NeuroGLIA Consortium: neuron-astroglia in brain function and dysfunction

Brief description and aims of work

Since 1998 a major research interest of our research group is to understand the pathogenesis, epileptogenesis and pharmacoresistance of focal epilepsy with particular attention to the role of neuron-glia dysfunctions. We use a multidisciplinary approach, through implementation of different techniques (histopathology, molecular biology and electrophysiology) using human material, as well as animal models and creating a collaborative team of specialists including neurology, neuropathology and neurobiology.

The overall aim is to further characterize the role of glial cells and the neuron-glia dysfunctions in the initiation and persistence of the inflammatory response associated with neuronal degeneration and hyperexcitability.

Specific aims are the following:

• To investigate and characterize at the cellular level the specific inflammatory pathways activated in different human disorders affecting both adult and developing human brain
• To evaluate in vivo the effects of modulation or inhibition of specific inflammatory pathways on neurodegeneration and hyperexcitability using animal models
• To define the impact of the sustained inflammatory reaction on pharmacoresistance in vitro and in vivo

Previous findings

In 2000 we showed the upregulation of specific glutamate receptor subtypes (metabotropic glutamate receptor subtype mGluR3 and mGluR5) in reactive rat astrocytes (1).

In 2001 we confirmed the overexpression of group I mGluRs in human reactive astrocytes in different disorders associated with gliosis (2,9,17). In a later study we found that mGluRs can regulate glutamate transporter expression (7).

Expression of mGluR5 protein in astrocytes
Confocal laser scanning microscopic merged images of GFAP (green) and mGluR5 (red) positive human astrocytes (A, B) in culture. A) shows GFAP-positive astroglial cells growing in minimally supplemented serum-free medium (SF) with a flat polygonal morphology and low expression of mGluR5. B) Addition to serum-free medium of specific growth factors (bFGF and EGF) (ADM) produced a highly branched, stellate morphology and increased the expression of mGluR5 IR (yellow) (7).

In 2002 we provided evidence that the helix-loop-helix proteins are expressed in reactive astrocytes and are involved in the enhanced proliferative potential of tumor astrocytes (4).

In 2003 we provided evidence that overexpression (in endothelial cells and astrocytes) of multidrug transporter proteins is a common mechanism underlying the pharmaco-resistance to antiepileptic drugs of different human focal lesion associated with epilepsy (11,12).

In 2004 we characterized the expression and glial distribution of high- and low-affinity neurotrophin receptors in human pathologies associated with epilepsy (13,14).

In 2005 our studies performed in cultures of human astrocytes revealed the existence of interactions between glial GluRs and the inflammatory response (15).

In 2006 we showed that inhibition of the multidrug transporter P-glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats (20).

In 2006-2007 we provided evidence of a prominent and persistent activation of the innate immune response in both experimental and human temporal lobe epilepsy (22,25).

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Overview of the neuropathological features of Ammon’s horn sclerosis in human (A,C,E,F,I) and experimental temporal lobe epilepsy (B,D,G,H,J).
Neuronal cell loss. A and B: neuronal nuclear protein (NeuN) staining showing the loss of pyramidal neurons in CA regions (CA1 and CA3) and in the hilus. Neuronal loss in experimental model (electrical stimulation) is more extensive in hilus than in CA regions.
Gliosis. C, D and E: Vimentin (Vim) staining showing prominent astrogliosis in the regions where neuronal cell loss occurs. F: human leukocyte antigen (HLA)-DR staining showing reactive migroglia cells in the hilar region. F: OX42 staining showing reactive microglia cells in experimental temporal lobe epilepsy. G: Vimentin staining shows reactive astrocytes (and blood vessels) in chronic epileptic rat CA3. H: Ox-42 staining shows microglial cells in chronic epileptic rat CA3 region.
Sprouting fibers. I: Dynorphin staining shows positivity in the molecular layer with a pattern similar to Timm’s staining in experimental temporal lobe epilepsy (J). Bar in B is 300 µm in A and C, 200 µm in B, 380 µm in D, 50µm in E and F, 8 µm in G and H, 250 µm in I and 120 µm in J. (Aronica and Gorter, Neuroscientist, 13(2):100-8)

