratory

 

Principal Investigator: José Abad Rodríguez Ph.D.

E-mail: jabadr@sescam.jccm.es

 

The Membrane Biology Laboratory (LBM) investigates the organization of the plasma membrane and its role in the nervous system. We study the role of glycans (in glycolipids and glycoproteins) and the proteins that bind them (lectins) in the differentiation of neurons, with special focus on the regeneration of the central nervous system and on neurological and neurodegenerative pathologies.

 

The scientific activity of the LBM was based on some notable milestones in the trajectory of the laboratory's PI, among which are the description of the influence of neuronal membrane cholesterol on the production of beta-amyloid peptide in Alzheimer's disease (12 ,17,18), or the establishment of the role of the membrane sialidase Neu3 in the determination, growth, and regeneration of axons, both in vitro and in vivo (10,16,19). Upon being established at the National Hospital for Paraplegics (HNP), the LBM has put ahead the importance of the galectin family (lectins that bind beta-galactosides) in some central functions of axonal physiology, such as axonal branching local stimulation by phosphorylated galectin-3 (14), the mechanism of galectin-4 and sulfatide-dependent axonal transport of glycoproteins (11), and the local inhibition of myelination in vitro by this same lectin (6), a function that, however, this lectin does not present in the myelination in vivo (2).

 

We have recently shown that in animal models such as LGalS4-KO mice, the lack of Galectin-4 that is mostly expressed in the intestine produces deficiencies in memory and learning, as well as alterations at the synaptic level that correlate with changes in the intestinal microbiota (publication in progress). This generated a new line of research in the laboratory, funded by a project from the Spanish National Research Agency, in which we address how intestinal alterations induced by the absence of galectins produce synaptic deficiencies through the “microbiota-intestine-brain” axis, and whether these also give rise to associated neurocognitive dysfunctions such as anxiety and depression.

 

To achieve our objectives, we work with in vitro systems such as primary cultures of neurons, astrocytes, oligodendrocytes and microglia, or cultures of established cell lines. Conventional, confocal and time-lapse microscopy are our most common cellular and tissue analysis techniques (immunohistochemistry). In addition, we currently develop new image analysis based on high-content microscopy, supported by artificial intelligence protocols in collaboration with the Microscopy Service of our center. We combine microscopic analyses with standard molecular biology (cloning, RT-PCR, etc.) and biochemical (immunoblot, ELISA, etc.) methods. A distinctive characteristic of our group is our specialization in the analysis of membranes and lipids, both in living cells (surface binding, copatching...), tissues (Fast Blue and Black Gold stains for myelin...), and in cell extracts (membrane fractionation, purification of rafts and synaptosomes, extraction and analysis of lipids/glycolipids). In this sense, we are currently developing lipidomic and metabolomic analyses of complex samples by LC-MS, in collaboration with the Proteomics service of our center. Likewise, we have set up several experimental systems to evaluate the cognitive function of rodents in the context of our research with the KO mice mentioned above, complemented with electrophysiological studies that we carry out in collaboration with the Experimental Neurophysiology group of the HNP and the Synaptic Plasticity Mechanisms group of the CBMSO-CSIC (Cantoblanco, Madrid).

 

Selected Publications

 

