Brain-derived neurotrophic factor protects neurons by stimulating mitochondrial function through protein kinase A.

Brain-derived neurotrophic factor (BDNF) stimulates dendrite outgrowth and synaptic plasticity by activating downstream protein kinase A (PKA) signaling. Recently, BDNF has been shown to modulate mitochondrial respiration in isolated brain mitochondria, suggesting that BDNF can modulate mitochondrial physiology. However, the molecular mechanisms by which BDNF stimulates mitochondrial function in neurons remain to be elucidated. In this study, we surmised that BDNF binds to the TrkB receptor and translocates to mitochondria to govern mitochondrial physiology in a PKA-dependent manner. Confocal microscopy and biochemical subcellular fractionation assays confirm the localization of the TrkB receptor in mitochondria. The translocation of the TrkB receptor to mitochondria was significantly enhanced upon treating primary cortical neurons with exogenous BDNF, leading to rapid PKA activation. Showing a direct role of BDNF in regulating mitochondrial structure/function, time-lapse confocal microscopy in primary cortical neurons showed that exogenous BDNF enhances mitochondrial fusion, anterograde mitochondrial trafficking, and mitochondrial content within dendrites, which led to increased basal and ATP-linked mitochondrial respiration and glycolysis as assessed by an XF24e metabolic analyzer. BDNF-mediated regulation of mitochondrial structure/function requires PKA activity as treating primary cortical neurons with a pharmacological inhibitor of PKA or transiently expressing constructs that target an inhibitor peptide of PKA (PKI) to the mitochondrion abrogated BDNF-mediated mitochondrial fusion and trafficking. Mechanistically, western/Phos-tag blots show that BDNF stimulates PKA-mediated phosphorylation of Drp1 and Miro-2 to promote mitochondrial fusion and elevate mitochondrial content in dendrites, respectively. Effects of BDNF on mitochondrial function were associated with increased resistance of neurons to oxidative stress and dendrite retraction induced by rotenone. Overall, this study revealed new mechanisms of BDNF-mediated neuroprotection, which entails enhancing mitochondrial health and function of neurons.

Mucosal Immunity and the Gut-Microbiota-Brain-Axis in Neuroimmune Disease.

Recent advances in next-generation sequencing (NGS) technologies have opened the door to a wellspring of information regarding the composition of the gut microbiota. Leveraging NGS technology, early metagenomic studies revealed that several diseases, such as Alzheimer's disease, Parkinson's disease, autism, and myalgic encephalomyelitis, are characterized by alterations in the diversity of gut-associated microbes. More recently, interest has shifted toward understanding how these microbes impact their host, with a special emphasis on their interactions with the brain. Such interactions typically occur either systemically, through the production of small molecules in the gut that are released into circulation, or through signaling via the vagus nerves which directly connect the enteric nervous system to the central nervous system. Collectively, this system of communication is now commonly referred to as the gut-microbiota-brain axis. While equally important, little attention has focused on the causes of the alterations in the composition of gut microbiota. Although several factors can contribute, mucosal immunity plays a significant role in shaping the microbiota in both healthy individuals and in association with several diseases. The purpose of this review is to provide a brief overview of the components of mucosal immunity that impact the gut microbiota and then discuss how altered immunological conditions may shape the gut microbiota and consequently affect neuroimmune diseases, using a select group of common neuroimmune diseases as examples.

Evidence-based practice strategies for faculty professional development in clinical translational research at Institutional Development Award (IDeA) state institutions in the Mountain West.

