A vitamin B12 deficiency (vit. B12 def.) is common in the elderly, because of changes in metabolism. Clinical studies have reported that a vit. B12 def. results in worse outcome after stroke, and the mechanisms through which a vit. B12 def. changes the brain requires further investigation. This study investigated the role of vit. B12 def. on stroke outcome and mechanisms using aged female mice. Eighteen-month-old females were put on a control or vit. B12 def. diet for 4 weeks, after which an ischemic stroke was induced in the sensorimotor cortex. After damage, motor function was measured, the animals were euthanized, and tissues were collected for analysis. Vit. B12 def. animals had increased levels of total homocysteine in plasma and liver, and choline levels were also increased in the liver. Vit. B12 def. animals had larger damage volume in brain tissue and more apoptosis. The cecum tissue pathway analysis showed dysfunction in B12 transport. The analysis of mitochondrial metabolomics in brain tissue showed reduced levels of metabolites involved in the TCA cycle in vit. B12 def. animals. Motor function after stroke was impaired in vit. B12 def. animals. A dietary vit. B12 def. impairs motor function through increased apoptosis and changes in mitochondrial metabolism in brain tissue.

The coordination of zinc by histone deacetylase inhibitors (HDACi), altering the bioavailability of zinc to histone deacetylases (HDACs), is key to HDAC enzyme inhibition. However, the ability of zinc binding groups (ZBGs) to alter intracellular free Zn⁺² levels, which may have far-reaching effects, has not been explored. Using two HDACis with different ZBGs, we documented shifts in intracellular free Zn⁺² concentrations that correlate with subsequent ROS production. Next, we assayed refolding and reactivation of the R175H mutant p53 protein in vitro to provide greater biological context as the activity of this mutant depends on cellular zinc concentration. The data presented demonstrates the differential activity of HDACi in promoting R175H response element (RE) binding. After cells are treated with HDACi, there are differences in R175H mutant p53 refolding and reactivation, which may be related to treatments. Collectively, we show that HDACis with distinct ZBGs differentially impact the intracellular free Zn⁺² concentration, ROS levels, and activity of R175H; therefore, HDACis may have significant activity independent of their ability to alter acetylation levels. Our results suggest a framework for reevaluating the role of zinc in the variable or off-target effects of HDACi, suggesting that the ZBGs of HDAC inhibitors may provide bioavailable zinc without the toxicity associated with zinc metallochaperones such as ZMC1.

Collective cell migration (CCM) is a coordinated process involving cell–cell and cell–environment interactions occurring in many physiological systems, including development, wound healing, and metastasis. Using zebrafish keratocytes as a wound healing model provides a unique system to investigate the interplay of matrix metalloproteinases (MMPs) in CCM. MMPs play an important role in CCM as they generate bioactive molecules that regulate proliferation, differentiation, angiogenesis, apoptosis, and cell migration. Secreted as pro-enzymes, MMPs must be activated, frequently by another MMP. As a group, MMPs have been reported to have a pro-migratory role during CCM, yet our data reveal that one MMP, MMP13, is not pro-migratory. Treatment of keratocytes with recombinant MMP13 resulted in a dose-dependent decrease in migration, reduced MMP13 activity, and increased MMP9 mRNA expression. Treatment with an MMP13-specific inhibitor resulted in a dose-dependent increase in migration with no change in the rate of cellular proliferation, an increase in total MMP activity, and increased MMP2 mRNA expression. Similarly, inhibition of MMP14 also resulted in a significant, dose-dependent decrease in migration. However, MMP14 inhibition resulted in both an increase in MMP2 mRNA expression and a decrease in MMP9 mRNA expression. The increase in MMP2 and/or MMP9 activity was observed on gel zymography for both treatments. Our data support the hypothesis that MMP13 is anti-migratory while MMP2, MMP9 and MMP14 have a pro-migratory effect on zebrafish keratocytes. Taken together, our results outline a novel inhibitory role for MMP regulation of CCM that has implications for many other processes in multicellular organisms.