Objectives Oxidative stress plays an important role in neuronal loss associated with mitochondrial dysfunction. Excess reactive oxygen species (ROS) production damages mitochondria, impairing neuronal energy metabolism. The damaged mitochondria promote aberrant ROS production leading to neuronal death. Fucoxanthin, a carotenoid with antioxidant properties primarily found in brown seaweeds, has been shown to protect mitochondria in various disease models. However, limited studies have demonstrated the mechanisms of fucoxanthin-mediated neuroprotection. In this study, we hypothesize that fucoxanthin regulates DJ-1, an oxidative stress sensing protein, and protects neurons against ROS-mediated mitochondrial dysfunction. Methods Rat primary hippocampal neurons were treated with fucoxanthin, hydrogen peroxide, or a combination of both. After 6 h of incubation, mitochondrial superoxide and the mitochondrial membrane potential were measured using mitoSOX and TMRM, respectively. Middle-aged male Sprague Dawley rats were supplemented with or without fucoxanthin (1 mg/kg, 5 d/w for 4 wk). After supplementation was completed, brain tissues were harvested, and DJ-1 protein levels were quantified using immunoblotting. Results Treatment with fucoxanthin decreased mitochondrial superoxide accumulation and prevented loss of mitochondrial membrane potential against ROS challenge in rat primary hippocampal neurons. Oral supplementation of fucoxanthin increased DJ-1 protein levels in the hippocampal tissues isolated from middle-aged rats. Conclusions We found that fucoxanthin treatment upregulates DJ-1 expression in the hippocampus in vivo and protects mitochondria during ROS challenges in primary hippocampal neurons in vitro. Our data suggest fucoxanthin has neuroprotective potential against ROS-associated mitochondrial dysfunction. Funding Sources RGC Program (University of Alabama); Crenshaw Research Fund (University of Alabama).

No abstract

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.

Objectives B-cell lymphoma-extra large (Bcl-xL) is a pro-survival protein localized to mitochondria and is also reported to support brain function by enhancing neuronal energy metabolism and synapse formation. We have previously shown that Bcl-xL is required for neurite outgrowth, and neurons lacking Bcl-xL were susceptible against neurotoxic challenges. In this study, we hypothesized that Bcl-xL supports maintaining neurite ATP by regulating mitochondrial motility. We thus tested if Bcl-xL depletion altered normal mitochondrial dynamics, neuronal energy retention, and neurite morphology. Methods Primary hippocampal neurons were transduced with either Bcl-xL shRNA or scrambled shRNA for 3 weeks. Mitochondria were labeled using mito-RFP BacMam2.0 and image sequences were obtained. Mitochondria motility parameters were quantified using KymoAnalyzer. Local ATP/ADP ratio was analyzed applying PercevalHR fluorescence biosensor, and neurite branches were quantified using Sholl analysis. We further tested viability of neurons against excitotoxicity applying calcein and propioduim iodin staining. Results Primary hippocampal neurons transduced with Bcl-xL shRNA decreased antero- and retrograde movement of mitochondria, lowered ATP/ADP ratio in neurites, and decreased length of neurites and number of branching points. Failure of achieving neurite complexity increased susceptibility of neurons to glutamate-induced excitotoxicity. Conclusions Primary hippocampal neurons transduced with Bcl-xL shRNA decreased antero- and retrograde movement of mitochondria, lowered ATP/ADP ratio in neurites, and decreased length of neurites and number of branching points. Failure of achieving neurite complexity increased susceptibility of neurons to glutamate-induced excitotoxicity. Funding Sources RGC Program (University of Alabama) Crenshaw Research Fund (University of Alabama) Sigma Xi Grants in Aid of Research (The National Academy of Sciences).

Objectives Neurite branching is necessary to achieve neurite complexity and synaptic plasticity. Therefore, understanding how neurons utilize intracellular energy to support neurite branching is key to elucidating cellular mechanisms of neuronal development. B-cell lymphoma extra large (Bcl-xL) is a pro-survival protein found in the mitochondria. Traditionally, Bcl-xL is known to block apoptotic pathway, yet increasing studies have demonstrated that Bcl-xL exhibits additional biological roles. Bcl-xL has been reported to enhance neuronal energy metabolism and synapse formation, and we have previously shown Bcl-xL to be essential for neurite outgrowth and Bcl-xL depletion increases susceptibility to hypoxia. In this study, we hypothesized that Bcl-xL supports neurite branching and maintains neurite ATP via regulation of mitochondrial motility. Methods Primary hippocampal neurons were transduced with either Bcl-xL shRNA or scrambled shRNA for 3 weeks. Mitochondria were labeled using mito-RFP BacMam2.0 and fluorescent and micrograph sequences were obtained. Mitochondrial motility parameters were then quantified using the KymoAnalyzer software. The ATP/ADP ratio was analyzed using the PercevalHR fluorescence biosensor to determine the effects of Bcl-xL depletion on energy retention. Neurite branching was quantified using Sholl analysis. Immunocytochemistry was performed to measure synapse formation. To measure susceptibility to excitotoxicity Fluorescent imaging using Fluo-4, propidium iodine, and calcein-AM was performed. Results We found that Bcl-xL depletion decreases antero- and retrograde movement of mitochondria. Bcl-xL depletion also lowered the ATP/ADP ratio in neurites and decreased the length and branching of neurites. Furthermore, Bcl-xL depletion impaired synapse formation and increased susceptibility to excitotoxicity. Conclusions This data suggests that Bcl-xL is essential in supporting neurite branching and energy retention by regulating mitochondrial motility. Bcl-xL may be a potential therapeutic target for brain disorders associated with abnormal or absent neurite development. Funding Sources Sigma Xi Grants in Aid of Research (The National Academy of Sciences).