Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

Mitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.

Adenosine A1 and A2A Receptors in the Brain: Current Research and Their Role in Neurodegeneration.

The inhibitory adenosine A1 receptor (A1R) and excitatory A2A receptor (A2AR) are predominantly expressed in the brain. Whereas the A2AR has been implicated in normal aging and enhancing neurotoxicity in multiple neurodegenerative diseases, the inhibitory A1R has traditionally been ascribed to have a neuroprotective function in various brain insults. This review provides a summary of the emerging role of prolonged A1R signaling and its potential cross-talk with A2AR in the cellular basis for increased neurotoxicity in neurodegenerative disorders. This A1R signaling enhances A2AR-mediated neurodegeneration, and provides a platform for future development of neuroprotective agents in stroke, Parkinson's disease and epilepsy.

Protein phosphatase role in adenosine A1 receptor-induced AMPA receptor trafficking and rat hippocampal neuronal damage in hypoxia/reperfusion injury.

Adenosine signaling via A1 receptor (A1R) and A2A receptor (A2AR) has shown promise in revealing potential targets for neuroprotection in cerebral ischemia. We recently showed a novel mechanism by which A1R activation with N(6)-cyclopentyl adenosine (CPA) induced GluA1 and GluA2 AMPA receptor (AMPAR) endocytosis and adenosine-induced persistent synaptic depression (APSD) in rat hippocampus. This study further investigates the mechanism of A1R-mediated AMPAR internalization and hippocampal slice neuronal damage through activation of protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B) using electrophysiological, biochemical and imaging techniques. Following prolonged A1R activation, GluA2 internalization was selectively blocked by PP2A inhibitors (okadaic acid and fostriecin), whereas inhibitors of PP2A, PP1 (tautomycetin), and PP2B (FK506) all prevented GluA1 internalization. Additionally, GluA1 phosphorylation at Ser831 and Ser845 was reduced after prolonged A1R activation in hippocampal slices. PP2A inhibitors nullified A1R-mediated downregulation of pSer845-GluA1, while PP1 and PP2B inhibitors prevented pSer831-GluA1 downregulation. Each protein phosphatase inhibitor also blunted CPA-induced synaptic depression and APSD. We then tested whether A1R-mediated changes in AMPAR trafficking and APSD contribute to hypoxia-induced neuronal injury. Hypoxia (20 min) induced A1R-mediated internalization of both AMPAR subunits, and subsequent normoxic reperfusion (45 min) increased GluA1 but persistently reduced GluA2 surface expression. Neuronal damage after hypoxia-reperfusion injury was significantly blunted by pre-incubation with the above protein phosphatase inhibitors. Together, these data suggest that A1R-mediated protein phosphatase activation causes persistent synaptic depression by downregulating GluA2-containing AMPARs; this previously undefined role of A1R stimulation in hippocampal neuronal damage represents a novel therapeutic target in cerebral ischemic damage.

Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus.

Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7-12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains.

Prolonged adenosine A1 receptor activation in hypoxia and pial vessel disruption focal cortical ischemia facilitates clathrin-mediated AMPA receptor endocytosis and long-lasting synaptic inhibition in rat hippocampal CA3-CA1 synapses: differential regulation of GluA2 and GluA1 subunits by p38 MAPK and JNK.

Activation of presynaptic adenosine A1 receptors (A1Rs) causes substantial synaptic depression during hypoxia/cerebral ischemia, but postsynaptic actions of A1Rs are less clear. We found that A1Rs and GluA2-containing AMPA receptors (AMPARs) form stable protein complexes from hippocampal brain homogenates and cultured hippocampal neurons from Sprague Dawley rats. In contrast, adenosine A2A receptors (A2ARs) did not coprecipitate or colocalize with GluA2-containing AMPARs. Prolonged stimulation of A1Rs with the agonist N(6)-cyclopentyladenosine (CPA) caused adenosine-induced persistent synaptic depression (APSD) in hippocampal brain slices, and APSD levels were blunted by inhibiting clathrin-mediated endocytosis of GluA2 subunits with the Tat-GluA2-3Y peptide. Using biotinylation and membrane fractionation assays, prolonged CPA incubation showed significant depletion of GluA2/GluA1 surface expression from hippocampal brain slices and cultured neurons. Tat-GluA2-3Y peptide or dynamin inhibitor Dynasore prevented CPA-induced GluA2/GluA1 internalization. Confocal imaging analysis confirmed that functional A1Rs, but not A2ARs, are required for clathrin-mediated AMPAR endocytosis in hippocampal neurons. Pharmacological inhibitors or shRNA knockdown of p38 MAPK and JNK prevented A1R-mediated internalization of GluA2 but not GluA1 subunits. Tat-GluA2-3Y peptide or A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine also prevented hypoxia-mediated GluA2/GluA1 internalization. Finally, in a pial vessel disruption cortical stroke model, a unilateral cortical lesion compared with sham surgery reduced hippocampal GluA2, GluA1, and A1R surface expression and also caused synaptic depression in hippocampal slices that was consistent with AMPAR downregulation and decreased probability of transmitter release. Together, these results indicate a previously unknown mechanism for A1R-induced persistent synaptic depression involving clathrin-mediated GluA2 and GluA1 internalization that leads to hippocampal neurodegeneration after hypoxia/cerebral ischemia.

Novel central nervous system drug delivery systems.

For decades, biomedical and pharmaceutical researchers have worked to devise new and more effective therapeutics to treat diseases affecting the central nervous system. The blood-brain barrier effectively protects the brain, but poses a profound challenge to drug delivery across this barrier. Many traditional drugs cannot cross the blood-brain barrier in appreciable concentrations, with less than 1% of most drugs reaching the central nervous system, leading to a lack of available treatments for many central nervous system diseases, such as stroke, neurodegenerative disorders, and brain tumors. Due to the ineffective nature of most treatments for central nervous system disorders, the development of novel drug delivery systems is an area of great interest and active research. Multiple novel strategies show promise for effective central nervous system drug delivery, giving potential for more effective and safer therapies in the future. This review outlines several novel drug delivery techniques, including intranasal drug delivery, nanoparticles, drug modifications, convection-enhanced infusion, and ultrasound-mediated drug delivery. It also assesses possible clinical applications, limitations, and examples of current clinical and preclinical research for each of these drug delivery approaches. Improved central nervous system drug delivery is extremely important and will allow for improved treatment of central nervous system diseases, causing improved therapies for those who are affected by central nervous system diseases.