Carlos Barcia

Evidence of active microglia in substantia nigra pars compacta of parkinsonian monkeys 1 year after MPTP exposure

Inflammatory changes have been found in Parkinsons disease, in humans intoxicated with the parkinsonian toxin MPTP, and in animal models of the disease. However, it is still not known whether inflammatory changes are responsible for active nerve cell death or if they have a protective role against neurodegeneration. In this study, we analyzed the glial reaction in the substantia nigra pars compacta (SNpc) and the striatum of monkeys rendered parkinsosian by chronic MPTP injections. At postmortem examination 1 year after the last MPTP injection, the density of astroglial cells and activated microglial cells in the SNpc, but not in the striatum, of MPTP‐intoxicated animals was significantly higher than in the two control animals. These data suggest that neurodegeneration was still active despite the absence of the agent triggering cell death and that the glial reaction is associated with long‐term neurodegeneration.

IFN-γ signaling, with the synergistic contribution of TNF-α, mediates cell specific microglial and astroglial activation in experimental models of Parkinson's disease.

To through light on the mechanisms underlying the stimulation and persistence of glial cell activation in Parkinsonism, we investigate the function of IFN-γ and TNF-α in experimental models of Parkinsons disease and analyze their relation with local glial cell activation. It was found that IFN-γ and TNF-α remained higher over the years in the serum and CNS of chronic Parkinsonian macaques than in untreated animals, accompanied by sustained glial activation (microglia and astroglia) in the substantia nigra pars compacta. Importantly, Parkinsonian monkeys showed persistent and increasing levels of IFN-γR signaling in both microglial and astroglial cells. In addition, experiments performed in IFN-γ and TNF-α KO mice treated with MPTP revealed that, even before dopaminergic cell death can be observed, the presence of IFN-γ and TNF-α is crucial for microglial and astroglial activation, and, together, they have an important synergistic role. Both cytokines were necessary for the full level of activation to be attained in both microglial and astroglial cells. These results demonstrate that IFN-γ signaling, together with the contribution of TNF-α, have a critical and cell-specific role in stimulating and maintaining glial cell activation in Parkinsonism.

Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses

ABSTRACT In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding β-galactosidase (β-gal) driven by a TetOn system containing the rtTASsM2 transactivator and the tTSKid repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-β-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-β-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-β-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-β-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.

Fms-Like Tyrosine Kinase 3 Ligand Recruits Plasmacytoid Dendritic Cells to the Brain

The lack of professional afferent APCs in naive brain parenchyma contributes to the systemic immune ignorance to Ags localized exclusively within the brain. Dendritic cells (DCs) appear within the brain as a consequence of inflammation, but no molecular mechanisms accounting for this influx have been described. In this study we demonstrate that Fms-like tyrosine kinase 3 ligand (Flt3L) recruits plasmacytoid DCs (pDCs; >50-fold; p < 0.001) to the brain parenchyma. These pDCs expressed IFN-α, the hallmark cytokine produced by pDCs, indicating recruitment and activation in situ of bona fide pDCs within the brain parenchyma. Flt3L did not increase the numbers of conventional DCs, macrophages, or B, T, NK, NKT, or microglial cells within the brain. Our data demonstrate that Flt3L reconstitutes a crucial afferent component of the immune response, namely, professional APCs within the brain parenchyma, and this could counteract the intrinsic systemic immune ignorance to Ags localized exclusively within the brain.

ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo

The role of microglial motility in the context of adult neurodegeneration is poorly understood. In the present work, we investigated the microanatomical details of microglia-neuron interactions in an experimental mouse model of Parkinsons disease following the intraperitoneal injection of MPTP. The specific intoxication of dopaminergic neurons induces the cellular polarization of microglia, leading to the formation of body-to-body neuron-glia contacts, called gliapses, which precede neuron elimination. Inhibiting ROCK/Cdc42-mediated microglial motility in vivo blocks the activating features of microglia, such as increased cell size and number of filopodia and diminishes their phagocyting/secreting domains, as the reduction of the Golgi apparatus and the number of microglia-neuron contacts has shown. High-resolution confocal images and three-dimensional rendering demonstrate that microglia engulf entire neurons at one-to-one ratio, and the microglial cell body participates in the formation of the phagocytic cup, engulfing and eliminating neurons in areas of dopaminergic degeneration in adult mammals.

Parkinson's disease and inflammatory changes

In 1988 McGeer and colleagues (Neurology 38, 1285–91) observed an activation of the microglia in substantia nigrapars compacta (SNpc) and striatum of brains from patients with Parkinsons disease. In the years that followed several studies performed in the cerebrospinal fluid and duringpost-mortem analysis in parkinsonian patients revealed increased levels of cytokines, suggesting the activation of a proinflammatory response. Moreover, Langston and his group described the presence of active microglia in the SNpc of three patients who had been exposed to MPTP several years before death. These results suggested that the inflammatory response may increase negative feed-back into the damaged area of the cerebral parenchyma, inducing an imbalance that could perpetuate and/or accelerate neuronal death over a period of years. Similar results have been obtained in parkinsonian monkeys, rats and mice. For these reasons, several groups have treated parkinsonian animals with different anti-inflammatory drugs and obtained promising results. However, it is still not known whether inflammatory changes are responsible for active nerve cell death or whether they play a protective role in neurodegeneration. These changes are putatively related to neuronal loss and suggest that anti-inflammatory treatment for parkinsonian patients could have beneficial effects in the progression of the disease by slowing down the process of neuronal loss.

