Inflammatory bowel disease induces pathological α-synuclein aggregation in the human gut and brain.

AIMS: According to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. METHODS: We have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of α-syn pathology. RESULTS: Our data support the existence of pathogenic α-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-α-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in α-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD. CONCLUSION: These results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating α-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration.

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.

Caspase signalling controls microglia activation and neurotoxicity.

Activation of microglia and inflammation-mediated neurotoxicity are suggested to play a decisive role in the pathogenesis of several neurodegenerative disorders. Activated microglia release pro-inflammatory factors that may be neurotoxic. Here we show that the orderly activation of caspase-8 and caspase-3/7, known executioners of apoptotic cell death, regulate microglia activation through a protein kinase C (PKC)-δ-dependent pathway. We find that stimulation of microglia with various inflammogens activates caspase-8 and caspase-3/7 in microglia without triggering cell death in vitro and in vivo. Knockdown or chemical inhibition of each of these caspases hindered microglia activation and consequently reduced neurotoxicity. We observe that these caspases are activated in microglia in the ventral mesencephalon of Parkinson's disease (PD) and the frontal cortex of individuals with Alzheimer's disease (AD). Taken together, we show that caspase-8 and caspase-3/7 are involved in regulating microglia activation. We conclude that inhibition of these caspases could be neuroprotective by targeting the microglia rather than the neurons themselves.

Metformin, besides exhibiting strong in vivo anti-inflammatory properties, increases mptp-induced damage to the nigrostriatal dopaminergic system.

Metformin is a widely used oral antidiabetic drug with known anti-inflammatory properties due to its action on AMPK protein. This drug has shown a protective effect on various tissues, including cortical neurons. The aim of this study was to determine the effect of metformin on the dopaminergic neurons of the substantia nigra of mice using the animal model of Parkinson's disease based on the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an inhibitor of the mitochondrial complex I. In vivo and in vitro experiments were used to study the activation of microglia and the damage of the dopaminergic neurons. Our results show that metformin reduced microglial activation measured both at cellular and molecular levels. Rather than protecting, metformin exacerbated dopaminergic damage in response to MPTP. Our data suggest that, contrary to other brain structures, metformin treatment could be deleterious for the dopaminergic system. Hence, metformin treatment may be considered as a risk factor for the development of Parkinson's disease.

Neuromelanin activates proinflammatory microglia through a caspase-8-dependent mechanism.

BACKGROUND: We have uncovered a caspase-dependent (caspase-8/caspase-3/7) signaling governing microglia activation and associated neurotoxicity. Importantly, a profuse non-nuclear activation of cleaved caspases 8 and 3 was found in reactive microglia in the ventral mesencephalon from subjects with Parkinson's disease, thus supporting the existence of endogenous factors activating microglia through a caspase-dependent mechanism. One obvious candidate is neuromelanin, which is an efficient proinflammogen in vivo and in vitro and has been shown to have a role in the pathogenesis of Parkinson's disease. Consequently, the goal of this study is to test whether synthetic neuromelanin activates microglia in a caspase-dependent manner. RESULTS: We found an in-vivo upregulation of CD16/32 (M1 marker) in Iba1-immunolabeled microglia in the ventral mesencephalon after neuromelanin injection. In vitro experiments using BV2 cells, a microglia-derived cell line, demonstrated that synthetic neuromelanin induced a significant chemotactic response to BV2 microglial cells, along with typical morphological features of microglia activation, increased oxidative stress and induction of pattern-recognition receptors including Toll-like receptor 2, NOD2, and CD14. Analysis of IETDase (caspase-8) and DEVDase (caspase-3/7) activities in BV2 cells demonstrated a modest but significant increase of both activities in response to neuromelanin treatment, in the absence of cell death. CONCLUSIONS: Caspase-8 inhibition prevented typical features of microglia activation, including morphological changes, a high rate of oxidative stress and expression of key proinflammatory cytokines and iNOS.

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.

Synergistic deleterious effect of chronic stress and sodium azide in the mouse hippocampus.

Alzheimer's disease is the most common cause of dementia in the elderly. Although the primary cause of the disease is presently unknown, to date several risk factors have been described. Evidence suggests that one of these risk factors could be chronic stress. The aim of this work is to demonstrate that chronic stress is able to induce Alzheimer's disease features after the administration of nontoxic doses of sodium azide. We found that chronic stress increases the levels of several proteins involved in Alzheimer's disease pathogenesis, such as presenilin 1, presenilin 2, and S100ß, besides inducing the aggregation of Tau, ubiquitin, and ß-amyloid proteins in the hippocampus. More important, our work shows a synergistic effect of stress and sodium azide treatment leading to significant neuronal death in the mouse hippocampus. Our results point out that chronic stress is a risk factor contributing to amplify and accelerate Alzheimer's disease features in the hippocampus.

