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Author(s): Rachel L. Batterham [1, 2]; Michael A. Cowley [2, 3, 4]; Caroline J. Small [1]; Herbert Herzog [5]; Mark A. Cohen [1]; Catherine L. Dakin [1]; Alison M. Wren [...]

Author(s): Michael A. Cowley [1, 2]; James L. Smart [1, 2]; Marcelo Rubinstein [3]; Marcelo G. Cerdán [3]; Sabrina Diano [4]; Tamas L. Horvath [4, 5]; Roger D. Cone (corresponding [...]

PCSK1 encodes an enzyme required for prohormone maturation into bioactive peptides. A striking number of single nucleotide polymorphisms and rare mutations in PCSK1 are associated with a range of clinical phenotypes. Infants bearing 2 copies of a catalytically inactivating mutation, such as G209R, exhibit life-threatening chronic diarrhea and subsequently develop systemic endocrinopathies. Using CRISPR/Cas9 technology, we have engineered a mouse model bearing a G209R missense mutation in exon 6 of the murine Pcsk1 locus. Most pups homozygous for the G209R mutation succumbed by day 2, and surviving pups were severely dwarfed. In homozygous (but not heterozygous) pups, blood glucose levels were significantly lower, accompanied by elevated plasma insulin-like immunoreactivity and accumulation of large quantities of unprocessed proinsulin in the pancreas. Peptide hormone processing was also aberrant in G209R mouse pituitary, with mature adrenocorticotropin levels markedly reduced in homozygotes, accompanied by a significant accumulation of proopiomelanocortin. We also observed a significant reduction in proprotein convertase 1/3 protein in the brains of G209R homozygous mice by Western blotting, while proprotein convertase 2 (PC2) levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-melanocyte-stimulating hormone were not impaired. Analysis of intestinal cell types indicated a modest reduction of enteroendocrine cells in G209R homozygotes. We suggest that the G209R Pcsk1 mouse model recapitulates many of the dramatic neonatal deficiencies of human patients with this homozygous mutation. [ABSTRACT FROM AUTHOR]

The melanocortin peptide receptor MC5-R is found in many exocrine tissues. Genetic manipulation by targeted mutation using mice was done to investigate the function of melanocortin in exocrine gland regulation. Mutant mice had significant reduction of sebaceous lipid production resulting in defective water repulsion. There are numerous other functions attributed to melanocortin in the coordinated regulation of exocrine function. The regulatory mechanisms appear to be mediated by MC5-R.

Dopamine (DA) is a neurotransmitter in brain pathways, and the human dopamine D4 receptor (D4R) has received much attention due to its high affinity for the atypical antipsychotic clozapine and its polymorphic nature. Mutant mice lacking the protein were produced and analyzed to clarify the role of D4R. The mice outperformed wild-type mice on the rotarod, displaying supersensitivity to ethanol, methamphetamine and cocaine. It is proposed that D4R modulates usual, coordinated and drug-stimulated motor behaviours.

A physiological role for [Beta]-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of [Beta]-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional [Mu]-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.