Microglial Caspase-3 is essential for modulating hippocampal neurogenesis.

Adult hippocampal neurogenesis (AHN) is a process involved in numerous neurodegenerative diseases. Many researchers have described microglia as a key component in regulating the formation and migration of new neurons along the rostral migratory stream. Caspase-3 is a cysteine-aspartate-protease classically considered as one of the main effector caspases in the cell death program process. In addition to this classical function, we have identified the role of this protein as a modulator of microglial function; however, its action on neurogenic processes is unknown. The aim of the present study is to identify the role of Caspase-3 in neurogenesis-related microglial functions. To address this study, Caspase-3 conditional knockout mice in the microglia cell line were used. Using this tool, we wanted to elucidate the role of this protein in microglial function in the hippocampus, the main region in which adult neurogenesis takes place. After the reduction of Caspase-3 in microglia, mutant mice showed a reduction of microglia in the hippocampus, especially in the dentate gyrus region, a region inherently associated to neurogenesis. In addition, we found a reduction in doublecortin-positive neurons in conditional Caspase-3 knockout mice, which corresponds to a reduction in neurogenic neurons. Furthermore, using high-resolution image analysis, we also observed a reduction in the phagocytic capacity of microglia lacking Caspase-3. Behavioral analysis using object recognition and Y-maze tests showed altered memory and learning in the absence of Caspase-3. Finally, we identified specific microglia located specifically in neurogenic niche positive for Galectin 3 which colocalized with Cleaved-Caspase-3 in control mice. Taken together, these results showed the essential role of Caspase-3 in microglial function and highlight the relevant role of this specific microglial phenotype in the maintenance of AHN in the hippocampus.

Hydroxytyrosol Decreases LPS- and α-Synuclein-Induced Microglial Activation In Vitro.

Neuroinflammation is a common feature shared by neurodegenerative disorders, such as Parkinson's disease (PD), and seems to play a key role in their development and progression. Microglia cells, the principal orchestrators of neuroinflammation, can be polarized in different phenotypes, which means they are able to have anti-inflammatory, pro-inflammatory, or neurodegenerative effects. Increasing evidence supports that the traditional Mediterranean dietary pattern is related to the reduction of cognitive decline in neurodegenerative diseases. A considerable intake of plant foods, fish, and extra virgin olive oil (EVOO), as well as a moderate consumption of red wine, all characteristic of the Mediterranean diet (MD), are behind these effects. These foods are especially rich in polyphenols, being the most relevant in the MD hydroxytyrosol (HT) and their derivatives present in EVOO, which have demonstrated a wide array of biological activities. Here, we demonstrate that HT is able to reduce the inflammation induced by two different stimuli: lipopolysaccharide and α-synuclein. We also study the possible molecular mechanisms involved in the anti-inflammatory effect of HT, including the study of nuclear factor kappa B (NF-кB), mitogen-activated protein kinases (MAPKs), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and inflammasome. Our data support the use of HT to prevent the inflammation associated with PD and shed light into the relationship between MD and this neurological disorder.

Evaluation of a method for murine monocyte isolation by bone marrow depletion.

The study of monocyte activation and differentiation has great applications in sepsis, chronic inflammatory diseases, and cancer studies. However, despite the existence of well-established protocols for monocyte purification from human blood, the isolation of murine monocytes that can be subsequently activated has not yet been fully optimized. Here we evaluate a recently developed commercial procedure for obtaining monocytes from the bone marrow based on immunomagnetic depletion of non-monocytic cells. Moreover, we compare the advantages and disadvantages of this approach relative to other existing procedures. We found that monocytes isolates generated using this technique had equal purity to those attained via depletion from peripheral blood; however, higher yields were achieved. Furthermore, isolates from this technique have lower levels of macrophage contamination than those reported in samples generated by culturing bone marrow extracts with macrophage colony-stimulating factor (M-CSF). In addition, we demonstrate that the purified monocytes are sensitive to lipopolysaccharide (LPS)-mediated activation and, therefore, are useful for studies aimed at elucidating the molecular mechanisms involved in monocyte activation and differentiation.

Role of dopamine in the recruitment of immune cells to the nigro-striatal dopaminergic structures.

