Proteome integral solubility alteration high-throughput proteomics assay identifies Collectin-12 as a non-apoptotic microglial caspase-3 substrate.

Caspases are a family of proteins mostly known for their role in the activation of the apoptotic pathway leading to cell death. In the last decade, caspases have been found to fulfill other tasks regulating the cell phenotype independently to cell death. Microglia are the immune cells of the brain responsible for the maintenance of physiological brain functions but can also be involved in disease progression when overactivated. We have previously described non-apoptotic roles of caspase-3 (CASP3) in the regulation of the inflammatory phenotype of microglial cells or pro-tumoral activation in the context of brain tumors. CASP3 can regulate protein functions by cleavage of their target and therefore could have multiple substrates. So far, identification of CASP3 substrates has been performed mostly in apoptotic conditions where CASP3 activity is highly upregulated and these approaches do not have the capacity to uncover CASP3 substrates at the physiological level. In our study, we aim at discovering novel substrates of CASP3 involved in the normal regulation of the cell. We used an unconventional approach by chemically reducing the basal level CASP3-like activity (by DEVD-fmk treatment) coupled to a Mass Spectrometry screen (PISA) to identify proteins with different soluble amounts, and consequently, non-cleaved proteins in microglia cells. PISA assay identified several proteins with significant change in their solubility after DEVD-fmk treatment, including a few already known CASP3 substrates which validated our approach. Among them, we focused on the Collectin-12 (COLEC12 or CL-P1) transmembrane receptor and uncovered a potential role for CASP3 cleavage of COLEC12 in the regulation of the phagocytic capacity of microglial cells. Taken together, these findings suggest a new way to uncover non-apoptotic substrates of CASP3 important for the modulation of microglia cell physiology.

Semisynthetic quercetin-quinone mitigates BV-2 microglia activation through modulation of Nrf2 pathway.

During brain ageing, microglia, the resident immune cells of the CNS, are immunologically activated and contribute to neuroinflammation, a vicious cycle that supports development of neurological disorders. Therapeutic approaches focus mainly on downregulation of their pro-inflammatory activated state that is associated with health benefits. Electrophilic compounds, such as natural quinones and their reduced pro-electrophilic precursors, flavonoids, represent a wide group of diverse substances with important biological effects. They can cause considerable cytotoxicity when used at higher dosages, but on the other hand, they have versatile health benefits at lower dosages. In this study, we investigated the cytotoxicity and prooxidant profile of synthetic conjugate of two electrophilic compounds, quercetin and 1,4-naphthoquinone, 4'-O-(2-chloro-1,4-naphthoquinone-3-yloxy) quercetin (CHNQ), and its attenuation of inflammatory responses and modulation of Nrf2 pathway in BV-2 microglial cells. CHNQ showed higher cytotoxicity than its precursors, accompanied by promotion of production of reactive oxygen species along with G2/M cell cycle arrest at higher concentrations tested. Nevertheless, at a lower non-toxic concentration, CHNQ, more significantly than did its precursors, downregulated LPS-stimulated microglia cells as documented by decreased iNOS, COX-2 and TNFα protein levels. Moreover, CHNQ most effectively upregulated expression of phase II antioxidant enzyme HO-1 and ß5 subunit of constitutive proteasome. The enhanced anti-inflammatory effect of CHNQ was accompanied by prominent increase in cytosolic expression of Nrf2 and c-Jun, however, induction effect on nuclear Nrf2 translocation was comparable to QUER. Moreover, a conditioned medium from activated BV-2 cells co-treated with quercetin and CHNQ maintained viability of neuron-like PC12 cells. The compounds tested did not show any disturbance of phagocytosis of live or dead PC12 cells. The present experimental data predict a preventive and therapeutic potential of semisynthetic derivative CHNQ in ageing and related pathologies, mediated by activation of proteins of the antioxidant response.

Protective Effect of Semisynthetic and Natural Flavonoid on Aged Rat Microglia-enriched Cultures.