Changes in vascularization in substantia nigra pars compacta of monkeys rendered parkinsonian

Summary.The degeneration of nigral dopaminergic neurons in Parkinson’s disease is believed to be associated with a glial reaction and inflammatory changes. In turn, local factors may induce changes in vascularization and contribute to neuronal vulnerability. Among these factors, Vascular Endothelial Growth Factor (VEGF) is released in adults under pathological conditions and is thought to induce angiogenesis.In order to determine whether changes in brain vasculature are observed in the affected brain regions in parkinsonism, we quantitatively analysed the VEGF-expressing cells and blood vessels in the substantia nigra of monkeys rendered parkinsonian by MPTP injection and compared the results with those obtained in control monkeys.Using stereological methods, we observed an increase in the number of VEGF-expressing neurons and an increase of the number of blood vessels and their volume occupying the substantia nigra pars compacta of monkeys rendered parkinsonian by chronic MPTP intoxication. These changes in vascularization may therefore modify the neuronal availability of blood nutrients, blood cells or toxic substances and neuronal susceptibility to parkinsonism.

Flt3L and TK gene therapy eradicate multifocal glioma in a syngeneic glioblastoma model

The disseminated characteristics of human glioblastoma multiforme (GBM) make it a particularly difficult tumor to treat with long-term efficacy. Most preclinical models of GBM involve treatment of a single tumor mass. For therapeutic outcomes to translate from the preclinical to the clinical setting, induction of an antitumor response capable of eliminating multifocal disease is essential. We tested the hypothesis that expression of Flt3L (human soluble FMS-like tyrosine kinase 3 ligand) and TK (herpes simplex virus type 1-thymidine kinase) within brain gliomas would mediate regression of the primary, treated tumor mass and a secondary, untreated tumor growing at a distant site from the primary tumor and the site of therapeutic vector injection. In both the single-GBM and multifocal-GBM models used, all saline-treated control animals succumbed to tumors by day 22. Around 70% of the animals bearing a single GBM mass treated with an adenovirus expressing Flt3L (AdFlt3L) and an adenovirus expressing TK (AdTK + GCV) survived long term. Approximately 50% of animals bearing a large primary GBM that were implanted with a second GBM in the contralateral hemisphere at the same time the primary tumors were being treated with AdFlt3L and AdTK also survived long term. A second multifocal GBM model, in which bilateral GBMs were implanted simultaneously and only the right tumor mass was treated with AdFlt3L and AdTK, also demonstrated long-term survival. While no significant difference in survival was found between unifocal and multifocal GBM-bearing animals treated with AdFlt3L and AdTK, both treatments were statistically different from the saline-treated control group (p < 0.05). Our results demonstrate that combination therapy with AdFlt3L and AdTK can eradicate multifocal brain tumor disease in a syngeneic, intracranial GBM model.

T cells' immunological synapses induce polarization of brain astrocytes in vivo and in vitro: a novel astrocyte response mechanism to cellular injury.

Background Astrocytes usually respond to trauma, stroke, or neurodegeneration by undergoing cellular hypertrophy, yet, their response to a specific immune attack by T cells is poorly understood. Effector T cells establish specific contacts with target cells, known as immunological synapses, during clearance of virally infected cells from the brain. Immunological synapses mediate intercellular communication between T cells and target cells, both in vitro and in vivo. How target virally infected astrocytes respond to the formation of immunological synapses established by effector T cells is unknown. Findings Herein we demonstrate that, as a consequence of T cell attack, infected astrocytes undergo dramatic morphological changes. From normally multipolar cells, they become unipolar, extending a major protrusion towards the immunological synapse formed by the effector T cells, and withdrawing most of their finer processes. Thus, target astrocytes become polarized towards the contacting T cells. The MTOC, the organizer of cell polarity, is localized to the base of the protrusion, and Golgi stacks are distributed throughout the protrusion, reaching distally towards the immunological synapse. Thus, rather than causing astrocyte hypertrophy, antiviral T cells cause a major structural reorganization of target virally infected astrocytes. Conclusions Astrocyte polarization, as opposed to hypertrophy, in response to T cell attack may be due to T cells providing a very focused attack, and thus, astrocytes responding in a polarized manner. A similar polarization of Golgi stacks towards contacting T cells was also detected using an in vitro allogeneic model. Thus, different T cells are able to induce polarization of target astrocytes. Polarization of target astrocytes in response to immunological synapses may play an important role in regulating the outcome of the response of astrocytes to attacking effector T cells, whether during antiviral (e.g. infected during HIV, HTLV-1, HSV-1 or LCMV infection), anti-transplant, autoimmune, or anti-tumor immune responses in vivo and in vitro.

The Involvement of Neuroinflammation and Kynurenine Pathway in Parkinson's Disease

Parkinsons disease (PD) is a common neurodegenerative disorder characterised by loss of dopaminergic neurons and localized neuroinflammation occurring in the midbrain several years before the actual onset of symptoms. Activated microglia themselves release a large number of inflammatory mediators thus perpetuating neuroinflammation and neurotoxicity. The Kynurenine pathway (KP), the main catabolic pathway for tryptophan, is one of the major regulators of the immune response and may also be implicated in the inflammatory response in parkinsonism. The KP generates several neuroactive compounds and therefore has either a neurotoxic or neuroprotective effect. Several of these molecules produced by microglia can activate the N-methyl-D-aspartate (NMDA) receptor-signalling pathway, leading to an excitotoxic response. Previous studies have shown that NMDA antagonists can ease symptoms and exert a neuroprotective effect in PD both in vivo and in vitro. There are to date several lines of evidence linking some of the KP intermediates and the neuropathogenesis of PD. Moreover, it is likely that pharmacological modulation of the KP will represent a new therapeutic strategy for PD.