The Mountain West Clinical Translational Research - Infrastructure Network (MW CTR-IN), established in 2013, is a research network of 13 university partners located among seven Institutional Development Award (IDeA) states targeting health disparities. This is an enormous undertaking because of the size of the infrastructure network (encompassing a third of the US landmass and spanning four time zones in predominantly rural and underserved areas, with populations that have major health disparities issues). In this paper, we apply the barriers, strategies, and metrics to an adapted educational conceptual model by Fink (2013). Applying this model, we used four tailored approaches across this regional infrastructure network to: (1) assess individual faculty specific needs, (2) reach out and engage with faculty, (3) provide customized services to meet the situational needs of faculty, and (4) utilize a "closed communication feedback loop" between Professional Development (PD) core and MW CTR-IN faculty within the context of their home institutional environment. Summary statement results from participating faculty show that these approaches were positive. Grounded in best educational practice approaches, we have an opportunity to refine and build from this sound foundation with implications for future use in other CTR-IN networks and institutions in the IDeA states.

Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.

Doxorubicin (DOX) is an aqueous soluble anthracycline therapeutic widely used in cancer treatment. Although DOX anti-cancer activity is dose-dependent, increased dosage enhances the risk of cardiotoxicity. Despite intensive investigation, the molecular basis of this undesirable side effect has yet to be established. In addition to serving as a DNA intercalation agent, DOX is known to bind to the signature mitochondrial phospholipid, cardiolipin (CL). Consistent with this, DOX associates with aqueous soluble nanoparticles, termed nanodisks (ND), comprised solely of CL and an apolipoprotein scaffold. Fluorescence microscopy analysis revealed that DOX uptake, and targeting to the nucleus of cultured hepatocarcinoma (HepG2) or breast cancer (MCF7) cells, was unaffected by its association with CL-ND. Subsequent studies revealed that free DOX and DOX-CL-ND were equivalent in terms of growth inhibition activity in both cell lines. By contrast, in studies with H9C2 cardiomyocytes, DOX-CL-ND induced a lesser concentration-dependent decline in cell viability than free DOX. Whereas incubation of H9C2 cardiomyocytes with free DOX caused a steep decline in maximal oxygen consumption rate, DOX-CL-ND treated cells were largely unaffected. The data indicate that association of DOX with CL-ND does not diminish its cancer cell growth inhibition activity yet confers protection to cardiomyocytes from DOX-induced effects on aerobic respiration. This study illustrates that interaction with CL plays a role in DOX-induced mitochondrial dysfunction and suggests CL-ND provide a tool for investigating the mechanistic basis of DOX-induced cardiotoxicity.

Multiplexing Seahorse XFe24 and ImageXpress® Nano Platforms for Comprehensive Evaluation of Mitochondrial Bioenergetic Profile and Neuronal Morphology.

The measurement of mitochondrial function has become imperative to understand and characterize diseases characterized by bioenergetic alterations. The advancement of automation and application of high-throughput technologies has propelled our understanding of biological complexity and facilitated drug discovery. Seahorse extracellular flux (XFe) technology measures changes in dissolved oxygen and proton concentration in cell culture media, providing kinetic measurements of oxidative phosphorylation and glycolytic metabolism. ImageXpress® Nano is an automated fluorescent microscope with the ability to perform high-content, fast, and robust imaging in multi-well formats. In this chapter, we present a comprehensive protocol to multiplex the Seahorse XFe24 analyzer with the ImageXpress® Nano high content imaging microscope to provide a comprehensive yet rigorous profile of bioenergetics and its correlation to neuronal function and morphology.

Intraperitoneal Administration of Forskolin Reverses Motor Symptoms and Loss of Midbrain Dopamine Neurons in PINK1 Knockout Rats.