The ROS-mediated lysosomal dysfunction and coinciding deterioration of mitochondrial function are thought to be the prominent mechanisms responsible for aging. Microglia, the resident macrophages in the central nervous system, were postulated to belong to the major targets vulnerable to these detrimental processes, acting as principal drivers in brain aging. The present study investigated the potential protective effect of the semisynthetic flavonoid 3'-O-(3-chloropivaloyl) quercetin (CPQ) and quercetin (Q) on microglia-enriched mixed brain cultures (MBCs) established from aged Wistar rats. Both flavonoids tested suppressed the development of lipofuscin-related autofluorescence in aged cells. Further ensuing protective effects included reduction of protein oxidation markers in aged cells. Moreover, unlike Q, CPQ significantly suppressed sensitivity of aged cells to stimulation of superoxide burst. Other activation markers, cellular hypertrophy and isolectin B4 binding, were also downregulated by treatment with both CPQ and Q. In conclusion, results of our study suggest that both flavonoids tested may protect microglia with a quite comparable efficacy against aging-related accumulated alterations. The protective mechanism can include interference with the ROS-mediated vicious cycles involving lysosomal dysfunction. Nevertheless, the lipophilized quercetin, CPQ, a compound with proposed enhanced biological availability compared to parent molecule, can represent an agent potentially useful for new effective pharmaceutical intervention against brain aging, overcoming the limitations of clinical applicability of quercetin.

Pyridoindole SMe1EC2 as cognition enhancer in ageing-related cognitive decline.

Synthetic pyridoindole-type substances derived from the lead compound stobadine represent promising agents in treatment of a range of pathologies including neurological disorders. The beneficial biological effects were suggested to be likely associated with their capacity to ameliorate oxidative damage. In our study, the effect of supplementation with the derivative of stobadine, SMe1EC2, on ageing-related cognitive decline in rats was investigated. The 20-months-old male Wistar rats were administered SMe1EC2 at a low dose, 0.5 mg/kg, daily during eight weeks. Morris water maze test was performed to assess the spatial memory performances. The cell-based assays of capacity of SMe1EC2 to modulate proinflammatory generation of oxidants by microglia were also performed. The rats treated with SMe1EC2 showed significantly increased path efficiency, significantly shorter time interval of successful trials and exerted also notably lower frequencies of clockwise rotations in the pool compared to non-supplemented aged animals. Mildly improved parameters included test durations, distances to reach the platform, time in periphery of the pool and overall rotations in the water maze. However, the pyridoindole SMe1EC2 did not show profound inhibitory effect on production of nitric oxide and superoxide by activated microglial cells. In conclusion, our study suggests that pyridoindole SMe1EC2, at low doses administered chronically, can act as cognition enhancing agent in aged rats. The protective mechanism less likely involves direct modulation of proinflammatory and prooxidant state of microglia, the prominent mediators of neurotoxicity in brain ageing and neurodegeneration.

Modulation of BV-2 microglia functions by novel quercetin pivaloyl ester.

Chronic inflammation in brain plays a critical role in major neurodegenerative diseases such as Alzheimer's, Parkinson's disease, stroke or multiple sclerosis. Microglia, resident macrophages and intristinc components of CNS, appear to be main effectors in this pathological process. Quercetin, a naturally occurring flavonoid, was proven to downregulate inflammatory genes in microglia. Synthetically modified quercetin, 3'-O-(3-chloropivaloyl) quercetin (CPQ), is assumed to possess better biological availability and enhanced antioxidant properties. In the present study, antineuroinflammatory capability of the novel compound CPQ was assessed in BV-2 microglial cells. Our data show that treatment with CPQ attenuated the production of the inflammatory mediators, nitric oxide (NO) and tumour necrosis factor-α (TNF-α), in LPS-stimulated microglia somewhat more efficiently than did quercetin (p > 0.05 for CPQ vs. quercetin-treated group). Also, protein level of inducible NO synthase (iNOS) in LPS-activated BV-2 microglia was to some extent more effectively supressed by CPQ than by unmodified flavonoid. In consistence with the extent of their effects on pro-inflammatory markers, CPQ and quercetin showed down-regulation of NFκB activation. This quercetin analogue caused also a decline in BV-2 microglia proliferation with interfering with cell cycle progression (p < 0.001 for CPQ vs. quercetin-treated group). However, CPQ did not remarkably affect cell viability. In addition, CPQ showed a minor better suppression of PMA-induced generation of superoxide than did quercetin. Neither CPQ nor quercetin influenced phagocytosis of BV-2 cells. These results point to the therapeutic potential of 3'-O-(3-chloropivaloyl)quercetin (CPQ) as a novel antiinflammatory drug in neurodegenerative diseases, mediating favourable modulation of pro-inflammatory functions of microglia.