Research indicates that inflammation and microglial activation are involved in the initiation and progression of Parkinson's disease (PD). Neuroinflammation contributes to the infiltration of peripheral immune cells and blood-brain barrier (BBB) leakage, linking peripheral and central inflammatory events in the pathogenesis of PD. Dopamine (DA) likely plays a role in this process. In the present study, the dopaminergic toxin 6-hydroxydopamine (6-OHDA) was used to damage dopaminergic neurons. Injection of 6-OHDA within the nigrostriatal pathway produced loss of astrocytes, disruption of the BBB, microglia activation and a reduction in osteopontin (OPN) immunoreactivity. Depletion of DA content by alpha-methylparatyrosine (α-MPT, a tyrosine hydroxylase inhibitor) reduced the infiltration of peripheral macrophages as well as the 6-OHDA-induced increase in microglial cells. DA could therefore be relevant in sustaining inflammation and lymphocyte recruitment induced by 6-OHDA, supporting DA implication in the degeneration of dopaminergic neurons induced by inflammatory processes.

Caspases playing in the field of neuroinflammation: old and new players.

Neuroinflammation is a complex immune response against the harmful effects of diverse stimuli within the central nervous system. Caspases are a family of intracellular cysteine proteases that mediate proteolytic events indispensable for transduction of signaling pathway-controlling biological phenomena such as apoptosis and inflammation. To date, 14 players have been identified in mammals. For many years, caspases were simply divided into 'apoptotic' and 'proinflammatory' caspases and this classification remains useful to some extent. However, increasing evidence indicates that many of these so-called apoptotic caspases also exert nonapoptotic functions. In addition, the role of certain members of the supposed inflammatory caspases in the inflammatory process per se has also been discussed. In this review, we highlight the role for 'apoptotic' and 'proinflammatory' caspases in the regulation of the inflammation response with a special focus on the central nervous system.

Peripheral inflammation increases the deleterious effect of CNS inflammation on the nigrostriatal dopaminergic system.

Evidence supports the role of inflammation in the development of neurodegenerative diseases. In this work, we are interested in inflammation as a risk factor by itself and not only as a factor contributing to neurodegeneration. We tested the influence of a mild to moderate peripheral inflammation (injection of carrageenan into the paws of rats) on the degeneration of dopaminergic neurons in an animal model based on the intranigral injection of lipopolysaccharide (LPS), a potent inflammatory agent. Overall, the treatment with carrageenan increased the effect of the intranigral injection of LPS on the loss of dopaminergic neurons in the SN along with all the other parameters studied, including: serum levels of the inflammatory markers TNF-α, IL-1ß, IL-6 and C-reactive protein; activation of microglia, expression of proinflammatory cytokines, the adhesion molecule ICAM and the enzyme iNOS, loss of astrocytes and damage to the blood brain barrier (BBB). The possible implication of BBB rupture in the increased loss of dopaminergic neurons has been studied using another Parkinson's disease animal model based on the intraperitoneal injection of rotenone. In this experiment, loss of dopaminergic neurons was also strengthened by carrageenan, without affecting the BBB. In conclusion, our data show that a mild to moderate peripheral inflammation can exacerbate the degeneration of dopaminergic neurons caused by a harmful stimulus.

Ulcerative colitis exacerbates lipopolysaccharide-induced damage to the nigral dopaminergic system: potential risk factor in Parkinson`s disease.

Peripheral inflammation could play a role in the origin and development of certain neurodegenerative disorders. To ascertain this possibility, a model of dopaminergic neurodegeneration based on the injection of the inflammatory agent lipopolysaccharide (LPS) within the substantia nigra was assayed in rats with ulcerative colitis (UC) induced by the ingestion of dextran sulphate sodium. We found an increase in the levels of inflammatory markers from serum (tumor necrosis factor-α, IL-1ß, IL-6 and the acute phase protein C-reactive protein) and substantia nigra (tumor necrosis factor-α, IL-1ß, IL-6, inducible nitric oxide synthase, intercellular adhesion molecule-1, microglial and astroglial populations) of rats with UC, as well as an alteration of the blood-brain barrier permeability and the loss of dopaminergic neurons. UC reinforced the inflammatory and deleterious effects of LPS. On the contrary, clodronate encapsulated in liposomes (ClodLip), which depletes peripheral macrophages, ameliorated the effect of LPS and UC. Peripheral inflammation might represent a risk factor in the development of Parkinson's disease.

The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination.

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining - a non-specific neuronal marker - and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre-synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold-labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up-regulation of cFos mRNA, apoptosis and accumulation of alpha-synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease-activated receptor 4 activation, as protease-activated receptor 4-activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.

Intracerebral VEGF injection highly upregulates AQP4 mRNA and protein in the perivascular space and glia limitans externa.