BACKGROUND: Parkinson's disease (PD) is a relentless, chronic neurodegenerative disease characterized by the progressive loss of substantia nigra (SN) neurons that leads to the onset of motor and non-motor symptoms. Standard of care for PD consists of replenishing the loss of dopamine through oral administration of Levodopa; however, this treatment is not disease-modifying and often induces intolerable side effects. While the etiology that contributes to PD is largely unknown, emerging evidence in animal models suggests that a significant reduction in neuroprotective Protein Kinase A (PKA) signaling in the SN contributes to PD pathogenesis, suggesting that restoring PKA signaling in the midbrain may be a new anti-PD therapeutic alternative. OBJECTIVE: We surmised that pharmacological activation of PKA via intraperitoneal administration of Forskolin exerts anti-PD effects in symptomatic PTEN-induced kinase 1 knockout (PINK1-KO), a bona fide in vivo model of PD. METHODS: By using a beam balance and a grip strength analyzer, we show that Forskolin reverses motor symptoms and loss of hindlimb strength with long-lasting therapeutic effects (> 5 weeks) following the last dose. RESULTS: In comparison, intraperitoneal treatment with Levodopa temporarily (24 h) reduces motor symptoms but unable to restore hindlimb strength in PINK1-KO rats. By using immunohistochemistry and an XF24e BioAnalyzer, Forskolin treatment reverses SN neurons loss, elevates brain energy production and restores PKA activity in SN in symptomatic PINK1-KO rats. CONCLUSION: Overall, our collective in vivo data suggest that Forskolin is a promising disease-modifying therapeutic alternative for PD and is superior to Levodopa because it confers long-lasting therapeutic effects.

Intranasal Administration of Forskolin and Noopept Reverses Parkinsonian Pathology in PINK1 Knockout Rats.

Parkinson's Disease (PD) is a brain-degenerative disorder characterized by a progressive loss of midbrain dopamine neurons. Current standard-of-care includes oral administration of Levodopa to address motor symptoms, but this treatment is not disease-modifying. A reduction in Protein Kinase A (PKA) signaling and neurotrophic support contributes to PD pathology. We previously showed that enhancing PKA activity in the brain via intraperitoneal administration of Forskolin in Parkinsonian rats (PINK1 knockout) abrogate motor symptoms and loss of midbrain dopamine neurons. Given that intraperitoneal administration is invasive, we hypothesized that intranasal administration of Forskolin and a second nootropic agent (Noopept) could reverse PD pathology efficiently. Results show that intranasal administration of a formulation (CNS/CT-001) containing Forskolin (10 µM) and Noopept (20 nM) significantly reversed motor symptoms, loss of hind limb strength, and neurodegeneration of midbrain dopamine neurons in PINK1-KO rats and is indistinguishable from wild-type (WT) rats; therapeutic effects associated with increased PKA activity and levels of BDNF and NGF in the brain. Intranasal administration of CNS/CT-001, but not Forskolin, significantly decreased the number of α-synuclein aggregates in the cortex of PINK1-KO rats, and is indistinguishable from WT rats. Overall, we show proof of concept that intranasal administration of CNS/CT-001 is a non-invasive, disease-modifying formulation for PD.

The Community of Bilingual English-Spanish Speakers Exploring Issues in Science and Health: Experiences During the COVID-19 Pandemic.

Language diversity is increasing in the United States. This growth has implications for language preference, cost, quality, and client outcomes in health services settings. However, language diversity among medical and allied health professionals is lacking. Education pipeline programs are a mechanism to prepare bi- and multi-lingual diverse students to enter health careers. The Community of Bilingual English-Spanish Speakers Exploring Issues in Science and Health (CBESS) is one such program. Through peer mentorship from Leadership Trainees (LT), and a multicomponent 17-month education curriculum, CBESS was designed to increase interest in STEM careers among English-Spanish bilingual high school youth. In 2020, the COVID-19 pandemic interrupted high school students' education and forced programs to innovate. CBESS was no exception. The most significant modifications were to a) expectations of SRs for a successful Summer Virtual Research Program (SVRP), b) LT roles, and c) scope and delivery of summer science content. A preliminary evaluation was conducted from data collected through pre-post surveys, process data, and focus groups. Among the outcomes were a significant increase in science knowledge among SVRP youth participants as well as no significant differences between cohort 1 and 2 suggesting that changes did not impede program goals. LTs highlighted skills needed and role of mentors. Adaptations were successful and will continue with the 2021 cohort.

Role of Cleaved PINK1 in Neuronal Development, Synaptogenesis, and Plasticity: Implications for Parkinson's Disease.