Distribution of C1q, C3c and C3d immunoreactivity in the hippocampus of control and TLE patients with hippocampal sclerosis
Sections are counterstained with hematoxylin. A and E: Control hippocampus, showing no detectable C1q immunoreactivity (IR) is observed in granule cell layer and hilus of the dentate gyrus (DG; A) or in the pyramidal neurons of CA1 (E).
B,C,D,F: Hippocampal sclerosis (HS). Panels B-D and F show prominent increased expression in reactive glial cells within the hippocampus (DG and CA1). Insert a in C: immunopositive glial cells with typical morphology of a reactive astrocyte. Insert b in C: merged image, showing co-localization (yellow) of C1q (red) with vimentin (green) in reactive astrocytes. D: Increased C1q staining is also observed around blood vessels. Both cells with the morphology of astrocytes (insert in D, arrow), as well as cell with the morphology of activated microglia/macrophages (insert in D, arrow-heads) are C1q positive. Inserts in F show high magnification of C1q positive cells (a) and the co-localization (yellow) of C1q (red) with HLA-DR (green) in cells of the microglia/macrophage lineage (b).
G-I: CA1 region in HS specimens showing C1q (G), C3c (H) and C3d (I) in residual pyramidal neurons (arrows) and reactive glial cells (arrow-heads). Scale bar in A, B, and E, 200 µm; C, D, and F, 80 µm; G-I, 50 µm. Granule cells layer (gcl); dentate gyrus (DG); hilus (H). (25)

Implications of our research

Data obtained from the new studies will lead to a better understanding of the multifactorial pathophysiology of human brain diseases that do not have a specific inflammatory pathophysiology but are characterized by neuron-glia dysfunctions and to the possible development of new pharmacological approaches that targets components of the inflammatory cascade for treatment of CNS human pathologies, improving the patient's quality of life.

