Biology subjects

Casas, A. I.

Publications and source records attributed to Casas, A. I..

5 recordsLinked to original sources

bioRxiv2020

Neuronal NOX4 knockdown alleviates pathological tau-related alterations in a humanized mouse model of tauopathy

Approximately 44 million people worldwide live with Alzheimers disease (AD) or a related form of dementia. Aggregates of the microtubule-associated protein tau are a common marker of these neurodegenerative diseases collectively termed as tauopathies. However, all therapeutic attempts based on tau have failed, suggesting that tau may only indicate a higher-level causal mechanism. For example, increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation. Here we show that type 4 NADPH oxidase (NOX), the most abundant isoform of the only dedicated reactive oxygen producing enzyme family, is upregulated in dementia and AD patients and in a humanized mouse model of tauopathy. Both global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP). Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevented cognitive decline, suggesting a direct and causal role for neuronal NOX4. Thus, NOX4 is a previously unrecognized causal, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/338954v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1dac496org.highwire.dtl.DTLVardef@1a07dd5org.highwire.dtl.DTLVardef@1a5653aorg.highwire.dtl.DTLVardef@198f3e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Time-dependent dual effect of NLRP3 inflammasome in brain ischemia

Background and purposePost-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1{beta} and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit. MethodsWT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. ResultsWe identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1{beta}, TNF-), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. ConclusionsThese findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

neuroscience↗

bioRxiv2018

Isoform-specific NADPH oxidase inhibition for pharmacological target validation

Unphysiological reactive oxygen species (ROS) formation is considered an important pathomechanism for several disease phenotypes with high unmet medical need. Therapeutically, antioxidants have failed multiple times. Instead, focusing on only disease-relevant, enzymatic sources of ROS appears to be a more promising and highly validated approach. Here the family of five NADPH oxidases (NOX) stands out as drug targets. Validation has been restricted, however, mainly to genetically modified rodents and is lacking in other species including human. It is thus unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five of the most advanced NOX inhibitors that indeed isoform selectivity can be achieved. NOX1 was most potently (IC50) targeted by ML171 (0.1 M); NOX2, by VAS2870 (0.7 M); NOX4, by M13 (0.01 M) and NOX5, by ML090 (0.01 M). Conditions need to be carefully controlled though as previously unrecognized non-specific antioxidant and assay artefacts may limit the interpretation of data and this included, surprisingly, one of the most advanced NOX inhibitors, GKT136901. As proof-of-principle that now also pharmacological and non-rodent target validation of different NOX isoforms is possible, we used a human blood-brain barrier model and NOX inhibitor panel at IC50 concentrations. The protective efficacy pattern of this panel confirmed the predominant role of NOX4 in stroke from previous genetic models. Our findings strongly encourage further lead optimization efforts for isoform-selective NOX inhibitors and clinical development and provide an experimental alternative when genetic validation of a NOX isoform is not an option.\n\nO_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC=\"FIGDIR/small/382226v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@1708996org.highwire.dtl.DTLVardef@15909daorg.highwire.dtl.DTLVardef@960159org.highwire.dtl.DTLVardef@328083_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

pharmacology and toxicology↗

bioRxiv2018

Calcium-dependent reactive oxygen formation and blood-brain barrier breakdown by NOX5 limits post-reperfusion outcome in stroke

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic options. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain-barrier. Its mechanism, however, is unknown. Here we identify type 5 NADPH oxidase (NOX5), a calcium-activated, reactive oxygen species (ROS)-forming enzyme as missing link. Using a humanised knock-in mouse model and in vitro in organotypic cultures, we find re-oxygenation or calcium overload to increase brain ROS levels in a NOX5-dependent manner. In vivo, post-ischemic ROS formation, infarct volume and functional outcomes were worsened in NOX5 knock-in mice. Of clinical and therapeutic relevance, in a human blood-barrier model pharmacological NOX inhibition also prevented acute re-oxygenation induced leakage. Our data therefore identify NOX5 as sufficient to induce acute post-reperfusion calcium-dependent blood-brain-barrier breakdown. We suggest urgent clinical validation by conducting protective post-stroke re-canalisation in the presence of a NOX inhibitor.

neuroscience↗

bioRxiv2018

Protein nitration is a physiological regulator of cardiac lactate dehydrogenase active site loop mobility and activity

Protein tyrosine nitration is a hallmark of oxidative stress related disease states, commonly detected as anti-nitrotyrosine immunoreactivity. The precise reactive oxygen sources, mechanisms of nitration as well as the modified target proteins and functional consequences, however, remain often unclear. Here we explore protein tyrosine nitration under basal conditions and find surprisingly physiologically nitrated proteins. Upon purifying a prominent physiologically nitrotyrosine immunopositive in hearts from mouse, rat and pig, we identify it as lactate dehydrogenase (LDH). Mechanistically, LDHs degree of basal nitration depended on two canonical sources, NO synthase (NOS) and myeloperoxidase (MPO), respectively. When validating the nitrated amino acid by MALDI-TOF mass spectrometry, we, surprisingly, located LDH nitration not to a tyrosine but the C-terminal tryptophan, Trp324. Molecular dynamics simulations suggested that Trp324 nitration restricts the interaction of the active site loop with the C-terminal -helix essential for activity. This prediction was confirmed by enzyme kinetics revealing an apparent lower Vmax of nitrated LDH, although yet unidentified concurrent oxidative modifications may contribute. Protein nitration is, thus, not a by definition disease marker but reflects also physiological signaling by eNOS/NO, MPO/nitrite and possibly other pathways. The commonly used assay of anti-nitrotyrosine immunoreactivity is apparently cross-reactive to nitrotryptophan requiring a reevaluation of the protein nitration literature. In the case of LDH, nitration of Trp324 is aggravated under cardiac metabolic stress conditions and functionally limits maximal enzyme activity. Trp324-nitrated LDH may serve both as a previously not recognized disease biomarker and possibly mechanistic lead to understand the metabolic changes under these conditions.

biochemistry↗