Chronic stress enhances microglia activation and exacerbates death of nigral dopaminergic neurons under conditions of inflammation.

BACKGROUND: Parkinson's disease is an irreversible neurodegenerative disease linked to progressive movement disorders and is accompanied by an inflammatory reaction that is believed to contribute to its pathogenesis. Since sensitivity to inflammation is not the same in all brain structures, the aim of this work was to test whether physiological conditions as stress could enhance susceptibility to inflammation in the substantia nigra, where death of dopaminergic neurons takes place in Parkinson's disease. METHODS: To achieve our aim, we induced an inflammatory process in nonstressed and stressed rats (subject to a chronic variate stress) by a single intranigral injection of lipopolysaccharide, a potent proinflammogen. The effect of this treatment was evaluated on inflammatory markers as well as on neuronal and glial populations. RESULTS: Data showed a synergistic effect between inflammation and stress, thus resulting in higher microglial activation and expression of proinflammatory markers. More important, the higher inflammatory response seen in stressed animals was associated with a higher rate of death of dopaminergic neurons in the substantia nigra, the most characteristic feature seen in Parkinson's disease. This effect was dependent on glucocorticoids. CONCLUSIONS: Our data demonstrate that stress sensitises midbrain microglia to further inflammatory stimulus. This suggests that stress may be an important risk factor in the degenerative processes and symptoms of Parkinson's disease.

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.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

Galectin-3, a rising star in modulating microglia activation under conditions of neurodegeneration.

The advent of high-throughput single-cell transcriptomic analysis of microglia has revealed different phenotypes that are inherently associated with disease conditions. A common feature of some of these activated phenotypes is the upregulation of galectin-3. Representative examples of these phenotypes include disease-associated microglia (DAM) and white-associated microglia (WAM), whose role(s) in neuroprotection/neurotoxicity is a matter of high interest in the microglia community. In this review, we summarise the main findings that demonstrate the ability of galectin-3 to interact with key pattern recognition receptors, including, among others, TLR4 and TREM2 and the importance of galectin-3 in the regulation of microglia activation. Finally, we discuss increasing evidence supporting the involvement of this lectin in the main neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, and stroke.

The Other Side of SARS-CoV-2 Infection: Neurological Sequelae in Patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread around the globe causing coronavirus disease 2019 (COVID-19). Because it affects the respiratory system, common symptoms are cough and breathing difficulties with fever and fatigue. Also, some cases progress to acute respiratory distress syndrome (ARDS). The acute phase of COVID-19 has been also related to nervous system symptoms, including loss of taste and smell as well as encephalitis and cerebrovascular disorders. However, it remains unclear if neurological complications are due to the direct viral infection of the nervous system, or they appear as a consequence of the immune reaction against the virus in patients who presented pre-existing deficits or had a certain detrimental immune response. Importantly, the medium and long-term consequences of the infection by SARS-CoV-2 in the nervous system remain at present unknown. This review article aims to give an overview of the current neurological symptoms associated with COVID-19, as well as attempting to provide an insight beyond the acute affectation.

Selective deletion of Caspase-3 gene in the dopaminergic system exhibits autistic-like behaviour.

Apoptotic caspases are thought to play critical roles in elimination of excessive and non-functional synapses and removal of extra cells during early developmental stages. Hence, an impairment of this process may thus constitute a basis for numerous neurological and psychiatric diseases. This view is especially relevant for dopamine due to its pleiotropic roles in motor control, motivation and reward processing. Here, we have analysed the effect of caspase-3 depletion on the development of catecholaminergic neurons and performed a wide array of neurochemical, ultrastructural and behavioural assays. To achieve this, we performed selective deletion of the Casp3 gene in tyrosine hydroxylase (TH)-expressing cells using Cre-loxP-mediated recombination. Histological evaluation of most relevant catecholaminergic nuclei revealed the ventral mesencephalon as the most affected region. Stereological analysis demonstrated an increase in the number of TH-positive neurons in both the substantia nigra and ventral tegmental area along with enlarged volume of the ventral midbrain. Analysis of main innervating tissues revealed a rather contrasting profile. In striatum, basal extracellular levels and potassium-evoked DA release were significantly reduced in mice lacking Casp3, a clear indication of dopaminergic hypofunction in dopaminergic innervating tissues. This view was sustained by analysis of TH-labelled dopaminergic terminals by confocal and electron microscopy. Remarkably, at a behavioural level, Casp3-deficient mice exhibited impaired social interaction, restrictive interests and repetitive stereotypies, which are considered the core symptoms of autism spectrum disorder (ASD). Our study revitalizes the potential involvement of dopaminergic transmission in ASD and provides an excellent model to get further insights in ASD pathogenesis.

