Cerium oxide nanoparticles with antioxidant properties ameliorate strength and prolong life in mouse model of amyotrophic lateral sclerosis.

Cerium oxide nanoparticles (CeNPs) neutralize reactive oxygen and nitrogen species. Since oxidative stress plays a role in amyotrophic lateral sclerosis (ALS) in humans and in the SOD1G93A mouse model of ALS, we tested whether administration of CeNPs would improve survival and reduce disease severity in SOD1G93A transgenic mice. Twice a week intravenous treatment of SOD1G93A mice with CeNPs started at the onset of muscle weakness preserved muscle function and increased longevity in males and females. Median survival after the onset of CeNP treatment was 33.0±3.7days (N=20), and only 22.0±2.5days in mice treated with vehicle, control injections (N=27; P=0.022). Since these citrate-EDTA stabilized CeNPs exhibited catalase and oxidase activity in cell-free systems and in in vitro models of ischemic oxidative stress, we hypothesize that antioxidant activity is the protective mechanism prolonging survival in the SOD1G93A mice.

Heat exposure in female rats elicits abnormal fear expression and cellular changes in prefrontal cortex and hippocampus.

Despite a twofold higher prevalence of fear-related disorders in women, the neurobiological factors that modulate and drive fear expression are rarely studied in female animals. Fear conditioning and extinction are useful tools for dissecting these mechanisms, and here we tested the effects of environmental manipulations - four days of exposure to 31°C temperatures in the animal housing facility - on fear learning and memory exclusively in female rats. We found that heat exposure disrupted freezing to tone during fear conditioning, and elicited enhanced freezing during extinction and extinction retrieval. We also performed immunohistochemistry for c-fos expression in the infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex during extinction retrieval, and found that heat exposure induced a switch from IL-dominated activity to PL-dominated activity. Finally, morphological analysis of spines in hippocampal CA3 neurons revealed an increase in spine head diameter in heat-exposed animals, which may partly underlie the persistent freezing observed in these animals. Together, our data show that heat exposure can induce changes at behavioral, physiological, and structural levels, and add to a woefully lacking body of literature on fear processes in female animals.

A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes.

While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.

Antioxidant Enzyme-Mimetic Activity and Neuroprotective Effects of Cerium Oxide Nanoparticles Stabilized with Various Ratios of Citric Acid and EDTA.

Cerium oxide (CeO2) nanoparticles (CeNPs) are potent antioxidants that are being explored as potential therapies for diseases in which oxidative stress plays an important pathological role. However, both beneficial and toxic effects of CeNPs have been reported, and the method of synthesis as well as physico-chemical, biological, and environmental factors can impact the ultimate biological effects of CeNPs. In the present study, we explored the effect of different ratios of citric acid (CA) and EDTA (CA/EDTA), which are used as stabilizers during synthesis of CeNPs, on the antioxidant enzyme-mimetic and biological activity of the CeNPs. We separated the CeNPs into supernatant and pellet fractions and used commercially available enzymatic assays to measure the catalase-, superoxide dismutase (SOD)-, and oxidase-mimetic activity of each fraction. We tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia to induce oxidative stress where the fluorescence indicator SYTOX green was used to assess cell death. Our results demonstrate that CeNPs stabilized with various ratios of CA/EDTA display different enzyme-mimetic activities. CeNPs with intermediate CA/EDTA stabilization ratios demonstrated greater neuroprotection in ischemic mouse brain slices, and the neuroprotective activity resides in the pellet fraction of the CeNPs. The neuroprotective effects of CeNPs stabilized with equal proportions of CA/EDTA (50/50) were also demonstrated in two other models of ischemia/reperfusion in mice and rats. Thus, CeNPs merit further development as a neuroprotective therapy for use in diseases associated with oxidative stress in the nervous system.

Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action.

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.

A Photoaffinity Labeling-Based Chemoproteomics Strategy for Unbiased Target Deconvolution of Small Molecule Drug Candidates.

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.

Dopamine D1 receptor activation rescues extinction impairments in low-estrogen female rats and induces cortical layer-specific activation changes in prefrontal-amygdala circuits.

Post-traumatic stress disorder (PTSD) is twice as common in women as in men; it is a major public health problem whose neurobiological basis is unknown. In preclinical studies using fear conditioning and extinction paradigms, women and female animals with low estrogen levels exhibit impaired extinction retrieval, but the mechanisms that underlie these hormone-based discrepancies have not been identified. There is much evidence that estrogen can modulate dopaminergic transmission, and here we tested the hypothesis that dopamine-estrogen interactions drive extinction processes in females. Intact male and female rats were trained on cued fear conditioning, and received an intraperitoneal injection of a D1 agonist or vehicle before extinction learning. As reported previously, females that underwent extinction during low estrogen estrous phases (estrus/metaestrus/diestrus (EMD)) froze more during extinction retrieval than those that had been in the high-estrogen phase (proestrus; PRO). However, D1 stimulation reversed this relationship, impairing extinction retrieval in PRO and enhancing it in EMD. We also combined retrograde tracing and fluorescent immunohistochemistry to measure c-fos expression in infralimbic (IL) projections to the basolateral area of the amygdala (BLA), a neural pathway known to be critical to extinction retrieval. Again we observed diverging, estrous-dependent effects; SKF treatment induced a positive correlation between freezing and IL-BLA circuit activation in EMD animals, and a negative correlation in PRO animals. These results show for the first time that hormone-dependent extinction deficits can be overcome with non-hormone-based interventions, and suggest a circuit-specific mechanism by which these behavioral effects occur.

Stress-induced cognitive dysfunction: hormone-neurotransmitter interactions in the prefrontal cortex.

The mechanisms and neural circuits that drive emotion and cognition are inextricably linked. Activation of the hypothalamic-pituitary-adrenal (HPA) axis as a result of stress or other causes of arousal initiates a flood of hormone and neurotransmitter release throughout the brain, affecting the way we think, decide, and behave. This review will focus on factors that influence the function of the prefrontal cortex (PFC), a brain region that governs higher-level cognitive processes and executive function. The PFC becomes markedly impaired by stress, producing measurable deficits in working memory. These deficits arise from the interaction of multiple neuromodulators, including glucocorticoids, catecholamines, and gonadal hormones; here we will discuss the non-human primate and rodent literature that has furthered our understanding of the circuitry, receptors, and signaling cascades responsible for stress-induced prefrontal dysfunction.