Role of Calcium in the Regulation of Chronic Stress-Induced Progression of Stress Granule Assembly in N2a Cells

The persistence of stress granules (SGs) and subsequent pathological inclusion are implicated in neurodegeneration. Although SGs formed under acute stress have been well investigated, the role of biophysical factors in the formation of persistent and large SGs in the context of such neurodegeneration remains unclear. Especially, it remains challenging to differentiate between calcium (Ca²⁺) and PERK (PKR-like ER kinase)-mediated SG maturation. In this work, we hypothesize that the maturation of SGs under chronic stress primarily occurs due to an interplay between PERK pathway activation and the accumulation of Ca²⁺. To examine this, we first demonstrate that chronic hypoxia induces the formation of large SGs only in the later phase. Next, we demonstrate that there is significant colocalization of Ca²⁺ and G3BP1 (Ras GTPase-activating protein-binding protein 1) within the late-phase SGs by live cell imaging using laser scanning confocal microscopy. Moreover, the specific inhibition of the L-type Ca²⁺ channel and PERK pathway indicates that Ca²⁺ overloading plays a major role in inducing large-sized SGs. Based on this, we further demonstrated that resveratrol treatment effectively suppressed the large SG by inhibiting <i>CAPN-2</i> (calpain-2) and <i>ATF4</i> (activating transcription factor 4). Our study highlights that ionic interactions assume critical importance in controlling SG size distribution under chronic hypoxia, and resveratrol can be a promising strategy for the reduction of large SGs.