Stroke is the third leading cause of death and the leading cause of adult disability in the industrialized nations. One of the consequences of stroke is blood-brain barrier (BBB) leakage and subsequent edema, which is one of the causes of mortality in this pathology. Aquaporin-4 (AQP4) is the most abundant water channel in the brain. Studies in AQP4 knock-out mice have shown a prominent role of this water channel in edema development and resolution after ischemia. Here we have studied changes in AQP4 mRNA and protein expression in response to vascular endothelial growth factor (VEGF), a potent angiogenic factor. VEGF administration highly upregulated AQP4 mRNA and protein in the ventral midbrain. Perfusion of the animals with FITC-albumin prior to sacrifice demonstrated localization of AQP4 protein in close proximity to the VEGF-induced new blood vessels. Expression levels of AQP4 mRNA were maximum 7 days after VEGF injection whereas our previous report showed that BBB leakage is resolved at this time point. Therefore, we speculate a positive role of AQP4 in edema resolution, which may partially explain the previously reported beneficial effects of delayed VEGF administration in ischemic rats. Our results provide new insights into the molecular changes in the edematous brain and may help in future therapeutical directions.

Blood-brain barrier disruption induces in vivo degeneration of nigral dopaminergic neurons.

We have evaluated the possibility that changes in the vascular system may constitute a contributing factor for the death of nigral dopaminergic neurons in Parkinson's disease. Thus, we have employed intranigral injections of vascular endothelial growth factor (VEGF), the most potent inducer of blood-brain barrier (BBB) permeability. A single dose of 1 mug of VEGF, chosen from a dose-response study, highly disrupted the BBB in the ventral mesencephalon in a time-dependent manner. A strong regional correlation between BBB disruption and loss of tyrosine hydroxylase-positive neurons was evident. Moreover, Fluoro-Jade B labelling showed the presence of dying neurons in the substantia nigra in response to VEGF injection. High number of TUNEL-positive nuclei was observed in this area along with activation of caspase 3 within nigral dopaminergic neurons. Analysis of the glial population demonstrated a strong inflammatory response and activation of astroglia in response to BBB disruption. We conclude that disruption of the BBB may be a causative factor for degeneration of nigral dopaminergic neurons.

Divergent regulatory mechanisms governing BDNF mRNA expression in cerebral cortex and substantia nigra in response to striatal target ablation.

We have studied the regulation of BDNF mRNA expression in the corticostriatal and nigrostriatal systems following neurotoxin ablation of striatal targets induced by quinolinic acid (QA) or 2S:2'R:3'R:-2-(2'3'-dicarboxycyclopropyl)glycine (DCG-IV) injections. Striatal lesions were verified by quantifying the loss of glutamic acid decarboxylase (GAD) mRNA expression. Levels of BDNF mRNA were analyzed at 6, 12, and 24 h postlesion. Both lesions paradigms highly induced BDNF mRNA in the ipsilateral cerebral cortex at 6 and 12 h postlesion to drop to control levels at 24 h postlesion. These inductions were mostly restricted to cortical layers II/III and V and ipsilateral insular and piriform cortices, which provide the main cortical inputs to the striatum. Analysis of neuronal activation on these areas demonstrated high levels of cFos mRNA in response to the excitotoxic striatal lesions. Dual labeling of cFos and BDNF mRNAs demonstrated a positive correlation between cortical neuronal activation and expression of BDNF mRNA. Consequently, expression of BDNF in cortical areas projecting to striatum is dependent on both target integrity and neuronal activity. Regulation of BNDF mRNA in substantia nigra, the second major source of BDNF to striatal cells, highly differed from that seen in cerebral cortex. Analysis of cellular expression alone or in combination of BDNF, cFos, tyrosine hydroxylase and GAD mRNAs demonstrated that expression of BDNF in substantia nigra is dependent on target integrity and independent of neuronal activity. In addition, we have studied regulatory mechanisms of BDNF mRNA in the subthalamic nucleus.

In vivo expression of aquaporin-4 by reactive microglia.

Aquaporin-4 (AQP4) is the most abundant aquaporin in the brain and it is widely accepted that this AQP is solely expressed by astrocytes and ependymal cells. AQP4 is particularly enriched in plasma membranes of ependymal cells and astrocyte membrane domains facing blood vessels and pia. AQP4 has gained much attraction due to its involvement in the physiopathology of brain edema, a major cause of death in humans. Consequently, it is of paramount importance to ascertain the phenotypic nature of AQP4 mRNA-expressing cells in the CNS before attempting future clinical studies aimed at minimizing the development of brain edema. We have used intranigral injections of lipopolysaccharide (LPS), a potent immunostimulant that causes disruption of the blood brain barrier, vasogenic edema, loss of reactive astrocytes and activation of microglial cells. These LPS-induced features are ideal for testing the possibility that reactive microglial cells express AQP4 in the adult brain. We have studied AQP4 at the mRNA and protein level. We provide strong evidence that reactive microglial cells highly express AQP4 mRNA and protein in response to LPS injections.

