Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring

Estudio inmunohistológico y electrofisiológico de la motoneurona en un modelo de transmisión pasiva de Esclerosis Lateral Amiotrófica

La Esclerosis Lateral Amiotrófica (ELA) es la enfermedad neuromotora humana más común. Los individuos que la padecen muestran disfunción y degeneración progresiva de motoneuronas cerebrales, de la protuberancia y de la médula espinal lo cual se manifiesta clínicamente como una debilidad muscular progresiva, parálisis y muerte dentro de los 3 a 5 años del diagnóstico. No existe una cura para esta enfermedad y su patofisiología permanece sin aclarar. Evidencias clínicas y experimentales apoyan la posibilidad de que un mecanismo autoinmune esté involucrado en la patogénesis de ELA. En trabajos anteriores de nuestro laboratorio se ha demostrado que la aplicación de inmunoglobulina G (IgG) de pacientes con ELA sobre terminales motores inducen una disfunción de la transmisión neuromuscular. Estudios animales han establecido que fisiológicamente la IgG y otras proteínas séricas endógenas son endocitadas por los terminales nerviosos de la placa neuromuscular. Estas proteínas pueden ser localizadas por técnicas inmunocitoquímicas en los somas donde se acumulan luego de ser transportadas por transporte axonal retrógrado. El objetivo de la primera parte de la tesis fue analizar si la captación de IgG es cuantitativamente diferente en el caso que la misma provenga de pacientes con ELA o de IgG controles (pacientes con otras enfermedades neurológicas o personas sanas). Para ello, hemos investigado la presencia de IgG humana de pacientes con ELA e IgG control en los somas de las motoneuronas de ratón. La IgG fue aplicada sobre los terminales motores del músculo elevador de la oreja del ratón, Levator auris longus, el cual está inervado por una rama del nervio facial. La fracción sérica de 7 pacientes con ELA, 6 sujetos controles y 3 pacientes con otras enfermedades neurológicas fue inyectada subcutáneamente dos veces por día durante 5 dias (50 μl cada vez de una solución de IgG 20 mg/ml). Secciones del tallo cerebral conteniendo el núcleo del facial fueron procesadas por inmunocitoquímica para detectar IgG humana y la inmunotinción fue cuantificada con un analizador de imágenes. Para todas las IgG consideradas, la tinción en las motoneuronas del núcleo del facial del lado ipsilateral al sitio de la inyección fue significativamente más intensa. En los animales tratados con IgG de ELA, la marca ipsilateral fue significativamente más alta que la encontrada en el lado ipsilateral de los animales inyectados con IgG control. Nuestros resultados son compatibles con el concepto de que las motoneuronas preferencialmente captan, transportan y/o acumulan IgG de ELA. La captación de anticuerpos patogénicos por terminales motores podrían tener un rol en la patogénesis de la enfermedad de la motoneurona. Para definir el posible rol de las IgG de ELA en la inducción de la disfunción de la transmisión neuromuscular, fue nuestro interés estudiar si la participación de canales de calcio voltaje dependientes (CCVD) puede ser alterada por la transmisión pasiva dichos anticuerpos. En la placa neuromuscular de humanos adultos y también en la placa de ratón, la transmisión sináptica está mediada por la entrada de calcio a través de CCVD del tipo P/Q. Otros canales de calcio, como los de tipo L y N, no están involucrados en la liberación de neurotransmisor. Sin embargo, en nervios en regeneración, la conducción de señales y la transmisión sináptica se hacen altamente sensibles a la nitrendipina, una dihidropiridina bloqueante de los canales de calcio de tipo L, sugiriendo que este canal estaría involucrado en la liberación de neurotransmisor. A fin de estudiar si la aplicación de IgG humana sobre los terminales motores induce cambios en la transmisión neuromuscular, IgG de pacientes con ELA y de sujetos controles fue inyectada subcutáneamente sobre el músculo Levator auris del ratón. La fracción sérica de 8 pacientes con ELA y 6 sujetos controles (3 sujetos sanos y 3 pacientes con otras enfermedades neurológicas) fue inyectada subcutáneamente dos veces por día durante 5 días (50 μl cada vez de una solución de IgG 20 mg/ml). Una semana o un mes después de la ultima inyección, los ratones fueron anestesiados y los músculos removidos en solución Ringer normal. El músculo fue montado en una cámara de registro y el nervio fue estimulado por medio de un electrodo de succión. Los potenciales de placa fueron registrados en por lo menos l5 fibras musculares distintas antes y después de la incubación con Nitrendipina l μM o toxinas polipeptídicas. El contenido cuántico de la respuesta evocada fue evaluado por el método de la varianza. No se observaron cambios en el contenido cuántico de la liberación de neurotransmisor. En los músculos tratados con IgG de ELA o IgG control, la liberación del neurotransmisor permaneció sensible a los bloqueantes de CCVD de tipo P/Q e insensibles al bloqueante de tipo N, como ocurre en los músculos sin