Chaperone-mediated autophagy (CMA) is a lysosomal-dependent proteolytic pathway recognized in mammals as significant for maintenance of cellular homeostasis during physiological and metabolic stress . The CMA mechanism has only recently been described as present in fish, thus its biological function is not well understood in these species. The objective of this study was to characterize the role of CMA as a metabolic regulator in rainbow trout through a loss-of-function approach. Chaperone-mediated autophagy was disrupted by CRISPR/Cas9-induced excision of the 9a exon within the lamp2a gene , thereby inhibiting the CMA rate-limiting process of protein substrate binding to the lysosomal membrane. Growth and nutrient metabolism and partitioning were characterized in wild type controls and lamp2a knockout (KO) rainbow trout during a high-carbohydrate nutritional challenge.
At 23 weeks post-hatch , KOs exhibited 20% greater body weight than controls ; this value increased to a 30% body weight difference after 7 weeks of consuming a high carbohydrate diet (Fig 1) . Gene expression analysis in brain suggested an increased appetite contributed to faster growth in KOs (P<0.05). Hepatic lipid profil e was also affected, with increased monounsaturated fatty acids and decreased polyunsaturated fatty acids in KOs relative to controls (P<0.01). Elevated expression of genes associated with lipid metabolism (P<0.05) suggesting an increased capacity for lipid synthesis in KOs . Hepatic proteome analysis indicated lamp2a KO affected the abundance of proteins linked with glucose, lipid, and glutathione metabolism, as well as mitochondrial respiration and proteasome activity (Fig 2,3).
Findings indicate that CMA is a central mechanism regulating growth , metabolism, energy production, and oxidative stress in rainbow trout. Further characterizing how CMA responds to metabolic, environmental, and immunological stressors will reveal how fish regulate cellular homeosta sis under metabolic stress and contribute to development of strategies that improve production efficiency.