Mitochondrial dysfunction plays a significant role in the pathogenesis of Parkinson's disease (PD). Consistent with this concept, loss of function mutations in the serine/threonine kinase- PINK1 (PTEN-induced putative kinase-1) causes autosomal recessive early onset PD. While the functional role of f-PINK1 (full-length PINK1) in clearing dysfunctional mitochondria via mitophagy is extensively documented, our understanding of specific physiological roles that the non-mitochondrial pool of PINK1 imparts in neurons is more limited. PINK1 is proteolytically processed in the intermembrane space and matrix of the mitochondria into functional cleaved products (c-PINK1) that are exported to the cytosol. While it is clear that posttranslational processing of PINK1 depends on the mitochondria's oxidative state and structural integrity, the functional roles of c-PINK1 in modulating neuronal functions are poorly understood. Here, we review the diverse roles played by c-PINK1 in modulating various neuronal functions. Specifically, we describe the non-canonical functional roles of PINK1, including but not limited to: governing mitochondrial movement, neuronal development, neuronal survival, and neurogenesis. We have published that c-PINK1 stimulates neuronal plasticity and differentiation via the PINK1-PKA-BDNF signaling cascade. In addition, we provide insight into how mitochondrial membrane potential-dependent processing of PINK1 confers conditional retrograde signaling functions to PINK1. Further studies delineating the role of c-PINK1 in neurons would increase our understanding regarding the role played by PINK1 in PD pathogenesis.

Coenzyme Q nanodisks counteract the effect of statins on C2C12 myotubes.

Depletion of coenzyme Q (CoQ) is associated with disease, ranging from myopathy to heart failure. To induce a CoQ deficit, C2C12 myotubes were incubated with high dose simvastatin. This resulted in a concentration-dependent inhibition of cell viability. Simvastatin-induced effects were prevented by co-incubation with mevalonic acid. When myotubes were incubated with 60 µM simvastatin, mitochondrial CoQ content decreased while co-incubation with CoQ nanodisks (ND) increased mitochondrial CoQ levels and improved cell viability. Incubation of myotubes with simvastatin also led to a reduction in oxygen consumption rate (OCR). When myotubes were co-incubated with simvastatin and CoQ ND, the decline in OCR was ameliorated. The data indicate that CoQ ND represent a water soluble vehicle capable of delivering CoQ to cultured myotubes. Thus, these biocompatible nanoparticles have the potential to bypass poor CoQ oral bioavailability as a treatment option for individuals with severe CoQ deficiency syndromes and/or aging-related CoQ depletion.

Cleaved PINK1 induces neuronal plasticity through PKA-mediated BDNF functional regulation.

Mutations in PTEN-induced kinase 1 (PINK1) lead to early onset autosomal recessive Parkinson's disease in humans. In healthy neurons, full-length PINK1 (fPINK1) is post-translationally cleaved into different lower molecular weight forms, and cleaved PINK1 (cPINK1) gets shuttled to the cytosolic compartments to support extra-mitochondrial functions. While numerous studies have exemplified the role of mitochondrially localized PINK1 in modulating mitophagy in oxidatively stressed neurons, little is known regarding the physiological role of cPINK1 in healthy neurons. We have previously shown that cPINK1, but not fPINK1, modulates the neurite outgrowth and the maintenance of dendritic arbors by activating downstream protein kinase A (PKA) signaling in healthy neurons. However, the molecular mechanisms by which cPINK1 promotes neurite outgrowth remain to be elucidated. In this report, we show that cPINK1 supports neuronal development by modulating the expression and extracellular release of brain-derived neurotrophic factor (BDNF). Consistent with this role, we observed a progressive increase in the level of endogenous cPINK1 but not fPINK1 during prenatal and postnatal development of mouse brains and during development in primary cortical neurons. In cultured primary neurons, the pharmacological activation of endogenous PINK1 leads to enhanced downstream PKA activity, subsequent activation of the PKA-modulated transcription factor cAMP response element-binding protein (CREB), increased intracellular production and extracellular release of BDNF, and enhanced activation of the BDNF receptor-TRKß. Mechanistically, cPINK1-mediated increased dendrite complexity requires the binding of extracellular BDNF to TRKß. In summary, our data support a physiological role of cPINK1 in stimulating neuronal development by activating the PKA-CREB-BDNF signaling axis in a feedforward loop.