Selected references on astrocyte work

1. Aronica E, van Vliet EA, Mayboroda OA, Troost D, da Silva FH, Gorter JA (2000) Upregulation of metabotropic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy. Eur. J. Neurosci. 12:2333-44.
2. Aronica E, Catania MV, Geurts J, Yankaya B, Troost D (2001) Immunohistochemical localization of group I and II metabotropic glutamate receptors in control and amyotrophic lateral sclerosis human spinal cord: upregulation in reactive astrocytes. Neuroscience 105:509-20.
3. Aronica E, van Vliet EA, Hendriksen E, Troost D, Lopes da Silva FH, Gorter JA (2001) Cystatin C, a cysteine protease inhibitor, is persistently up-regulated in neurons and glia in a rat model for mesial temporal lobe epilepsy. Eur. J. Neurosci. 14:1485-91.
4. Aronica E, Vandeputte DA, van Vliet EA, Lopes da Silva FH, Troost D, Gorter JA (2001) Expression of Id proteins increases in astrocytes in the hippocampus of epileptic rats. Neuroreport 12:2461-5.
5. Aronica E, Yankaya B, Troost D, van Vliet EA, Lopes da Silva FH, Gorter JA (2001) Induction of neonatal sodium channel II and III alpha-isoform mRNAs in neurons and microglia after status epilepticus in the rat hippocampus. Eur. J. Neurosci. 13:1261-6.
6. Vandeputte DA, Troost D, Leenstra S, Ijlst-Keizers H, Ramkema M, Bosch DA, Baas F, Das NK, Aronica E (2002) Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors. Glia 38:329-38.
7. Aronica E, Gorter JA, Ijlst-Keizers H, Rozemuller AJ, Yankaya B, Leenstra S, Troost D (2003) Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins. Eur. J. Neurosci. 17:2106-18.
8. Aronica E, Ozbas-Gerçeker F, Redeker S, Ramkema M, Spliet WG, van Rijen PC, Leenstra S, Gorter JA, Troost D (2004) Expression and cellular distribution of high- and low-affinity neurotrophin receptors in malformations of cortical development. Acta Neuropathol. 108:422-34.
9. Aronica E, Gorter JA, Jansen GH, van Veelen CW, van Rijen PC, Leenstra S, Ramkema M, Scheffer GL, Scheper RJ, Troost D (2003) Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors. Neuroscience 118:417-29.
10. Aronica E, Troost D, Rozemuller AJ, Yankaya B, Jansen GH, Isom LL, Gorter JA (2003) Expression and regulation of voltage-gated sodium channel beta1 subunit protein in human gliosis-associated pathologies. Acta Neuropathol. 105:515-23.
11. Aronica E, Gorter JA, Ramkema M, Redeker S, Ozbas-Gerçeker F, van Vliet EA, Scheffer GL, Scheper RJ, van der Valk P, Baayen JC, Troost D, Ozbas-Gerçerer F (2004) Expression and cellular distribution of multidrug resistance-related proteins in the hippocampus of patients with mesial temporal lobe epilepsy. Epilepsia 45:441-51.
12. Ozbas-Gerçeker F, Gorter JA, Redeker S, Ramkema M, van der Valk P, Baayen JC, Ozgüç M, Saygi S, Soylemezoglu F, Akalin N, Troost D, Aronica E (2004) Neurotrophin receptor immunoreactivity in the hippocampus of patients with mesial temporal lobe epilepsy. Neuropathol. Appl. Neurobiol. 30:651-64.
13. Aronica E, Gorter JA, Rozemuller AJ, Yankaya B, Troost D (2005) Activation of metabotropic glutamate receptor 3 enhances interleukin (IL)-1beta-stimulated release of IL-6 in cultured human astrocytes. Neuroscience 130:927-33.
14. Aronica E, Boer K, Redeker S, Spliet WG, van Rijen PC, Troost D, Gorter JA (2007) Differential expression patterns of chloride transporters, Na⁺-K⁺-2Cl^--cotransporter and K⁺-Cl^--cotransporter, in epilepsy-associated malformations of cortical development. Neuroscience 145:185-96.
15. Geurts JJ, Wolswijk G, Bö L, van der Valk P, Polman CH, Troost D, Aronica E (2003) Altered expression patterns of group I and II metabotropic glutamate receptors in multiple sclerosis. Brain 126:1755-66.
16. Gorter JA, van Vliet EA, Aronica E, Breit T, Rauwerda H, Lopes da Silva FH, Wadman WJ (2006) Potential new antiepileptogenic targets indicated by microarray analysis in a rat model for temporal lobe epilepsy. J. Neurosci. 26:11083-110.
17. Ravizza T, Boer K, Redeker S, Spliet WG, van Rijen PC, Troost D, Vezzani A, Aronica E (2006) The IL-1beta system in epilepsy-associated malformations of cortical development. Neurobiol. Dis. 24:128-43.
18. van Vliet EA, van Schaik R, Edelbroek PM, Redeker S, Aronica E, Wadman WJ, Marchi N, Vezzani A, Gorter JA (2006) Inhibition of the multidrug transporter P-glycoprotein improves seizure control in phenytoin-treated chronic epileptic rats. Epilepsia 47:672-80.
19. Boer K, Spliet WG, van Rijen PC, Redeker S, Troost D, Aronica E (2006) Evidence of activated microglia in focal cortical dysplasia. J. Neuroimmunol. 173:188-95.
20. van Vliet EA, da Costa Araújo S, Redeker S, van Schaik R, Aronica E, Gorter JA (2007) Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy. Brain 130:521-34.

Selected collaborations,
past and present

• Dr. Jan A. Gorter and Prof. W. Wadman · Neurobiology, UVA, SEIN
• Dr. Annamaria Vezzani · Neuroscience, Mario Negri, Milan, Italy
• Dr. Peter Crino · Neurology, University of Pennsylvania, USA
• Dr. Sanjay Sisodiya and Maria Thom · Neurology, University College London, UK
• Dr. Albert Becker · Dept of Neuropathology, University of Bonn Medical Center