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 α-tocotrienol (TCT) will prevent ∆N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with α-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. α-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.

The brain requires vast amounts of energy to carry out neurotransmission; indeed, it is responsible for approximately one-fifth of the body’s energy consumption. Therefore, in order to understand functions of brain cells under both normal and pathological conditions, it is critical to elucidate dynamics of intracellular energy. The mitochondrion is the key intercellular organelle that controls neuronal energy and survival. Numerous studies have reported a correlation between altered mitochondrial function and brain-associated diseases; thus mitochondria may serve as a promising target for treating these conditions. In this chapter, we will discuss the mechanisms of mitochondrial production, movement, and degradation in order to understand accessibility of energy during physiological and pathological conditions of the brain. While research targeting molecular dynamics is promising, translation into clinical relevance based on bench research is challenging. For these reasons, we will also summarize lifestyle factors, including interventions and chronic comorbidities that disrupt mitochondrial dynamics. By determining lifestyle factors that are readily accessible, we can propose a new viewpoint for a synergistic and translational approach for neuroprotection.

Objectives Alpha-tocotrienol (α-TCT), a form of vitamin E, is a lipophilic antioxidant with neuroprotective properties. We recently reported that α-TCT treatment prevents oxidative stress-induced proteolytic cleavage of B-cell lymphoma-extra large (Bcl-xL), a pro-survival mitochondrial protein necessary during neuronal growth. However, it is still unclear if α-TCT exhibits beneficial effects during the physiological development of neurons. In this study, we hypothesized that chronic α-TCT treatment advances the development of primary hippocampal neurons by improving mitochondrial function. Methods Primary rat hippocampal neurons were grown in neurobasal media with or without α-TCT for three weeks, and media was replaced with conditioned media containing fresh α-TCT every week. Intracellular α-TCT levels were quantified using HPLC-MS, and intracellular ATP and mitochondrial superoxide levels were determined using luciferase and mitoSOX, respectively. Neurite morphology was examined by Sholl analysis. mRNA and protein levels of Bcl-xL were quantified using qPCR and immunoblotting, respectively. Results Primary hippocampal neurons grown in media containing α-TCT had increased intracellular α-TCT levels and decreased mitochondrial superoxide. Treatment with α-TCT increased mRNA and protein levels of Bcl-xL, neuronal ATP, and the number of neurite branches in primary hippocampal neurons. Conclusions We found that primary rat hippocampal neurons treated with α-TCT developed advanced neurite complexity. We suggest that α-TCT treatment improves mitochondrial function via upregulation of Bcl-xL, supporting normal neuron development. Funding Sources RGC Program (University of Alabama); Crenshaw Research Fund (University of Alabama).

Objectives Neurite outgrowth is a foundational process in brain development and recovery from brain injury. Assembly of the cytoskeleton and formation of new synapses during neurite outgrowth requires an abundance of energy. We have reported that the mitochondrial protein Bcl-xL is necessary for neurite outgrowth and arborization. However, Bcl-xL undergoes post-translational cleavage during oxidative stress resulting in a product that impairs mitochondrial function. Our recent publication demonstrated that treatment with alpha-tocotrienol, an antioxidant member of the vitamin E family, prevents cleavage of Bcl-xL and protects neurons from oxidative stress. In this study, we hypothesize that treatment with alpha-tocotrienol improves mitochondrial function to support the energy demanding processes of growth and development in the neurons. Methods Primary hippocampal neurons were grown in neurobasal media with or without alpha-tocotrienol for 3 weeks. Then, the number of neurite branches was quantified applying Sholl analysis. We also assayed the ATP/ADP ratio at neurites using the PercevalHR fluorescence biosensor. mRNA and protein levels of total Bcl-xL and cleaved Bcl-xL were measured using real time PCR and immunoblotting. Results Neurons grown with alpha-tocotrienol achieved more advanced neurite complexity than the control group. Treatment with alpha-tocotrienol enhanced both total ATP and local neurite ATP levels in primary hippocampal neurons. Furthermore, we found that alpha-tocotrienol Increased mRNA and protein levels of Bcl-xL without enhancing post-translational cleavage of Bcl-xL, consistent with our previous study. Conclusions Our data show that alpha-tocotrienol improves mitochondria-mediated ATP production by enhancing Bcl-xL to support metabolically demanding processes in neurons. We suggest a novel function of alpha-tocotrienol in normal physiological development of the brain. This study also suggests a potential therapeutic role of alpha-tocotrienol in brain diseases associated with neurite injury. Funding Sources RGC Program (University of Alabama) Crenshaw Research Fund (University of Alabama).

Objectives Neurite outgrowth is a pivotal process of brain development and recovery after brain injury. This metabolically demanding process requires assembly of the cytoskeleton and formation and maintenance of synapses. We have recently found that treatment with alpha-tocotrienol, an antioxidant and a member of the vitamin E family, prevents loss of mitochondrial inner membrane potential during oxidative stress. In this study, we hypothesize that the mitochondrion is the central target of alpha-tocotrienol-mediated neuroprotection, and treatment with alpha-tocotrienol may improve neuronal energy metabolism and promote neurite outgrowth. Methods Primary hippocampal neurons were grown in neurobasal media with or without alpha-tocotrienol for 3 weeks. Then, the morphological development of neurites, including polarity and arborization, was analyzed. We also assayed the ATP: ADP ratio in neurites using PercevalHR fluorescence biosensor after treatment with alpha-tocotrienol. Results Neurons grown with alpha-tocotrienol achieved neuronal polarity prior to the control group, and alpha-tocotrienol treated neurons showed longer and more branched neurites compared to the control group. Treatment with alpha-tocotrienol enhanced ATP levels in primary hippocampal neurons. Conclusions Our data show that alpha-tocotrienol improves mitochondria-mediated ATP production and supports the metabolically demanding process of neurite growth. This study also suggests that alpha-tocotrienol may be beneficial for recovery after brain injuries associated with neurite loss. Funding Sources RG14811 (University of Alabama).