Downregulation of Plasma Membrane Ca2+ ATPase driven by tyrosine hydroxylase-Gal4 reduces Drosophila lifespan independently of effects in neurons.

In Drosophila melanogaster, several Gal4 drivers are used to direct gene/RNAi expression to different dopaminergic neuronal clusters. We previously developed a fly model of Parkinson's disease, in which dopaminergic neurons had elevated cytosolic Ca2+ due to the expression of a Plasma Membrane Ca2+ ATPase (PMCA) RNAi under the thyroxine hydroxylase (TH)-Gal4 driver. Surprisingly, TH-Gal4>PMCARNAi flies died earlier compared to controls and showed swelling in the abdominal area. Flies expressing the PMCARNAi under other TH drivers also showed such swelling and shorter lifespan. Considering that TH-Gal4 is also expressed in the gut, we proposed to suppress the expression specifically in the nervous system, while maintaining the activation in the gut. Therefore, we expressed Gal80 under the direction of the panneuronal synaptobrevin (nSyb) promoter in the context of TH-Gal4. nSyb-Gal80; TH-Gal4>PMCARNAi flies showed the same reduction of survival as TH-Gal4>PMCARNAi flies, meaning that the phenotype of abdomen swelling and reduced survival could be due to the expression of the PMCARNAi in the gut. In perimortem stages TH-Gal4>PMCARNAi guts had alteration in the proventriculi and crops. The proventriculi appeared to lose cells and collapse on itself, and the crop increased its size several times with the appearance of cellular accumulations at its entrance. No altered expression or phenotype was observed in flies expressing PMCARNAi in the dopaminergic PAM cluster (PAM-Gal4>PMCARNAi). In this work we show the importance of checking the global expression of each promoter and the relevance of the inhibition of PMCA expression in the gut.

Understanding the role of the blood brain barrier and peripheral inflammation on behavior and pathology on ongoing confined cortical lesions.

BACKGROUND: Inflammation in the Central Nervous System (CNS) is associated with blood brain barrier (BBB) breakdown during the early stages of Multiple Sclerosis (MS), indicating a facilitated entry of waves of inflammatory cells from the circulation to the CNS. In the progressive forms of MS, as the lesion becomes chronic, the inflammation remains trapped within the CNS compartment forming the slow evolving lesion, characterized by low inflammation and microglia activation at the lesions edges. The chronic expression of interleukin 1ß (IL-1ß) in the cortex induces BBB breakdown, demyelination, neurodegeneration, microglial/macrophage activation and impaired cognitive performance. The latter can be improved, as long as the BBB recovers and the lesion presents low inflammation. Here, we study the effects of peripheral inflammation on cortical central lesions after the restoration of the BBB, in order to elucidate the role of the peripheral inflammation on these lesions with intact BBB, as it occurs in the progressive forms of MS. MATERIALS AND METHODS: Cortical lesions and peripheral inflammation were induced by the chronic expression of IL-1ß using an adenovector. We performed histological, immunohistochemistry on brain tissue and behavioural analyses. RESULTS: The effects of the chronic expression of IL-1ß in the cortex resolved within 56 days. However, peripheral and sustained inflammation re-opened the BBB, allowing the reappearance of the neuroinflammatory processes within the cortical lesions, increased demyelination and neurodegeneration, and an increase of the behavioral symptoms, such as cognitive impairment and anxiety-like symptoms. CONCLUSIONS: The early treatment of peripheral inflammatory processes should be considered in order to protect the brain from exacerbation of the ongoing neurodegenerative process.

Plasma membrane calcium ATPase downregulation in dopaminergic neurons alters cellular physiology and motor behaviour in Drosophila melanogaster.

The accumulation of Ca2+ and its subsequent increase in oxidative stress is proposed to be involved in selective dysfunctionality of dopaminergic neurons, the main cell type affected in Parkinson's disease. To test the in vivo impact of Ca2+ increment in dopaminergic neurons physiology, we downregulated the plasma membrane Ca2+ ATPase (PMCA), a pump that extrudes cytosolic Ca2+ , by expressing PMCARNAi in Drosophila melanogaster dopaminergic neurons. In these animals, we observed major locomotor alterations paralleled to higher cytosolic Ca2+ and increased levels of oxidative stress in mitochondria. Interestingly, although no overt degeneration of dopaminergic neurons was observed, evidences of neuronal dysfunctionality were detected such as increases in presynaptic vesicles in dopaminergic neurons and in the levels of dopamine in the brain, as well as presence of toxic effects when PMCA was downregulated in the eye. Moreover, reduced PMCA levels were found in a Drosophila model of Parkinson's disease, Parkin knock-out, expanding the functional relevance of PMCA reduction to other Parkinson's disease-related models. In all, we have generated a new model to study motor abnormalities caused by increments in Ca2+ that lead to augmented oxidative stress in a dopaminergic environment, added to a rise in synaptic vesicles and dopamine levels.

Environmental enrichment improves cognitive symptoms and pathological features in a focal model of cortical damage of multiple sclerosis.