tratar. En contraste, las IgG de 5 pacientes distintos de ELA indujeron una reducción significativa en el contenido cuántico de la respuesta evocada luego de la aplicación de Nitrendipina. Esto indicaría que una sensibilidad nueva a este bloqueante de CCVD aparece en los terminales tratados con IgG de pacientes con ELA. Estos resultados sugieren la participación del canal de calcio voltaje dependiente de tipo L en el proceso que llevaría a la muerte neuronal inducida por las inmunoglobulinas de ELA.Amyotrophic lateral sclerosis (ALS) is the most common human motor neuron disease. Affected individuals show progressive dysfunction and degeneration of brain, brainstem and spinal cord motor neurons, clinically manifested as progressive muscular weakness, paralysis and death within a mean of 3 years. There is no cure available for this disease and its pathophysiology remains unclear. Clinical and experimental evidence support an autoimmune etiopathogenesis for ALS. Previous studies in our laboratory have shown that local application of immunoglobulin G (IgG) of ALS patients onto nerve terminals induces dysfunction in neuromuscular transmission. In animal studies, it has been established that endogenous IgG and other circulating serum proteins can be taken up by motor nerve terminals. They can be detected by immunocitochemistry in the motoneurons somas where they accumulate following retrograde axonal transport. We investigated the presence of human ALS and control IgG in the soma of mice motoneurons. IgG was applied onto motor nerve terminals by injections on the levator auris longus muscle which is innervated by a branch of the facial nerve. The seric IgG fraction of 7 ALS patients, 6 control subjects and 3 patients with other neurological diseases was injected subcutaenously, twice a day for 5 days, on the surface of the left Levator auris longus muscle of adult mice (50 μl of 20 mg/ml IgG in PBS per application). Sections of the brainstem containing the facial nuclei were immunoprocessed to detect human IgG. For all lgG tested, motoneuron labeling was significantly more intense in the facial nucleus ipsilateral to the site of injection. In ALS-IgG-treated animals, ipsilateral labeling was significantly stronger than that found on the ipsilateral side of control IgG-treated animals. Our results are compatible with the concept that motoneurons preferentially take up, transport and/or accumulate ALS-IgG. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease. At both the mature mouse and human neuromuscular junction, synaptic transmission is mediated by Ca² entry through voltage-sensitive calcium channels (VSCCs) of the P/Q-type family. Other calcium channels, like the L- and N-type, are not involved in neurotransmitter release. However. in regenerating nerves, signal conduction and synaptic transmission become highly sensitive to Nitrendipine, a dihydropyridine antagonist of L-type calcium channel, suggesting that this type of calcium channel mediate transmitter release. Therefore, to further define the possible role of ALS IgG in the induction of NMJ dysfunction, it was our interest to study if the participation of other types of VSCCs in transmitter release can be induced by passively transferring these antibodies, as it was found in regenerating NMJ. In order to search for early changes induced by the application of human IgG onto the motor nerve terminals, IgG from ALS patients and control subjects was injected on the mice Levator auris muscle. The seric IgG fraction of 8 ALS patients and 6 control subjects (3 healthy subjects and 3 with other neurological diseases) was injected subcutaenously, twice a day for 5 days, on the surface of the left Levator auris longus muscle of adult mice (50 μl of 20 mg/ml IgG in PBS per application). A week or a month after the last injection, mice were anesthetized and the muscle removed in Normal Ringer solution. The muscle was mounted in a recording chamber and the nerve was stimulated by a suction electrode. The end plate potentials were recorded from at least 15 different muscle fibers before and after the incubation with l μM Nitrendipine or polipeptidic toxins. The mean quantal content of the evoked response was evaluated by the coefficient of variation method. No changes in quantal content of transmitter release were observed. In control and ALS IgG treated muscles, neurotransmitter release remained sensitive to P/Q-type and insensitive to N-type VSCC blockers as in untreated muscles. In contrast, IgG from 5 of 8 different ALS patients induced a significant reduction in the quantal content of the evoked response afler the incubation with Nitrendipine, indicating that a novel sensitivity to this calcium channel blocker appears in these motor nerve terminals. These results suggest the involvement of L-type VSCC in the process leading to neuronal death induced by ALS IgG.Fil:Fratantoni, Silvina Andrea. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Multi-omics profile of the mouse dentate gyrus after kainic acid-induced status epilepticus

