Posttreatment Strategy Against Hypoxia and Ischemia Based on Selective Targeting of Nonnuclear Estrogen Receptors with PaPE-1

Abstract

Newly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERβ, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3β. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

Amorfrutin B Protects Mouse Brain Neurons from Hypoxia/Ischemia by Inhibiting Apoptosis and Autophagy Processes Through Gene Methylation- and miRNA-Dependent Regulation

“…The adequacy of the implemented models has been confirmed by the use of specific inhibitors targeting apoptosis (caspase-8, -9, -3/6, GSK3β, JNK, p38 MAPK) and autophagy (mTOR, ULK1, ULK2, USP10, USP13) that diminished hypoxia/ischemia-induced neurotoxicity. Moreover, the contribution of apoptosis- and autophagy-dependent signaling to the development of hypoxic/ischemic injuries in brain neurons has been supported by other studies, including ours [ 28 , 35 – 49 ].…”

“…JC-10 is a lipophilic dye that enters mitochondria and reversibly changes its color from green to orange, which is detected as an increase in membrane potential. JC-10 dye-loading solution was added to the cell plate and incubated with the cells for 1 h at 37 °C as previously described [ 28 ]. After adding Assay Buffer B to the dye-loading plate, the fluorescence was monitored at λ ex = 540 nm/λ em = 590 nm and λ ex = 490 nm/λ em = 525 nm with an Infinite M200pro microplate reader (Tecan Mannedorf, Switzerland).…”

Emerging Evidence on Membrane Estrogen Receptors as Novel Therapeutic Targets for Central Nervous System Pathologies

“…Pretreatment with PaPE-1 (subcutaneous pellets) reduced the brain infarct volume and leukocyte infiltration into the ischemic brain in mice subjected to MCAO. Our recent publication provided a “proof of concept” confirming the ability of posttreatment with PaPE-1 to provide neuroprotection against hypoxic and ischemic injuries (a 6 h delay of initiation of hypoxia/ischemia) [ 74 ]. The mechanism of PaPE-1 neuroprotection relied on inhibition of apoptosis and ROS formation as well as restoration of cellular metabolic activity.…”

“…This is probably the reason that interest was recently directed toward non-GPER-dependent nonnuclear ERs, namely, mERα and mERβ. Additionally, no serious controversies have been recognized with respect to the actions mediated by mERα and mERβ in neuronal tissue [ 74 ].…”

Targeting the non-classical estrogen pathway in neurodegenerative diseases and brain injury disorders

“…Treatment with G-1 improved neuronal survival after brain ischemia, reduced infarct size, neuronal injury and improved neuroinflammation and immunosuppression after experimentally induced stroke and cerebral ischemia ( 104 , 111 , 112 ). Furthermore, treatment with other non-classical pathway activators, such as PaPE-1 and the SERM bazedoxifene, protected neurons against ischemic brain damage in rodents and in neuronal culture, potentially through the MAPK/ERK1/2 signaling pathway ( 113 , 114 ).…”