Multiple Sclerosis (MS) is a neuroinflammatory disease affecting white and grey matter, it is characterized by demyelination, axonal degeneration along with loss of motor, sensitive and cognitive functions. MS is a heterogeneous disease that displays different clinical courses: relapsing/remitting MS (RRMS), and MS progressive forms: primary progressive (PPMS) and secondary progressive (SPMS). Cortical damage in the progressive MS forms has considerable clinical relevance due to its association with cognitive impairment and disability progression in patients. One treatment is available for the progressive forms of the disease, but none are specific for cognitive deficits. We developed an animal model that reflects most of the characteristics of the cortical damage, such as cortical neuroinflammation, demyelination, neurodegeneration and meningeal inflammation, which was associated with cognitive impairment. Cognitive rehabilitation, exercise and social support have begun to be evaluated in patients and animal models of neurodegenerative diseases. Environmental enrichment (EE) provides exercise as well as cognitive and social stimulation. EE has been demonstrated to exert positive effects on cognitive domains, such as learning and memory, and improving anxiety-like symptoms. We proposed to study the effect of EE on peripherally stimulated cortical lesion induced by the long term expression of interleukin IL-1ß (IL-1ß) in adult rats. Here, we demonstrated that EE: 1) reduces the peripheral inflammatory response to the stimulus, 2) ameliorates cognitive deficits and anxiety-like symptoms, 3) modulates neurodegeneration, demyelination and glial activation, 4) regulates neuroinflammation by reducing the expression of pro-inflammatory cytokines and enhancing the expression of anti-inflammatory ones. Our findings correlate with the fact that EE housing could be considered an effective non- pharmacological therapeutic agent that can synergistically aid in the rehabilitation of the disease.

A new focal model resembling features of cortical pathology of the progressive forms of multiple sclerosis: Influence of innate immunity.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of unknown aetiology that causes neurological disabilities in young adults. MS displays different clinical patterns, including recurrent episodes with remission periods ("relapsing-remitting MS" (RRMS)), which can progress over several years to a secondary progressive form (SPMS). However, 10% of patients display persistent progression at the onset of disease ("primary progressive MS" (PPMS)). Currently, no specific therapeutic agents are available for the progressive forms, mainly because the underlying pathogenic mechanisms are not clear and because no animal models have been specifically developed for these forms. The development of MS animal models is required to clarify the pathological mechanisms and to test novel therapeutic agents. In the present work, we overexpressed interleukin 1 beta (IL-1ß) in the cortex to develop an animal model reflecting the main pathological hallmarks of MS. The treated animals presented with neuroinflammation, demyelination, glial activation, and neurodegeneration along with cognitive symptoms and MRI images consistent with MS pathology. We also demonstrated the presence of meningeal inflammation close to cortical lesions, with characteristics similar to those described in MS patients. Systemic pro-inflammatory stimulation caused a flare-up of the cortical lesions and behavioural symptoms, including impairment of working memory and the appearance of anxiety-like symptoms. Our work demonstrated induced cortical lesions, reflecting the main histopathological hallmarks and cognitive impairments characterizing the cortical pathology described in MS patients with progressive forms of the disease.

Fibulin-2 is a key mediator of the pro-neurogenic effect of TGF-beta1 on adult neural stem cells.

Transforming growth factor beta 1 (TGF-beta1), an anti-inflammatory cytokine, has been shown to have pro-neurogenic effects on adult Neural Stem Cells (aNSC) from the dentate gyrus and in vivo models. Here, we expanded the observation of the pro-neurogenic effect of TGF-beta1 on aNSC from the subventricular zone (SVZ) of adult rats and performed a functional genomic analysis to identify candidate genes to mediate its effect. 10 candidate genes were identified by microarray analysis and further validated by qRT-PCR. Of these, Fibulin-2 was increased 477-fold and its inhibition by siRNA blocks TGF-beta1 pro-neurogenic effect. Curiously, Fibulin-2 was not expressed by aNSC but by a GFAP-positive population in the culture, suggesting an indirect mechanism of action. TGF-beta1 also induced Fibulin-2 in the SVZ in vivo. Interestingly, 5 out of the 10 candidate genes identified are known to interact with integrins, paving the way for exploring their functional role in adult neurogenesis. In conclusion, we have identified 10 genes with putative pro-neurogenic effects, 5 of them related to integrins and provided proof that Fibulin-2 is a major mediator of the pro-neurogenic effects of TGF-beta1. These data should contribute to further exploring the molecular mechanism of adult neurogenesis of the genes identified and the involvement of the integrin pathway on adult neurogenesis.

Chronic systemic IL-1ß exacerbates central neuroinflammation independently of the blood-brain barrier integrity.

