Estrogen receptors (ERs) are believed to be ligand-activated transcription factors belonging to the nuclear receptor superfamily, which on ligand binding translocate into the nucleus and activate gene transcription. To date, two ERs have been identified: ERα and ERβ. ERα plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ERβ is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ERβ localizes to the mitochondria. In immunocytochemistry studies, ERβ was detected with two ERβ antibodies and found to colocalize almost exclusively with a mitochondrial marker in rat primary neuron, primary cardiomyocyte, and a murine hippocampal cell line. The colocalization of ERβ and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ERβ into the nucleus on 17β-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ERβ, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS/PAGE. The 50,000–65,000 M _r band was digested with trypsin and subjected to matrix-assisted laser desorption/ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ERβ. In summary, this study demonstrated that ERβ is localized to mitochondria, suggesting a role for mitochondrial ERβ in estrogen effects on this important organelle.

RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.

Substantial evidence now exists that intrinsic free-radical scavenging contributes to the receptor-independent neuroprotective effects of estrogens. This activity is inherently associated with the presence of a phenolic A-ring in the steroid. We report a previously unrecognized antioxidant cycle that maintains the “chemical shield” raised by estrogens against the most harmful reactive oxygen species, the hydroxyl radical (•OH) produced by the Fenton reaction. In this cycle, the capture of •OH was shown to produce a nonphenolic quinol with no affinity to the estrogen receptors. This quinol is then rapidly converted back to the parent estrogen via an enzyme-catalyzed reduction by using NAD(P)H as a coenzyme (reductant) and, unlike redox cycling of catechol estrogens, without the production of reactive oxygen species. Due to this process, protection of neuronal cells against oxidative stress is also possible by quinols that essentially act as prodrugs for the active hormone. We have shown that the quinol obtained from a 17β-estradiol derivative was, indeed, able to attenuate glutamate-induced oxidative stress in cultured hippocampus-derived HT-22 cells. Estrone quinol was also equipotent with its parent estrogen in reducing lesion volume in ovariectomized rats after transient middle carotid artery occlusion followed by a 24-h reperfusion. These findings may establish the foundation for a rational design of neuroprotective antioxidants focusing on steroidal quinols as unique molecular leads.

Understanding the control of sleep–wake states by the basal forebrain (BF) poses a challenge due to the intermingled presence of cholinergic, GABAergic, and glutamatergic neurons. All three BF neuronal subtypes project to the cortex and are implicated in cortical arousal and sleep–wake control. Thus, nonspecific stimulation or inhibition studies do not reveal the roles of these different neuronal types. Recent studies using optogenetics have shown that “selective” stimulation of BF cholinergic neurons increases transitions between NREM sleep and wakefulness, implicating cholinergic projections to cortex in wake promotion. However, the interpretation of these optogenetic experiments is complicated by interactions that may occur within the BF. For instance, a recentin vitrostudy from our group found that cholinergic neurons strongly excite neighboring GABAergic neurons, including the subset of cortically projecting neurons, which contain the calcium-binding protein, parvalbumin (PV) (Yang et al., 2014). Thus, the wake-promoting effect of “selective” optogenetic stimulation of BF cholinergic neurons could be mediated by local excitation of GABA/PV or other non-cholinergic BF neurons. In this study, using a newly designed opto-dialysis probe to couple selective optical stimulation with simultaneousin vivomicrodialysis, we demonstrated that optical stimulation of cholinergic neurons locally increased acetylcholine levels and increased wakefulness in mice. Surprisingly, the enhanced wakefulness caused by cholinergic stimulation was abolished by simultaneous reverse microdialysis of cholinergic receptor antagonists into BF. Thus, our data suggest that the wake-promoting effect of cholinergic stimulation requires local release of acetylcholine in the basal forebrain and activation of cortically projecting, non-cholinergic neurons, including the GABAergic/PV neurons.SIGNIFICANCE STATEMENTOptogenetics is a revolutionary tool to assess the roles of particular groups of neurons in behavioral functions, such as control of sleep and wakefulness. However, the interpretation of optogenetic experiments requires knowledge of the effects of stimulation on local neurotransmitter levels and effects on neighboring neurons. Here, using a novel “opto-dialysis” probe to couple optogenetics andin vivomicrodialysis, we report that optical stimulation of basal forebrain (BF) cholinergic neurons in mice increases local acetylcholine levels and wakefulness. Reverse microdialysis of cholinergic antagonists within BF prevents the wake-promoting effect. This important result challenges the prevailing dictum that BF cholinergic projections to cortex directly control wakefulness and illustrates the utility of “opto-dialysis” for dissecting the complex brain circuitry underlying behavior.

Most peptides do not enter the central nervous system because of their hydrophilic character and the presence of peptidolytic enzymes in the lipoidal blood-brain barrier. To achieve brain delivery of a peptide conjugate, an opioid peptide (enkephalin) was placed in a molecular environment that disguises its peptide nature and provides biolabile, lipophilic functions to penetrate the blood-brain barrier by passive transport. The strategy also incorporates a 1,4-dihydrotrigonellinate targetor that undergoes an enzymatically mediated oxidation to a hydrophilic, membrane-impermeable trigonellinate salt. The polar targetorpeptide conjugate that is trapped behind the lipoidal blood-brain barrier is deposited in the central nervous system. Analgesia was observed with "packaged" enkephalin but not with the unmodified peptide or lipophilic peptide precursors.