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.

Microglia: Agents of the CNS Pro-Inflammatory Response.

The pro-inflammatory immune response driven by microglia is a key contributor to the pathogenesis of several neurodegenerative diseases. Though the research of microglia spans over a century, the last two decades have increased our understanding exponentially. Here, we discuss the phenotypic transformation from homeostatic microglia towards reactive microglia, initiated by specific ligand binding to pattern recognition receptors including toll-like receptor-4 (TLR4) or triggering receptors expressed on myeloid cells-2 (TREM2), as well as pro-inflammatory signaling pathways triggered such as the caspase-mediated immune response. Additionally, new research disciplines such as epigenetics and immunometabolism have provided us with a more holistic view of how changes in DNA methylation, microRNAs, and the metabolome may influence the pro-inflammatory response. This review aimed to discuss our current knowledge of pro-inflammatory microglia from different angles, including recent research highlights such as the role of exosomes in spreading neuroinflammation and emerging techniques in microglia research including positron emission tomography (PET) scanning and the use of human microglia generated from induced pluripotent stem cells (iPSCs). Finally, we also discuss current thoughts on the impact of pro-inflammatory microglia in neurodegenerative diseases.

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.

Reformulating Pro-Oxidant Microglia in Neurodegeneration.

In neurodegenerative diseases, microglia-mediated neuroinflammation and oxidative stress are central events. Recent genome-wide transcriptomic analyses of microglial cells under different disease conditions have uncovered a new subpopulation named disease-associated microglia (DAM). These studies have challenged the classical view of the microglia polarization state's proinflammatory M1 (classical activation) and immunosuppressive M2 (alternative activation). Molecular signatures of DAM and proinflammatory microglia (highly pro-oxidant) have shown clear differences, yet a partial overlapping gene profile is evident between both phenotypes. The switch activation of homeostatic microglia into reactive microglia relies on the selective activation of key surface receptors involved in the maintenance of brain homeostasis (a.k.a. pattern recognition receptors, PRRs). Two relevant PRRs are toll-like receptors (TLRs) and triggering receptors expressed on myeloid cells-2 (TREM2), whose selective activation is believed to generate either a proinflammatory or a DAM phenotype, respectively. However, the recent identification of endogenous disease-related ligands, which bind to and activate both TLRs and TREM2, anticipates the existence of rather complex microglia responses. Examples of potential endogenous dual ligands include amyloid ß, galectin-3, and apolipoprotein E. These pleiotropic ligands induce a microglia polarization that is more complicated than initially expected, suggesting the possibility that different microglia subtypes may coexist. This review highlights the main microglia polarization states under disease conditions and their leading role orchestrating oxidative stress.

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.

Peripheral Inflammation Enhances Microglia Response and Nigral Dopaminergic Cell Death in an in vivo MPTP Model of Parkinson's Disease.

The impact of systemic inflammation in nigral dopaminergic cell loss remains unclear. Here, we have investigated the role of peripheral inflammation induced by systemic lipopolysaccharide (LPS) administration in the MPTP-based model of Parkinson's disease. Brain inflammation, microglia and astroglia activation, disruption of the blood-brain barrier (BBB) and integrity of the nigrostriatal dopaminergic system were evaluated in response to i.p. injection of LPS, MPTP or the combination of both. Our results showed that combinative treatment exacerbates microglia activation and enhances (i) the appearance of galectin-3-positive microglia, recently identified as microglial disease-associated phenotypic marker, (ii) the up-regulation of pro-inflammatory cytokines, (iii) the occurrence of A1 neurotoxic astrocytes, (iv) the breakdown of the BBB, and (v) the loss of dopaminergic neurons in the substantia nigra. Microglia activation was triggered earlier than other degenerative events, suggesting that over-activation of microglia (including different polarization states) may induce dopaminergic neuron loss by itself, initiating the endless cycle of inflammation/degeneration. Our study revitalizes the importance of peripheral inflammation as a potential risk factor for Parkinson's disease and raises the possibility of using new anti-inflammatory therapies to improve the course of neurodegenerative diseases, including those directly aimed at modulating the deleterious activity of disease-associated microglia.