Mitochondrial PKA Is Neuroprotective in a Cell Culture Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive decline. In hippocampal neurons, the pathological features of AD include the accumulation of extracellular amyloid-beta peptide (Aß) accompanied by oxidative stress, mitochondrial dysfunction, and neuron loss. A decrease in neuroprotective Protein Kinase A (PKA) signaling contributes to mitochondrial fragmentation and neurodegeneration in AD. By associating with the protein scaffold Dual-Specificity Anchoring Protein 1 (D-AKAP1), PKA is targeted to mitochondria to promote mitochondrial fusion by phosphorylating the fission modulator dynamin-related protein 1 (Drp1). We hypothesized that (1) a decrease in the endogenous level of endogenous D-AKAP1 contributes to decreased PKA signaling in mitochondria and that (2) restoring PKA signaling in mitochondria can reverse neurodegeneration and mitochondrial fragmentation in neurons in AD models. Through immunohistochemistry, we showed that endogenous D-AKAP1, but not other mitochondrial proteins, is significantly reduced in primary neurons treated with Aß42 peptide (10µM, 24 h), and in the hippocampus and cortex from asymptomatic and symptomatic AD mice (5X-FAD). Transiently expressing wild-type, but not a PKA-binding deficient mutant of D-AKAP1, was able to reduce mitochondrial fission, dendrite retraction, and apoptosis in primary neurons treated with Aß42. Mechanistically, the protective effects of D-AKAP1/PKA are moderated through PKA-mediated phosphorylation of Drp1, as transiently expressing a PKA phosphomimetic mutant of Drp1 (Drp1-S656D) phenocopies D-AKAP1's ability to reduce Aß42-mediated apoptosis and mitochondrial fission. Overall, our data suggest that a loss of D-AKAP1/PKA contributes to mitochondrial pathology and neurodegeneration in an in vitro cell culture model of AD.

Molecular Mechanism by which Cobra Venom Cardiotoxins Interact with the Outer Mitochondrial Membrane.

Cardiotoxin CTII from Najaoxiana cobra venom translocates to the intermembrane space (IMS) of mitochondria to disrupt the structure and function of the inner mitochondrial membrane. At low concentrations, CTII facilitates ATP-synthase activity, presumably via the formation of non-bilayer, immobilized phospholipids that are critical in modulating ATP-synthase activity. In this study, we investigated the effects of another cardiotoxin CTI from Najaoxiana cobra venom on the structure of mitochondrial membranes and on mitochondrial-derived ATP synthesis. By employing robust biophysical methods including 31P-NMR and 1H-NMR spectroscopy, we analyzed the effects of CTI and CTII on phospholipid packing and dynamics in model phosphatidylcholine (PC) membranes enriched with 2.5 and 5.0 mol% of cardiolipin (CL), a phospholipid composition that mimics that in the outer mitochondrial membrane (OMM). These experiments revealed that CTII converted a higher percentage of bilayer phospholipids to a non-bilayer and immobilized state and both cardiotoxins utilized CL and PC molecules to form non-bilayer structures. Furthermore, in order to gain further understanding on how cardiotoxins bind to mitochondrial membranes, we employed molecular dynamics (MD) and molecular docking simulations to investigate the molecular mechanisms by which CTII and CTI interactively bind with an in silico phospholipid membrane that models the composition similar to the OMM. In brief, MD studies suggest that CTII utilized the N-terminal region to embed the phospholipid bilayer more avidly in a horizontal orientation with respect to the lipid bilayer and thereby penetrate at a faster rate compared with CTI. Molecular dynamics along with the Autodock studies identified critical amino acid residues on the molecular surfaces of CTII and CTI that facilitated the long-range and short-range interactions of cardiotoxins with CL and PC. Based on our compiled data and our published findings, we provide a conceptual model that explains a molecular mechanism by which snake venom cardiotoxins, including CTI and CTII, interact with mitochondrial membranes to alter the mitochondrial membrane structure to either upregulate ATP-synthase activity or disrupt mitochondrial function.

