Francisco Ros-Bernal

Microglial glucocorticoid receptors play a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism

Among the pathogenic processes contributing to dopaminergic neuron (DN) death in Parkinson disease (PD), evidence points to non–cell-autonomous mechanisms, particularly chronic inflammation mounted by activated microglia. Yet little is known about endogenous regulatory processes that determine microglial actions in pathological states. We examined the role of glucocorticoid receptors (GRs), activated by glucocorticoids released in response to stress and known to regulate inflammation, in DN survival. Overall GR level was decreased in substantia nigra of PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. GR changes, specifically in the microglia after MPTP treatment, revealed a rapid augmentation in the number of microglia displaying nuclear localization of GR. Mice with selective inactivation of the GR gene in macrophages/microglia (GRLysMCre) but not in DNs (GRDATCre) showed increased loss of DNs after MPTP intoxication. This DN loss in GRLysMCre mice was not prevented by corticosterone treatment, in contrast to the protection observed in control littermates. Moreover, absence of microglial GRs augmented microglial reactivity and led to their persistent activation. Analysis of inflammatory genes revealed an up-regulation of Toll-like receptors (TLRs) by MPTP treatment, particularly TLR9, the level of which was high in postmortem parkinsonian brains. The regulatory control of GR was reflected by higher expression of proinflammatory genes (e.g., TNF-α) with a concomitant decrease in anti-inflammatory genes (e.g., IL-1R2) in GRLysMCre mice. Indeed, in GRLysMCre mice, alterations in phosphorylated NF-κB levels indicated its protracted activation. Together, our data indicate that GR is important in curtailing microglial reactivity, and its deregulation in PD could lead to sustained inflammation-mediated DN injury.

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.

Implications of glial nitric oxide in neurodegenerative diseases.

Nitric oxide (NO) is a pleiotropic janus-faced molecule synthesized by nitric oxide synthases (NOS) which plays a critical role in a number of physiological and pathological processes in humans. The physiological roles of NO depend on its local concentrations, as well as its availability and the nature of downstream target molecules. Its double-edged sword action has been linked to neurodegenerative disorders. Excessive NO production, as the evoked by inflammatory signals, has been identified as one of the major causative reasons for the pathogenesis of several neurodegenerative diseases. Moreover, excessive NO synthesis under neuroinflammation leads to the formation of reactive nitrogen species and neuronal cell death. There is an intimate relation between microglial activation, NO and neuroinflammation in the human brain. The role of NO in neuroinflammation has been defined in animal models where this neurotransmitter can modulate the inflammatory process acting on key regulatory pathways, such as those associated with excitotoxicity processes induced by glutamate accumulation and microglial activation. Activated glia express inducible NOS and produce NO that triggers calcium mobilization from the endoplasmic reticulum, activating the release of vesicular glutamate from astroglial cells resulting in neuronal death. This change in microglia potentially contributes to the increased age-associated susceptibility and neurodegeneration. In the current review, information is provided about the role of NO, glial activation and age-related processes in the central nervous system (CNS) that may be helpful in the isolation of new therapeutic targets for aging and neurodegenerative diseases.

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.

CCL2-expressing astrocytes mediate the extravasation of T lymphocytes in the brain. Evidence from patients with glioma and experimental models in vivo.

CCL2 is a chemokine involved in brain inflammation, but the way in which it contributes to the entrance of lymphocytes in the parenchyma is unclear. Imaging of the cell type responsible for this task and details on how the process takes place in vivo remain elusive. Herein, we analyze the cell type that overexpresses CCL2 in multiple scenarios of T-cell infiltration in the brain and in three different species. We observe that CCL2+ astrocytes play a part in the infiltration of T-cells in the brain and our analysis shows that the contact of T-cells with perivascular astrocytes occurs, suggesting that may be an important event for lymphocyte extravasation.

