Browsing by Author "Lackey, Kimberly H."

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    Alpha-tocotrienol enhances arborization of primary hippocampal neurons via upregulation of Bcl-xL
    (Pergamon, 2022) Park, Han-A; Crowe-White, Kristi M.; Ciesla, Lukasz; Scott, Madison; Bannerman, Sydni; Davis, Abigail U.; Adhikari, Bishnu; Burnett, Garrett; Broman, Katheryn; Ferdous, Khondoker Adeba; Lackey, Kimberly H.; Licznerski, Pawel; Jonas, Elizabeth A.; University of Alabama Tuscaloosa; Yale University
    Alpha-tocotrienol ( a-TCT) is a member of the vitamin E family. It has been reported to protect the brain against various pathologies including cerebral ischemia and neurodegeneration. However, it is still unclear if a-TCT exhibits beneficial effects during brain development. We hypothesized that treatment with a-TCT improves intracellular redox homeostasis supporting normal development of neurons. We found that primary hippocampal neurons isolated from rat feti grown in a-TCT-containing media achieved greater levels of neurite complexity compared to ethanol-treated control neurons. Neurons were treated with 1 mu M aTCT for 3 weeks, and media were replaced with fresh a-TCT every week. Treatment with a-TCT increased a-TCT levels (26 pmol/mg protein) in the cells, whereas the control neurons did not contain a-TCT. a-TCT-treated neurons produced adenosine triphosphate (ATP) at a higher rate and increased ATP retention at neurites, supporting formation of neurite branches. Although treatment with a-TCT alone did not change neuronal viability, neurons grown in a-TCT were more resistant to death at maturity. We further found that messenger RNA and protein levels of B-cell lymphoma-extra large (Bcl-xL) are increased by a-TCT treatment without inducing posttranslational cleavage of Bcl-xL. Bcl-xL is known to enhance mitochondrial energy production, which improves neuronal function including neurite out-growth and neurotransmission. Therefore alpha-TCT-mediated Bcl-xL upregulation may be the central mechanism of neuroprotection seen in the alpha-TCT-treated group. In summary, treat-ment with alpha-TCT upregulates Bcl-xL and increases ATP levels at neurites. This correlates with increased neurite branching during development and with protection of mature neu-rons against oxidative stress. (C) 2022 Elsevier Inc. All rights reserved.
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    Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons
    (MDPI, 2020) Park, Han-A; Mnatsakanyan, Nelli; Broman, Katheryn; Davis, Abigail U.; May, Jordan; Licznerski, Pawel; Crowe-White, Kristi M.; Lackey, Kimberly H.; Jonas, Elizabeth A.; University of Alabama Tuscaloosa; Yale University
    B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to N-Bcl-xL, and accumulation of N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of N-Bcl-xL. We further proposed that the application of an antioxidant with neuroprotective properties such as alpha-tocotrienol (TCT) will prevent N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with alpha-TCT, glutamate, or a combination of both. Glutamate challenge significantly increased cytosolic and mitochondrial ROS and N-Bcl-xL levels. N-Bcl-xL accumulation was accompanied by intracellular ATP depletion, loss of mitochondrial membrane potential, and cell death. alpha-TCT prevented loss of mitochondrial membrane potential in hippocampal neurons overexpressing N-Bcl-xL, suggesting that N-Bcl-xL caused the loss of mitochondrial function under excitotoxic conditions. Our data suggest that production of ROS is an important cause of N-Bcl-xL formation and that preventing ROS production may be an effective strategy to prevent N-Bcl-xL-mediated mitochondrial dysfunction and thus promote neuronal survival.
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    Bcl-xL Is Required by Primary Hippocampal Neurons during Development to Support Local Energy Metabolism at Neurites
    (MDPI, 2021) Jansen, Joseph; Scott, Madison; Amjad, Emma; Stumpf, Allison; Lackey, Kimberly H.; Caldwell, Kim A.; Park, Han-A; University of Alabama Tuscaloosa
    Simple Summary B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic protein that regulates energy metabolism in neurons. In this study, we found that primary hippocampal neurons transduced with Bcl-xL shRNA or treated with a pharmacological inhibitor of Bxl-xL had a decrease in the population of motile mitochondria. Primary hippocampal neurons lacking Bcl-xL failed to retain ATP at their neurites, which hindered the formation of complex neurite arbors, and ultimately had enhanced vulnerability to excitotoxic challenge. B-cell lymphoma-extra large (Bcl-xL) is a mitochondrial protein known to inhibit mitochondria-dependent intrinsic apoptotic pathways. An increasing number of studies have demonstrated that Bcl-xL is critical in regulating neuronal energy metabolism and has a protective role in pathologies associated with an energy deficit. However, it is less known how Bcl-xL regulates physiological processes of the brain. In this study, we hypothesize that Bcl-xL is required for neurite branching and maturation during neuronal development by improving local energy metabolism. We found that the absence of Bcl-xL in rat primary hippocampal neurons resulted in mitochondrial dysfunction. Specifically, the ATP/ADP ratio was significantly decreased in the neurites of Bcl-xL depleted neurons. We further found that neurons transduced with Bcl-xL shRNA or neurons treated with ABT-263, a pharmacological inhibitor of Bcl-xL, showed impaired mitochondrial motility. Neurons lacking Bcl-xL had significantly decreased anterograde and retrograde movement of mitochondria and an increased stationary mitochondrial population when Bcl-xL was depleted by either means. These mitochondrial defects, including loss of ATP, impaired normal neurite development. Neurons lacking Bcl-xL showed significantly decreased neurite arborization, growth and complexity. Bcl-xL depleted neurons also showed impaired synapse formation. These neurons showed increased intracellular calcium concentration and were more susceptible to excitotoxic challenge. Bcl-xL may support positioning of mitochondria at metabolically demanding regions of neurites like branching points. Our findings suggest a role for Bcl-xL in physiological regulation of neuronal growth and development.
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    Linker-free conjugation and specific cell targeting of antibody functionalized iron-oxide nanoparticles
    (Royal Society of Chemistry, 2014) Xu, Yaolin; Baiu, Dana C.; Sherwood, Jennifer A.; McElreath, Meghan R.; Qin, Ying; Lackey, Kimberly H.; Otto, Mario; Bao, Yuping; University of Alabama Tuscaloosa; University of Wisconsin Madison
    Specific targeting is a key step to realize the full potential of iron oxide nanoparticles in biomedical applications, especially tumor-associated diagnosis and therapy. Here, we developed anti-GD2 antibody conjugated iron oxide nanoparticles for highly efficient neuroblastoma cell targeting. The antibody conjugation was achieved through an easy, linker-free method based on catechol reactions. The targeting efficiency and specificity of the antibody-conjugated nanoparticles to GD2-positive neuroblastoma cells were confirmed by flow cytometry, fluorescence microscopy, Prussian blue staining and transmission electron microscopy. These detailed studies indicated that the receptor-recognition capability of the antibody was fully retained after conjugation and the conjugated nanoparticles quickly attached to GD2-positive cells within four hours. Interestingly, longer treatment (12 h) led the cell membrane-bound nanoparticles to be internalized into cytosol, either by directly penetrating the cell membrane or escaping from the endosomes. Last but importantly, the uniquely designed functional surfaces of the nanoparticles allow for easy conjugation of other bioactive molecules.