Psychological distress and lack of PINK1 promote bioenergetics alterations in peripheral blood mononuclear cells.

Psychological distress induces oxidative stress and alters mitochondrial metabolism in the nervous and immune systems. Psychological distress promotes alterations in brain metabolism and neurochemistry in wild-type (WT) rats in a similar manner as in Parkinsonian rats lacking endogenous PTEN-induced kinase 1 (PINK1), a serine/threonine kinase mutated in a recessive forms of Parkinson's disease. PINK1 has been extensively studied in the brain, but its physiological role in peripheral tissues and the extent to which it intersects with the neuroimmune axis is not clear. We surmised that PINK1 modulates the bioenergetics of peripheral blood mononuclear cells (PBMCs) under basal conditions or in situations that promote oxidative stress as psychological distress. By using an XF metabolic bioanalyzer, PINK1-KO-PBMCs showed significantly increased oxidative phosphorylation and basal glycolysis compared to WT cells and correlated with motor dysfunction. In addition, psychological distress enhanced the glycolytic capacity in PINK1-KO-PBMCs but not in WT-PBMCs. The level of antioxidant markers and brain-derived neurotrophic factor were altered in PINK1-KO-PBMCs and by psychological distress. In summary, our data suggest that PINK1 is critical for modulating the bioenergetics and antioxidant responses in PBMCs whereas lack of PINK1 upregulates compensatory glycolysis in response to oxidative stress induced by psychological distress.

Assembly and Characterization of Biocompatible Coenzyme Q10 -Enriched Lipid Nanoparticles.

Coenzyme Q10 (CoQ10 ) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ10 was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ10 nanodisks (ND), contain 1.0 mg CoQ10 /5 mg PtdCho/2 mg apoA-I (97% CoQ10 solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ10 ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ10 into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ10 ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ10 . Incubation of cultured cells with CoQ10 ND, but not empty ND, resulted in a significant increase in the CoQ10 content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ10 in lipid nanoparticles has been developed. The availability of CoQ10 ND provides a novel means to investigate biochemical aspects of CoQ10 uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.

Psychological Stress Phenocopies Brain Mitochondrial Dysfunction and Motor Deficits as Observed in a Parkinsonian Rat Model.

Psychological distress is a public health issue as it contributes to the development of human diseases including neuropathologies. Parkinson's disease (PD), a chronic, progressive neurodegenerative disorder, is caused by multiple factors including aging, mitochondrial dysfunction, and/or stressors. In PD, a substantial loss of substantia nigra (SN) neurons leads to rigid tremors, bradykinesia, and chronic fatigue. Several studies have reported that the hypothalamic-pituitary-adrenal (HPA) axis is altered in PD patients, leading to an increase level of cortisol which contributes to neurodegeneration and oxidative stress. We hypothesized that chronic psychological distress induces PD-like symptoms and promotes neurodegeneration in wild-type (WT) rats and exacerbates PD pathology in PINK1 knockout (KO) rats, a well-validated animal model of PD. We measured the bioenergetics profile (oxidative phosphorylation and glycolysis) in the brain by employing an XF24e Seahorse Extracellular Flux Analyzer in young rats subjected to predator-induced psychological distress. In addition, we analyzed anxiety-like behavior, motor function, expression of antioxidant enzymes, mitochondrial content, and neurotrophic factors brain-derived neurotrophic factor (BDNF) in the brain. Overall, we observed that psychological distress diminished up to 50% of mitochondrial respiration and glycolysis in the prefrontal cortex (PFC) derived from both WT and PINK1-KO rats. Mechanistically, the level of antioxidant proteins, mitochondrial content, and BDNF was significantly altered. Finally, psychological distress robustly induced anxiety and Parkinsonian symptoms in WT rats and accelerated certain symptoms of PD in PINK1-KO rats. For the first time, our collective data suggest that psychological distress can phenocopy several aspects of PD neuropathology, disrupt brain energy production, as well as induce ataxia-like behavior.