  1. Abad-Rodríguez J, Brocca ME, Higuero AM. Glycans and Carbohydrate-Binding/Transforming Proteins in Axon Physiology. Adv. Neurobiol. 2023; 29:185-217 doi: 10.1007/978-3-031-12390-0_7
  2. Brocca ME, Mora-Rubio A, Alonso-Calviño E, Fernández-López E, Díez-Revuelta N, Martos-Puñal D, Aguilar J, Higuero AM, Abad-Rodríguez J*. Normal Cortical Myelination in Galectin-4-Deficient Mice. Cells. 2022;11(21):3485. doi: 10.3390/cells11213485
  3. Kutzner TJ, Higuero AM, Süßmair M, Hingar M, Kaltner H, Lindner I, Kopitz J, Abad-Rodríguez J, Reusch D, Gabius HJ. What Happens If a Human Galectin Enters the Endoplasmic Reticulum? Methods Mol Biol. 2022; 2442:247-288. doi: 10.1007/978-1-0716-2055-7_15.
  4. Habermann FA, Kaltner H, Higuero AM, García Caballero G, Ludwig AK, C Manning J, Abad-Rodríguez J, Gabius HJ. What Cyto- and Histochemistry Can Do to Crack the Sugar Code. Acta Histochem Cytochem. 2021; 54(2):31-48. doi: 10.1267/ahc.21-00017
  5. Ledeen RW., Kopitz J., Abad-Rodríguez J., Gabius HJ. Glycan Chains of Gangliosides: Functional Ligands for Tissue Lectins (Siglecs/Galectins). Prog Mol Biol Transl Sci. 2018;156:289-324. doi: 10.1016/bs.pmbts.2017.12.004
  6. Díez-Revuelta N.,Higuero A.M.,Velasco S, Peñas-de-la-Iglesia M., Gabius HJ, Abad-Rodríguez J. Neurons define non-myelinated axon segments by the regulation of galectin-4-containing axon membrane domains. Sci Rep. 2017; 25;7(1):12246. doi: 10.1038/s41598-017-12295-6.
  7. Higuero A.M., Díez-Revuelta N., Abad-Rodríguez J. The sugar code in neuronal physiology. Histochem Cell Biol. 2017; 147(2):257-267 doi:10.1007/s00418-016-1519-3
  8. Abad-Rodríguez J, Díez-Revuelta N. Axon glycoprotein routing in nerve polarity, function, and repair. TIBS July 2015; 40(7):385-396 doi: 10.1016/j.tibs.2015.03.015.
  9. Oliviero A.; Carrasco-Lopez M.C.; Campolo M.; Perez-Borrego Y.A., Soto-León V., Javier Gonzalez-Rosa; Alonso M Higuero; Bryan A Strange; Jose Abad-Rodriguez; Foffani G. Safety study of transcranial static magnetic field stimulation (tSMS) of the human cortex. Brain Stimul. 2015 May-Jun;8(3):481-5 doi: 10.1016/j.brs.2014.12.002.
  10. Kappagantula S., Andrews M.R., Cheah M., Abad-Rodríguez J., Dotti C.G., Fawcett J.W. Neu-3 Sialidase-mediated ganglioside conversion is necessary for axon regeneration and is blocked in CNS axons. J. Neurosci.  2014, 34(7):2477-2492; doi:10.1523/jneurosci.4432-13.2014.
  11. Velasco S, Díez-Revuelta N, Hernández-Iglesias T, Kaltner H, André S, Gabius HJ, Abad-Rodríguez J* Neuronal Galectin-4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering. J. Neurochem. 2013 125(1):49 - 62. doi: 10.1111/jnc.12148.
  12. Abad-Rodríguez J*. ApoE isoform-related behavioral defects. Is chronic cholesterol loss-driven membrane disorganization behind? Exp. Neurol. 2013, 241:1-4. doi: 10.1016/j.expneurol.2012.12.002.
  13. De Cárcer G., Escobar B., Higuero A., García L., Ansón A., Pérez G., Mollejo M., Manning G., Meléndez B., Abad-Rodríguez J., and Malumbres M. Plk5, a Polo-box domain-only protein with specific roles in neuron differentiation and glioblastoma suppression. Mol. Cell. Biol. 2011, 31: 1225-39.
  14. Díez-Revuelta N., Velasco S., André S., Kübler D., Gabius H.J. and Abad-Rodríguez J*. Phosphorylation of adhesion/growth-regulatory human Galectin-3 leads to the induction of axonal branching by local membrane L1/ERM redistribution.  J Cell Sci. 2010 Mar 1;123: 671-81.
  15. Higuero AM, Sánchez-Ruiloba L, Doglio LE, Portillo F, Abad-Rodríguez J, Dotti CG, Iglesias T. Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development. J. Biol. Chem. 2010 Jan 8; 285(2):1343-57.
  16. Santos Da Silva J, Hasegawa T, Miyagi T, Dotti CG. and Abad-Rodríguez J*.  Asymmetric membrane ganglioside sialidase activity specifies axonal fate. Nature Neurosci. 2005, May; 8(5): 606-15.
  17. Abad-Rodríguez J, Ledesma MD, Craessaerts K, Perga S, Medina M, Delacourte A, Dingwall C, De Strooper B and Dotti CG. Neuronal membrane cholesterol loss enhances amyloid peptide generation. J. Cell Biol. 2004 Dec 6;167(5):953-60; doi: 10.1083/jcb.200404149
  18. LedesmaMD*, Abad-RodríguezJ.*, Galvan C, Biondi E., NavarroP., DelacourteA, DingwallC, and DottiCG. Raft disorganization leads to reduced plasmin activity in Alzheimer’s disease brains. EMBO Rep. 2003 Dec; 4(12):1190-6; doi: 10.1038/sj.embor.7400021; (* equal contribution)
  19.  Abad-Rodríguez J, Piddini E, Hasegawa T, Miyagi T, Dotti CG. Plasma membrane ganglioside sialidase regulates axonal growth and regeneration in hippocampal neurons in culture. J. Neurosci. 2001 Nov 1;21(21):8387-95, Doi: 10.1523/JNEUROSCI.21-21-08387.2001

