br Introduction Proprotein convertase subtilisin kexin type
Introduction Proprotein convertase subtilisin/kexin type 1 (PCSK1), encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity (Jackson et al., 1997). Homozygous or compound heterozygous mutations are associated with severe forms of obesity (Farooqi et al., 2007; Jackson et al., 1997; Martin et al., 2013; O\'Rahilly et al., 1995). In addition, common nonsynonymous variants in PCSK1 confer risk of obesity (Benzinou et al., 2008). PC1/3 is essential for processing prohormones and neuropeptides in neuroendocrine tissues encompassing hypothalamus, pituitary, adrenal glands, and pancreatic islets (Jackson et al., 2003; Zhu et al., 2002). PC1/3 substrates include propeptides for pro-opiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), progrowth-hormone releasing hormone (GHRH), prothyrotropin-releasing hormone, proinsulin, and proglucagon (Creemers et al., 2006; Jackson et al., 2003; O\'Rahilly et al., 1995; Paquet et al., 1996; Zhu et al., 2002). PC1/3 deficiency results in several endocrinopathies, including growth hormone and adrenal insufficiency, hypogonadism, hypothyroidism, and hyperproinsulinemia in both humans and rodents (Jackson et al., 2003; O\'Rahilly et al., 1995; Zhu et al., 2002). In vitro functional assays of PC1/3 bioactivity suggest that most mutant carboxypeptidase identified in PC1/3-deficient patients affect the enzymatic activity of PC1/3 by influencing either the stability of PCSK1 mRNA or the production and secretion of mature PC1/3 (Farooqi et al., 2007; Martin et al., 2013). In the hypothalamus, the prohormone convertases, PC1/3 and PC2, function proximally to another proteolytic enzyme, carboxypeptidase E (CPE), to mediate the processing of POMC and other neuropeptides (Figure 1A). PC1/3 preferentially hydrolyzes the dibasic proteolytic cleavage site, KR, on the POMC propeptide to generate intermediate peptides including pro-adrenocorticotropic hormone (pro-ACTH), ACTH, and β-lipotropin (β-LPH) (Zhou et al., 1993). PC2 and CPE participate in downstream processing of ACTH and β-LPH into active α-melanocyte stimulating hormone (αMSH) and β-endorphin (βEP), respectively (Figure 1A) (Wardlaw, 2011). Importantly, αMSH is the endogenous agonist at the melanocortin-4 receptor (MC4R) leading to its activation and reduced food intake (Marsh et al., 1999). In PC1/3-deficient patients, concentrations of circulating POMC and ACTH intermediates are significantly increased, while levels of ACTH are unchanged or decreased, consistent with impaired POMC processing in the pituitary (O\'Rahilly et al., 1995). In PC1/3 null mice, the levels of Pomc transcript and protein in both hypothalamus and pituitary are significantly increased. Pituitary ACTH is undetectable and αMSH production is unchanged or decreased, consistent with impaired POMC processing in the hypothalamus and pituitary (Pan et al., 2005; Zhu et al., 2002). In PC1/3N222D/N222D mice, hypothalamic αMSH peptide is reduced, but POMC protein levels are not changed. And, in these animals, plasma concentrations of mature ACTH are not affected (Lloyd et al., 2006). These findings implicate important functions of PC1/3 in hypothalamic and pituitary POMC processing, with likely mutation- and even species-specific molecular compensations for reductions in the enzyme\'s activity. Moreover, prolylcarboxypeptidase (PRCP) functions as an enzyme to inactivate αMSH by removing one amino acid from its C terminus and is expressed in the hypothalamic neurons that send efferents to areas where αMSH is released (Wallingford et al., 2009). Inhibition of PRCP activity or deletion of Prcp in mice decreases food intake and protects the mice from obesity induced by a high-fat diet (Wallingford et al., 2009). Recently, we and another group reported in vitro differentiation protocols for the generation of hypothalamic neurons from human pluripotent stem cells (hPSCs) (Merkle et al., 2015; Wang et al., 2015). To investigate the impact of PCSK1 loss of function in a relevant human cell type, we differentiated PCSK1 short hairpin RNA (shRNA) knockdown and CRISPR-Cas9 knockout hESCs into neurons that closely resemble those of the arcuate nucleus (ARC) (Schneeberger et al., 2013) of the hypothalamus (Wang et al., 2015, 2016). Here, we assess POMC processing-specific cellular and molecular phenotypes in PC1/3-deficient cells and further examine the neuromolecular physiology of PCSK1 hypomorphic mutations. We find that the molecular phenotypes of hESC-derived hypothalamic neurons recapitulate those seen in the mouse. Interestingly, the upregulation of POMC production and consequences of downstream processing enzymes appear to compensate for the loss of PCSK1 in hESC-derived hypothalamic neurons, maintaining the production of αMSH and βEP.