Miguel Angel Burguillos

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.

Microchannel Acoustophoresis does not Impact Survival or Function of Microglia, Leukocytes or Tumor Cells

Background The use of acoustic forces to manipulate particles or cells at the microfluidic scale (i.e. acoustophoresis), enables non-contact, label-free separation based on intrinsic cell properties such as size, density and compressibility. Acoustophoresis holds great promise as a cell separation technique in several research and clinical areas. However, it has been suggested that the force acting upon cells undergoing acoustophoresis may impact cell viability, proliferation or cell function via subtle phenotypic changes. If this were the case, it would suggest that the acoustophoresis method would be a less useful tool for many cell analysis applications as well as for cell therapy. Methods We investigate, for the first time, several key aspects of cellular changes following acoustophoretic processing. We used two settings of ultrasonic actuation, one that is used for cell sorting (10 Vpp operating voltage) and one that is close to the maximum of what the system can generate (20 Vpp). We used microglial cells and assessed cell viability and proliferation, as well as the inflammatory response that is indicative of more subtle changes in cellular phenotype. Furthermore, we adapted a similar methodology to monitor the response of human prostate cancer cells to acoustophoretic processing. Lastly, we analyzed the respiratory properties of human leukocytes and thrombocytes to explore if acoustophoretic processing has adverse effects. Results BV2 microglia were unaltered after acoustophoretic processing as measured by apoptosis and cell turnover assays as well as inflammatory cytokine response up to 48 h following acoustophoresis. Similarly, we found that acoustophoretic processing neither affected the cell viability of prostate cancer cells nor altered their prostate-specific antigen secretion following androgen receptor activation. Finally, human thrombocytes and leukocytes displayed unaltered mitochondrial respiratory function and integrity after acoustophoretic processing. Conclusion We conclude that microchannel acoustophoresis can be used for effective continuous flow-based cell separation without affecting cell viability, proliferation, mitochondrial respiration or inflammatory status.

Apoptosis-inducing factor mediates dopaminergic cell death in response to LPS-induced inflammatory stimulus: evidence in Parkinson's disease patients.

We show that intranigral lipopolysaccharide (LPS) injection, which provokes specific degeneration of DA neurons, induced caspase-3 activation in the rat ventral mesencephalon, which was mostly associated with glial cells. In contrast, nigral DA neurons exhibited AIF nuclear translocation in response to LPS. A significant decrease of the Bcl-2/Bax ratio in nigral tissue after LPS injection was observed. We next developed an in vitro co-culture system with the microglial BV2 and the DA neuronal MN9D murine cell lines. The silencing of caspase-3 or AIF by small interfering RNAs exclusively in the DA MN9D cells demonstrated the key role of AIF in the LPS-induced death of DA cells. In vivo chemical inhibition of caspases and poly(ADP-ribose)polymerase-1, an upstream regulator of AIF release and calpain, proved the central role of the AIF-dependent pathway in LPS-induced nigral DA cell death. We also observed nuclear translocation of AIF in the ventral mesencephalon of Parkinsons disease subjects.

The executioners sing a new song: killer caspases activate microglia

Activation of microglia and inflammation-mediated neurotoxicity are suggested to have key roles in the pathogenesis of several neurodegenerative disorders. We recently published an article in Nature revealing an unexpected role for executioner caspases in the microglia activation process. We showed that caspases 8 and 3/7, commonly known to have executioner roles for apoptosis, can promote microglia activation in the absence of death. We found these caspases to be activated in microglia of PD and AD subjects. Inhibition of this signaling pathway hindered microglia activation and importantly reduced neurotoxicity in cell and animal models of disease. Here we review evidence suggesting that microglia can have a key role in the pathology of neurodegenerative disorders. We discuss possible underlying mechanisms regulating their activation and neurotoxic effect. We focus on the provocative hypothesis that caspase inhibition can be neuroprotective by targeting the microglia rather than the neurons themselves.

The role of Galectin-3 in α-synuclein-induced microglial activation

BackgroundParkinsons disease (PD) is the most prevalent neurodegenerative motor disorder. The neuropathology is characterized by intraneuronal protein aggregates of α-synuclein and progressive degeneration of dopaminergic neurons within the substantia nigra. Previous studies have shown that extracellular α-synuclein aggregates can activate microglial cells, induce inflammation and contribute to the neurodegenerative process in PD. However, the signaling pathways involved in α-synuclein-mediated microglia activation are poorly understood. Galectin-3 is a member of a carbohydrate-binding protein family involved in cell activation and inflammation. Therefore, we investigated whether galectin-3 is involved in the microglia activation triggered by α-synuclein.ResultsWe cultured microglial (BV2) cells and induced cell activation by addition of exogenous α-synuclein monomers or aggregates to the cell culture medium. This treatment induced a significant increase in the levels of proinflammatory mediators including the inducible Nitric Oxide Synthase (iNOS), interleukin 1 Beta (IL-1β) and Interleukin-12 (IL-12). We then reduced the levels of galectin-3 expression using siRNA or pharmacologically targeting galectin-3 activity using bis-(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)-sulfane. Both approaches led to a significant reduction in the observed inflammatory response induced by α-synuclein. We confirmed these findings using primary microglial cells obtained from wild-type and galectin-3 null mutant mice. Finally, we performed injections of α-synuclein in the olfactory bulb of wild type mice and observed that some of the α-synuclein was taken up by activated microglia that were immunopositive for galectin-3.ConclusionsWe show that α-synuclein aggregates induce microglial activation and demonstrate for the first time that galectin-3 plays a significant role in microglia activation induced by α-synuclein. These results suggest that genetic down-regulation or pharmacological inhibition of galectin-3 might constitute a novel therapeutic target in PD and other synucleinopathies.

Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats

A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial- and muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi.

Regulation of caspase-3 processing by cIAP2 controls the switch between pro-inflammatory activation and cell death in microglia

The activation of microglia, resident immune cells of the central nervous system, and inflammation-mediated neurotoxicity are typical features of neurodegenerative diseases, for example, Alzheimers and Parkinsons diseases. An unexpected role of caspase-3, commonly known to have executioner role for apoptosis, was uncovered in the microglia activation process. A central question emerging from this finding is what prevents caspase-3 during the microglia activation from killing those cells? Caspase-3 activation occurs as a two-step process, where the zymogen is first cleaved by upstream caspases, such as caspase-8, to form intermediate, yet still active, p19/p12 complex; thereafter, autocatalytic processing generates the fully mature p17/p12 form of the enzyme. Here, we show that the induction of cellular inhibitor of apoptosis protein 2 (cIAP2) expression upon microglia activation prevents the conversion of caspase-3 p19 subunit to p17 subunit and is responsible for restraining caspase-3 in terms of activity and subcellular localization. We demonstrate that counteracting the repressive effect of cIAP2 on caspase-3 activation, using small interfering RNA targeting cIAP2 or a SMAC mimetic such as the BV6 compound, reduced the pro-inflammatory activation of microglia cells and promoted their death. We propose that the different caspase-3 functions in microglia, and potentially other cell types, reside in the active caspase-3 complexes formed. These results also could indicate cIAP2 as a possible therapeutic target to modulate microglia pro-inflammatory activation and associated neurotoxicity observed in neurodegenerative disorders.

Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype

Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.

Preservation of aortic root architecture and properties using a detergent-enzymatic perfusion protocol.

Aortic valve degeneration and dysfunction is one of the leading causes for morbidity and mortality. The conventional heart-valve prostheses have significant limitations with either life-long anticoagulation therapeutic associated bleeding complications (mechanical valves) or limited durability (biological valves). Tissue engineered valve replacement recently showed encouraging results, but the unpredictable outcome of tissue degeneration is likely associated to the extensive tissue processing methods. We believe that optimized decellularization procedures may provide aortic valve/root grafts improved durability. We present an improved/innovative decellularization approach using a detergent-enzymatic perfusion method, which is both quicker and has less exposure of matrix degenerating detergents, compared to previous protocols. The obtained graft was characterized for its architecture, extracellular matrix proteins, mechanical and immunological properties. We further analyzed the engineered aortic root for biocompatibility by cell adhesion and viability in vitro and heterotopic implantation in vivo. The developed decellularization protocol was substantially reduced in processing time whilst maintaining tissue integrity. Furthermore, the decellularized aortic root remained bioactive without eliciting any adverse immunological reaction. Cell adhesion and viability demonstrated the scaffolds biocompatibility. Our optimized decellularization protocol may be useful to develop the next generation of clinical valve prosthesis with a focus on improved mechanical properties and durability.

Galectin-3 released in response to traumatic brain injury acts as an alarmin orchestrating brain immune response and promoting neurodegeneration

Traumatic brain injury (TBI) is currently a major cause of morbidity and poor quality of life in Western society, with an estimate of 2.5 million people affected per year in Europe, indicating the need for advances in TBI treatment. Within the first 24 h after TBI, several inflammatory response factors become upregulated, including the lectin galectin-3. In this study, using a controlled cortical impact (CCI) model of head injury, we show a large increase in the expression of galectin-3 in microglia and also an increase in the released form of galectin-3 in the cerebrospinal fluid (CSF) 24 h after head injury. We report that galectin-3 can bind to TLR-4, and that administration of a neutralizing antibody against galectin-3 decreases the expression of IL-1β, IL-6, TNFα and NOS2 and promotes neuroprotection in the cortical and hippocampal cell populations after head injury. Long-term analysis demonstrated a significant neuroprotection in the cortical region in the galectin-3 knockout animals in response to TBI. These results suggest that following head trauma, released galectin-3 may act as an alarmin, binding, among other proteins, to TLR-4 and promoting inflammation and neuronal loss. Taking all together, galectin-3 emerges as a clinically relevant target for TBI therapy.