 

 

Laboratory Members

 

José Abad Rodríguez: PI; Ph.D. Sciences (Biochemistry and Molecular Biology) Universidad Autónoma de Madrid (Spain).

http://www.linkedin.com/pub/jose-abad-rodriguez/1b/137/5ab

 

Alonso Higuero Romero: Staff Scientist; Ph.D. Biochemistry, Molecular Biology and Biomedicine. Universidad Autónoma de Madrid (Spain).

 

Natalia Díez Revuelta: Staff Scientist; Ph.D. Biochemistry, Molecular Biology. Universidad Complutense de Madrid (Spain).

http://www.linkedin.com/pub/natalia-d%C3%ADez-revuelta/35/10a/128

 

María Elvira Brocca: Contracted Scientist; Ph.D. Human Biochemistry. Universidad de Buenos Aires (Argentina).

 

Arancha Mora Rubio: Ph.D Student. Bs.C. Biochemistry. Universidad de Castilla La Mancha (Spain). Ms.C. Translational Biomedical Research. Universidad de Córdoba (Spain)

 

Juan Carlos Guirado Calatrava: Contracted Scientist (programe “Yo investigo”). Bs.C. Biology. Universidad de Castilla La Mancha (Spain). Ms.C.. Genetics and Evolution, Ms.C. Advances in Agrarian Biology and Aquaculture. Universidad de Granada (Spain)

 

David Martos Puñal: Staff Technician. Superior Technician in Clinical Diagnosis Laboratory. IES Juanelo Turriano (Spain). 

 

Carlos García Rodríguez: Contracted Technician. Bs.C. Biology (Universidad de Sevilla). Ms.C. in Sanitary Biotechnology. Universidad Pablo de Olavide (Spain). Superior Technician in Clinical Diagnosis Laboratory. Medac (Spain)

 

 

 

Research Lines

  

- Role of galectins in the nervous system

Using carbohydrate-binding proteins (galectins), obtained by recombinant technology, we have developed a set of experimental systems to study the effect of carbohydrate interactions on axonal growth, guidance and regeneration, as well as their role in myelination. . For example, we have shown that the phosphorylated form of galectin-3 regulates axon branching, and that galectin-4 determines the axonal transport of glycoproteins associated with axon growth such as NCAM-L1. These and other results in this line have identified several of the galectins studied as potential tools for nerve regeneration.

 

- Regulation of brain functions by intestinal glycans and lectins. The “microbiota-gut-brain” axis

In this line we address how intestinal alterations induced by the absence of galectins produce changes in the microbiota and associated immunological and metabolic disorders that give rise to neurocognitive dysfunctions such as anxiety and depression, and that signal through the microbiota-gut-brain axis.

 

Ongoing Project

 

- Memory Disorders, Anxiety and Depression Induced by an Altered Microbiota-Gut-Brain Axis. From “-Omics” to Behaviour (MoodMax). Agencia Estatal de Investigación (Ref. PID2021_125428OB100). 09/2022-08/2026. PI José Abad Rodríguez.