Peripheral circulating cytokines are involved in immune to brain communication and systemic inflammation is considered a risk factor for flaring up the symptoms in most neurodegenerative diseases. We induced both central inflammatory demyelinating lesion, and systemic inflammation with an interleukin-1ß expressing adenovector. The peripheral pro-inflammatory stimulus aggravated the ongoing central lesion independently of the blood-brain barrier (BBB) integrity. This model allows studying the role of specific molecules and cells (neutrophils) from the innate immune system, in the relationship between central and peripheral communication, and on relapsing episodes of demyelinating lesions, along with the role of BBB integrity.

The degenerating substantia nigra as a susceptible region for gene transfer-mediated inflammation.

Parkinson's disease (PD) is characterized by the progressive degeneration of neurons in the substantia nigra pars compacta (SN). The naïve SN is highly susceptible to inflammation. In addition, microglial activation in the degenerating SN displays distinct characteristics that increase the reactivity of the region towards inflammatory stimuli. On the other hand, gene therapy for PD has recently move forward into clinical settings, with PD being the neurodegenerative disorder with the highest number of Phase I/II gene therapy clinical trials approved and completed. These clinical trials are not targeting the SN, but this region is a certain candidate for future gene therapy interventions. Here, the unique immune-related properties of the degenerating SN in the context of a putative gene therapy intervention are reviewed. Several variables affecting the host response to gene delivery such as vector type and dosage, age and stage of disease of patients, and method of gene delivery and transgene used are discussed. Finally, approaches to diminish the risk of immune-mediated toxicity by gene transfer in the SN are presented.

Nigral neurodegeneration triggered by striatal AdIL-1 administration can be exacerbated by systemic IL-1 expression.

Neuroinflammation has been proposed as an important component of Parkinson's Disease (PD) aetiology and/or progression. However, the inflammatory components and the mechanisms underlying their effects are only partially known. By injecting an adenovirus expressing IL-1 in the striatum, we provoked progressive neurodegeneration of dopaminergic cells in the substantia nigra, motor symptoms and microglial activation. All these effects were attenuated by an anti-inflammatory treatment. Interestingly, peripheral inflammatory stimuli exacerbated IL-1beta induced neurodegeneration and the central inflammatory reaction. These data provide evidence that central, chronic IL-1beta expression can trigger and systemic IL-1beta exacerbate nigral neurodegeneration and highlight the functional relevance of this cytokine in PD.

Central and systemic IL-1 exacerbates neurodegeneration and motor symptoms in a model of Parkinson's disease.

Parkinson's disease is a neurodegenerative disorder with uncertain aetiology and ill-defined pathophysiology. Activated microglial cells in the substantia nigra (SN) are found in all animal models of Parkinson's disease and patients with the illness. Microglia may, however, have detrimental and protective functions in this disease. In this study, we tested the hypothesis that a sub-toxic dose of an inflammogen (lipopolysaccharide) can shift microglia to a pro-inflammatory state and exacerbate disease progression in an animal model of Parkinson's disease. Central lipopolysaccharide injection in a degenerating SN exacerbated neurodegeneration, accelerated and increased motor signs and shifted microglial activation towards a pro-inflammatory phenotype with increased interleukin-1beta (IL-1beta) secretion. Glucocorticoid treatment and specific IL-1 inhibition reversed these effects. Importantly, chronic systemic expression of IL-1 also exacerbated neurodegeneration and microglial activation in the SN. In vitro, IL-1 directly exacerbated 6-OHDA-triggered dopaminergic toxicity. In vivo, we found that nitric oxide was a downstream molecule of IL-1 action and partially responsible for the exacerbation of neurodegeneration observed. Thus, IL-1 exerts its exacerbating effect on degenerating dopaminergic neurons by direct and indirect mechanisms. This work demonstrates an unequivocal association between IL-1 overproduction and increased disease progression, pointing to inflammation as a risk factor for Parkinson's disease and suggesting that inflammation should be efficiently handled in patients to slow disease progression.

Progressive neurodegeneration and motor disabilities induced by chronic expression of IL-1beta in the substantia nigra.

The functional role of the long-lasting inflammation found in the substantia nigra (SN) of Parkinson's disease (PD) patients and animal models is unclear. Proinflammatory cytokines such as interleukin-1beta (IL-1beta) could be involved in mediating neuronal demise. However, it is unknown whether the chronic expression of cytokines such as IL-1beta in the SN can alter neuronal vitality. The aim of this study was to investigate the effects of the chronic expression of IL-1beta in the adult rat SN using a recombinant adenovirus expressing IL-1beta. The chronic expression of IL-1beta for 60 days induced dopaminergic cell death in the SN and unilateral akinesia starting only at 21 days post-injection. Microglial cell activation and inflammatory cell infiltrate were associated with dopaminergic cell death and motor disabilities. Astrocytic activation was delayed and associated with scar formation. The chronic expression of a single proinflammatory cytokine as IL-1beta in the SN elicited most of the characteristics of PD, including progressive dopaminergic cell death, akinesia and glial activation. Our data suggest that IL-1beta per se is able to mediate inflammatory-mediated toxic effects in the SN if its expression is sustained. This model will be helpful to identify possible therapeutic targets related to inflammation-derived neurodegeneration in the SN.