Another-regulin (ALN) is a newly identified micropeptide that binds and inhibits the sarco/endoplasmic reticulum calcium ATPase (SERCA), a pump responsible for maintaining intracellular calcium homeostasis. In neurons, calcium dyshomeostasis is associated with oxidative stress and reactive oxygen species (ROS) accumulation, leading to endoplasmic reticulum (ER) stress and cell death in diseases like Alzheimer’s disease (AD) and stroke. Unlike other SERCA-regulating micropeptides which show tissue specific expression, ALN demonstrates ubiquitous expression across tissues, and it is the predominant SERCA regulator in neurons. Thus, ALN may have a uniquely important role in neuronal calcium homeostasis. To investigate ALN regulatory mechanisms, we quantified the relative affinity of ALN for SERCA enzymatic states using fluorescence resonance energy transfer (FRET). We found that ALN preferentially binds the E1-Ca 2+ -ATP and E2 states and shows lower affinity for the E1P state. This state preference is a unique pattern compared to other micropeptides. Knockdown (KD) of ALN in hippocampal neuron-derived HT22 cells resulted in decreased ROS production, as quantified using the ROS indicator H2DCFDA. This is consistent with previous studies that suggest increased SERCA activity is protective against cell stress. Indeed, a trypan blue viability assay showed that the reduced cell stress resulted in improved hippocampal HT22 resistance to hypoxia. Overall, the data suggest that enhanced SERCA activity reduces neuronal oxidative stress and improves hypoxia tolerance. We hypothesize that although ALN is important for physiological regulation of SERCA, it may play a pathophysiological role under conditions of hypoxia. Ongoing studies will explore the possible role of ALN in exacerbating neuronal cell stress in the context of ischemic stroke.
Emily Neeb (Sun,) studied this question.