Endoplasmic reticulum stress in forebrain subfornical organ excitatory neurons during diet‐induced obesity

Abstract

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in the brain are emerging as key mechanisms in obesity‐related cardiovascular and metabolic diseases. For example, we recently demonstrated that inhibition of brain ER stress rescues hypertension and hepatic steatosis during high fat diet (HFD) feeding. Additional findings pointed to robust HFD‐induced ER stress in the subfornical organ (SFO), a circumventricular nucleus located outside of the blood‐brain‐barrier that is crucial for cardiometabolic regulation. The neuron population and anatomical location within the SFO that experiences ER stress during obesity remains unknown. Based on this, we comprehensively investigated UPR activation in the SFO during diet‐induced obesity. Six wk old male C57Bl/6J mice were placed on a HFD (n=4) or remained on normal chow (n=5) for 11 wks. Using serial cryosections, immunohistochemistry revealed marked upregulation of the ER chaperone glucose‐regulated peptide 78 kDa (GRP78) in the SFO of HFD fed mice, relative to normal chow controls (positive cells/section: 102±8 vs 177±9, normal chow vs HFD, p<0.05). This was evident throughout the rostral to caudal extent of the SFO (fold normal chow: rostral, 1.4±0.2; medial, 1.8±0.2; caudal, 1.8±0.1; all p<0.05). Similar findings were obtained when examining the ER chaperone protein disulfide isomerase (e.g. positive cells/section: 75±8 vs 107±4, normal chow vs HFD, p<0.05). Accumulating evidence indicates involvement of the PERK‐eIF2α pathway (protein kinase R like endoplasmic reticulum kinase‐eukaryotic initiation factor 2α) in cardiometabolic diseases and in line with this, the number of PERK expressing cells (cells/section: 32±10 vs 89±5, normal chow vs HFD, p<0.05) and downstream phosphorylated eIF2α positive cells (cells/section: 51±12 vs 125±24, normal chow vs HFD, p<0.05) was markedly elevated in the SFO following HFD feeding. The SFO can be anatomically subdivided into “core” and “shell” zones, which have distinct efferent projections to downstream nuclei involved in fluid, cardiovascular, and energy homeostasis; UPR activation was evident in both the core and shell zones. Given that a significant portion of SFO neurons that project to cardiometabolic nuclei are glutamatergic, we next investigated HFD‐induced ER stress in excitatory SFO neurons using double immunohistochemistry for UPR markers and the neuronal indicator calcium/calmodulin‐dependent kinase II (CAMKII; n=7/group). While the number of CAMKII positive neurons in the SFO was similar between HFD and normal chow groups, UPR activation was clearly evident in excitatory neurons during obesity as indicated by increased co‐localization of CAMKII with GRP78 (46+2 vs 59+3%, normal chow vs HFD, p<0.05), PDI (30+1 vs 49+3%, normal chow vs HFD, p<0.05), and phosphorylated eIF2α (40+2 vs 55+3%, normal chow vs HFD, p<0.05). Collectively, these findings indicate that obesity is associated with widespread anatomical ER stress throughout the SFO, including within excitatory neurons – suggesting that SFO ER stress during obesity has the potential to influence diverse downstream nuclei and thus physiological processes.Support or Funding Information1R01DK117007, 1R01HL141393