A Pilot STEM Curriculum Designed to Teach High School Students Concepts in Biochemical Engineering and Pharmacology.

The creation of technology that affords for the design of artificial enzymes is a new branch of biochemical engineering with the objective to solve the looming global catastrophe including food shortages, energy crisis, novel diseases, climate change and environmental degradation. However, the development of science and technology that will lead to the design of artificial enzymes depends on availability of scientists with a broad range of expertise including chemistry and physics of chemical bonding, structural biochemistry of macromolecular interactions, theoretical physics and mathematics with the focus on computer modeling of dynamic docking of macromolecules. Our previous experience in university STEM education led us to conclude that in order to train future scientists with a broad expertise in STEM, it is critical for high school students to learn interdisciplinary concepts of STEM courses at an earlier age. In this article, we describe the first phase of a STEM project that involved introducing students to STEM curriculum designed to steer high school students' interest towards biochemical engineering and pharmacology. In addition, we present the outline of the STEM curriculum, along with user-friendly tutorials of AutoDock Vina, AutoDock Tools and PyMol programs that we designed to teach secondary STEM students computer modeling and docking of macromolecules. STEM high school students performed multiple exercises to understand how the potential pharmacological agents, cardiotoxins from cobra venom, interact with mitochondrial phospholipids in order to gain a deep understanding of elevated biophysical and biochemical concepts in protein drug interactions with biomembranes. We also present the results of evaluative assessments that tested students' knowledge and skills that students gained following the completion of our pilot STEM course. In brief, the assessment results showed that the students successfully acquired a high level of understanding in structural biophysics and biochemistry. Importantly, this paper provides strong proof-of-concept that our pilot STEM curriculum can be successfully integrated in the traditional American and Chinese high school classroom. The curriculum and tutorials presented in this article could be used by college and high school teachers and students in STEM classes and to support undergraduate university courses in Pharmacology, Inorganic and Organic Chemistry, Biochemistry and Structural Biology for classroom instructions and homework assignments.

G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells.

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in ß-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.

Naja mossambica mossambica Cobra Cardiotoxin Targets Mitochondria to Disrupt Mitochondrial Membrane Structure and Function.

Cobra venom cardiotoxins (CVCs) can translocate to mitochondria to promote apoptosis by eliciting mitochondrial dysfunction. However, the molecular mechanism(s) by which CVCs are selectively targeted to the mitochondrion to disrupt mitochondrial function remains to be elucidated. By studying cardiotoxin from Naja mossambica mossambica cobra (cardiotoxin VII4), a basic three-fingered S-type cardiotoxin, we hypothesized that cardiotoxin VII4 binds to cardiolipin (CL) in mitochondria to alter mitochondrial structure/function and promote neurotoxicity. By performing confocal analysis, we observed that red-fluorescently tagged cardiotoxin rapidly translocates to mitochondria in mouse primary cortical neurons and in human SH-SY5Y neuroblastoma cells to promote aberrant mitochondrial fragmentation, a decline in oxidative phosphorylation, and decreased energy production. In addition, by employing electron paramagnetic resonance (EPR) and protein nuclear magnetic resonance (¹H-NMR) spectroscopy and phosphorescence quenching of erythrosine in model membranes, our compiled biophysical data show that cardiotoxin VII4 binds to anionic CL, but not to zwitterionic phosphatidylcholine (PC), to increase the permeability and formation of non-bilayer structures in CL-enriched membranes that biochemically mimic the outer and inner mitochondrial membranes. Finally, molecular dynamics simulations and in silico docking studies identified CL binding sites in cardiotoxin VII4 and revealed a molecular mechanism by which cardiotoxin VII4 interacts with CL and PC to bind and penetrate mitochondrial membranes.