Temporal lobe epilepsy (TLE) can develop from alterations in hippocampal structure and circuit characteristics, and can be modeled in mice by administration of kainic acid (KA). Adult neurogenesis in the dentate gyrus (DG) contributes to hippocampal functions and has been reported to contribute to the development of TLE. Some of the phenotypical changes include neural stem and precursor cells (NPSC) apoptosis, shortly after their birth, before they produce hippocampal neurons. Here we explored these early phenotypical changes in the DG 3 days after a systemic injection of KA inducing status epilepticus (KA-SE), in mice. We performed a multi-omics experimental setup and analyzed DG tissue samples using proteomics, transcriptomics and microRNA profiling techniques, detecting the expression of 2327 proteins, 13401 mRNAs and 311 microRNAs. We here present a description of how these data were obtained and make them available for further analysis and validation. Our data may help to further identify and characterize molecular mechanisms involved in the alterations induced shortly after KA-SE in the mouse DG

Functional activity of digested 38B8 mAb in the TrkB NFAT reporter gene assays.

<p>CellSensor® TrkB-NFAT-bla CHO-K1 cells were stimulated with (A) BDNF, mAb 38B8, 38B8 Fab or 38B8 F(ab’)2 over the indicated concentration range for 5 hours (agonist mode) or (B) 38B8 Fab for 1 hour prior to 0.3 nM BDNF stimulation for 4 hours (antagonist mode) before beta-lactamase assay was performed as described in Methods. % of control (maximal BDNF concentration  =  9 nM) values were plotted for the indicated concentrations of each ligand (n = 2 ± SD for each data point).</p

mHTT-induced neuronal toxicity: 7,8-dihydroxyflavone and LM22A-4 pharmacology.

<p>Using our primary rat cortico-striatal co-culture system we stimulated with 7,8-dihydroxyflavone, LM22A-4 or BDNF over the indicated concentration range according to the mHTT-induced co-culture protocol (A), as described in Methods. % Rescue (Normalized to in-plate controls; 1 nM BDNF (100% Rescue) and vehicle (0% Rescue)) values were plotted for striatal neurons over the indicated concentrations of each ligand (n = 6 ± SD for each data point). Rat primary cortico-striatal cells (non-transfected) were stimulated with 7,8-dihydroxyflavone (B) or LM22A-4 (C) using the indicated concentrations for 15 minutes and profiled by western blot. BDNF- (10 nM) mediated TrkB phosphorylation validated the experimental system.</p

A Monoclonal Antibody TrkB Receptor Agonist as a Potential Therapeutic for Huntington’s Disease

<div><p>Huntington’s disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for <i>in vivo</i> proof of concept studies in transgenic HD models.</p></div

TrkB signaling and assay cascades.

<p>(A) TrkB signalling cascade showing proximal and distal assay measurement points. Red arrows represent BDNF-induced trans-phosphorylation events within the intracellular tyrosine kinase domains. Changes in TrkB phosphorylation/activation detected by (1) Invitrogen CellSensor®; (2) DiscoveRx PathHunter®; and (3) MSD® pAKT assays. (B) Screening cascade used to characterize TrkB modulators.</p

TrkB antibody panel and associated activities.

#<p>Reactivity as described by suppliers.</p

Functional activity of TrkB mAbs in the primary cortico-striatal neuronal co-culture system.

<p>Rat primary cortico-striatal co-cultures were stimulated with BDNF, mAb 38B8 and mAb 29D7 and profiled by (A) western blot; agonists tested at 10 nM over the indicated incubation times using non-transfected co-cultures; or (B) mHTT-induced co-culture protocol over the indicated concentration range, as described in the Materials and Methods. For western blot, lysates were prepared and run on SDS-PAGE gels and transferred to membranes that were then hybridized to the corresponding primary antibodies, as described in the Methods and Materials. For mHTT-induced co-culture assay, % Rescue (Normalized to in-plate controls; 0.22 nM BDNF (100% Rescue) and vehicle (0% Rescue)) values were plotted for striatal neurons over the indicated concentrations of each ligand (n = 6 ± SD for each data point). Data demonstrates activation of TrkB phosphorylation signal transduction cascade and rescue of mHTT-induced neuronal toxicity by TrkB mAbs.</p