Minocycline reduces the lipopolysaccharide-induced inflammatory reaction, peroxynitrite-mediated nitration of proteins, disruption of the blood-brain barrier, and damage in the nigral dopaminergic system.

We have evaluated the potential neuroprotectant activity of minocycline in an animal model of Parkinson's disease induced by intranigral injection of lipopolysaccharide. Minocycline treatment was very effective in protecting number of nigral dopaminergic neurons and loss of reactive astrocytes at 7 days postlesion. Evaluation of microglia revealed that minocycline treatment highly prevented the lipopolysaccharide-induced activation of reactive microglia as visualized by OX-42 and OX-6 immunohistochemistry. Short-term RT-PCR analysis demonstrated that minocycline partially prevented the lipopolysaccharide-induced increases of mRNA levels for interleukin-1alpha and tumor necrosis factor-alpha. In addition, lipopolysaccharide highly induced protein nitration as seen by 3-nitrotyrosine immunoreactivity in the ventral mesencephalon. Minocycline treatment strongly diminished the extent of 3-nitrotyrosine immunoreactivity. We also found a direct correlation between location of IgG immunoreactivity-a marker of blood-brain barrier disruption-and neurodegenerative processes including death of nigral dopaminergic cells and reactive astrocytes. There was also a precise spatial correlation between disruption of blood-brain barrier and 3-nitrotyrosine immunoreactivity. We discuss potential involvement of lipopolysaccharide-induced formation of peroxynitrites and cytokines in the pathological events in substantia nigra in response to inflammation. If inflammation is proved to be involved in the ethiopathology of Parkinson's disease, our data support the use of minocycline in parkinsonian patients.

Thrombin induces in vivo degeneration of nigral dopaminergic neurones along with the activation of microglia.

Seven days after the injection of different concentrations of thrombin into the nigrostriatal pathway, a strong macrophage/microglial reaction was observed in the substantia nigra (SN), indicated by immunostaining, using OX-42 and OX-6 antibodies, and by the induction of iNOS, IL-1alpha, Il-1beta and TNF-alpha. Moreover, selective damage to dopaminergic neurones was produced after thrombin injection, evidenced by loss of tyrosine hydroxylase immunostaining and tyrosine hydroxylase mRNA-expressing cell bodies, and the unaltered transcription of glutamic acid decarboxylase mRNA in the SN and striatum. These thrombin effects could be produced by its ability to induce the activation of microglia described in in vitro studies, and are in agreement with the effects described for other proinflammatory compounds. Thrombin effects are produced by its biological activity since they almost disappeared when thrombin was heat-inactivated or injected along with its inhibitor alpha-NAPAP. Thrombin is a multi-functional serine protease rapidly produced from prothrombin at the sites of tissue injury, and also upon breakdown of the blood-brain barrier, which strongly suggests it could easily enter into the CNS. These results could have special importance in some degenerative processes of the nigrostriatal dopaminergic system.

Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats.

Recent studies have implicated chronic elevated exposures to environmental agents, such as metals (e.g. manganese, Mn) and pesticides, as contributors to neurological disease. Eighteen-month-old rats received intraperitoneal injections of manganese chloride (6 mg Mn/kg/day) or equal volume of saline for 30 days in order to study the effect of manganese on the dopamine- and GABA-neurons. The structures studied were substantia nigra, striatum, ventral tegmental area, nucleus accumbens and globus pallidus. First, we studied the enzymatic activity of mitochondrial complex II succinate dehydrogenase (SDH). We found an overall decrease of SDH in the different brain areas analyzed. We then studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra and ventral tegmental area following Mn treatment. Correspondingly, TH immunoreactivity was increased in substantia nigra and ventral tegmental area. Manganese treatment significantly decreased GAD mRNA levels in individual GABAergic neurons in globus pallidus but not in striatum. We also quantified the density of glial fibrillary acidic protein (GFAP)-labeled astrocytes and OX-42 positive cells. Reactive gliosis in response to Mn treatment occurred only in striatum and substantia nigra and the morphology of the astrocytes was different than in control animals. These results suggest that the nigrostriatal system could be specifically damaged by manganese toxicity. Thus, changes produced by manganese treatment on 18-month-old rats could play a role in the etiology of Parkinson's disease.