7-Nitroindazole down-regulates dopamine/DARPP-32 signaling in neostriatal neurons in a rat model of Parkinson's disease

Neuronal nitric oxide synthase (nNOS) is involved in the regulation of diverse intracellular messenger systems in the brain. Nitric Oxide (NO) contributes to inducing signaling cascades that involve a complex pattern of phosphorylation of DARPP-32 (in Thr-34), which controls the phosphoproteins involved in neuronal activation. However, the role of NO in the pathophysiology of Parkinsons disease (PD) and its effect in striatal neurons have been scarcely explored. In the present work, we investigate the effects of a nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI) in the nigrostriatal pathway of striatal 6-hydroxydopamine (6-OHDA) lesioned rats. Our quantitative histological findings show that treatment with 7-NI significantly reduced 6-OHDA-induced dopaminergic damage in the dorsolateral striatum and Substantia Nigra pars compacta (SNpc). Moreover, 6-OHDA lesioned rats show a significant increase of nNOS(+) and Phospho-Thr34-DARPP-32(+) cells, accompanied by a consequent decrease of total DARPP-32(+) cells, which suggests an imbalance of NO activity in the DA-depleted striatum, which is also reflected in behavioral studies. Importantly, these effects are reverted in the group treated with 7-NI. These results show a clear link between the state of phosphorylation of DARPP-32 and parkinsonism, which is regulated by nNOS. This new evidence suggests a prominent role for nitric oxide in the neurotransmitter balance within the basal ganglia in the pathophysiology of experimental parkinsonism.

Persistent phagocytic characteristics of microglia in the substantia nigra of long-term Parkinsonian macaques

Patients with Parkinsons disease show persistent microglial activation in the areas of the brain where the degeneration of dopaminergic neurons takes place. The reason for maintaining this activated state is still unknown, but it is thought that this persistent microglial activation may contribute to the degeneration of dopaminergic neurons. In this study, we report the microanatomical details of microglia and the relationship between microglia and neurons in the substantia nigra pars compacta of Parkinsonian monkeys years after insult with MPTP. We observed that microglial cells appear polarized toward dopaminergic neurons in MPTP-treated macaques compared to untreated animals and present clear phagocytic characteristics, such as engulfing gliaptic contacts, an increase in Golgi apparatus protein machinery and ball-and-chain phagocytic buds. These results demonstrate that activated microglia maintain phagocytic characteristics years after neurotoxin insult, and phagocytosis may be a key contributor to the neurodegenerative process.

MPTP administration increases plasma levels of acute phase proteins in non-human primates (Macaca fascicularis)

Parkinsons disease (PD) is characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc). Parkinsonian patients and animal models of PD show inflammatory phenomena such as microglial activation and cytokine production that could modulate the progression of the disease, since they play a crucial role in the degenerative process. Since acute phase proteins (APPs) are involved in a number of homeostatic alterations and inflammatory processes, we analyzed the levels of APPs in primates before and after treatment with MPTP. A significant increase in C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (HP) levels after MPTP treatment. These results demonstrate that MPTP induces a systemic generalized inflammatory reaction after specific dopaminergic neurotoxicity insult, suggesting that the inflammatory process in Parkinsonism may affect other immune-inflammatory responses outside the brain.

Evidence of oligodendrogliosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism

V. Annese, C. Barcia, F. Ros‐Bernal, A. Gómez, C. M. Ros, V. De Pablos, E. Fernández‐Villalba, M.‐E. De Stefano and M. T. Herrero (2013) Neuropathology and Applied Neurobiology39, 132–143

Designing serious games for learning support in medicine studies: A specific method to elicit and formalize requirements

In Spain, the new Medicine degrees of the European Higher Education Area (EHEA) have incorporated Information Technologies (IT) subjects to develop horizontal competences in their curriculum. Medicine Studies are costly for Higher Education (HE) institutions and, then, every cost-effective educational innovation is welcomed. There are different general approaches for designing and developing serious games in general, but specific target disciplines as Medicine (educational and professional training), lack of tailor-made methodologies, even in eliciting requirements under the Requirements Engineering (RE) framework, as a subfield of Software Engineering. In medical higher education, the main objective of using games is to engage learners in complex problem spaces that replicate real world situations, as it happens in hospital emergencies, surgery operations and so on, without the constraints and risks of the day to day practice (patient injuries, health complications and death). Therefore, the development of serious HE games is a complex, time consuming and costly process, especially for learning medical skills. In this Work in Progress, we address the problem of lacking suitable methodologies in the design and development processes of educational serious games for HE medical environments. Our proposal is a semi-formal and simple method to elicit and formalize the specific teaching needs identified in subjects or practices of Medicine degrees, in order to design and develop serious games based on sound educational principles and theories and on RE rules.