Phosphoinositide 3-kinase gamma (PI3K gamma) expressed in immune cells is linked to neuroinflammation in several neurological diseases. However, the expression and role of PI3K gamma in preclinical traumatic brain injury (TBI) have not been investigated. In WT mice, we found that TBI induced rapid and extensive expression of PI3K gamma in neurons within the perilesional cortex and the ipsilateral hippocampal subfields (CA1, CA3), which peaked between 1 and 3 days and declined significantly 7 days after TBI. Intriguingly, the induction of neuronal PI3K gamma in these subregions of the brain spatiotemporally coincided with both the TBI-induced activation of the neuronal ER stress pathway (p-eIF2 alpha, ATF4, and CHOP) and neuronal cell death (marked by TUNEL-positive neurons) 3 days after TBI. Further, we show that the absence of PI3K gamma in knockout mice profoundly reduced the TBI-induced activation of the ER stress pathway and neuronal cell death. White matter disruption is a better predictor of long-term clinical outcomes than focal lesion size. We show that PI3K gamma deficiency not only reduced brain tissue loss but also alleviated white matter injury (determined by axonal injury and demyelination) up to 28 days after TBI. Importantly, PI3K gamma-knockout mice exhibited greater functional recovery including forepaw use, sensorimotor balance and coordination, and spatial learning and memory up to 28 days after TBI. These results unveil a previously unappreciated role for neuronal PI3K gamma in the regulation of ER stress associated with neuronal cell death, white matter damage, and long-term functional impairment after TBI.

Osteocalcin is an osteoblast-specific secreted protein that has been associated with endocrine roles in multiple aspects of energy metabolism. We examined whether undercarboxylated osteocalcin (ucOC) downregulates pancreatic lipase (PNLIP) expression in pancreatic acinar cells and then identified the downstream signaling pathway involved. We previously demonstrated that beta adrenergic blockade attenuates body weight/fat mass gain in high-fat diet-fed mice and that this effect is associated with decreased PNLIP expression in pancreatic acinar cells. In the present study, we first confirmed that the serum ucOC level is inversely correlated with PNLIP expression, i.e., mice exhibiting high serum levels of ucOC showed low PNLIP levels in the pancreas. In in vitro experiments using primary pancreatic acinar and 266-6 cells, ucOC downregulated PNLIP expression. cAMP/PICA signaling inhibitors significantly reversed ucOC-induced downregulation of PNLIP expression. ucOC promoted the phosphorylation of CAMP response element-binding protein 2 (ATF4). Overexpression of ATF4 significantly suppressed PNLIP expression. Knockdown of ATF4 by siRNA reversed the ucOC-induced down regulation of PNLIP expression. A luciferase reporter assay showed that ucOC suppressed PNLIP promoter transactivation. Chromatin immunoprecipitation and a luciferase reporter assay demonstrated that ATF4 directly bound to the CRE on the mouse PNLIP promoter and suppressed PNLIP transactivation. Knockdown of G-protein coupled receptor 6A (Gprc6a), a candidate receptor for mediating the response to ucOC in the bone-pancreas endocrine loop, by siRNA reversed the downregulating effect of ucOC on PNLIP expression. Taken together, ucOC downregulates pancreatic lipase expression in a cAMP/protein kinase A/ATF4-dependent manner. Gprc6a is a potential osteocalcin-sensing receptor that regulates PNLIP expression in pancreatic acinar cells.